Modulation of gut microbiota to treat neurodegenerative disorders

Abstract

Disclosed herein are methods and compositions useful for ameliorating motor deficiency and neuroinflammation in a subject in need thereof, such as a subject having a neurodegenerative disorder, such as Parkinson's disease. Also disclosed are methods and compositions useful for diagnosing neurodegenerative disorders, such as Parkinson's disease.

Description

[0001] This application is a divisional application of patent application No. 201780045705.7, filed on May 22, 2017, entitled "Regulating Gut Microbiota to Treat Neurodegenerative Diseases".

[0002] Related applications

[0003] This application claims priority under 35 U.S. SC §119(e) to U.S. Provisional Application No. 62 / 340408, filed May 23, 2016; U.S. Provisional Application No. 62 / 370,578, filed August 3, 2016; and U.S. Provisional Application No. 62 / 443952, filed January 9, 2017. The contents of each of these related applications are expressly and wholly incorporated herein by reference.

[0004] Statement on federal government-funded research and development

[0005] This invention was developed with government support from the National Institutes of Health (NIH) grant number NS085910. The U.S. government holds certain rights to this invention. Technical Field

[0006] This disclosure generally relates to the field of diagnosis and treatment of neurodegenerative disorders (such as Parkinson's disease). Background Technology

[0007] Neurological dysfunction underlies many human diseases. Behavioral, psychotic, and neurodegenerative disorders typically manifest as signature neuropathologies within the central nervous system (CNS). One neuropathology, amyloidosis, is caused by the abnormal aggregation of specific neuronal proteins that disrupt the function of many cells. Affected tissues often contain insoluble protein aggregates exhibiting altered conformations, a feature believed to contribute to an estimated 50 different human diseases (Sacchettini and Kelly, 2002). Neurodegenerative amyloid disorders, including Alzheimer's disease, Huntington's disease, and Parkinson's disease (PD), are associated with a variety of different amyloid proteins (Brettschneider et al., 2015). PD is the second most common neurodegenerative disease in the United States, affecting an estimated 1 million people and 1% of the U.S. population over 60 years of age (Nalls et al., 2014). Worldwide, approximately 3 million patients and caregivers are affected by the often debilitating symptoms of PD, involving motor deficits including tremor, muscle rigidity, bradykinesia, and gait impairment. This is a multifactorial disease with a strong environmental component, as less than 10% of cases are hereditary (Nalls et al., 2014). The accumulation of α-synuclein (αSyn) is considered pathogenic in a family of diseases known as synucleinopathies, including PD, multiple system atrophy, and Lewy body disease (Brettschneider et al., 2015; Luk et al., 2012; Prusiner et al., 2015). αSyn accumulation is a gradual process, leading to oligomerization and the accumulation of intransient fibrils within neurons. Dopaminergic neurons in the substantia nigra pars compacta (SNpc) appear to be particularly susceptible to αSyn aggregates. Dopamine modulators are first-line treatments for PD; however, treatment can have serious side effects and often loses its effectiveness (Jenner, 2008). Safe and effective treatments need to be found to address the growing burden of PD in an aging population, a paradoxical consequence of humanity's achievements in extending lifespan. Summary of the Invention

[0008] This document discloses methods and compositions that can be used to improve motor deficits and neuroinflammation in subjects in need, such as those with neurodegenerative diseases (e.g., Parkinson's disease). Methods and compositions that can be used to diagnose neurodegenerative diseases (e.g., Parkinson's disease) are also disclosed.

[0009] Some embodiments provide methods for treating neurodegenerative diseases in subjects, wherein the method includes modulating the composition of the gut microbiota in subjects with neurodegenerative diseases. Some embodiments provide methods for delaying or reducing the likelihood of developing neurodegenerative diseases in subjects, wherein the method includes modulating the composition of the gut microbiota in subjects at risk of developing neurodegenerative diseases. In some embodiments, neurodegenerative diseases are synucleinopathies, such as Parkinson's disease (PB), dementia with Lewy body disease, multiple system atrophy, or any combination thereof.

[0010] Some embodiments provide methods for improving motor deficits in subjects in need (e.g., subjects with synucleinopathies, including Parkinson's disease), wherein the method includes modulating the composition of the gut microbiota in the subject. Some embodiments provide methods for reducing microglial activation in subjects in need, wherein the method includes modulating the composition of the gut microbiota in the subject. Some embodiments provide methods for reducing α-synuclein (αSyn) aggregates in subjects in need, wherein the method includes modulating the composition of the gut microbiota in the subject. In some embodiments, the method promotes the clearance of insoluble αSyn protein aggregates, reduces αSyn protein aggregation, or both. In some embodiments, the method includes measuring the rate and / or level of αSyn aggregation in the subject, measuring the rate and / or level of clearance of insoluble αSyn protein aggregates in the subject, or a combination thereof. In some embodiments, the method measures the rate and / or level of αSyn aggregation in the subject's brain, the rate and / or level of clearance of insoluble αSyn protein aggregates in the subject's brain, or a combination thereof. In some embodiments, the method further includes measuring the rate and / or level of αSyn aggregation in the subject after adjusting the composition of the gut microbiota, measuring the rate and / or level of clearance of insoluble αSyn protein aggregates in the subject, or a combination thereof.

[0011] Some implementations provide methods for reducing neuroinflammation in subjects in need, wherein the method includes adjusting the composition of the gut microbiota in the subject.

[0012] The desired subject in the methods disclosed herein may be, for example, a subject suffering from neurodegenerative amyloidosis (e.g., synucleinopathy). Non-limiting examples of synucleinopathy include Parkinson's disease, dementia with Lewy body disease, multiple system atrophy, and any combination thereof. In some embodiments, the desired subject is a subject suffering from Parkinson's disease. In some embodiments, the methods disclosed herein may further include identifying the desired subject, wherein the desired subject has abnormal levels of α-synuclein (αSyn) aggregation. In some embodiments, identifying the desired subject includes measuring the rate and / or level of αSyn aggregation in the subject, measuring the rate and / or level of clearance of insoluble αSyn protein aggregates in the subject, or a combination thereof. In some embodiments, measuring the rate and / or level of αSyn aggregation in the subject's brain, measuring the rate and / or level of clearance of insoluble αSyn protein aggregates in the subject's brain, or a combination thereof. In some embodiments, the method further includes measuring the rate and / or level of αSyn aggregation in the subject after adjusting for gut microbiota composition, measuring the rate and / or level of clearance of insoluble αSyn protein aggregates in the subject, or a combination thereof. In some embodiments, the method improves one or more physical impairments in the subject. In some embodiments, the method improves one or more GI functions in the subject. In some embodiments, the method alleviates constipation in the subject. In some embodiments, the motor deficit is tremor, muscle rigidity, bradykinesia, impaired gait, or any combination thereof.

[0013] In some embodiments, the methods disclosed herein can restore the composition of the gut microbiota in a subject to normal levels. In some embodiments, adjusting the composition of the gut microbiota in a subject includes administering one or more antibiotics to the subject. The antibiotics may be natural, synthetic, or semi-synthetic. One or more antibiotics may include, for example, ampicillin, vancomycin, neomycin, gentamicin, erythromycin, teicoplanin, doxycycline, tetracycline, norfloxacin, ciprofloxacin, octament, cephalexin (e.g., Keflex), penicillin, ampicillin, kanamycin, rifamycin, rifaximin, neomycin, metronidazole, or any combination thereof. The antibiotics may be administered orally, intravenously, rectally, or in combination thereof. In some embodiments, one or more antibiotics do not contain rifampin and / or minocycline. In some embodiments, adjusting the composition of the gut microbiota in a subject includes administering one or more inhibitors of PD-enhancing microbial metabolites to the subject. In some implementations, adjusting the composition of the gut microbiota in a subject includes administering the subject an antibody against one or more PD-enhancing microbial metabolites, an antibody against an intermediate used in the in vivo synthesis of one or more PD-enhancing microbial metabolites, or an antibody against a substrate used in the in vivo synthesis of one or more PD-enhancing microbial metabolites.

[0014] In some embodiments, modulating the composition of the gut microbiota in a subject includes administering an inhibitor of an enzyme involved in the synthesis of one or more PD-enhancing microbial metabolites in vivo. One or more PD-enhancing microbial metabolites may include, for example, one or more fatty acids, their salts or esters, or any combination thereof. In some embodiments, one or more PD-enhancing microbial metabolites include one or more short-chain fatty acids (SCFAs), their salts or esters, or any combination thereof. In some embodiments, one or more PD-enhancing microbial metabolites include one or more medium-chain fatty acids, one or more long-chain fatty acids, salts or esters of medium-chain fatty acids, salts or esters of long-chain fatty acids, or any combination thereof. In some embodiments, one or more PD-enhancing microbial metabolites include SCFA acetate / ester, SCFA propionate / ester, SCFA butyrate / ester, or any combination thereof.

[0015] In some embodiments, modulating the composition of the gut microbiota in a subject includes enhancing the levels of one or more PD-protective bacterial species in the subject. In some embodiments, modulating the composition of the gut microbiota in a subject includes administering a composition comprising one or more PD-protective bacterial species to the subject. At least one of the one or more PD-protective bacterial species may belong to, for example, the families Lachnospiraceae, Rikenellaceae, Peptostreptococcaceae, Clostridium, Enterococcus, Clostridium, Bacteroides, or Butyricicoccus sp. In some embodiments, the composition is a probiotic composition, a nutraceutical composition, a pharmaceutical composition, or any combination thereof.

[0016] In some embodiments, modulating the composition of the gut microbiota in subjects includes fecal transplantation, microbiota conventionalization, microbial colonization, gut microbiota reconstruction, probiotic treatment, or combinations thereof. In some embodiments, modulating the composition of the gut microbiota in subjects includes reducing the level of one or more PD-enhancing bacterial species in the subjects. At least one of the one or more PD-enhancing bacterial species may belong to, for example, the genera *Proteus* sp., *Bilophila* sp., *Roseburiasp.*, *Pseudoramibacter Eubacterium*, or the family Veillonellaceae. In some embodiments, at least one of the one or more PD-enhancing bacterial species is an SCFA-producing bacterium. Non-limiting examples of SCFA-producing bacteria include bacteria belonging to the KEGG family K00929, K01034, and K01035.

[0017] In some implementations, adjusting the composition of the gut microbiota in subjects includes introducing gut microbiota from healthy subjects into subjects to be treated.

[0018] Some embodiments provide methods for treating neurodegenerative diseases in a subject, wherein the method includes one or more of the following: administering antibiotics to the subject; and administering an inhibitor of PD-enhancing microbial metabolites to the subject. Some embodiments provide methods for delaying or reducing the likelihood of neurodegenerative disease onset in a subject, wherein the method includes one or more of the following: administering antibiotics to the subject; and administering an inhibitor of PD-enhancing microbial metabolites to the subject. Neurodegenerative diseases can be, for example, synucleinopathies (e.g., Parkinson's disease, dementia with Lewy body disease, multiple system atrophy, or combinations thereof).

[0019] Some embodiments provide methods for improving Parkinson's disease symptoms in subjects in need, wherein the method includes one or more of the following: administering an antibiotic to the subject; administering an anti-inflammatory agent to the subject; and administering an inhibitor of one or more PD-enhancing microbial metabolites to the subject. In some embodiments, the anti-inflammatory agent and antibiotic are not minocycline. In some embodiments, Parkinson's disease symptoms include impaired motor function, increased α-synuclein (αSyn) aggregation, abnormal microglial activation, or any combination thereof. In some embodiments, Parkinson's disease symptoms include tremor, bradykinesia, rigidity, postural and balance impairment, loss of spontaneous movement, speech impairment, writing impairment, or any combination thereof. In some embodiments, the inhibitor of PD-enhancing microbial metabolites is an antibody against one or more PD-enhancing microbial metabolites, an antibody against an intermediate used in the in vivo synthesis of one or more PD-enhancing microbial metabolites, an antibody against a substrate used in the in vivo synthesis of one or more PD-enhancing microbial metabolites, an inhibitor of an enzyme involved in the in vivo synthesis of one or more PD-enhancing microbial metabolites, or a combination thereof. In some embodiments, the subject or subject in need has not received antibiotic treatment. In some embodiments, the method does not include administering any antibiotics to the subject or subject in need. In some embodiments, the subject or subject in need has not been treated with rifampin and / or minocycline. In some embodiments, the subject or subject in need has not received any antibiotic treatment for at least 12 hours, 1 day, 5 days, 10 days, or 20 days prior to adjusting the gut microbiota composition or other treatments. In some embodiments, the subject or subject in need has not received any antibiotic treatment for at least 12 hours, 1 day, 5 days, 10 days, or 20 days after adjusting the gut microbiota composition or other treatments. In some embodiments, the method further includes determining the presence and / or level of one or more PD-associated bacterial species in the subject to identify subjects in need.

[0020] Some embodiments provide a method for diagnosing Parkinson's disease in a subject, wherein the method includes determining the presence and / or level of one or more PD-associated bacterial species in the subject, whereby the presence and / or abnormal level of one or more PD-associated bacterial species indicates that the subject is at risk of developing Parkinson's symptoms or has Parkinson's symptoms. In some embodiments, at least one of the one or more PD-associated bacterial species belongs to the genus Proteus, genus Bilitrophus, genus Roselle, genus Pseudoramibacter Eubacterium, or family Veillonaceae. In some embodiments, the presence and / or level of one or more PD-associated bacterial species in the subject's intestine is determined. In some embodiments, the presence and / or abnormal level of one or more PD-associated bacterial species indicates that the subject is at risk of developing Parkinson's disease. In some embodiments, the presence and / or abnormal level of one or more PD-associated bacterial species indicates that the subject has Parkinson's disease. In some embodiments, Parkinson's disease is primary or idiopathic Parkinson's disease, secondary or acquired Parkinson's disease, hereditary Parkinson's disease, Parkinson plus syndrome, or multisystem degeneration, or any combination thereof. In some implementations, Parkinson's disease is referred to as Parkinson's syndrome. In some implementations, the subject is an adult.

[0021] Some embodiments provide compositions comprising one or more PD-protective bacterial species. In some embodiments, the composition does not contain PD-enhancing bacterial species. In some embodiments, at least one of the one or more PD-protective bacterial species belongs to the families Trichophyceae, Rikenbacteraceae, Peptostreptococciaceae, Clostridiumceae, Enterococcus, Clostridium, Bacteroides, or Clostridium butyricum. In some embodiments, the composition does not contain bacterial species belonging to at least one of Proteus, Biliophilus, Roselle, Pseudoramibacterium, and Veillonaceae. In some embodiments, the composition does not contain bacterial species belonging to Proteus, Biliophilus, Roselle, Pseudoramibacterium, and Veillonaceae. In some embodiments, the composition does not contain pathogenic Clostridium bacteria. In some embodiments, at least one of the one or more PD-protective bacterial species is viable bacteria. In some embodiments, the one or more PD-protective bacterial species are live bacteria. The composition may be, for example, a probiotic composition, a nutritional medicine composition, a pharmaceutical composition, or any combination thereof. In some embodiments, the composition is a pharmaceutical composition comprising one or more pharmaceutically acceptable carriers.

[0022] Some embodiments provide pharmaceutical compositions comprising an inhibitor of a PD-enhancing microbial metabolite and one or more pharmaceutically acceptable carriers. The PD-enhancing microbial metabolite can be any microbial metabolite, such as a fatty acid or its salts or esters. In some embodiments, the PD-enhancing microbial metabolite is a short-chain fatty acid (SCFA), a medium-chain fatty acid, a long-chain fatty acid, a salt or ester of a short-chain fatty acid, a salt or ester of a medium-chain fatty acid, or a salt or ester of a long-chain fatty acid. The inhibitor of the PD-enhancing microbial metabolite can be, for example, an antibody against the PD-enhancing microbial metabolite, an antibody against an intermediate used in the in vivo synthesis of the PD-enhancing microbial metabolite, an antibody against a substrate used in the in vivo synthesis of the PD-enhancing microbial metabolite, an inhibitor of an enzyme involved in the in vivo synthesis of the PD-enhancing microbial metabolite, or a combination thereof. Attached Figure Description

[0023] Figure 1 This is a schematic diagram illustrating the need for signaling from the gut microbiota in neuroinflammatory responses and the hallmark gastrointestinal and α-synuclein-dependent motor deficits in a Parkinson's disease (PD) model. Gut microbiota promote α-synuclein-mediated motor deficits and brain pathology, and gut bacterial depletion reduces microglial activation. Short-chain fatty acid (SCFA) treatment in animals modulates microglia and enhances PD pathophysiology, and human gut microbiota from PD patients induces enhanced motor dysfunction in mice.

[0024] Figures 2A-2F This study illustrates the role of gut microbiota in promoting motility and gastrointestinal dysfunction. Animals were tested at 12–13 weeks of age. N = 4–6. Error bars represent the mean and standard error of three trials per animal. Data represent two trials. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001. Abbreviations: SPF, specific pathogen-free; GF, germ-free; WT, wild-type; ASO, Thy1-α-synuclein genotype. Figure 2A The study showed that, compared to wild-type littermates (SPF-WT), ASO animals with complex microbiomes (SPF-ASO) required significantly more time to cross challenging beams, a measure of gross motor function. Figure 2B The study showed that, compared to wild-type littermates, ASO animals with a complex microbiota exhibited increased time spent descending along the pole, another measure of gross motor function. Figure 2C This shows that, compared to SPF-WT mice, removal of adhesive from the bridge of the nose (a test of fine motor control) was impaired in SPF-ASO mice. Figure 2D The study showed that the hindlimb grasping reflex (a measure of striatal dysfunction) was defective in SPF-ASO mice. Figures 2A-2D It was also shown that 12-13 week old ASO mice (GF-ASO) and wild-type mice (GF-WT) reacquired under sterile conditions exhibited reduced defects in beam crossing, rod descent, adhesive removal, and hind limb grasping. Figures 2E-2F The study showed that at 12-13 weeks of age, a significant reduction in total fecal pellet output was observed in SPF-ASO animals, while fecal output remained unchanged in GF-ASO animals. Figure 2E This shows the time-dependent process of fecal output in a new environment within 15 minutes. Figure 2F This shows the total number of fecal pellets produced within 15 minutes. See also: Figures 3A-3K .

[0025] Figures 3A-3K The images show the body weight of SPF and GF animals and the analysis of aged mice (with...). Figures 2A-2F (Related). N = 4–6, error bars represent mean and standard error. Data from 3 trials per animal used for movement testing. Data represent 2 trials. 0.1 > p > 0.05; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001. SPF = Specific Pathogen Free; GF = Sterile; WT = Wild Type; ASO = Thy1-α-synuclein genotype. Figure 3A The results showed that 12-13 week old GF-ASO mice did not show any difference in body weight compared to 12-13 week old SPF-ASO animals. Figure 3B The study showed that both SPF-ASO and GF-ASO animals exhibited deficiencies in the inverted grid assay, a measure of limb strength based on the time taken to fall from an inverted grid. Figures 3C-3G The study showed that at a later age (24-25 weeks), SPF-ASO animals exhibited a progressive decline in motor function, which was significantly delayed in GF-ASO animals. Figure 3C The time it takes for 24-25 week old animals to cross the beam is shown. Figure 3D The time it takes for 24-25 week old animals to descend the pole is shown. Figure 3E The time it takes for the adhesive to be removed from the bridge of the nose in animals aged 24–25 weeks is shown. Figure 3F The results show the hindlimb grasping reflex scores of animals aged 24-25 weeks. Figure 3G The weight of animals aged 24-25 weeks is shown. Figures 3H-3I The study showed that at 24-25 weeks of age, a significant decrease in total fecal pellet output was observed in SPF-ASO animals, while fecal output remained unchanged in GF-ASO animals. Figure 3H and Figure 3I ). Figure 3H This illustrates the time-dependent process of fecal output in 24-25 week old animals in a new environment within 15 minutes. Figure 3IThe total fecal pellets produced by 24-25 week old animals within 15 minutes are shown. Figure 3J The results showed that the fecal pellets produced by 12-13 week old SPF-ASO mice contained reduced water content compared to 12-13 week old GF-ASO mice, revealing a reduction in GI deficiency in GF animals. Figure 3K Principal component analysis (PCoA) is shown for compiling all motor function scores from SPF-WT, SPF-ASO, GF-WT, and GF-ASO cohorts. Compiling all motor phenotypes into the PCoA reveals a striking segregation in the SPF-ASO group, while the GF-ASO animal cluster is more similar to the WT mice.

[0026] Figures 4A-4G This study shows an increase in αSyn pathology in mice with gut microbiota. Tissues were collected from 12–13-week-old mice. N = 3–4. Error bars represent mean and standard error. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. Abbreviations: SPF, specific pathogen-free; GF, germ-free; WT, wild-type; ASO, Thy1-α-synuclein genotype. See also Figures 5A-5H . Figure 4A and Figure 4B This shows the significant aggregation of αSyn in the caudoputamen (CP) and substantia nigra (SN) of ASO animals observed under SPF conditions. Figure 4A Representative images of the caudate capsid (CP) from SPF-ASO or GF-ASO animals are shown, stained with aggregation-specific αSyn antibody, phosphate-Ser129-αSyn antibody, and Neurotrace / Nissl. Figure 4B Representative images of the substantia nigra (SN) from SPF-ASO or GF-ASO animals are shown, stained as above. Figure 4C Representative protein blots of triton soluble and insoluble brain homogenates (immunostaining with anti-αSyn antibody to quantify αSyn aggregation). Figure 4D and Figure 4E Significantly less insoluble αSyn was observed in the brains of GF-ASO animals. Figure 4D The optical density quantification of anti-αSyn protein blots is shown for all αSyn staining. Figure 4E The optical density method for quantifying anti-αSyn protein by comparing the ratio of insoluble to soluble αSyn staining is shown. Figure 4F and Figure 4G The study showed that the levels of αSyn transcripts and proteins in the inferior midbrain and CP were similar between SPF-ASO and GF-ASO animals. Figure 4F The qRT-PCR analysis of human αSyn in the CP or midbrain (Mid) is shown. Figure 4G ELISA analysis of total αSyn present in homogenates from the CP or midbrain (Mid) is shown.

[0027] Figures 5A-5H This demonstrates that gut microbiota promotes region-specific αSyn pathology (and...) Figures 4A-4G and Figures 6A-6H (related), and Figure 5I (qPCR analysis of bdnf and ddit4 in CD11b+ cells derived from brain homogenate) showed that neuroprotective levels of Bdnf and the cell cycle marker Ddit4 were upregulated in GF animals. Animals were tested at 12–13 weeks of age. N = 3–4, error bars represent mean and standard error. *p ≤ 0.05. SPF = specific pathogen-free; GF = sterile; WT = wild-type; ASO = Thy1-α-synuclein genotype. Figures 5A-5C Showing relative to Figures 4C-4E A similar reduction in αSyn aggregation was observed in GF-ASO animals. Figure 5A Aggregation-specific αSyn dot imprints of homogenates from the caudate putamen (CP) and midbrain (Mid) of SPF-ASO and GF-ASO animals are shown. Figure 5B and Figure 5C The optical density method was used to quantify the dotted imprints of the midbrain under (B) CP or (C). Figures 5D-5H The study showed region-specificity of αSyn aggregation observed: in the frontal cortex (FC), GF-ASO animals showed less αSyn aggregation compared to SPF animals; while in the cerebellum (CB), almost equal amounts of αSyn were observed in SPF and GF mice. Figure 5D Representative images of the frontal cortex (FC) from SPF-ASO or GF-ASO animals are shown, stained with aggregation-specific αSyn antibody, phosphate-Ser129-αSyn antibody, and Neurotrace / Nissl. Figure 5E Representative images of the cerebellum (CB) from SPF-ASO or GF-ASO animals are shown, stained as above. Figure 5F Dot blot images of FC or CB homogenates derived from SPF-ASO and GF-ASO animals are shown (immunostained with aggregation-specific αSyn antibody). Figure 5G and Figure 5H Densitometry quantification of dot imprints from (G)FC and (H)CB is shown.

[0028] Figures 6A-6HThis illustrates αSyn-dependent microglia activation induced by the microbiota. Tissues were collected from 12–13-week-old mice. N = 3–4 (20–60 cells analyzed per region per animal); error bars represent mean and standard error. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001. Abbreviations: SPF, specific pathogen-free; GF, germ-free; WT, wild-type; ASO, Thy1-α-synuclein genotype. See also Figures 5A-5I . Figures 6A-6C The results show that, compared to SPF-WT animals, GF-WT mice exhibit an increased number of microglia and a greater total branch length in CP and SN mice. Figure 6A Representative 3D reconstructions of Iba1-stained microglia residing in the caudate capsid (CP) of SPF-WT, SPF-ASO, GF-WT, and GF-ASO animals are shown. Figure 6B The parameters of CP-resident microglia are shown: diameter, number of branching points, and total branch length. Figure 6C The parameters of microglia residing in the substantia nigra (SN) are shown: diameter, number of branching points, and total branch length. Figure 6D and Figure 6E The results showed that, compared to GF-ASO mice, tissue homogenates from the CP and midbrain of SPF-ASO mice contained significantly increased levels of pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Figure 6D The ELISA analysis shows the presence of TNF-α and IL-6 in the homogenate from CP. Figure 6E ELISA analysis of TNF-α and IL-6 present in homogenates from the lower midbrain (Mid). Figure 6F qPCR analysis of tnfa and il6 in CD11b+ cells derived from brain homogenate revealed increased expression of tnfα and il6 in SPF-ASO animals, which was almost absent in GF animals. Figure 6G and Figure 6H The study showed that the neuroinflammatory response was region-specific, with increased microglial cell diameter and TNF-α production in FC, but not in CB. Figure 6G The diameter of microglia residing in the frontal cortex (FC) or cerebellum (CB) is shown. Figure 6H ELISA analysis of TNF-α present in homogenates from FC or CB is shown.

[0029] Figures 7A-7IPostnatal microbial signals are shown to promote locomotor and gastrointestinal dysfunction. Animals were tested at 12 - 13 weeks of age. N = 6 - 12; error bars represent mean and standard error compiled from 3 trials per animal and 20 - 60 microglia analyzed from 2 independent cohorts or per region. #0.05 < p < 0.1; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001. Abbreviations: SPF, specific pathogen free; GF, germ free; Abx, antibiotic treated; Ex - GF, recolonized germ free animals; WT, wild type; ASO, Thy1 - alpha - synuclein genotype. See also Figures 8A-8O . Figure 7A Schematic of the time course of animal handling and testing. Figures 7B-7E Animals treated with antibiotics (Abx) are shown to exhibit minimal αSyn - dependent locomotor dysfunction, very similar to mice born under GF conditions. Figures 7B-7E Postnatal colonization (Ex - GF) of previously GF animals is also shown to recapitulate the genotype effect observed in SPF mice, with mice overexpressing αSyn showing significant locomotor dysfunction. Figure 7B Time across the beam apparatus is shown. Figure 7C Time down the pole is shown. Figure 7D Time to remove nasal adhesive is shown. Figure 7E Hindlimb grasp reflex score is shown. Figure 7F and Figure 7G GI function (measured by fecal output) is also shown to be significantly improved in Abx - treated animals, while Ex - GF mice show an αSyn - dependent reduction in total fecal output. Figure 7F Time course of fecal output in a novel environment over 15 minutes is shown. Figure 7G Total fecal pellets produced over 15 minutes are shown. Figure 7H and Figure 7I In the transgenic ASO line, the soma diameter of microglia from Ex - GF animals is shown to increase, comparable to that in SPF mice. Figure 7H and Figure 7I However, Abx - ASO animals are also shown to carry microglia with diameters similar to those of GF animals. Figure 7H Representative 3D reconstructions of Iba1 - stained microglia resident in the caudate - putamen (CP) of Abx - ASO or Ex - GF - ASO animals are shown. Figure 7I Diameter of microglia resident in the CP or substantia nigra (SN) is shown.

[0030] Figures 8A-8OThis illustrates SCFA alterations and increased αSyn pathology in Abx, Ex-GF, and SCFA mice (as shown in the original text). Figures 7A-7I and Figures 9A-9H (Related). Animals were tested at 12-13 weeks of age. N=3-6, 20-60 microglia were analyzed per region. Error bars represent the mean and standard error. *p≤0.05; **p≤0.01; ***p≤0.001; ****p≤0.0001. Abbreviations: SPF = Specific Pathogen Free; GF = Germ-Free; Abx = Antibiotic-treated animals; Ex-GF = Recolonized Germ-Free animals; SCFA = Short-Chain Fatty Acid-treated animals; WT = Wild Type; ASO = Thy1-α-synuclein genotype. Figure 8A Lower fecal SCFA concentrations were observed in animals treated with GF and Abx compared to SPF mice. Figure 8A In this study, the fecal concentrations of acetate, propionate, and butyrate were normalized using soluble chemical oxygen demand (sCOD). Figures 8B-8C The study showed that within the affected brain regions (i.e., CP and SN), microglia in animals treated with SCFA exhibited a morphology indicating increased activation, similar to cells from Ex-GF and SPF mice, compared to untreated mice. However, animals treated with Abx showed microglia morphology similar to those in GF animals. Figure 8B The parameters of microglia residing in the caudate capsid (CP) are shown: number of branch points and total branch length. Figure 8C The parameters of microglia residing in the substantia nigra (SN) are shown: number of branching points and total branch length. Figure 8D and Figure 8E The study showed that changes in microglial cell diameter were also observed in FC, but not in CB, indicating that this is a region-specific response. Figure 8D The diameter of microglia residing in the frontal cortex (FC) is shown. Figure 8E The diameter of microglia residing in the cerebellum (CB) is shown. Figures 8F-8O The results show that αSyn accumulates in mice given SCFA, similar to Ex-GF animals, compared to untreated and Abx-treated mice. Figures 8F-8I Representative images of CP, SN, FC, and CB from Abx-ASO, Ex-GF-ASO, or SCFA-ASO animals, stained with aggregation-specific αSyn antibody, phosphate-Ser129-αSyn antibody, and Neurotrace / Nissl, are shown. Figure 8J Dotted images of homogenates of CP, midbrain (Mid), FC, and CB from Abx-ASO, Ex-GF-ASO, and SCFA-ASO animals are shown (immunostained with aggregation-specific αSyn antibody). Figures 8K-8NDensitometry quantification of dotted imprints from (K)CP, (L) subbrain, (M)FC, and (N)CB is shown. Figure 8O The protein blots of αSyn derived from Triton soluble and insoluble fractions of CP homogenates from Abx-ASO and SCFA-ASO animals are shown.

[0031] Figures 9A-9H This study demonstrates that SCFA promotes αSyn-stimulated microglial activation and motor dysfunction. Animals were tested at 12–13 weeks of age. N = 6–12; error bars represent the mean and standard error of data from three trials per animal, compiled from two independent cohorts or 20–60 microglial cells per region. For clarity, data are expressed using data from… Figures 7A-7I Comparative plotting data. *p≤0.05; **p≤0.01; ***p≤0.001; ****p≤0.0001. Abbreviations: SPF, specific pathogen-free; GF, sterile; SCFA, short-chain fatty acid treatment; WT, wild type; ASO, Thy1-α-synuclein genotype. See also Figures 8A-8O , Figures 10A-10M and Figures 11A-11H . Figure 9A and Figure 9B The study showed that within the affected brain regions (i.e., CP and SN), microglia in animals treated with SCFA exhibited a morphology indicating increased activation, similar to cells from Ex-GF and SPF mice, compared to untreated mice. However, animals treated with Abx showed microglia morphology similar to those in GF animals. Figure 9A A representative 3D reconstruction of Iba1-stained microglia residing in the caudate shell nucleus (CP) of wild-type or ASO animals treated with SCFA is shown. Figure 9B The diameter of microglia residing in the CP or substantia nigra (SN) is shown. Figures 9C-9F The results show that, compared to untreated GF-ASO animals, SCFA-ASO mice exhibited significantly impaired performance in several motor tasks, including impaired beam crossing, descent along a pole, and hind limb reflexes (comparison between GF-ASO mice and SCFA-ASO mice). Figure 9C The time for crossing the beam apparatus is shown. Figure 9D The time taken to descend along the rod is shown. Figure 9E The time required to remove the nasal adhesive is shown. Figure 9F The scores for the hindlimb grasping reflex are shown. Figure 9G and Figure 9H The study also showed that GI deficiency was observed in transgenic animals treated with SCFA. Figure 9G This shows the time-dependent process of fecal output in a new environment within 15 minutes. Figure 9H The total number of fecal pellets produced within 15 minutes is shown.

[0032] Figures 10A-10M This shows that SCFA does not directly change αSyn aggregation (and) Figures 9A-9H (Related). Animals were tested at 12–13 weeks of age. N = 6–12; error bars represent the mean and standard error of three trials per animal. Data were compiled from two independent cohorts and, for clarity, from [source missing]. Figures 7A-7I Comparative plots. *p≤0.05; **p≤0.01; ***p≤0.001; ****p≤0.0001. Abbreviations: SPF = Specific Pathogen Free; GF = Sterile; HK = Heat-Inactivated Bacteria Treatment; WT = Wild Type; ASO = Thy1-α-Synuclein Genotype. Figures 10A-10G The results showed that neither SCFA alone nor in mixtures of SCFAs promoted the aggregation of human αSyn in vitro within a certain concentration range. Figures 10A-10C The concentration shown is as indicated by the presence of ( Figure 10A Sodium acetate, ( Figure 10B Sodium propionate or ( Figure 10C αSyn aggregation kinetics measured by ThT fluorescence in the presence of sodium butyrate. Figure 10D and Figure 10E The αSyn aggregation kinetics are shown as measured by ThT fluorescence in the presence of a separate SCFA mixture. Figure 10D SCFA Mix 1 - 29.6 mM acetate, 11 mM propionate, and 18.5 mM butyrate; SCFA Mix 2 - 88.8 mM propionate, 33 mM propionate, and 55.5 mM butyrate; Figure 10E SCFA Mix 3 - 0.4 mM acetate, 0.15 mM propionate and 0.24 mM butyrate; SCFA Mix 4 - 0.8 mM acetate, 0.3 mM propionate and 0.47 mM butyrate; SCFA Mix 5 - 2.0 mM acetate, 0.74 mM propionate and 1.18 mM butyrate. Figure 10F and Figure 10G The time to reach half-maximum fluorescence intensity is shown for individual SCFA treatments or SCFA mixtures, respectively. N=3, and bars represent the mean and standard error. Figure 10H and Figure 10I This demonstrates that neither SCFA alone nor in mixture form does alter the overall structure of αSyn amyloid fibrils within a certain concentration range. Figure 10H and Figure 10I Representative atomic force microscopy results from the final products of the above αSyn aggregation determination are shown, with and without SCFA and with SCFA Mix 1. Figures 10J-10M The results showed that oral treatment of GF animals with heat-inactivated bacteria did not induce motor defects, indicating that the bacteria need to be metabolically active. Figure 10J Shows the time across the beam. Figure 10K Shows the time of descent along the pole. Figure 10L Shows the time of removal of nasal adhesive. Figure 10M Shows the hindlimb reflex score.

[0033] Figures 11A-11H Shows that minocycline reduces SCFA-induced αSyn motor deficits and pathology (associated with Figures 9A-9H ). Animals were tested at 12 - 13 weeks of age. N = 6 - 12; error bars represent mean and standard error from 3 trials per animal. Data were compiled from 2 independent cohorts. 0.05 < p < 0.1; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001. Abbreviations: SPF = specific pathogen free; GF = germ free; WT = wild type; ASO = Thy1-α-synuclein genotype. Figures 11A-11H Shows that oral treatment of SCFA-fed animals with the anti-inflammatory compound minocycline is sufficient to reduce TNF-α production, reduce αSyn aggregation and improve motor function without altering transgene expression. Figure 11A Shows ELISA analysis of TNFα present in the caudate putamen (CP) and midbrain (Mid). Figure 11B Shows dot blot images of CP and midbrain (Mid) homogenates with respect to aggregated-specific αSyn. Figure 11C Shows densitometric quantification of dot blots from CP and midbrain (Mid). Figure 11D Shows qPCR analysis of human snca expression in whole brain homogenates. Figure 11E Shows the time across the beam apparatus. Figure 11F Shows the time of descent along the pole. Figure 11G Shows the time of removal of nasal adhesive. Figure 11H Shows the hindlimb grasp reflex score.

[0034] Figures 12A-12E Shows microbiome dysregulation in PD patient samples after transplantation into germ-free mice. N = 3 - 6, at more than 3 time points after colonization. Error bars represent mean and standard error. ***p ≤ 0.001, 999 permutations. Abbreviations: HC, germ-free mice colonized with fecal microbiota from healthy controls; PD, germ-free mice colonized with fecal microbiota from Parkinson's disease patients; WT, wild type; ASO, Thy1-α-synuclein genotype. See also Figures 13A-13E . Figure 12A And Figure 12B Shows that in PCoA-based unweighted UniFrac, the recipient animal groups most resemble the profiles of their respective human donors. Figure 12AUnweighted UniFrac principal coordinate analysis of the microbial communities of human donors (large circles) and recipient mice (small circles) is shown. Each donor and recipient sample is matched by shadow. Figure 12B Unweighted and weighted UniFrac analyses of the microbial community in recipient animals based on donor identity are shown. Figure 12C and Figure 12D The results show that humanized mouse groups from PD donors are significantly more similar to each other compared to communities transplanted from healthy donors, a trend that persists when stratified by genetic background. Significant differences exist between healthy and PD donors in the ASO background compared to WT recipients, indicating the influence of genotype on microbial community structure. Figure 12C Unweighted and weighted UniFrac analyses of the microbial community in recipient animals based on mouse genotypes are shown. Figure 12D This shows a comparison of unweighted and weighted UniFrac analyses of the microbial community in the recipient animals. Figure 12E This section shows several genera that were altered in animals colonized with a microbiome derived from a PD donor compared to healthy controls. Taxonomical analysis of the changes in each genera between PD and healthy donors is presented, along with the genotype of the recipient mouse. The left column indicates the percentage of statistically significant differences observed; the right column indicates the fold change between PD and healthy donors. "*" indicates a non-statistically significant difference.

[0035] Figures 13A-13E This demonstrates alterations in microbial metabolic pathways in humanized animals (and) Figures 12A-12E (Related). N = 3-6, for KEGG analysis, more than 3 time points after colonization; for SCFA abundance, N = 21-24. Error bars represent the mean and standard error. *p: <0.05; **p: <0.01; ***p: <0.001. Figure 13A The Bray-Curtis distance comparison between humanized groups is shown. Figure 13B and Figure 13C Comparisons of Bray-Curtis mean distances are shown between the same donor and different donors, or between wild-type (WT) and Thy1-α-synuclein (ASO) genotypes. Figure 13D The PICRUSt analysis of specific KEGG families involved in SCFA production is shown, with light circles indicating microorganisms derived from healthy controls and dark circles indicating microorganisms derived from PD. Figure 13E Fecal concentrations and relative abundances of acetate, propionate, and butyrate from humanized animals are shown, normalized to soluble chemical oxygen demand. Compiled from 6 independent donor pairs; HC = healthy controls; PD = Parkinson's disease.

[0036] Figures 14A-14GShows microbiota from PD patients inducing enhanced αSyn-mediated motor deficits. Animals were tested at 12 - 13 weeks of age. N = 3 - 6, error bars represent mean and standard error from 3 trials per animal. #0.05 < p < 0.1; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001. Abbreviations: HC, germ-free mice colonized with fecal microbiota from healthy controls; PD, germ-free mice colonized with fecal microbiota from Parkinson's disease patients; WT, wild type; ASO, Thy1-α-synuclein genotype. See also Figures 15A-15G and Table 2. Figures 14A-14F Shows consistency among 4 out of 6 pairs (pairs #1, 3, 4, and 5), microbiota from individuals with PD promotes enhanced αSyn-mediated motor dysfunction. Figures 14A-14F Shows time to cross a beam, time to descend a pole, time to remove nose adhesive, and hindlimb grip reflex scores of mice humanized with microbiota from PD patients or matched healthy controls. Figure 14G Compilation of performance data from all groups shows that in 3 out of 4 tests used in this study, microbiota from PD patients induced increased motor impairment in ASO animals compared to microbiota from healthy controls. Compilation of all independent cohorts in each motor task: beam crossing, pole descent, adhesive removal, and hindlimb grip reflex scores, grouped by health status of fecal donor.

[0037] Figures 15A-15G Shows body weight and fecal output of humanized animals (correlated with Figures 14A-14G ). Animals were tested at 12 - 13 weeks of age. N = 3 - 6, error bars represent mean and standard error from 3 trials per animal. 0.05 < p < 0.1; *p ≤ 0.05; **p ≤ 0.01. Abbreviations: HC = germ-free mice colonized with fecal microbiota from healthy controls; PD = germ-free mice colonized with fecal microbiota from Parkinson's disease patients; WT = wild type; ASO = Thy1-α-synuclein genotype. Figures 15A-15F Shows that recipient animals showed little change in body weight and GI function as measured by fecal output. Body weight and fecal output 15 minutes after placement in a new environment for each PD and HC humanized pair. ( Figure 15A ) Pair #1; ( Figure 15B ) Pair #2; ( Figure 15C ) Pair #3; ( Figure 15D ) Pair #4; ( Figure 15E ) Pair #5; ( Figure 15F ) Pair #6. Figure 15G Shows principal component analysis of compiled motor function among humanized animals. Figure 15GAll motor functions depicted by PCoA revealed striking global differences between animals colonized with microbiota from PD donors compared to animals colonized with gut bacteria derived from healthy individuals. Detailed Implementation

[0038] Reference is made to the accompanying drawings, which form part of this document, in the following detailed description. In the drawings, similar symbols generally identify similar components unless the context otherwise requires. The illustrative embodiments described in the detailed description, drawings, and claims are not intended to be limiting. Other embodiments and changes may be utilized without departing from the spirit or scope of the subject matter set forth herein. It will be readily understood that the aspects of this disclosure as generally described herein and illustrated in the drawings can be arranged, substituted, combined, separated, and designed in a variety of different configurations, all of which are expressly contemplated herein.

[0039] definition

[0040] Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. See, for example, Singleton et al., Dictionary of Microbiology and Molecular Biology 2 nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). For the purposes of this disclosure, the following terms are defined as follows.

[0041] As used herein, the term "subject" refers to an animal, such as a vertebrate, preferably a mammal. The term "mammal" is defined as an individual belonging to the class Mammalia, including but not limited to humans, domesticated and farm animals, as well as zoo animals, sports animals, or pet animals, such as sheep, dogs, horses, cats, or cows. In some embodiments, the subject is a mouse or rat. In some embodiments, the subject is a human.

[0042] As used herein, the term "treatment" refers to an intervention (e.g., a clinical intervention) in response to a disease, symptom, or physical condition exhibited by a patient, particularly one with a neurodegenerative disease (e.g., Parkinson's disease). Treatment objectives may include, but are not limited to, one or more of the following: symptom relief or prevention; slowing or stopping the progression or worsening of the disease, symptom, or condition; and remission of the disease, symptom, or condition. In some embodiments, "treatment" refers to both therapeutic treatment and preventative or protective measures. Subjects requiring treatment include those already affected by a disease or symptom or undesirable physical condition, and those for whom prevention of the disease or symptom or undesirable physical condition is desired. For example, in some embodiments, treatment may reduce, alleviate, or eradicate symptoms of a disease. As used herein, the term "prevention" refers to any activity that reduces the burden on an individual who subsequently exhibits symptoms of Parkinson's disease. This can be carried out at primary, secondary, and / or tertiary prevention levels, wherein: a) primary prevention avoids the development of symptoms / conditions / conditions; b) secondary prevention activities target the early stages of treatment for conditions / conditions / symptoms, thereby increasing the opportunity for intervention to prevent the progression of conditions / conditions / symptoms and the onset of symptoms; and c) tertiary prevention reduces the negative impact of established conditions / conditions / symptoms by, for example, restoring function and / or reducing any conditions / conditions / symptoms or related complications.

[0043] A pharmaceutically acceptable carrier is one that is non-toxic to cells or mammals exposed to it at the doses and concentrations used. A pharmaceutically acceptable carrier can be, but is not limited to, organic or inorganic excipients, solid or liquid excipients, suitable for the chosen mode of administration (e.g., oral or injectable) and given in the form of conventional pharmaceutical formulations (e.g., solids such as tablets, granules, powders, capsules; and liquids such as solutions, emulsions, suspensions, etc.). Physiologically acceptable carriers are typically aqueous pH buffers, such as phosphate buffers or citrate buffers. Physiologically acceptable carriers may also contain one or more of the following: antioxidants, including ascorbic acid; low molecular weight (less than about 10 residues) peptides and proteins, such as serum albumin, gelatin, and immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids; carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium; and nonionic surfactants, such as Tween. TM Polyethylene glycol (PEG) and Pluronics TM Auxiliary agents, stabilizers, emulsifiers, lubricants, binders, pH adjusters, isotonic agents, and other conventional additives can also be added to the carrier.

[0044] Pharmaceutically acceptable or suitable carriers may include other compounds known to be beneficial to gastrointestinal impairment (e.g., antioxidants such as vitamin C, vitamin E, selenium, or zinc); or food compositions. Food compositions may be, but are not limited to, milk, yogurt, curd, cheese, fermented milk, milk-based fermented products, ice cream, fermented grain-based products, milk-based powders, infant formula, tablets, liquid bacterial suspensions, dry oral supplements, or wet oral supplements.

[0045] Therapeutic or preventative agents may include “medicines.” As used herein, “medicine” means a therapeutic or diagnostic agent, including any substance other than food used to prevent, diagnose, alleviate, treat, or cure a disease. Stedman’s Medical Dictionary, 25th edition (1990). A medicine may include any substance disclosed in at least one of the following publications: The Merck Index, 12th edition (1996); Pei-Show Juo, Concise Dictionary of Biomedicine and Molecular Biology (1996); US Pharmacopeia Dictionary, 2000 edition; and Physician’s Desk Reference, 2001 edition. In some embodiments, a therapeutic agent is one of the embodiments of the compositions described herein. In some embodiments, the medicine used in the therapeutic system is placed, embedded, encapsulated, or otherwise incorporated into a delivery matrix.

[0046] As used in this article, "nutraceutical" refers to food substances (as fortified foods or dietary supplements) that provide health benefits. Nutraceutical foods are not subject to the same testing and regulations as drugs.

[0047] As used herein, the term "probiotics" refers to live microorganisms that, when administered in adequate amounts, confer health benefits to a host. Probiotics can be obtained in foods and dietary supplements (e.g., but not limited to capsules, tablets, and powders). Non-limiting examples of foods containing probiotics include dairy products such as yogurt, fermented and unfermented milk, smoothies, butter, cream, hummus, kombucha, salad dressings, miso, tempeh, nutrition bars, and some fruit juices and soy beverages.

[0048] As used herein, the term "metabolite" refers to any molecule involved in metabolism. A metabolite can be a product, substrate, or intermediate in a metabolic process. For example, a metabolite can be a primary metabolite, a secondary metabolite, an organic metabolite, or an inorganic metabolite. Metabolites include, but are not limited to, fatty acids, amino acids, peptides, acylcarnitines, monosaccharides, oligosaccharides, lipids and phospholipids, prostaglandins, hydroxyeicosatetraenoic acid, hydroxyoctadecadienoic acid, steroids, bile acids, glycolipids, and phospholipids. In some embodiments, the metabolite is a microbial metabolite, which is produced by microorganisms to, for example, regulate their own growth and development, promote beneficial interactions with other organisms, and inhibit harmful organisms. Microbial metabolites can be, for example, small molecule quantitative compounds (<2,500 Da). In some embodiments, the metabolite is an analogue of a microbial metabolite. In some embodiments, microbial metabolites and their analogues include short-chain fatty acids (SCFAs), medium-chain fatty acids, and long-chain fatty acids, as well as salts and esters of short-chain fatty acids, medium-chain fatty acids, and long-chain fatty acids. Non-limiting examples of fatty acids include SCFA acetates, propionates, and butyrates.

[0049] As used herein, the term "antibody" includes polyclonal antibodies, monoclonal antibodies (including full-length antibodies containing the Fc region of immunoglobulins), antibody compositions with multi-epitope specificity, multispecific antibodies (e.g., bispecific antibodies), diabodies, and single-chain molecules and antibody fragments (e.g., Fab or F(ab')2 and Fv). For information on the structure and properties of different classes of antibodies, see, for example, Basic and Clinical Immunology, 8th ed., Daniel P. Sties, Abba I. Terr, and Tristram G. Parsolw, Appleton & Lange, Norwalk, Conn., 1994, p. 71 and Chapter 6.

[0050] Neurodegenerative diseases

[0051] Neurological dysfunction is the basis of many human diseases. Behavioral disorders, psychotic disorders, and neurodegenerative disorders typically display signature neuropathologies within the central nervous system (CNS). One neuropathology, amyloidosis, is caused by the abnormal aggregation of specific neuronal proteins that disrupt the function of many cells. Affected tissues often contain aggregates of insoluble proteins exhibiting altered conformations, a feature believed to contribute to an estimated 50 different human diseases. Neurodegenerative amyloid disorders, including Alzheimer's disease, Huntington's disease, and Parkinson's disease (PD), are associated with amyloid proteins. PD is the second most common neurodegenerative disease in the United States, affecting an estimated 1 million people and 1% of the U.S. population over 60 years of age. Worldwide, approximately 3 million patients and caregivers are affected by PD's often debilitating symptoms, involving motor deficits including tremor, muscle rigidity, bradykinesia, and impaired gait. It is a multifactorial disorder with a strong environmental component, as less than 10% of cases are hereditary. The accumulation of α-synuclein (αSyn) is considered pathogenic in a family of diseases known as synucleinopathies, including Parkinson's disease (PD), multiple system atrophy, and Lewy body disease. αSyn accumulation is a gradual process leading to oligomerization and the accumulation of non-transient fibrils within neurons. Dopaminergic neurons in the substantia nigra pars compacta (SNpc) appear to be particularly susceptible to αSyn aggregates. Dopamine modulators are first-line treatments for PD; however, treatment can have serious side effects and often loses its effectiveness. Safe and effective treatments are needed to address the growing burden of PD in an aging population.

[0052] Peripheral influences have been implicated in the onset and / or progression of diseases affecting the brain (Dinan and Cryan, 2015). Bidirectional communication between the gut and brain has been proposed in anxiety, depression, nociceptive disorders, and autism spectrum disorders (ASD) (Mayer et al., 2014; Schroeder and Backhed, 2016; Sharon et al., 2016). Gastrointestinal (GI) physiology and motility are influenced by signals generated locally in the gut and from the CNS. Neurotransmitters, immune signals, hormones, and neuropeptides produced in the gut may, in turn, affect the brain (Selkrig et al., 2014; Wall et al., 2014).

[0053] Humans are permanently colonized by microorganisms on almost all surfaces exposed to the environment, with the majority residing in the gastrointestinal tract. The microbiome can have profound effects on neurodevelopment and the central nervous system (CNS). Germ-free (GF) mice and antibiotic-treated specific pathogen-free (SPF) mice exhibit altered hippocampal neurogenesis, leading to impaired spatial and object recognition. The microbiome regulates the expression of serotonin receptors (5-HT1A), brain-derived neurotrophic factor (BDNF), and NMDA receptor subunit 2 (NR2A). GF mice exhibit altered cortical myelination and impaired blood-brain barrier function. Furthermore, the microbiome promotes gut and circulating serotonin production in mice and influences anxiety, hyperactivity, and cognition. Fecal and mucosal gut microbiota differ between individuals with Parkinson's disease (PD) and healthy controls.

[0054] Gut bacteria control the differentiation and function of immune cells in the gut, periphery, and brain. Subjects with Parkinson's disease (PD) exhibit enteritis and GI abnormalities, such as constipation that often precedes motor deficits. Braak hypothesized that abnormal αSyn accumulation begins in the gut and spreads to the brain via the vagus nerve in a prion-like manner. This view is supported by pathophysiological evidence: αSyn inclusion bodies appear earlier in the enteric nervous system (ENS) as well as in the glossopharyngeal and vagus nerves, and individuals with vagus nerve transection have a reduced risk of PD. Furthermore, injecting αSyn fibrils into the intestinal tissue of healthy rodents is sufficient to induce pathology in the vagus nerve and brainstem.

[0055] Treatment of neurodegenerative diseases

[0056] As disclosed in this article, modulating the composition of the gut microbiota in subjects in need (e.g., by reducing / depleting PD-enhancing microbes in the subject's gut microbiota, introducing healthy microbes, or both) can provide benefits in delaying PD progression in subjects.

[0057] This document discloses methods for treating neurodegenerative diseases in subjects in need (e.g., patients with neurodegenerative diseases), methods for delaying or reducing the likelihood of neurodegenerative disease onset, and methods for improving motor deficits. In some embodiments, the methods include modulating the composition of the gut microbiota in the subject. In some embodiments, the methods also improve one or more physical impairments in the subject. The methods may, for example, improve one or more GI functions in the subject, or relieve constipation in the subject. Non-limiting examples of motor deficits include tremor, muscle rigidity, bradykinesia, impaired gait, and any combination thereof. In some embodiments, the neurodegenerative disease is a synucleinopathy, including but not limited to primary or idiopathic Parkinson's disease, secondary or acquired Parkinson's disease, hereditary Parkinson's disease, Parkinson's plus syndromes, or multisystem degeneration, and any combination thereof.

[0058] In some embodiments, the method includes identifying a subject in need, wherein the subject in need is a subject with abnormal levels of α-synuclein (αSyn) aggregation (e.g., abnormally high levels of αSyn aggregation). In some embodiments, the method includes measuring the rate and / or level of αSyn aggregation in the subject (e.g., in the subject's brain), measuring the rate and / or level of clearance of insoluble αSyn protein aggregates in the subject (e.g., in the subject's brain), or a combination thereof. Measuring the rate and / or level of αSyn aggregation in the subject (e.g., in the subject's brain), measuring the rate and / or level of clearance of insoluble αSyn protein aggregates in the subject (e.g., in the subject's brain), or a combination thereof, may be performed at different time points, such as before, during, and / or after adjusting the composition of the subject's gut microbiota.

[0059] This document also provides methods for reducing microglial activation in subjects in need, methods for reducing α-synuclein (αSyn) aggregates in subjects in need, and methods for reducing neuroinflammation in subjects in need. In some embodiments, the subjects in need suffer from neurodegenerative diseases, such as synucleinopathies (e.g., Parkinson's disease, dementia with Lewy body disease, multiple system atrophy, or any combination thereof). In some embodiments, the subjects suffer from PD. In some embodiments, the methods include modulating the composition of the gut microbiota in the subjects. The methods may, for example, promote the clearance of insoluble αSyn protein aggregates (e.g., clearance rate, clearance amount, or both), reduce the aggregation of αSyn proteins (e.g., aggregation rate, aggregation amount, or both), or both. In some embodiments, the methods include measuring the rate and / or level of αSyn aggregation in the subject (e.g., in the subject's brain), measuring the rate and / or level of clearance of insoluble αSyn protein aggregates in the subject (e.g., in the subject's brain), or a combination thereof. Measuring the rate and / or level of αSyn aggregation in subjects (e.g., in the subject's brain), measuring the rate and / or level of clearance of insoluble αSyn protein aggregates in subjects (e.g., in the subject's brain), or combinations thereof, can be performed at different time points, such as before, after, and / or after adjusting the composition of the subject's gut microbiota.

[0060] Subjects in need may be those with or at risk of developing a neurodegenerative disease, such as neurodegenerative amyloidosis, including but not limited to Alzheimer's disease, Huntington's disease, Parkinson's disease, or any combination thereof. In some embodiments, subjects in need are those with or at risk of developing a synucleinopathy, such as Parkinson's disease, dementia with Lewy body disease, multiple system atrophy, or any combination thereof. In some embodiments, subjects in need are those with or at risk of developing Parkinson's disease. Non-limiting examples of neurodegenerative diseases include primary or idiopathic Parkinson's disease, secondary or acquired Parkinson's disease, hereditary Parkinson's disease, Parkinson's plus syndromes, or multiple system degeneration, and any combination thereof.

[0061] As described herein, in some embodiments of the methods disclosed herein, after adjusting for gut microbiota composition in subjects, the gut microbiota composition in the subjects is restored to normal levels. As used herein, “normal level” of gut microbiota composition refers to the gut microbiota composition level in non-PD subjects (e.g., healthy subjects). Those skilled in the art will understand that variability in gut microbiota composition can exist among non-PD (e.g., healthy) individuals, and that normal levels can be established as representative of gut microbiota composition in non-PD populations or healthy subject populations for comparison. Various criteria can be used to determine the inclusion and / or exclusion of specific subjects in a reference population, including but not limited to the subject’s age (e.g., reference subjects may be in the same age group as subjects requiring treatment) and the subject’s sex (e.g., reference subjects may be of the same sex as subjects requiring treatment).

[0062] As described in this article, modulating the composition of the gut microbiota in subjects can be achieved through a variety of methods, including but not limited to fecal transplantation, microbiota routine, microbial colonization, gut microbiota reconstruction, probiotic treatment, antibiotic treatment, or combinations thereof.

[0063] In some embodiments, modulating the composition of the gut microbiota in a subject includes administering one or more antibiotics to the subject. At least one of the one or more antibiotics may be, for example, ampicillin, vancomycin, neomycin, gentamicin, erythromycin, or any combination thereof. In some embodiments, antibiotic treatment does not include administering rifampin and / or minocycline to the subject. Modulating the composition of the gut microbiota in a subject can also be achieved, for example, by administering an inhibitor of a PD-enhancing microbial metabolite to the subject. Examples of inhibitors of PD-enhancing microbial metabolites include, but are not limited to, antibodies against one or more PD-enhancing microbial metabolites, antibodies against intermediates used in the in vivo synthesis of one or more PD-enhancing microbial metabolites, antibodies against substrates used in the in vivo synthesis of one or more PD-enhancing microbial metabolites, and inhibitors of one or more enzymes involved in the in vivo synthesis of one or more PD-enhancing microbial metabolites.

[0064] As used herein, the term "PD-enhancing microbial metabolite" refers to a microbial metabolite at elevated levels in subjects with PD or any pathological condition with one or more symptoms of Parkinson's disease, compared to subjects without PD or any pathological condition without one or more symptoms of Parkinson's disease (e.g., healthy subjects). For example, the level of circulating microbial metabolites in subjects with PD can be altered compared to non-PD subjects. The level of microbial metabolites can be altered in, for example, the blood, feces, serum, plasma, body fluids (e.g., cerebrospinal fluid, pleural fluid, amniotic fluid, semen, or saliva) and / or urine of PD subjects. Non-limiting examples of PD-enhancing microbial metabolites include one or more fatty acids, their salts or esters, or any combination thereof. Fatty acids can be, for example, short-chain fatty acids, medium-chain fatty acids, or long-chain fatty acids. In some embodiments, PD-enhancing microbial metabolites comprise one or more short-chain fatty acids (SCFAs), their salts or esters, or any combination thereof. In some embodiments, PD-enhancing microbial metabolites comprise SCFA acetate, SCFA propionate, SCFA butyrate, or any combination thereof.

[0065] Modifying the composition of the gut microbiota in a subject may include, for example, increasing the level of one or more PD-protective bacterial species in the subject. In some embodiments, modifying the composition of the gut microbiota in a subject includes administering the subject a composition comprising one or more PD-protective bacterial species. As used herein, the term "PD-protective bacterial species" refers to a type of bacteria present in the gut microbiota of a subject that can protect the subject from developing PD or a pathological condition with one or more Parkinson's disease symptoms, slow disease progression in subjects with PD or a pathological condition with one or more Parkinson's disease symptoms, alleviate the condition of PD or a pathological condition with one or more Parkinson's disease symptoms, alleviate at least one symptom of PD, or a combination thereof. In some embodiments, PD-protective bacterial species are present only in non-PD subjects (e.g., in the subject's gut) but not in subjects with PD or a pathological condition with one or more Parkinson's disease symptoms. In some embodiments, PD-protective bacterial species are present at significantly lower levels (e.g., no more than 50%, no more than 25%, no more than 10%, or no more than 5% or less) in subjects with PD or a pathological condition having one or more Parkinson's symptoms (e.g., in the subject's gut) compared to non-PD subjects (e.g., healthy subjects). Non-limiting examples of PD-protective bacterial species include species belonging to the families Trichophytonceae, Rikenbacteriaceae, Peptostreptococciaceae, Clostridiumceae, Enterococcus, Clostridium, Bacteroides, or Clostridium butyricum. In some embodiments, the composition is a probiotic composition, a nutritional medicine composition, a pharmaceutical composition, or any combination thereof.

[0066] In some embodiments, adjusting the composition of the gut microbiota in subjects includes reducing the level of one or more PD-enhancing bacterial species in the subject. As used herein, the term "PD-enhancing bacterial species" refers to a bacterial species present (alone or in combination with one or more other bacterial species) in the gut microbiota of a subject that can increase the likelihood of a subject developing PD or a pathological condition with one or more Parkinson's disease symptoms, accelerate the onset of PD or a pathological condition with one or more Parkinson's disease symptoms, increase the severity of PD or a pathological condition with one or more Parkinson's disease symptoms, worsen at least one symptom of PD, or a combination thereof. In some embodiments, PD-enhancing bacterial species are present only in subjects with PD or a pathological condition with one or more Parkinson's disease symptoms (e.g., in the subject's gut), but not in non-PD subjects (e.g., healthy subjects). In some embodiments, PD-enhancing bacterial species are present at significantly lower levels (e.g., no more than 50%, no more than 25%, no more than 10%, or no more than 5% or less) in non-PD subjects (e.g., in the subject's gut) compared to subjects with PD or a pathological condition with one or more Parkinson's disease symptoms. In some embodiments, the PD-enhancing bacterial species are those belonging to the genera *Proteus*, *Biliobacterium*, *Roseidonella*, *Pseudoramibacterium*, or *Veillonaceae*. In some embodiments, the PD-enhancing bacterial species are SCFA-producing bacteria, such as SCFA-producing bacteria belonging to the KEGG family K00929, K01034, or K01035.

[0067] In some implementations, adjusting the composition of the gut microbiota in subjects includes introducing gut microbiota from healthy subjects into subjects to be treated.

[0068] Some embodiments provide methods for treating neurodegenerative diseases in subjects, the method comprising one or more of the following: administering an antibiotic to the subject; and administering an inhibitor of a PD-enhancing microbial metabolite to the subject. Some embodiments provide methods for delaying or reducing the likelihood of neurodegenerative disease onset in subjects, the method comprising one or more of the following: administering an antibiotic to the subject; and administering an inhibitor of a PD-enhancing microbial metabolite to the subject. Some embodiments provide methods for improving Parkinson's disease symptoms in subjects in need, the method comprising one or more of the following: administering an antibiotic to the subject; administering an anti-inflammatory agent to the subject; and administering one or more inhibitors of a PD-enhancing microbial metabolite to the subject. The antibiotic may not be rifampin or minocycline. Neurodegenerative diseases may be, for example, neurodegenerative amyloidosis, including but not limited to Alzheimer's disease, Huntington's disease, Parkinson's disease, or any combination thereof. In some embodiments, neurodegenerative diseases are synucleinopathies, such as Parkinson's disease, dementia with Lewy body disease, multiple system atrophy, or any combination thereof.

[0069] As described herein, in some embodiments, the anti-inflammatory agent may be a synthetic nonsteroidal anti-inflammatory drug (NSAID), such as acetylsalicylic acid, diclofenac, indomethacin, oxamethacin, ibuprofen, indoprofen, naproxen, ketoprofen, mefamanic acid, metamizole, piroxicam, and celecoxib. In some embodiments, the anti-inflammatory agent is a pro-hormone that regulates the inflammatory process, including but not limited to pro-hormone converting enzyme 1, pro-proopiomelanocortin, pro-B-type natriuretic peptide, and pro-SMR1 hormone. In some embodiments, the anti-inflammatory agent is an enzyme with anti-inflammatory activity, including but not limited to bromelain, papain, serrapeptidase, and proteolytic enzymes, such as secretin (a mixture of trypsin, amylase, and lipase). In some embodiments, the anti-inflammatory agent is a peptide with anti-inflammatory activity, including but not limited to inhibitors of phospholipase A2, such as anti-inflammatory 1 (a peptide corresponding to amino acid residues 246-254 of lipocortin); anti-inflammatory 2 (a peptide corresponding to amino acid residues 39-47 of globin); S7 peptide (which inhibits the interaction between interleukin-6 and its receptor); RP1 (an isoprenoid protein inhibitor); and similar peptides. In some embodiments, the anti-inflammatory peptide is cortistatin (a cyclic neuropeptide associated with somatostatin); or a peptide corresponding to the N-terminal fragment of SV-IV protein, the conserved regions of E-selectin, L-selectin, and P-selectin, etc. Other non-limiting examples of anti-inflammatory agents include collagen hydrolysates and milk micronutrient concentrates (e.g., MicroLactin RTM, available from Stolle Milk Biologics, Inc., Cincinnati, Ohio), milk protein hydrolysates, casein hydrolysates, whey protein hydrolysates, and plant protein hydrolysates. In some embodiments, the anti-inflammatory agent is a plant extract with anti-inflammatory properties, including but not limited to extracts from the following plants: blueberry, boswella, black catechu, and Chinese skullcap, celery seed, chamomile, cherry, dewclaw, eucalyptus, evening primrose, ginger, hawthorn fruit, horsetail, Kalopanax pictu bark, licorice root, turmeric, white willow, willow bark, and yucca.

[0070] In some implementations, the anti-inflammatory agents and antibiotics are not minocycline. Non-limiting examples of Parkinson's disease symptoms include impaired motor function, increased αSyn aggregation, abnormal microglial activation, tremor, bradykinesia, muscle rigidity, impaired posture and balance, loss of spontaneous movement, speech impairment, writing impairment, and any combination thereof.

[0071] Inhibitors of PD-enhancing microbial metabolites may be, for example, antibodies against one or more PD-enhancing microbial metabolites, antibodies against intermediates used in the in vivo synthesis of one or more PD-enhancing microbial metabolites, antibodies against substrates used in the in vivo synthesis of one or more PD-enhancing microbial metabolites, or inhibitors of enzymes involved in the in vivo synthesis of one or more PD-enhancing microbial metabolites.

[0072] In the methods described herein, adjusting the composition of the gut microbiota in a subject and / or administering an inhibitor of PD-enhancing microbes to the subject may be performed alone or in combination with one or more other therapies (e.g., antibiotic therapy and anti-inflammatory therapy); or alone to achieve therapeutic efficacy. For example, in some embodiments of the methods disclosed herein, the subject or subject in need has not received antibiotic and / or anti-inflammatory treatment. In some embodiments, the method does not include administering any antibiotics and / or anti-inflammatory agents to the subject or subject in need. In some embodiments, the subject or subject in need has not been treated with rifampin and / or minocycline. In some embodiments, the subject or subject in need has not received any antibiotic and / or anti-inflammatory treatment for at least 1 hour, at least 6 hours, at least 12 hours, at least 18 hours, at least 1 day, at least 2 days, at least 3 days, at least 5 days, at least 10 days, or at least 20 days prior to adjusting the composition of the gut microbiota or other treatments (e.g., administering an inhibitor of one or more PD-enhancing microbe metabolites). In some implementations, subjects or subjects in need are not receiving any antibiotic and / or anti-inflammatory treatment for at least 1 hour, at least 6 hours, at least 12 hours, at least 18 hours, at least 1 day, at least 2 days, at least 3 days, at least 5 days, at least 10 days, or at least 20 days after adjusting the composition of the gut microbiota or other treatments (e.g., administration of one or more inhibitors of PD-enhancing microbial metabolites).

[0073] In some embodiments of the method disclosed herein, the method further includes determining the presence and / or level of one or more PD-associated bacterial species in a subject to identify subjects requiring treatment.

[0074] Diagnosis of PD

[0075] As disclosed in this article, identifying specific PD-associated gut microbiota in subjects can provide a diagnosis of PD and / or serve as a biomarker of disease progression severity prior to the onset of severe motor symptoms.

[0076] A method for diagnosing Parkinson's disease in a subject is provided. In some embodiments, the method includes determining the presence and / or level of one or more PD-associated bacterial species in the subject, whereby the presence and / or abnormal level of one or more PD-associated bacterial species indicates that the subject is at risk of developing or has Parkinson's disease symptoms. In some embodiments, the presence and / or level of one or more PD-associated bacterial species in the subject's gut is determined.

[0077] As used herein, the term "PD-associated bacterial species" refers to bacterial species whose levels are altered in the gut microbiota of subjects with PD or those with pathological conditions exhibiting one or more symptoms of Parkinson's disease, compared to subjects without PD or those without such pathological conditions (e.g., healthy subjects). In some embodiments, the levels of PD-associated bacterial species are elevated in the gut microbiota of subjects with PD or those with such pathological conditions, compared to subjects without PD or those without such pathological conditions (e.g., healthy subjects). In some embodiments, the levels of PD-associated bacterial species are decreased in the gut microbiota of subjects with PD or those with such pathological conditions, compared to subjects without PD or those without such pathological conditions (e.g., healthy subjects). In some embodiments, PD-associated bacterial species are present only in subjects with PD or those with such pathological conditions (e.g., in the gut of the subject), but not in non-PD subjects (e.g., healthy subjects). In some embodiments, PD-associated bacterial species are present at significantly lower levels (e.g., no more than 50%, no more than 25%, or no more than 10%) in non-PD subjects (e.g., in the subject's intestine) compared to subjects with PD or pathological conditions exhibiting one or more Parkinson's symptoms. In some embodiments, PD-associated bacterial species are those belonging to the genera *Proteus*, *Biliobacterium*, *Roseidonella*, *Pseudoramibacterium*, or *Veillonaceae*. In some embodiments, PD-associated bacterial species are SCFA-producing bacteria, such as SCFA-producing bacteria belonging to the KEGG family K00929, K01034, or K01035.

[0078] The presence and / or abnormal levels of one or more PD-associated bacterial species can indicate that a subject is at risk of developing Parkinson's disease or that the subject has Parkinson's disease. Parkinson's disease can be, for example, primary or idiopathic Parkinson's disease, secondary or acquired Parkinson's disease, hereditary Parkinson's disease, Parkinson's plus syndromes, or multisystem degeneration, or any combination thereof. In some embodiments, Parkinson's disease is Parkinson's syndrome. In some embodiments, the subject is an adult.

[0079] Composition

[0080] The disclosure herein includes compositions comprising one or more PD-protective bacterial species. Non-limiting examples of PD-protective bacterial species include bacterial species belonging to the families Trichophyceae, Rikenbacteraceae, Peptostreptococciaceae, Clostridiumceae, Enterococcus, Clostridium, Bacteroides, or Clostridium butyricum. In some embodiments, the composition does not contain PD-enhancing bacterial species. For example, in some embodiments, the composition does not contain bacterial species belonging to at least one of Proteus, Biliophilus, Roselle, Pseudoramibacter Eubacterium, and Veillonaceae. In some embodiments, the composition does not contain bacterial species belonging to Proteus, Biliophilus, Roselle, Pseudoramibacter Eubacterium, and Veillonaceae.

[0081] The type of composition can vary; for example, the composition can be a probiotic composition, a nutritional medicine composition, a pharmaceutical composition, or any combination thereof. In some embodiments, the composition is a food, beverage, or food supplement. In some embodiments, the composition is a pharmaceutical composition comprising one or more pharmaceutically acceptable carriers. In some embodiments, the pharmaceutical composition comprises an acid suppressant, an antacid, an H2 antagonist, a proton pump inhibitor, or a combination thereof. The composition can be in various forms; for example, the composition can be in a lyophilized, pulverized, or powdered formulation; in a suspension (e.g., a suspension in which bacteria are suspended); a liquid culture; or lyophilized, pulverized, or powdered; or as a pharmaceutical composition dissolved in, for example, saline.

[0082] The composition may be in the following forms: capsules, tablets, slushes, or powders, or combinations thereof. The composition may include, for example, dairy products such as yogurt, fermented and unfermented milk, smoothies, butter, cream, hummus, kombucha, salad dressings, miso, fermented black beans, nutrition bars, and some fruit juices and soy beverages. In some embodiments, the composition is formulated as enteric-coated capsules, enteric-coated microcapsules, powders suitable for rehydration, nasoduodenal infusions, or for delivery as enemas or colonoscopy infusions; or as a pharmaceutical composition added to foods, food additives, dairy-based products, soy-based products or derivatives thereof, jellies, or yogurt.

[0083] This document also discloses pharmaceutical compositions comprising inhibitors of PD-enhancing microbial metabolites. PD-enhancing microbial metabolites may be, for example, fatty acids, or salts or esters thereof. In some embodiments, the PD-enhancing microbial metabolites are short-chain fatty acids (SCFAs), medium-chain fatty acids, long-chain fatty acids, salts or esters of short-chain fatty acids, salts or esters of medium-chain fatty acids, or salts or esters of long-chain fatty acids. Inhibitors may be, for example, antibodies against PD-enhancing microbial metabolites, antibodies against intermediates used in the in vivo synthesis of PD-enhancing microbial metabolites, antibodies against substrates used in the in vivo synthesis of PD-enhancing microbial metabolites, or inhibitors of enzymes involved in the in vivo synthesis of PD-enhancing microbial metabolites. The pharmaceutical composition may comprise, for example, one or more pharmaceutically acceptable excipients. The pharmaceutical composition may be used to treat a variety of conditions / diseases, including but not limited to neurodegenerative conditions (such as Parkinson's disease).

[0084] Pharmaceutically acceptable prodrugs for pharmaceutical compositions and treatment methods using such pharmaceutically acceptable prodrugs are also provided. The term "prodrug" refers to a precursor of a specified compound that, upon administration to a subject, is produced in vivo through a chemical or physiological process (e.g., solvent degradation or enzymatic cleavage) or under physiological conditions (e.g., conversion to a pharmaceutical agent upon being brought to physiological pH). A "pharmaceuticalally acceptable prodrug" is a non-toxic and biologically tolerable prodrug that is biologically suitable for administration to a subject. Illustrative methods for selecting and preparing suitable prodrug derivatives are described, for example, in Bundgaard, Design of Prodrugs (Elsevier Press, 1985).

[0085] Pharmaceutically active metabolites of the pharmaceutical composition and their use in the methods of the invention are also provided. "Pharmaceutically active metabolites" refers to the pharmacologically active products of the metabolism of a compound or its salts in vivo. Prodrugs and active metabolites of the compound can be determined using conventional techniques known or available in the art. See, for example, Bertolini et al., J. Med. Chem. 1997, 40, 2011-2016; Shan et al., J. Pharm. Sci. 1997, 86(7), 765-767; Bagshawe, Drug Dev. Res. 1995, 34, 220-230; Bodor, Adv. Drug Res. 1984, 13, 255-331; Bundgaard, Design of Prodrugs (Elsevier Press, 1985); and Larsen, Design and Application of Prodrugs, Drug Design and Development (Krogsgaard-Larsen et al., eds., Harwood Academic Publishers, 1991).

[0086] Any suitable formulation of the compounds described herein can be prepared. See Remington's Pharmaceutical Sciences, (2000) Hoover, J.E. editor, 20th edition, Lippincott Williams and Wilkins Publishing Company, Easton, Pa., pp. 780-857. The formulation is selected to suit a suitable route of administration. Some routes of administration are oral, parenteral, inhalation, topical, rectal, nasal, oral, vaginal, via an implanted reservoir, or other methods of drug administration. Administration of the compound in salt form may be suitable if the compound is sufficiently basic or acidic to form a stable, non-toxic acid salt or alkaline salt. Examples of pharmaceutically acceptable salts are addition salts of organic acids that form physiologically acceptable anions, such as toluenesulfonates, methanesulfonates, acetates, citrates, malonates, tartrates, succinates, benzoates, ascorbic acid salts, α-ketoglutarate, and α-glycerophosphates. Suitable inorganic salts can also be formed, including hydrochlorides, sulfates, nitrates, bicarbonates, and carbonates. Pharmaceutically acceptable salts are obtained using standard methods well known in the art, such as by reacting a sufficiently basic compound (e.g., an amine) with a suitable acid (providing a physiologically acceptable anion). Alkali metal (e.g., sodium, potassium, or lithium) or alkaline earth metal (e.g., calcium) salts of carboxylic acids can also be prepared.

[0087] When the compound under consideration is administered in a pharmaceutical composition, it is considered that the compound can be formulated together with pharmaceutically acceptable excipients and / or carriers in a mixture. For example, the compound under consideration can be administered orally as a neutral compound or as a pharmaceutically acceptable salt, or intravenously in a physiological saline solution. Conventional buffers (such as phosphates, bicarbonates, or citrates) can be used for this purpose. Those skilled in the art can modify the formulation to provide a variety of formulations for a particular route of administration within the scope taught in the specification. In particular, the compound under consideration can be modified to make it more soluble in water or other media, which can be readily accomplished, for example, by minor modifications (salt formation, esterification, etc.) within the capabilities of those skilled in the art. Modifying the route of administration and dosing regimen of a particular compound to manage the pharmacokinetics of the compounds of the present invention to obtain the maximum beneficial effect in the patient is also within the capabilities of those skilled in the art.

[0088] The pharmaceutical compositions described herein may be soluble in organic solvents such as chloroform, dichloromethane, ethyl acetate, ethanol, methanol, isopropanol, acetonitrile, glycerol, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, or any combination thereof. In some embodiments, the formulation is prepared by mixing the pharmaceutical agent with a pharmaceutically acceptable carrier. In some embodiments, the formulation may be prepared using a method comprising the following steps: a) dissolving the pharmaceutical agent in a water-soluble organic solvent, a nonionic solvent, a water-soluble lipid, cyclodextrin, a vitamin (such as tocopherol), a fatty acid, a fatty acid ester, a phospholipid, or a combination thereof to provide a solution; b) adding a saline or buffer solution containing 1-10% carbohydrate solution. The carbohydrate may include, for example, dextran. The pharmaceutical compositions obtained using the methods of the present invention are stable and suitable for animal and clinical use.

[0089] Non-limiting examples of water-soluble organic solvents used in the methods of this invention include, but are not limited to, polyethylene glycol (PEG), alcohols, acetonitrile, N-methyl-2-pyrrolidone, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, or combinations thereof. Examples of alcohols include, but are not limited to, methanol, ethanol, isopropanol, glycerol, or propylene glycol.

[0090] Non-limiting examples of water-soluble nonionic surfactants used in the methods of the present invention include, but are not limited to, those mentioned above. EL, polyethylene glycol modified (polyoxyethyleneglycerol triricinoleate 35), hydrogenated RH40, hydrogenation RH60, PEG-succinate, polysorbate 20, polysorbate 80 HS (polyethylene glycol 660 12-hydroxystearate), sorbitan monooleate, poloxamer, (ethoxylated almond oil) (capryl-caproyl polyethylene glycol-8-glyceride) (glycerides) (PEG 6 caprylic / caprylic glyceride), glycerol, glycerol-polysorbate, or combinations thereof.

[0091] Non-limiting examples of water-soluble lipids used in the methods of this invention include, but are not limited to, vegetable oils, triglycerides, vegetable oils, or combinations thereof. Examples of lipid oils include, but are not limited to, castor oil, polyoxyethylene castor oil, corn oil, olive oil, cottonseed oil, peanut oil, peppermint oil, safflower oil, sesame oil, soybean oil, hydrogenated rapeseed oil, hydrogenated soybean oil, triglycerides of coconut oil, palm seed oil, and their hydrogenated forms or combinations thereof.

[0092] Non-limiting examples of fatty acids and fatty acid esters used in the methods of the present invention include, but are not limited to, oleic acid, monoglycerides, diglycerides, mono- or di-fatty acid esters of PEG, or combinations thereof.

[0093] Non-limiting examples of cyclodextrins used in the methods of this invention include, but are not limited to, α-cyclodextrin, β-cyclodextrin, hydroxypropyl-β-cyclodextrin, or sulfobutyl ether-β-cyclodextrin.

[0094] Non-limiting examples of phospholipids used in the methods of this invention include, but are not limited to, soybean phosphatidylcholine or distearate phosphatidylglycerol and their hydrogenated forms or combinations thereof.

[0095] Those skilled in the art can modify the formulations within the scope taught in the specification to provide multiple formulations for a particular route of administration. For example, compounds can be modified to make them more soluble in water or other media. Modifying the route of administration and dosing regimen of a particular compound to manage the pharmacokinetics of the compounds of the present invention to obtain the maximum beneficial effect in the patient is also within the capabilities of those skilled in the art.

[0096] The pharmaceutical compositions disclosed herein can be administered orally, parenterally, by inhalation, topically, rectally, nasally, orally, vaginally, via implanted reservoirs, or by other methods of drug administration. As used herein, the term "parenteren" includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques.

[0097] Sterile injectable compositions (such as sterile injectable aqueous or oily suspensions) can be formulated using suitable dispersants or wetting agents and suspending agents according to techniques known in the art. Sterile injectable formulations can also be sterile injectable solutions or suspensions in non-toxic, parenteral diluents or solvents. Acceptable media and solvents include mannitol, water, Ringer's solution, and isotonic sodium chloride solution. Suitable carriers and other pharmaceutical composition components are generally sterile.

[0098] In addition, sterile fixed oils are routinely used as solvents or suspension media (such as synthetic monoglycerides or diglycerides). Fatty acids (such as oleic acid and its glyceride derivatives) can be used to prepare injectable formulations, just as pharmaceutically acceptable oils (such as olive oil or castor oil, especially their polyoxyethylated forms). These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, or carboxymethyl cellulose or similar dispersants. Various emulsifiers or bioavailability enhancers commonly used to prepare pharmaceutically acceptable solid, liquid, or other dosage forms can also be used for formulation purposes.

[0099] Compositions for oral administration can be in any orally acceptable dosage form, including but not limited to tablets, capsules, emulsions, aqueous suspensions, dispersants, and solutions. In the case of tablets for oral use, common carriers include lactose and corn starch. Lubricants (such as magnesium stearate) may also be added. For oral administration in capsule form, useful diluents include lactose and dried corn starch. When administered orally in aqueous suspensions or emulsions, the active ingredient can be suspended or dissolved in an oil phase mixed with an emulsifier or suspending agent. If desired, certain sweeteners, flavoring agents, or coloring agents may be added. Nasal aerosols or inhalation compositions can be prepared according to techniques well known in the pharmaceutical formulation field and can be formulated as solutions, for example, in saline, using suitable preservatives (e.g., benzyl alcohol), absorption enhancers to improve bioavailability, and / or other solubilizers or dispersants known in the art.

[0100] Example

[0101] Some aspects of the implementation methods discussed above are disclosed in more detail in the following embodiments, which do not limit the scope of this disclosure in any way.

[0102] Experimental Materials and Methods

[0103] The following experimental materials and methods were used in Examples 1-8 described below.

[0104] Table 1. Reagents and Resources

[0105]

[0106]

[0107]

[0108] mice

[0109] Thy1-αSyn animals with a heterozygous female BDF1 background regarding the Thy1-α-synuclein transgene on the X chromosome were bred with wild-type male BDF1 mice to produce male ASO and WT littermates used in the study. Male BDF1 were bred by crossing female C57BL / 6 with male DBA / 2 (Charles River, Hollister, CA). Breeding pairs were replenished every 6 months with transgenic females and newly generated BDF1 males. Germ-free (GF) Thy1-αSyn breeding pairs were generated by cesarean section and newly generated males every 6 months. Microbiologically germ-free animals were raised by GF Swiss-Webster dams following surgical removal of the uterus and pup birth. SPF animals, antibiotic-treated animals, and Ex-GF animals were housed in autoclaved, ventilated, microisolated cages. GF ​​and SCFA-treated animals were housed in open-top cages with flexible membrane separators and maintained as microbiologically germ-free. Microbiological asepticity is confirmed on a bi-weekly basis by the following:

[0110] 16S rRNA PCR of fecal DNA was performed, and fecal pellets were plated anaerobically on Brucella blood agar and aerobically on tryptic soyblood agar. Regardless of colonization status, all animals receiving autoclaved food (LabDiet Laboratory Autoclavable Diet 5010, St. Louis, MO) and water (free access) were kept under the same 12-hour light-dark cycle and housed in the same facility. From 5–6 weeks of age until 12–13 weeks of age, antibiotic-treated animals were given ampicillin (1 g / L; Sigma Aldrich, St. Louis, MO), vancomycin (0.5 g / L; Sagent Pharmaceuticals, Schaumburg, IL), neomycin (0.5 g / L; Fisher Scientific), gentamicin (100 mg / L; Sigma Aldrich), and erythromycin (10 mg / L; Sigma Aldrich) in their drinking water. Ex-GF animals were generated by colonizing cecal contents from three wild-type BDF1 males resuspended in sodium bicarbonate buffer prior to oral gravidarization. From 5-6 weeks of age until 12-13 weeks of age, SCFA-treated animals were provided with drinking water containing sodium acetate (67.5 mM; Sigma Aldrich), sodium propionate (25 mM; Sigma Aldrich), and sodium butyrate (40 mM; Sigma Aldrich). Minocycline (Arcos Organics) was administered at 2 g / L in drinking water (free access) from 5-6 weeks of age until 12-13 weeks of age, along with SCFA. GF animals treated with heat-inactivated bacteria were provided with ~5 × 10⁶ bacteria in drinking water (free access). 8 Escherichia coli MC4100 grown in CFU / mL lysogen broth (LB) (from Matthew Chapman, U. of Michigan) (washed twice in phosphate-buffered saline and boiled for 45 minutes).

[0111] Human donors and standards

[0112] Human donors were selected from patients seen at the Rush University Atonic-Motor Disorders Clinic. PD was diagnosed according to the UK Brain Bank criteria. Exclusion criteria for PD subjects included: atypical or secondary Parkinson's disease; use of probiotics or antibiotics within three months prior to sample collection; use of NSAIDs; primary gastrointestinal pathology; history of chronic GI (including IBD and celiac disease); unstable medical, neurological, or psychotic conditions; low platelet count (<80kJ); irremediable prolonged PT (>15 seconds); or a history of bleeding that would impede biopsy. All patients had normal mucosa in the rectum and sigmoid colon as determined by sigmoidoscopy and by H&E histology. Healthy controls were matched as closely as possible to PD patients. Inclusion criteria for healthy subjects included: normal physical examination and blood tests; no history of digestive complaints, symptoms, or illness; no neurodegenerative diseases; and no use of probiotics, antibiotics, NSAIDs, or prescription medications for at least three months prior to sample collection.

[0113] Motor function and gastrointestinal tests

[0114] All motor function assessments, except for those performed on humanized animals, were conducted in the same gnotobiotic animal facility. Humanized animals were tested in a laminar flow biosafety cabinet within the same facility. Motor function was assessed in all animals between hours 7 and 9 of the light phase. All tests were performed in a manner similar to that described in Fleming et al., 2004, Early and progressive sensorimotor anomalies in mice overexpressing wild-type human alpha-synuclein, J. Neurosci. 24, 9434-9440 (the contents of which are incorporated herein by reference in their entirety). Beam crossing was performed first, followed by approximately 1 hour of rest and testing of descent along the beam. Adhesive removal and hind limb scoring were performed the following day. Fecal output was performed within 3 days and immediately before tissue collection.

[0115] Liang Crossing

[0116] A 1-meter acrylic beam (Stark's Plastics, Forest Park, OH) was constructed into four segments 0.25 μm long. Each segment had a narrowing width (3.5 cm, 2.5 cm, 1.5 cm, and 0.5 cm), with a 1 cm overhang placed 1 cm below the beam surface. The widest segment served as the loading platform for the animals, while the narrowest end was placed in the rearing cage. Animals were trained for two days to cross the beam length before testing. On day 1, animals underwent one trial in which the rearing cage was placed near the loading platform, and the animals were guided forward along the narrowing beam. Animals underwent two additional trials in which limited or no assistance was provided to encourage forward movement and stability on the beam. On day 2, animals underwent three trials of crossing the beam, generally without assistance in forward movement. On day 3, the time was recorded for the animals crossing from the loading platform to the rearing cage during the three trials. Timing begins when the animals place their forelimbs on a 2.5cm segment and ends when one forelimb reaches the rearing cage.

[0117] Descending along the pole

[0118] A 1cm diameter, 0.5m long pole, lined with a non-adhesive shelf liner to enhance animal grip, was placed in the enclosure. Animals were trained for two days to descend from the top of the pole into the enclosure. On day 1, the animals underwent three trials: the first trial with the animal headfirst at one-third of the distance above the floor; the second trial at two-thirds; and the third trial with the animal at the top. On day 2, the animals were given three more trials descending headfirst from the top of the pole. On the test day, the animal was headfirst at the top of the pole, and the descent back into the enclosure was timed. Timing began when the animal was released and ended when one hind leg reached the bottom of the enclosure.

[0119] Adhesive removal

[0120] Place a 1 / 4” circular adhesive tag (Avery, Glendale, CA) on the bridge of the nose between the nostrils and the forehead. Place the animals in their cages (remove cage mates) and time the complete removal of the tag. Record the results for the animals in 3 trials.

[0121] Hindlimb grasping reflex score

[0122] Similar to that described in Zhang et al., 2014, Motor impairments, striatal degeneration, and altered dopamine-glutamate interplay in mice lacking PSD-95. J. Neurogenet. 28, 98-111 (Zhang et al.'s work is incorporated herein by reference in its entirety), the animal was gently lifted upwards by the midsection of the tail and observed for approximately 5-10 seconds. The animal was assigned a score of 0, 1, 2, or 3 based on the degree of inward grasping of the hind limbs. A score of 0 (representing no grasping) was given to animals that had free movement of all limbs and extended them outwards. A score of 1 was assigned to animals that grasped one hind limb inwards during restraint or if both legs exhibited partial inward grasping. A score of 2 was given if both legs grasped inwards but still showed some flexibility in most observations. A score of 3 was assigned if the animal exhibited complete paralysis of the hind limbs with immediate inward grasping and showed no signs of flexibility.

[0123] Inverted grid

[0124] Place the animal in the center of a 30cm x 30cm sieve with 1cm wide mesh. Invert the sieve head to tail and place it on a support approximately 40cm above an open cage with a deep bedding layer. Time the animal until it is released or held for 60 seconds.

[0125] fecal output

[0126] Animals were removed from their cages and placed in 12cm × 25cm translucent cylinders. Fecal pellets were counted every 5 minutes for a cumulative total of over 15 minutes. Principal component analysis of all motor functions was performed using MATLAB software (MathWorks) based on behavioral data collected from subjects performing at least three tasks. The data was centered and standardized (s = 1) before running the pca function. Only PC1 and PC2 (representing 70.5% of variance) were plotted using the corresponding factor loadings for each subject.

[0127] Immunostaining and microglial reconstitution

[0128] Animals were sedated with pentobarbital and thoroughly perfused with phosphate-buffered saline. The brain was dissected, and the hemispheres were fixed in 4% (w / w) paraformaldehyde. 50 mm sagittal sections were produced using a vibratory microtome. Free-floating sections were stained with anti-aggregation / fibrillary αSyn MJFR1 (1:1000; rabbit; AbCam, Cambridge UK), anti-phosphoric acid Ser129αSyn (1:1000; mouse; Biolegend, San Diego, CA), and Neurotrace (Life Technologies, Carlsbad, CA), or with anti-Iba1 (1:1000; rabbit; Wako, Richmond, YA), followed by staining with anti-mouse IgG-AF488 and anti-rabbit IgG-AF546 (1:1000; Life Technologies). Sections were mounted on a ProFade Diamond (Life Technologies) microscope and imaged on a Zeiss LSM800 confocal microscope with a 10X objective. Two to three fields of view were imaged per region for each animal, and the images were compiled into ImageJ software for analysis. For microglia reconstruction, as described in Erny et al., 2015, Host microbiota constantly control maturation and function of microglia in the CNS.Nat.Neurosci.18, 965-977 (Erny et al.'s work is incorporated herein by reference), Z-stacking was imaged in 1 mm steps, followed by analysis using Imaris software. Semi-automated reconstruction and processing of microglia cell bodies were performed, whereby the experimenter specified individual cell bodies and the software quantified diameter, dendrite length, and branching points from each given cell body. Two to sixty cells were analyzed per region per animal. CD11b enrichment and qPCR analysis were performed.

[0129] The perfused whole brain was homogenized in PBS by passing it through a 100 mm sieve filter. Myelin fragments were removed using magnetic separation with myelin removal beads (Miltenyi Biotec, San Diego, CA) according to the manufacturer's instructions. CD11b enrichment was similarly performed using magnetic enrichment with microglia microbeads (Miltenyi Biotec, San Diego, CA) according to the manufacturer's instructions. Typically, more than 90% of the enriched cells were CD11b positive by immunofluorescence microscopy. For RNA analysis, anatomical tissue (frontal cortex, caudate putamen, hypomidbrain, and cerebellum) or CD11b-enriched cell pellets were lysed in Trizol for DirectZol RNA extraction (Zymo Research, Irvine, CA). cDNA was generated using the iScript cDNA Synthesis Kit (BioRad, Hercules, CA). qRT-PCR was performed on an AB 7900ht instrument using primers from Primer Bank targeting specific target genes, using SybrGreen master mix (Applied Biosystems, Foster City, CA), and quantified relative to gapdh as DDCT (primers listed in Table 1 above). Cytokine and αSyn ELISA and Western blotting were also performed.

[0130] Tissue homogenates were prepared in RIPA buffer containing a mixture of protease inhibitors (Thermo Fisher, Pittsburgh, PA) and diluted in PBS. TNF-α and IL-6 ELISA (eBioscience, San Diego, CA) and αSyn ELISA (Thermo Fisher) were performed according to the manufacturer's instructions. For dot blot quantification of αSyn fibrils, 1 mg of tissue homogenate from a designated region was spotted onto a 1 mL aliquot placed on a 0.45 mm nitrocellulose membrane. For Western blotting of Triton X soluble and insoluble fractions, the brain hemispheres were homogenized in RIPA buffer containing 1% Triton X-100 and centrifuged at 15 kJ / g at 4 °C for 60 min to precipitate insoluble proteins from the Triton X soluble supernatant. As previously described in Klucken et al. (2006, Clinical and biochemical correlates of insoluble alpha-synuclein in dementia with Lewy bodies. Acta Neuropathol. 111, 101-108, the contents of which are incorporated herein by reference in their entirety), the insoluble fraction was dissolved in 10% sodium dodecyl sulfate. 5 mg of each fraction was separated by 4%–20% SDS-PAGE (Thermo Fisher) and imprinted onto PVDF membranes. All membranes were blocked with 5% skim milk powder in Tris-buffered saline containing 0.1% Tween 20. Anti-aggregation alpha-Syn antibody (1:2000; rabbit; Abcam) or anti-alpha-Syn (1:1000; mouse; BD) was diluted in skim milk and incubated overnight at 4°C. The membranes were probed with anti-rabbit or anti-mouse IgG HRP (1:1000; Cell Signaling Technology). All blots were detected on a BioRad GelDoc XR using the Clarity chemiluminescent substrate (BioRad). Densitometry was performed using ImageJ software.

[0131] αSyn aggregation assay

[0132] For in vitro aggregation kinetics, as described in Chorell et al., 2015, Bacterial chaperones CsgE and CsgC differentially modulate human α-synuclein amyloid formation via transient contacts. PLoS ONE 10, e0140194 (the entire contents of Chorell et al. are incorporated herein by reference), 70 mM αSyn was purified and incubated in phosphate-buffered saline (0.01 M phosphate buffer, 0.0027 M potassium chloride, 0.137 M sodium chloride, pH 7.4) in the presence of 12 mM thioflavin T (ThT; Sigma Aldrich) and increased concentrations of SCFA. For each experiment, a non-binding 96-well plate (Corning #3881) with half the area was used, and 2 mm diameter glass beads were added to each well to accelerate aggregation. ThT fluorescence signals were recorded at 37°C under intermittent oscillation conditions using a Fluostar OPTIMA microplate reader (BMG Labtech) with an excitation filter of 440 ± 10 nm and an emission filter of 490 ± 10 nm. Kinetic curves were normalized to the fluorescence maximum and the time to reach the quantified half-maximum intensity. For atomic force microscopy (AFM) imaging, samples were diluted with ultrapure water to a total protein concentration of ~3 mM, and 50 ml was pipetted onto freshly cut mica and allowed to dry. Samples were imaged in air in intermittent contact mode using a modular scanning probe microscope, the NTEGRA Prima (NT-MDT), with a gold-coated single-crystal silicon cantilever (spring constant approximately 5.1 N / m) at a resonant frequency of approximately 150 kHz. AFM images were processed using Gwyddion open-source software.

[0133] SCFA Extraction and Analysis

[0134] Fecal samples were collected from 12-week-old animals. Each fecal pellet was mixed with 1 mL of sterile 18 U deionized water. The fecal pellet-water mixture was homogenized by mixing at 3200 rpm for 5 min and centrifuged at 13,000 rpm for 15 min at 4 °C. The supernatant was filtered through a 0.2 mm PVDF membrane (Pall Life Sciences) using an Acrodisc LC 13 mm sterile syringe filter. The filtrate was used for high-performance liquid chromatography (HPLC) analysis. Short-chain fatty acids (SCFAs) were analyzed using HPLC (LC-20AT, Shimadzu) equipped with a carbohydrate column (Aminex HPX-87H column, Biorad) and a photodiode array detector (PDA, Shimadzu). The eluent was 5 mM H₂SO₄, fed at a flow rate of 0.6 mL / min, and the column temperature was 50 °C. The run time was 60 min. A standard curve was generated by diluting 10 mM standard solutions of volatile fatty acids (acetic acid, butyric acid, formic acid, valeric acid, isovaleric acid, hexanoic acid, isohexanoic acid, and heptanoic acid) to 50 nM–5000 nM. The concentrations of SCFAs were normalized to soluble chemical oxygen demand (COD). The sCOD value in fecal supernatant was measured using a high-range (20–1500 mg / L) Hach COD digestion tube (Hach Company, Loveland) as recommended by the manufacturer. The wavelength used for COD measurement with a Hach spectrophotometer was 620 nm.

[0135] Microbiome property analysis

[0136] Fecal pellets were collected from animals in groups 1–3, based on genotype and donor, at days 7, 14, 21, and 49 post-fecal transplantation. Samples were sequenced according to the Earth Microbiome Project protocol as described by Gilbert et al., 2014, The Earth Microbiome project: successes and aspirations. BMC Biol. 12, 69 (Gilbert et al. are incorporated herein by reference in their entirety). Briefly, DNA was extracted using the MoBio Power soil kit (Carlsbad, CA), and the V4 region of the 16S rRNA gene was amplified using barcode primers described by Walters et al., 2015. Sequencing was performed using Illumina MiSeq. For Greengenes (described in McDonald et al., 2012), published in August 2013, in QIIME 1.9 (described in Caporaso et al., 2010), operational taxonomic units (OTUs) were selected as closed references using SortMeRNA 2.0 (described in Kopylova et al., 2012). For α and β diversity calculations, the table was selected to have 7500 sequences per sample. Differential abundance was performed on tables filtered to exclude samples with fewer than 7500 sequences. Weighted and unweighted UniFrac (described in Lozupone and Knight, 2005) distances were calculated in QIIME 1.9. Principal Coordinate Analysis (PCoA) predictions were visualized using Emperor 0.9.4 (described in Vazquez-Baeza et al., 2013). Functional repertoires were predicted using PICRUSt 1.0 (described in Langille et al., 2013); Bray Curtis distance comparisons were used to predict functional repertoires. Significance was tested using permanova in scikit-bio 0.4.2 and permutation t-tests in QIIME 1.9, each with 999 permutations. Differential abundance was calculated using genus-level taxonomic units and KEGG-based relative abundance (all counts offset by one). One-way ANOVA was performed using ANCOM in scikit-bio 0.4.2 (described in Mandal et al., 2015) with a Bonferroni-corrected α of 0.1 as the rejection threshold. Mice colonized with samples from healthy or PD donors were compared in BDF1 or Thy1-αSyn genetic backgrounds. The taxonomic units that are significantly different between the two groups are compared and classified as: significant in both; significant only in the Thy1-αSyn background; or significant in the BDF1 background.The graph was generated using Seaborn 0.7.0.

[0137] Quantitative and statistical analysis

[0138] Microbiome statistics are described in detail above. Excluding these, the dataset was analyzed in GraphPad Prism 6 software. Paired comparisons were generated using a two-tailed t-test. Group comparisons were generated using one-way ANOVA. The definitions of p-value, n-value, center and dispersion measurements are indicated in the relevant legend for each graph.

[0139] Data and software availability

[0140] 16S sequencing data and metadata can be obtained online via the QIITA website (https: / / qiita.ucsd.edu / ) using study accession number #10483 and via the EMBL ENA database (http: / / www.ebi.ac.uk / ena) using study accession number #ERP019564.

[0141] Example 1

[0142] Gut microbiota promotes motility and GI dysfunction

[0143] This embodiment uses ASO animals with complex microbiota and wild-type animals to demonstrate that gut microbiota promotes motility and GI dysfunction.

[0144] Thy1-αSyn (overexpressing α-synuclein [ASO]) mice exhibit progressive deficits in fine and gross motor function, as well as intestinal motility deficits. Evidence has linked unregulated αSyn expression in humans to a higher risk of PD, providing an epidemiological basis for the Thy1-αSyn mouse model. Deficits in coordinated motor tasks become apparent by 12 weeks of age. Motor function was measured using four tests, as previously validated in this model: beam crossing, descent along a bar, nasal adhesive removal, and hindlimb grasping reflex (as described in Fleming et al., 2004, Early and progressive sensorimotoranomalies in mice overexpressing wild-type human alpha-synuclein. J. Neurosci. 24, 9434-9440, the contents of which are incorporated herein by reference in their entirety).

[0145] Compared to wild-type littermates (SPF-WT), 12-13 week old ASO animals with a complex microbiome (SPF-ASO) required significantly more time to cross challenging beams and also showed increased time for descent along the beam, two measures of gross motor function. Figure 2A and Figure 2B Compared to SPF-WT mice, the removal of adhesive from the bridge of the nose (a test of fine motor control) was impaired in SPF-ASO mice. Figure 2C The hindlimb grasping reflex (a measure of striatal dysfunction) is defective in SPF-ASO mice. Figure 2D ).

[0146] To assess the contribution of gut bacteria, ASO mice (GF-ASO) and wild-type mice (GF-WT) were re-acquired under sterile conditions. Surprisingly, 12-13 week old GF-ASO animals exhibited reduced deficiencies in beam crossing, rod descent, adhesive removal, and hind limb grasping. Figures 2A-2D In fact, the motor function tasks performed by GF-ASO mice were similar to the performance levels of WT animals in many cases. GF-ASO mice did not show a difference in body weight compared to SPF-ASO animals. Figure 3A Both SPF-ASO and GF-ASO animals showed defects in the inverted grid assay (a measure of limb strength). Figure 3B Therefore, the results of exercise tests do not depend on weight or physical strength.

[0147] At a later age (24-25 weeks), SPF-ASO animals exhibited a progressive decline in motor function. Figures 3C-3G This significantly delayed ( ) in GF-ASO animals. Figures 3C-3G No consistent differences in motor tasks were observed in GF-WT and SPF-WT animals, providing evidence for gene-microbiome interactions. This is because, in PD, motor dysfunction in this mouse model co-occurred with reduced GI function and constipation.

[0148] At 12-13 weeks of age and 24-25 weeks of age, a significant decrease in total fecal pellet output was observed in SPF-ASO animals, while fecal output remained unchanged in GF-ASO animals. Figure 2E , Figure 2F , Figure 3H and Figure 3I Furthermore, compared to GF-ASO mice, SPF-ASO mice produced fecal pellets with reduced water content. Figure 3JTogether, they revealed a reduction in GI deficiency in GF animals. In fact, compiling all motor phenotypes into principal component analysis (PCoA) showed a striking deviation in the SPF-ASO group, while the GF-ASO animal clusters were more similar to WT mice. Figure 3K ).

[0149] The data presented in Example 1 demonstrate that the presence of gut microbiota promotes signature motor and intestinal dysfunction in preclinical PD models.

[0150] Example 2

[0151] αSyn pathology requires gut microbiota

[0152] This example demonstrates that αSyn pathology is increased in mice with gut microbiota.

[0153] Motor deficits in PD are consistent with αSyn aggregation. Immunofluorescence microscopy was used to visualize αSyn inclusions in the mouse brain using antibodies that recognize only conformation-specific αSyn aggregates and fibrils. Under SPF conditions, significant aggregation of αSyn was observed in the caudate putamen (CP) and substantia nigra (SN) of ASO animals. Figure 4A and Figure 4B This refers to the substantia nigra-striatal pathway, a brain region affected in both mouse models and human PD. Surprisingly, GF-ASO mice showed significantly fewer αSyn aggregates ( Figure 4A and Figure 4B To quantify αSyn aggregation, Western blotting of brain extracts was performed. Figure 4C Significantly lower levels of insoluble α-Syn were observed in the brains of GF-ASO animals. Figures 4C-4E To further confirm these findings, dotted imprint analysis was performed on the aggregated αSyn in the CP and the lower midbrain (where the SN is located). A similar reduction in αSyn aggregation was observed in GF-ASO animals. Figures 5A-5C ).

[0154] Region-specific aggregation of αSyn was observed: in the frontal cortex (FC), GF-ASO animals showed less αSyn aggregation compared to SPF animals; while in the cerebellum (CB), almost equal amounts of αSyn were observed in SPF and GF mice. Figures 5D-5H To ensure that these findings do not reflect differences in transgene expression, the levels of αSyn transcripts and proteins in the midbrain and CP were determined to be similar between SPF-ASO and GF-ASO animals. Figure 4F and Figure 4G ).

[0155] In summary, these data suggest that the microbiome regulates pathways that promote αSyn aggregation and / or prevent the clearance of insoluble protein aggregates.

[0156] Example 3

[0157] αSyn-dependent microglia activation by microbiota

[0158] This example demonstrates αSyn-dependent microglia activation mediated by the microbiome.

[0159] The microbiome regulates immune development in the CNS, and αSyn aggregates activate immune cells, including brain-resident microglia. Microglia undergo significant morphological changes upon activation, transforming from elongated cell bodies with multiple branching extensions to round, amoeba-like cells with fewer branches. In situ 3D reconstructions of individual microglia from confocal fluorescence microscopy revealed that wild-type GF animals possess microglia distinct from SPF animals. In CP and SN, compared to SPF-WT animals, GF-WT mice showed an increased number of microglia and a greater total branch length (…). Figures 6A-6C These morphological features indicate that microglia in GF animals are maturation arrested and / or have reduced activation, confirming recent reports of gut bacteria influencing immune cells in the brain.

[0160] Extending these observations to disease models, microglia from SPF-ASO mice showed a significantly increased cell body diameter and fewer, shorter processes compared to GF-ASO mice. Figures 6A-6C Compared to GF-ASO mice, tissue homogenates from the CP and lower midbrain of SPF-ASO mice contained significantly increased levels of pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Figure 6D and Figure 6E Both cytokines were elevated in the brains of PD patients. Gene expression analysis of RNA from enriched CD11b+ cells (primarily microglia) revealed increased expression of Tnfα and Il6 in SPF-ASO animals, which was almost absent in GF animals. Figure 6F As observed in previous studies, such as Erny et al., 2015; and Matcovitch-Natan et al., 2016, levels of the neuroprotective Bdnf and the cell cycle marker Ddit4 were upregulated in GF animals. Figure 5I The neuroinflammatory response is region-specific; microglia diameter and TNF-α production are increased in FC, but not in CB. Figure 6G and Figure 6H ).

[0161] In summary, these findings support the hypothesis that gut microbes promote αSyn-dependent activation of microglia in specific brain regions involved in disease.

[0162] Example 4

[0163] Postnatal microbial signaling regulation αSyn-dependent pathophysiology

[0164] This example demonstrates the αSyn-dependent pathophysiology of microbial signaling regulation after birth.

[0165] The microbiome influences neurological outcomes during pregnancy and in adulthood via active gut-to-brain signaling. To differentiate these mechanisms, SPF animals were treated with a mixture of antibiotics to deplete their microbiome after birth. Figure 7A Conversely, 5-6 week old GF mice were colonized with a complex microbiota from SPF-WT animals. Figure 7A Notably, antibiotic-treated (Abx) animals exhibited very little αSyn-dependent motor dysfunction, very similar to mice born under GF conditions. Figures 7B-7E Postnatal colonization (Ex-GF) in previously observed GF animals reiterated the genotypic effects observed in SPF mice, with mice overexpressing αSyn exhibiting significant motor dysfunction. Figures 7B-7E ).

[0166] GI function (as measured by fecal output) was also significantly improved in Abx-treated animals, while Ex-GF mice showed an αSyn-dependent decrease in total fecal output. Figure 7F and Figure 7G Furthermore, in the transgenic ASO line, the cell body diameter of microglia from Ex-GF animals increased, comparable to that in SPF mice. Figure 7H and Figure 7I However, Abx-ASO animals have microglia with diameters similar to those of GF animals. Figure 7H and Figure 7I ).

[0167] In summary, these data suggest that, although the role of microglia during prenatal neurodevelopment cannot be ruled out, modulation of microglia activation in adulthood contributes to αSyn-mediated motor dysfunction and neuroinflammation, indicating active gut-brain signaling by the microbiome.

[0168] Example 5

[0169] SCFA is sufficient to promote αSyn-mediated neuroinflammation.

[0170] This example demonstrates that SCFA is sufficient to promote αSyn-mediated neuroinflammation.

[0171] Gut bacteria can regulate microglial cell activation during viral infection by producing microbial metabolites (i.e., short-chain fatty acids, SCFAs). For example... Figure 8A As shown, lower fecal SCFA concentrations were observed in animals treated with GF and Abx compared to SPF mice. To investigate whether SCFAs affected neuroimmune responses in PD mouse models, GF-ASO and GF-WT animals were treated with a mixture of SCFA acetate, propionate, and butyrate (while the animals were kept microbiologically sterile), and fecal SCFA concentrations were significantly restored. Figure 8A Within the affected brain regions (i.e., CP and SN), microglia in animals treated with SCFA showed morphology indicating increased activation, similar to cells from Ex-GF and SPF mice, compared to untreated mice. Figure 9A , Figure 9B , Figure 8B and Figure 8C See also Figures 6A-6H and Figures 7A-7I Compared to GF-WT animals treated with SCFA (SCFA-WT), microglia from GF-ASO mice fed with SCFA (SCFA-ASO) showed significantly larger diameters, accompanied by reduced branch length and total number. However, Abx-treated animals exhibited microglia morphology similar to GF animals. Figure 9B , Figure 8B and Figure 8C See also Figures 6A-6H and Figures 7A-7I Changes in microglial cell diameter were also observed in FC, but not in CB, suggesting this is a region-specific response. Figure 8D and Figure 8E ).

[0172] Corresponding to microglia morphology, αSyn accumulated in mice given SCFA, similar to Ex-GF animals, compared to untreated and Abx-treated mice. Figures 8F-8I Notably, postnatal signals derived from microbes were observed to induce increased αSyn aggregation in both CP and SN. Figure 8F and Figure 8G ), while no observable differences were found in FC and CB. Figure 8H and Figure 8I This was confirmed by quantification and Western blotting. Figure 8J-Figure 8O Neither standalone nor mixed forms of SCFA promoted the aggregation of human α-Syn in vitro within a certain concentration range. Figures 10A-10G It does not change the overall structure of αSyn amyloid fibrils. Figure 10H and Figure 10I ).

[0173] In summary, these data suggest that SCFA promotes αSyn aggregation in vivo, independent of direct molecular interactions.

[0174] Example 6

[0175] SCFA is sufficient to promote motor defects

[0176] This example demonstrates that SCFA promotes αSyn-stimulated microglial cell activation and motor dysfunction.

[0177] To explore the link between microbial metabolites and motor symptoms in the Thy1-αSyn model, GF mice were treated with a mixture of SCFAs starting at 5–6 weeks of age, and motor function was assessed at 12–13 weeks of age. Compared to untreated GF-ASO mice, SCFA-ASO mice showed significantly impaired performance in several motor tasks. Figures 9C-9F ), including impaired beam crossing, descent along the pole, and hind limb reflexes (comparing GF-ASO mice with SCFA-ASO mice). All effects induced by SCFA were genotype-specific in Thy1-αSyn mice. GI deficiency was also observed in SCFA-treated transgenic animals. Figure 9G and Figure 9H Oral treatment of GF animals with heat-inactivated bacteria did not induce motor defects. Figures 10J-10M This indicates that the bacteria need to possess metabolic activity. Furthermore, oral treatment of SCFA-fed animals with the anti-inflammatory compound minocycline was sufficient to reduce TNF-α production, decrease αSyn aggregation, and improve motor function without altering transgene expression. Figures 11A-11H ).

[0178] In summary, these data suggest that the microbiome actively produces metabolites (such as SCFAs) that are required for microglia activation and αSyn aggregation, which contribute to motor dysfunction in preclinical models of PD.

[0179] Example 7

[0180] PD microbiome dysregulation

[0181] This example demonstrates the dysregulation of the PD microbiome.

[0182] Altered microbiomes have been observed in PD patients. In this embodiment, fecal samples were collected from six human subjects diagnosed with PD and six matched healthy controls (Table 2) to determine whether human gut microbiota affected disease outcomes when transferred to GF mice. To limit confounding effects, only new-onset, treatment-naive PD patients with healthy gut histology were selected, in addition to the relevant inclusion and exclusion criteria (Table 2).

[0183] Table 2. Demographic characteristics of human donors (PD and healthy controls) (and) Figures 12A-12E and Figures 14A-14G (Related). Demographic data for fecal donors for each PD and healthy donor pair. UPDRS = Uniform Parkinson's Disease Rating Scale; HY stage = Hoehn and Yahr scale; BMI = Body Mass Index.

[0184]

[0185] Fecal microbiota from PD patients or controls were transplanted into various groups of GF recipient animals via oral intensive feeding. Fecal pellets were collected from “humanized” mice, bacterial DNA was extracted, and 16S rRNA was sequenced. Sequences were annotated as operational taxonomic units (OTUs) using closed reference selection against the Greengenes database, and metagenomic functions were predicted using PICRUSt. In PCoA-based unweighted UniFrac (Lozupone and Knight, 2005), the recipient animal groups were most similar to their respective human donor profiles ( Figure 12A and Figure 12B Notably, the disease state of the donor had a strong influence on the microbiome within the recipient mice. Humanized mice from PD donors were significantly more similar to each other compared to those from healthy donors, a trend that persisted when stratified by genetic background. Figure 12C and Figure 12D Furthermore, significant differences existed between healthy and PD donors in the ASO background compared to wild-type (WT) receptors, indicating the influence of genotype on microbial community structure. Figure 12C and Figure 12D ).

[0186] Several genera were identified that showed alterations in the microbiota colonized with PD donors compared to healthy controls. Figure 12E ) and the altered KEGG pathways between these groups as indicated by the Bray-Curtis distance ( Figures 13A-13C In mice with a PD microbiome, increased abundance of OTUs included Proteus, Biliophilus, and Roselle, accompanied by the loss of members of the Trichophyceae, Rikenaceae, and Peptostreptococci families, as well as the Clostridium butyricum family. Figure 13E Interestingly, some taxa changed only in ASO animals (e.g., *Proteus*, *Biliobacterium*, and *Trichophyton* families), while others showed significant changes independent of mouse genotypes (e.g., *Roseidon*, *Rikenaceae*, and *Enterococcus*). Figure 13EInterestingly, the abundance of three SCFA-producing KEGG family members (K00929, butyrate kinase; and K01034 and K01035, acetate-CoA / acetoacetate-CoA transferase α and β) increased in mice receiving fecal microbiota from PD donors. Figure 13D Furthermore, compared to animals colonized with microbes from healthy controls, animals receiving a microbiome derived from a PD donor exhibited a significantly altered SCFA profile, with lower concentrations of acetate and higher relative abundances of propionate and butyrate. Figure 13E ).

[0187] In summary, these data demonstrate that differences in the fecal microbiome between PD patients and controls can be maintained after transfer to mice. Furthermore, αSyn overexpression caused significant alterations in gut microbiome characteristics after transplantation.

[0188] Example 8

[0189] Gut microbiota derived from PD promotes motor dysfunction

[0190] This example demonstrates that gut microbiota derived from PD promotes motor dysfunction.

[0191] To assess the function of the microbiome, motor function was tested in the humanized animal groups derived from each donor pair. Consistency was found among four of the six pairs (pairs #1, #3, #4, and #5), indicating that the microbiome from individuals with PD promoted enhanced αSyn-mediated motor dysfunction. Figures 14A-14F Compared to genotype-matched recipient mice with gut bacteria from healthy controls, ASO animals colonized with PD microbiota showed significantly impaired beam crossing, rod descent, and nasal adhesive removal. On the other hand, hindlimb reflex scores generally did not differ between donors. Interestingly, microbiota from a single sample did not induce significant genotype effects in the beam crossing and rod descent tasks (for #2, ...). Figure 14B This reflects the need to address potential heterogeneity within populations through robust cohort studies. No significant effects on locomotor function were observed in WT recipient animals colonized with microbiota from either donor group. Figures 14A-14F This finding in preclinical mouse models suggests that the PD microbiota contributes to disease symptoms in genetically susceptible hosts. Furthermore, as measured by fecal output, recipient animals showed little change in body weight and GI function. Figures 15A-15F Compilation of performance data from all groups revealed that, among the four tests used in this study, the microbiota from PD patients induced increased motor impairment in ASO animals compared to the microbiota from healthy controls. Figure 14GIn fact, all motor functions depicted by PCoA revealed striking global differences between animals colonized with microbiota from PD donors compared to those colonized with gut bacteria derived from healthy individuals. Figure 15G ).

[0192] In summary, these data (which show that gut bacteria from PD patients improve motor deficits in mouse models compared to healthy controls) provide evidence of the functional contribution of the microbiome to synucleinopathy.

[0193] As described in this article, the gut microbiota influences neurodevelopment, regulates behavior, and promotes neuropathic disorders. Without being bound by any particular theory, the functional link between gut bacteria and neurodegenerative diseases remains to be explored. Synucleinopathies are characterized by the aggregation of the protein α-synuclein (αSyn), which typically leads to motor dysfunction, such as Parkinson's disease (PD). As shown in Examples 1-8, using mice overexpressing αSyn, the gut microbiota was found to be required for motor deficits, microglial activation, and αSyn pathology. Antibiotic treatment alleviated pathophysiology in adult animals, while microbial recolonization promoted pathophysiology in adult animals, indicating that postnatal signaling between the gut and brain modulates the disease. Oral administration of specific microbial metabolites to germ-free mice promoted neuroinflammation and motor symptoms. Furthermore, colonization of αSyn-overexpressing mice with the microbiota from PD-affected patients enhanced physical damage compared to microbiota grafts from healthy human donors. These findings suggest that gut bacteria regulate motor impairment in mice, while alterations in the human microbiome represent risk factors for PD.

[0194] Example 9

[0195] Treatment of Parkinson's Disease (PD)

[0196] This example illustrates the treatment for patients with PD.

[0197] Determine the rate of α-Syn aggregation or the level of α-Syn aggregation (e.g., the amount of α-Syn aggregates) or both in the subject. Abnormal rates of α-Syn aggregation or abnormal levels of α-Syn aggregation (e.g., the amount of α-Syn aggregates) or both in the subject indicate that the subject has PD. Adjust the composition of the gut microbiota in the subject. It is expected that adjusting the composition of the gut microbiota in the subject will alleviate one or more symptoms of PD, such as improving one or more motor deficits.

[0198] In at least some of the previously described embodiments, one or more elements used in one embodiment may be used interchangeably in another embodiment, unless such substitution is technically impractical. Those skilled in the art will understand that various other omissions, additions, and modifications can be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and variations are intended to fall within the scope of the subject matter defined by the appended claims.

[0199] Regarding the use of virtually any plural and / or singular terms in this document, those skilled in the art can convert from plural to singular and / or from singular to plural as appropriate to the context and / or application. For clarity, various singular / plural permutations may be explicitly described herein.

[0200] Those skilled in the art will understand that, generally, the terms used herein, and particularly those used in the appended claims (e.g., the text of the appended claims), are intended to be “open-ended” terms (e.g., the term “comprising” should be interpreted as “including but not limited to”; the term “having” should be interpreted as “having at least”; the term “including” should be interpreted as “including but not limited to”, etc.). Those skilled in the art will further understand that if there is an intention to elaborate a particular number of introduced claim recitations, this intention will be explicitly stated in the claim, and the absence of such recitations will not indicate this intention. For example, to aid understanding, the appended claims may contain the use of the introductory phrases “at least one” and “one or more” to introduce the claim recitations. However, the use of these phrases should not be construed as implying that a claim reference introduced by the indefinite article "a" or "an" will limit any particular claim containing such an introductory claim reference to embodiments containing only one such reference, even when the same claim contains the introductory phrase "one or more" or "at least one" and indefinite articles such as "a" or "an" (e.g., "a" and / or "an" should be interpreted as meaning "at least one" or "one or more"); the same applies to the use of definite articles for introductory claim references. Furthermore, even when a specific number of introductory claim references are explicitly cited, those skilled in the art will recognize that such references should be interpreted as indicating at least the number cited (e.g., a reference with only "two references" without other modifiers indicates at least two references or more). Furthermore, when using conventions such as "at least one of A, B, and C," such a structure is generally intended to convey the meaning of the convention as would be understood by a person skilled in the art (e.g., "a system having at least one of A, B, and C" includes, but is not limited to, a system having only A, a system having only B, a system having only C, a system with A and B together, a system with A and C together, a system with B and C together, and / or a system with A, B, and C together, etc.). When using conventions such as "at least one of A, B, or C," such a structure is generally intended to convey the meaning of the convention as would be understood by a person skilled in the art (e.g., "a system having at least one of A, B, or C" includes, but is not limited to, a system having only A, a system having only B, a system with only C, a system with A and B together, a system with A and C together, a system with B and C together, and / or a system with A, B, and C together, etc.). A person skilled in the art will further understand that, whether in the specification, claims, or drawings, any transition words and / or phrases that present two or more alternative terms should be understood to contemplate the possibility of including one, any, or both of these terms. For example, the phrase “A or B” would be understood to include the possibility of “A” or “B” or “A and B”.

[0201] Furthermore, where features or aspects of this disclosure are described in the manner of the Markush group, those skilled in the art will recognize that this disclosure is therefore also described in the manner of any individual member or subgroup of the Markush group.

[0202] As those skilled in the art will understand, for any and all purposes, as provided in writing, all scopes disclosed herein also include any and all possible subscopes and combinations thereof. Any listed scope can be readily considered to adequately describe the same scope divided into at least two, three, four, five, ten, etc., equal parts, and such division into at least two, three, four, five, ten, etc., equal parts is effective. As a non-limiting example, the scopes discussed herein can be readily divided into lower thirds, middle thirds, and upper thirds, etc. As those skilled in the art will also understand, all language such as “up to,” “at least,” “greater than,” “less than,” etc., includes the cited numbers and relates to scopes that can subsequently be divided into subscopes as discussed above. Finally, as those skilled in the art will understand, a scope includes each individual member. Thus, for example, a group having 1-3 items means a group having 1, 2, or 3 items. Similarly, a group having 1-5 items means a group having 1, 2, 3, 4, or 5 items, etc.

[0203] While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for illustrative purposes and not restrictive, and the true scope and spirit are indicated by the appended claims.

Claims

1. A method for treating synucleinopathies in a subject, the method comprising modulating the composition of the gut microbiota in a subject in need thereof, wherein, The subject has synucleinopathy.

2. A method for delaying or reducing the likelihood of onset of synucleinopathy in a subject, the method comprising modulating the composition of the gut microbiota in a subject in need thereof, wherein, The subject is at risk of developing synucleinopathy.

3. A method for improving motor deficits in a subject, the method comprising modulating the composition of the gut microbiota in a subject in need of improvement of motor deficits.

4. The method of any one of claims 1-3, comprising identifying a subject in need thereof, wherein, The subject in need has an abnormal level of alpha-synuclein (aSyn) aggregation.

5. The method of claim 4, wherein, Identifying the subject in need comprises measuring the rate and / or level of aSyn aggregation in the subject, measuring the rate and / or level of clearance of insoluble aSyn protein aggregates in the subject, or a combination thereof.

6. The method of claim 5, wherein, Measuring the rate and / or level of aSyn aggregation in the brain of the subject, measuring the rate and / or level of clearance of insoluble aSyn protein aggregates in the brain of the subject, or a combination thereof.

7. The method of any one of claims 4-6, further comprising measuring the rate and / or level of aSyn aggregation in the subject, measuring the rate and / or level of clearance of insoluble aSyn protein aggregates in the subject, or a combination thereof, after modulating the composition of the gut microbiota in the subject.

8. The method of any one of claims 1-7, wherein, The method improves one or more physical impairments in the subject.

9. The method of any one of claims 1-7, wherein, The method improves one or more GI functions in the subject.

10. The method of any one of claims 1-7, wherein, The method alleviates constipation in the subject.

Images