Methods and Uses of Microbiota Compositions, Components, or Metabolites for Treating Neurodegenerative Diseases

Abstract

Disclosed are methods and compositions (e.g., comprising one or more microbial strains, one or more components, one or more metabolites, or combinations thereof) for treating neurodegenerative diseases, disorders, and conditions.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 245,648, filed September 17, 2021, and No. 63 / 330,148, filed April 12, 2022, the entire contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Many neurodegenerative diseases, disorders, or conditions, including but not limited to amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Huntington's disease (HD), and Parkinson's disease (PD), can cause degeneration of nerve cells and affect physical and / or mental function. Currently, there are no effective treatments for such diseases, including ALS, PD, or AD, and finding new drugs or treatment methods is a priority. Summary of the Invention

[0003] The present disclosure provides insight that the compositions (e.g., microbiota compositions) described herein may be used to treat a disease, disorder, or condition (e.g., of the nervous system (e.g., a neurodegenerative disease, disorder, or condition (e.g., ALS, AD, PD, HD, etc.)) in a subject (e.g., a mammal (e.g., a human, a mouse, etc.)). Among other things, the present disclosure describes techniques that may be used to treat, prevent, and / or reduce the risk of a disease, disorder, or condition (e.g., of the nervous system). In some embodiments, the present disclosure describes compositions and methods for evaluating the effect of administering such compositions (e.g., microbiota compositions described herein) to a subject and / or for identifying or characterizing the effect and / or modulation of levels of metabolites or metabolome in a subject upon administration of such compositions. In some embodiments, metabolites that may be modulated may be associated with a particular disease, disorder, or condition. In some embodiments, such techniques may be useful for identifying metabolite level differences in a particular subject (e.g., a patient) or population (e.g., before and after administration of a disclosed composition). Thus, the present disclosure also provides techniques that may be useful for identifying and / or evaluating the properties and effects of the disclosed compositions in specific subjects (e.g., patients) and / or populations, and thus providing subject-specific information on how to treat a disease, disorder, or condition (e.g., of the nervous system) in an individual subject or individual population. For example, in some embodiments, the techniques provided herein may be useful for treating and / or preventing a disease, disorder, or condition (e.g., of the nervous system) by identifying a subject-specific composition based on the metabolome in a subject-specific sample and administering a disclosed composition (e.g., a subject-specific composition) (e.g., to modulate the metabolome of the subject). Thus, the techniques described herein may be useful as therapeutics and tools for reducing the risk of certain diseases, disorders, or conditions (e.g., of the nervous system) and for treating and / or preventing such diseases, disorders, or conditions.

[0004] Among other things, the disclosure provides methods of treating or preventing a neurodegenerative disease, disorder, or condition. In some embodiments, the method comprises administering a composition comprising one or more microbial strains, components thereof (e.g., microbial components), or metabolites thereof (e.g., microbial metabolites (e.g., derived from a source other than the microbial strain (e.g., synthetically derived), derived from the microbial strain) to a subject in need thereof. In some embodiments, the method comprises administering a composition comprising one or more metabolites to the subject. In some embodiments, the method comprises administering a composition comprising one or more microbial metabolites to the subject. In some embodiments, the neurodegenerative disease, disorder, or condition is amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), or Huntington's disease (HD). In some embodiments, the neurodegenerative disease, disorder, or condition is ALS.

[0005] In some embodiments, the subject is an animal. In some embodiments, the subject is a mammal, for example, a mammal experiencing or susceptible to a disease, disorder, or condition described herein. In some embodiments, the animal is a vertebrate, for example, a mammal such as a non-human primate (especially a higher primate), sheep, dog, rodent (e.g., mouse or rat), guinea pig, goat, pig, cat, rabbit, or cow. In some embodiments, the animal is a non-mammal, such as a chicken, amphibian, reptile, or invertebrate. In some embodiments, the subject is a human.

[0006] In some embodiments, the subject suffers from or is susceptible to one or more neurodegenerative diseases, disorders, or conditions described herein. In some embodiments, the subject exhibits one or more symptoms of one or more neurodegenerative diseases, disorders, or conditions. In some embodiments, the subject has been diagnosed with one or more neurodegenerative diseases, disorders, or conditions described herein. In some embodiments, the subject is undergoing or has undergone a certain therapy to diagnose and / or treat one or more neurodegenerative diseases, disorders, or conditions.

[0007] In some embodiments, one or more microbial strains are derived from an animal microbiota. In some embodiments, one or more microbial strains are derived from a mammalian microbiota. In some embodiments, one or more microbial strains are derived from a human microbiota. In some embodiments, the human microbiota is a subject's microbiota. In some embodiments, the human microbiota is administered to maintain or regulate the subject's microbiota.

[0008] In some embodiments, the one or more components or metabolites (e.g., of one or more microbial strains, microbial metabolites (e.g., derived from a source other than a microbial strain (e.g., synthetically derived), derived from a microbial strain) are selected from Appendix 1, Appendix 3, or Appendix 4. In some embodiments, the metabolites may be derived from one or more microbial strains. In some embodiments, the metabolites may be derived from a source that is not a microbial strain, e.g., synthetically produced. In some embodiments, the one or more metabolites (e.g., microbial metabolites of one or more microbial strains, (e.g., derived from a source other than a microbial strain (e.g., synthetically derived), derived from a microbial strain) are or comprise bile acids. In some embodiments, the one or more metabolites (e.g., microbial metabolites of one or more microbial strains, (e.g., derived from a source other than a microbial strain (e.g., synthetically derived), derived from a microbial strain) are selected from Appendix 1, Appendix 3, or Appendix 4. In some embodiments, the metabolites may be derived from one or more microbial strains. In some embodiments, the one or more metabolites (e.g., microbial metabolites of one or more microbial strains, (e.g., derived from a source other than a microbial strain (e.g., synthetically derived), derived from a microbial strain) are selected from Appendix 1, Appendix 3, or Appendix 4. In some embodiments, the one or more components or metabolites are butyrylcamitine, theobromine, p-hydroxyphenylpyruvic acid, propionic acid, picolinic acid, 2-hydroxy-4 methylvaleric acid, N6-acetylcysteine, urocanic acid, N5-ethylglutamine, trigonelline, stachydrine, ectoine, 5-hydroxylysine, arginine (arg), cholic acid, 2-(4-hydroxyphenyl)propionic acid, N-acetyltryptophan, hydroxyproline, argininosuccinic acid, glutamic acid (Glu), sarcosine, 5-methoxyindoleacetic acid, indole-3-lactic acid, isovalerylalanine, N-acetylleucine, 1-methylhistidine, N-acetylphenylalanine, proline (Pro), or any combination thereof.In some embodiments, the one or more components or metabolites are 4-hydroxyphenylpyruvate, ectoine, gramine, N-acetyl-L-phenylalanine, nepsilon-acetyl-L-lysine, stachydrine, trigonelline, 3-ureidopropionic acid, theobromine, hippuric acid, imidazolepropionic acid, NG-methyl-L-arginine, transurocanic acid, N-acetyl-L-leucine, sarcosine, isobutyrylcalcine, acetyl-L-phenylalan ... Nitine, b-Hydroxyisovaleric acid, L-Theanine / N5-Ethylglutamine, 5-Hydroxylysine, Phenaceturic acid, Betaine, Hydroxyproline, Picolinic acid, 2-Aminoadipic acid, Glycerophosphocholine, Carnitine, Glycerol 3-phosphate, Argininosuccinic acid, Creatine, Terephthalic acid, Homocitrulline, Mucic acid, Homocysteine ​​sulfinic acid, Trimethyllysine, Spermidine, Glyoxylic acid, XA0013 C6H6O4S, 3-indoxyl sulfate, nicotinamide, N-formylglycine, ureidoglycolate, N-methylproline, glucaric acid, butyrylcarnitine, methionine sulfoxide, carboxymethyllysine, glycolic acid, phenaceturic acid, diethanolamine, phosphorylcholine, guanidinosuccinic acid, N-acetylhistidine, glyceric acid, S-methylmethionine, cysteine ​​glutathione disulfide, kynurenine, N-acetylphenylalanine, threonic acid, malic acid, 7,8-dihydrobiopterin, homovanillic acid, taurocholic acid, 5-methoxyindoleacetic acid, butyrate, b-hydroxyisovaleric acid, 2-oxoglutaric acid, N-acetyltryptophan, thiaproline, hypotaurine, cholic acid, acetoacetic acid, ethanolamine, guanidoacetic acid, S-sulfocysteine, myristic acid C14:0 XA0027, or any combination thereof.

[0009] In some embodiments, the one or more microbial strains are or include Gluconacetobacter hansenii, Terrisporobacter glycolicus, Coprococcus sp., Lactobacillus plantarum, Clostridium butyricum, Paenibacillus sp., Veillonella sp., Bifidobacterium sp., Bacillus subtilis, Acidaminococcus sp., or combinations thereof. In some embodiments, the one or more microbial strains are or include Gluconacetobacter hanseni, Terrisporobacter glycolicus, Coprococcus sp., Lactobacillus plantarum, Veillonella sp., Bifidobacterium sp., or combinations thereof. In some embodiments, the one or more microbial strains are or include Gluconacetobacter hanseni, Terrisporobacter glycolicus, Coprococcus catus, Lactobacillus plantarum, Veillonella atypica, Bifidobacterium breve, or a combination thereof. In some embodiments, the one or more microbial strains are or include Bacillus subtilis.

[0010] In some embodiments, the composition comprises two or more microbial strains. In some embodiments, the composition comprises five or more microbial strains. In some embodiments, the composition comprises ten or more microbial strains.

[0011] In some embodiments, the composition is administered topically, orally, subcutaneously, intravenously, intramuscularly, intracerebrally, intrathecally, intrarectally, ophthalmically, intravitreally, or suprachoroidally. In some embodiments, the composition is administered orally. In some embodiments, the composition is administered intracerebrally.

[0012] In some embodiments, the composition is formulated as a syrup, liquid, tablet, lozenge, gummy, capsule, powder, gel, film, injectable, or eye drops.

[0013] In some embodiments, each microbial strain of the one or more microbial strains is 1 ~10 15 In some embodiments, each of the one or more microbial strains is present in the composition at a concentration of at least 10 CFU. 6 In some embodiments, each of the one or more microbial strains in the composition is present in the composition at a concentration of 10 CFU. 1 Colony forming units (CFU) ~10 20 In some embodiments, each of the one or more microbial strains in the composition comprises 10 1 Colony forming units (CFU) ~10 15 In some embodiments, each of the one or more microbial strains in the composition comprises 10 6 CFU~10 15 In some embodiments, each of the one or more microbial strains in the composition comprises about 10 1 CFU~10 15 CFU, or about 10 2 CFU~10 14 CFU, or about 10 3 CFU~10 13 CFU, or about 10 4 CFU~10 13 CFU, or about 10 5 CFU~10 12 CFU, or about 10 6 CFU~10 11 CFU, or about 10 7 CFU~10 10 CFU, or about 10 8 CFU~10 9 CFU, or about 10 5 CFU~10 10 CFU, or about 10 8 CFU~10 12In some embodiments, each microbial strain of the one or more microbial strains in the composition comprises at least about 10 1 , 5×10 1 , 10 2 , 5×10 2 , 10 3 , 5×10 3 , 10 4 , 5×10 4 , 10 5 , 5×10 5 , 10 6 , 5×10 6 , 10 7 , 5×10 7 , 10 8 , 5×10 8 , 10 9 , 5×10 9 , 10 10 , 5×10 10 , 10 11 , 5×10 11 , 10 12 In some embodiments, each of the one or more microbial strains in the composition comprises at most about 10 15 , 5×10 14 , 10 14 , 5×10 13 , 10 13 , 5×10 12 , 10 12 , 5×10 11 , 10 11 , 5×10 10 , 10 10 , 5×10 9 , 10 9 , 5×10 8 , 10 8 In some embodiments, each microbial strain of the one or more microbial strains in the composition comprises the same number of CFU. In some embodiments, some microbial strains of the one or more microbial strains in the composition comprise different numbers of CFU.

[0014] The present disclosure includes, inter alia, compositions for treating or for use in treating a neurodegenerative disease, disorder, or condition comprising one or more microbial strains, components thereof, or metabolic products thereof. In some embodiments, the compositions described herein comprise one or more metabolic products (e.g., metabolic products derived from a microbial strain, derived from a source other than the microbial strain (e.g., synthetically derived), etc.), and the compositions are for treating a neurodegenerative disease, disorder, or condition.

[0015] The present disclosure provides a composition comprising one or more microbial strains selected from Gluconacetobacter hansenii, Terrisporobacter glycolicus, Coprococcus sp., Lactobacillus plantarum, Clostridium butyricum, Paenibacillus sp., Veillonella sp., Bifidobacterium sp., Bacillus subtilis, Acidaminococcus sp., or combinations thereof. In some embodiments, the composition comprises one or more microbial strains selected from Gluconacetobacter hanseni, Terrisporobacter glycolicus, Coprococcus sp., Lactobacillus plantarum, Veillonella atypica, Bifidobacterium sp., or combinations thereof. In some embodiments, the composition comprises a microbial strain. In some embodiments, the microbial strain is Bacillus subtilis. In some embodiments, the composition comprises at least two microbial strains selected from the group consisting of Gluconacetobacter hansenii, Terrisporobacter glycolicus, Coprococcus sp., Lactobacillus plantarum, Clostridium butyricum, Paenibacillus sp., Veillonella sp., Bifidobacterium sp., Bacillus subtilis, Acidaminococcus sp., or combinations thereof. In some embodiments, the composition comprises at least two microbial strains selected from the group consisting of Gluconacetobacter hanseni, Terrisporobacter glycolicus, Coprococcus sp., Lactobacillus plantarum, Veillonella atypica, Bifidobacterium sp., or combinations thereof.In some embodiments, the composition comprises at least five microbial strains selected from the group consisting of Gluconacetobacter hansenii, Terrisporobacter glycolicus, Coprococcus sp., Lactobacillus plantarum, Clostridium butyricum, Paenibacillus sp., Veillonella sp., Bifidobacterium sp., Bacillus subtilis, Acidaminococcus sp., or combinations thereof. In some embodiments, the composition comprises at least five microbial strains selected from the group consisting of Gluconacetobacter hanseni, Terrisporobacter glycolicus, Coprococcus sp., Lactobacillus plantarum, Veillonella atypica, Bifidobacterium sp., or combinations thereof. In some embodiments, the composition comprises or consists of Gluconacetobacter hansenii, Terrisporobacter glycolicus, Coprococcus sp., Lactobacillus plantarum, Clostridium butyricum, Paenibacillus sp., Veillonella sp., Bifidobacterium sp., Bacillus subtilis, Acidaminococcus sp. In some embodiments, the composition comprises or consists of Gluconacetobacter hanseni, Terrisporobacter glycolicus, Coprococcus sp., Lactobacillus plantarum, Veillonella atypica, Bifidobacterium sp.

[0016] In some embodiments, the compositions described herein comprise one or more metabolic products (e.g., metabolic products derived from a source other than a microbial strain (e.g., synthetically derived), derived from a microbial strain, etc.), and the compositions are for treating a neurodegenerative disease, disorder, or condition.

[0017] In some embodiments, the composition is for topical, oral, subcutaneous, intravenous, intramuscular, intracerebral, intrathecal, rectal, intraocular, intravitreal, or suprachoroidal administration. In some embodiments, the composition is for oral administration. In some embodiments, the composition is for intracerebral administration.

[0018] The present disclosure provides that the compositions described herein are for use in modulating one or more metabolic products (e.g., microbial metabolic products, (e.g., derived from a source other than a microbial strain (e.g., synthetically derived), derived from a microbial strain) in a subject.

[0019] The present disclosure provides that the compositions described herein are for use in regulating one or more characteristics in a subject.In some embodiments, the one or more characteristics are or include: (i) cell survival level, (ii) nucleic acid or protein level or activity, or a form thereof, (iii) microgliosis, (iv) astrocytosis, (v) ATP level, (vi) proteasome function, (vii) lysosomal function, (viii) oxidative stress, or (ix) inflammation.

[0020] The present disclosure provides that the compositions described herein are for use in characterizing the ability of one or more microbial strains to modulate one or more metabolic products (e.g., microbial metabolic products, (e.g., derived from a source other than the microbial strain (e.g., synthetically derived), derived from the microbial strain)) in a subject.

[0021] The disclosure provides that the use of the compositions described herein is for treating or ameliorating a neurodegenerative disease, disorder, or condition in a subject, wherein the disease, disorder, or condition is associated with one or more metabolites (e.g., microbial metabolites, (e.g., derived from a source other than a microbial strain (e.g., synthetically derived), derived from a microbial strain). The disclosure further provides that the compositions described herein are for use in treating or preventing or ameliorating a neurodegenerative disease, disorder, or condition, comprising one or more components or metabolites, which may be selected from Appendix 1, Appendix 3, or Appendix 4. In some embodiments, the use of the compositions described herein is for treating or ameliorating a disease, disorder, or condition. In some embodiments, the use of the compositions described herein is for treating or ameliorating a disease, disorder, or condition selected from ALS, AD, PD, or HD. In some embodiments, the use of the compositions described herein is for treating or ameliorating ALS.

[0022] The disclosure provides a method of screening microbial strains comprising contacting the microbial strain with a culture comprising a neuronal cell or neuronal cell line that models a neurodegenerative disease, disorder, or condition, and determining whether the microbial strain alters a characteristic of the culture, where the characteristic is associated with the neurodegenerative disease, disorder, or condition.

[0023] In some embodiments, the determining step comprises comparing the characteristic before and after performance of the contacting step, hi some embodiments, the determining step comprises comparing the characteristic to an equivalent reference after the contacting step.

[0024] In some embodiments, the comparable reference is a historical reference. In some embodiments, the comparable reference is a negative control reference. In some embodiments, the comparable reference is a positive control reference.

[0025] In some embodiments, the characteristic is a level of cell survival. In some embodiments, the characteristic is a level or activity of a nucleic acid or protein, or a form thereof. In some embodiments, the characteristic is or comprises microgliosis. In some embodiments, the characteristic is or comprises astrocytosis. In some embodiments, the characteristic is or comprises ATP levels. In some embodiments, the characteristic is or comprises proteasome function. In some embodiments, the characteristic is or comprises lysosomal function. In some embodiments, the characteristic is or comprises oxidative stress.

[0026] In some embodiments, the microbial strain or metabolites alter one or more characteristics of the culture. In some embodiments, the one or more characteristics are associated with a neurodegenerative disease, disorder, or condition described herein. In some embodiments, the one or more characteristics are or include (i) levels of cell survival, (ii) levels or activities of nucleic acids or proteins, or forms thereof, (iii) microgliosis, (iv) astrocytosis, (v) ATP levels, (vi) proteasome function, (vii) lysosomal function, (viii) oxidative stress, or (ix) inflammation.

[0027] The present disclosure provides a method comprising administering a composition comprising one or more microbial strains, components thereof, or metabolites thereof to a subject in need thereof. In some embodiments, the present disclosure provides a method comprising administering a composition comprising one or more components or metabolites to a subject in need thereof. In some embodiments, the metabolites can be derived from one or more microbial strains. In some embodiments, the metabolites can be derived from a source that is not a microbial strain, e.g., synthetically produced.

[0028] In some embodiments, the microbial strain may alter a characteristic of interest. In some embodiments, the characteristic is a level of cell survival. In some embodiments, the characteristic is a level or activity of a nucleic acid or protein, or a form thereof. In some embodiments, the characteristic is or includes microgliosis. In some embodiments, the characteristic is or includes astrocytosis. In some embodiments, the characteristic is or includes ATP levels. In some embodiments, the characteristic is or includes proteasome function. In some embodiments, the characteristic is or includes lysosomal function. In some embodiments, the characteristic is or includes oxidative stress.

[0029] In some embodiments, the microbial strain may alter one or more characteristics of a subject, in some embodiments, the one or more characteristics is or includes (i) levels of cell survival, (ii) levels or activity of nucleic acids or proteins or forms thereof, (iii) microgliosis, (iv) astrocytosis, (v) ATP levels, (vi) proteasome function, (vii) lysosomal function, (viii) oxidative stress, or (ix) inflammation.

[0030] In some embodiments the feature is associated with a neurodegenerative disease, disorder, or condition.

[0031] The present disclosure provides a method of characterizing a microbial strain comprising adding the microbial strain to a culture comprising a neuronal cell or neuronal cell line that models a neurodegenerative disease, disorder, or condition, and determining whether the microbial strain affects the level of one or more characteristics of the neuronal cell or neuronal cell line, where the one or more characteristics are associated with the neurodegenerative disease, disorder, or condition.

[0032] The present disclosure provides a method of manufacturing a pharmaceutical treatment comprising characterizing one or more microbial strains, components, or metabolites thereof, comprising the steps of adding one or more microbial strains to a culture comprising neuronal cells or neuronal cell lines that model a neurodegenerative disease, disorder, or condition, and determining whether the one or more microbial strains affect the level of one or more characteristics of the neuronal cells or neuronal cell lines, where the one or more characteristics are associated with the neurodegenerative disease, disorder, or condition.

[0033] The disclosure provides methods of producing pharmaceutical treatments that include adding one or more microbial strains, components, or metabolic products thereof (e.g., metabolic products derived from different sources (e.g., microbial strains, synthetic, etc.)) to a syrup, liquid, tablet, troche, gummy, capsule, powder, gel, film, injectable, or eye drop.

[0034] The present disclosure provides methods of evaluating a microbial strain for an ability to affect one or more characteristics of the culture, comprising adding the microbial strain to a culture comprising neuronal cells or a neuronal cell line that models a neurodegenerative disease, disorder, or condition, and determining whether the microbial strain affects the level of one or more characteristics of the neuronal cells or neuronal cell line, where the one or more characteristics are associated with the neurodegenerative disease, disorder, or condition.

[0035] In some embodiments, the method further comprises determining a level of one or more characteristics of the neuronal cells or neuronal cell line in the culture prior to adding the microbial strain to the culture, determining a level of the same one or more characteristics of the neuronal cells or neuronal cell line in the culture after adding the microbial strain to the culture, and comparing the level of the one or more characteristics determined prior to adding the microbial strain to the level of the one or more characteristics determined after adding the microbial strain.

[0036] In some embodiments, the one or more characteristics include (i) cell viability, (ii) levels or activity of nucleic acids or proteins or forms thereof, (iii) microgliosis, (iv) ATP levels, (v) inflammation, (vi) astrocytosis, (vii) proteasome function, (viii) lysosomal function, (ix) oxidative stress, or (x) a combination thereof.

[0037] The present disclosure provides that the compositions described herein are for use in the treatment or prevention of a neurodegenerative disease, disorder, or condition that includes one or more microbial strains, components thereof, or metabolic products thereof. In some embodiments, the compositions described herein are for use in the treatment or prevention of a neurodegenerative disease, disorder, or condition that includes one or more metabolic products (e.g., metabolic products derived from a microbial strain, derived from a source other than the microbial strain (e.g., synthetically derived).

[0038] The present disclosure provides that the compositions described herein are for use in the treatment or prevention of a neurodegenerative disease, disorder, or condition comprising one or more microbial strains, components thereof, or metabolites thereof, and the one or more components or metabolites of the one or more microbial strains are selected from Appendix 1, Appendix 3, or Appendix 4. The present disclosure further provides that the compositions described herein are for use in the treatment or prevention of a neurodegenerative disease, disorder, or condition comprising one or more components or metabolites, which may be selected from Appendix 1, Appendix 3, or Appendix 4.

[0039] In some embodiments, the metabolites may be derived from one or more microbial strains. In some embodiments, the metabolites may be derived from a source that is not a microbial strain, e.g., synthetically produced. In some embodiments, the one or more components or metabolites (e.g., of one or more microbial strains) are bile acids. In some embodiments, the one or more components or metabolites (e.g., of one or more microbial strains) are tauroursodeoxycholic acid. In some embodiments, the one or more components or metabolites are butyrylcamitine, theobromine, p-hydroxyphenylpyruvic acid, propionic acid, picolinic acid, 2-hydroxy-4 methylvaleric acid, N6-acetylcysine, urocanic acid, N5-ethylglutamine, trigonelline, stachydrine, ectoine, 5-hydroxylysine, arginine (Arg), cholic acid, 2-(4-hydroxyphenyl)propionic acid, N-acetyltryptophan, hydroxyproline, argininosuccinic acid, glutamic acid (Glu), sarcosine, 5-methoxyindoleacetic acid, indole-3-lactic acid, isovalerylalanine, N-acetylleucine, 1-methylhistidine, N-acetylphenylalanine, proline (Pro), or any combination thereof.In some embodiments, the one or more components or metabolites are 4-hydroxyphenylpyruvate, ectoine, gramine, N-acetyl-L-phenylalanine, nepsilon-acetyl-L-lysine, stachydrine, trigonelline, 3-ureidopropionic acid, theobromine, hippuric acid, imidazolepropionic acid, NG-methyl-L-arginine, transurocanic acid, N-acetyl-L-leucine, sarcosine, isobutyrylcalcine, acetyl-L-phenylalan ... Nitine, b-Hydroxyisovaleric acid, L-Theanine / N5-Ethylglutamine, 5-Hydroxylysine, Phenaceturic acid, Betaine, Hydroxyproline, Picolinic acid, 2-Aminoadipic acid, Glycerophosphocholine, Carnitine, Glycerol 3-phosphate, Argininosuccinic acid, Creatine, Terephthalic acid, Homocitrulline, Mucic acid, Homocysteine ​​sulfinic acid, Trimethyllysine, Spermidine, Glyoxylic acid, XA0013 C6H6O4S, 3-indoxyl sulfate, nicotinamide, N-formylglycine, ureidoglycolate, N-methylproline, glucaric acid, butyrylcarnitine, methionine sulfoxide, carboxymethyllysine, glycolic acid, phenaceturic acid, diethanolamine, phosphorylcholine, guanidinosuccinic acid, N-acetylhistidine, glyceric acid, S-methylmethionine, cysteine ​​glutathione disulfide, kynurenine, N-acetylphenylalanine, threonic acid, malic acid, 7,8-dihydrobiopterin, homovanillic acid, taurocholic acid, 5-methoxyindoleacetic acid, butyrate, b-hydroxyisovaleric acid, 2-oxoglutaric acid, N-acetyltryptophan, thiaproline, hypotaurine, cholic acid, acetoacetic acid, ethanolamine, guanidoacetic acid, S-sulfocysteine, myristic acid C14:0 XA0027, or any combination thereof.

[0040] In some embodiments, the compositions described herein are for use in the treatment or prevention of a neurodegenerative disease, disorder, or condition involving one or more microbial strains, components thereof, or metabolic products thereof.

[0041] In some embodiments, the composition comprises one or more microbial strains selected from Gluconacetobacter hansenii, Terrisporobacter glycolicus, Coprococcus sp., Lactobacillus plantarum, Clostridium butyricum, Paenibacillus sp., Veillonella sp., Bifidobacterium sp., Bacillus subtilis, Acidaminococcus sp., or combinations thereof. In some embodiments, the composition described herein is for the use described herein and comprises one or more microbial strains selected from Gluconacetobacter hanseni, Terrisporobacter glycolicus, Coprococcus sp., Lactobacillus plantarum, Veillonella atypica, Bifidobacterium sp., or combinations thereof. In some embodiments, the composition described herein is for the use described herein and comprises a microbial strain. In some embodiments, the composition described herein is for the use described herein and the microbial strain is Bacillus subtilis. In some embodiments, the compositions described herein are for the uses described herein and comprise at least two microbial strains selected from the group consisting of Gluconacetobacter hansenii, Terrisporobacter glycolicus, Coprococcus sp., Lactobacillus plantarum, Clostridium butyricum, Paenibacillus sp., Veillonella sp., Bifidobacterium sp., Bacillus subtilis, Acidaminococcus sp., or combinations thereof.In some embodiments, the compositions described herein are for the uses described herein and comprise at least two microbial strains selected from the group consisting of Gluconacetobacter hanseni, Terrisporobacter glycolicus, Coprococcus sp., Lactobacillus plantarum, Veillonella atypica, Bifidobacterium sp., or combinations thereof. In some embodiments, the compositions described herein are for the uses described herein and comprise at least five microbial strains selected from the group consisting of Gluconacetobacter hansenii, Terrisporobacter glycolicus, Coprococcus sp., Lactobacillus plantarum, Clostridium butyricum, Paenibacillus sp., Veillonella sp., Bifidobacterium sp., Bacillus subtilis, Acidaminococcus sp., or combinations thereof. In some embodiments, the compositions described herein are for the uses described herein and comprise at least five microbial strains selected from the group consisting of Gluconacetobacter hanseni, Terrisporobacter glycolicus, Coprococcus sp., Lactobacillus plantarum, Veillonella atypica, Bifidobacterium sp., or combinations thereof.In some embodiments, the compositions described herein are for the uses described herein and comprise or consist of Gluconacetobacter hansenii, Terrisporobacter glycolicus, Coprococcus sp., Lactobacillus plantarum, Clostridium butyricum, Paenibacillus sp., Veillonella sp., Bifidobacterium sp., Bacillus subtilis, Acidaminococcus sp. In some embodiments, the compositions described herein are for the uses described herein and comprise or consist of Gluconacetobacter hanseni, Terrisporobacter glycolicus, Coprococcus sp., Lactobacillus plantarum, Veillonella atypica, Bifidobacterium.

[0042] The present disclosure provides an injectable comprising a composition described herein.

[0043] The present disclosure provides dietary supplements comprising the compositions described herein.

[0044] The present disclosure provides kits comprising the compositions described herein for use in treating or preventing a neurodegenerative disease, disorder, or condition.

[0045] The present disclosure provides, inter alia, a method of treating amyloid plaques or reducing plaque burden, plaque number, or plaque size in a subject diagnosed with a neurodegenerative disease, disorder, or condition. In some embodiments, the methods described herein include administering a composition comprising one or more microbial strains or microbial components thereof to a subject in need thereof. In some embodiments, the methods described herein include administering a composition comprising one or more microbial metabolites to a subject in need thereof. In some embodiments, the neurodegenerative disease, disorder, or condition is amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), or Huntington's disease (HD). In some embodiments, the neurodegenerative disease, disorder, or condition is AD.

[0046] In some embodiments, the amyloid plaques are located in the subject's brain. In some embodiments, the amyloid plaques are located in the cortex of the subject's brain. In some embodiments, the amyloid plaques are located in the hippocampus of the subject's brain. In some embodiments, the amyloid plaques are located in the cortex and hippocampus of the subject's brain.

[0047] In some embodiments, microglia in the subject's brain are reduced after administering a composition described herein. In some embodiments, microglia located within amyloid plaques in the subject's brain are reduced after administering a composition described herein.

[0048] In some embodiments, plaque burden is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%. In some embodiments, plaque number is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%. In some embodiments, plaque size is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%.

[0049] In some embodiments, after the administering step, the subject exhibits stabilization or improvement of one or more symptoms associated with the neurodegenerative disease, disorder, or condition. In some embodiments, the one or more symptoms associated with the neurodegenerative disease, disorder, or condition are or include cognition, memory, reasoning, mood swings, personality changes, anxiety, aggression, hallucinations, delusions, paranoia, restlessness, agitation, muscle contractions, or duration of concentration.

[0050] These and other aspects encompassed by the present disclosure are described in more detail below and in the claims.

[0051] definition The scope of the present invention is defined by the claims attached hereto, and is not limited by any particular embodiment described herein. Those skilled in the art will recognize, upon reading this specification, various modifications that may be equivalent to such described embodiments or otherwise fall within the scope of the claims. Generally, terms used herein follow their understood meanings within the art, unless expressly indicated otherwise. Explicit definitions of certain terms are provided below, and the meaning of these and other terms in specific instances throughout this specification will be clear to those skilled in the art from the context.

[0052] The use of ordinal terms such as "first," "second," "third," etc. in the claims to modify claim elements does not, in itself, convey any priority, precedence, or ordering of the elements of one claim relative to another claim, or the temporal order in which acts of a method are performed, but is merely used as a label to distinguish the elements of a claim within one claim having a particular name from elements within another claim having the same name (in the absence of the use of the ordinal terminology).

[0053] The articles "a" and "an" as used herein should be understood to include plural referents unless clearly indicated to the contrary. A claim or description including "or" between one or more members of a group is considered satisfied if one, more than one, or all of the members of the group are present in, employed in, or otherwise relevant to a given product or process, unless clearly indicated to the contrary or otherwise evident from the context. In some embodiments, exactly one member of a group is present in, employed in, or otherwise relevant to a given product or process. In some embodiments, more than one or all members of a group are present in, employed in, or otherwise relevant to a given product or process. It should be understood that the present invention encompasses all modifications, combinations, and permutations of one or more limitations, elements, clauses, descriptive terms, etc. from one or more of the enumerated claims that are introduced into another claim that is dependent on the same base claim (or any other claim, if relevant), unless otherwise indicated or unless it would be apparent to one of ordinary skill in the art that a contradiction or inconsistency would result. When elements are presented as a list (e.g., in a Markush group or similar format), it is understood that each subgroup of elements is also disclosed, and any element(s) can be removed from the group. In general, when an embodiment or aspect is referred to as "comprising" certain elements, features, etc., it should be understood that a particular embodiment or aspect "consists" or "consists essentially of" such elements, features, etc. For purposes of brevity, the embodiments have not been specifically described in so many words in all instances herein. It should also be understood that any embodiment or aspect may be expressly excluded from the claims, regardless of whether a specific exclusion is described herein.

[0054] Administration: As used herein, the term "administration" typically refers to administration of a composition to a subject or system to achieve delivery of the agent to the subject or system. In some embodiments, the agent is or is included in a composition, and in some embodiments, the agent is produced through metabolism of the composition or one or more components thereof. Those skilled in the art will recognize various routes that may be utilized for administration to a subject, e.g., a human, in the appropriate circumstances. For example, in some embodiments, administration may be intraocular, oral, parenteral, topical, etc. In some specific embodiments, administration may be bronchial (e.g., by intratracheal instillation), oral, dermal (e.g., may be or include one or more of topical to the dermis, intradermal, interdermal, transdermal, etc.), enteral, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, in a specific organ (e.g., intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, vitreous, etc. In many embodiments provided by the present disclosure, administration is oral administration. In some embodiments, administration may involve only a single dose. In some embodiments, administration may involve application of a fixed number of doses. In some embodiments, administration may involve administration that is intermittent (e.g., multiple doses separated in time) and / or periodic (e.g., individual doses separated by a common period of time) administration. In some embodiments, administration may involve continuous administration (e.g., perfusion) for at least a selected period of time. Administration of cells may be by any suitable route that results in delivery to a desired location in a subject, where at least a portion of the delivered cells or components of cells remain viable. The period of survival of cells after administration to a subject may be as short as a few hours, e.g., 24 hours, to as long as days or years, i.e., long-term engraftment. In some embodiments, administration involves delivery of a bacterial extract or preparation that includes one or more bacterial metabolic products and / or by-products, but is completely devoid of viable bacterial cells.

[0055] Analog: As used herein, the term "analog" refers to a substance that shares one or more specific structural features, elements, components, or moieties with a reference substance. Typically, an "analog" exhibits significant structural similarity with the reference substance, e.g., sharing a core or consensus structure, but differs in certain individual respects. In some embodiments, an analog is a substance that can be generated from a reference substance, e.g., by chemical manipulation of the reference substance. In some embodiments, an analog is a substance that can be generated through the implementation of a synthetic process that is substantially similar (e.g., shares multiple steps) to that which generates the reference substance. In some embodiments, an analog is generated or can be generated through the implementation of a synthetic process that is different from that used to generate the reference substance.

[0056] Approximately: When applied to one or more values ​​of interest, includes values ​​that are similar to a stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values ​​that are within ±10% (more or less) of a stated reference value, unless otherwise stated or otherwise clear from the context (except where such number exceeds 100% of the possible values).

[0057] Equivalent: As used herein, the term "equivalent" refers to two or more agents, entities, situations, sets of conditions, subjects, etc. that may not be identical to each other, but are sufficiently similar to allow comparisons between them, so that a person skilled in the art will understand that conclusions can be reasonably drawn based on observed differences or similarities. In some embodiments, equivalent sets of conditions, situations, individuals, or populations are characterized by a number of substantially identical characteristics and one or a few altered characteristics. A person skilled in the art will understand what degree of identity is required in any given situation for two or more such agents, entities, situations, sets of conditions, etc. to be considered equivalent in context. For example, a person skilled in the art will understand that sets of situations, individuals, or populations are equivalent to each other when they are characterized by a sufficient number and type of substantially identical characteristics to warrant a reasonable conclusion that the results obtained under or with the different sets of situations, individuals, or populations, or the differences in the observed phenomena, are caused by or indicate variations in the characteristics that are diverse.

[0058] Conservative: As used herein, refers to the case when describing a conservative amino acid substitution, which involves the replacement of an amino acid residue with another amino acid residue having a side chain R group with similar chemical properties (e.g., charge or hydrophobicity). Generally, a conservative amino acid substitution does not substantially change the desired functional property of a protein, e.g., the ability of a receptor to bind a ligand. Examples of groups of amino acids having side chains with similar chemical properties include aliphatic side chains such as glycine (Gly, G), alanine (Ala, A), valine (Val, V), leucine (Leu, L), and isoleucine (Ile, I); aliphatic hydroxyl side chains such as serine (Ser, S) and threonine (Thr, T); amide-containing side chains such as asparagine (Asn, N) and glutamine (Gln, Q); aromatic side chains such as phenylalanine (Phe, F), tyrosine (Tyr, Y), and tryptophan (Trp, W); basic side chains such as lysine (Lys, K), arginine (Arg, R), and histidine (His, H); acidic side chains such as aspartic acid (Asp, D) and glutamic acid (Glu, E); and sulfate-containing side chains such as cysteine ​​(Cys, C) and methionine (Met, M). Conservative amino acid substitutions include, for example, valine / leucine / isoleucine (Val / Leu / Ile, V / L / I), phenylalanine / tyrosine (Phe / Tyr, F / Y), lysine / arginine (Lys / Arg, K / R), alanine / valine (Ala / Val, A / V), glutamate / aspartate (Glu / Asp, E / D), and asparagine / glutamine (Asn / Gln, N / Q). In some embodiments, a conservative amino acid substitution can be the substitution of any naturally occurring residue in a protein with alanine, for example, as used in alanine scanning mutagenesis. In some embodiments, conservative substitutions are made that have a positive value in the PAM250 log-likelihood matrix, as disclosed in Gonnet, GH et al., 1992, Science 256:1443-1445, which is incorporated herein by reference in its entirety. In some embodiments, the substitutions are reasonably conservative substitutions, and the substitutions have non-negative values ​​in the PAM250 log-likelihood matrix. [Table 1]

[0059] Control: as used herein refers to the art-understood meaning of "control," which is a standard against which results are compared. Typically, a control is used to enhance the integrity of an experiment by isolating a variable in order to make conclusions about such a variable. In some embodiments, a control is a reaction or assay that is performed simultaneously with a test reaction or assay to provide a comparison target. "Control" also includes "control animals." A "control animal" can have a modification described herein, a different modification described herein, or no modification (i.e., a wild-type animal). In one experiment, the "test" (i.e., the variable being tested) is applied. In a second experiment, the "control," the variable being tested, is not applied. In some embodiments, a control is a historical control (i.e., a control of a previously performed test or assay, or a previously known amount or result). In some embodiments, a control is or includes a printed or otherwise kept record. A control can be a positive or negative control.

[0060] Determining, Measuring, Evaluating, Assessing, Assaying, and Analyzing: Determining, measuring, evaluating, assessing, assaying, and analyzing are used interchangeably herein to refer to any form of measurement, including determining whether an element is present. These terms include both quantitative and / or qualitative determinations. Assaying can be relative or absolute. "Assaying for the presence of" can be determining the amount of something present and / or determining whether it is present or absent.

[0061] Dosage form: Those skilled in the art will understand that the term "dosage form" may be used to refer to a physically discrete unit of an agent (e.g., a therapeutic agent) for administration to a subject. Typically, each such unit contains a predetermined amount of the agent. In some embodiments, such an amount is a unit dosage (or a whole fraction thereof) appropriate for administration according to a dosing regimen that has been determined to correlate with a desired or beneficial outcome when administered to a relevant population (i.e., in a therapeutic dosing regimen). Those skilled in the art will understand that the total amount of a therapeutic composition or agent to be administered to a particular subject is determined by one or more attending physicians and may involve administration of multiple dosage forms.

[0062] Dosage regimen: Those skilled in the art will understand that the term "dosage regimen" may be used to refer to a set of unit doses (typically more than one) administered individually to a subject, typically separated by a period of time. In some embodiments, a given drug has a recommended dosing regimen that may involve one or more doses. In some embodiments, the dosing regimen includes multiple doses, each of which is separated in time from the other doses. In some embodiments, the individual doses are separated from each other by periods of the same length, and in some embodiments, the dosing regimen includes multiple doses, and at least two different periods separating the individual doses. In some embodiments, all doses in the dosing regimen are the same unit dose amount. In some embodiments, different doses in the dosing regimen are different amounts. In some embodiments, the dosing regimen includes a first dose in a first dose amount, followed by one or more additional doses in a second dose amount that is different from the first dose amount. In some embodiments, the dosing regimen includes a first dose in a first dose amount, followed by one or more additional doses in a second dose amount that is the same as the first dose amount. In some embodiments, the dosing regimen correlates with a desired or beneficial outcome when administered across a relevant population.

[0063] Engineered: In general, the term "engineered" refers to the aspect of being manipulated by the hand of man. For example, a cell or organism is considered "engineered" when it has been manipulated such that its genetic information is changed (e.g., new genetic material not previously present is introduced, for example, by transformation, mating, somatic cell hybridization, transfection, transduction, or other mechanisms, or previously present genetic material is changed or removed, for example, by substitution or deletion mutations, or by mating protocols). As is common practice and understood by those skilled in the art, the progeny of an engineered polynucleotide or cell is typically still referred to as "engineered", despite the actual manipulation performed on the previous entity.

[0064] Excipient: as used herein refers to an inactive (e.g., non-therapeutic) agent that may be included in a pharmaceutical composition, for example, to provide or contribute to a desired consistency or stabilizing effect. In some embodiments, suitable pharmaceutical excipients may include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, nonfat dry milk, glycerol, propylene, glycol, water, ethanol, and the like.

[0065] Functional: As used herein, a "functional" biomolecule is a biomolecule in a form in which it exhibits the property and / or activity for which it is characterized. A biomolecule can have two functions (i.e., bifunctional) or many functions (i.e., multifunctional).

[0066] Gene: as used herein refers to a DNA sequence in a chromosome that codes for a product (e.g., an RNA product and / or a polypeptide product). In some embodiments, a gene includes coding sequences (i.e., sequences that code for a particular product). In some embodiments, a gene includes non-coding sequences. In some particular embodiments, a gene may include both coding (e.g., exon) and non-coding (e.g., intron) sequences. In some embodiments, a gene may include one or more regulatory sequences (e.g., promoters, enhancers, etc.) and / or intron sequences that may, for example, control or affect one or more aspects of gene expression (e.g., cell type specific expression, inducible expression, etc.). For purposes of clarity, it should be noted that the term "gene" as used in this disclosure generally refers to a portion of a nucleic acid that codes for a polypeptide or a fragment thereof, and that the term may optionally encompass regulatory sequences as will be clear to one of skill in the art from the context. This definition is not intended to exclude the application of the term "gene" to non-protein-coding expression units, but rather to clarify that in most cases, the term as used herein refers to a nucleic acid that codes for a polypeptide.

[0067] Improve, increase, enhance, inhibit, or reduce: As used herein, the terms "improve," "increase," "enhance," "inhibit," "reduce," or their grammatical equivalents, refer to a value that is relative to a baseline or other reference measurement. In some embodiments, the value is statistically significantly different from the baseline or other reference measurement. In some embodiments, a suitable reference measurement may be or include a measurement in a particular system (e.g., in a single individual) under otherwise comparable conditions in the absence (e.g., before and / or after) of a particular agent or treatment, or in the presence of a suitable comparable reference agent. In some embodiments, a suitable reference measurement may be or include a measurement in a comparable system that is known or expected to respond in a particular way in the presence of the relevant agent or treatment. In some embodiments, a suitable reference is a negative reference, and in some embodiments, a suitable reference is a positive reference.

[0068] Isolated: As used herein, refers to substances and / or entities that are (1) separated from at least some of the components with which they were originally produced (whether in nature and / or in an experimental setting) and / or (2) designed, produced, prepared, and / or manufactured by the hand of man. In some embodiments, isolated substances or entities may be concentrated, and in some embodiments, isolated substances or entities may be pure. In some embodiments, isolated substances and / or entities may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater than about 99% of other components with which they were originally associated. In some embodiments, an isolated agent is about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components. In some embodiments, as will be understood by those skilled in the art, a substance may still be considered "enriched," "isolated," or "pure" after being combined with certain other components, such as, for example, one or more carriers or excipients (e.g., buffers, solvents, water, etc.), and in such embodiments, the isolation or purity rate of the substance is calculated without including such carriers or excipients. Those skilled in the art are aware of various techniques for isolating (e.g., concentrating or purifying) a substance or agent (e.g., using one or more of fractionation, extraction, precipitation, or other separation).

[0069] Level: As used herein, the term "level" refers to a scale of the amount or quantity of a substance (e.g., a metabolite). In some embodiments, the level may simply be the presence or absence of a substance. The level of a substance may be expressed in multiple ways or formats. For example, in some embodiments, the level is expressed as a percentage (%), a measure of weight (e.g., mg, μg, ng, etc.), a measure of concentration (e.g., mg / mL, μg / mL, ng / mL, etc.), a measure of volume (e.g., mL, μL, nL, etc.), % change, etc.

[0070] Metabolite: As used herein, the term "metabolite" refers to a substance (e.g., a small molecule, a macromolecule, an organic compound, or an inorganic compound) that is made or used during metabolism. Metabolism is generally understood as the process by which a substance (e.g., a food, a drug, a chemical, a cell, or a tissue) is chemically broken down. In some embodiments, a metabolite is an end product. In some embodiments, a metabolite is an intermediate. Exemplary metabolic products are provided herein, for example, in Appendices 1-1, 1-3, and 3. Exemplary metabolic pathways are provided herein, for example, in Appendices 1-2. In some embodiments, a metabolite can be produced or made by an organism. In some embodiments, a metabolite can be produced or made by a microorganism (e.g., a microbial strain). In some embodiments, a microbial metabolite is produced or made by a microbial strain. In some embodiments, a metabolite can be produced or made naturally (e.g., by an organism (e.g., a microorganism (e.g., a microbial strain)). In some embodiments, a metabolite can be produced or made synthetically (e.g., from a source that is not a microbial strain (e.g., synthetically produced)).

[0071] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to a composition in which an active agent is formulated with one or more pharma- ceutically acceptable carriers. In some embodiments, the active agent is present in an amount of unit dose suitable for administration in a treatment regimen that exhibits a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, the pharmaceutical composition may be specially formulated for administration in solid or liquid form, including those adapted for the following: intraocular, intravitreal, suprachoroidal, oral, subcutaneous, intravenous, intramuscular, intracerebral, intrathecal administration, e.g., drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., tablets targeted for oral, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue, capsules, powders, etc. In some embodiments, the active agent is or may include a cell or population of cells (e.g., a culture, e.g., a culture of an ellagitannin enzyme synthesizing (EES) microorganism). In some embodiments, the active agent may be or comprise an extract or component of a cell or population of cells (e.g., a culture). In some embodiments, the active agent may be or comprise an isolated, purified, or pure compound. In some embodiments, the active agent may have been synthesized in vitro (e.g., via chemical and / or enzymatic synthesis). In some embodiments, the active agent may be or comprise a natural product (whether isolated from its natural source or synthesized in vitro).

[0072] Pharmaceutically acceptable: As used herein, the term "pharmaceutical acceptable," which may be used, for example, in reference to a carrier, diluent, or excipient used to formulate a pharmaceutical composition disclosed herein, means the carrier, diluent, or excipient is compatible with the other ingredients of the composition and not deleterious to the recipient thereof.

[0073] Pharmaceutically acceptable carrier: As used herein, the term "pharmaceutical acceptable carrier" refers to a pharma- ceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, that is involved in carrying or transporting the subject compounds from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the subject (e.g., patient). Some examples of materials which can function as pharma- ceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; excipients such as powdered tragacanth, malt, gelatin, talc, cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar-agar, buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, pH buffer solutions, polyesters, polycarbonates, and / or polyanhydrides, and other non-toxic compatible substances employed in pharmaceutical formulations.

[0074] Prebiotic: As used herein, "prebiotic" refers to an ingredient that enables or promotes specific changes in both composition and / or activity in the gastrointestinal microflora that may (or may not) confer a benefit to the host. In some embodiments, the prebiotic may include one or more of the following: The prebiotic includes pome fruit extract, berry extract, and walnut extract.

[0075] Prevention: The term "prevention" as used herein refers to a delay in the onset and / or a reduction in the frequency and / or severity of one or more symptoms of a particular disease, disorder, or condition. In some embodiments, prevention is assessed on a population basis such that an agent is considered to "prevent" a particular disease, disorder, or condition if a statistically significant reduction in the onset, frequency, and / or intensity of one or more symptoms of the disease, disorder, or condition is observed in a population susceptible to the disease, disorder, or condition. In some embodiments, prevention may be considered complete, for example, when the onset of a disease, disorder, or condition has been delayed for a predetermined period of time.

[0076] Reference: as used herein describes a standard or control against which a comparison is made. For example, in some embodiments, an agent, animal, individual, population, sample, sequence, or value of interest is compared to a reference or control agent, animal, individual, population, sample, sequence, or value. In some embodiments, the reference or control is tested and / or determined substantially simultaneously with the test or determination of interest. In some embodiments, the reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as will be understood by those of skill in the art, the reference or control is determined or characterized under conditions or circumstances equivalent to those being evaluated. Those of skill in the art will understand when sufficient similarity exists to justify reliance on and / or comparison to a particular potential reference or control. In some embodiments, the reference is a negative control reference, and in some embodiments, the reference is a positive control reference.

[0077] Risk: As understood from the context, "risk" of a disease, disorder, and / or condition refers to the likelihood that a particular individual will develop the disease, disorder, and / or condition. In some embodiments, the risk is expressed as a percentage. In some embodiments, the risk is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or up to 100%. In some embodiments, the risk is expressed relative to the risk associated with a reference sample or a group of reference samples. In some embodiments, the reference sample or a group of reference samples has a known risk of the disease, disorder, condition, and / or event. In some embodiments, the reference sample or a group of reference samples is derived from an individual comparable to the particular individual. In some embodiments, the relative risk is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.

[0078] Sample: As used herein, the term "sample" typically refers to an aliquot of material obtained from or derived from a source of interest. In some embodiments, the source of interest is a biological or environmental source. In some embodiments, the source of interest may be or include a cell, or an organism such as a microorganism, a plant, or an animal (e.g., a human). In some embodiments, the source of interest is or includes a biological tissue or fluid. In some embodiments, the biological tissue or fluid may be or include amniotic fluid, aqueous humor, peritoneal fluid, bile, bone marrow, blood, breast milk, cerebrospinal fluid, earwax, chyle, chyme, ejaculate, endolymph, exudate, feces, gastric acid, gastric juice, lymph, mucus, pericardial fluid, perilymph, peritoneal fluid, pleural fluid, pus, catarrhal secretions, saliva, sebum, semen, serum, smegma, phlegm, synovial fluid, sweat, tears, urine, vaginal secretions, vitreous humor, vomit, plasma, mucus, digestive fluids, stool, and / or combinations or component(s) thereof. In some embodiments, the biological fluid may be or include intracellular fluid, extracellular fluid, intravascular fluid (plasma), interstitial fluid, lymph, and / or transcellular fluid. In some embodiments, the biological fluid may be or include phytoexudates. In some embodiments, the biological tissue or sample may be obtained, for example, by aspirate, biopsy (e.g., fine needle or tissue biopsy), swab (e.g., oral, nasal, skin, or vaginal swab), scraping, surgery, washing or lavage (e.g., bronchoalveolar epithelium, duct, nasal, ocular, oral, uterine, vaginal, or other washing or lavage). In some embodiments, the biological sample is or includes cells obtained from an individual. In some embodiments, the sample is a "primary sample" obtained directly from the source of interest by any suitable means. In some embodiments, as will be clear from the context, the term "sample" refers to a preparation obtained by processing the primary sample (e.g., by removing one or more components of the primary sample and / or by adding one or more agents to the primary sample), for example, by filtering using a semi-permeable membrane.Such a "processed sample" may include, for example, nucleic acids or proteins extracted from a sample or obtained by subjecting the primary sample to one or more techniques, such as amplification or reverse transcription of nucleic acids, isolation and / or purification of certain components, etc.

[0079] Small molecule: As used herein, the term "small molecule" refers to a small organic or inorganic molecule with a molecular weight below about 3,000 Daltons. Generally, a small molecule can have a molecular weight less than 3,000 Daltons (Da). The small molecule can be, for example, at least about 100 Da to about 3,000 Da (e.g., about 100 to about 3,000 Da, about 100 to about 2500 Da, about 100 to about 2,000 Da, about 100 to about 1,750 Da, about 100 to about 1,500 Da, about 100 to about 1,250 Da, about 100 to about 1,000 Da, about 100 to about 750 Da, about 100 to about 500 Da, about 200 to about 1500, about 500 to about 1000, about 300 to about 1000 Da, or about 100 to about 250 Da).

[0080] Subject: As used herein, the term "subject" refers to an individual to whom the provided treatment is administered. In some embodiments, the subject is an animal. In some embodiments, the subject is a mammal, for example, a mammal experiencing or susceptible to a disease, disorder, or condition described herein. In some embodiments, the animal is a vertebrate, for example, a mammal such as a non-human primate (especially a higher primate), sheep, dog, rodent (e.g., mouse or rat), guinea pig, goat, pig, cat, rabbit, or cow. In some embodiments, the animal is a non-mammal, such as a chicken, an amphibian, a reptile, or the invertebrate model C. elegans. In some embodiments, the subject is a human. In some embodiments, the subject is afflicted with or susceptible to one or more diseases, disorders, or conditions described herein. In some embodiments, the subject exhibits one or more symptoms of one or more diseases, disorders, or conditions described herein. In some embodiments, the subject has been diagnosed with one or more diseases, disorders, or conditions described herein. In some embodiments, the subject is undergoing or has undergone a particular treatment to diagnose and / or treat a disease, disorder, or condition, hi another embodiment, the subject is an experimental animal or surrogate animal as a disease model.

[0081] Substantially: As used herein, refers to a qualitative state of exhibiting the full or nearly full extent or degree of a desired characteristic or property. Those skilled in the biological arts will understand that biological and chemical phenomena rarely, if ever, proceed to completion and / or completeness or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0082] Therapeutic regimen: "Therapeutic regimen," as that term is used herein, refers to a dosing regimen, the administration of which across a relevant population can be correlated with a desired or beneficial therapeutic outcome.

[0083] Therapeutically effective amount: as used herein, refers to the amount at which it is administered that produces the desired effect. In some embodiments, the term refers to an amount that is sufficient to treat a disease, disorder, and / or condition when administered to a population suffering from or susceptible to the disease, disorder, and / or condition according to a treatment regimen. In some embodiments, a therapeutically effective amount is an amount that reduces the incidence and / or severity and / or delays the onset of one or more symptoms of the disease, disorder, and / or condition. Those skilled in the art will appreciate that the term "therapeutically effective amount" does not actually require that successful treatment be achieved in a particular individual. Rather, a therapeutically effective amount may be an amount that, when administered to a subject (e.g., a patient) in need of such treatment, provides a particular desired pharmacological response in a significant number of subjects. In some embodiments, reference to a therapeutically effective amount may be a reference to an amount measured in one or more specific tissues (e.g., tissues affected by a disease, disorder, or condition) or fluids (e.g., blood, saliva, serum, sweat, tears, urine, etc.). One of skill in the art will appreciate that in some embodiments, a therapeutically effective amount of a particular agent or therapy may be formulated and / or administered in a single dose, hi some embodiments, a therapeutically effective agent may be formulated and / or administered in multiple doses, e.g., as part of a dosing regimen.

[0084] Treatment: As used herein, the term "treatment" (also "treat" or "treating") refers to any administration of a therapy that partially or completely relieves, ameliorates, alleviates, inhibits, delays the onset, reduces the severity, and / or reduces the incidence of one or more symptoms, characteristics, and / or causes of a particular disease, disorder, and / or condition. In some embodiments, such treatment may be treatment of a subject who does not show signs of the relevant disease, disorder, and / or condition and / or a subject who shows only early signs of the disease, disorder, and / or condition. Alternatively, or in addition, such treatment may be treatment of a subject who shows one or more established signs of the relevant disease, disorder, and / or condition. In some embodiments, the treatment may be treatment of a subject who has been diagnosed as suffering from the relevant disease, disorder, and / or condition. In some embodiments, the treatment may be treatment of a subject who is known to have one or more susceptibility factors that are statistically correlated with an increased risk of developing the relevant disease, disorder, and / or condition. [Brief description of the drawings]

[0085] [Figure 1A] Fluorescence data representing microgliosis in each of the four study groups of mice (G1, G2, G3, and G4) are shown. (A) Plots show the fluorescence intensity of microgliosis observed in each group relative to vehicle-treated wild-type mice (i.e., mock-treated wild-type mice in group G1). (B) Representative photomicrographs show fluorescently stained cells in the spinal cord for each of the four groups. [Figure 1B]Fluorescence data representing microgliosis in males, females, and each of the four study groups (G1, G2, G3, and G4) of male and female mice are shown. Image quantification was performed using ImageJ analysis software. The mean of the fluorescence values ​​from the G1 group was calculated, and the % change in fluorescence compared to the mean of the G1 group was plotted. Scatter dot blots were generated in GraphPad Prism. Scatter dot blots show mean ± SEM values. Each dot represents the % fluorescence of a single image compared to the G1 group. Statistical analysis was performed in GraphPad Prism. Data sets were analyzed by one-way ANOVA followed by Dunnett's test. A level of p<0.05 was considered statistically significant. Compared to the male G2 group, male G3 or male G4 had significantly different levels, respectively (*p=0.04 and **p=0.001). Compared to the female G2 group, female G3 or female G4, respectively, had significantly different levels (****p<0.0001). [Figure 2A] Fluorescence data representing astrocytosis in each of the four study groups of mice (G1, G2, G3, and G4) are shown. (A) Plots show the fluorescence intensity of astrocytosis observed in each group relative to vehicle-treated wild-type mice (i.e., mock-treated wild-type mice in group G1). (B) Representative photomicrographs show fluorescently stained cells in the spinal cord for each of the four groups. [Figure 2B] Fluorescence data representing astrocytosis in males, females, and each of the four study groups (G1, G2, G3, and G4) of male and female mice are shown. Image quantification was performed using ImageJ analysis software. The mean of the fluorescence values ​​from the G1 group was calculated, and the % change in fluorescence compared to the mean of the G1 group was plotted. Scatter dot blots were generated in GraphPad Prism. Scatter dot blots show mean ± SEM values. Each dot represents the % fluorescence of a single image compared to the G1 group. Statistical analysis was performed in GraphPad Prism. Data sets were analyzed by one-way ANOVA followed by Dunnett's test. A level of p<0.05 was considered statistically significant. Compared to the female G2 group, male or female G3 and male or female G4, respectively, had significantly different GFAP levels (****p<0.0001). [Diagram 3] Percent ATP levels of each group relative to wild-type vehicle-treated wild-type males, females, and male and female mice (i.e., mock-treated wild-type mice in group G1) are shown. ATP measurements were performed on apical spinal cord protein homogenates using Promega CellTiter-Glo. Scatter dot blots were generated in GraphPad Prism. Each dot represents one spinal cord sample as described in Table 3. Data are expressed as mean ± SEM. Percent ATP levels were calculated by comparing luminescence values ​​to the mean of the G1 group. Statistical analysis was performed in GraphPad Prism. Data sets were analyzed by one-way ANOVA followed by Dunnett's test. A level of p<0.05 was considered statistically significant. Compared to the G1 group, G3 and G4 had significantly different ATP levels (*p=0.03, **p=0.0029, ***p=0.0003, and ****p<0.0001). ns indicates no statistically significant difference between groups G1 and G2. [Figure 4] Plasma NF-L levels for each group are shown for wild-type vehicle-treated wild-type males, females, and male and female mice (i.e., mock-treated wild-type mice in group G1). Plasma NF-L levels from animals were determined using an NF-L ELISA kit. Scatter dot blots were made in GraphPad Prism. Each dot represents a plasma sample from an individual animal. Data are presented as mean ± SEM. Statistical analysis was performed in GraphPad Prism. Data sets were analyzed by one-way ANOVA followed by Dunnett's test. A level of p<0.05 was considered statistically significant. Compared to the G2 group, G3 and G4 had significantly different NF-L levels (*p=0.02, **p=0.0074, ***p=0.0003, and ****p<0.0001). ns indicates no statistically significant difference between female G2 and G4 groups. [Diagram 5]Figure 1 shows the innervated neuromuscular (NMJ) integrity of each group relative to wild-type vehicle-treated wild-type males, females, and male and female mice (i.e., mock-treated wild-type mice in group G1). Each dot in the plot represents an NMJ from at least four animals. NMJ integrity was measured by determining the overlap between presynaptic (vesicular acetylcholine transporter, VAChT) and postsynaptic (α-bungarotoxin, α-BTX, which binds to the nicotinic acetylcholine transporter). Box plots were generated in GraphPad Prism. The center line in the box plot represents the mean %. SEM was plotted for each group. Each dot represents a single NMJ where the % overlap between VAChT and a-BTX was determined. Statistical analysis was performed in GraphPad Prism. Data sets were analyzed by one-way ANOVA followed by Dunnett's test. A level of p<0.05 was considered statistically significant. The innervated NMJs observed in the male or female G2 groups were significantly different compared to the male G1 or female G2 groups, respectively (****p<0.0001). In contrast, the innervated NMJs in the male G3 or male G4 or female G3 or female G4 groups were significantly different from the male G2 or female G2 groups, respectively (****p<0.0001). [Figure 6A]Data representing proteasome function in each of the four study groups (G1, G2, G3, and G4) of male, female, and male and female mice are shown. The plots show the values ​​of PSMD11 protein levels normalized using the respective b-actin loading control observed in each group relative to vehicle-treated wild-type male, female, and male and female mice (i.e., mock-treated wild-type mice in group G1). The respective b-actin loading control was used to normalize the protein bands detected by Western blot. Scatter dot blots were generated in GraphPad Prism. Scatter dot blots show mean ± SEM values. Each dot in the figure represents a relative value compared to the G1 group mean of a single spinal cord lysate as described in Table 3 in Appendix 5. Statistical analysis was performed in GraphPad Prism. Data sets were analyzed by one-way ANOVA followed by Dunnett's test. A level of p<0.05 was considered statistically significant. Compared to the male G2 group, PSMD11 levels in male G3 or G4 were significantly different (***p=0.0002 and ****p<0.0001). PSMD11 protein levels were not statistically significant between female G1 and G2 groups, or G2 and G3 groups, or G2 and G4 groups. ns indicates no statistical significance. However, when male and female data were combined, significant differences were found between G2 and G3 groups and G2 and G4 groups (*p=0.02, ****p<0.0001). [Figure 6B]Data representing proteasome function in each of the four study groups (G1, G2, G3, and G4) of male, female, and male and female mice are shown. Plots show the values ​​of proteasome activity observed in each group relative to vehicle-treated wild-type male, female, and male and female mice (i.e., mock-treated wild-type mice in group G1). Proteasome activity was measured using Suc-LLVY-AMC substrate. The mean of the fluorescence values ​​from the G1 group was calculated, and the % change in fluorescence compared to the mean of the G1 group was plotted. Scatter dot blots were made in GraphPad Prism. Scatter dot blots show mean ± SEM values. Statistical analysis was performed in GraphPad Prism. Data sets were analyzed by one-way ANOVA followed by Dunnett's test. A level of p<0.05 was considered statistically significant. Compared to the G1 group, the G2 group had significantly different levels (*p=0.0377). In contrast, G3 and G4 had significantly different levels of proteasome activity compared to the G2 group (**p<0.01). Proteasome activity was not statistically significant between female G1 and G2 groups, or G2 and G3 groups, or G2 and G4 groups. ns indicates no statistical significance. However, when male and female data were combined, significant differences were found between G1 and G2 groups, or G2 and G3 groups, or G2 and G4 groups (*p=0.02 and **p=0.009). [Figure 7]Data representing lysosomal function in each of the four study groups of mice (G1, G2, G3, and G4) are shown. (A) Plots show values ​​of LAMP2A protein levels normalized using the respective b-actin loading control observed in each group relative to vehicle-treated wild-type mice (i.e., mock-treated wild-type mice in group G1). The respective b-actin loading control was used to normalize protein bands detected by Western blot. Raw data were organized and sorted in Excel. See Appendix 5 for raw data. Scatter dot blots were generated in GraphPad Prism. Each dot in the figure represents a single spinal cord lysate as described in Table 3 in Appendix 5. Data are expressed as mean ± SEM. Statistical analysis was performed in GraphPad Prism. Data sets were analyzed by one-way ANOVA followed by Dunnett's test. A level of p<0.05 was considered statistically significant. Compared to the male G2 group, males G3 and G4 had significantly different levels of LAMP2A (*p=0.03, ***p=0.0002, and ****p<0.0001). LAMP2A protein levels were statistically significant between female G1 and G2, but not between female G2 and G3 or G2 and G4 groups. ns indicates no statistical significance. However, when male and female data were combined, significant differences were found between G1 and G2, G2 and G3, and G2 and G4 groups (*p=0.04 and ****p<0.0001). [Figure 8] Data from the beam walking test are shown. (A) Plots show the beam walking test results for each of the four study groups (G1, G2, G3, and G4) of the study conducted on SOD1 male mice. (B) Plots show the beam walking test results for each of the four study groups (G1, G2, G3, and G4) of the study conducted on SOD1 female mice. [Figure 9]Data from the P100 rotarod test are shown. (A) Plots show the P100 rotarod test results for each of the four test groups (G1, G2, G3, and G4) of the test performed on SOD1 male mice. (B) Plots show the P100 rotarod test results for each of the four test groups (G1, G2, G3, and G4) of the test performed on SOD1 female mice. [Figure 10] Data from grip strength testing are shown. (A) Plots show grip strength test results (measured in grams of force) for each of the four test groups (G1, G2, G3, and G4) of studies performed on SOD1 male mice. (B) Plots show grip strength test results (measured in grams of force) for each of the four test groups (G1, G2, G3, and G4) of studies performed on SOD1 female mice. [Figure 11] Figure 1 shows metabolomic analysis of CT6-treated wild-type male and female mice. Plots show % change in metabolite levels relative to vehicle-treated mice for 660 metabolites identified in mouse plasma (blue: male mice, orange: female mice). [Figure 12] Figure 1 shows metabolomic analysis of CT6-treated wild-type male mice. Plots show % change in metabolite levels relative to vehicle-treated male mice for approximately 660 to approximately 700 metabolites identified in mouse plasma. [Figure 13] Figure 1 shows metabolomic analysis of CT6-treated wild-type female mice. Plots show % change in metabolite levels relative to vehicle-treated female mice for approximately 660 to approximately 700 metabolites identified in mouse plasma. [Figure 14] The top 25 metabolite sets in descending order of increased enrichment ratio in CT6-treated wild-type male mice are shown. [Figure 15] The top 25 metabolite sets in descending order of increased enrichment ratio in CT6-treated wild-type female mice are shown. [Figure 16]Figure 1 shows metabolites increased in CT6-treated wild-type male and female mice. (A) Plot shows % change in metabolite levels for metabolites increased in CT6-treated wild-type male and female mice relative to vehicle-treated mice. (B) Plot shows the top 25 metabolite sets and their enrichment ratios for metabolites increased in CT6-treated wild-type male and female mice. [Figure 17] Figure 1 shows metabolites decreased in CT6-treated wild-type male and female mice. (A) Plot shows % change in metabolite levels relative to vehicle-treated mice for metabolites decreased in CT6-treated wild-type male and female mice. (B) Plot shows the top 25 metabolite set and its enrichment ratio for metabolites decreased in CT6-treated wild-type male and female mice. [Figure 18A] Shows the relevance of bile acids in various neurodegenerative diseases including ALS, AD, PD, and HD. [Figure 18B] Shown is a list of bile acids and their % change (ie, increased or decreased bile acid levels) in CT6-treated wild-type male and female mice relative to vehicle-treated mice. [Figure 19A] The role of NO in various aspects of neuronal function, including but not limited to neurotransmission, neuroplasticity, cerebral microcirculation, inflammation, oxidative stress, and the like, is shown. [Figure 19B] Shown are positive and negative controls observed in the nitric oxide (NO) assay, with assays performed in duplicate using clear 96-well plates. [Figure 19C] 1 shows the results of a nitric oxide (NO) assay, plotting % nitrite levels compared to untreated SIMA9 cells for various metabolites and controls. Yellow bars represent statistically significant results. [Figure 20] (A) Amyloid plaque burden and (B) plaque size in the cortical regions of the brain in treated animals. Groups: G2=untreated, G3=CT10, G4=CT10m, G5=CT10x. [Figure 21](A) Amyloid plaque burden and (B) plaque size in the hippocampal region of the brain in treated animals. Groups: G2=untreated, G3=CT10, G4=CT10m, G5=CT10x. [Figure 22] (A) Amyloid plaque burden and (B) plaque size in both cortical and hippocampal regions of the brain in treated animals performed in separate experiments. Groups: G2=untreated, G3=CT10, G4=CT10m, G5=CT10x. [Diagram 23] Plots of binned amyloid particle size within the cortical region of the brain in treated animals are shown for each of the groups G2=untreated, G3=CT10, G4=CT10m, G5=CT10x. [Figure 24] Plots of binned amyloid particle size within the hippocampal region of the brain in treated animals are shown for each of the groups G2=untreated, G3=CT10, G4=CT10m, G5=CT10x. [Diagram 25] For each of the groups G2=untreated, G3=CT10, G4=CT10m, G5=CT10x, plots of binned amyloid particle size in both the cortical and hippocampal regions of the brain in treated animals performed in separate experiments are shown. [Figure 26] Images of stained microglia in brain sections are shown with images adjusted to the same intensity scale. [Figure 27] Plots of microglial staining in (A) the cortical region of the brain, (B) the hippocampal region of the brain, and (C) the cortical and hippocampal regions of the brain in treated animals for each of the groups G2=untreated, G3=CT10, G4=CT10m, and G5=CT10x are shown. Microglial staining cells and processes were measured in brain tissue sections using Iba1 / AIF-1 antibody. [Figure 28] Images of stained microglia associated with amyloid plaques in brain sections. Images were adjusted to the same intensity scale. [Figure 29]For each of groups G2=untreated, G3=CT10, G4=CT10m, G5=CT10x, plots of microglia associated with amyloid plaques in (A) the cortical region of the brain, (B) the hippocampal region of the brain, and (C) the cortical and hippocampal regions of the brain in treated animals are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0086] Neurodegenerative diseases, disorders, and conditions Neurodegenerative diseases, disorders, and conditions are an umbrella term for a series of diseases, disorders, and conditions that primarily affect neurons in the human brain. These are untreatable and debilitating conditions that result in the progressive degeneration and / or death of nerve cells. This leads to problems with movement (called ataxia) or problems with mental function (called dementia). Some examples of neurodegenerative diseases, disorders, and conditions include ALS, PD, AD, and HD.

[0087] Amyotrophic lateral sclerosis (ALS): ALS, also known as Lou Gehrig's disease, is the most fatal progressive neurodegenerative disease characterized by a marked loss of motor neurons (MNs) within the primary motor cortex, brainstem, and spinal cord. The loss of motor neurons disrupts basic foundational movements such as breathing, typically causing death in patients within 2-5 years of diagnosis. The progressive deterioration of motor function in patients severely disrupts the patient's ability to breathe, necessitating some form of respiratory support for the patient's survival. Other symptoms may also include muscle weakness of the hands, arms, legs, or swallowing muscles. Some patients (e.g., FTD-ALS) may also develop frontotemporal dementia.

[0088] According to the ALS Association, approximately 5,600 people are diagnosed with ALS each year in the United States. The incidence of ALS is 2 per 100,000, and it is estimated that as many as 30,000 Americans may have the disease at any given time.

[0089] Two forms of ALS have been described: sporadic ALS (SALS), which is the most common form of ALS in the United States, accounting for 90-95% of all diagnosed cases, and familial ALS (FALS), which occurs primarily in familial lineages with dominant inheritance and accounts for only approximately 5-10% of all cases in the U.S. SALS and FALS are clinically indistinguishable.

[0090] Pathological studies have found that disturbances in several cellular processes occur after disease onset, including increased ER stress, generation of free radicals (i.e., reactive oxygen species (ROS)), mitochondrial dysfunction, protein aggregation, apoptosis, inflammation, and glutamate excitotoxicity, especially in motor neurons (MNs).

[0091] The causes of ALS are complex and heterogeneous. In general, ALS is considered a complex genetic disorder in which multiple genes in combination with environmental exposures confer susceptibility to individuals. 2+ / Zn 2+ More than a dozen genes have been found to be associated with ALS, including superoxide dismutase), TDP-43 (TARDBP, TAR DNA-binding protein 43), FUS (sarcoma fusion / sarcoma translocation), ANG (angiogenin), ATXN2 (ataxin-2), valosin-containing protein (VCP), OPTN (optineurin), and an expansion of a noncoding GGGGCC hexanucleotide repeat in chromosome 9 open reading frame 72 (C9ORF72). However, the exact mechanism of motor neuron degeneration remains elusive.

[0092] Currently, there is no therapeutic treatment for ALS. The only FDA approved drug is riluzole, which antagonizes glutamate response and reduces the pathological development of ALS. However, only about 3 months of life extension has been reported for early stage ALS patients, and no therapeutic benefit has been observed for late stage ALS patients, indicating a lack of therapeutic options for patients (Bensimon G et al., J Neurol. 2002, 249, 609-615). Therefore, new therapeutic strategies that can effectively prevent the progression of the disease are needed.

[0093] Parkinson's Disease (PD): PD is a neurodegenerative movement disorder characterized by resting tremor, rigidity, bradykinesia, and postural instability. PD symptoms are classically due to dopamine depletion and degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc). However, additional neural circuits are affected and non-motor symptoms are often present, suggesting systemic pathology. There is strong evidence that mitochondrial dysfunction is a key event in the disease process. It has been reported that PD-associated mutations and mitochondrial dynamics are interrelated. PD-associated mutations can disrupt mitochondrial dynamics, and the outcome of these mutations can be regulated by mitochondrial dynamics.

[0094] In one embodiment, effective treatment of PD is determined by a reduction in the dose of pharmacological treatment, such as L-dopa, required to maintain adequate control of PD symptoms, hi another embodiment, the effectiveness of treatment is monitored using the Unified Parkinson's Disease Rating Scale (UPDRS), known in the art.

[0095] PD is the most common movement-related disorder among mid- or late-life diseases, affecting approximately 6.2 million people worldwide (Global Burden of Disease Study 2015. Lancet 388, 1545-1602 (2016)). PD is characterized by the accumulation of α-synuclein inclusions in neurons, as well as the degeneration and / or loss of dopaminergic neurons. The major clinical symptoms of PD include slow movement, resting tremor, rigidity, and postural instability (Pires, AO, et al., Therapeutics. Prog. Neurobiol. (2017); doi:10.1016 / j.pneurobio.2017.04.006). The majority of PD cases are sporadic with unknown origin, although mutations in several genes have been associated with rare familial forms of the disease. Several lines of evidence implicate defects in mitochondrial respiration in the etiology and pathogenesis of PD. First, MPTP, an inhibitor of complex I of the electron transport chain, can induce PD (Nicklas, WJ, et al., Life Sci. 36, 2503-2508 (1985); Ramsay, RR, et al., Biochem Biophys Res Commun 135, 269-275 (1986)). Inhibition of complex I leads to a decrease in mitochondrial ATP production, an increase in the production of mitochondrial-derived reactive oxygen species (ROS), and activation of the mitochondria-dependent apoptotic pathway. Second, postmortem studies of PD patients have found elevated levels of oxidative stress markers / products in dopaminergic neurons (Yoritaka, A. et al. Proceedings of the National Academy of Sciences 93, 2696-2701 (1996); Floor, E, & Wetzel, MG, JNeurochem 70, 268-275 (2002); Zhang, J. et al., Am. J Pathol. 154, 1423-1429 (1999)).Third, a 30% reduction in mitochondrial complex I activity was observed in the brain and peripheral tissues of PD patients (Schapira, AHV, et al., The Lancet 333, 1269 (1989); Parker, WD, et al., Ann. Neural. 26, 719-723 (1989)). Fourth, neurotoxins such as rotenone, paraquat, and 6-hydroxydopamine (6-OHDA) induce mitochondrial dysfunction that leads to PD-related phenotypes in animal models (Tieu, K, Cold Spring Harb Perspect Med 1, a009316-a009316 (2011)). Finally, PD-associated genes such as α-synuclein, LRRK2 (leucine-rich repeat kinase 2), parkin, PINK1, and DJ-1 affect mitochondrial dynamics, trafficking, autophagy, and quality control (Moore, DJ, et al., Annu. Rev. Neurosci. 28, 57-87 (2005); Robinson, PA, Expert Review of Proteomics, 7(4), 565-578).

[0096] All cells require mitochondria for their energy needs, including neurons, which are critically dependent on proper mitochondrial function. Neurons have high metabolic activity and are heavily dependent on mitochondria for their bioenergetic needs. Several factors make neurons in general, and dopaminergic neurons in particular, susceptible to degeneration. These include ROS (resulting from dopamine metabolism and mitochondrial dysfunction), low endogenous antioxidant levels, and high levels of iron and calcium (known to promote ROS formation) (Dias, V, et al, Journal of Parkinson's Disease, 3(4), 461-491 (2013)). Furthermore, neural tissue contains high levels of polyunsaturated fatty acids, which are prone to lipid peroxidation and the generation of toxic products (Liu, X, et al, Joumal of Biological Chemistry, 283(50), 34887-34895 (2008)). Mitochondrial dysfunction, whether a primary or secondary cause, is a promising potential therapeutic target. Ageing is the greatest risk factor for PD (Collier, TJ, et al, Nature Reviews. Neuroscience, 12(6), 359-366 (2011)), and therefore, with increasing life expectancy worldwide (GBD 2013, Lancet 385, 117 (2015)), the number of people affected by PD will rise considerably in the near future. Thus, there is a significant clinical unmet need for new therapeutic approaches that can be used not only to slow down PD but also as a preventative measure for the aging population.

[0097] Although most of the early research on PD focused entirely on brain pathology, the gastrointestinal (GI) system is now recognized as an important source of pathogenesis in PD (Mukherjee, A, et al, Journal of Gastroenterology, 22(25), 5742-5752 (2016); Pellegrini, C, et al, Neurogastroenterology & Motility, 28(12), 1781-1791 (2016); Kuo, YM, et al, Human Molecular Genetics, 19, 1, 1633-1650 (2010)). GI symptoms such as constipation affect approximately 80% of PD patients, and idiopathic constipation is an important risk factor for PD (Noyce, AJ, et al, Anals of Neurology, 72, 893-901 (2012)). In PD, constipation is associated with accumulation of alpha-synuclein in the enteric nervous system (Cersosimo, MG, and Benarroch, EE, Neurobiology of Disease, 46, 559-564 (2012)), intestinal inflammation, and increased intestinal permeability (Devos, D, et al, Neurobiology of Disease, 50, 42-48 (2013)). Furthermore, intestinal mucosal inflammation is thought to lead to synuclein accumulation in enteric nerves, which can then spread in a prion-like manner to the central nervous system via autonomic connections (Braak, H, et al, Journal of Neurotransmission, 110, 517-536 (2003); Hawkes, CH, et al, Neuropathology and Applied Neurobiology, 33, 599-614 (2007); Hawkes, CH, et al, International Symposium of Olfaction and Taste, 1170, 615-622 (2009)).Many of the GI tract changes are observed even before the onset of neuronal symptoms (Verbaan, D, et al, Neurology, 69, 333-341 (2007)), and therefore the pathogenic mechanisms of PD may act primarily through the GI tract (Shannon, KM, et al, Mov. Disord. 27, 716-719 (2012); Kieburtz, K, and Wunderle, KB, Mov. Disord., 28, 8-13 (2013)).

[0098] Alzheimer's Disease (AD): AD is a fatal neurodegenerative disease characterized by progressive decline in memory and cognitive function. Early-onset familial AD, associated with mutations in the APP or γ-secretase genes, accounts for less than 5% of all cases, while 95% of sporadic or late-onset AD have an unknown etiology (Masters, CL et al., Nat Rev Dis Primers C 15056 (2015)). The pathological hallmark of AD is the accumulation of extracellular senile plaques and intracellular neurofibrillary tangles (NFTs) in the brains of AD patients. Senile plaques consist of β-amyloid peptide (Aβ) as the main component, while NFTs consist of abnormal fibrillar forms of the microtubule-associated protein tau as the main component (Nelson, PT et al., J. Neuropathol. Exp. Neurol. 71, 362-381 (2012)). Aβ accumulation and NFTs are thought to indicate neuronal dysfunction and impending neuronal destruction (Nelson, PT et al., J. Neuropathol. Exp. Neurol. 71, 362-381 (2012)).

[0099] Mitochondrial dysfunction and defects in energy metabolism have been consistently observed in human AD patients (Halliwell, B, J Neurochem, 97, 1634-1658 (2006); Cheignon, C. et al., Redox Biol 14, 450-464 (2018)). Aβ and tau pathology are strongly associated with mitochondrial dysfunction in AD. Aβ and tau directly affect mitochondrial function, causing reduced ATP production, increased reactive oxygen species (ROS) production, decreased oxygen consumption, and decreased mitochondrial complex I and IV function (Muller, WE, et al., Mol. Neurobiol. 41, 159-171 (2010)). Mitochondrial dysfunction has been found to be an early event in AD. In sporadic AD, a progressive increase in oxidative stress with age has been shown to cause Aβ deposition and NFT formation (Moreira, PI, et al., Biochim. Biophys. Acta 1802, 2-10 (2010)). This may lead to a continuous cycle of events in which Aβ and tau exacerbate mitochondrial dysfunction, leading to rapidly progressive AD symptoms.

[0100] Approximately one-third of drugs currently used in the clinic were initially isolated from plants or microorganisms. Although chemical synthesis of compounds has dominated the pharmaceutical industry as a source for identifying novel therapeutics, bioprospecting natural sources such as plants and microorganisms continue to play an important role as therapeutic agents. Recent studies have begun to explore the human microbiome as a source of drugs (Donia, MS, and Fischbach, MA, Science 349, 1254766-1254766 (2015)). The human body is home to a community of benign, commensal, commensal, and pathogenic microorganisms, collectively known as the microbiome. These microorganisms can regulate host brain function and behavior via the gut-brain axis, as well as the production of several metabolites, such as GABA, glutamate, and serotonin.

[0101] Dysfunction of microbiota-derived metabolite signaling may contribute to neurological disorders, including AD (Sharon, G, et al, Cell 167, 915-932 (2016); Hill, JM, Front Neurol 5, 43 (2014)). In summary, the microbiota presents a rich, untapped resource for deriving novel neuroprotective compounds or live biotherapeutics with relevance to AD.

[0102] The present disclosure provides compositions (e.g., microbiota compositions) and methods that inhibit one or more of the events or processes that occur in a neurodegenerative disease, disorder, or condition. The present disclosure is based, in part, on the discovery that one or more microbial strains or compositions comprising one or more microbial strains are particularly suitable as therapeutic agents for neurodegenerative diseases, disorders, or conditions.

[0103] Huntington's Disease (HD): HD is a monogenic, fatal neurodegenerative disease characterized by progressive chorea, neuropsychiatric and cognitive impairment. HD is known to be caused by an autosomal dominant polyglutamine-coding triplet (CAG) repeat expansion in the N-terminus of the huntingtin (HTT) protein. This repeat expansion results in a toxic gain of function of HTT, ultimately leading to striatal neurodegeneration that progresses to widespread brain atrophy. Symptoms typically appear between the ages of 35 and 44 years, with a life expectancy of 10 to 25 years after onset. Interestingly, the length of the HTT expansion correlates with both the age of onset and the rate of disease progression, with longer expansions being linked to greater disease severity. In a small percentage of the HD population (approximately 6%), disease onset occurs from 2 to 20 years of age with the appearance of an akinetic-rigid syndrome. These cases tend to progress faster than the late-onset variety and are classified as juvenile or Westphal variant HD. Approximately 35,000-70,000 patients are estimated to suffer from HD in the United States and Europe today. Currently, only symptom relief and supportive therapies are available to treat HD, and no cure has yet been identified. Eventually, individuals with HD succumb to other complications, such as other illnesses (e.g., pneumonia, heart failure, etc.), shortness of breath, choking, or physical injury from falls.

[0104] The mechanism by which CAG-expanded HTT leads to neurotoxicity is not fully understood. Huntingtin protein is expressed in all cells, but its concentration is highest in the brain. The normal function of HTT is unknown, but in the brains of HD patients, HTT aggregates into abnormal intranuclear inclusions. It is currently believed that this process of misfolding and aggregation, together with associated protein intermediates (i.e., soluble species and toxic N-terminal fragments), leads to neurotoxicity.

[0105] Microbial preparation(s) and / or component(s) The present disclosure provides a system and method for evaluating, characterizing, and identifying one or more microbial strains of a microbiota. For example, the present disclosure provides a system and method for evaluating, characterizing, and identifying one or more microbial strains of a microbiota having one or more capabilities. Such a system and method can be useful for evaluating, characterizing, and identifying one or more microbial strains that affect the health of humans, livestock, and / or pets. In some embodiments, one or more microbial strains affect the health of humans, livestock, and / or pets by regulating their respective metabolome, cell viability, ATP levels, one or more other parameters or characteristics (e.g., of an organ of interest), or a combination thereof, to prevent, treat, or reduce the risk of a disease, disorder, or condition. For example, the technology described herein may result in modulation of the metabolome, improved cell survival, increased ATP levels, modulation of one or more other parameters or characteristics (e.g., microgliosis, astrocytosis, proteasome function, lysosomal function, inflammation, levels or activity of nucleic acids or proteins or forms thereof, etc.), or combinations thereof in a subject, which results in a decrease in the production of toxic components and / or components that are indicative of or are markers of cellular damage in the subject (e.g., in the blood of the subject) (e.g., increased blood levels of neurofilament light chain protein (NF-L)).

[0106] The present disclosure also provides systems and methods for producing pharmaceutical compositions that include evaluating, characterizing, and identifying one or more microbial strains of a microbiota.

[0107] In some embodiments, the method includes evaluating, characterizing, and identifying one or more microbial strains from a snake, lizard, fish, or bird microbiota. In some embodiments, the method includes evaluating, characterizing, and identifying one or more microbial strains from a mammalian microbiota. The mammalian microbiota can be a canine, feline, equine, bovine, ovine, caprine, or porcine microbiota. In some embodiments, the microbiota used in the system or method described herein can prevent or treat a disease or condition.

[0108] Microbiota can be isolated from any system or tissue of an organism that supports microbial growth. For example, the microbiota can be skin microbiota, oral microbiota, nasal microbiota, gastrointestinal microbiota, brain microbiota, lung microbiota, or urogenital microbiota. A list of exemplary microbial strains found in gastrointestinal microbiota is included in Table 1 below. Those skilled in the art will understand that microbiota samples can be obtained by various methods known in the art. For example, skin, oral, nasal, lung, or urogenital microbiota samples can be obtained using swabs or tissue scrapes. In some embodiments, gastrointestinal microbiota can be sampled from feces. Skin microbiota, oral microbiota, nasal microbiota, gastrointestinal microbiota, brain microbiota, lung microbiota, or urogenital microbiota samples can be obtained via biopsy.

[0109] In some embodiments, the microbiota is the microbiota of a healthy individual or an individual who does not suffer from or is not at risk of developing a particular disease or disorder.In some embodiments, the microbiota is the microbiota of an individual who suffers from or is at risk of developing a particular disease, disorder, or condition.In some embodiments, the microbiota is the microbiota of an individual who is known to suffer from a particular disease, disorder, or condition.In some embodiments, the human microbiota is the microbiota of a human who has an unknown risk of one or more diseases, disorders, or conditions.

[0110] In some embodiments, the microbiota is a reference microbiota. The reference microbiota may be the microbiota of a healthy individual or an individual who is not suffering from or at risk of developing a particular disease, disorder, or condition. In some cases, the reference microbiota may be derived from the same individual as the microbiota to be evaluated or characterized, but obtained at a different time. In some cases, the reference microbiota may be derived from the same individual as the microbiota to be evaluated or characterized, but obtained from a different system or tissue.

[0111] In some embodiments, individual microbial strains or combinations of microbial strains may be evaluated, characterized, or identified in relative abundance that differs from that of one or more such strains found in the microbiota. For example, the effect of modulating a cell or organism in response to a single strain may be evaluated, characterized, or identified using in vitro methods (e.g., mammalian cells) or in vivo methods using a mammal (e.g., mouse, human, etc.) described herein. In some embodiments, the effect of modulating a cell or organism to treat, prevent, or reduce the risk of, for example, a disease, disorder, or condition (e.g., a neurodegenerative disease, disorder, or condition described herein) may be evaluated, characterized, or identified using in vitro methods (e.g., mammalian cells) or in vivo methods using a mammal (e.g., mouse, human, etc.) described herein. In some embodiments, the effect of modulating a cell or organism to treat, prevent, or reduce the risk of a disease, disorder, or condition (e.g., a neurodegenerative disease, disorder, or condition described herein) by, for example, modulating one or more metabolites of the cell or organism, one or a characteristic or parameter of the cell or organism (e.g., cell survival, microgliosis, astrocytosis, proteasome function, lysosomal function, inflammation, ATP levels, levels or activities of nucleic acids or proteins or forms thereof, etc.), or a combination thereof, can be evaluated, characterized, or identified using in vitro methods (e.g., mammalian cells) or in vivo methods using mammals (e.g., mice, humans, etc.) described herein. As another example, the effect of modulating a cell or organism (e.g., the level of one or more metabolites) to treat, prevent, or reduce the risk of a disease, disorder, or condition described herein in response to two microbial strains can be evaluated, characterized, or identified together using the methods described herein.

[0112] Microbial strain extracts, components, or compounds can also be evaluated, characterized, or identified together using the methods described herein. In some cases, microbial strain extracts, components, or compounds that are determined to treat, prevent, or reduce the risk of disease, disorder, or condition in organisms (e.g., mammals) described herein can be evaluated, characterized, or identified. Evaluating, characterizing, or identifying microbial strain extracts, components, or compounds that treat, prevent, or reduce the risk of disease, disorder, or condition in organisms (e.g., mammals) can provide additional information about potential biomarkers, targets, or protective agents in microbiota.

[0113] A variety of techniques are known in the art that can be used to prepare extracts of microbial strains and / or to isolate or process extracts, components, or compounds therefrom (e.g., to isolate and / or purify one or more components or compounds therefrom). To name just a few, such techniques can include, for example, one or more of organic extraction, vacuum concentration, chromatography, etc.

[0114] Biological effects assessment The present disclosure provides insight that the compositions (e.g., microbiota compositions) described herein can be used to treat, prevent, and / or reduce the risk of a disease, disorder, or condition in an organism (e.g., a mammal (e.g., a human)) by contacting the organism with the composition(s) (e.g., feeding the composition to the organism, administering to the organism). In some embodiments, the organism can be afflicted with or at risk of a disease, disorder, or condition (e.g., a mammalian disease, disorder, or condition). To determine whether one or more compositions treat, prevent, or reduce the risk of a disease, disorder, or condition (e.g., a neurodegenerative disease, disorder, or condition), the level of one or more metabolites can be observed, measured, or evaluated in a sample contacted with one or more compositions. For example, the level of one or more metabolites can be observed, measured, or evaluated in a sample at different times (e.g., before administration of the composition, after administration of the composition, during administration of the composition, etc.). To determine whether one or more compositions treat, prevent, or reduce the risk of a disease, disorder, or condition (e.g., a neurodegenerative disease, disorder, or condition), one or more characteristics or parameters can be observed, measured, or evaluated in a sample contacted with one or more compositions. For example, one or more characteristics or parameters can be observed, measured, or evaluated in a sample at different times (e.g., before administration of the composition, after administration of the composition, during administration of the composition, etc.).

[0115] In some embodiments, the methods described herein utilize a first sample and a second sample. In some embodiments, the first sample is a reference sample. In some embodiments, the reference sample may be a sample obtained from a subject contacted with (e.g., administered or provided with) a composition, e.g., a CT10 composition, a CT6 composition, or a CT6m composition. In some embodiments, the reference sample may be a sample obtained from a subject contacted with (e.g., administered or provided with) a composition, e.g., a CT10 composition, a CT6 composition, or a CT6m composition, at a first time point. In some embodiments, the reference sample may be a sample obtained from a subject before contacting (e.g., administered or provided with) a composition, e.g., a CT10 composition, a CT6 composition, or a CT6m composition. In some embodiments, the reference sample may be a sample obtained from a healthy individual. In some embodiments, the reference sample may be a sample obtained from an individual who may be suffering from or at risk for a disease, disorder, or condition (e.g., a neurodegenerative disease, disorder, or condition). In some embodiments, the reference sample is a control sample. In some embodiments, the reference sample is a negative control sample. In some embodiments, the reference sample is a positive control sample. In some embodiments, the reference sample can be a historical reference (e.g., a value across a control sample). In some embodiments, the reference sample can be from a printed publication (e.g., a textbook, a journal, etc.).

[0116] In some embodiments, the second sample may be a test sample. In some embodiments, the test sample may be a sample obtained from a subject contacted with (e.g., administered or provided with) a composition, such as a CT10 composition, a CT6 composition, or a CT6m composition. In some cases, the subject (e.g., a patient or a population) may be suffering from or at risk of a disease, disorder, or condition (e.g., a neurodegenerative disease, disorder, or condition). In some cases, the subject (e.g., a patient or a population) may have an unknown risk of one or more diseases, disorders, or conditions described herein. In some embodiments, the test may be a sample obtained from a subject contacted with (e.g., administered or provided with) a composition, such as a CT10 composition, a CT6 composition, or a CT6m composition, at a second time point.

[0117] In some embodiments, the methods described herein include comparing one or more metabolite levels (e.g., metabolome), or one or more parameters or features (e.g., cell viability, microgliosis, astrocytosis, proteasome function, lysosomal function, inflammation, ATP levels, levels or activity of nucleic acids or proteins or forms thereof, etc.) obtained from a test sample together with one or more metabolite levels (e.g., metabolome), or one or more parameters or features (e.g., cell viability, microgliosis, astrocytosis, proteasome function, lysosomal function, inflammation, ATP levels, levels or activity of nucleic acids or proteins or forms thereof, etc.) obtained from a reference sample. In some embodiments, by comparing one or more metabolite levels, parameters, or features obtained from a test sample with one or more metabolite levels, parameters, or features obtained from a reference sample, a composition described herein can be evaluated, characterized, or identified as useful for treating, preventing, or reducing the risk of a disease, disorder, or condition described herein (e.g., a neurodegenerative disease, disorder, or condition). In some embodiments, by comparing one or more metabolite levels, parameters, or characteristics obtained from a test sample with one or more metabolite levels, parameters, or characteristics obtained from a reference sample, it can be determined that the compositions disclosed herein increase the severity or incidence of a disease, disorder, or condition phenotype. In some embodiments, by comparing one or more metabolite levels, parameters, or characteristics obtained from a test sample with one or more metabolite levels, parameters, or characteristics obtained from a reference sample, it can be determined that the compositions disclosed herein reduce the severity or incidence of a disease, disorder, or condition phenotype. In some embodiments, by comparing one or more metabolite levels, parameters, or characteristics obtained from a test sample with one or more metabolite levels, parameters, or characteristics obtained from a reference sample, it can be determined that the compositions disclosed herein do not affect the severity or incidence of a disease, disorder, or condition phenotype.In some embodiments, it may be determined that a composition disclosed herein prevents a disease, disorder, or condition phenotype by comparing one or more metabolite levels, parameters, or characteristics obtained from a test sample with one or more metabolite levels, parameters, or characteristics obtained from a reference sample.

[0118] The present disclosure also provides the recognition that the compositions and methods provided herein can be used to monitor the progression of a disease, disorder, or condition (e.g., a neurodegenerative disease, disorder, or condition) in an individual. For example, if a metabolite level, parameter, or feature determined to increase the severity of a disease, disorder, or condition (e.g., cell viability, microgliosis, astrocytosis, proteasome function, lysosomal function, inflammation, ATP levels, levels or activities of nucleic acids or proteins or forms thereof, etc.) decreases in relative amount, it may indicate that the disease, disorder, or condition is attenuated, for example, by a treatment or immune response.

[0119] The present disclosure also provides insight that the compositions and methods provided herein can be used to tailor treatments (e.g., therapeutics, nutraceuticals, and / or probiotics) to individual patients. In some embodiments, the compositions and methods provided herein can provide "personalized" therapy. In some cases, metabolite levels, features, or parameters (e.g., cell viability, microgliosis, astrocytosis, proteasome function, lysosomal function, inflammation, ATP levels, levels or activities of nucleic acids or proteins or forms thereof, etc.) within an individual can be evaluated, characterized, or identified to determine whether the individual has a disease, disorder, or condition. Based on the results, the individual can be treated with one or more compositions to adjust metabolite levels (i.e., its metabolome), features, or parameters. In some cases, this will affect the disease, disorder, or condition that the individual suffers from or is at risk of developing. For example, if an individual is determined to have relatively small levels of one or more metabolites determined to reduce the severity of a disease, disorder, or condition, administration to the individual of one or more compositions (or extracts, components, or compounds thereof) determined to reduce the severity of the disease, disorder, or condition may attenuate the severity of the disease or condition in the individual.

[0120] The present disclosure provides insight that the compositions and methods provided herein may be used recursively to treat, prevent, or ameliorate a disease, disorder, or condition. In some embodiments, for example, one or more compositions disclosed herein may be administered (e.g., fed, injected, etc.) to a subject after determining the effect of one or more compositions on the subject's metabolite levels, or after determining the effect of one or more compositions on the subject's characteristics or parameters (e.g., cell viability, microgliosis, astrocytosis, proteasome function, lysosomal function, inflammation, ATP levels, levels or activity of nucleic acids or proteins or forms thereof, etc.). In some embodiments, the composition may be administered once. In some embodiments, the composition may be administered more than once. In some embodiments, the composition may be administered daily, weekly, biweekly, monthly, bimonthly, etc. In each of these cases, changes in the levels or characteristics or parameters of one or more metabolites may be monitored. In some embodiments, changes in the levels or characteristics or parameters of one or more metabolites (e.g., metabolome) may be monitored prior to administration of the composition. In some embodiments, changes in the levels of one or more metabolites (eg, the metabolome), or characteristics or parameters, may be monitored following administration of the composition.

[0121] Pharmaceutical Compositions Compositions are provided herein that include individual microbial strains, or combinations of microbial strains, their metabolites, their extracts, or their components. In some embodiments, the compositions include individual microbial strains or combinations of microbial strains from mammalian microbiota, their metabolites, their extracts, and / or their components that have been assessed, identified, characterized, or assayed using the methods described herein. In some embodiments, the compositions provided herein include one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more microbial strains from mammalian microbiota, their extracts, their metabolites, and / or their components that have been assessed, identified, characterized, or assayed using the methods described herein.

[0122] Compositions comprising one or more components or metabolites are also provided herein.In some embodiments, the components or metabolites in the compositions herein are derived from a source that is not a microbial strain, for example, are synthetically produced.In some embodiments, the components or metabolites in the compositions may be identified from a microbial strain, but are independent of the microbial strain and are not produced by the microbial strain, for example, they can be synthetically produced.

[0123] In some embodiments, the compositions provided herein comprise two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more microbial strains listed in Table 1 below. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8]

[0124] In some embodiments, the compositions provided herein include Gluconacetobacter hansenii, Terrisporobacter glycolicus, Coprococcus sp., Lactobacillus plantarum, Clostridium butyricum, Paenibacillus sp., Veillonella sp., Bifidobacterium sp., Bacillus subtilis, Acidaminococcus sp., or a combination thereof. In some embodiments, the compositions include at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or all of Gluconacetobacter hansenii, Terrisporobacter glycolicus, Coprococcus sp., Lactobacillus plantarum, Clostridium butyricum, Paenibacillus sp., Veillonella sp., Bifidobacterium sp., Bacillus subtilis, and Acidaminococcus sp. In some embodiments, for example, the composition includes all of Gluconacetobacter hansenii, Terrisporobacter glycolicus, Coprococcus sp., Lactobacillus plantarum, Clostridium butyricum, Paenibacillus sp., Veillonella sp., Bifidobacterium sp., Bacillus subtilis, and Acidaminococcus sp., and may be referred to by different names, including, but not limited to, CT10 composition, CT10 cocktail, and the like.

[0125] In some embodiments, the compositions provided herein include Gluconacetobacter hanseni, Terrisporobacter glycolicus, Coprococcus sp., Lactobacillus plantarum, Veillonella sp., Bifidobacterium sp., or combinations thereof. In some embodiments, the compositions include at least two, at least three, at least four, at least five, or all of Gluconacetobacter hanseni, Terrisporobacter glycolicus, Coprococcus sp., Lactobacillus plantarum, Veillonella sp., and Bifidobacterium sp. In some embodiments, for example, the compositions include all of Gluconacetobacter hanseni, Terrisporobacter glycolicus, Coprococcus sp., Lactobacillus plantarum, Veillonella sp., and Bifidobacterium sp., and may be referred to by different names, including, but not limited to, CT6 composition, CT6 cocktail, and the like. In some embodiments, the compositions provided herein include Gluconacetobacter hanseni, Terrisporobacter glycolicus, Coprococcus catus, Lactobacillus plantarum, Veillonella atypica, Bifidobacterium breve, or a combination thereof. In some embodiments, the compositions include at least two, at least three, at least four, at least five, or all of Gluconacetobacter hanseni, Terrisporobacter glycolicus, Coprococcus catus, Lactobacillus plantarum, Veillonella atypica, and Bifidobacterium breve.In some embodiments, for example, the composition includes all of Gluconacetobacter hanseni, Terrisporobacter glycolicus, Coprococcus catus, Lactobacillus plantarum, Veillonella atypica, and Bifidobacterium breve, and may be referred to by different names, including, but not limited to, CT6 composition, CT6 cocktail, etc.

[0126] In some embodiments, the compositions provided herein comprise one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more metabolites. Metabolites that may be assessed, identified, characterized, or assayed for and / or included in the compositions disclosed herein include, for example, those listed in the appendices filed herewith (e.g., Appendix 1-1, 1-2, 1-3, 2, 3, 4, or 5).

[0127] In some embodiments, the metabolite can be butyrylcamitine, theobromine, p-hydroxyphenylpyruvic acid, propionic acid, picolinic acid, 2-hydroxy-4 methylvaleric acid, N6-acetylcysine, urocanic acid, N5-ethylglutamine, trigonelline, stachydrine, ectoine, 5-hydroxylysine, arginine (Arg), cholic acid, 2-(4-hydroxyphenyl)propionic acid, N-acetyltryptophan, hydroxyproline, argininosuccinic acid, glutamic acid (Glu), sarcosine, 5-methoxyindoleacetic acid, indole-3-lactic acid, isovalerylalanine, N-acetylleucine, 1-methylhistidine, N-acetylphenylalanine, proline (Pro), or any combination thereof.

[0128] In some embodiments, the metabolite is 4-hydroxyphenylpyruvate, ectoine, gramine, N-acetyl-L-phenylalanine, nepsilon-acetyl-L-lysine, stachydrine, trigonelline, 3-ureidopropionic acid, theobromine, hippuric acid, imidazolepropionic acid, NG-methyl-L-arginine, transurocanic acid, N-acetyl-L-leucine, sarcosine, isobutyrylcarnitine, b-hydroxyisovaleric acid, L-theanine / N5-ethylglutamine, 5-hydroxylysine, phenaceturic acid, betaine, hydroxyproline, picolinic acid, 2-aminoadipic acid, glycerophosphocholine, carnitine, glycerol 3-phosphate, argininosuccinic acid, creatine, terephthalic acid, homocitrulline, mucic acid, homocysteine ​​sulfinic acid, trimethyllysine, spermidine, glyoxylic acid, XA0013 C6H6O4S, 3-indoxyl sulfate, nicotinamide, N-formylglycine, ureidoglycolate, N-methylproline, glucaric acid, butyrylcarnitine, methionine sulfoxide, carboxymethyllysine, glycolic acid, phenaceturic acid, diethanolamine, phosphorylcholine, guanidinosuccinic acid, N-acetylhistidine, glyceric acid, S-methylmethionine, cysteine ​​glutathione disulfide, kynurenine, N-acetylphenylalanine, threonic acid, malic acid, 7,8-dihydrobiopterin, homovanillic acid, taurocholic acid, 5-methoxyindoleacetic acid, butyrate, b-hydroxyisovaleric acid, 2-oxoglutaric acid, N-acetyltryptophan, thiaproline, hypotaurine, cholic acid, acetoacetic acid, ethanolamine, guanidoacetic acid, S-sulfocysteine, myristic acid C14:0 XA0027, or any combination thereof.

[0129] In some embodiments, the individual microbial strains or combinations of microbial strains are derived from the microbiota of a killed (e.g., heat-killed) mammal. Alternatively, in some embodiments, the individual microbial strains or combinations of microbial strains derived from the microbiota of a mammal may be viable or contain living cells.

[0130] In some embodiments, the one or more microbial strains include viable or living individual microbial strains or combinations of microbial strains, for example, from a mammalian microbiota.

[0131] In some embodiments, the one or more microbial strains include, for example, viable or living individual microbial strains or combinations of microbial strains derived from a mammalian microbiota, and include, and / or are formulated through the use of, one or more cell cultures and / or supernatants or pellets thereof, and / or powders formed therefrom, as described herein.

[0132] In some embodiments, the compositions for use according to the present disclosure are, for example, pharmaceutical compositions for administration (e.g., topical, oral, subcutaneous, intravenous, intramuscular, intracerebral, intrathecal, rectal (e.g., rectal intubation), intraocular, intravitreal, or suprachoroidal administration) to a mammal (e.g., a human). Pharmaceutical compositions typically include an active agent (e.g., an individual or combination of microbial strains from a mammalian microbiota, an extract thereof, and / or a component thereof) and a pharmaceutically acceptable carrier. Certain exemplary pharmaceutically acceptable carriers include, for example, saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration.

[0133] In some embodiments, pharmaceutical compositions for use according to the present disclosure may include and / or be administered in conjunction with one or more supplementary active compounds, and in certain embodiments, such supplementary active agents may be ginger, curcumin, probiotics (e.g., probiotic strains of one or more of the following genera: Lactobacillus, Bifidobacterium, Saccharomyces, Enterococcus, Streptococcus, Pediococcus, Leuconostoc, Bacillus, and / or Escherichia coli (Fijan, Int J Environ Res Public Health. 2014, incorporated herein by reference in its entirety). May;11(5):4745-4767), prebiotics (non-digestible food ingredients that help support the growth of probiotic bacteria, e.g., fructans such as fructooligosaccharides (FOS) and inulin, galactans such as galactooligosaccharides (GOS), dietary fibers such as resistant starch, pectin, beta-glucans, and xylooligosaccharides) (see Hutkins et al., Curr Opin Biotechnol. 2016 Feb;37:1-7, incorporated herein by reference in its entirety), and combinations thereof.

[0134] In some embodiments, the prebiotics include fructooligosaccharides, inulin, isomaltooligosaccharides, lactylol, lactosucrose, lactulose, soybean oligosaccharides, transgalactooligosaccharides, xylooligosaccharides, seaweed, or combinations thereof. In some embodiments, the prebiotics include seaweed. In some embodiments, the prebiotics include pome fruit extract, berry extract, and walnut extract.

[0135] In some embodiments, the probiotic composition may be formulated for oral administration. In some embodiments, the probiotic composition may be a food, beverage, feed composition, or dietary supplement. In some embodiments, the ellagitanin composition, the enzyme composition, or both, may be a liquid, syrup, tablet, lozenge, gummy, capsule, powder, gel, or film. In some embodiments, the probiotic composition is an enteric coated formulation.

[0136] In some embodiments, the probiotic comprises a prebiotic, which in some embodiments comprises fructooligosaccharides, inulin, isomaltooligosaccharides, lactylol, lactosucrose, lactulose, soybean oligosaccharides, transgalactooligosaccharides, xylooligosaccharides, seaweed, pome fruit extract, berry extract, and walnut extract, or a combination thereof.

[0137] A pharmaceutical composition is typically formulated to be compatible with its intended route of administration. Examples of routes of administration include topical, oral, subcutaneous, intravenous, intramuscular, intracerebral, intrathecal, rectal (e.g., rectal intubation), intraocular, intravitreal, or suprachoroidal administration. Methods for formulating suitable pharmaceutical compositions are known in the art, see, for example, Remington: The Science and Practice of Pharmacy, 21st ed., 2005, and the books in the series Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY), which are incorporated herein by reference in their entirety. Oral compositions generally include an inert diluent or an edible carrier (e.g., a pharma- ceutically acceptable diluent, a pharma- ceutically acceptable carrier). In some embodiments, oral formulations can be or include syrups, liquids, tablets, lozenges, gummies, capsules, such as gelatin capsules, powders, gels, films, and the like, to name just a few. Similarly, ophthalmic compositions (e.g., for intraocular, intravitreal, or suprachoroidal administration) may include various additives, such as inert diluents or carriers (e.g., pharma- ceutically acceptable diluents, pharma-ceutically acceptable carriers), viscosity enhancers, permeation enhancers, cyclodextrins, and the like. Examples of viscosity enhancers include hydroxymethylcellulose, hydroxyethylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, and polyalcohols. Examples of permeation enhancers include chelating agents, preservatives, surfactants, bile salts, benzalkonium chloride, polyoxyethylene glycol ethers (lauryl, stearyl, and oleyl), ethylenediaminetetraacetic acid sodium salt, sodium taurocholate, saponin, and Cremophor EL, and the like. For example, in some embodiments, the ophthalmic formulation may be or include a suspension, an emulsion (e.g., water-in-oil or oil-in-water), a nanocarrier, (e.g., nanoparticles, nanosuspensions, liposomes, nanomicelles, dendrimers, and the like), an ointment, a gel, an eye drop, and the like.The brain composition (e.g., for intracerebral or intrathecal administration) may include an inert diluent or carrier, and / or an additive. In some embodiments, the brain composition does not include a preservative. In some embodiments, the brain composition is sterile.

[0138] In some embodiments, pharma- ceutical compatible binders and / or adjuvant materials can be included as part of the pharmaceutical composition. In some particular embodiments, the pharmaceutical composition can contain, for example, any one or more of the following inactive ingredients or compounds of a similar nature: binders such as microcrystalline cellulose, gum tragacanth, or gelatin; excipients such as starch or lactose; disintegrants such as alginic acid, Primogel, or corn starch; lubricants such as magnesium stearate or sterotes; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or flavorings such as peppermint, methyl salicylate, or orange flavoring. In some embodiments, the composition can be taken as is, or sprinkled on or mixed into food or liquid (such as water). In some embodiments, compositions that can be administered to a mammal described herein can be or can include ingestible items (e.g., foods or beverages) that contain (e.g., are supplemented with) individual or combinations of microbial strains from the mammal's microbiota, extracts thereof, and / or components thereof.

[0139] In some embodiments, the food product can be or include one or more of bars, candies, baked goods, cereals, savory snacks, pasta, chocolate, and other solid foods, liquid or semi-solid foods, including yogurt, soups, and stews, and beverages, such as smoothies, shakes, juices, and other carbonated or non-carbonated beverages. In some embodiments, the food product is prepared by the subject by mixing individual or combinations of microbial strains from a mammal's microbiota, extracts thereof, and / or components thereof.

[0140] The compositions can be included in a kit, container, pack, or dispenser together with instructions for administration or use in the methods described herein.

[0141] Those of skill in the art will understand upon reading this disclosure that in some embodiments, the compositions (e.g., pharmaceutical compositions) described herein can be or include one or more cells, tissues, or organisms (e.g., plant or microbial cells, tissues, or organisms) that produce (e.g., have produced and / or are producing) the relevant compound.

[0142] Those skilled in the art will appreciate that in some embodiments, techniques for preparing and / or preparing compositions and / or preparations (particularly for preparing pharmaceutical compositions) may include one or more steps of evaluating or characterizing the compound, preparation, or composition, e.g., as part of quality control. In some embodiments, if an assayed material does not meet predetermined specifications for relevant evaluation, it is discarded. In some embodiments, if such assayed material meets predetermined specifications, it continues to be processed as described herein.

[0143] In some embodiments, the pharmaceutical compositions provided herein can promote colonization of individual microbial strains or combinations of microbial strains from a mammalian microbiota, particularly microbial strain(s) that have been identified, characterized, or evaluated as reducing the severity or incidence of a mammalian disease, disorder, or condition in a mammal suffering from or at risk for the mammalian disease, disorder, or condition. In some embodiments, the pharmaceutical compositions provided herein can attenuate colonization of individual microbial strains or combinations of microbial strains from a mammalian microbiota, particularly microbial strain(s) that have been identified, characterized, or evaluated as increasing the severity or incidence of a mammalian disease, disorder, or condition in a mammal suffering from or at risk for the mammalian disease, disorder, or condition. In some embodiments, the pharmaceutical compositions provided herein are capable of promoting colonization of individual microbial strains or combinations of microbial strains from the microbiota of a mammal, particularly a microbial strain(s) that have been identified, characterized, or evaluated as not affecting the severity or incidence of a mammalian disease, disorder, or condition, but that have been identified, characterized, or evaluated as being capable of outcompeting one or more microbial strains that have been identified, characterized, or evaluated as increasing the severity or incidence of a mammalian disease, disorder, or condition, in a mammal suffering from or at risk for the mammalian disease, disorder, or condition.

[0144] In some embodiments, each of the one or more microbial strains in the composition is at least 10 1 Colony forming units (CFU) ~10 20 In some embodiments, each of the one or more microbial strains in the composition comprises 10 1 Colony forming units (CFU) ~10 15 In some embodiments, each of the one or more microbial strains in the composition comprises 10 6 CFU~10 15 In some embodiments, each of the one or more microbial strains in the composition comprises about 101 CFU~10 15 CFU, or about 10 2 CFU~10 14 CFU, or about 10 3 CFU~10 13 CFU, or about 10 4 CFU~10 13 CFU, or about 10 5 CFU~10 12 CFU, or about 10 6 CFU~10 11 CFU, or about 10 7 CFU~10 10 CFU, or about 10 8 CFU~10 9 CFU, or about 10 5 CFU~10 10 CFU, or about 10 8 CFU~10 12 In some embodiments, each of the one or more microbial strains in the composition comprises at least about 10 1 , 5×10 1 , 10 2 , 5×10 2 , 10 3 , 5×10 3 , 10 4 , 5×10 4 , 10 5 , 5×10 5 , 10 6 , 5×10 6 , 10 7 , 5×10 7 , 10 8 , 5×10 8 , 10 9 , 5×10 9 , 10 10 , 5×10 10 , 10 11 , 5×10 11 , 10 12 In some embodiments, each of the one or more microbial strains in the composition comprises at most about 10 15 , 5×10 14 , 10 14 , 5×10 13 , 10 13 , 5×10 12 , 1012 , 5×10 11 , 10 11 , 5×10 10 , 10 10 , 5×10 9 , 10 9 , 5×10 8 , 10 8 In some embodiments, each of the one or more microbial strains in the composition comprises the same number of CFUs. In some embodiments, some of the one or more microbial strains in the composition comprise different numbers of CFUs.

[0145] In some embodiments, the composition comprises a total of 10 1 CFU~10 20 In some embodiments, the composition comprises a total of 10 6 CFU~10 15 In some embodiments, the composition comprises about 10 1 CFU~10 20 CFU, or about 10 5 CFU~10 15 CFU, or about 10 5 CFU~10 12 CFU, approximately 10 5 CFU~10 10 CFU, or about 10 8 CFU~10 12 In some embodiments, the composition may comprise about 10 CFU of one or more microbial strains. 1 CFU~10 15 CFU, or about 10 2 CFU~10 14 CFU, or about 10 3 CFU~10 13 CFU, or about 10 4 CFU~10 13 CFU, or about 10 5 CFU~10 12 CFU, or about 10 6 CFU~10 11 CFU, or about 10 7 CFU~10 10 CFU, or about 10 8 CFU~10 9CFU, or about 10 5 CFU~10 10 CFU, or about 10 8 CFU~10 12 In some embodiments, the composition may comprise at least about 10 CFU of one or more microbial strains. 1 , 5×10 1 , 10 2 , 5×10 2 , 10 3 , 5×10 3 , 10 4 , 5×10 4 , 10 5 , 5×10 5 , 10 6 , 5×10 6 , 10 7 , 5×10 7 , 10 8 , 5×10 8 , 10 9 , 5×10 9 , 10 10 , 5×10 10 , 10 11 , 5×10 11 , 10 12 In some embodiments, the composition may contain at most about 10 CFU of one or more microbial strains. 15 , 5×10 14 , 10 14 , 5×10 13 , 10 13 , 5×10 12 , 10 12 , 5×10 11 , 10 11 , 5×10 10 , 10 10 , 5×10 9 , 10 9 , 5×10 8 , 10 8 The culture may contain one or more microbial strains at or below CFU.

[0146] In some embodiments, a pharmaceutical composition is tailored to a particular mammal (e.g., a particular human, e.g., a patient) based on the microbiota of that mammal (e.g., a human). In some embodiments, a pharmaceutical composition is specific to the microbiota of an individual mammal (e.g., a human). In some embodiments, a pharmaceutical composition is specific to the microbiota of a population of mammals (e.g., humans). A population of mammals can include, but is not limited to, a family, mammals in the same geographic location (e.g., neighborhood, city, state, or country), mammals having the same disease or condition, mammals of a particular age or age range, mammals consuming a particular diet (e.g., foods, food sources, or caloric intake).

[0147] Treatment methods The present disclosure recognizes that the compositions described herein can be useful for treating a subject. The methods provided by the present disclosure include methods for treating certain diseases, disorders, and conditions. In some embodiments, the relevant disease, disorder, and condition can be or include a neurodegenerative disease, disorder, or condition. In some embodiments, the neurodegenerative disease, disorder, or condition can be ALS, AD, PD, or HD.

[0148] Generally, the methods of treatment provided by the present disclosure involve administering to a subject in need of, or determined to be in need of, such treatment a therapeutically effective amount of a composition described herein, alone or in combination with other compositions and / or treatments.

[0149] In some embodiments, the methods of treatment provided herein are prophylactic or preventative, e.g., may be administered to a subject prior to the display of noticeable symptoms and / or prior to exposure to a particular anticipated trigger associated with a neurodegenerative disease, disorder, or condition described herein. In some embodiments, the methods of treatment provided herein are therapeutic, e.g., may be administered to a subject following the onset of noticeable symptoms associated with a neurodegenerative disease, disorder, or condition.

[0150] In some embodiments, the methods of treatment provided are administered to a subject that is a mammal, e.g., a mammal experiencing a disease, disorder, or condition described herein, and in some embodiments the subject is a human or non-human animal subject, e.g., an ape, a cat, a monkey, or a pig.

[0151] In many embodiments, treatment involves ameliorating at least one symptom of a disease, disorder, or condition associated with a neurodegenerative disease, disorder, or condition. In some embodiments, the method of treatment can be prophylactic.

[0152] In some embodiments, the methods can include administration of a therapeutically effective amount of a composition disclosed herein before, during (e.g., simultaneously with), or after administration of a treatment expected to be associated with a neurodegenerative disease, disorder, or condition.

[0153] In some embodiments, a subject receiving a treatment described herein may be undergoing and / or may have been undergoing other therapies (e.g., pharmacological treatments / therapies, surgery, etc.) that may be intended to treat, for example, one or more symptoms of a disease, disorder, or condition described herein (e.g., a neurodegenerative disease, disorder, or condition), and thus the provided compositions are administered in combination with such other therapies (i.e., treatments) to treat the relevant disease, disorder, or condition.

[0154] In some embodiments, the compositions described herein may be administered in a form containing one or more pharma- ceutically acceptable carriers. Suitable carriers have been described previously and vary with the desired form and mode of administration of the composition. For example, pharma- ceutically acceptable carriers may include diluents or excipients, such as fillers, binders, wetting agents, disintegrants, surfactants, glidants, and lubricants. Typically, carriers may be solids (including powders), liquids, or any combination thereof. Each carrier is preferably "acceptable" in the sense that it is compatible with the other ingredients in the composition and not harmful to the subject. Carriers may be biologically acceptable and inert (e.g., allowing the composition to maintain the viability of biological materials until delivered to the appropriate site).

[0155] Tablets, pills, capsules, troches, and the like can contain any of the following ingredients, or compounds of a similar nature: binders such as microcrystalline cellulose, gum tragacanth, or gelatin, excipients such as starch or lactose, disintegrants such as alginic acid, primogel, or corn starch, lubricants such as magnesium stearate or sterotes, glidants such as colloidal silicon dioxide, sweeteners such as sucrose or saccharin, or flavorings such as peppermint, methyl salicylate, orange flavoring, or other suitable flavors. These are for purposes of example only and are not intended to be limiting.

[0156] Oral compositions can include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with an excipient and used in the form of tablets, lozenges, drops, troches, or capsules, such as gelatin capsules. Oral compositions can also be prepared by combining the compositions of the present disclosure with food. In some embodiments, the microorganism (e.g., one or more microbial strains) can be formulated into food. Some non-limiting examples of food that can be used with the methods and compositions described herein include popsicles, cheese, cream, chocolate, milk, meat, beverages, pickled vegetables, kefir, miso, sauerkraut, and the like. In other embodiments, the food may be juice, soft drinks, tea drinks, beverage preparations, jelly drinks, and functional drinks, alcoholic beverages such as beer, carbohydrate-containing foods such as rice foods, noodles, bread, and pasta, paste products such as fish, ham, sausage, seafood paste products, curry, foods with thick starchy sauces, and retort pouch products such as Chinese soup, dairy products such as soup, milk, milk drinks, ice cream, and yogurt, fermented products such as fermented soybean paste, fermented beverages, and pickles, soy products, various confectionery products including biscuits, cookies, and the like, candy, chewing gum, gummies, jellies, cream caramel, and cold desserts including frozen desserts, instant foods such as instant soups and instant miso soup, and the like. It is preferred that the food preparation does not require cooking after mixing with the microbial strain(s) to avoid killing any microorganisms. In one embodiment, the food used for administration is chilled, for example iced, flavored water. In certain embodiments, the food product is not a potentially allergenic food (e.g., not soy, wheat, peanut, tree nut, dairy, egg, shellfish, or fish). Pharmaceutically compatible binding agents, and / or adjuvant materials can be included as part of the composition.

[0157] Ophthalmic formulations (e.g., for intraocular, intravitreal, or suprachoroidal administration) can include inert diluents or carriers. For the purpose of ophthalmic therapeutic administration, active compounds can be incorporated with excipients and used in the form of suspensions, emulsions (e.g., water-in-oil or oil-in-water), nanocarriers, (e.g., nanoparticles, nanosuspensions, liposomes, nanocells, dendrimers, etc.), ointments, gels, eye drops, etc. In some embodiments, administration of such formulations is topical (e.g., eye drops). In some embodiments, administration of such formulations is via injection (e.g., intravitreal, suprachoroidal, etc.).

[0158] Brain preparations (e.g., for intracerebral or intrathecal administration) can include inert diluents or carriers. For the purpose of brain therapeutic administration, active compounds can be incorporated with excipients and used in the form of suspensions, emulsions (e.g., water-in-oil or oil-in-water), nanocarriers, (e.g., nanoparticles, nanosuspensions, liposomes, nanocells, dendrimers, etc.), ointments, gels, etc. In some embodiments, administration of such formulations is local (e.g., ointments). In some embodiments, administration of such formulations is via injection (e.g., intracerebral, intrathecal, etc.).

[0159] In some such embodiments, the compositions described herein are administered to a subject according to a dosing schedule that achieves the administration of cells into the microbiota of the subject.In some embodiments, the compositions are administered to a subject in a single dose.In some embodiments, the compositions are administered to a subject in multiple doses.In some embodiments, a single dose of the composition is administered to a subject twice a day, daily, weekly, or monthly.

[0160] In some embodiments, each of the one or more microbial strains in a dose is 10 1 ~10 15 In some embodiments, each of the one or more microbial strains in a dose comprises 10 6 ~10 15In some embodiments, each of the one or more microbial strains in a dose contains the same number of CFU. In some embodiments, some of the one or more microbial strains in a dose contain different numbers of CFU.

[0161] In some embodiments, a single dose of one or more microbial strains comprises a total of 10 6 ~10 15 In some embodiments, a single dose of one or more microbial strains comprises a total of 10 7 ~10 15 In some embodiments, a single dose of the one or more microbial strains comprises between 5 and 200 billion CFU. In some embodiments, a single dose of the one or more microbial strains comprises between 5 and 50 billion CFU. In some embodiments, a single dose of the one or more microbial strains comprises between 5 and 20 billion CFU. In some embodiments, a single dose of the one or more microbial strains comprises between 5 and 100 billion CFU. In some embodiments, a single dose of the one or more microbial strains comprises between 100 and 200 billion CFU.

[0162] In some embodiments, efficacy can be evaluated by measuring the degree of cellular oxidative stress in biological samples before and after administration of the compositions described herein.The degree of cellular oxidative stress can be evaluated, for example, by measuring the expression of oxidative stress biomarkers, such as reactive oxygen species (ROS) levels, or lipid, protein, and nucleic acid damage levels, or by determining the ratio of oxidized to reduced forms of one or more biomarkers.Since high levels of oxidative stress can be cytotoxic, the degree of oxidative stress can be measured by evaluating the concentration of intracellular proteins present in the systemic circulation from inflamed or lysed cells (e.g., neuronal cells). EXAMPLES

[0163] Thorough details of the objectives, mouse models used, studies performed, and results of Examples 1-5 are listed in Appendix 5 submitted herewith.

[0164] Example 1: Evaluating the efficacy of microbiota compositions in a male mouse model of ALS Thorough details of the objectives, mouse model used, studies performed, and results of this example are listed in Appendix 5 submitted herewith.

[0165] This example provides an evaluation of the efficacy of microbiota compositions, specifically, CT6 and CT6m compositions, in an in vivo male mouse model of ALS.

[0166] Mouse model: Superoxide dismutase (SOD), also known as superoxide dismutase 1 or SOD1, is an enzyme that is encoded by the SOD1 gene in humans and is involved in apoptosis and familial ALS. SOD1-G93A (or G93A-SOD1, or SOD1 G93A A .) transgene was designed with a mutant human SOD1 gene (with a single amino acid substitution of glycine to alanine at codon 93) driven by its endogenous human SOD1 promoter. This transgene was injected into fertilized B6SJLF1 mouse eggs to obtain founder animals. Transgenic mice on a mixed B6SJL genetic background were sent to The Jackson Laboratory.

[0167] ALS-SOD1 mice with the aforementioned genetic background that were 49–63 days old were used for all experiments. This mouse model is the most well-received transgenic mouse with a four-fold increase in SOD activity and displays a phenotype similar to ALS in humans. Wild-type mice were used as controls in all experiments.

[0168] Study: Male mice were divided into 4 groups of 13-15 mice per group and provided with sham (DPBS) or microbiota composition (CT6 or CT6m, composition details listed in Tables 2 and 3 below) by daily oral gavage. Groups were: (i) G1: wild type mice treated with DPBS, (ii) G2: SOD1 transgenic mice treated with DPBS, (iii) G3: SOD1 transgenic mice treated with CT6 composition, and (iv) G4: SOD1 transgenic mice treated with CT6m composition. Animals were sacrificed when they reached a NeuroScore (NS) of 4 (see Appendix 5 for details) and then survival, histological and biochemical analyses were performed. [Table 3] [Table 4]

[0169] result: Microgliosis in the spinal cord: Tissue samples from the lumbar spinal cord of mice in each group were examined for microgliosis, an important marker of neuroinflammation, by anti-IBA1 staining and fluorescent imaging. As shown in Figure 1AA, Figure 1AB, and Figure 1B, mice in group 2, i.e., mock-treated SOD1 mice, showed higher levels of microgliosis compared to wild-type mice in group 1. Furthermore, treatment with the CT6 and CT6m microbiota cocktail (i.e., mice in groups 3 and 4) showed reduced microgliosis, as evidenced by reduced fluorescence intensity in lumbar spinal cord tissues of mice in groups 3 and 4. Indeed, treatment with the CT6 and CT6m cocktail resulted in the restoration of microglial levels to near normal (compared to wild-type mice in group 1) from the 1.5-fold increase seen in the ALS-SOD1 control group 2.

[0170] Astrocytosis in the spinal cord: Tissue samples from the lumbar spinal cord of mice in each group were examined for astrocytosis, another important marker of neuroinflammation, by anti-GFAP staining and fluorescent imaging. As shown in Figure 2AA, Figure 2AB, and Figure 2B, mice in group 2, i.e., mock-treated SOD1 mice, showed significantly higher levels of astrocytosis compared to wild-type mice in group 1. Furthermore, treatment with the CT6 and CT6m microbiota cocktail (i.e., mice in groups 3 and 4) showed reduced astrocytosis, as evidenced by reduced fluorescence intensity in lumbar spinal cord tissues of mice in groups 3 and 4. Indeed, treatment with the CT6 and CT6m cocktail resulted in the restoration of astrocyte levels to near normal (compared to wild-type mice in group 1) from the 3-fold increase seen in the ALS-SOD1 control group 2.

[0171] ATP production in the apical spinal cord: Spinal cord lysates from the apical spinal cord of mice in each group were examined for ATP levels. Spinal cord protein lysates were prepared using PhosphoSafe™ Extraction Reagent (EMD Millipore, Cat. 71296) and mechanically homogenized using bead-filled Lysing Matrix D tubes (MP Biomedicals, Illkirch, France). ATP production is an important marker of neurodegeneration, as individuals with neurodegenerative diseases, disorders, or conditions, such as ALS, PD, and AD, are known to have damaged mitochondria that lead to reduced ATP production. As shown in Figure 3, treatment with the CT6 and CT6m microbiota cocktail (i.e., mice in groups 3 and 4) showed approximately 2-fold increased production of ATP compared to wild-type mice in group 1 and ALS-SOD1 mice in group 2.

[0172] Plasma neurofilament light chain protein (NF-L) levels: Plasma from mice in each group was examined for neurofilament light chain (NF-L) protein levels. Death of motor neurons in the spinal cord is associated with an increased risk of developing SOD1 G93AIt is a salient feature of the model. NF-L is a product of axonal and neuronal damage that enters the bloodstream. NF-L is a well-validated biomarker for assessing the progression of neurodegenerative diseases, with higher plasma NF-L levels associated with neuronal death and increased disease severity (see Loeffler et al., Front Neurosci 14,579,2020). ALS, PD, and AD patients show increased blood levels of NF-L. Plasma NF-L levels of mice in each of the four groups were determined according to the protocol of the NF-L ELISA kit (Abbexa, abx154439). As shown in Figure 4, compared to wild-type mice in group 1, plasma levels of NF-L were significantly higher in SOD1 mice in group 2 than in wild-type mice in group 3. G93A The plasma levels of SOD1 in CT6- or CT6m-treated mice in groups 3 and 4 were significantly higher than those in wild-type mice (approximately 1.8-fold higher than the plasma levels in wild-type mice), indicating significantly higher death of motor neurons. However, CT6- or CT6m-treated mice in groups 3 and 4 showed significantly higher levels of SOD1 compared with mock-treated mice. G93A As can be seen in Figure 4, NF-L levels in the microbiota composition-treated mice (i.e., groups 3 and 4) were reduced on average from 1.8-fold (in mock-treated mice in group 2) to 1.35-fold the plasma levels in wild-type mice.

[0173] NMJ Innervation of the Tibialis Anterior Muscle: Neuromuscular (NMJ) integrity is a key focus for neuromuscular activity. This is the site for the transmission of action potentials from nerve to muscle. CT6 or CT6m administration increased SOD1 expression in the tibialis anterior muscle. G93A It was observed that mock-treated SOD1 T cells improved NMJ integrity in mice. A significant reduction in overlap between presynaptic (vesicular acetylcholine transporter, VAChT) and postsynaptic (α-bungarotoxin, α-BTX, which binds to the nicotinic acetylcholine transporter) receptors was observed in group 2 compared to wild-type mice in group 1. G93AA significant increase in overlap between VAChT and a-BTX was observed in CT6- or CT6m-treated animals in groups 3 and 4, suggesting a decrease in NMJ innervation. This result suggests that CT6 or CT6m administration preserves NMJ innervation (see FIG. 5).

[0174] Each dot in Figure 5 represents NMJs from at least 4 animals. The fluorescence intensity of each VAChT and a-BTX was measured and the ratio between them was used to calculate the % overlap. The center line of the box plot represents the mean value. ****p<0.0001. As shown in Figure 5, treatment with CT6 and CT6m significantly increased the NMJs compared to untreated SOD1 in Group 2. G93A This resulted in a two-fold increase in the levels of innervated NMJs compared to mice.

[0175] Proteasome function in the spinal cord: Neurons depend on the ubiquitin-proteasome system (UPS) and autophagy-associated lysosomal degradation for protein degradation and removal. The UPS is the major intracellular proteolytic system involved in maintaining protein turnover and selectively removing damaged proteins (Glickman and Ciechanover, Physiol Rev 82, 373-428, 2002). Notably, ubiquitin-rich protein inclusions are frequently observed in ALS patients (Migheli et al., Neurosci Lett 114, 5-10, 1990), while proteasome activity is downregulated by SOD1 in ALS. G93AIt is significantly reduced and / or impaired in the spinal cord of mouse models (Kabashi et al., J Neurochem 105, 2353-2366, 2008). Inhibition of proteasome activity increases SOD1 aggregates, whereas restoring proteasome function reduces the levels of protein aggregates (Puttaparthi et al., J Neurochem 87, 851-860, 2003). Therefore, improving or restoring proteasome function as a mechanism to reduce the accumulation of misfolded proteins is an attractive therapeutic approach. Upregulation of regulatory subunits of the proteasome, such as PSMD11, has been shown to increase proteasome assembly and functional activity, resulting in the clearance of polyubiquitinated substrates (Vilchez et al., Nature 489, 263-268, 2012).

[0176] To determine whether CT6 or CT6m treatment affects proteasome function, PSMD11 protein levels were quantified by Western blot in the spinal cord of SOD1-G93A mice. Spinal cord protein lysates were prepared using PhosphoSafe™ extraction reagent (EMD Millipore, Cat. 71296) and mechanically homogenized using bead-filled Lysing Matrix D tubes (MP Biomedicals, Illkirch, France). Protein concentrations were determined by the Bradford method using BioRad Protein assay reagent. Thirty mg of total protein was mixed with SDS sample buffer and boiled for 10 min. Samples were electrophoresed on custom-made SDS-polyacrylamide Bis-tris gels (4–12%) using MOPS or MES running buffer and subsequently transferred onto PVDF membranes (Invitrogen) using iBlot2 (Invitrogen). Membranes were blocked for 1 h using Odyssey blocking buffer and incubated overnight with PSMD11 specific antibody (cat. no. NBP1-30252, Novus Biologicals) at 1:1000 dilution under shaking conditions at 4°C. The next day, membranes were washed extensively in TBST (TBS+Tween 20) and incubated in HRP-conjugated secondary antibody (cat. no. 7076, Cell signaling) at 1:2000 dilution at room temperature for 1 h. Densitometric quantification of immunoblots was performed by GeneTools from Syngene after visualization with GBox Mini (SYNGENE). Target bands were normalized using respective b-actin loading controls. Each dot in the plot in Figure 6A represents pooled spinal cord lysates from two animals. PSMD11 protein levels were significantly higher in SOD1 compared to wild-type mice. G93A However, in spinal cord lysates of CT6- or CT6m-treated mice, SOD1 expression was significantly decreased compared with that of mock-treated mice. G93A Mice treated with CT6 or CT6m had significantly increased PSMD11 protein levels compared to SOD1 G93APSMD11 protein levels in spinal cord lysates of mice were at least 1.5- to 2-fold higher than the levels in wild-type mice. Statistical analysis was performed in GraphPad Prism. Data sets were analyzed by one-way ANOVA followed by Dunnett's test. Data are expressed as mean ± SEM. A level of p<0.05 was considered statistically significant. Compared to group 2 (i.e., G2), group 3 (i.e., G3) and group 4 (i.e., G4) had significantly elevated levels of PSMD11 (***p=0.0002 and ****p<0.0001).

[0177] To confirm that the increase in PSMD11 protein levels also corresponds to a concomitant increase in proteasome function, a proteasome substrate cleavage assay was performed. Suc-Leu-Leu-Val-Tyr-AMC (S-LLVY-AMC) is a fluorescent substrate that becomes fluorescent upon cleavage by the 20S proteasome. Spinal cord protein lysates were prepared and spinal cords were obtained by immersing the tissue in PhosphoSafe buffer (EMD Millipore) in tubes containing Lysing Matrix D (MP Biomedicals, Illkirch, France). Protein concentration was determined by the Bradford method using BioRad Protein assay reagent. 90 mg of total protein was added to assay buffer (T-PER containing 5 mM Suc-LLVY-AMC and 1 mM ATP). The enzymatic reaction was incubated at 37 °C / 5% CO 2The incubation was started at 4°C. Fluorescence was measured after 2 hours using a microplate reader (Promega discoverer, Promega corp). Excitation was set at 360 nm and emission at 450 nm. Assay buffer containing 5 mM Suc-LLVY-AMC was used as blank. Proteasome activity was calculated as (sample fluorescence value-blank fluorescence value). The mean of the fluorescence values ​​from group 1 (i.e., G1) was calculated and the % change in fluorescence compared to the mean of the G1 group was plotted. Statistical analysis was performed in GraphPad Prism. Data sets were analyzed by one-way ANOVA followed by Dunnett's test. Data are expressed as mean ± SEM. A level of p<0.05 was considered statistically significant. As shown in Figure 6B, compared to the G1 group, the G2 group had significantly lower proteasome activity (*p=0.0377). In contrast, groups G3 and G4 had significantly elevated levels of proteasome activity compared to group G2 (**p<0.01). These results suggest that CT6 and CT6m treatment improves proteasome activity. As can be seen from Figures 6A and 6B, treatment with CT6 and CT6m restored proteasome functional activity in the spinal cord to the level in wild-type mice.

[0178] Lysosomal function in the spinal cord: As mentioned above, neurons depend on the ubiquitin-proteasome system (UPS) and autophagy-associated lysosomal degradation for protein degradation and removal. While the UPS targets ubiquitin-conjugated proteins for removal, the lysosomal pathway targets long-lived proteins and damaged organelles. Dysregulation of protein degradation is involved in the pathogenesis of ALS (Root et al., Neurobiology of Disease 154, 105360, 2021) (Rubinsztein, Nature 443, 780-786, 2006). ALS is characterized by the presence of cytoplasmic inclusions or protein aggregates in affected motor neurons, which indicate reduced protein degradation. Lysosomal degradation of cytoplasmic aggregates or inclusions is crucial for neuronal growth and survival. Disruption of lysosomal function is also sufficient to cause neurodegeneration. Lysosome-associated membrane protein type 2 (LAMP2A) is a key protein required for the proper functioning of lysosomes and is the receptor for chaperone-mediated autophagy with which substrate proteins interact and are then transported into the lysosomal lumen for degradation.

[0179] To determine whether CT6 or CT6m treatment affects lysosomal function, LAMP2A protein levels were quantified by Western blot in the spinal cord of SOD1-G93A mice. Protein lysates were prepared using PhosphoSafe™ extraction reagent (EMD Millipore, Cat. 71296) and mechanically homogenized using bead-filled Lysing Matrix D tubes (MP Biomedicals, Illkirch, France). Protein concentrations were determined by the Bradford method using BioRad Protein assay reagent. Thirty mg of total protein was mixed with SDS sample buffer and boiled for 10 min. Samples were electrophoresed on custom-made SDS-polyacrylamide Bis-tris gels (4–12%) using MOPS or MES running buffer and subsequently transferred onto PVDF membranes (Invitrogen) using iBlot2 (Invitrogen). Membranes were blocked for 1 h using Odyssey blocking buffer and incubated overnight with LAMP2A specific antibody (cat. no. A0593, Abclonal) at 1:2000 dilution under shaking conditions at 4°C. The next day, membranes were washed extensively in TBST (TBS+Tween 20) and incubated in HRP-conjugated secondary antibody (cat. no. 7074, Cell signaling) at 1:2000 dilution at room temperature for 1 h. Densitometric quantification of immunoblots was performed by GeneTools from Syngene after visualization with GBox Mini (SYNGENE). Target bands were normalized using respective b-actin loading controls. Each dot in the plots in Figure 7 represents pooled spinal cord lysates from two animals. LAMP2A protein levels were significantly higher in SOD1 compared to wild-type mice. G93A However, in spinal cord lysates of CT6- or CT6m-treated mice, SOD1 expression was significantly decreased compared with that of mock-treated mice. G93A Mice treated with CT6 or CT6m had significantly increased LAMP2A protein levels compared to SOD1 mice. G93ALAMP2A protein levels in spinal cord lysates of mice were restored to the levels in wild-type mice. Statistical analysis was performed with GraphPad Prism. Data sets were analyzed by one-way ANOVA followed by Dunnett's test. Data are expressed as mean ± SEM. A level of p<0.05 was considered statistically significant. As shown in Figure 7, compared with the G2 group, G3 and G4 had significantly elevated levels of LAMP2A (***p=0.0002 and ****p<0.0001). This result suggests that CT6 and CT6m treatment increased lysosomal activity and that treatment with CT6 and CT6m restored lysosomal activity in the spinal cord to the levels in wild-type mice.

[0180] Example 2: Evaluating the efficacy of microbiota compositions in a female mouse model of ALS For example, thorough details of the mouse model used, the studies conducted, and the results of this example are listed in Appendix 5 submitted herewith.

[0181] The study described in Example 1 is repeated in female SOD1-G93A transgenic mice. The results of this study are analyzed as described in Example 1.

[0182] Example 3: Physical Performance Test to Evaluate the Efficacy of Microbiota Compositions in an ALS Mouse Model Thorough details of the objectives, mouse model used, studies performed, and results of this example are listed in Appendix 5 submitted herewith.

[0183] This example provides an evaluation of the efficacy of microbiota compositions, specifically CT6 and CT6m compositions, in an in vivo mouse model of ALS using a physical performance test.

[0184] Mouse model: The mouse model described in Example 1 was used in this study. RESEARCH: Physical performance testing is important for evaluating the effects of physical activity interventions in people with neurodegenerative diseases, disorders, or conditions (e.g., ALS, PD, AD, HD, dementia, etc.).

[0185] Male and female mice were each divided into four groups of 13-15 mice per group and provided with sham (DPBS) or microbiota composition (CT6 or CT6m, composition details listed in Tables 2 and 3 above) by daily oral gavage for a known number of consecutive days. The groups were: (i) G1: wild type mice treated with DPBS, (ii) G2: SOD1 transgenic mice treated with DPBS, (iii) G3: SOD1 transgenic mice treated with CT6 composition, and (iv) G4: SOD1 transgenic mice treated with CT6m composition. After treatment, each group underwent physical performance tests including beam walking test, P100 rotator test, and grip strength test.

[0186] result: Beam walking: The "beam walking test" or "balance beam test" is used to analyze rodent gait in a testing environment that challenges the ability to maintain balance, considering that the animal must cross a high beam with a narrow diameter. This test is used to evaluate motor coordination, especially of the hind limbs. The animal is placed at one corner of a narrow beam and is asked to walk across the narrow beam from one end to the other three times. The number of foot slips encountered in each trial and the time taken to cross the beam are recorded.

[0187] The beam walking test was performed on male mice aged 66-80 days. Mice were treated for 17 consecutive days, after which mice in each of the four study groups were subjected to the beam walking test. The results of this test are shown in FIG. 8A. The results showed that wild-type mice in G1 passed the test about 51.1% of the time. In contrast, mock-treated SOD1 mice (i.e., G2 mice) passed the test only about 41% of the time. Treatment with the CT6 and CT6m compositions (i.e., G3 and G4) resulted in higher pass rates of about 59% and 46%, respectively, significantly higher than G2 mock-treated SOD1 mice.

[0188] Similarly, the beam walking test was performed on 71-80 day old female mice. Mice were treated for 22 consecutive days, after which mice in each of the four study groups were subjected to the beam walking test. The results of this test are shown in FIG. 8B. The results showed that G1 wild type mice passed the test about 64.1% of the time. Mock treated SOD1 mice (i.e., G2 mice) also passed the test about 61.5% of the time. However, treatment with CT6 and CT6m compositions (i.e., G3 and G4) resulted in significantly higher pass rates of about 84.8% and 80.9%, respectively, significantly higher than G1 wild type and G2 mock treated SOD1 mice.

[0189] Thus, the microbiota compositions disclosed herein are helpful in improving motor coordination, which is a major concern in patients with neurodegenerative diseases, disorders, or conditions.

[0190] P100 Rotarod: The "Rotarod test" is frequently used in the early stages of drug development to eliminate drugs that may later cause subtle dysfunction. The Rotarod test is a performance-based test on a long cylindrical rotating rod in which forced motor activity is applied by rodents. In this test, rodents are placed on a long cylindrical rod that rotates along its long axis. The speed of the rod can be kept constant or accelerated. However, if the speed is constant, some animals with poor coordination will fall at the start, while for those that stay on, the test starts immediately to measure endurance rather than coordination itself. In the accelerated version of the test, the latency of the animal to fall (in seconds) is recorded as the rodent falls off the rod onto a plate placed below. The length of time the animal stays on this rotating rod is a measure of its balance, coordination, body condition, and motor planning.

[0191] The rotarod test was performed on male mice aged 66-80 days. The mice were treated for 17 consecutive days, after which the mice in each of the four study groups were subjected to the rotarod test. The speed of the rod was kept constant. The results of this test are shown in FIG. 9A. The results showed that G1 wild type mice stayed on the rod for nearly 130-150 seconds compared to mock-treated SOD1 mice (i.e., G2 mice), which stayed on the rod for only about 75-80 seconds. Treatment with the CT6 and CT6m compositions (i.e., G3 and G4) resulted in a longer time on the rod of about 110 seconds and 90 seconds, respectively, higher than the G2 mock-treated SOD1 mice.

[0192] Similarly, the rotarod test was performed on 71-80 day old female mice. The mice were treated for 22 consecutive days, after which mice in each of the four study groups were subjected to the rotarod test. The results of this test are shown in FIG. 9B. The results showed that G1 wild type mice stayed on the rod for nearly 160-165 seconds compared to mock treated SOD1 mice (i.e., G2 mice) that stayed on the rod for only about 100-105 seconds. Treatment with CT6 and CT6m compositions (i.e., G3 and G4) resulted in a longer time on the rod of about 140 seconds and 135 seconds, respectively, higher than G2 mock treated SOD1 mice.

[0193] Thus, the microbiota compositions disclosed herein help improve balance, coordination, physical condition, and motor planning, all of which are major concerns in patients with neurodegenerative diseases, disorders, or conditions.

[0194] Grip strength: The "Grip Bar Strength Test" or "Grip Strength Test" is the most commonly used in vivo test to assess limb strength loss caused by pathological progression. It is a simple and rapid non-invasive method used to assess forelimb / hindlimb muscle strength in vivo. The animal is lowered onto a platform and allowed to grip a horizontal metal bar or grid with its forelimbs / hindlimb, then pulled backwards by the experimenter until it releases its grip. The bar or grid is attached to a transducer, which measures the force generated while pulling the animal. Forelimb and hindlimb assessments can be measured simultaneously using a dual sensor model, or in separate trials using a single stand model. Values ​​are expressed in pounds, kilograms, grams, or newtons.

[0195] Grip strength testing was performed on male mice aged 76-90 days. Mice were treated for 27 consecutive days, after which mice in each of the four study groups were subjected to grip strength testing. The results of this test are shown in FIG. 10A. The results showed that G1 wild type mice exhibited a grip strength of about 210 grams compared to mock treated SOD1 mice (i.e., G2 mice) which exhibited a grip strength of about 150 grams. Treatment with CT6 and CT6m compositions (i.e., G3 and G4) resulted in higher grip strengths of about 180 grams and 185 grams, respectively, higher than G2 mock treated SOD1 mice.

[0196] Similarly, grip strength tests were performed on 76-90 day old female mice. Mice were treated for 27 consecutive days, after which mice in each of the four study groups were subjected to grip strength tests. The results of this test are shown in FIG. 10B. The results showed that wild type mice in G1 exhibited grip strength of about 200 grams compared to mock treated SOD1 mice (i.e., G2 mice), which exhibited grip strength of about 100 grams. Treatment with CT6 and CT6m compositions (i.e., G3 and G4) did not result in a statistically significant increase in grip strength over G2 mock treated SOD1 mice. The fact that no increase in grip strength was observed does not provide any clue as to whether CT6 and CT6m compositions can or would be expected to improve, for example, limb strength or other symptoms associated with a neurodegenerative disease, disorder, or condition, at least because the SOD1 mice used in this analysis model an invasive and fast moving neurodegenerative condition. Thus, for example, the effects of CT6 and CT6m compositions on limb strength or other symptoms associated with a neurodegenerative disease, disorder, or condition may be more pronounced and / or detectable in minimally invasive models or human subjects (e.g., human subjects having a mild or moderate neurodegenerative disease, disorder, or condition).

[0197] Thus, the microbiota compositions disclosed herein are useful in ameliorating limb strength loss, which is a concern in patients with neurodegenerative diseases, disorders, or conditions.

[0198] Example 4: Effect of Microbiota Therapy (MBT) on the Metabolome of Wild-Type Mice This example provides an evaluation of the effect of microbiota therapy (MBT) on the metabolome of wild-type male and female mice.

[0199] Mouse model: Wild-type male and female mice were used in this study. Study: 10 male and 10 female mice were divided into two groups each. One group was provided with sham (DPBS) and the other group was provided with CT6 microbiota composition (details of composition listed in Table 2) by daily oral gavage for 21 days. The groups were: (i) G1: wild type mice treated with DPBS, and (ii) wild type mice treated with CT6 composition. After 21 days of treatment, terminal bleeds were sent for metabolomic analysis.

[0200] Results: Metabolomic analysis identified 660 metabolites, of which 436 metabolites were increased by >1% in CT6-treated animals compared to mock-treated animals (see FIG. 11). FIG. 12 shows the results of metabolomic analysis of CT6-treated wild-type male mouse plasma, plotting the % change in metabolite levels relative to vehicle-treated mice. Similarly, FIG. 13 shows the results of metabolomic analysis of CT6-treated wild-type female mouse plasma, plotting the % change in metabolite levels relative to vehicle-treated mice. FIG. 14 and FIG. 15 also provide enrichment ratios for the top 25 metabolites enriched in male and female wild-type mice treated with the CT6 composition, respectively. Additionally, FIG. 16A and FIG. 16B show the results of metabolomic analysis of CT6-treated wild-type male and female mouse plasma, plotting the % change in metabolite levels relative to vehicle-treated mice for metabolites increased in male and female mice. Figure 16B also provides the enrichment ratios of 25 metabolites that were increased in both male and female wild-type mice treated with the CT6 composition. Similarly, Figures 17A and 17B show the results of metabolomics analysis of CT6-treated wild-type male and female mouse plasma, plotting the % change in metabolite levels relative to vehicle-treated mice for metabolites that were decreased in male and female mice. Figure 17B also provides the enrichment ratios of 25 metabolites that were decreased in both male and female wild-type mice treated with the CT6 composition. In addition, Appendix 4 lists the % change in metabolites in CT6-treated male and female mice compared to mock-treated male and female mice, respectively.

[0201] As can be seen from Figures 11-17, multiple classes of metabolites are modulated upon treatment with the microbiota compositions disclosed herein (e.g., CT6). One such class that is modulated is bile acids. As shown in Figure 18A, bile acids are known to be associated with various neurodegenerative diseases, disorders, or conditions, including ALS, PD, AD, and HD. Bile acids are also important components of neuronal pathways known to be disrupted in patients with neurodegenerative diseases, disorders, or conditions (Ackerman, HD and Gerhard, GS, Frontiers in Aging Neuroscience, 8, 263 (2016)). Figure 18B shows a list of bile acids modulated in male and female mice upon treatment with the CT6 composition, along with the % change in bile acid metabolites in CT6-treated wild-type male and female mice relative to vehicle-treated mice. Thus, treatment with the microbiota compositions disclosed herein that modulate the levels of bile acids in a subject can provide a new method of treatment for various neurodegenerative diseases, disorders, or conditions.

[0202] Example 5: Nitric oxide assay to discover metabolites that modulate neuroinflammation This example provides an assay for determining metabolites that modulate neuroinflammation.

[0203] Study: SIM-A9 microglial cell line was purchased from ATCC and cultured in DMEM:F12 medium supplemented with 10% heat-inactivated FBS, 5% heat-inactivated horse serum, and 1% L-glutamine. Cells were maintained at 37 °C and 5% CO in an incubator. 2 All experiments were performed using only passage 3-7 cells. For 6-well plates, cells were cultured at 1 × 10 using a total volume of 3 ml (300,000 cells total). 5 Cells were seeded at a density of 10 ... 2After 6 hours of incubation at 100° C., one of the two wells for each metabolite was treated with 1 μg / ml lipopolysaccharide (LPS) in water. In the other well, water without LPS was added. Control wells were also treated with either water containing LPS or water without LPS. After 16 hours of LPS treatment, approximately 800 μL of conditioned medium was collected from each well using a 1 mL syringe and filtered using a 0.22 micron PES syringe filter.

[0204] For the nitric oxide (NO) assay, 50 μl of conditioned medium was placed in duplicate on a clear 96-well plate. The NO assay was initiated by adding 50 μl of NO assay solution (A-103 Nitric Oxide Assay Kit, BMR Services) to each well (see FIG. 19B). The plate was incubated for 15 minutes at room temperature, shielded from light. The medium used to grow SIMA9 cells was used for background measurements. The absorbance was measured at 560 nm using a microplate reader (Promega discoverer, Promega corp). To correct for background absorbance, the sample or control absorbance values ​​were subtracted from the background value of the culture medium. 1 μM nitrite dissolved in SIM-A9 cell culture medium was used as a positive control. SIM-A9 cell culture medium without nitrite was used as a negative control for the assay. The average absorbance of the duplicate wells for each condition was calculated. NO levels were calculated as follows: NO level %=[(sample NO absorbance value / control NO absorbance value)*100]

[0205] Results: Figure 19A shows the role of NO in various aspects of neuronal function, including but not limited to neurotransmission, neuroplasticity, cerebral microcirculation, inflammation, oxidative stress, etc. Figure 19C plots the results of the NO assay. Specifically, it shows the % nitrite levels compared to untreated SIMA9 cells for various metabolites and controls. Yellow bars represent statistically significant results, and these yellow bars and corresponding metabolites are involved in regulating neuroinflammation.

[0206] Example 6: Evaluation of the efficacy of microbiota compositions in treating amyloid plaque deposits in the Tg2576 mouse model This example provides an evaluation of the efficacy of microbiota compositions, specifically, CT10, CT10m, and CT10x compositions, in an in vivo mouse model for treating amyloid plaque deposits.

[0207] Mouse model: 9-month-old Tg2576 mice were used for these experiments. This mouse model is one of the most popular transgenic mouse models that overexpresses a mutant form of amyloid precursor protein (APP) (isoform 695) with the Swedish mutation (KM670 / 671NL), leading to increased levels of amyloid beta (Aβ) and ultimately amyloid plaques. Wild-type mice were used as controls in all experiments.

[0208] Study: Tg2576 mice were divided into 5 groups of 15 mice per group and provided with sham (DPBS) or microbiota composition (CT10, CT10m, or CT10x, composition details listed in Tables 4, 5, and 6 below) by daily oral gavage for 6 months. The groups were: (i) G1: wild type mice treated with DPBS, (ii) G2: Tg2576 transgenic mice treated with DPBS, (iii) G3: Tg2576 transgenic mice treated with CT10 composition, (iv) G4: Tg2576 transgenic mice treated with CT10m composition, and (v) G5: Tg2576 transgenic mice treated with CT10x composition. Animals were sacrificed at the end of the study and histological analysis was then performed. [Table 5] [Table 6] [Table 7]

[0209] method: Sectioning: Paraffin-embedded brains were sectioned on a microtome and sections were mounted on superimposed microscope slides. All animals from all groups were sectioned on the same day (staining set). Sections were dried overnight and immunostained.

[0210] Immunostaining: Separate antibody staining was performed for each of (i) amyloid plaque staining, (ii) microglia staining, and (iii) amyloid plaque-microglia overlap. Brain sections were dewaxed and serially rehydrated. Antigen unmasking (recovery) was performed in citric acid solution with steam treatment. Nonspecific antibody binding was blocked in a blocking solution of 5% normal horse serum and 0.5% Triton X100 in 1x PBS. Primary and secondary antibody solutions were prepared in blocking solution. Sections were incubated overnight at 4°C in primary antibody solutions consisting of (i) amyloid plaque staining: anti-6E10, (ii) microglia staining: anti-IbaA1 / AIF-1, or (iii) amyloid plaque-microglia overlap: anti-6E10 + anti-IbaA1 / AIF-1. Sections were washed with 1x PBS and incubated for 1 hour at room temperature in secondary antibody solution consisting of: (i) amyloid plaque staining: IgG H&L conjugated to Alexa Fluor 647, (ii) microglia staining: IgG H&L conjugated to Alexa Fluor 488, or (iii) amyloid plaque-microglia overlap: IgG H&L conjugated to Alexa Fluor 647, IgG H&L conjugated to Alexa Fluor 488. Sections were mounted on glass coverslips using Vectashield® PLUS Antifade Mounting Medium with DAPI. Slides were sealed and stored at 4°C until imaging.

[0211] Image Acquisition: Stained slides were imaged on a Zeiss Axiovision microscope using a 10x objective. Images were collected using Micromanager software. An image grid was defined to collect the entire section using multiple images with 10% overlap between individual images. The same imaging settings were used for all sections within each staining set.

[0212] process: Image processing was performed using the FIJI image processing package, a distribution of ImageJ2. Images were saved and processed in TIFF format. Single images of each section were reconstructed from the individual gridded image panels using the Grid / Collection Stitching plugin. Each single section image was scaled to 25% of the original resolution using the scale function and bilinear interpolation. Scaled images were visually inspected and the sectioning depth through the hippocampus and cortex was determined by setting the minimum and maximum values ​​(min / max set) to the same value for all images in the staining set. Min / max set adjustments were performed separately for each immunostained secondary antibody and DAPI independently. The hippocampus and dorsal part of the cortex (isocortex) were outlined as regions of interest (ROI) for analysis. Scaled images were converted to 8-bit and processed using the subtract background command with a rolling ball radius of 100 pixels, followed by median filtering with a size of 2 pixels.

[0213] Quantification of amyloid burden and plaque size: Quantification was performed using the FIJI image processing package, a distribution of ImageJ2. Amyloid plaques were identified by thresholding 6E10 processed images with the autothreshold command using maximum entropy. The analyze particles command calculated the size of each plaque and the area fraction of each ROI covered by plaque. Plaque size and area fraction data were saved as comma separated value files. Analysis and data visualization were performed with R statistical computing software.

[0214] statistics: Plaque burden and plaque size of individual animals were calculated as the average of multiple stained slides when more than one slide was used. Plaque burden and plaque size of treatment groups were calculated as the average of individual animal values, and SEM was also calculated. Microglial recruitment of individual animals was calculated as the average of multiple stained slides when more than one slide was used. Microglial recruitment of treatment groups was calculated as the average of individual animal values, and SEM was also calculated. Microglial association of individual animals within amyloid plaques was calculated from one stained slide as the average of all plaques of one individual. Microglial association of treatment groups within amyloid plaques was calculated as the average of individual animal values, and SEM was also calculated. Differences in group values ​​were assessed using one-way ANOVA followed by one-tailed Dunnett's test for reduction in values ​​compared to transgenic vehicle control (group 2). Because the effect of treatment group on plaque size was previously assessed using ANOVA and Dunnett's test, the binned plaque size estimates were evaluated using Student's t-test with multiple hypothesis correction using the Benjimini-Hochberg method. Differences were considered statistically significant when p<0.05.

[0215] result: Amyloid plaques in the cortex: Amyloid plaque burden and plaque size in the cortex were measured in treated animals. Amyloid plaques were measured in the cortex of brain tissue sections using 6E10 antibody. Plaque burden was measured as the percentage of cortical area with amyloid plaques (Figure 20A). Plaque size was measured as the size in pixel area of ​​amyloid plaques (Figure 20B). Colored bars indicate the mean value for each group (n=6 or 7 animals), and error bars indicate the standard error of the mean. Values ​​for each animal were determined by calculating the mean value from one or two tissue sections from independent staining experiments. Group comparisons were performed using ANOVA (p value at the top left of the plot), followed by a one-tailed Dunnett's test using G2 as the control comparison group (brackets and numbers above the bar). p<0.05 was considered statistically significant. As can be seen from Figures 20A and 20B, amyloid plaque burden and size was reduced in the cortex when mice were treated with the microbiota compositions, specifically, the CT10, CT10m, and CT10x compositions.

[0216] Furthermore, amyloid plaques measured after treatment in the cortical regions of the brain were binned by their plaque area. Colored bars indicate the mean values ​​for each group (n=6 or 7 animals), and error bars indicate the standard error of the mean. Values ​​for each animal were determined by calculating the mean values ​​from one or two tissue sections from independent staining experiments. Group comparisons were performed using Student's t-tests and p-values ​​corrected for multiple hypothesis testing. p<0.05 was considered statistically significant. Figure 23 shows that amyloid plaques binned into different sizes in the cortex were reduced in animals treated with the microbiota composition.

[0217] Amyloid plaques in the hippocampus: Amyloid plaque burden and plaque size in the hippocampus of treated animals were measured. Amyloid plaques were measured in the hippocampus of brain tissue sections using 6E10 antibody. Plaque burden was measured as the percentage of hippocampal area with amyloid plaques (Figure 21A). Plaque size was measured as the size in pixel area of ​​amyloid plaques (Figure 21B). Colored bars indicate the mean value for each group (n=6 or 7 animals), and error bars indicate the standard error of the mean. Values ​​for each animal were determined by calculating the mean value from one or two tissue sections from independent staining experiments. Group comparisons were performed using ANOVA (p value at the top left of the plot), followed by a one-tailed Dunnett's test using G2 as the control comparison group (brackets and numbers above the bar). p<0.05 was considered statistically significant. As can be seen from FIG. 21A and FIG. 21B, amyloid plaque burden and size was reduced in the hippocampus when mice were treated with the microbiota compositions, specifically, the CT10, CT10m, and CT10x compositions.

[0218] Furthermore, the amyloid plaques measured after treatment in the hippocampus region of the brain were binned according to their plaque area. The colored bars indicate the mean value of each group (n=6 or 7 animals), and the error bars indicate the standard error of the mean. The values ​​of each animal were determined by calculating the mean value from one or two tissue sections from independent staining experiments. Group comparisons were performed using Student's t-test and p-values ​​corrected for multiple hypothesis testing. p<0.05 was considered statistically significant. Figure 24 shows that amyloid plaques binned into different sizes in the hippocampus were reduced in animals treated with the microbiota composition.

[0219] Amyloid plaques in the cortex and hippocampus: Amyloid plaque burden and plaque size were measured in both the cortex and hippocampus in treated animals. Amyloid plaques were measured in both the cortex and hippocampus in brain tissue sections using 6E10 antibody. Plaque burden was measured as the percentage of cortical area with amyloid plaques (Figure 22A). Plaque size was measured as the size in pixel area of ​​amyloid plaques (Figure 22B). Colored bars indicate the mean value for each group (n=6 or 7 animals), and error bars indicate the standard error of the mean. Values ​​for each animal were determined by calculating the mean value from one or two tissue sections from independent staining experiments. Group comparisons were performed using ANOVA (p value at the top left of the plot), followed by a one-tailed Dunnett's test using G2 as the control comparison group (brackets and numbers above the bar). p<0.05 was considered statistically significant. As can be seen from Figures 22A and 22B, amyloid plaque burden and size was reduced in both the cortex and hippocampus when mice were treated with the microbiota compositions, specifically, the CT10, CT10m, and CT10x compositions.

[0220] Furthermore, the amyloid plaques measured after treatment in both the cortical and hippocampal regions of the brain were binned according to their plaque area. The colored bars indicate the mean value for each group (n=6 or 7 animals), and the error bars indicate the standard error of the mean. The values ​​for each animal were determined by calculating the mean value from one or two tissue sections from independent staining experiments. Group comparisons were performed using Student's t-test and p-values ​​corrected for multiple hypothesis testing. p<0.05 was considered statistically significant. Figure 25 shows that amyloid plaques binned into different sizes in the cortex and hippocampus were reduced in animals treated with the microbiota composition.

[0221] Example 7: Evaluation of the efficacy of microbiota compositions on microglial recruitment and association within amyloid plaques in the Tg2576 mouse model This example provides an evaluation of the efficacy of microbiota compositions, specifically CT10, CT10m, and CT10x compositions, in an in vivo mouse model of microglial recruitment and association within amyloid plaques.

[0222] Mouse model: The mouse model described in Example 6 was used in this study.

[0223] Study: The study described in Example 6 was carried out in this experiment.

[0224] Methods: The method described in Example 6 specific for microglial recruitment and association within amyloid plaques was carried out in this experiment.

[0225] Quantification of microglial recruitment and association with amyloid plaques: Quantification was performed using the FIJI image processing package, a distribution of ImageJ2. Maximum entropy was used to identify microglia by thresholding the processed Iba1 stained images with the autothreshold command. The analyze particles command calculated the area fraction of each ROI covered by Iba1 stained cells. For microglia associated with amyloid plaques, the processed Iba1 images were masked with the processed amyloid plaque images to calculate the area fraction of each plaque covered by Iba1 cells in each ROI. Area fraction data were saved as comma separated value files. Analysis and data visualization were performed with R statistical computing software.

[0226] result: Microglia in the cortex: Microglial staining was performed in the cortex in treated animals as described above in Example 6. Using Iba1 / AIF-1 antibody, microglial stained cells and processes were measured in the cortex of brain tissue sections. Figure 26 shows images of stained microglia in brain sections with images adjusted to the same intensity scale. Iba1 area was measured as the percentage of cortical area with Iba1 positive staining area (Figure 27A). In the figure, the colored bars indicate the mean value for each group (n=6 or 7 animals) and the error bars indicate the standard error of the mean. Values ​​for each animal were determined by calculating the mean value from one or two tissue sections from independent staining experiments. Group comparisons were performed using ANOVA (p value at the top left of the plot) followed by a one-tailed Dunnett's test using G2 as the control comparison group (brackets and numbers above the bar). p<0.05 was considered statistically significant. As seen in Figure 27A, microglia were reduced in the cortex when mice were treated with the microbiota composition, specifically, the CT10x composition. It is hypothesized that the effects observed with the CT10x composition are the greatest because these compositions contain a higher amount of microbial strains compared to the CT10 and CT10m compositions.

[0227] In addition, microglia were measured within amyloid plaques in the cortex in treated animals. Microglia were measured in the cortex of brain tissue sections using 6E10 (amyloid) Iba1 / AIF-1 (microglia) antibody. Iba1 area was measured as the percentage of cortical area with Iba1 positive staining area (Figure 29A). In Figure 29A, colored bars indicate the mean value for each group (n=6 or 7 animals) and error bars indicate the standard error of the mean. Values ​​for each animal were determined by calculating the mean value from one or two tissue sections from independent staining experiments. Group comparisons were performed using ANOVA (p value at the top left of the plot) followed by a one-tailed Dunnett's test using G2 as the control comparison group (brackets and numbers above the bar). p<0.05 was considered statistically significant. As can be seen from FIG. 29A, microglia within amyloid plaques in the cortical regions of the brain were significantly reduced when mice were treated with the microbiota compositions, specifically, the CT10, CT10m, and CT10x compositions.

[0228] Microglia in the hippocampus: Microglial staining was performed in the hippocampus in treated animals as described above in Example 6. Using Iba1 / AIF-1 antibody, microglial stained cells and processes were measured in the cortex of brain tissue sections. Iba1 area was measured as the percentage of cortical area with Iba1 positive staining area (Figure 27B). In the figure, the colored bars indicate the mean value of each group (n=6 or 7 animals), and the error bars indicate the standard error of the mean. Values ​​for each animal were determined by calculating the mean value from one or two tissue sections from independent staining experiments. Group comparisons were performed using ANOVA (p value at the top left of the plot), followed by a one-tailed Dunnett's test using G2 as the control comparison group (brackets and numbers above the bar). p<0.05 was considered statistically significant. As observed from Figure 27B, microglia were reduced in the hippocampus when mice were treated with the microbiota composition, specifically with the CT10x composition. It is hypothesized that the greatest efficacy is observed with the CT10x compositions because these compositions contain a higher amount of microbial strains compared to the CT10 and CT10m compositions.

[0229] In addition, microglia were measured within amyloid plaques in the hippocampus in treated animals. Microglia were measured in the hippocampus of brain tissue sections using 6E10 (amyloid) Iba1 / AIF-1 (microglia) antibody. Iba1 area was measured as the percentage of cortical area with Iba1 positive staining area (Figure 29B). In Figure 29B, colored bars indicate the mean value for each group (n=6 or 7 animals) and error bars indicate the standard error of the mean. Values ​​for each animal were determined by calculating the mean value from one or two tissue sections from independent staining experiments. Group comparisons were performed using ANOVA (p value at the top left of the plot) followed by a one-tailed Dunnett's test using G2 as the control comparison group (brackets and numbers above the bar). p<0.05 was considered statistically significant. As seen in Figure 29B, microglia in amyloid plaques in the hippocampus region of the brain were reduced when mice were treated with microbiota compositions, specifically, CT10m and CT10x compositions.It is hypothesized that the effects observed with CT10m and CT10x compositions are the highest because these compositions contain a higher amount of microbial strains compared to the CT10 composition.

[0230] Microglia in the cortex and hippocampus: Microglial staining was performed in both the cortex and hippocampus in treated animals as described above in Example 6. Using Iba1 / AIF-1 antibody, microglial stained cells and processes were measured in the cortex of brain tissue sections. Iba1 area was measured as the percentage of the cortical area with Iba1 positive staining area (Figure 27C). In the figure, the colored bars indicate the mean value for each group (n=6 or 7 animals), and the error bars indicate the standard error of the mean. Values ​​for each animal were determined by calculating the mean value from one or two tissue sections from independent staining experiments. Group comparisons were performed using ANOVA (p value at the top left of the plot), followed by a one-tailed Dunnett's test using G2 as the control comparison group (brackets and numbers above the bar). p<0.05 was considered statistically significant. As observed from Figure 27C, microglia were reduced in the cortex and hippocampus when mice were treated with the microbiota composition, specifically with the CT10m and CT10x compositions. It is hypothesized that the greatest efficacy is observed with the CT10m and CT10x compositions because these compositions contain a higher amount of microbial strains compared to the CT10 composition.

[0231] In addition, microglia were measured within amyloid plaques in the cortex and hippocampus in treated animals. Microglia were measured in the cortex and hippocampus of brain tissue sections using 6E10 (amyloid) Iba1 / AIF-1 (microglia) antibody. Iba1 area was measured as the percentage of cortical area with Iba1 positive staining area (Figure 29C). In Figure 29C, colored bars indicate the mean value for each group (n=6 or 7 animals) and error bars indicate the standard error of the mean. Values ​​for each animal were determined by calculating the mean value from one or two tissue sections from independent staining experiments. Group comparisons were performed using ANOVA (p value at the top left of the plot) followed by a one-tailed Dunnett's test using G2 as the control comparison group (brackets and numbers above the bar). p<0.05 was considered statistically significant. As can be seen from FIG. 29C, microglia within amyloid plaques in the cortical and hippocampal regions of the brain were significantly reduced when mice were treated with the microbiota compositions, specifically, the CT10, CT10m, and CT10x compositions.

[0232] Other embodiments It will be understood by those skilled in the art that various changes, modifications, and improvements to the present disclosure will be readily suggested to those skilled in the art. Such changes, modifications, and improvements are intended to be part of this disclosure and are intended to be within the spirit and scope of the present invention. Therefore, the foregoing description and drawings are by way of example only, and any inventions described in this disclosure, as further described in detail by the following claims, are within the spirit and scope of the present invention.

[0233] One of ordinary skill in the art will understand the typical standard deviation or error attributable to values ​​obtained from the assays or other processes described herein. Publications, websites, and other reference materials referenced herein to describe the background of the invention and to provide additional details regarding its practice are hereby incorporated by reference in their entirety.

[0234] While embodiments of the invention have been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate, but not to limit, the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0235] Equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the invention is not intended to be limited to the above Description, but rather is as set forth in the following claims. [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4] [Table 8-5] [Table 9-1] [Table 9-2] [Table 9-3] [Table 9-4] [Table 9-5] Appendix 1-3 Metabolites of interest C10H10O3S C32H30O12 C6H10O8 1-Methyl-4-imidazole acetic acid 1-Methyladenosine 1-Methylhistamine 1-Methylhistidine 3-Methylhistidine 1-Methylhistidine; 3-Methylhistidine 1-Methylnicotinamide 1-Palmitoyl-glycero-3-phosphocholine-1 1,2-Dipalmitoyl-glycero-3-phosphoethanolamine-1 1,2-Dipalmitoyl-glycero-3-phosphoethanolamine-2 11-amino-undecanoic acid 15(S)-HETE 17α-hydroxyprogesterone 17α-hydroxyprogesterone-2 Deoxycorticosterone-2 1H-imidazole-4-propionic acid 1H-Imidazole-4-propionic acid; 1-Methyl-4-imidazoleacetic acid 2-(beta-D-glucosyl)-sn-glycerol 2-Aminoadipic Acid 2-Aminoisobutyric acid 2-Aminobutyric acid 2-Arachidonoylglycerol 2-Diethylaminoethanol 2-Hydroxy-4-methylvaleric acid 2-Hydroxybutyric Acid 2-Hydroxyglutaric acid 2-Hydroxyisobutyric Acid 2-Hydroxyvaleric acid 2-Keto-glutaric acid 2-Methylserine 2-Oxoarginine 2-Oxoglutaric Acid 2-Oxoisovaleric acid 2-oxoisovaleric acid; 2-oxoisovaleric acid 2-Phosphoglyceric acid 2-Phosphoglyceric acid; 3-Phosphoglyceric acid 2-Thiopheneacetic acid 2,3-Diphosphoglyceric Acid 2,6-Diaminopimelic Acid 2'-Deoxycytidine 2'-Deoxyuridine 20α-hydroxyprogesterone 21-Deoxycortisol-2 21-Hydroxypregnenolone 3-(4-hydroxyphenyl)propionic acid 2-(4-hydroxyphenyl)propionic acid 3-(3-hydroxyphenyl)propionic acid 3-(4-hydroxyphenyl)propionic acid;2-(4-hydroxyphenyl)propionic acid;3-(3-hydroxyphenyl)propionic acid;Tropic acid;3-(2-hydroxyphenyl)propionic acid;m-Ethoxybenzoic acid;3-phenyllactic acid;p-Methoxyphenylacetic acid 3-cis-hydroxy-b,e-caroten-3'-one 3-Guanidinopropanoate 3-Hydroxy-2-methyl-4-pyrone 3-Hydroxy-3-methylglutaric acid 3-Hydroxy-3',4'-didehydro-β,γ-carotene 3-Hydroxybutyric Acid 3-Hydroxybutyric acid; 2-Hydroxybutyric acid; 2-Hydroxyisobutyric acid 3-Hydroxybutyric acid; 2-Hydroxyisobutyric acid 3-Hydroxyglutaric acid 3-Hydroxytetradecanoic acid-1 3-Indoxyl Sulfate 3-Mercaptolactic Acid 3-Methylhistamine; 1-Methylhistamine 3-Methylhistidine; 1-Methylhistidine 3-Oxocholic Acid 3-Phosphoglyceric acid 3-Ureidopropionic acid 3,4-Dihydroxyhydrocinnamic acid;Homovanillic acid;Hydroxyphenyllactic acid 4-Acetamidobutanoic acid 4-Guanidinobutyric Acid 4-Hydroxyquinoline 4-Methyl-2-oxovaleric acid 3-Methyl-2-oxovaleric acid 2-Oxohexanoic acid 4-Methyl-2-oxovaleric acid;3-Methyl-2-oxovaleric acid;2-Oxohexanoic acid 4-Oxypyrrolidine-2-carboxylic acid 5-Amino-4-hydroxynaphthalene-1,3-disulfonic acid 5-Amino-4-oxovaleric acid 5-Hydroxyindoleacetic acid 5-Hydroxylysine 5-Hydroxypentanoic acid;fA-Hydroxyisovaleric acid;2-Hydroxyvaleric acid 5-Hydroxytryptophan 5-Isopropyl-2'-deoxyuridine triphosphate 5-Methoxyindoleacetic acid; Indole-3-lactic acid 5-Methyl-2'-deoxycytidine 5-Oxoproline 5α-Cholestan-3-one-1 5α-Cholestan-3-one-2 5α-Pregnan-3,20-dione 6-Hydroxyhexanoic acid 6-Hydroxyhexanoic acid; 2-Hydroxy-4-methylvaleric acid 6-Hydroxynicotinic Acid 7-Dehydrocholesterol 7-Dehydrocholesterol 7-Dehydrocholesterol-2 Desmosterol-2 7-Dehydrocholesterol-3 Desmosterol-3 7-Methoxy-2-methylisoflavone 7-Methylguanine 7-Methylguanine; 3-Methylguanine 7,8-Dihydrobiopterin 7,8-Dihydroneopterin Abietic acid Abietic acid-1 Abietic acid-3 AC(10:0) AC(12:0)-1 AC(12:0)-2 AC(12:1) AC(12:1)-1 AC(12:1)-3 AC(13:1) AC(13:1)-1 AC(14:0)-1 AC(14:0)-2 AC(14:1)-1 AC(14:1)-2 AC(14:1)-3 AC(14:1)-4 AC(14:2)-1 AC(14:2)-2 AC(14:2)-3 AC(14:3)-1 AC(14:3)-2 AC(14:3)-3 AC(14:3)-4 AC(15:0)-1 AC(15:0)-2 AC(16:1) AC(16:2)-1 AC(16:2)-2 AC(17:0)-1 AC(17:0)-2 AC(17:1) AC(18:0) AC(18:1) AC(18:2)-1 AC(18:2)-2 AC(20:0) AC(20:1) AC(22:0) Acetohydroxamic acid;Gly ADMA;SDMA ADP ADP-ribose AEA(22:6) Ala ala ser / gly thr Aminoacetone AMP AMP; dGMP Anandamide ANDS(C-SCOPE IS) Anserine Arachidic acid Arachidonic acid Arg Argininosuccinic Acid Ascorbic acid Asiatic Acid Asiatic Acid-1 Asn Asp Asp Asp Pro Ser Asp Gly His Asp Asp Leu Asn Arg Asp-Pro ATP Baicalein Behenic acid Betaine Betaine aldehyde + H2O Betulinic Acid Betulinic Acid-2 Biopterin Biotin Butyrylcarnitine C;C C3H8N4O C4H7NO4 C5H12N2O2 C5H6O7 C6H10O8 C6H11NO2 C6H12N2O3 C7H9N3O2 C8H17NO C8H18N2O3 C9H18N2O cAMP Campesterol Carbachol Carboxymethyl Lysine Carnitine Carnosine Carnosine;His-Ala;Ala-His Chenodeoxycholic acid cholesterol Cholesterol sulfate Cholic acid Colin cis-11-eicosenoic acid Cis-11,14-eicosadienoic acid-1 Cis-11,14-eicosadienoic acid-2 cis-4,7,10,13,16,19-docosahexaenoic acid Cis-5,8,11,14,17-eicosapentaenoic acid EPA Cis-8,11,14-eicosatrienoic acid Cis-Aconitic Acid Citric acid Citrulline Corosolic acid Cortexolone Corticosterone 21-Deoxycortisol-1 Cortisol 18-Hydroxycorticosterone Humulon Creatine Creatinine Crotonic Acid CSA; Cyclodopa glucoside Cyclohexylamine Cys Cys Csy Asn Asn Cystathionine Cysteine ​​Glutathione Disulfide Cystine Cytidine Cytosine Daidzein Dansylic acid (C-SCOPE IS) Deoxycholic Acid Dethiobiotin dGDP;ADP dGTP; ATP Diethanolamine Dimethylaminoethanol DOPA DPA;DPA Dyphylline Ectoine Erucic Acid Ethanolamine Ethanolamine Phosphate Ethyl arachidonate Ethyl Glucuronide FA 16:1 FA C18:1 FA(12:0) FA(13:0) FA(14:1) FA(14:1)-2 FA(14:2)-1 FA(14:2)-2 FA(14:3) FA(15:0) FA(15:0)-1 FA(15:1) FA(15:1)-2 FA(16:2)-1 FA(16:2)-2 FA(16:3 FA(16:3)-2 FA(17:0) FA(17:1) FA(17:2) FA(17:3) FA(19:0) FA(19:0)-1 FA(19:1) FA(19:2) FA(20:3) FA(22:2) FA(22:3)-1 FA(22:3)-2 FA(22:4)-1 FA(22:4)-2 FA(22:5)-1 FA(22:5)-2 FA(24:0) FA(24:2) FA(24:4) FA(24:5)-1 FA(24:5)-2 Flavanones Formiminoglutamic Acid Formononetin Fucosyltryptophan Fucosyl-lysine Fumaric acid fA-Ala fA-Butyrobetaine fA-Glu-Gly fA-Glu-Met fA-Glu-Phe fA-Glu-Ser fA-Glu-Tyr fA-Glu-Val-Gly GABA GABA; 3-aminoisobutyric acid Galactosyl Hydroxylysine Galacturonic acid-1 Glucuronic acid-1 Galacturonic acid; Glucuronic acid Gamma-Glu-Gln GDP Genistein Gln Glu Glu;isoglutamic acid;threo-fA-methylaspartic acid;N-methylaspartic acid;N-acetylserine Glucaric acid Gluconic acid Gluconolactone Glucosamine Glucosamine-6-sulfate Glucose-6-phosphate Glucosyl-glycerol Glutaric acid Methylsuccinic acid Glutaric acid; Methylsuccinic acid Glutathione (GSSG)_Divalent Gly Gly-Lys Gly-Ala Gly-Asp Gly-Asp; Asp-Gly Gly-Leu;N-acetyl-lysine;Val-Ala;Ala-Val;Leu-Gly Glyceric acid Glycerol Glycerol-3-phosphate Glycerophosphocholine Glycitein Glycophenodeoxycholic acid Glycocholic acid Glycodeoxycholic acid Glyoxylic Acid GMP Guanidinosuccinic acid Guanidoacetic acid Gulonolactone; Gluconolactone Hecogenin Henicosylic acid 19-Methylarachidic Acid Heptadecanoic acid-1 FA(17:0)-1 Heptadecanoic acid-2 FA(17:0)-2 Heptanoic acid Hexanoic acid hippuric acid Hippuric acid (benzylglycine) His His Pro Ser Val Arg Tyr Thr His-Asp histamine Homoarginine Homoproline Betaine Homocarnosine Homocitrulline Homocysteine ​​sulfinic acid Homoserine Homovanillic acid Hydroxyphenyl Lactic Acid Hydroxyindoles Hydroxyoctanoic acid Hydroxyprogesterone caproate Hydroxyproline Hydroxytetradecanoic acid Hyodeoxycholic acid Hypotaurine IDP Ile Ile;Leu;Alloisoleucine Ile;Leu;fA-Leucine;Alloisoleucine;6-Aminohexanoic acid Imidazole-4-acetic acid Imidazole Lactate IMP Indole-3-acetic acid Indole-3-carboxaldehyde Indole-3-lactic acid-1 5-Methoxyindoleacetic acid-1 Indole-3-lactic acid; 5-Methoxyindoleacetic acid Indole-3-propionic acid Indole-3-propionic acid (IPA) Inosine 2',3'-cyclic phosphate cIMP Isethionic Acid Isobutyrylcarnitine; Butyrylcarnitine Isocitric Acid Isoglutamic acid Isoliquiritigenin-1 Isoliquiritigenin-2 Isoliquiritigenin-3 Isonicotinamide; Nicotinamide Isovalerylalanine-2 N-acetyl-leucine-2 Isovalerylalanine; N-Acetylleucine Isovalerylcarnitine Kynurenic acid Kynurenine Lactamide Lactic acid Lanosterol Lauric Acid Leu Leukotriene B4 Linoleic acid Linolenic Acid Linoleylethanolamide Liquiritigenin Lithocholic acid Luteolin Lys Lys-Asp Lys-Val Malic acid Mannosamine MCA Met Methionine sulfone Methionine sulfoxide Methylmalonic acid; Succinic acid Morpholine Mucic acid Myristic acid Myristic Acid 14:0 Myristoleic acid N-(1-deoxy-1-fructosyl)valine N-Acetyl-Beta-Alanine N-Acetyl-β-Alanine N-Acetylalanine N-Acetylalanine; N-acetyl-fA-alanine N-Acetylasparagine N-acetylaspartic acid N-Acetylgalactosamine; N-Acetylglucosamine; N-Acetylmannosamine N-Acetylglucosamine N-Acetylglutamic Acid N-Acetylglutamine N-Acetylglycine N-Acetylhistidine N-Acetyl Leucine N-Acetyl Lysine N-Acetylmethionine N-acetylneuraminic acid N-Acetylornithine N-Acetylphenylalanine N-acetyltryptophan N-Acetyl Tyrosine N-Carbamylglutamic Acid N-Carboxymethylserine N-Ethylmaleimide + H2O N-Formyl Aspartic Acid N-Formylglycine N-Formylmethionine N-glycolylneuraminic acid N-Hydroxy-L-tryptophan N-Methylethanolamine phosphate N-Methylproline N,N-Dimethylglycine N'-Formylkynurenine N1-Acetylspermidine N1-Acetylspermidine; N8-Acetylspermidine N1-Methyl-4-pyridone-5-carboxamide N1-Methylguanosine N5-Ethylglutamine N5-Ethylglutamine; N-Acetylornithine N6-Acetyl Lysine N6-Methyl-2'-deoxyadenosine N6-Methyllysine N6,N6,N6-Trimethyllysine Naringenin Nervonic Acid Nerbonylcarnitine Propylbetaine (triethylamine) Nf-Formylkynurenine Nicotinamide No match Norophthalmic Acid Norvaline; 2-Amino-2-methylbutyric acid; 5-Aminovaleric acid; Val Nω-Methylarginine O-Acetylcarnitine O-Acetylhomoserine o-Coumaric acid p-Coumaric acid o-Hydroxybenzoic acid Oleanolic Acid Oleic Acid Oleoylethanolamide AEA(18:1) Ophthalmic Acid Ornithine Orotidine; Uridine; Pseudouridine p-Hydroxyphenylpyruvic acid p-Hydroxyphenylpyruvic acid; Caffeic acid Palmitic acid Palmitoleic acid Palmitoylcarnitine Palmitoylethanolamide Pantothenic acid Penicillamine Penicillamine;Met Pentadecanoic acid Phe Phe Met His Glu Phe Phe Trp Trp Phe-Thr Phenaceturic Acid Phenol Phenyl sulfate Phenylpyruvic Acid Phosphocreatine Phosphoenolpyruvate Phosphorylcholine Picolinic Acid Pipecolic Acid Pipecolic acid;N-methylproline;1-aminocyclopentanecarboxylic acid Piperidine Pro Pro-Gly;Gly-Pro Progesterone Proline Betaine Propionylcarnitine XC0061 Propionylcarnitine;XC0061 Prostaglandin E1-1 Prostaglandin D1-1 Prostaglandin E1-2 Prostaglandin D1-2 Putrescine Pyridoxal Pyrrolidine Pyruvate Retinol Vitamin A Retinol-2 Riboflavin Ribose-5-phosphate Ribulose-5-phosphate Ribulose-5-phosphate; Ribose-1-phosphate; Xylulose-5-phosphate Ricinoleic Acid Ricinoleic Acid 18:1 Hydroxy Ricinoleic acid-2 Ricinoleic acid-3 S-Acetyldihydrolipoamide (XC0086) S-Adenosylhomocysteine S-Adenosylmethionine S-Carboxymethylcysteine S-Methylcysteine S-Methylglutathione S-Methylmethionine S-Sulfocysteine Sarcosine SDMA Sedoheptulose-7-phosphate Ser Ser Ala / Thr gly Ser Glu Pro Thr Asp Pro Serotonin Sitosterol Spermidine Spermine Sphinganine Sphingomyelin (d18:1 / 16:0)-1 Sphingomyelin (d18:1 / 16:0)-2 Sphingomyelin (d18:1 / 18:0)-1 Sphingomyelin (d18:1 / 18:0)-2 Sphingosine Stearic acid Stearidonic acid Stearoylethanolamide Stigmasterol-1 Stigmasterol-2 Succinic acid Sulfaguanidine (C-SCOPE IS) Sulfolitocholylglycine Taurine Taurochenodeoxycholic acid Taurocholic acid Taurodeoxycholic acid Taurolithocholic Acid Tauroursodeoxycholic acid Terephthalic Acid Theobromine; Aminophylline; Paraxanthine Thiamine Thiamine phosphate Thr Thr Ala Ala Thr Asp or Ser Glu Threo-3-hydroxyaspartic acid Threo-3-hydroxyaspartic acid-1 Threo-3-hydroxyaspartic acid-2 Threonate Thymidine Thyroxine Trans-Glutaconic Acid Trans-glutaconic acid; Itaconic acid Trichosyl acid Trigonelline Trilaurin Trimesic acid; Trimesic acid Trimethylamine Trimethylamine N-oxide Trimethylaminoacetone Trp Tyr UDP-galactose UDP-glucose Uracil urea uric acid Uridine Uridine; Pseudouridine Urocanic acid Ursodeoxycholic acid Val XA0005 XA0008 XA0009 XA0011 XA0017 XA0019 XA0023 XA0026 XA0033 XA0034 XA0037 XA0039 XA0052 Xanthosine XC0016 XC0039 XC0040 XC0047 XC0049 XC0054;XC0055;fA-Glu-Gly XC0056 XC0060 XC0063 XC0064 XC0065 XC0067 XC0070 XC0075 XC0088 XC0094 XC0101 XC0103 XC0107;fA-Glu-Gln XC0114;fA-Glu-His XC0117 XC0118 XC0119 XC0120 XC0126 XC0133 XC0135 XC0138 XC0139 XC0140 Zeaxanthin Alpha-tocopherol Alpha-tocopherol acetate β-Ala β-estradiol 17α-estradiol β-Hydroxyisovaleric Acid γ-Butyrobetaine γ-Glu-Ala γ-Glu-Arg γ-Glu-Asn γ-Glu-Asp γ-Glu-Citrulline γ-Glu-Gln γ-Glu-Glu γ-Glu-Gly γ-Glu-His γ-Glu-Leu γ-Glu-Lys γ-Glu-Met γ-Glu-ornithine γ-Glu-Phe γ-Glu-Ser γ-Glu-Taurine γ-Glu-Thr γ-Glu-Trp γ-Glu-Tyr γ-Glu-Val γ-Glu-Val-Gly Gamma-tocopherol [Table 10-1] [Table 10-2] [Table 10-3] [Table 10-4] [Table 10-5] [Table 11] [Table 12-1] [Table 12-2] [Table 12-3] [Table 12-4] [Table 13-1] [Table 13-2] [Table 14-1] [Table 14-2] [Table 14-3] [Table 14-4] [Table 14-5] [Table 14-6] Appendix 5 research report Efficacy study in SOD1 mice [Table 15] This document contains confidential and / or proprietary information and is subject to confidentiality protection. Any dissemination, distribution, or duplication of this document by anyone other than the designated recipient is strictly prohibited. If you have received this document in error, please disregard this communication and delete it. table of contents TIFF2024536771000045.tif246162 TIFF2024536771000046.tif246162 TIFF2024536771000047.tif245159 TIFF2024536771000048.tif851581.General Information 1.1 Research objectives The aim of this study was to investigate the therapeutic potential of two experimental microbial test substances, CT6 and CT6m, in the SOD1-G93A (SOD1) transgenic mouse model of ALS. In this study, equal numbers of SOD1 males and SOD1 females were used to compare the effects of CT6 or CT6m treatment in improving both motor function and pathophysiological features of ALS with vehicle-treated SOD1 animals. Vehicle-treated, age-matched non-transgenic (ntg) littermates were included as an additional control group for behavioral motor function assessment. In total, 100 animals (70 SOD1 and 30 ntg) were enrolled in the study, and animals were randomized into different treatment groups based on body weight. For SOD1 males, there were three different age cohorts that differed in age with an interval of 7 to 14 days. For females, where the age variation was only 7 days, there were only two different age cohorts. Mice were treated daily by oral gavage with Dulbecco's phosphate-buffered saline (D-PBS) as vehicle or one of two different microbial cocktails, CT6 and CT6m, for 3.5 months. Males were started on treatment between 49-63 days of age, whereas females started treatment after reaching 49-56 days of age. In addition to collecting fecal pellets from all animals at p115 and p125, fecal pellets were also collected from several moribund mice before they were euthanized due to disease progression (i.e., ALS-TDI neurological scoring of 4). To test the efficacy of CT6 or CT6m treatment, several behavioral tests were performed to evaluate motor function, including the rotarod test, grip test, balance beam test, and beam walking test. The first motor function test was a grip test 10 days after the start of daily treatment. The second grip test was performed 26 days after the start of treatment, and the final grip test was performed when the mice reached postnatal day p115. The balance beam test was performed 17 days after the start of treatment for males and 22 days after the start of treatment for females, and a modified version of the balance beam test, the beam walking test, was performed for p125-day-old mice. The first rotarod test was performed for all animals 30 consecutive days after treatment. The second and third rotarod tests were performed when the animals reached p100 and p120 days of age, respectively. Body weights were recorded twice a week until the animals reached approximately p90 days of age, the typical number of days when disease onset becomes observable. To assess disease progression, mice were scored daily according to the ALS-TDI scoring criteria, and body weights were recorded daily as well. Animals were euthanized when a humane endpoint of neurological score 4 was reached. All SOD1 transgenic animals were euthanized, followed by non-transgenic wild-type littermates. Animals were sacrificed and blood, brain, spinal cord, gastrocnemius and tibialis anterior muscles of each mouse were collected. Blood was processed to plasma. Brains and apical portions of spinal cord were frozen on dry ice for biochemical analysis. Lumbar spinal cord was postfixed and embedded in cryomolds for subsequent immunohistological analysis. For biochemical analysis, 10 animals from each of the four groups were collected and investigated in five samples (total of 20 samples). Inflammatory markers were measured in apical spinal homogenates by Eve technology. Also, ATP content was measured in apical spinal homogenates using a Promega kit. Furthermore, proteasome protein PSMD11 levels were measured by Western blot analysis. In addition, proteasome functional activity was assessed using a fluorescent substrate. Also, the concentration of LAMP2A lysosomal membrane protein was measured in apical spinal homogenates. Furthermore, neurofilament light chain (NF-L) concentrations in plasma from terminal blood draws [i.e. one time point] were also measured using a NF-L ELISA kit by Abbexa. Histologically, 4–7 lumbar spinal cords per group were cryosectioned (10–15 sections each). Five sections per animal were subsequently used for quantitative immunofluorescence labeling of astrocytes (glial fibrillary acidic protein, GFAP) and microglia (ionized calcium-binding adaptor molecule 1, IBA1). 1.2 Test system and justification of the test system Human SOD1 G93A Transgenic mice carrying the mutation (Gurney et al., 1994) display a phenotype similar to human ALS patients and are used as a reliable familial ALS (fALS) model to evaluate the efficacy of new chemical entities. The route of administration and dosage were selected based on literature analysis of microbiota products. We performed an extensive literature analysis but found no studies testing this microbial combination. Therefore, our study does not unnecessarily duplicate previous studies. 1.3 Responsibilities [Table 16] 1.4 Research name and schedule [Table 17] 1.5 Regulatory Compliance This study was conducted as a non-Good Laboratory Practice (GLP) study. All animal experiments were approved by the Avastus Institutional Animal Care and Use Committee (IACUC) under protocol number APS19-011 and conformed to the U.S. Animal Welfare Act and the Guidelines for the Care and Use of Laboratory Animals set by the National Institutes of Health. All personnel working with mice were approved by the Avastus IACUC. 2. Animal Welfare - Overview of Outbreaks CT6 and CTm were well tolerated by the animals. Six early deaths occurred throughout the study (Table 1). Two male mice (#901 and #904) were euthanized due to severe wounds from fighting. One male mouse (#902) was found dead, but the cause of death is unknown because the animal husbandry staff discarded the body before autopsy. Another male mouse (#916) was found dead at the start of the study, despite being closely monitored as he exhibited decreased mobility with shivering. However, no autopsy was performed as the animal husbandry staff discarded the body. One female mouse (#994) exhibited labored breathing, along with a hunched posture and decreased body temperature, within one week of the study beginning. The animal was euthanized due to its morbidity, and autopsy revealed that the mouse (#994) had abnormally small, almost nonexistent lungs. [Table 18] 3. Test Sample basis: The test samples used in the study were CT6 and CT6m. CT6 was at a concentration of 1 × 10 9 The combination of six bacterial strains was selected based on their ability to improve one or more of the following goals or functions: improved mitochondrial function, resilience to oxidative stress, suppression of neuroinflammation, M1-M2 microglial polarization, improved proteasome function, improved lysosomal function, and HDAC inhibition. These goals or functions were selected based on their involvement or perturbation in ALS. CT6m was used in combination with CT6m at a concentration of 1 × 10 per strain. 9CFU / ml of four bacterial strains and 3 × 10 9 In combination with two bacterial strains (Gluconacetobacter hansenii and Veillonella atypica) at CFU / ml, the concentrations of these two bacteria were increased to test whether there was an additive beneficial effect with the increased dose. [Table 19] The test samples used in this study were produced under non-GMP conditions at Marvelbiome. The test samples are proprietary strains maintained and cultured as follows: All bacterial master stock strains (MBL) are maintained at -80°C at Marvelbiome. Working bacterial libraries (WBL) were prepared from MBL stocks as required as follows: An inoculated strain from a vial of MBL was streaked onto selective media using standard microbiological methods and cultured in appropriate growth conditions until isolated single colonies appeared. One isolated single colony was then grown in appropriate selective media for 48 hours and a glycerol stock was prepared and stored at -80°C. Before opening a new WBL for use as starting material for production, purity was confirmed by streaking onto specific media. Gluconacetobacter hansenii was grown in selective medium consisting of 5% peptone, 5% yeast extract, and 5% glucose under aerobic conditions with vigorous shaking for 48 hours at 30°C. When the absorbance measured at 600 nm reached an optical density of 1.5, the bacterial cells were harvested by centrifugation. The harvested moist bacterial pellet was resuspended in Dulbecco's phosphate buffered saline and serially diluted. The serial dilutions were placed on selective medium for 48 hours at 30°C. The colonies formed were counted and determined to be 1 x 10, the concentration in the final test sample composition. 9 CFU (colony forming units) / ml or 3 x 10 9 The dilution factor required to achieve CFU / ml was determined. Bifidobacterium breve was grown in selective MRS medium (Anaerobic Culture System, AS-609) for 24 hours at 37°C with vigorous shaking under anaerobic conditions. Bacterial cells were harvested by centrifugation when the absorbance measured at 600 nm reached an optical density of 1.5. The harvested moist bacterial pellet was resuspended in Dulbecco's phosphate buffered saline and serially diluted. The serial dilutions were plated on selective medium at 37°C for 24 hours under anaerobic conditions. The colonies formed were counted and determined to be 1×10 6 colonies in the final test sample composition at a concentration of 1×10 6 colonies. 9 The dilution factor required to achieve CFU / ml was determined. Terrisporobacter glycolicus was grown in selective enriched Clostridia medium (aerobic culture system, catalog number: AS-6062) under anaerobic conditions for 48 hours at 37°C. Bacterial cells were harvested by centrifugation when the absorbance measured at 600 nm reached an optical density of 1.5. The harvested moist bacterial pellet was resuspended in Dulbecco's phosphate buffered saline and serially diluted. The serial dilutions were plated on selective medium under anaerobic conditions for 48 hours at 37°C. The colonies formed were counted and determined to be 1×10 in the final test sample composition at a concentration of 1×10 9 The dilution factor required to achieve CFU / ml was determined. Veillonella atypica was grown in 90% enriched Clostridia medium (Anaerobic Culture System, Catalog No. AS-6062) and 10% yeast casitone fatty acid with carbohydrate medium (Anaerobic Culture System, Catalog No. AS-6080) under anaerobic conditions for 24 hours at 37°C. Bacterial cells were harvested by centrifugation when the absorbance measured at 600 nm reached an optical density of 1.5. The harvested moist bacterial pellet was resuspended in Dulbecco's phosphate buffered saline and serially diluted. The serial dilutions were plated on selective media under anaerobic conditions for 24 hours at 37°C. The colonies formed were counted and counted to determine the concentration of 1 x 10 in the final test sample composition. 9 CFU / ml or 3 × 10 9 The dilution factor required to achieve a concentration of CFU / ml was determined. Coprococcus catus was grown in 90% enriched Clostridia medium (Anaerobic Culture System, Catalog No. AS-6062) and 10% yeast casitone fatty acid with carbohydrate medium (Anaerobic Culture System, Catalog No. AS-6080) under anaerobic conditions for 24 hours at 37°C. Bacterial cells were harvested by centrifugation when the absorbance measured at 600 nm reached an optical density of 1.5. The harvested moist bacterial pellet was resuspended in Dulbecco's phosphate buffered saline and serially diluted. The serial dilutions were plated on selective media under anaerobic conditions for 48 hours at 37°C. The colonies formed were counted and determined to be 1×10 6 cells / mL in the final test sample composition at a concentration of 1×10 6 cells / mL. 9 The dilution factor required to achieve CFU / ml was determined. Lactobacillus plantarum was grown in selective MRS medium (Anaerobic Culture System, AS-609) for 24 hours at 37°C with vigorous shaking under anaerobic conditions. Bacterial cells were harvested by centrifugation when the absorbance measured at 600 nm reached an optical density of 1.5. The harvested moist bacterial pellet was resuspended in Dulbecco's phosphate buffered saline and serially diluted. The serial dilutions were plated on selective medium at 37°C for 24 hours under anaerobic conditions. The colonies formed were counted and determined to be 1×10 in the final test sample composition at a concentration of 1×10 9 The dilution factor required to achieve CFU / ml was determined. 3.1 Test sample 1: CT6 [Table 20] 3.2 Test sample 2: CT6m [Table 21] The required number of vials containing CT6 and CT6m were thawed to room temperature (RT) before oral gavage of the mice and discarded after treatment. 4.Animal management 4.1 Animal Licensing and Sourcing A license was obtained from Northwestern University to obtain the rights and permission to use the B6SJL-Tg(SOD1*G93A)1Gur / J (referred to herein as TgSOD1) mouse model of ALS. All TgSOD1 mice and non-Tg (non-carrier, wild-type littermates) used in these studies were F1 hybrids purchased from Jackson Laboratories (Stock number: 002726). 4.2 Containment Animals were housed (2-3 per cage) in disposable cages with Innorichment™-supplied corncob bedding (Innovive Disposable IVC Rodent Caging System®) and two additional amenities (i.e., cardboard hutch and nest). Cages were racked on an Innorack® caging system, and climate control and airflow were controlled for all cages in a housing room maintained at 21 ± 4°C and 50% ± 20% relative humidity. Housing rooms were maintained on an automatic timer with a 12-hour light / 12-hour dark cycle with no dim lights. Mice were provided with dry pelleted standard rodent chow Rodent Laboratory Chow 5001 and sterile water ad libitum. All animals used in this study were purchased from Jackson Laboratories and arrived on the same day. Mice were acclimated for 2 weeks before handling and were checked daily by animal care staff and experimenters. Animals that displayed aggressive behavior toward cage mates were separated and housed alone. 4.3 Identification Animals were identified by uniquely numbered metal ear tags and treatment groups were differentiated using a sticker color identification method on the cage cards. Each cage card contained relevant information such as protocol number and protocol PI, study number, vendor, animal arrival date and date of birth, and sex. Additional information such as group number and treatment type were also annotated on the cage cards as a further measure to prevent mis-dosing of animals. 4.4 Lifetime data collection and observation All data and observations were recorded in a laboratory notebook and electronic records were recorded using LabCat software, which is specifically designed to set up study protocols and collect all in vivo data and test results. Recording data in LabCat allows for an audit trail and time stamp of all data and observations recorded. 4.5 Group Allocation Only animals in apparent good health were included in the study. At the start of the study, mice were randomly assigned to groups according to body weight. 4.6 Health and cage observations Prior to beginning the in vivo phase of the experiment, the health status of each individual animal was assessed. Any significant observations in the cages during the study were recorded and immediately reported to the Principal Investigator, who determined the subsequent action (e.g., euthanasia). Body weights were recorded twice weekly until the animals reached age p60, and were then monitored and recorded at least 5 days per week, if not daily. To assess disease progression, mice were scored daily after reaching age P60 according to the ALS-TDI scoring criteria. Animals were euthanized when the humane endpoint, neurological score 4 (section 4.5.1), was reached. All SOD1 transgenic animals were euthanized, followed by non-transgenic wild-type littermates. 4.6.1 Neurological Scoring Criteria for ALS TDI Definitions: Methods for Implementing the Neurological Scoring System (NeuroScore) Each mouse is assessed daily using three different conditions / parameters: a) tail suspension test, b) gait analysis by walking test, and if paresis is observed, c) measurement of the righting reflex while the mouse is lying down to identify the onset of paresis. 1. For the tail suspension test, the mouse is held for 1-2 seconds with its tail approximately 1.5 inches from the base of the tail suspended over the wire top of the home cage, away from the food container, while observing the hind limbs. The tail suspension test is repeated three times and the most consistent result is recorded. 2. For the walking test, place the mouse on a clean surface with some traction and a walking distance of 25 cm (e.g., a paper towel taped to prevent slipping). Allow the mouse to walk a total of 75 cm (3 times the length of the paper towel) and observe its gait. 3. In the "righting reflex" test, the mouse is placed on its left or right side and a stopwatch is used to measure the time it takes to right itself unaided from the lying position (left or right). Note: This test is only given one trial. Determine the NS for each hind paw (left or right) separately on a scale from 0 to 4. NOTE: For example, a score of 0, 1 indicates an NS of 0 for the left hind paw and an NS of 1 for the right hind paw. A series of observations are performed and a neurological score (0-4) is assigned based on the criteria outlined in the ALS TDI. ALS-TDI Clinical Scoring Criteria **Each animal receives two scores, one for each hind paw, per scoring period / day. [Table 22] 4.7 Early cancellation In general, animals showing signs of moribundity, chronic pain or distress, or any of the following clinical symptoms were euthanized: severe weight loss (chronic weight loss of more than 20% plus general decreased behavior or persistent weight loss), severe cachexia, severe lethargy, severe immobility, severe dehydration, respiratory distress, severe diarrhea, severe abdominal enlargement, severe jaundice or anemia, uncontrollable bleeding from body orifices, persistent self-mutilation, severe large and / or deep skin lesions resistant to treatment (e.g., dermatitis, wounds). 5. Materials and Methods 5.1 Animals and Experimental Overview [Table 23] 5.2 Treatment 5.2.1 Male SOD1 study Group 1: 15 non-Tg (wild-type) males were administered 10 ml / kg of Dulbecco's phosphate-buffered saline (DPBS) by oral gavage daily from postnatal days p46–p63 until the end of the study. Group 2: 14 TgSOD1 males were administered 10 ml / kg DPBS daily by oral gavage starting from days p46–p63 until ALS-TDI NS reached 4. Group 3: 13 TgSOD1 males were administered 10 ml / kg of CT6 daily by oral gavage starting from days p46–p63 until an NS score of 4 was reached. Group 4: 13 TgSOD1 males were administered 10 ml / kg CT6max (hereafter referred to as CT6m) daily by oral gavage starting from days p46–p63 until an NS score of 4 was reached. 5.2.2 Female SOD1 study Group 1: 13 non-Tg (wild type) females were administered 10 ml / kg DPBS daily by oral gavage starting from days p46-p52 until the end of the study. Group 2: 13 TgSOD1 females were administered 10 ml / kg DPBS daily by oral gavage starting from days p46–p52 until they became moribund (e.g., ALS-TDI neurological score (NS) reached 4). Group 3: 13 TgSOD1 females were administered 10 ml / kg of CT6 daily by oral gavage starting from days p46–p52 until an NS score of 4 was reached. Group 4: 14 TgSOD1 females were administered 10 ml / kg of CT6max (hereafter referred to as CT6m) daily by oral gavage starting from days p46–p52 until an NS score of 4 was reached. 5.3 Experimental overview [Figure 1] TIFF2024536771000057.tif601645.4 Fecal collection Fecal pellets were collected from all animals at p115 and p120, in addition to some moribund mice, before they were euthanized due to disease progression (i.e., ALS-TD neurological scoring of 4). Each mouse was removed from its home cage and placed separately in a clean (previously washed with an additional 70% ethanol) and empty (no bedding material) cage for individual pellet harvest. Feces from each animal was collected in individual tubes. Feces were harvested with clean, sterile forceps and flash frozen on dry ice. Tubes were placed on dry ice during pellet collection. As many pellets as possible were collected. Vials containing collected fecal pellets were stored at a target temperature of -80°C. 5.5 Behavioral assessment 5.5.1 Grip strength Grip dynamometers allow neuromuscular studies in rodents by determining the maximum force exerted by an animal. This test is included in the Functional Observational Scale (FOB) to screen for neurobehavioral toxicity. In this context, the grip strength test is used to measure the maximum peak force exerted by an animal. The animal is placed on a grip dynamometer (BIOSEB In Vivo Research Instrument, Grip Strength Test Model GT3) equipped with a horizontally positioned metal grid device, the animal is held by its tail and lowered towards the device. The animal is able to grasp the metal grid, which is then pulled backwards in a horizontal plane. The force applied to the grid just before the animal releases it is recorded as the peak tension. The animal's maximum muscle strength is recorded. 5.5.2 Balance beam test The balance beam test is a behavioral test to assess motor coordination, especially of the hind limbs. The balance beam is 53 cm long and has a wooden dowel (1.1 cm diameter) fixed between two support posts with black box platforms at each end. The beam is positioned 43 cm above a 5 cm thick padded surface, which acts as a cushion. For each trial, the animal is placed in the center of the dowel beam and released. The trial ends when the mouse places both forepaws on the black box or the animal falls. The maximum time for each trial is 30 s, alternating between animals to allow them to rest before performing the next trial, with each animal performing three trials. [Figure 2] TIFF2024536771000058.tif911645.5.3 Rotarod The rotarod test was performed using a PC RotaRod-IV model from AccuScan Instruments, Inc., equipped with a computer equipped with AccuScan Fusion 3.5 software for real-time data acquisition. The data obtained here were output as text or Excel files. For the rotarod test, each mouse performed three consecutive trials, each for a maximum of 180 seconds at 10 rpm. 5.5.4 Beam walking test The beam walking test is a modified version of the balance beam test to address the development of progressive paralysis in aged SOD1 animals, preventing the animal from balancing on or grasping a thin round wooden dowel. For the beam walking test, the balance beam test was reconfigured by switching from a 1.1 cm (diameter) wooden dowel to a flat wooden beam 53 cm long, 1.9 cm wide, with a single black box platform at one end. An additional canopy catch was attached approximately 10 cm below the beam to reduce the stress of falling. For each round, the animal was positioned at the end of the beam between the two lines. The maximum time for each round was 60 s, alternating between animals to allow them to rest before performing the next round, with each animal performing three rounds. The trial ended when the mouse passed the marked finish line or the animal fell. Animals not crossing the finish line within 60 s were considered "timed out" and the distance traveled on the beam was recorded. [Figure 3] TIFF2024536771000059.tif781645.6 Tissue sampling Mice were euthanized by carbon dioxide asphyxiation, and once it was confirmed that the animals were completely euthanized, they were immediately exsanguinated by cardiac puncture using a 25-gauge needle attached to a 1 ml syringe. The needle was removed and the blood sample was transferred to a MiniCollect® K3EDTA (potassium ethylenediaminetetraacetate) blood tube. The tube was inverted completely to help distribute the EDTA evenly and prevent clotting. The blood sample was centrifuged at 10,000×g for 10 minutes at 4° C., and the collection tube was kept on ice. Plasma was transferred to a pre-labeled 1.7 ml Eppendorf tube, frozen on dry ice, and stored at −80° C. To collect the brain, the head was cut at the base of the skull around the C1 and C2 regions. The skull was carefully dissected on a cooled surface and the brain was removed. The brain hemispheres were further dissected on a cooled surface, snap frozen on liquid nitrogen, and stored at −80° C. The other hemisphere was fixed by immersion in 4% paraformaldehyde in phosphate buffer (PFA, pH=7.4) overnight at 4°C. After denervation, the entire spinal column was excised and bisected by making a transverse cut below the caudal rib to separate the lumbar region from the upper or thoracic region. Both the upper and lumbar spinal cords were flushed from the spinal column by hydraulic extrusion with ice-cold PBS using a 10 ml syringe with a 23-gauge blunt needle. The lumbar spinal cord was immersed in ice-cold 4% paraformaldehyde and the upper spinal cord was snap frozen in liquid nitrogen in a 1.7 ml Eppendorf tube. Both gastrocnemius muscles of each animal were dissected on a chilled surface. One side was further dissected on a chilled surface, flash frozen on liquid nitrogen, and stored at -80°C. The other side was fixed by immersion in 4% paraformaldehyde in phosphate buffer (PFA, pH=7.4) overnight at 4°C. 5.7 Histological evaluation 5.7.1 Tissue preparation For cryoprotection, the fixed 4% PFA overnight immersed tissue was rinsed in ice-cold PBS and then transferred to a 30% sucrose (w / v) solution in PBS and then maintained at 4 °C until the tissue was submerged in the sucrose solution (24-72 h). A plastic mold (Fisher Scientific) was used to embed the spinal cord using OCT freezing medium (Fisher Healthcare), and the tissue was oriented so that both transverse and longitudinal sections could be achieved in a single cross-section. Frozen tissue blocks were stored at -80 °C until all tissue samples were ready for cryosectioning. 5.7.2 Disconnection A Leica CM3050s cryostat was used to cryosection 7 μM thick spinal cord sections, 10 μM thick anterior tibial sections, or 10 μM thick gastrocnemius muscle sections, which were mounted on supercharged microscope slides (Fisher Scientific). 5.7.3 Immunofluorescence Protocol: IBA1+GFAP+DAPI For immunostaining, lumbar sections were first washed in PBS three times for 5 min each to remove the freezing medium, followed by incubation in 5% normal horse serum in 1x PBS supplemented with a blocking solution of 0.5% Triton X100 (Vector Laboratories, Inc, Cat. S-2000) for 1 h. The same blocking solution was used to prepare both the primary and secondary antibody solutions. They were then incubated overnight at 4°C in a primary antibody cocktail containing chicken anti-GFAP (Abeam, Cat. No. ab4674) diluted 1:1000, and anti-IBA1 / AIF-1 (E404W) rabbit monoclonal antibody (Cell Signaling, Cat. No. 17198) diluted 1:200. After overnight primary antibody incubation, slides were thoroughly washed in 1x PBS three times for 5 min each before starting the secondary antibody incubation. The secondary antibody cocktail consisted of goat anti-chicken IgY H&L Alexa Fluor® 647 (Abcam, ab150175) diluted 1:500 and goat anti-rabbit IgG H&L Alexa Fluor® 488 (Abcam, ab150077) diluted 1:1000. Sections were incubated for 1 hour at room temperature protected from light and then washed thoroughly in 1x PBS. Nuclei were stained using Vectashield® PLUS Antifade Mounting Medium with DAPI (Vector, Cat# H-2000) and glass coverslips were mounted to the tissue sections on microscope slides. Protocol: VAChT + a-bungarotoxin + DAPI For immunostaining, tibialis anterior muscle sections were first washed in PBS three times for 5 min each to remove the freezing medium, followed by incubation for 1 h in 5% normal horse serum (Vector Laboratories, Inc, Cat. S-2000) in 1x PBS supplemented with a blocking solution of 0.5% Triton X100. The same blocking solution was used to prepare both the primary and secondary antibody solutions. The tissue was then incubated in a cocktail containing goat polyclonal anti-VAChT (EMD Millipore, Cat. No. ABN 100) diluted 1:500, and a-bungarotoxin conjugated to Alex Fluor488 (Invitrogen, Cat. No. B13422) diluted 1:1000. After incubation at room temperature for 1 h, slides were washed extensively in 1x PBS three times for 5 min each before starting the secondary antibody incubation. The secondary antibody used was Donkey anti-Goat IgG Alexa Fluor® 568 (Invitrogen, Cat. No. 2304269) diluted 1:1000. Slides were incubated for 1 hour at room temperature protected from light and then washed thoroughly in 1x PBS. Nuclei were stained using Vectashield® PLUS Antifade Mounting Medium with DAPI (Vector, Cat. No. H-2000) and glass coverslips were mounted to the tissue sections on the microscope slides. Protocol:CD68+DAPI For immunostaining, the lumbar sections were first washed in PBS three times for 5 min each to remove the freezing medium, followed by incubation in 5% normal horse serum (Vector Laboratories, Inc, Cat. S-2000) in 1x PBS supplemented with a blocking solution of 0.5% Triton X100 for 1 h. The same blocking solution was used to prepare both the primary and secondary antibody solutions. The tissues were then incubated overnight at 4°C in rabbit anti-CD68 (Abcam, Cat. No. ab125212) diluted 1:500. After overnight incubation with primary antibody, the slides were washed extensively in 1x PBS three times for 5 min each before starting the secondary antibody incubation. The secondary antibody cocktail consisted of donkey anti-rabbit IgG Alexa Fluor® 647 (Abcam, ab150175) diluted 1:500 and goat anti-rabbit IgG H&L Alexa Fluor® 488 (Abcam, ab150075) diluted 1:500. Sections were incubated for 1 hour at room temperature protected from light and then washed thoroughly in 1x PBS. Nuclei were stained using Vectashield® PLUS Antifade Mounting Medium with DAPI (Vector, Cat# H-2000) and glass coverslips were mounted to tissue sections on microscope slides. 5.7.4 Imaging The same environment was maintained for all groups, and images were acquired using a Zeiss Axiovision microscope (Carl Zeiss). 5.7.5 Quantification Quantification of images was performed using ImageJ analysis software as previously described. Data are expressed as a percentage of mock-treated wild-type mice. Raw data were cleaned and sorted in Excel, then transferred to GraphPad Prism for statistical analysis and graph generation. 5.8 Biochemical evaluation 5.8.1 Sample preparation - spinal cord Spinal cord protein homogenates were prepared using PhosphoSafe™ extraction reagent (EMD Millipore, Cat. No. 71296) and mechanically homogenized using bead-filled Lysing Matrix D tubes (MP Biomedicals, III-kirch, France). Protein concentrations were determined by the Bradford method using BioRad Protein assay reagent. Because protein concentrations from individual animals were too low, spinal cord lysates were combined as follows (Table 3). [Table 24] 5.8.1.1 SDS-PAGE, Western Blot, and Imaging Protocol for determining PSMD11 protein concentration from spinal cord lysates 30 μg of total protein was mixed with SDS sample buffer and boiled for 10 min. Samples were electrophoresed on custom-made SDS polyacrylamide Bis-Tris gels (4-12%) using MOPS or MES running buffer and then transferred onto PVDF membranes (Invitrogen) using iBlot2 (Invitrogen). Membranes were blocked for 1 h using Odyssey blocking buffer and incubated with PSMD11 specific antibody (catalog no. NBP1-30252, Novus Biologicals) at a dilution of 1:1000 overnight at 4°C under shaking conditions. The next day, membranes were washed extensively in TBST (TBS+Tween 20) and incubated in HRP-conjugated secondary antibody (catalog no. 7076, Cell Signaling) at a dilution of 1:2000 for 1 h at room temperature. Protocol for measuring LAMP2A protein concentration from spinal cord lysates 30 μg of total protein was mixed with SDS sample buffer and boiled for 10 min. Samples were electrophoresed on custom-made SDS polyacrylamide Bis-Tris gels (4-12%) using MOPS or MES running buffer and then transferred onto PVDF membranes (Invitrogen) using iBlot2 (Invitrogen). Membranes were blocked for 1 h using Odyssey blocking buffer and incubated with a specific antibody for LAMP2A (catalog no. A0593, Abclonal) at a dilution of 1:2000 overnight at 4°C under shaking conditions. The next day, membranes were washed extensively in TBST (TBS+Tween 20) and incubated in HRP-conjugated secondary antibody (catalog no. 7074, Cell Signaling) at a dilution of 1:2000 for 1 h at room temperature. 5.8.1.2 Quantification Densitometric quantification of immunoblots was performed by Syngene's GeneTools after visualization with GBox Mini (SYNGENE). Target bands were normalized using the respective β-actin loading controls. 5.8.2 Proteasome functional assays Suc-Leu-Leu-Val-Tyr-AMC (S-LLVY-AMC) is a fluorescent substrate that emits fluorescence upon cleavage by the 20S proteasome. 90 μg of total protein prepared from spinal cord homogenates was added to the assay buffer (T-PER containing 5 mM Suc-LLVY-AMC and 1 mM ATP). The incubation was performed at 37°C / 5% CO 2 The enzymatic reaction was started with 500 ng / mL. Fluorescence was measured after 2 hours using a microplate reader (Promega discoverer, Promega corp). Excitation was set at 360 nm and emission at 450 nm. Assay buffer containing 5 mM Suc-LLVY-AMC was used as blank. Proteasome activity was calculated as follows (fluorescence value of sample-fluorescence value of blank). The average of the fluorescence values ​​from the G1 group was calculated and the % change in fluorescence compared to the average of the G1 group was plotted. 5.8.3 Detection of inflammatory markers Cytokine concentrations were assessed in spinal cord homogenates using the Mouse Cytokine / Chemokine 31-Plex Discovery Assay (Eve Technologies, Calgary, AB, Canada). 5.8.4 Detection of NF concentration in plasma A mouse neurofilament light (NEFL) ELISA kit from Abbexa was used for the analysis. Terminal plasma, 8-9 animals per group. Samples were diluted in assay buffer (1:2:5 for plasma) and analyzed according to the manufacturer's protocol. Briefly, after dilution, 100 μl of sample was added to precoated wells, sealed and incubated for 1 h at 37°C. After discarding the liquid, 100 μl of reagent A standard solution was added to each well. The plate was sealed with a cover and incubated for 1 h at 37°C. The wells were washed 3 times with 1x wash buffer and 100 μl of detection reagent B standard solution was added to each well. The plate was incubated for 30 min at 37°C. After 5 washes with wash buffer, 90 μl of TMB substrate was added to each well and incubated for 20 min at 37°C in the dark. 50 μl of stop reagent was added to each well and the plates were read at 450 nm on a microplate reader (Promega discoverer, Promega corp). Data was evaluated against the calibration curve provided in the kit and expressed as pg / ml of plasma. 5.8.5 Determination of ATP Levels from Spinal Cord Lysates 90 μg of total protein made from spinal cord homogenates was added to Promega CellTiter-Glo and incubated for 1 h at room temperature protected from light. Luminescence was measured using a microplate reader (Promega discoverer, Promega Corp). The average of duplicate wells for each sample was calculated and the average blank value was subtracted to obtain the sample luminescence value. The average G1 luminescence value was calculated from all G1 sample values. % ATP was calculated as follows: [(sample luminescence value / average G1 luminescence value) / 100]. 5.9 Statistics Statistical analysis was performed in Graph Pad Prism 9.0.0. Most data sets of studies were analyzed by one-way ANOVA followed by Dunnett's test, except for the rotarod experiment, where a mixed-effects analysis was performed on the mean duration data. Data are expressed as mean ± SEM. A level of p < 0.05 was considered statistically significant. 6. Results and Discussion 6.1 Weight Weight loss is a common feature of disease progression in ALS patients, and is similar in the SOD1-G93A mouse model of ALS. In this study, body weight was recorded twice a week until the animals reached age p60, after which they were monitored and recorded at least 5 days a week, if not daily. While non-Tg (wild type) mice showed a gradual expected increase in body weight with age, TgSODI animals showed a continued loss of body weight as a function of disease manifested as progression and worsening of hindlimb and paresis. Animals treated with CT6 or CT6m showed no difference in body weight compared to vehicle-treated SOD1 mice, indicating that treatment did not benefit in preventing weight loss, as the mice showed worsening paresis. Notably, both male and female SOD1 mice treated with CT6 or CT6m did not show accelerated or worsening weight loss compared to vehicle-treated animals throughout the study, demonstrating that continued daily treatment with CT6 or CT6m was well tolerated by the mice. [Figure 4] TIFF2024536771000061.tif91164 Figure 4: CT6 and CT6m treatments do not change the body weight of TgSODI mice, (a) the body weight of non-Tg and TgSODI males remains unchanged from the first day of treatment, indicated as day 0, until the animals are euthanized due to disease severity. There was no significant difference in body weight between any of the TgSODI1 animals during the treatment period. The only notable difference in body weight was seen between non-TgSOD1 and TgSOD1 animals, regardless of the type of treatment. A similar trend in body weight change is also observed in the female cohort, regardless of the type of treatment, (b) where no significant difference exists between any of the TgSOD1 animals. 6.2 Survival [Figure 5] TIFF2024536771000062.tif951646.3 Behavioral assessment To determine whether treatment with CT6 or CT6m would lead to improved function, several behavioral studies were performed: grip strength, balance beam, rotarod, and beam walking tests were performed. 6.3.1 Grip strength Grip dynamometers allow the study of neuromuscular function in rodents by determining the maximum force exerted by the animal. Grip strength was measured in females and males at three different time points. Administration of CT6 or CT6m for 27 consecutive days was found to significantly improve grip strength in females, although this difference was not significant at later time points. For males, no significant differences were found between vehicle-treated groups or CT6- or CT6m-treated groups. Furthermore, individual grip strength measurements taken at three different time points were analyzed, but no significant differences were found (Figures 6-13). [Figure 6] TIFF2024536771000063.tif84164[Figure 7] TIFF2024536771000064.tif771646.3.2 Balance beam test The balance beam test is a behavioral test to assess motor coordination specifically of the hind limbs. [Figure 8] TIFF2024536771000065.tif126164[Figure 9] TIFF2024536771000066.tif1311646.3.3 Rotarod The rotarod test was performed on females and males at three different time points. In p79-93 day old animals treated with CT6, there was a significant improvement in the mean duration the animals stayed on the rod, but this difference was not significant at later time points. For females, when the rotarod data was plotted by mean duration, no differences were found between CT6 or CT6m treated females compared to those of vehicle treated SOD1 males or SOD1 females at any time point. Also, when the data was plotted by the maximum time the animals spent on the rod, no significant differences were found between CT6 or CT6m treated males or females compared to those of vehicle treated SOD1 males or females at any time point. Furthermore, the individual rotarod measurements collected at the three different time points were analyzed and no significant differences were found (see Figure 10 and Appendix section 7.2). [Figure 10] TIFF2024536771000067.tif240164[Figure 11] TIFF2024536771000068.tif2401646.3.4 Beam walking test The beam walking test is a modified version of the balance beam test to address the development of progressive paralysis in aged SOD1 animals, which prevents animals from balancing on or grasping a thin round wooden dowel. The beam walking test was performed on p125 females. [Figure 12] TIFF2024536771000069.tif2401646.4 Histological evaluation 6.4.1 Microgliocytosis and astrocytosis Administration of CT6 or CT6m increases SOD1 G93A Microglial and astroglial immune responses are reduced in the spinal cord of mice. Neuroinflammation is a prominent feature in ALS animal models (Brettschneider et al., 2012). Neuroinflammation is seen in the spinal cord of human ALS patients and in SOD1 G93AThese include microgliosis and astrocytosis, which are significantly increased in SOD1 mice (Brettschneider et al., 2012) (Lewis et al., 2014). Microglial pathology in ALS is determined using Iba1 and CD68 markers. IBA-1 is an actin-binding protein expressed in both resting and activated microglia. CD68 is a pan-macrophage marker that is also expressed in phagocyte microglia. Glial fibrillary acidic protein (GFAP) is a type III intermediate filament protein expressed by astrocytes and used as a marker for astrocytosis. We assessed whether administration of CT6 or CT6m affected the immunoreactivity of astroglia (GFAP+ labeled cells), microglia (IBA1+ labeled cells), and microglia (CD68+ labeled cells) in lumbar spinal cord sections from SOD1 males and SOD1 females (Figures 25-29). Compared to wild-type mice, mock-treated SOD1 G93A A significant increase in the fluorescence intensity of IBA1+, GFAP+, or CD68+ was observed in the lumbar spinal cord of male or female mice (Figures 25 to 29). We found that administration of CT6 or CT6m reduced the expression of mock-treated SOD1 G93A We found that the fluorescence intensity of microglia and astroglia was significantly reduced compared to male and female mice (FIGS. 25 to 29). [Figure 13] TIFF2024536771000070.tif125164Figure 13: Immunofluorescence images of spinal cord sections stained with anti-IBA1. Images were taken under the same conditions for all groups with a Zeiss Axiovision microscope (Carl Zeiss). The white lines in the images indicate the scale bars. [Figure 14] TIFF2024536771000071.tif99164 Figure 14: Image quantification was performed using ImageJ analysis software. The mean of the fluorescence values ​​from the G1 group was calculated and the % change in fluorescence compared to the mean of the G1 group was plotted. Raw data was organized and sorted in Excel (see Appendix for raw data). Scatter dot blots were generated in GraphPad Prism. Scatter dot blots show mean ± SEM values. Each dot represents the percentage of fluorescence of a single image compared to that of the G1 group. Statistical analysis was performed in GraphPad Prism. Data sets were analyzed by one-way ANOVA followed by Dunnett's test. A level of p<0.05 was considered statistically significant. Compared to the male G2 group, the male G3 group or the male G4 group had significantly different levels, respectively (*p=0.04 and **p=0.001). Compared to the female G2 group, the female G3 group or the female G4 group, respectively, had significantly different levels (****p<0.0001). [Figure 15] TIFF2024536771000072.tif112164Figure 15: Immunofluorescence images of spinal cord sections stained with anti-GFAP. Images were taken under the same conditions for all groups with a Zeiss Axiovision microscope (Carl Zeiss). The white lines in the images indicate the scale bars. [Figure 16] TIFF2024536771000073.tif105164 Figure 16: Image quantification was performed using ImageJ analysis software. The mean of the fluorescence values ​​from the G1 group was calculated and the % change in fluorescence compared to the mean of the G1 group was plotted. Raw data was organized and sorted in Excel (see Appendix for raw data). Scatter dot blots were generated in GraphPad Prism. Scatter dot blots show mean ± SEM values. Each dot represents the percentage of fluorescence of a single image compared to that of the G1 group. Statistical analysis was performed in GraphPad Prism. Data sets were analyzed by one-way ANOVA followed by Dunnett's test. A level of p<0.05 was considered statistically significant. Compared to the male or female G2 group, the male or female G3 group and the male or female G4 group, respectively, had significantly different GFAP levels (****p<0.0001). [Figure 17] TIFF2024536771000074.tif135164 Figure 17: Image quantification was performed using ImageJ analysis software. The mean of the fluorescence values ​​from the G1 group was calculated and the % change in fluorescence compared to the mean of the G1 group was plotted. Raw data was organized and sorted in Excel (see Appendix for raw data). Scatter dot blots were generated in GraphPad Prism. Scatter dot blots show mean ± SEM values. Each dot represents the percentage of fluorescence of a single image compared to that of the G1 group. Statistical analysis was performed in GraphPad Prism. Data sets were analyzed by one-way ANOVA followed by Dunnett's test. A level of p<0.05 was considered statistically significant. Compared to the male G2 group, the male G3 group and the male G4 group, respectively, had significantly different GFAP levels (**p<0.0028, ****p<0.0001). 6.4.2 Neuromuscular junction integrity Administration of CT6 or CT6m increased SOD1 G93A NMJ integrity was improved in mouse tibialis anterior muscles. Loss of the neuromuscular junction (NMJ) is a key pathological feature in human ALS patients as well as in animal models (Fischer et al., 2004) (Clark et al., 2016). NMJ denervation is a good marker of disease progression, since denervation of the NMJ is observed even before the onset of symptoms (Fischer et al., 2004). To assess whether CT6 or CT6m affected the NMJ, we analyzed the overlap between presynaptic (vesicular acetylcholine transporter:VAChT) and postsynaptic (fluorescently labeled α-bungarotoxin:α-BTX binding to nicotinic acetylcholine receptors) in the tibialis anterior muscle. A significant decrease in the overlap between presynaptic (vesicular acetylcholine transporter:VAChT) and postsynaptic (fluorescently labeled α-bungarotoxin:α-BTX binding to nicotinic acetylcholine receptors) suggests increased denervation in mock-treated SOD1 compared to that in wild-type mice. G93A In contrast, CT6- or CT6m-treated animals showed a significant increase in overlap between VAChT and α-BTX, suggesting a decrease in denervation. This result suggests that administration of CT6 or CT6m maintains innervation of the NMJ. [Figure 18] TIFF2024536771000075.tif105164 Figure 18: NMJ integrity was measured by determining the overlap between presynaptic (vesicular acetylcholine transporter: VAChT) and postsynaptic (fluorescently labeled α-bungarotoxin: α-BTX binding to nicotinic acetylcholine receptors). Raw data were organized and sorted in Excel (see Appendix for raw data). Box plots were generated in GraphPad Prism. The center line of the box plot represents the mean %. SEM is plotted per group. Each dot represents a single NMJ where the % overlap between VAChT and α-BTX was determined. Statistical analysis was performed in GraphPad Prism. Data sets were analyzed by one-way ANOVA followed by Dunnett's test. A level of p<0.05 was considered statistically significant. The innervated NMJs observed in the male or female G2 group were significantly different compared to the male G1 or female G2 group, respectively (****p<0.0001). In contrast, the innervated NMJs in the male G3 or male G4 or female G3 or female G4 groups were significantly different from the male G2 or female G2 group, respectively (****p<0.0001). 6.4.3 Number of motor neurons [Figure 19] TIFF2024536771000076.tif101646.5 Biochemical evaluation 6.5.1 Proteasome function Administration of CT6 or CT6m increases SOD1 G93A Proteasome function was restored in the spinal cord of male mice. The ubiquitin proteasome system (UPS) is a major intracellular proteolytic system involved in maintaining protein turnover and selectively removing damaged proteins (Glickman and Ciechanover, 2002). Ubiquitin-rich protein inclusions are observed in ALS patients (Migheli et al., 1990). Proteasome activity is regulated by the SOD1 G93ASOD1 aggregates are significantly decreased in the spinal cord of the model (Kabashi et al., 2008). When proteasome activity was inhibited, SOD1 aggregates increased, and when proteasome function was restored, aggregates decreased (Puttaparthi et al., 2003). Thus, there is a need to identify therapies that increase proteasome function and thereby reduce the accumulation of misfolded proteins. It has been shown that upregulating regulatory subunits of the proteasome, such as PSMD11, increases proteasome assembly and functional activity, leading to the clearance of polyubiquitinated substrates (Vilchez et al., 2012). We found that PSMD11 protein concentrations were significantly increased in SOD1 G93A We found that SOD1 expression was significantly decreased in spinal cord lysates of male mice. However, CT6- or CT6m-treated male mice showed no significant difference from mock-treated SOD1 expression. G93A Male mice had significantly higher PSMD11 protein concentrations than female mice. G93A The PSMD11 protein concentration in spinal cord lysates from male mice was comparable to that of wild-type male mice. G93A Animals and mock-treated SOD1 G93A Animals, CT6-treated SOD1 G93A Animals and CT6m-treated SOD1 G93A There were no statistically significant differences between animals. To confirm that the increased PSMD11 protein concentration in males also corresponded to a concomitant increase in proteasome function, we performed a proteasome substrate cleavage assay. Suc-Leu-Leu-Val-Tyr-AMC (S-LLVY-AMC) is a fluorescent substrate that fluoresces upon cleavage by the 20S proteasome. Compared to wild-type male animals, SOD1 G93A Male mice had significantly lower proteasome activity in spinal cord lysates (*p=0.0377). In contrast, CT6 or CT6m treated SOD1 G93A Spinal cord lysates from male mice were analyzed using DPBS-treated SOD1 G93ACompared to male mice, the level of proteasome activity was significantly increased (**p<0.01). This result suggests that CT6 and CT6m treatment improves proteasome activity. [Figure 20] TIFF2024536771000077.tif97164 Figure 20: Spinal cord samples were electrophoresed on an SDS-Polyacrylamide-Bis-tris polyacrylamide gel (4-12%) and transferred onto a PVDF membrane. PSMD11 was detected using a specific antibody. Actin was used as a loading control. Immunoblots were visualized using GBox Mini (SYNGENE). [Figure 21] TIFF2024536771000078.tif101164 Figure 21: Target bands detected in Figure 32 were normalized using the respective β-actin loading control. Raw data were collated and sorted in Excel (see Appendix for raw data). Scatter dot blots were generated in GraphPad Prism. Scatter dot blots show mean ± SEM values. Each dot in the figure represents a relative value compared to the G1 group mean of a single spinal cord lysate as described in Table 3. Statistical analysis was performed in GraphPad Prism. Data sets were analyzed by one-way ANOVA followed by Dunnett's test. A level of p<0.05 was considered statistically significant. PSMD11 concentrations in male G3 or G4 groups were significantly different compared to male G2 group (***p=0.0002 and ****p<0.0001). PSMD11 protein concentrations were not statistically significant between female G1, G2 and G3 groups, or G2 and G4 groups (ns indicates no statistical significance), but when male and female data were combined, significant differences were observed between G2 and G3 groups, and G2 and G4 groups (*p=0.02 and ****p<0.0001). [Figure 22] TIFF2024536771000079.tif98165 Figure 22: Proteasome activity was measured using Suc-LLVY-AMC substrate. The mean of the fluorescence values ​​from the G1 group was calculated and the % change in fluorescence compared to the mean of the G1 group was plotted. Raw data was organized and sorted in Excel (see Appendix for raw data). Scatter dot blots were generated in GraphPad Prism. Scatter dot blots show mean ± SEM values. Statistical analysis was performed in GraphPad Prism. Data sets were analyzed by one-way ANOVA followed by Dunnett's test. A level of p<0.05 was considered statistically significant. Compared to the G1 group, the levels of the G2 group were significantly different (*p=0.0377). In contrast, the proteasome activity levels of the G3 and G4 groups were significantly different compared to the G2 group (**p<0.01). Proteasome activity was not significantly different between female G1 and G2, G2 and G3, or G2 and G4 groups (ns indicates no statistical significance), but when male and female data were combined, significant differences were observed between G1 and G2, G2 and G3, and G2 and G4 groups (*p=0.02 and **p=0.009). 6.5.2 Lysosomal function Administration of CT6 or CT6m increases SOD1 G93A Prisosomal function was restored in the spinal cord of male mice. Neurons rely on the ubiquitin proteasome system (UPS) and autophagy-associated lysosomal degradation for protein degradation and removal. The UPS targets ubiquitin-conjugated proteins for protein removal, whereas the lysosomal pathway targets long-lived proteins and damaged organelles. Dysregulation of protein degradation is involved in the pathogenesis of ALS (Root et al., 2021) (Rubinsztein, 2006). ALS is characterized by the presence of cytoplasmic inclusions or protein aggregates in affected motor neurons, indicating impaired protein degradation. Lysosomal degradation of cytoplasmic aggregates or inclusions is essential for neuronal growth and survival. Disruption of lysosomal function is also sufficient to cause neurodegeneration. Lysosome-associated membrane protein type 2 (LAMP2A) is a key protein required for proper lysosome function and is a receptor for chaperone-mediated autophagy with which substrate proteins interact and are then delivered to the lumen of the lysosome for degradation. We found that LAMP2A protein levels were significantly higher in SOD1 mice than in wild-type male mice. G93A We found that SOD1 expression was significantly decreased in spinal cord lysates of CT6- or CT6m-treated male mice. However, SOD1 expression was significantly decreased in spinal cord lysates of CT6- or CT6m-treated male mice compared with mock-treated mice. G93A Compared with male mice, CT6- or CT6m-treated female mice had significantly higher LAMP2A protein concentrations. G93A LAMP2A protein concentrations in spinal cord lysates from female mice were significantly higher than those in mock-treated SOD1 mice. G93A However, when the male and female data were combined, mock-treated SOD1 G93A In animals, CT6 or CT6m treated SOD1 G93A The concentration of LAMP2A protein was significantly increased in the animals. [Figure 23] TIFF2024536771000080.tif103164 Figure 23: Spinal cord samples were electrophoresed on an SDS-Bis-tris polyacrylamide gel (4-12%) and transferred onto a PVDF membrane. LAMP2A was detected using a specific antibody. Actin was used as a loading control. Immunoblots were visualized using GBox Mini (SYNGENE). [Figure 24] TIFF2024536771000081.tif103164 Figure 24: Target bands detected in Figure 34 were normalized using the respective β-actin loading control. Raw data were organized and sorted in Excel. For raw data, see Appendix. Scatter dot blots were generated in GraphPad Prism. Each dot in the figure represents a single spinal cord lysate listed in Table 3. Data are expressed as mean ± SEM. Statistical analysis was performed in GraphPad Prism. Data sets were analyzed by one-way ANOVA followed by Dunnett's test. Data sets were analyzed by one-way ANOVA followed by Dunnett's test. Compared to male G2 group, male G3 and G4 groups have significantly different concentrations of LAMP2A (*p=0.03, ***p=0.0002 and ****p<0.0001). The concentration levels of LAMP2A protein were statistically significant between female G1 and G2 groups, but not between female G2 and G3 groups, or between female G2 and G4 groups (ns indicates no statistical significance). However, when the male and female data were combined, significant differences were found between G1 and G2 groups, G2 and G3 groups, and G2 and G4 groups (*p=0.04 and ****p=0.0001). 6.5.3 Inflammatory markers Administration of CT6 or CT6m increases SOD1 G93A Decreased concentrations of proinflammatory cytokines / chemokines in the spinal cord of To evaluate the effect of CT6 or CT6m treatment on inflammation, we measured the concentrations of proinflammatory cytokines and chemokines in spinal cord lysates of male and female mice using the Mouse Cytokine / Chemokine 31-Plex Discovery Assay (Eve Technologies, Calgary, AB, Canada). Levels of TNF-α, IL-6, and RANTES (Regulated upon Activation, Normal T Cell Expressed and Secreted) were elevated in plasma and in the cerebrospinal fluid of ALS patients (Sekizawa et al., 1998) (Rentzos et al., 2007) (Chen et al., 2018). RANTES, also called CCL5, was found to be upregulated in the lumbar spinal cord of SOD1 mice (Komine et al., 2018). We found that CT6 or CT6m treatment led to significant changes in the concentrations of TNF-α and RANTES, but not in other cytokines (see Appendix for detailed changes in spinal cord cytokine concentrations). [Figure 25] TIFF2024536771000082.tif108164 Figure 25: Quantification of IL-6 levels in the spinal cord. For raw data, see Appendix. Scatter dot blots were generated in GraphPad Prism. Data are presented as mean ± SEM. Each dot represents a replicate of a spinal cord sample described in Table 3. Statistical analysis was performed in GraphPad Prism. Data sets were analyzed by one-way ANOVA followed by Dunnett's test. Data sets were analyzed by one-way ANOVA followed by Dunnett's test. A level of p<0.05 was considered statistically significant. IL-6 showed significant differences in the male G2 or female G2 groups compared to the male G1 or female G1 groups, respectively (***p=0.0001), but there were no significant differences between the male or female G2 groups and the male or female G3 or G4 groups. [Figure 26] TIFF2024536771000083.tif107164 Figure 26: Each dot represents a replicate of a spinal cord sample listed in Table 3. For raw data, see Appendix. Scatter dot blots were generated in GraphPad Prism. Data are presented as mean ± SEM. Statistical analysis was performed in GraphPad Prism. Data sets were analyzed by one-way ANOVA followed by Dunnett's test. Data are presented as mean ± SEM. A level of p<0.05 was considered statistically significant. Compared to the male G2 group, the male G3 and G4 groups had significantly different concentrations (***p=0.0009 and **p=0.0067). ns indicates no statistical significance between the female G2 and G3 groups, or the female G2 and G4 groups, or the female G1 and G2 groups. In addition, TNF-α was significantly increased in the G2 group (males and females combined) compared to the G1 group (p=0.01), but no significant differences were observed between the G2 group (males and females combined) and the G3 group (males and females combined), or between the G2 group and the G4 group. [Figure 27] TIFF2024536771000084.tif108164 Figure 27: Each dot represents a replicate of a spinal cord sample listed in Table 3. For raw data, see Appendix. Scatter dot blots were generated in GraphPad Prism. Data are presented as mean ± SEM. Statistical analysis was performed in GraphPad Prism. Data sets were analyzed by one-way ANOVA followed by Dunnett's test. Data are presented as mean ± SEM. A level of p<0.05 was considered statistically significant. Compared to the male G2 group, the G3 and G4 groups had significantly different RANTES concentrations (*p=0.01 and ****p=0.0001). ns indicates no statistical significance between the G2 and G3 groups or the G2 and G4 groups. 6.5.4 Plasma NF-L concentration Administration of CT6 or CT6m increases SOD1 G93A Decreased plasma NF-L concentration in mice Spinal cord motor neuron death is mediated by SOD1 G93AA striking feature of the model is that neurofilament light chain (NF-L) is a well-validated biomarker to assess the progression of neurodegenerative diseases (Loeffler et al., 2020). Serum NF-L concentrations increase with neuronal death (Loeffler et al., 2020). Plasma NF-L concentrations from animals were determined using an NF-L ELISA kit. Compared to wild-type male or female mice, plasma concentrations of NF-L were significantly higher than SOD1, which indicates motor neuron death. G93A The expression of SOD1 in male and female mice was significantly higher than that in mice treated with CT6 or CT6m. G93A Compared with male or female mice, serum levels of NF-L were significantly lower, suggesting less motor neuron death. [Figure 28] TIFF2024536771000085.tif106164 Figure 28: Plasma NF-L concentrations from animals were determined using a NF-L ELISA kit. For raw data, see Appendix. Scatter dot blots were generated in GraphPad Prism. Each dot represents a plasma sample from an individual animal. Data are presented as mean ± SEM. Statistical analysis was performed in GraphPad Prism. Data sets were analyzed by one-way ANOVA followed by Dunnett's test. A level of p<0.05 was considered statistically significant. Compared to the G2 group, NF-L concentrations in the G3 and G4 groups were significantly different (*p=0.02, **p=0.0074, ***p=0.0003, and ****p<0.0001). ns indicates no statistical significance between the G2 and G4 groups in females. 6.5.5 ATP levels in the spinal cord Administration of CT6 or CT6m increases SOD1 G93A Increased ATP levels in mice Mitochondrial dysfunction is associated with numerous neurodegenerative diseases, including ALS. Mitochondrial dysfunction has been observed in human ALS patients and ALS animal models (Smith et al., 2019). Therefore, we measured the production of mitochondrial ATP in spinal cord homogenates. Although no statistically significant differences were observed in ATP levels in vehicle-treated SOD1 males or females compared to wild-type males or females, we found that ATP levels in SOD1 males or females treated with CT6 or CT6m were significantly higher compared to vehicle-treated SOD1 males or females, or wild-type males or females. [Figure 29] TIFF2024536771000086.tif105164 Figure 29: ATP was measured on apical spinal cord protein homogenates using Promega CellTiter-Glo. For raw data, see Appendix. Scatter dot blots were generated in GraphPad Prism. Each dot represents each spinal cord sample listed in Table 3. Data are expressed as mean ± SEM. % ATP levels were calculated by comparing luminescence values ​​to the mean of the G1 group. Statistical analysis was performed in GraphPad Prism. Data sets were analyzed by one-way ANOVA followed by Dunnett's test. A level of p<0.05 was considered statistically significant. Compared to the G1 group, ATP concentrations in G3 and G4 were significantly different (*p=0.03, **p=0.0029, ***p=0.0003, and ****p<0.0001). ns indicates no statistical significance between female G1 and G2 groups. 7. Attachments 7.1 References: Brettschneider, J., Toledo, JB, Van Deerlin, VM, Elman, L., McCluskey, L., Lee, VM-Y., and Trojanowski, JQ(2012).Microglial activation correlates with disease progression and upper motor neuron clinical symptoms in amyotrophic lateral sclerosis.PLoS One 7,e39216. Chen, X., Hu, Y., Cao, Z., Liu, Q., and Cheng, Y. (2018). Cerebrospinal Fluid Inflammatory Cytokine Aberrations in Alzheimer's Disease, Parkinson's Disease and Amyotrophic Lateral Sclerosis: A Systematic Review and Meta-Analysis. Front Immunol 9, 2122. Clark,JA,Southam,KA,Blizzard,CA,King,AE,and Dickson,TC(2016).Axonal degeneration, distal collateral branching and neuromuscular junction architecture alterations occur prior to symptom onset in the SOD1(G93A)mouse model of amyotrophic lateral sclerosis.J Chem Neuroanat 76,35-47. Fischer,LR,Culver,DG,Tennant,P.,Davis,AA,Wang,M.,Castellano-Sanchez,A.,Khan,J.,Polak,MA,and Glass,JD(2004).Amyotrophic lateral sclerosis is a distal axonopathy: evidence in mice and man.Exp Neurol 185,232-240. Glickman, M.H., and Ciechanover, A.(2002).The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction.Physiol Rev 82,373-428. Gurney,ME,Pu,H.,Chiu,AY,Dal Canto,MC,Polchow,CY,Alexander,DD,Caliendo,J.,Hentati,A.,Kwon,YW,and Deng,HX(1994).Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation.Science 264,1772-1775. Kabashi,E.,Agar,JN,Hong,Y.,Taylor,DM,Minotti,S.,Figlewicz,DA,and Durham,HD(2008).Pro-teasomes remain intact,but show early focal alteration in their composition in a mouse model of amyotrophic lateral sclerosis.J Neurochem 105,2353-2366. Komine,O.,Yamashita,H.,Fujimori-Tonou,N.,Koike,M.,Jin,S.,Moriwaki,Y.,Endo,F.,Watanabe,S.,Uematsu,S.,Akira,S.,et al.(2018).Innate immune adaptor TRIF deficiency accelerates disease progression of ALS mice with accumulation of aberrantly activated astrocytes.Cell Death Differ 25,2130-2146. Lewis, KE, Rasmussen, AL, Bennett, W., King, A., West, AK, Chung, RS, and Chuah, Ml(2014).Microglia and motor neurons during disease progression in the SOD1G93A mouse model of amyotrophic lateral sclerosis:changes in arginasel and inducible nitric oxide synthase. J Neuroinflammation 11,55. Loeffler, T., Schilcher, I., Flunkert, S., and Hutter-Paier, B. (2020). Neurofilament-Light Chain as Biomarker of Neurodegenerative and Rare Diseases With High Translational Value. Front Neurosci 14, 579. Migheli, A., Autilio-Gambetti, L., Gambetti, P., Mocellini, C., Vigliani, MC, and Schiffer, D. (1990). Ubiquitinated filamentous inclusions in spinal cord of patients with motor neuron disease. Neurosci Lett 114, 5-10. Puttaparthi, K., Wojcik, C., Rajendran, B., DeMartino, GN, and Elliott, JL (2003). Aggregate formation in the spinal cord of mutant SOD1 transgenic mice is reversible and mediated by proteasomes. J Neuro-chem 87, 851-860. Rentzos, M., Nikolaou, C., Rombos, A., Boufidou, F., Zoga, M., Dimitrakopoulos, A., Tsoutsou, A., and Vassilopoulos, D. (2007). RANTES levels are elevated in serum and cerebrospinal fluid in patients with amyotrophic lateral sclerosis. Amyotroph Lateral Scler 8, 283-287. Root, J., Merino, P., Nuckols, A., Johnson, M., and Kukar, T. (2021). Lysosome dysfunction as a cause of neurodegenerative diseases: Lessons from frontotemporal dementia and amyotrophic lateral sclerosis. Neurobiology of Disease 154, 105360. Rubinsztein DC(2006).The roles of intracellular protein-degradation pathways in neurodegeneration.Nature 443,780-786. Sekizawa,T.,Openshaw,H.,Ohbo,K.,Sugamura,K.,Itoyama,Y.,and Niland,JC(1998).Cerebrospinal fluid interleukin 6 in amyotrophic lateral sclerosis:immunological parameters and comparison with inflammatory and non-inflammatory central nervous system diseases.J Neurol Sci 154,194-199. Smith, EF, Shaw, PJ, and De Vos, KJ (2019). The role of mitochondria in amyotrophic lateral sclerosis. Neurosci Lett 710, 132933. Vilchez, D., Morante, I., Liu, Z., Douglas, PM, Merkwirth, C., Rodrigues, APC, Manning, G., and Dillin, A. (2012). RPN-6 determines C. elegans longevity under proteotoxic stress conditions. Nature 489, 263-268. 7.2 Behavioral research: individual data 7.2.1 Grip strength: individual data from male studies [Table 25] [Table 26] [Table 27] [Table 28] [Table 29] [Table 30] 7.2.2 Grip strength: individual data from the female study [Table 31] [Table 32] [Table 33] [Table 34] [Table 35] [Table 36] 7.2.3 Rotarod: individual data from male studies [Table 37] [Table 38] [Table 39] [Table 40] [Table 41] [Table 42] 7.2.4 Rotarod: individual data from female studies [Table 43] [Table 44] [Table 45] [Table 46] [Table 47] [Table 48] 7.3 Histology 7.3.1 IBA1 [Table 49] [Table 50] 7.3.2 GFAP [Table 51] [Table 52] 7.3.3 CD68 [Table 53] 7.3.3.2 Female research 7.3.4 MNJ research [Table 54] [Table 55]

Table 56

Table 57

Table 58

Table 59

Table 60

Table 61

Table 62

Table 63

Table 64

Table 65

Table 66

Table 67

Table 68

Table 69

Table 70

Table 71

Table 72

Table 73

Table 74

Table 75

Table 76

Table 77

Table 78

Table 79

Table 80

Table 81

Table 82

Table 83

Table 84

Table 85

Table 86

Table 87

Table 88

Table 89

Claims

1. A pharmaceutical composition for treating or preventing a neurodegenerative disease, disorder, or condition in a subject in need thereof, comprising: The pharmaceutical composition comprises one or more microbial strains or microbial components thereof, or one or more microbial metabolites.

2. 2. The pharmaceutical composition of claim 1, wherein the neurodegenerative disease, disorder, or condition is amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), or Huntington's disease (HD).

3. 2. The pharmaceutical composition of claim 1, wherein the neurodegenerative disease, disorder, or condition is ALS.

4. The pharmaceutical composition of claim 1 , wherein the subject is a mammal.

5. The pharmaceutical composition of claim 1 , wherein the subject is a human.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the one or more microbial strains are derived from the microbiota of a mammal.

7. The pharmaceutical composition according to any one of claims 1 to 5, wherein the one or more microbial strains are derived from the human microbiome.

8. The pharmaceutical composition of claim 7 , wherein the human microbiota is the microbiota of the subject.

9. 9. The pharmaceutical composition of claim 8, wherein the pharmaceutical composition maintains or modulates the microbiota of the subject.

10. 6. The pharmaceutical composition of any one of claims 1 to 5, wherein the one or more microbial components or microbial metabolites are selected from Appendix 1, Appendix 3, or Appendix 4.

11. 6. The pharmaceutical composition of any one of claims 1 to 5, wherein the one or more microbial metabolic products is or comprises a bile acid.

12. 6. The pharmaceutical composition of any one of claims 1 to 5, wherein the one or more microbial metabolites is or comprises tauroursodeoxycholic acid.

13. 6. The pharmaceutical composition of any one of claims 1 to 5, wherein the one or more microbial components or microbial metabolites are butyrylcamitine, theobromine, p-hydroxyphenylpyruvic acid, propionic acid, picolinic acid, 2-hydroxy-4-methylvaleric acid, N6-acetyllysine, urocanic acid, N5-ethylglutamine, trigonelline, stachydrine, ectoine, 5-hydroxylysine, arginine (arg), cholic acid, 2-(4-hydroxyphenyl)propionic acid, N-acetyltryptophan, hydroxyproline, argininosuccinic acid, glutamic acid (Glu), sarcosine, 5-methoxyindoleacetic acid, indole-3-lactic acid, isovalerylalanine, N-acetylleucine, 1-methylhistidine, N-acetylphenylalanine, proline (Pro), or any combination thereof.

14. The one or more microbial components or microbial metabolites include 4-hydroxyphenylpyruvate, ectoine, gramine, N-acetyl-L-phenylalanine, nepsilon-acetyl-L-lysine, stachydrine, trigonelline, 3-ureidopropionic acid, theobromine, hippuric acid, imidazolepropionic acid, NG-methyl-L-arginine, trans-urocanic acid, N-acetyl-L-leucine, sarcosine, and isobutyrylcarnitine. , b-hydroxyisovaleric acid, L-theanine / N5-ethylglutamine, 5-hydroxylysine, phenaceturic acid, betaine, hydroxyproline, picolinic acid, 2-aminoadipic acid, glycerophosphocholine, carnitine, glycerol 3-phosphate, argininosuccinic acid, creatine, terephthalic acid, homocitrulline, mucic acid, homocysteine ​​sulfinic acid, trimethyllysine, spermidine, glyoxylic acid, XA0013 C6H6O4S, 3-indoxyl sulfate, nicotinamide, N-formylglycine, ureidoglycolate, N-methylproline, glucaric acid, butyrylcarnitine, methionine sulfoxide, carboxymethyllysine, glycolic acid, phenaceturic acid, diethanolamine, phosphorylcholine, guanidinosuccinic acid, N-acetylhistidine, glyceric acid, S-methylmethionine, cysteine ​​glutathione disulfide, kynurenine, N-acetylphenylalanine, threonic acid, malic acid, 7,8-dihydrobiopterin, homovanillic acid, taurocholic acid, 5-methoxyindoleacetic acid, butyrate, b-hydroxyisovaleric acid, 2-oxoglutaric acid, N-acetyltryptophan, thiaproline, hypotaurine, cholic acid, acetoacetic acid, ethanolamine, guanidoacetic acid, S-sulfocysteine, myristic acid C14:0 XA0027, or any combination thereof.

15. 6. The pharmaceutical composition of any one of claims 1 to 5, wherein the one or more microbial strains are or comprise Gluconacetobacter hansenii, Terrisporobacter glycolicus, Coprococcus sp., Lactobacillus plantarum, Clostridium butyricum, Paenibacillus sp., Veillonella sp., Bifidobacterium sp., Bacillus subtilis, Acidaminococcus sp., or a combination thereof.

16. 6. The pharmaceutical composition of any one of claims 1 to 5, wherein the one or more microbial strains are or comprise Gluconacetobacter hansenii, Terrisporobacter glycolicus, Coprococcus sp., Lactobacillus plantarum, Veillonella sp., Bifidobacterium sp., or a combination thereof.

17. 6. The pharmaceutical composition of any one of claims 1 to 5, wherein the one or more microbial strains are or comprise Gluconacetobacter hansenii, Terrisporobacter glycolicus, Coprococcus catus, Lactobacillus plantarum, Veillonella atypica, Bifidobacterium breve, or a combination thereof.

18. 6. The pharmaceutical composition of any one of claims 1 to 5, wherein the one or more microbial strains are or comprise Bacillus subtilis.

19. The pharmaceutical composition according to any one of claims 1 to 5, wherein the pharmaceutical composition comprises two or more microbial strains.

20. The pharmaceutical composition according to any one of claims 1 to 5, wherein the pharmaceutical composition comprises five or more microbial strains.

21. The pharmaceutical composition according to any one of claims 1 to 5, wherein the pharmaceutical composition comprises 10 or more microbial strains.

22. 6. The pharmaceutical composition of any one of claims 1 to 5, wherein the pharmaceutical composition is administered topically, orally, subcutaneously, intravenously, intramuscularly, intracerebrally, intrathecally, intrarectally, ophthalmically, intravitreally, or suprachoroidally.

23. 23. The pharmaceutical composition of claim 22, wherein the pharmaceutical composition is administered orally.

24. 23. The pharmaceutical composition of claim 22, wherein the pharmaceutical composition is administered intracerebrally.

25. 6. The pharmaceutical composition of any one of claims 1 to 5, wherein the pharmaceutical composition is formulated as a syrup, liquid, tablet, troche, gummy, capsule, powder, gel, film, injectable, or eye drop.

26. Each microbial strain of the one or more microbial strains is １ ~10 １５ 6. The pharmaceutical composition of claim 1, wherein the virion is present in the pharmaceutical composition at a concentration of CFU.

27. wherein each microbial strain of said one or more microbial strains is at least 10 ６ 6. The pharmaceutical composition of claim 1, wherein the virion is present in the pharmaceutical composition at a concentration of CFU.

28. The pharmaceutical composition described in claim 1, wherein the pharmaceutical composition regulates one or more microbial metabolic products in the subject.

29. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition regulates one or more characteristics in the subject.

30. 1. A method for screening a microbial strain, comprising: contacting the microbial strain with a culture comprising a neuronal cell or neuronal cell line that models a neurodegenerative disease, disorder, or condition; and determining whether the microbial strain has altered a characteristic of the culture, wherein the characteristic is associated with the neurodegenerative disease, disorder, or condition.

31. A composition comprising: The composition comprises one or more microbial strains or microbial components thereof, or one or more microbial metabolic products, and is administered to a subject in need thereof.

32. 1. A method for characterizing a microbial strain, comprising: adding the microbial strain to a culture comprising a neuronal cell or neuronal cell line that models a neurodegenerative disease, disorder, or condition; and determining whether the microbial strain affects the level of one or more characteristics of the neuronal cell or neuronal cell line, wherein the one or more characteristics are associated with the neurodegenerative disease, disorder, or condition.

33. 1. A method for producing a pharmaceutical composition comprising characterizing one or more microbial strains, microbial components, or microbial metabolic products thereof, comprising: adding the one or more microbial strains to a culture comprising neuronal cells or a neuronal cell line that models a neurodegenerative disease, disorder, or condition; determining whether the one or more microbial strains affect the level of one or more characteristics of the neuronal cell or neuronal cell line, wherein the one or more characteristics are associated with the neurodegenerative disease, disorder, or condition.

34. A method for producing a pharmaceutical composition, comprising: The method comprises adding one or more microbial strains or components, or one or more microbial metabolites to a syrup, liquid, tablet, lozenge, gummy, capsule, powder, gel, film, injectable, or eye drop.

35. 1. A method for evaluating a microbial strain for its ability to affect one or more characteristics of a culture, comprising: adding the microbial strain to the culture comprising a neuronal cell or neuronal cell line that models a neurodegenerative disease, disorder, or condition; and determining whether the microbial strain affects the level of one or more characteristics of the neuronal cell or neuronal cell line, wherein the one or more characteristics are associated with the neurodegenerative disease, disorder, or condition.

36. An injection comprising the pharmaceutical composition of claim 1.

37. A dietary supplement comprising the pharmaceutical composition of claim 1.

38. A kit comprising the pharmaceutical composition of claim 1.

39. 1. A pharmaceutical composition for treating amyloid plaques or reducing plaque burden, plaque number, or plaque size in a subject diagnosed with a neurodegenerative disease, disorder, or condition, comprising: The pharmaceutical composition comprises one or more microbial strains or microbial components thereof, or one or more microbial metabolites.