Controlling the gut microbiota to treat neurodegenerative disorders

Abstract

This application discloses methods and compositions that can be used to improve movement disorders and neuroinflammation in subjects in need thereof, for example, subjects suffering from a neurodegenerative disorder (e.g., Parkinson's disease). Additionally, methods and compositions that can be used to diagnose neurodegenerative disorders such as Parkinson's disease are disclosed.

Description

[Technical field]

[0001] Related Applications This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 62 / 340,408, filed May 23, 2016, U.S. Provisional Application No. 62 / 370,578, filed August 3, 2016, and U.S. Provisional Application No. 62 / 443,952, filed January 9, 2017, the contents of which are expressly incorporated by reference in their entireties.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under Grant No. NS085910 awarded by the National Institutes of Health. The United States Government has certain rights in this invention.

[0003] The present disclosure relates generally to the fields of diagnosis and treatment of neurodegenerative diseases (e.g., Parkinson's disease). [Background technology]

[0004] Neurological dysfunction is the basis of many human diseases. Behavioral, psychiatric, and neurodegenerative disorders often present with characteristic neuropathologies in the central nervous system (CNS). Amyloidosis, one of these neuropathologies, arises from the abnormal aggregation of certain neuronal proteins and disrupts various cellular functions. Insoluble protein aggregates with altered conformation are often found in affected tissues, and structural changes in these aggregates are thought to be a contributing factor in an estimated 50 human diseases (Sacchettini and Kelly, 2002). Amyloid-driven neurodegenerative disorders, including Alzheimer's disease, Huntington's disease, and Parkinson's disease (PD), are associated with various amyloid proteins (Brettschneider et al., 2015). Among these diseases, PD is the second most prevalent neurodegenerative disease in the United States, affecting an estimated 1 million people, or 1% of the U.S. population aged 60 years or older (Nalls et al., 2014). Approximately 3 million PD patients and their caregivers worldwide suffer from PD, a disease that is often debilitating and characterized by movement disorders such as tremor, rigidity, bradykinesia, and gait disturbance. PD is a multifactorial disease that is strongly influenced by environmental factors, with genetic predisposition accounting for less than 10% of cases (Nalls et al., 2014). Furthermore, α-synuclein (αSyn) aggregation is thought to be pathogenic in a group of disorders collectively referred to as synucleinopathies, including PD, multiple system atrophy, and Lewy body diseases (Brettschneider et al., 2015; Luk et al., 2012; Prusiner et al., 2015). αSyn aggregation progresses in a stepwise manner, leading to the accumulation of oligomers and non-transient fibrils within neurons. Dopaminergic neurons in the substantia nigra pars compacta (SNpc) appear to be particularly vulnerable to damage caused by the action of αSyn aggregates. Dopamine-modulating drugs are the first-line treatment for PD, but they can cause significant side effects and often lose efficacy (Jenner, 2008). In an ever-aging society, there is a need to find safe and effective treatments to address the growing burden of PD, seemingly at odds with the increased life expectancy achieved. Summary of the Invention [Means for solving the problem]

[0005] The present application discloses methods and compositions that can be used to improve movement disorders and neuroinflammation in subjects in need thereof, such as subjects suffering from a neurodegenerative disorder (e.g., Parkinson's disease).Furthermore, the present application discloses methods and compositions that can be used to diagnose neurodegenerative disorders, such as Parkinson's disease.

[0006] In some embodiments, a method for treating a neurodegenerative disorder in a subject is provided, comprising regulating the composition of gut microbiota in a subject suffering from a neurodegenerative disorder and in need of treatment for the neurodegenerative disorder. In some embodiments, a method for delaying or reducing the likelihood of developing a neurodegenerative disorder in a subject is provided, comprising regulating the composition of gut microbiota in a subject at risk of developing a neurodegenerative disorder and in need of delaying or reducing the likelihood of developing the neurodegenerative disorder. In some embodiments, the neurodegenerative disorder is a synucleinopathy, such as Parkinson's disease (PD), dementia with Lewy bodies, multiple system atrophy, or any combination thereof.

[0007] In some embodiments, a method of improving a movement disorder in a subject in need of such improvement (e.g., a subject with a synucleinopathy, including Parkinson's disease) is provided, comprising modulating a composition of the gut microbiota of the subject. In some embodiments, a method of reducing microglial activation in a subject in need of such reduction is provided, comprising modulating a composition of the gut microbiota of the subject. In some embodiments, a method of reducing α-synuclein (αSyn) aggregates in a subject in need of such reduction is provided, comprising modulating a composition of the gut microbiota of the subject. In some embodiments, the method can promote the clearance of insoluble αSyn protein aggregates, reduce αSyn protein aggregation, or both. In some embodiments, the method can include measuring the rate and / or extent of aggregation of αSyn in the subject, measuring the rate and / or extent of clearance of insoluble αSyn protein aggregates in the subject, or a combination thereof. In some embodiments, the aggregation rate and / or degree of αSyn in the brain of the subject, the clearance rate and / or degree of insoluble αSyn protein aggregates in the brain of the subject, or a combination thereof is measured. In some embodiments, the method further comprises measuring the aggregation rate and / or degree of αSyn in the subject, measuring the clearance rate and / or degree of insoluble αSyn protein aggregates in the subject, or a combination thereof, after adjusting the composition of the gut microbiota of the subject.

[0008] In some embodiments, a method of reducing neuroinflammation in a subject in need thereof is provided, the method comprising modulating the composition of the gut microbiota in the subject.

[0009] In the methods disclosed herein, the subject in need of the method may be, for example, a subject suffering from an amyloid-induced neurodegenerative disorder (e.g., synucleinopathy). Synucleinopathy includes, but is not limited to, Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, and any combination thereof. In some embodiments, the subject in need of the method is a subject suffering from Parkinson's disease. In some embodiments, the methods disclosed herein further include identifying a subject in need of the method, the subject in need of the method exhibiting an α-synuclein (αSyn) aggregation disorder. In some embodiments, identifying a subject in need of the method includes measuring the aggregation rate and / or degree of αSyn in the subject, measuring the removal rate and / or degree of insoluble αSyn protein aggregates in the subject, or a combination thereof. In some embodiments, the aggregation rate and / or degree of αSyn in the subject's brain, the removal rate and / or degree of insoluble αSyn protein aggregates in the subject's brain, or a combination thereof is measured. In some embodiments, the method further comprises measuring the aggregation rate and / or degree of αSyn in the subject after adjusting the composition of the gut microbiota of the subject, measuring the clearance rate and / or degree of insoluble αSyn protein aggregates in the subject, or a combination thereof. In some embodiments, the method can improve one or more physical disorders of the subject. In some embodiments, the method can improve one or more gastrointestinal functions of the subject. In some embodiments, the method can relieve constipation in the subject. In some embodiments, the movement disorder is tremor, rigidity, bradykinesia, gait disorder, or any combination thereof.

[0010] In some embodiments, the methods disclosed herein can restore the composition of the gut microbiota of the subject to normal levels. In some embodiments, modulating the composition of the gut microbiota of the subject includes administering one or more antibiotics to the subject. The antibiotics can be natural antibiotics, synthetic antibiotics, or semi-synthetic antibiotics. The one or more antibiotics can include, for example, ampicillin, vancomycin, neomycin, gentamicin, erythromycin, teicoplanin, doxycycline, tetracycline, norfloxacin, ciprofloxacin, augmentin, cephalexin (e.g., Keflex), penicillin, ampicillin, kanamycin, rifomycin, rifaximin, neomycin, metronidazole, or any combination thereof. The antibiotics can be administered orally, intravenously, rectally, or any combination thereof. In some embodiments, the one or more antibiotics do not include rifampicin and / or minocycline. In some embodiments, modulating the composition of the gut microbiota of the subject comprises administering to the subject an inhibitor against one or more microbial metabolites that exacerbate Parkinson's disease (PD). In some embodiments, modulating the composition of the gut microbiota of the subject comprises administering to the subject an antibody against one or more microbial metabolites that exacerbate PD, an antibody against an in vivo synthetic intermediate of one or more microbial metabolites that exacerbate PD, or an antibody against an in vivo synthetic substrate of one or more microbial metabolites that exacerbate PD.

[0011] In some embodiments, modulating the composition of the gut microbiota of the subject comprises administering to the subject an inhibitor against an enzyme involved in the in vivo synthesis of one or more microbial metabolites that exacerbate Parkinson's disease (PD). The one or more microbial metabolites that exacerbate PD may comprise, for example, one or more fatty acids, their salts or esters, or any combination thereof. In some embodiments, the one or more microbial metabolites that exacerbate PD comprise one or more short chain fatty acids (SCFAs), their salts or esters, or any combination thereof. In some embodiments, the one or more microbial metabolites that exacerbate PD comprise 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, the one or more microbial metabolites that exacerbate PD comprise short chain fatty acid (SCFA) acetate, short chain fatty acid (SCFA) propionate, short chain fatty acid (SCFA) butyrate, or any combination thereof.

[0012] In some embodiments, modulating the composition of the gut microbiota of the subject comprises increasing the amount of one or more bacterial species that are protective against Parkinson's disease (PD) in the subject. In some embodiments, modulating the composition of the gut microbiota of the subject comprises administering to the subject a composition comprising one or more bacterial species that are protective against PD. At least one of the one or more bacterial species that are protective against PD may be, for example, a bacterial species belonging to the family Lachnospiraceae, Rikenellaceae, family Peptostreptococcaceae, family Clostridiaceae, genus Enterococcus, genus Clostridium, genus Bacteroides or genus Butyricicoccus. In some embodiments, the composition is a probiotic composition, a nutritional composition, a pharmaceutical composition, or any combination thereof.

[0013] In some embodiments, modulating the composition of the gut microbiota of the subject comprises fecal transplantation, microbiota normalization, microbial colonization, gut microbiota reconstruction, probiotic treatment, or a combination thereof. In some embodiments, modulating the composition of the gut microbiota of the subject comprises reducing the amount of one or more bacterial species that exacerbate Parkinson's disease (PD) in the subject. At least one of the one or more bacterial species that exacerbate PD may be, for example, a bacterial species belonging to the genus Proteus, Bilophila, Roseburia, Pseudoramibacter, Eubacterium, or Veillonellaceae. In some embodiments, at least one of the one or more bacterial species that exacerbate PD is a short chain fatty acid (SCFA) producing bacterium. Examples of SCFA-producing bacteria include, but are not limited to, bacteria belonging to the KEGG families K00929, K01034, and K01035.

[0014] In some embodiments, modulating the composition of the subject's gut microbiota comprises introducing into the subject undergoing treatment gut microbiota obtained from a healthy subject.

[0015] In some embodiments, a method for treating a neurodegenerative disorder in a subject is provided, comprising one or more of administering an antibiotic to the subject; and administering an inhibitor against a microbial metabolite that exacerbates Parkinson's disease (PD) to the subject. In some embodiments, a method for delaying or reducing the likelihood of developing a neurodegenerative disorder in a subject is provided, comprising one or more of administering an antibiotic to the subject; and administering an inhibitor against a microbial metabolite that exacerbates Parkinson's disease (PD) to the subject. The neurodegenerative disorder may be, for example, a synucleinopathy (e.g., Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, or a combination thereof).

[0016] In some embodiments, a method for improving Parkinson's disease-like symptoms in a subject in need of such improvement is provided, comprising one or more of the following: administering an antibiotic to the subject; administering an anti-inflammatory agent to the subject; and administering an inhibitor against one or more microbial metabolites that exacerbate Parkinson's disease (PD) to the subject. In some embodiments, the anti-inflammatory agent and the antibiotic are not minocycline. In some embodiments, the Parkinson's disease-like symptoms include motor dysfunction, enhanced aggregation of α-synuclein (αSyn), abnormal activation of microglia, or any combination thereof. In some embodiments, the Parkinson's disease-like symptoms include tremor, bradykinesia, muscle rigidity, abnormal posture and balance, loss of motility, impaired speech, impaired writing, or any combination thereof. In some embodiments, the inhibitor of a microbial metabolite that exacerbates PD is an antibody against one or more microbial metabolites that exacerbate PD, an antibody against an in vivo synthetic intermediate of one or more microbial metabolites that exacerbate PD, an antibody against an in vivo synthetic substrate of one or more microbial metabolites that exacerbate PD, an inhibitor against an enzyme involved in the in vivo synthesis of one or more microbial metabolites that exacerbate PD, or a combination thereof. In some embodiments, the subject or the subject in need of the method has not been treated with an antibiotic. In some embodiments, the method does not include administering an antibiotic to the subject or the subject in need of the method. In some embodiments, the subject or the subject in need of the method has not been treated with rifampicin and / or minocycline. In some embodiments, the subject or the subject in need of the method has not been treated with an antibiotic for at least 12 hours, 1 day, 5 days, 10 days, or 20 days prior to performing the adjustment of the composition of the gut microbiota or other treatment. In some embodiments, the subject or a subject in need of the method is not treated with an antibiotic for at least 12 hours, 1 day, 5 days, 10 days, or 20 days after modulating the composition of the gut microbiota or other treatment.In some embodiments, the method further comprises identifying a subject in need of the method by determining the presence and / or amount of one or more bacterial species associated with PD in the subject.

[0017] In some embodiments, a method for diagnosing Parkinsonism in a subject is provided, comprising measuring the presence and / or amount of one or more bacterial species associated with Parkinson's disease (PD) in the subject, wherein the presence and / or abnormal amount of one or more bacterial species associated with PD indicates that the subject is at risk of developing or suffering from symptoms of Parkinsonism. In some embodiments, at least one of the one or more bacterial species associated with PD is a bacterial species belonging to the genus Proteus, Bilophila, Roseburia, Pseudoramibacter, Eubacterium, or Veillonellaceae. In some embodiments, the presence and / or amount of one or more bacterial species associated with PD is measured in the intestine of the subject. In some embodiments, the presence and / or abnormal amount of one or more bacterial species associated with PD indicates that the subject is at risk of developing Parkinsonism. In some embodiments, the presence and / or abnormal amount of one or more bacterial species associated with PD indicates that the subject suffers from Parkinsonism. In some embodiments, the Parkinsonism is primary or idiopathic Parkinsonism, secondary or acquired Parkinsonism, hereditary Parkinsonism, Parkinson plus syndrome or multisystem degeneration, or any combination thereof. In some embodiments, the Parkinsonism is Parkinson's disease. In some embodiments, the subject is an adult.

[0018] In some embodiments, a composition is provided that includes one or more bacterial species that are protective against Parkinson's disease (PD). In some embodiments, the composition does not include a bacterial species that exacerbates PD. In some embodiments, at least one of the one or more bacterial species that are protective against PD is a bacterial species belonging to the family Lachnospiraceae, Rikenellaceae, family Peptostreptococcaceae, family Clostridiaceae, genus Enterococcus, genus Clostridium, genus Bacteroides, or genus Butyricicoccus. In some embodiments, the composition does not include at least one of the following bacterial species: Proteus, Bilophila, Roseburia, Pseudoramibacter, Eubacterium, and Veillonellaceae. In some embodiments, the composition does not include at least one of the following bacterial species: Proteus, Bilophila, Roseburia, Pseudoramibacter, Eubacterium, or Veillonellaceae. In some embodiments, the composition does not include at least one of the following bacterial species: Proteus, Bilophila, Roseburia, Pseudoramibacter, Eubacterium, or Veillonellaceae. In some embodiments, the composition does not include pathogenic Clostridium bacteria. In some embodiments, at least one of the one or more bacterial species that are protective against PD is a live bacterium. In some embodiments, the one or more bacterial species that are protective against PD is a live bacterium. The composition may be, for example, a probiotic composition, a nutritional composition, a pharmaceutical composition, or any combination thereof. In some embodiments, the composition is a pharmaceutical composition that includes one or more pharma- ceutically acceptable carriers.

[0019] In some embodiments, a pharmaceutical composition is provided that includes an inhibitor against a microbial metabolite that exacerbates Parkinson's disease (PD) and one or more pharma- ceutically acceptable carriers. The microbial metabolite that exacerbates PD may be any microbial metabolite, for example, a fatty acid or a salt or ester thereof. In some embodiments, the microbial metabolite that exacerbates PD 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 against a microbial metabolite that exacerbates PD may be, for example, an antibody against a microbial metabolite that exacerbates PD, an antibody against an intermediate of in vivo synthesis of a microbial metabolite that exacerbates PD, an antibody against a substrate of in vivo synthesis of a microbial metabolite that exacerbates PD, an inhibitor against an enzyme involved in in vivo synthesis of a microbial metabolite that exacerbates PD, or a combination thereof. [Brief description of the drawings]

[0020] [Figure 1] Schematic showing that signals from gut microbes are required for neuroinflammatory responses and characteristic gut-mediated α-synuclein-dependent motor impairment in Parkinson's disease (PD) models. Gut microbes promote α-synuclein-mediated motor impairment and brain disease, and depletion of gut bacteria reduces microglial activation. In mice treated with short-chain fatty acids (SCFAs), SCFAs alter microglia, thereby enhancing pathological functional changes associated with PD. Additionally, human gut microbiota from PD patients enhances motor dysfunction in mice.

[0021] [Diagram 2]Figures 2A-2F show that gut microbes exacerbate motility and gastrointestinal dysfunction. Mice aged 12-13 weeks were used in the study. n=4-6. Error bars indicate the mean and standard error of three trials per mouse. Data are representative of two experiments. *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 shows that ASO mice with complex microbiota (SPF-ASO) took significantly longer to cross than wild-type littermate mice (SPF-WT) in the beam test, which indicates overall locomotor function. Figure 2B shows that ASO mice with complex microbiota took longer to descend than wild-type littermate mice in the pole test, another test indicating overall locomotor function. Figure 2C shows that SPF-ASO mice showed impaired motor control compared to SPF-WT mice in a test to remove adhesive tape from the bridge of the nose to examine fine motor control. Figure 2D shows that SPF-ASO mice showed abnormalities in a hindlimb hugging reflex test to examine striatal dysfunction. Figures 2A-2D also show that 12-13 week-old ASO mice (GF-ASO) and wild-type mice (GF-WT) separately generated in a germ-free condition showed improved motor impairment in the beam test, pole test, adhesive tape removal test, and hindlimb hugging reflex test. Figures 2E-2F show that at 12-13 weeks of age, no change was observed in the amount of feces excreted in GF-ASO mice, whereas a significant decrease was observed in the total number of fecal pellets excreted in SPF-ASO mice. Figure 2E shows the time course of the amount of feces excreted when placed in a novel environment for 15 minutes. Figure 2F shows the total number of fecal pellets excreted in 15 minutes. See also Figures 3A-3K.

[0022] [Diagram 3]Figures 3A-3K show the body weights of SPF and germ-free (GF) mice (related to Figures 2A-2F), as well as the analysis of mice at further ages. N=4-6. Error bars indicate the mean and standard error. Motor function tests are shown from three trials per mouse. Data are representative of two experiments. 0.1>p>0.05; *p≦0.05; **p≦0.01; ***p≦0.001; ****p≦0.0001. SPF=specific pathogen free; GF=germ-free; WT=wild type; ASO=Thy1-α-synuclein genotype. Figure 3A shows that there was no difference in body weight between 12-13 week old GF-ASO mice and 12-13 week old SPF-ASO mice. Figure 3B shows that SPF-ASO and GF-ASO mice showed impaired motor function in the wire hang test, which measures grip strength, as measured by the time it took them to fall from an upside-down wire mesh. Figures 3C-3G show that with age (24-25 weeks), SPF-ASO mice showed a progressive decline in motor function, but this progressive decline was significantly delayed in GF-ASO mice. Figure 3C shows the beam crossing time of 24-25 week-old mice. Figure 3D shows the pole descent time of 24-25 week-old mice. Figure 3E shows the time it took 24-25 week-old mice to remove the adhesive tape from the bridge of their nose. Figure 3F shows the hindlimb hug reflex score of 24-25 week-old mice. Figure 3G shows the body weight of 24-25 week-old mice. Figures 3H-3I show that at 24-25 weeks of age, no change was observed in the amount of feces excreted in GF-ASO mice, whereas a significant decrease was observed in the total number of fecal pellets excreted in SPF-ASO mice (Figures 3H and 3I). Figure 3H shows the time course of fecal pellets excreted when 24-25 week-old mice were placed in a novel environment for 15 minutes. Figure 3I shows the total number of fecal pellets excreted in 15 minutes by 24-25 week-old mice. Figure 3J shows that the water content of fecal pellets excreted by 12-13 week-old SPF-ASO mice was lower than that of 12-13 week-old GF-ASO mice, revealing that gastrointestinal disorders were improved in germ-free (GF) mice.Figure 3K shows a principal component analysis (PCoA) of all motor scores from the SPF-WT, SPF-ASO, GF-WT and GF-ASO groups. When all motor phenotypes were combined in the PCoA, only the SPF-ASO group significantly deviated, and the GF-ASO mice clustered closely with the WT mice.

[0023] [Figure 4]Figures 4A-4G show that αSyn pathology is increased in mice with gut microbiota. Tissues were collected from 12-13 week old mice. n=3-4. Error bars indicate 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. Figures 4A and 4B show that significant aggregation of αSyn was observed in the caudate putamen (CP) and substantia nigra (SN) of ASO mice in SPF conditions. Figure 4A shows representative photographs of the caudate putamen (CP) of SPF-ASO or GF-ASO mice stained with αSyn aggregate-specific antibody, phosphorylated Ser129-αSyn antibody and Neurotrace (Nissl staining). Figure 4B shows representative photographs of the substantia nigra (SN) of SPF-ASO or GF-ASO mice stained as described above. Figure 4C shows representative Western blots in which Triton-soluble and Triton-insoluble fractions of brain homogenates were immunostained with anti-αSyn antibody to quantify αSyn aggregation. Figures 4D and 4E show that the amount of insoluble αSyn was significantly lower in the brains of GF-ASO mice. Figure 4D shows the results of densitometry of Western blots stained with anti-αSyn antibody and integrating the concentration of all αSyn. Figure 4E shows the results of densitometry of Western blots stained with anti-αSyn antibody and calculating the ratio of insoluble αSyn fraction to soluble αSyn fraction. Figure 4F and Figure 4G show that the amount of αSyn transcripts and protein in the subcapsular midbrain and caudate putamen (CP) were similar between SPF-ASO and GF-ASO mice. Figure 4F shows the results of qRT-PCR analysis of human αSyn in the caudate putamen (CP) or subcapsular midbrain (Mid). Figure 4G shows the results of ELISA analysis of total αSyn in homogenates obtained from the caudate putamen (CP) or subcapsular midbrain (Mid).

[0024] [Diagram 5]Figures 5A-5H show (in relation to Figures 4A-2G and Figures 6A-6H) that gut microbes promote αSyn pathology in a region-specific manner. Figure 5I shows the results of qPCR analysis of bdnf and ddit4 expression in CD11b+ cells obtained from brain homogenates, showing that the expression levels of Bdnf, which has neuroprotective effects, and Ddit4, a cell cycle marker, were upregulated in germ-free (GF) mice. Mice aged 12-13 weeks were used in the study. N=3-4. Error bars indicate the mean and standard error. *p≦0.05. SPF=specific pathogen free; GF=germ-free; WT=wild type; ASO=Thy1-α-synuclein genotype. Figures 5A-5C show that, similar to Figures 4C-4E, a reduction in αSyn aggregation was observed in GF-ASO mice. Figure 5A shows dot blots that specifically detected αSyn aggregates in homogenates of the caudate putamen (CP) and the lower midbrain (Mid) from SPF-ASO and GF-ASO mice. Figure 5B and Figure 5C show the results of densitometric measurements of dot blots of (B) caudate putamen (CP) or (C) lower midbrain homogenates. Figure 5D-5H show that region-specific αSyn aggregation was observed, with less αSyn aggregation in the frontal cortex (FC) in GF-ASO mice than in SPF mice, whereas αSyn aggregation in the cerebellum (CB) was approximately the same amount between SPF and GF mice. Figure 5D shows representative photographs of the frontal cortex (FC) of SPF-ASO or GF-ASO mice stained with αSyn aggregate-specific antibodies, phosphorylated Ser129-αSyn antibodies, and Neurotrace (Nissl staining). Figure 5E shows representative photographs of the cerebellum (CB) of SPF-ASO or GF-ASO mice stained as described above. Figure 5F shows dot blot images of frontal cortex (FC) or cerebellar (CB) homogenates from SPF-ASO and GF-ASO mice immunostained with an αSyn aggregate-specific antibody. Figure 5G and Figure 5H show the results of densitometric measurements of dot blots of (G) frontal cortex (FC) homogenates and (H) cerebellar (CB) homogenates.

[0025] [Figure 6]Figures 6A-6H show that the microbiota activates microglia in an αSyn-dependent manner. Tissues were collected from 12-13 week old mice. n=3-4 (20-60 cells analyzed per region per mouse). Error bars indicate 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 show that compared to SPF-WT mice, GF-WT mice had higher numbers of microglia in the caudate putamen (CP) and substantia nigra (SN) and had longer total length microglial processes. Figure 6A shows representative 3D reconstructions of microglia in the caudate putamen (CP) of SPF-WT, SPF-ASO, GF-WT and GF-ASO mice stained with Iba1 antibody. Figure 6B shows the diameter, number of branches and total length of processes as parameters of microglia in the caudate putamen (CP). Figure 6C shows the diameter, number of branches and total length of processes as parameters of microglia in the substantia nigra (SN). Figure 6D and Figure 6E show that tissue homogenates obtained from the caudate putamen (CP) and lower midbrain of SPF-ASO mice showed a significant increase in the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) compared with those of GF-ASO mice. Figure 6D shows the results of ELISA analysis of TNF-α and IL-6 in homogenates obtained from the caudate putamen (CP). Figure 6E shows ELISA analysis of TNF-α and IL-6 in homogenates from the lower midbrain (Mid). Figure 6F shows qPCR analysis of tnfa and il6 in CD11b+ cells from brain homogenates, showing increased expression of Tnfa and Il6 in SPF-ASO mice but little expression in germ-free (GF) mice. Figures 6G and 6H show that the neuroinflammatory response appears to be region specific, as microglial diameters increased and TNF-α production increased in the frontal cortex (FC) but not in the cerebellum (CB).Figure 6G shows the diameter of microglia present in the frontal cortex (FC) or cerebellum (CB), and Figure 6H shows the results of ELISA analysis of TNF-α in homogenates obtained from the frontal cortex (FC) or cerebellum (CB).

[0026] [Figure 7]Figures 7A - 7I show that signals from microbiota after birth exacerbate motor and gastrointestinal dysfunctions. Mice at 12 - 13 weeks of age were used in the tests. n = 6 - 12. Error bars indicate the mean and standard error when three trials were performed per mouse, data pooled from two independent groups, or data pooled from analyzing 20 - 60 microglia 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: Germ - Free mice colonized with microbiota; WT: Wild - type; ASO: Thy1 - α - synuclein genotype. See also Figures 8A - 8O. Figure 7A shows an overview of the treatment and tests on mice over a time axis. Figures 7B - 7E show that antibiotic - treated (Abx) mice showed little αSyn - dependent motor dysfunction and had results very similar to those of mice born in a germ - free (GF) state. Also, Figures 7B - 7E show that genotype - specific effects as observed in SPF mice were shown in germ - free mice colonized with microbiota after birth (Ex - GF), and that mice overexpressing αSyn showed significant motor dysfunction. Figure 7B shows the cross - time of the beam test apparatus. Figure 7C shows the pole descent time. Figure 7D shows the time required to remove the adhesive tape from the nose. Figure 7E shows the hindlimb clasping reflex score. Figures 7F and 7G show that in antibiotic - treated (Abx) mice, there was also a significant improvement in gastrointestinal function evaluated from fecal excretion, but in Ex - GF mice, the total fecal volume decreased in an αSyn - dependent manner. Figure 7F shows the time course of fecal output when placed in a new environment for 15 minutes. Figure 7G shows the total number of fecal pellets excreted in 15 minutes. Figures 7H and 7I show that among transgenic ASO lines, in Ex - GF mice, the diameter of the microglial cell body increased and was comparable to that of microglia in SPF mice. Also, Figures 7H and 7I show that, unlike Ex - GF mice, the microglia in Abx - ASO mice had a diameter comparable to that of microglia in germ - free (GF) mice.Figure 7H shows representative 3D reconstructions of microglia in the caudate putamen (CP) of Abx-ASO or Ex-GF-ASO mice stained with Iba1 antibody, and Figure 7I shows the diameters of microglia in the CP or substantia nigra (SN).

[0027] [Figure 8]Figures 8A-8O show (in relation to Figures 7A-7I and Figures 9A-9H) that administration of short chain fatty acids (SCFAs) alters microglial morphology and increases αSyn pathology in antibiotic (Abx)-treated mice, Ex-GF mice and SCFA-treated mice. Mice aged 12-13 weeks were used for the study. N=3-6. 20-60 microglia were analyzed per region. Error bars indicate 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 mice; Ex-GF=germ-free mice colonized with microbiota; SCFA=short chain fatty acid-treated mice; WT=wild type; ASO=Thy1-α-synuclein genotype. Figure 8A shows that fecal SCFA concentrations were observed to be lower in germ-free (GF) and antibiotic (Abx)-treated mice compared to SPF mice. In Figure 8A, fecal concentrations of acetate, propionate, and butyrate were normalized by soluble chemical oxygen demand (sCOD). Figures 8B-8C show that in affected brain regions (i.e., caudate putamen (CP) and substantia nigra (SN)), microglia from SCFA-treated mice exhibited morphological features indicative of increased activation compared to those from untreated mice, and exhibited morphological features very similar to those of microglia from Ex-GF and SPF mice. In contrast, microglia from mice treated with antibiotics (Abx) exhibited morphological features similar to those of germ-free (GF) mice. Figure 8B shows the number of branches and the total length of processes as parameters of microglia present in the caudate putamen (CP). Figure 8C shows the number of branches and the total length of processes as parameters of microglia present in the substantia nigra (SN). Figures 8D and 8E show that changes in microglial diameter were also observed in the frontal cortex (FC) but not in the cerebellum (CB), indicating a region-specific response. Figure 8D shows the diameter of microglia present in the frontal cortex (FC). Figure 8E shows the diameter of microglia present in the cerebellum (CB). Figures 8F-8O show that SCFA-treated mice showed aggregation of αSyn compared to untreated and antibiotic (Abx)-treated mice, and the extent of aggregation was comparable to Ex-GF mice.Figures 8F-8I show representative photographs of the caudate putamen (CP), substantia nigra (SN), frontal cortex (FC) and cerebellum (CB) of Abx-ASO, Ex-GF-ASO or SCFA-ASO mice stained with αSyn aggregate-specific antibody, phosphorylated Ser129-αSyn antibody and Neurotrace (Nissl staining). Figure 8J shows dot blot images of homogenates of the caudate putamen (CP), lower midbrain (Mid), frontal cortex (FC) and cerebellum (CB) obtained from Abx-ASO, Ex-GF-ASO and SCFA-ASO mice immunostained with αSyn aggregate-specific antibody. Figures 8K-8N show the results of densitometric measurements of dot blots of homogenates of (K) caudate putamen (CP), (L) lower midbrain, (M) frontal cortex (FC) and (N) cerebellum (CB). Figure 8O shows the results of Western blot detection of αSyn in Triton-soluble and Triton-insoluble fractions of caudate-putamen (CP) homogenates obtained from Abx-ASO and SCFA-ASO mice.

[0028] [Figure 9]Figures 9A-9H show that αSyn-stimulated microglial activation and motor dysfunction are promoted by SCFAs. Mice aged 12-13 weeks were used in the study. N=6-12. Error bars indicate the mean and standard error of three trials per mouse, and are a summary of data from two independent groups or from 20-60 microglia analyzed per area. For ease of understanding, data are plotted together with the controls used in Figures 7A-7I. *p≦0.05; **p≦0.01; ***p≦0.001; ****p≦0.0001. Abbreviations: SPF: specific pathogen free; GF: germ-free; SCFA: short-chain fatty acid-treated; WT: wild type; ASO: Thy1-α-synuclein genotype. See also Figures 8A-8O, 10A-10M and 11A-11H. Figure 9A and Figure 9B show that in the affected regions of the brain (i.e., the caudate putamen (CP) and substantia nigra (SN)), microglia from SCFA-treated mice exhibited morphological features indicative of increased activation compared to those from untreated mice, and exhibited morphological features similar to those of microglia from Ex-GF and SPF mice. In contrast, microglia from mice treated with antibiotics (Abx) exhibited morphological features similar to those of germ-free (GF) mice. Figure 9A shows representative 3D reconstructions of microglia present in the caudate putamen (CP) of wild-type or SCFA-treated ASO mice stained with Iba1 antibody. Figure 9B shows the diameter of microglia present in the caudate putamen (CP) or substantia nigra (SN). Figures 9C-9F show that SCFA-ASO mice exhibited significant functional impairments in several motor tasks compared to untreated GF-ASO mice, specifically, they exhibited impairments in the beam test, pole test, and hindlimb embrace reflex (comparison of SCFA-ASO mice to GF-ASO mice). Figure 9C shows the time to cross the beam test experimental apparatus. Figure 9D shows the time to descend the pole. Figure 9E shows the time required to remove the adhesive tape from the nose. Figure 9F shows the hindlimb hug reflex score. Figure 9G and Figure 9H show that gastrointestinal disorders were also observed in transgenic mice treated with SCFA. Figure 9G shows the time course of fecal volume when placed in a novel environment for 15 minutes.FIG. 9H shows the total number of fecal pellets excreted over a 15-minute period.

[0029] [Figure 10]Figures 10A-10M show (related to Figures 9A-9H) that SCFAs do not directly alter αSyn aggregation. Mice aged 12-13 weeks were used in the study. N=6-12. Error bars indicate the mean and standard error of three trials per mouse. Data are pooled from two independent groups and plotted together with the controls used in Figures 7A-7I for ease of understanding. *p≦0.05; **p≦0.01; ***p≦0.001; ****p≦0.0001. Abbreviations: SPF=specific pathogen free; GF=germ-free; HK=heat-killed bacteria treatment; WT=wild type; ASO=Thy1-α-synuclein genotype. Figures 10A-10G show that various concentrations of SCFAs, either alone or in the form of a mixture, did not promote the aggregation of human αSyn in vitro. Figures 10A-10C show the aggregation kinetics of αSyn measured by ThT fluorescence in the presence of (Figure 10A) sodium acetate, (Figure 10B) sodium propionate, or (Figure 10C) sodium butyrate at the concentrations indicated in the graphs. Figures 10D and 10E show the aggregation kinetics of αSyn measured by ThT fluorescence in the presence of each SCFA mixture. FIG. 10D: SCFA mixture 1-29.6 mM acetate, 11 mM propionate, and 18.5 mM butyrate; SCFA mixture 2-88.8 mM acetate, 33 mM propionate, and 55.5 mM butyrate; FIG. 10E: SCFA mixture 3-0.4 mM acetate, 0.15 mM propionate, and 0.24 mM butyrate; SCFA mixture 4-0.8 mM acetate, 0.3 mM propionate, and 0.47 mM butyrate; SCFA mixture 5-2.0 mM acetate, 0.74 mM propionate, and 1.18 mM butyrate. FIG. 10F and FIG. 10G show the time to reach half the maximum fluorescence intensity value for each SCFA treatment or each SCFA mixture treatment. N=3. Error bars indicate the mean and standard error. Figures 10H and 10I show that various concentrations of SCFAs, either alone or in the form of a mixture, did not change the overall structure of αSyn amyloid fibrils. Figures 10H and 10I show representative atomic force microscopy images of the end products obtained in the αSyn aggregation assay in the absence of SCFAs or in the presence of SCFA Mixture 1.Figures 10J - 10M showed that no motor impairment was induced in germ - free (GF) mice orally administered heat - killed bacteria. From this result, it was suggested that active metabolism of intestinal bacteria is required for the induction of motor impairment. Figure 10J shows the beam crossing time. Figure 10K shows the pole - descent time. Figure 10L shows the time required to remove the adhesive tape from the nose. Figure 10M shows the hind - limb clasping reflex score.

[0030] [Figure 11] Figures 11A - 11H (related to Figures 9A - 9H) show that SCFA - induced α - Syn - mediated motor impairment and lesions are alleviated by minocycline. Mice at 12 - 13 weeks of age were used in the test. N = 6 - 12. Error bars indicate the mean value and standard error when three trials were performed per mouse. Data obtained from two independent groups were pooled. 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 show that oral administration of the anti - inflammatory compound minocycline alone to mice given SCFA was sufficient to reduce the production of TNF - α, decrease the aggregation of α - Syn, improve motor function, and the expression level of the foreign gene did not change. Figure 11A shows the results of ELISA analysis of TNF - α in the caudate putamen (CP) or midbrain (Mid). Figure 11B shows the dot - blot images specifically detecting α - Syn aggregates in the caudate putamen (CP) homogenate and midbrain (Mid) homogenate. Figure 11C shows the results of densitometric concentration measurement of the dot - blots of the caudate putamen (CP) homogenate and midbrain (Mid) homogenate with a densitometer. Figure 11D shows the results of qPCR analysis of the expression of human snca in the whole - brain homogenate. Figure 11E shows the crossing time of the beam test apparatus. Figure 11F shows the pole - descent time. Figure 11G shows the time required to remove the adhesive tape from the nose. Figure 11H shows the hind - limb clasping reflex score.

[0031] [Figure 12]Figures 12A-12E show microbiome perturbations (dysbiosis) observed after transplantation of PD patient samples into germ-free mice. n=3-6. Measurements were made at three time points after microbiota colonization. Error bars show mean and standard error. ***p≦0.001, 999 permutations. Abbreviations: HC: germ-free mice colonized with fecal microbes from healthy controls; PD: germ-free mice colonized with fecal microbes from Parkinson's disease patients; WT: wild type; ASO: Thy1-α-synuclein genotype. See also Figures 13A-13E. Figures 12A and 12B show that in unweighted UniFrac analysis visualized in PCoA, the recipient mouse groups showed profiles closest to those of their corresponding human donors. Figure 12A shows the unweighted UniFrac-based principal coordinate analysis between the microbial communities of the human donors (large circles) and the recipient mice (small circles). Each donor and recipient sample data pair is differentiated by color shading. Figure 12B shows unweighted and weighted UniFrac analysis of the recipient mouse microbial community based on donor ID. Figures 12C and 12D show that the microbial communities of humanized mice transplanted from PD donors were significantly more similar to each other, and that the microbial communities of mice transplanted from healthy donors and humanized mice transplanted from PD donors were less similar, even when classified based on genetic background. Compared to WT recipient mice, the ASO line showed significant differences between mice transplanted from healthy donors and mice transplanted from PD donors, suggesting that genotype influences microbial community composition. Figure 12C shows unweighted and weighted UniFrac analysis of the recipient mouse microbial community based on mouse genotype. FIG. 12D shows a comparison of the results of unweighted and weighted UniFrac analyses of the microbial communities of recipient mice.Figure 12E shows that changes in various bacterial genera were identified in mice colonized with microbiota from PD donors compared to healthy controls. Changes in each bacterial genus between PD and healthy donors were analyzed at the taxon level as a function of recipient mouse genotype. The left bar graph shows the measured percentage and significant difference; the right bar graph shows the fold change between PD and healthy donors. "*" indicates no statistically significant difference.

[0032] [Figure 13] Figures 13A-13E show (related to Figures 12A-12E) that microbial metabolic pathways are altered in humanized mice. N=3-6. KEGG analysis was performed at three time points after microbiota colonization. SCFA abundance was tested at N=21-24. Error bars indicate mean and standard error. *p:<0.05; **p:<0.01; ***p:<0.001. Figure 13A shows a comparison of Bray-Curtis distances between humanized mouse groups. Figure 13B shows a comparison of the average Bray-Curtis distance within the same donor to the average Bray-Curtis distance between different donors, and Figure 13C shows a comparison of the average Bray-Curtis distance within wild type (WT) to Thy1-α-synuclein (ASO) genotypes. Figure 13D shows PICRUSt analysis of specific KEGG families involved in the production of SCFAs (light circles = microbes from healthy controls, dark circles = microbes from PD). Figure 13E shows fecal concentrations and relative abundance of acetate, propionate and butyrate in humanized mice normalized by soluble chemical oxygen demand. Data are from six independent donor pairs. HC = healthy controls; PD = Parkinson's disease.

[0033] [Figure 14]Figures 14A - 14G show that the microbiota from PD patients exacerbates αSyn - mediated motor impairment. Mice at 12 - 13 weeks of age were used in the study. n = 3 - 6. Error bars indicate the mean and standard error when three trials were performed per mouse. #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 obtained from healthy controls; PD: Germ - free mice colonized with fecal microbiota obtained from Parkinson's disease patients; WT: Wild - type; ASO: Thy1 - α - synuclein genotype. See also Figures 15A - 15G and Table 2. Figures 14A - 14F show that in 4 out of 6 pairs (Pair No.1, No.3, No.4, and No.5), αSyn - mediated motor dysfunction was consistently exacerbated by the microbiota obtained from each PD patient donor. Figures 14A - 14F show the beam - crossing time, pole - descent time, adhesive - tape removal time from the nose, and hind - limb clasping reflex score of humanized mice with microbiota from PD patients or humanized mice with microbiota from healthy controls matched to PD patients. Figure 14G shows that when the functional data of all groups were aggregated, it was revealed that the microbiota from PD patients exacerbated the motor impairment of ASO mice in 3 out of 4 tests used in this study compared to the case when microbiota from healthy controls was colonized. The results of each motor task, including the beam test, pole test, adhesive - tape removal test, and hind - limb clasping reflex score, in all independent groups were summarized according to the health status of the fecal donor.

[0034] [Figure 15]Figures 15A-15G show the body weight and fecal excretion of humanized mice (in relation to Figures 14A-14G). Mice at 12-13 weeks of age were used in the study. N = 3-6. Error bars indicate the mean value and standard error when three trials were performed per mouse. 0.05 < p < 0.1; *p ≤ 0.05; **p ≤ 0.01. Abbreviations: HC = germ-free mice colonized with fecal microbiota obtained from healthy controls; PD = germ-free mice colonized with fecal microbiota obtained from Parkinson's disease patients; WT = wild type; ASO = Thy1-α-synuclein genotype. Figures 15A-15F show that there were few changes in the body weight of recipient mice and little change was observed in gastrointestinal function measured by fecal output. Body weight and fecal output when each pair of PD humanized mice and healthy control (HC) humanized mice were placed in a new environment for 15 minutes are shown. (Figure 15A) Pair No. 1, (Figure 15B) Pair No. 2, (Figure 15C) Pair No. 3, (Figure 15D) Pair No. 4, (Figure 15E) Pair No. 5, (Figure 15F) Pair No. 6. Figure 15G shows a principal component analysis aggregating the motor functions among each humanized mouse. As shown in Figure 15G, when all motor functions were visualized by PCoA, significant and extensive differences were shown between mice colonized with the microbiota from PD donors and mice colonized with the gut bacteria from healthy individuals.

Modes for Carrying Out the Invention

[0035] In the following detailed description, reference is made to the accompanying drawings, which form a part of this specification. Unless otherwise indicated, similar symbols in the accompanying drawings generally indicate similar components. The illustrative embodiments described in the detailed description, drawings, and claims are not intended to limit the invention. Also, other embodiments are possible, and other changes are possible, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure as generally described herein and illustrated in the drawings can be adjusted, substituted, combined, and separated into various alternative configurations. All such configurations are expressly contemplated herein.

[0036] Definition of Terms Unless otherwise specified, technical and scientific terms used herein shall have the meanings commonly understood by those skilled in the art to which the present invention belongs.See, for example, Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd 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 purposes of disclosure of the present invention, the following terms are defined as follows:

[0037] As used herein, a "subject" is an animal, such as a vertebrate, preferably a mammal. A "mammal" is defined as an individual belonging to the class Mammalia, including, but not limited to, humans, domestic and farm animals, zoo animals, sport animals, and 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.

[0038] As used herein, "treatment" refers to an intervention (e.g., clinical intervention) made in response to a disease, disorder, or physiological condition exhibited by a patient, particularly a patient suffering from a neurodegenerative disease (e.g., Parkinson's disease). The objectives of treatment may include, but are not limited to, one or more of the following: alleviation or prevention of symptoms; delaying or arresting the progression or worsening of a disease, disorder, or condition; and remission of a disease, disorder, or condition. In some embodiments, "treatment" refers to both therapeutic and prophylactic treatment. Subjects in need of treatment include those already affected by a disease, disorder, or undesirable physiological condition, as well as those in whom a disease, disorder, or undesirable physiological condition needs to be prevented. For example, in some embodiments, treatment may reduce, alleviate, or eliminate symptoms of a disease. As used herein, "prevention" refers to any action that reduces the burden of an individual developing Parkinson's disease-like symptoms later. This prevention may be performed at primary, secondary, and / or tertiary stages. a) Primary prevention avoids the onset of symptoms / disorders / conditions. b) Secondary prevention actions are taken in the early stages of treatment of a condition / disorder / symptom, preventing its progression and manifestation by providing early intervention, c) Tertiary prevention actions are taken to reduce the negative impact of an already existing condition / disorder / symptom, for example by restoring function and / or reducing any condition / disorder / symptom or associated complications.

[0039] A "pharmacologically acceptable" carrier is one that is not toxic to cells or mammals at the dosages and concentrations used therein. "Pharmaceutically acceptable" carriers include, but are not limited to, organic or inorganic solid or liquid additives suitable for the selected method of use, such as oral application or injection. "Pharmaceutically acceptable" carriers are administered in the form of conventional pharmaceutical formulations, including solid formulations such as tablets, granules, powders, and capsules, and liquid formulations such as solutions, emulsions, and suspensions. In many cases, physiologically acceptable carriers are aqueous pH buffers, such as phosphate buffers and citrate buffers. Physiologically acceptable carriers also include antioxidants, such as ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, and immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids; sugars, such as glucose, mannose, and dextrin; chelating agents, such as EDTA; sugar alcohols, such as mannitol and sorbitol; salt-forming counterions, such as sodium; and Tween. TM , Polyethylene glycol (PEG), Pluronic TM In addition, conventional additives such as auxiliary agents, stabilizers, emulsifiers, lubricants, binders, pH adjusters / controllers, and isotonicity agents may be added to the carrier.

[0040] The pharma- ceutically acceptable or suitable carriers include other compounds known to be beneficial to the compromised gastrointestinal tract (e.g., antioxidants such as vitamin C, vitamin E, selenium, zinc, etc.); or food compositions, including but not limited to milk, yogurt, curds, cheese, fermented milk, fermented milk-based products, ice cream, fermented cereal-based products, milk powder, infant formula, tablets, bacterial suspensions, dry oral supplements, or wet oral supplements.

[0041] The therapeutic or protective agent may include a "drug". As used herein, a "drug" refers to a therapeutic or diagnostic agent and includes any substance other than food that is used to prevent, diagnose, mitigate, treat or cure disease (Stedman's Medical Dictionary, 25th Edition (1990)). A "drug" may also include any substance disclosed in at least one of 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, one of the embodiments of the composition described herein is a therapeutic agent. In some embodiments, the drug used in the therapeutic system of the present invention is supported, embedded or encapsulated in a delivery matrix, or incorporated therein.

[0042] As used herein, "nutraceutical" refers to food that has a health benefit (either as a fortified food or a dietary supplement). Testing and regulation of nutritional functional foods is different from that of pharmaceuticals.

[0043] As used herein, "probiotics" refers to live microorganisms that, when administered in adequate amounts, provide health benefits to the host.Probiotics are available in the form of food and dietary supplements, including, but not limited to, capsules, tablets and powders.Foods that contain probiotics include, but are not limited to, dairy products, such as yogurt, fermented and unfermented milk, smoothies, butter, cream, hummus, kombucha, salad dressings, miso, tempeh, nutritional bars, and some juices and soy drinks.

[0044] As used herein, the term "metabolite" refers to any molecule involved in metabolism. A metabolite may be a product, substrate, or intermediate in a metabolic process. For example, a metabolite may 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 acids, hydroxyoctadecadienoic acids, steroids, bile acids, glycolipids, and phospholipids. In some embodiments, the metabolite is a microbial metabolite, e.g., a metabolite produced by a microorganism for the purposes of controlling the growth and development of the microorganism itself, promoting beneficial interactions with other organisms, and inhibiting organisms harmful to the microorganism. A microbial metabolite may be, for example, a low molecular weight compound (<2,500 Da). In some embodiments, a microbial metabolite is an analog of a microbial metabolite. In some embodiments, microbial metabolites and analogs thereof include short chain fatty acids (SCFAs), medium chain fatty acids and long chain fatty acids; and salts and esters of short chain fatty acids, medium chain fatty acids and long chain fatty acids. Fatty acids include, but are not limited to, SCFAs acetate, propionate and butyrate.

[0045] As used herein, the term "antibody" includes polyclonal antibodies, monoclonal antibodies (including full-length antibodies having an Fc region of an immunoglobulin), antibody compositions having specificity for multiple epitopes, multispecific antibodies (e.g., bispecific antibodies, diabodies, and single-chain molecules), and antibody fragments (e.g., Fab or F(ab')2 and Fv). For the structure and properties of various types of antibodies, see, for example, Chapter 6 on page 71 of Basic and Clinical Immunology, 8th Edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton & Lange, Norwalk, Conn., 1994.

[0046] Neurodegenerative disorders Neurological dysfunction is the basis of many human diseases. Behavioral, psychiatric, and neurodegenerative disorders often present with characteristic neuropathologies in the central nervous system (CNS). One type of neuropathology, amyloidosis, arises from the abnormal aggregation of certain neural proteins and disrupts various cellular functions. Insoluble protein aggregates with altered conformation are often found in affected tissues, and structural changes in these aggregates are thought to be a contributing factor in an estimated 50 human diseases. Amyloid-driven neurodegenerative disorders, including Alzheimer's disease, Huntington's disease, and Parkinson's disease (PD), are associated with amyloid proteins. Among these diseases, PD is the second most prevalent neurodegenerative disease in the United States, affecting an estimated 1 million people, or 1% of the U.S. population aged 60 years or older. Approximately 3 million PD patients and their caregivers worldwide suffer from PD, a disease that often leads to debilitating symptoms, including movement disorders such as tremor, rigidity, bradykinesia, and gait disturbances. PD is a multifactorial disease strongly influenced by environmental factors, with genetic factors accounting for less than 10% of cases. Furthermore, aggregation of α-synuclein (αSyn) is thought to be pathogenic in a group of diseases collectively known as synucleinopathies (including PD, multiple system atrophy, and Lewy body diseases). αSyn aggregation progresses in a stepwise manner, resulting in the accumulation of oligomers and non-transient fibrils within neurons. Dopaminergic neurons in the substantia nigra pars compacta (SNpc) appear to be particularly vulnerable to damage caused by the action of αSyn aggregates. Dopamine modulators are the first-line treatment for PD, but they can cause significant side effects and often lose efficacy. There is a need to find safe and effective treatments to address the increasing burden of PD in an ever-aging society.

[0047] Peripheral events have been reported to influence the onset and / or progression of diseases that affect the brain (Dinan and Cryan, 2015). Bidirectional communication between the gut and the brain has been suggested in anxiety, depression, nociception and autism spectrum disorders (ASD) (Mayer et al., 2014; Schroeder and Backhed, 2016; Sharon et al., 2016). Gastrointestinal physiology and motility are influenced by signals generated locally in the gut and from the central nervous system (CNS). Neurotransmitters, immune system signaling, hormones and neuropeptides generated in the gut can subsequently affect the brain (Selkrig et al., 2014; Wall et al., 2014).

[0048] In the human body, microbes are permanently colonized on virtually every surface exposed to the external environment, the majority of which reside within the gastrointestinal tract. The gut microbiota can have profound effects on neural development and the central nervous system (CNS). Germ-free (GF) mice and specific pathogen-free (SPF) mice treated with antibiotics have altered neurogenesis in the hippocampus, resulting in abnormalities in spatial and object recognition. The gut microbiota controls the expression of 5-hydroxytryptamine receptors (5-HT1A), brain-derived neurotrophic factor (BDNF) and NMDA receptor subunit 2 (NR2A). Germ-free (GF) mice exhibit altered myelination of the cerebral cortex and abnormalities in blood-brain barrier function. Furthermore, the mouse gut microbiota promotes the production of gut and blood serotonin, affecting anxiety, hyperactivity and cognition. Additionally, gut microbes present in feces and in the gut mucosa differ between PD patients and healthy controls.

[0049] Gut bacteria can control the differentiation and function of immune cells in the intestine, periphery, and brain. PD patients with dysmotility often have gastrointestinal abnormalities, such as constipation, and intestinal inflammation for years before the onset of dysmotility. Braak hypothesized that abnormal accumulation of αSyn begins in the intestine and spreads to the brain via the vagus nerve in a manner similar to prions. This concept is supported by pathophysiological evidence that αSyn inclusions appear early in the enteric nervous system (ENS) and in the glossopharyngeal and vagus nerves, and that vagotomized individuals are at lower risk for PD. Furthermore, it has been shown that the disease can be induced in the vagus nerve and brainstem simply by injecting αSyn fibers into the intestinal tissue of healthy rodents.

[0050] Treatment of neurodegenerative disorders As disclosed herein, in a subject in need of modulation of the composition of the gut microbiota, the composition of the gut microbiota can be modulated, for example, by reducing or removing microorganisms in the gut microbiota that exacerbate PD, or by introducing a microbiota from a healthy individual, or by both, thereby providing the benefit of slowing the progression of PD in the subject.

[0051] The present specification further discloses a method of treating a neurodegenerative disorder in a subject (e.g., a patient with a neurodegenerative disorder) in need of such treatment, a method of delaying or reducing the likelihood of developing a neurodegenerative disorder in a subject (e.g., a patient with a neurodegenerative disorder) in need of such treatment, and a method of improving a movement disorder in a subject (e.g., a patient with a neurodegenerative disorder) in need of such treatment. In some embodiments, the method comprises regulating the composition of the gut microbiota of the subject. In some embodiments, the method can further improve one or more physical disorders of the subject. The method can, for example, improve one or more gastrointestinal functions of the subject and can relieve constipation in the subject. The movement disorder can include, but is not limited to, tremor, rigidity, bradykinesia, gait disorder, and any combination thereof. In some embodiments, the neurodegenerative disorder is a synucleinopathy, including, but not limited to, primary or idiopathic parkinsonism, secondary or acquired parkinsonism, hereditary parkinsonism, Parkinson-plus syndrome or multiple system degeneration, and any combination thereof.

[0052] In some embodiments, the method includes identifying a subject in need of the method, the subject in need of the method being a subject exhibiting an alpha-synuclein (αSyn) aggregation disorder, e.g., abnormally high levels of αSyn aggregates. In some embodiments, the method includes measuring the rate and / or extent of αSyn aggregation in the subject (e.g., the brain of the subject), measuring the rate and / or extent of clearance of insoluble αSyn protein aggregates in the subject (e.g., the brain of the subject), or a combination thereof. Measuring the rate and / or extent of αSyn aggregation in the subject (e.g., the brain of the subject), measuring the rate and / or extent of clearance of insoluble αSyn protein aggregates in the subject (e.g., the brain of the subject), or a combination thereof, can be performed at various times, e.g., before, simultaneously with, and / or after modulating the composition of the gut microbiota of the subject.

[0053] The present invention further provides a method of reducing microglial activation in a subject in need of reducing microglial activation, a method of reducing alpha-synuclein (αSyn) aggregates in a subject in need of reducing αSyn aggregates, and a method of reducing neuroinflammation in a subject in need of reducing neuroinflammation. In some embodiments, the subject in need of these methods is a subject suffering from a neurodegenerative disease, such as a synucleinopathy (e.g., Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, or any combination thereof). In some embodiments, the subject suffers from PD. In some embodiments, the method includes modulating the composition of the gut microbiota of the subject. The method can, for example, promote the removal of insoluble αSyn protein aggregates (e.g., increase the removal rate, amount, or both of insoluble αSyn protein aggregates), reduce the aggregation of αSyn protein (e.g., reduce the aggregation rate, amount, or both of αSyn protein), or both. In some embodiments, the method comprises measuring the rate and / or extent of aggregation of αSyn in the subject (e.g., the brain of the subject), measuring the rate and / or extent of clearance of insoluble αSyn protein aggregates in the subject (e.g., the brain of the subject), or a combination thereof. Measuring the rate and / or extent of aggregation of αSyn in the subject (e.g., the brain of the subject), measuring the rate and / or extent of clearance of insoluble αSyn protein aggregates in the subject (e.g., the brain of the subject), or a combination thereof can be performed at various time points, for example, before, simultaneously with and / or after modulating the composition of the gut microbiota of the subject.

[0054] The subject in need of the method may be a subject suffering from or at risk of developing a neurodegenerative disorder, such as, for example, an amyloid-induced neurodegenerative disorder, including, but not limited to, Alzheimer's disease, Huntington's disease, Parkinson's disease, or any combination thereof. In some embodiments, the subject in need of the method is a subject suffering from or at risk of developing a synucleinopathy, such as, for example, Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, or any combination thereof. In some embodiments, the subject in need of the method is a subject suffering from or at risk of developing Parkinson's disease. Neurodegenerative disorders include, but are not limited to, primary or idiopathic parkinsonism, secondary or acquired parkinsonism, hereditary parkinsonism, Parkinson plus syndrome, or multiple system degeneration, and any combination thereof.

[0055] As described herein, in some embodiments of the method disclosed herein, the composition of the gut microbiota of the subject is adjusted to restore the composition of the gut microbiota of the subject to a normal level. As used herein, the "normal level" of the composition of the gut microbiota refers to the same state as the composition of the gut microbiota of a subject not suffering from PD (e.g., a healthy subject). Those skilled in the art will appreciate that the composition of the gut microbiota may vary among subjects not suffering from PD (e.g., a healthy subject), and that the normal level can be established as a representative of the composition of the gut microbiota of a population not suffering from PD, i.e., a healthy subject population, and can be used as a comparison control. Various criteria can be used to select a particular subject to be included in the reference population and / or to exclude a particular subject from the reference population, including, but not limited to, the age of the subject (e.g., the reference subject may be included in the same age group as the subject in need of treatment), and the gender of the subject (e.g., the reference subject may be the same gender as the subject in need of treatment).

[0056] As described herein, modulation of the composition of the gut microbiota of the subject can be achieved by a variety of methods, including, but not limited to, fecal transplantation, microbiota normalization, microbiota colonization, gut microbiota reconstitution, probiotic treatment, antibiotic treatment, or a combination thereof.

[0057] In some embodiments, adjusting the composition of the gut microbiota of the subject includes administering one or more antibiotics to the subject. The one or more antibiotics may be, for example, ampicillin, vancomycin, neomycin, gentamicin, erythromycin, or any combination thereof. In some embodiments, the antibiotic treatment does not include administering rifampicin and / or minocycline to the subject. Adjusting the composition of the gut microbiota of the subject can also be, for example, by administering an inhibitor against a microbial metabolite that exacerbates PD to the subject. Examples of inhibitors against a microbial metabolite that exacerbates PD include, but are not limited to, antibodies against one or more microbial metabolites that exacerbate PD, antibodies against in vivo synthetic intermediates of one or more microbial metabolites that exacerbate PD, antibodies against in vivo synthetic substrates of one or more microbial metabolites that exacerbate PD, and inhibitors against one or more enzymes involved in the in vivo synthesis of one or more microbial metabolites that exacerbate PD.

[0058] As used herein, a "microbial metabolite that exacerbates PD" refers to a microbial metabolite that is increased in a subject with PD or a subject with a pathological condition with one or more Parkinson's disease-like symptoms compared to a subject without PD or a subject without a pathological condition with one or more Parkinson's disease-like symptoms (e.g., a healthy subject). For example, the amount of a microbial metabolite in the circulatory system may be different between a subject with PD and a subject without PD. The amount of a microbial metabolite may be altered, for example, in the blood, feces, serum, plasma, body fluids (e.g., cerebrospinal fluid, pleural fluid, amniotic fluid, semen, or saliva) and / or urine of a subject with PD. The microbial metabolite that exacerbates PD includes, but is not limited to, one or more fatty acids, salts or esters thereof, or any combination thereof. The fatty acid may be, for example, a short-chain fatty acid, a medium-chain fatty acid, or a long-chain fatty acid. In some embodiments, the microbial metabolites that exacerbate PD include one or more short chain fatty acids (SCFAs), their salts or esters, or any combination thereof. In some embodiments, the microbial metabolites that exacerbate PD include the SCFA acetate, the SCFA propionate, the SCFA butyrate, or any combination thereof.

[0059] Modulating the composition of the gut microbiota of the subject may, for example, include increasing the amount of one or more bacterial species that have a protective effect against Parkinson's disease (PD) in the subject. In some embodiments, modulating the composition of the gut microbiota of the subject includes administering to the subject a composition comprising one or more bacterial species that have a protective effect against Parkinson's disease (PD). As used herein, "bacterial species that have a protective effect against Parkinson's disease (PD)" refers to a bacterial species that, when present in the gut microbiota of a subject, can protect the subject from developing Parkinson's disease (PD) or a pathological condition accompanied by one or more Parkinson's disease-like symptoms, can delay disease progression in a subject suffering from PD or a pathological condition accompanied by one or more Parkinson's disease-like symptoms, can improve the pathology of PD or a pathological condition accompanied by one or more Parkinson's disease-like symptoms, can alleviate at least one symptom of PD, or achieve a combination thereof. In some embodiments, the bacterial species that are protective against PD are present only in subjects not suffering from PD (e.g., in the gut of the subject) and are absent in subjects suffering from PD or a pathological condition associated with one or more Parkinson's-like symptoms. In some embodiments, the amount of bacterial species that are protective against PD present in a subject suffering from PD or a pathological condition associated with one or more Parkinson's-like symptoms (e.g., in the gut of the subject) is significantly less (e.g., 50% or less, 25% or less, 10% or less, or 5% or less) than in subjects not suffering from PD (e.g., healthy subjects). Bacterial species that are protective against PD include, but are not limited to, bacterial species belonging to the family Lachnospiraceae, Rikenellaceae, Peptostreptococcaceae, Clostridiaceae, genera Enterococcus, Clostridium, Bacteroides, or Butyricicoccus.In some embodiments, the composition is a probiotic composition, a nutritional composition, a pharmaceutical composition, or any combination thereof.

[0060] In some embodiments, regulating the composition of the gut microbiota of the subject comprises reducing the amount of one or more bacterial species that exacerbate PD in the subject. As used herein, "Parkinson's disease (PD)-exacerbating bacterial species" refers to a bacterial species that, when present (alone or with one or more other bacterial species) in the gut microbiota of a subject, can increase the likelihood that the subject will develop Parkinson's disease (PD) or a pathological condition with one or more Parkinson's disease-like symptoms, can promote the onset of PD or a pathological condition with one or more Parkinson's disease-like symptoms, can increase the severity of PD pathology or a pathological condition with one or more Parkinson's disease-like symptoms, can worsen at least one symptom of PD, or a combination thereof. In some embodiments, the PD-exacerbating bacterial species is present only in a subject (e.g., the gut of the subject) that suffers from PD or a pathological condition with one or more Parkinson's disease-like symptoms, and is not present in a subject (e.g., a healthy subject) that does not suffer from PD. In some embodiments, the amount of bacterial species present in a subject not suffering from PD (e.g., in the intestine of the subject) that exacerbates PD is significantly less (e.g., 50% or less, 25% or less, 10% or less, or 5% or less) than that of a subject suffering from PD or a pathological condition with one or more Parkinson's disease-like symptoms. In some embodiments, the bacterial species that exacerbates PD is a bacterial species belonging to the genera Proteus, Bilophila, Roseburia, Pseudoramibacter, Eubacterium, or Veillonellaceae. In some embodiments, the bacterial species that exacerbates PD is a short chain fatty acid (SCFA)-producing bacterium, for example, a SCFA-producing bacterium belonging to the KEGG family K00929, K01034, or K01035.

[0061] In some embodiments, modulating the composition of the subject's gut microbiota comprises introducing into the subject undergoing treatment gut microbiota obtained from a healthy subject.

[0062] In some embodiments, a method for treating a neurodegenerative disorder in a subject is provided, comprising one or more of administering an antibiotic to the subject; and administering an inhibitor against a microbial metabolite that exacerbates Parkinson's disease (PD) to the subject. In some embodiments, a method for delaying or reducing the likelihood of developing a neurodegenerative disorder in a subject is provided, comprising one or more of administering an antibiotic to the subject; and administering an inhibitor against a microbial metabolite that exacerbates Parkinson's disease (PD) to the subject. In some embodiments, a method for improving Parkinson's disease-like symptoms in a subject in need of improvement is provided, comprising one or more of administering an antibiotic to the subject; administering an anti-inflammatory agent to the subject; and administering an inhibitor against one or more microbial metabolites that exacerbate Parkinson's disease (PD) to the subject. The antibiotic may not be rifampicin or may not be minocycline. The neurodegenerative disorder may be, for example, an amyloid-induced neurodegenerative disorder, including, but not limited to, Alzheimer's disease, Huntington's disease, Parkinson's disease, or any combination thereof. In some embodiments, the neurodegenerative disorder is a synucleinopathy, for example, Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, or any combination thereof.

[0063] As discussed 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, mefenamic acid, metamizole, piroxicam, celecoxib, and the like. In some embodiments, the anti-inflammatory agent is a prohormone that regulates the inflammatory process, such as, but not limited to, prohormone convertase 1, proopiomelanocortin, the prohormone B-type natriuretic peptide, the SMR1 prohormone, and the like. In some embodiments, the anti-inflammatory agent is an enzyme with anti-inflammatory properties, such as, but not limited to, bromelain, papain, serrapeptidase, proteolytic enzymes, such as pancreatin (a mixture of trypsin, amylase, and lipase), and the like. In some embodiments, the anti-inflammatory agent is a peptide having an anti-inflammatory effect, and includes, but is not limited to, phospholipase A2 inhibitors, such as antiflavin-1 (a peptide corresponding to the amino acid sequence of 246-254 of lipocortin); antiflavin-2 (a peptide corresponding to the amino acid sequence of 39-47 of uteroglobin); S7 peptide (a peptide that inhibits the interaction between interleukin-6 and interleukin-6 receptor); RP1 (a prenylated protein inhibitor); and peptides similar thereto. In some embodiments, the anti-inflammatory peptide is cortistatin, a cyclic neuropeptide related to somatostatin, a peptide corresponding to the N-terminal fragment of SV-IV protein, a conserved region of E-selectin, L-selectin, and P-selectin, and the like. Other anti-inflammatory agents include, but are not limited to, collagen hydrolysates, milk micronutrient concentrates (e.g., MicroLactin®; available from Stolle Milk Biologics, Inc., Cincinnati, Ohio), milk protein hydrolysates, casein hydrolysates, whey protein hydrolysates, and vegetable protein hydrolysates.In some embodiments, the anti-inflammatory agent is a plant extract having anti-inflammatory properties, including, but not limited to, blueberry extract, boswellia extract, acacia catechu extract, scutellaria extract, celery seed extract, chamomile extract, cherry extract, hornwort extract, eucalyptus extract, evening primrose extract, ginger extract, hawthorn fruit extract, horsetail extract, hawthorn bark extract, licorice extract, turmeric extract, white willow extract, willow bark extract, and yucca extract.

[0064] In some embodiments, the anti-inflammatory agent and the antibiotic are not minocycline.Parkinson's disease-like symptoms include, but are not limited to, motor dysfunction, enhanced aggregation of αSyn, abnormal activation of microglia, tremor, bradykinesia, muscle rigidity, abnormal posture and balance, loss of motility, speech disorder, writing disorder, and any combination thereof.

[0065] The inhibitor against a microbial metabolite that aggravates PD may be, for example, an antibody against one or more microbial metabolites that aggravate PD, an antibody against an in vivo synthetic intermediate of one or more microbial metabolites that aggravate PD, an antibody against an in vivo synthetic substrate of one or more microbial metabolites that aggravate PD, or an inhibitor against an enzyme involved in the in vivo synthesis of one or more microbial metabolites that aggravate PD.

[0066] In the methods described herein, the adjustment of the composition of the gut microbiota of the subject and / or the administration of an inhibitor against microbial metabolites that exacerbate PD to the subject may be performed alone, or may be performed in combination with one or more other therapies, such as antibiotic therapy or anti-inflammatory therapy; or may be performed alone to achieve therapeutic effect.For example, in some embodiments of the methods disclosed herein, the subject or the subject in need of the method has not been treated with an antibiotic and / or an anti-inflammatory agent.In some embodiments, the method does not include administering an antibiotic and / or an anti-inflammatory agent to the subject or the subject in need of the method.In some embodiments, the subject or the subject in need of the method has not been treated with rifampicin and / or minocycline. In some embodiments, the subject or subject in need of the method has not been treated with an antibiotic and / or an anti-inflammatory agent 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 modulating the composition of the gut microbiota or otherwise treating (e.g., administering one or more inhibitors of a microbial metabolite that exacerbates PD). In some embodiments, the subject or subject in need of the method has not been treated with an antibiotic and / or an anti-inflammatory agent 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 modulating the composition of the gut microbiota or otherwise treating (e.g., administering one or more inhibitors of a microbial metabolite that exacerbates PD).

[0067] In some of the embodiments of the methods disclosed herein, the methods further comprise identifying a subject in need of said treatment by determining the presence and / or amount of one or more bacterial species associated with PD in said subject.

[0068] Diagnosis of Parkinson's Disease As disclosed herein, by identifying specific PD-associated gut microbes in a subject, PD can be diagnosed before the onset of severe motor symptoms and / or such PD-associated gut microbes can be used as markers of disease progression severity.

[0069] Methods for diagnosing Parkinsonism in a subject are provided. In some embodiments, the methods include measuring the presence and / or amount of one or more bacterial species associated with Parkinson's disease (PD) in the subject, and the presence and / or abnormal amount of one or more bacterial species associated with PD indicates that the subject is at risk of developing or suffers from symptoms of Parkinsonism. In some embodiments, the presence and / or amount of one or more bacterial species associated with PD is measured in the intestine of the subject.

[0070] As used herein, a "Parkinson's disease (PD)-associated bacterial species" is a bacterial species whose abundance is altered in the gut microbiota of a subject suffering from PD or a pathological condition with one or more Parkinson's disease-like symptoms, compared to a subject not suffering from PD or a subject not suffering from a pathological condition with one or more Parkinson's disease-like symptoms (e.g., a healthy subject). In some embodiments, a "PD-associated bacterial species" is whose abundance is increased in the gut microbiota of a subject suffering from PD or a pathological condition with one or more Parkinson's disease-like symptoms, compared to a subject not suffering from PD or a subject not suffering from a pathological condition with one or more Parkinson's disease-like symptoms (e.g., a healthy subject). In some embodiments, a "PD-associated bacterial species" is whose abundance is decreased in the gut microbiota of a subject suffering from PD or a pathological condition with one or more Parkinson's disease-like symptoms, compared to a subject not suffering from PD or a subject not suffering from a pathological condition with one or more Parkinson's disease-like symptoms (e.g., a healthy subject). In some embodiments, the PD-associated bacterial species is present only in subjects suffering from PD or a pathological condition with one or more Parkinson's-like symptoms (e.g., in the intestines of the subjects) and is absent in subjects not suffering from PD (e.g., healthy subjects). In some embodiments, the amount of the PD-associated bacterial species in subjects not suffering from PD (e.g., in the intestines of the subjects) is significantly less (e.g., 50% or less, 25% or less, or 10% or less) than in subjects suffering from PD or a pathological condition with one or more Parkinson's-like symptoms. In some embodiments, the PD-associated bacterial species is a bacterial species belonging to the genera Proteus, Bilophila, Roseburia, Pseudoramibacter, Eubacterium, or Veillonellaceae. In some embodiments, the PD-associated bacterial species is a short chain fatty acid (SCFA)-producing bacterium, for example, a SCFA-producing bacterium belonging to the KEGG family K00929, K01034, or K01035.

[0071] The presence and / or abnormal amount of one or more bacterial species associated with PD can indicate that the subject is at risk of developing parkinsonism or that the subject suffers from parkinsonism.The parkinsonism can be, for example, primary or idiopathic parkinsonism, secondary or acquired parkinsonism, hereditary parkinsonism, Parkinson plus syndrome or multisystem degeneration, or any combination thereof.In some embodiments, the parkinsonism is Parkinson's disease.In some embodiments, the subject is an adult.

[0072] composition The present specification further discloses a composition comprising one or more bacterial species that have a protective effect against Parkinson's disease (PD). Bacterial species that have a protective effect against PD include, but are not limited to, bacterial species belonging to the family Lachnospiraceae, Rikenellaceae, family Peptostreptococcaceae, family Clostridiaceae, genus Enterococcus, genus Clostridium, genus Bacteroides or genus Butyricicoccus. In some embodiments, the composition does not contain bacterial species that exacerbate PD. For example, in some embodiments, the composition does not include a bacterial species belonging to at least one of the genera Proteus, Bilophila, Roseburia, Pseudoramibacter, Eubacterium, and Veillonellaceae. In some embodiments, the composition does not include a bacterial species belonging to the genera Proteus, Bilophila, Roseburia, Pseudoramibacter, Eubacterium, or Veillonellaceae.

[0073] The composition may be in various types of compositions, such as a probiotic composition, a nutritional composition, a pharmaceutical composition, or any combination thereof. In some embodiments, the composition is a food, a beverage, or a dietary supplement. In some embodiments, the composition is a pharmaceutical composition comprising one or more pharma- ceutically acceptable carriers. In some embodiments, the pharmaceutical composition comprises a gastric acid suppressant, an antacid, an H2 antagonist, a proton pump inhibitor, or a combination thereof. The composition may be in various forms, such as a lyophilized, pulverized, or powdered formulation; a suspension (e.g., a suspension in which the bacteria is suspended); a liquid culture, or a lyophilized, pulverized, or powdered liquid; or a pharmaceutical composition dissolved in, for example, saline.

[0074] The composition may be in the form of capsules, tablets, ice slurries, 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, tempeh, nutritional bars, and some juices and soy drinks.In some embodiments, the composition is formulated as a dosage form for delivery in the form of enteric capsules, enteric microcapsules, powder suitable for reconstitution, nasoduodenal infusion, or enema or colonoscopic infusion; or as a pharmaceutical composition to be added to food, food additives, dairy-based products, soy-based products, or their derivatives, or jellies or yogurt.

[0075] The present specification further discloses a pharmaceutical composition comprising an inhibitor against a microbial metabolite that aggravates PD. The microbial metabolite that aggravates PD may be, for example, a fatty acid or a salt or ester thereof. In some embodiments, the microbial metabolite that aggravates PD 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 may be, for example, an antibody against a microbial metabolite that aggravates PD, an antibody against an intermediate of in vivo synthesis of a microbial metabolite that aggravates PD, an antibody against a substrate of in vivo synthesis of a microbial metabolite that aggravates PD, or an inhibitor against an enzyme involved in in vivo synthesis of a microbial metabolite that aggravates PD. The pharmaceutical composition may, for example, include one or more pharma- ceutically acceptable additives. The pharmaceutical composition may be used to treat various disorders / diseases, including, but not limited to, neurodegenerative disorders (such as Parkinson's disease).

[0076] Further provided are pharma- ceutically acceptable prodrugs of said pharmaceutical compositions and methods of treatment using such pharma- ceutically acceptable prodrugs. "Prodrug" refers to a precursor of a given compound, which, after administration to a subject, undergoes a chemical or physiological process, such as solvolysis or enzymatic degradation, in vivo to produce the given compound, or is exposed to physiological conditions to produce the given compound (e.g., a prodrug that is exposed to physiological pH and converted to a given drug). A "pharmaceutically acceptable prodrug" is a prodrug that is non-toxic and biologically acceptable, or biologically suitable for administration to a subject. Methods for selecting and preparing suitable prodrug derivatives are exemplified, for example, in Bundgaard, Design of Prodrugs (Elsevier Press, 1985).

[0077] Further provided are pharma- ceutically active metabolites of said pharmaceutical compositions and the use of such metabolites in the methods of the present invention. "Pharmaceutically active metabolites" refer to pharmacologically active in vivo metabolites of a compound or its salt. Prodrugs and active metabolites of a compound may be identified using conventional techniques known or available in the art. See, e.g., 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).

[0078] The compounds described herein can be prepared in any suitable formulation. For an overview, see pages 780-857 of Remington's Pharmaceutical Sciences, (2000) Hoover, JE editor, 20th edition, Lippincott Williams and Wilkins Publishing Company, Easton, Pa. A formulation suitable for the appropriate route of administration is selected. Routes of administration include oral, parenteral, inhalation, topical, rectal, nasal, buccal, vaginal, implanted reservoir, or other dosing methods. When a compound is sufficiently acidic or basic to form a stable non-toxic acid or base salt, it may be appropriate to administer the compound in the form of a salt. Pharmaceutically acceptable salts include organic acid addition salts formed with acids that generate physiologically acceptable anions, such as tosylate, methanesulfonate, acetate, citrate, malonate, tartrate, succinate, benzoate, ascorbylates, α-ketoglutarate, and α-glycerophosphate. Suitable inorganic salts may also be formed, including hydrochlorides, sulfates, nitrates, bicarbonates, and carbonates. Pharmaceutically acceptable salts may be obtained using standard methods known in the art, for example, by reacting a sufficiently basic compound, such as an amine, with a suitable acid that generates a physiologically acceptable anion to generate a pharma-ceutically acceptable salt. Alkali metal (e.g., sodium, potassium, or lithium) carboxylates or alkaline earth metal (e.g., calcium) carboxylates may also be formed.

[0079] When the compounds described herein are administered in the form of a pharmaceutical composition, the compounds can be formulated by mixing with pharma- ceutically acceptable additives and / or carriers. For example, the compounds described herein can be administered orally as neutral compounds or pharma- ceutically acceptable salts, or can be administered intravenously in saline. For this purpose, conventional buffers such as phosphate buffer, bicarbonate buffer, citrate buffer, etc. can be used. Those skilled in the art will be able to modify the formulations within the teachings of the present specification to provide various formulations suitable for specific routes of administration. In particular, the compounds described herein may be modified to improve their solubility in water or other bases, which can be easily achieved, for example, by minor modifications (such as salt formation, esterification, etc.), and such methods are well known to those skilled in the art. Furthermore, those skilled in the art are also well known to be able to control the pharmacokinetics of the compounds of the present invention and maximize the beneficial effects on patients by modifying the route and dosing schedule of a particular compound.

[0080] The pharmaceutical compositions described herein may be soluble in organic solvents, such as chloroform, dichloromethane, ethyl acetate, ethanol, methanol, isopropanol, acetonitrile, glycerin, N,N-dimethylformamide, N,N-dimethylacetamide, dimethylsulfoxide, or any combination thereof. In some embodiments, the formulation is prepared by mixing the agent with a pharma- ceutically acceptable carrier. In some embodiments, the formulation may be prepared using a method comprising: a) dissolving the agent described herein in a water-soluble organic solvent, a non-ionic solvent, a water-soluble lipid, a cyclodextrin, a vitamin (such as tocopherol), a fatty acid, a fatty acid ester, a phospholipid, or a combination thereof to provide a solution; and b) adding saline or a buffer containing 1-10% sugar solution. The sugar may include, for example, dextrose. The pharmaceutical compositions obtained using the methods of the present invention are stable and useful in veterinary and clinical applications.

[0081] The water-soluble organic solvents used in the method of the present invention include, but are not limited to, polyethylene glycol (PEG), alcohols, acetonitrile, N-methyl-2-pyrrolidone, N,N-dimethylformamide, N,N-dimethylacetamide, dimethylsulfoxide, or combinations thereof. Alcohols include, but are not limited to, methanol, ethanol, isopropanol, glycerin, or propylene glycol.

[0082] Water-soluble nonionic surfactants for use in the methods of the present invention include, but are not limited to, CREMOPHOR® EL, polyethylene glycol modified CREMOPHOR® (polyoxyethylene glycerol triricinoleate 35), hydrogenated CREMOPHOR® RH40, hydrogenated CREMOPHOR® RH60, PEG succinate, polysorbate 20, polysorbate 80, SOLUTOL® HS (polyethylene glycol 660 12-hydroxystearate), sorbitan monooleate, poloxamer, LABRAFIL® (ethoxylated persic oil), LABRASOL® (caprylcaproyl macrogol-8-glyceride), GELUCIRE® (glycerol esters), SOFTIGEN® (PEG-6 caprylic acid glyceride), glycerin, glycol-polysorbates, and combinations thereof.

[0083] The water-soluble lipid used in the method of the present invention includes, but is not limited to, vegetable oil, triglyceride, vegetable-derived oil, or combinations thereof.Lipid oil includes, but is not limited to, castor oil, polyoxyl castor oil, corn oil, olive oil, cottonseed oil, peanut oil, peppermint oil, safflower oil, sesame oil, soybean oil, hydrogenated vegetable oil, hydrogenated soybean oil, coconut oil triglyceride, palm kernel oil triglyceride, and hydrogenated versions thereof, or combinations thereof.

[0084] Fatty acids and fatty acid esters for use in the methods of the present invention include, but are not limited to, oleic acid, monoglycerides, diglycerides, mono fatty acid esters of PEG, di fatty acid esters of PEG, or combinations thereof.

[0085] Cyclodextrins for use in the methods of the present invention include, but are not limited to, α-cyclodextrin, β-cyclodextrin, hydroxypropyl-β-cyclodextrin, or sulfobutylether-β-cyclodextrin.

[0086] Phospholipids for use in the methods of the present invention include, but are not limited to, soybean phosphatidylcholine, distearoylphosphatidylglycerol, and hydrogenated versions thereof, or combinations thereof.

[0087] Those skilled in the art will be able to modify the formulations within the scope of the teachings of this specification to provide various formulations suitable for specific administration routes. For example, the compounds described herein may be modified to improve their solubility in water or other bases. Furthermore, those skilled in the art will be aware that the pharmacokinetics of the compounds of the present invention can be controlled and the beneficial effects on patients can be maximized by modifying the administration route and dosing schedule of a particular compound.

[0088] The pharmaceutical compositions disclosed herein may be administered orally, parenterally, by inhalation, topically, rectally, nasally, buccal, vaginally, via an implanted reservoir, or by any other method. As used herein, "parenterally" includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, intracranial, or infusion.

[0089] Sterile injectable compositions (e.g., sterile injectable aqueous or oily suspensions) may be prepared according to techniques known in the art using suitable dispersants or wetting agents and suspending agents. Sterile injectable preparations may be sterile injectable solutions or suspensions in non-toxic parenterally acceptable diluents or solvents. Examples of parenterally acceptable carriers and solvents that can be used include mannitol, water, Ringer's solution, and isotonic saline. Suitable carriers and other pharmaceutical composition ingredients are usually sterilized.

[0090] In addition, sterile fixed oils (e.g. synthetic monoglycerides or diglycerides) are also conventionally used as solvents or suspension media. As pharmaceutically acceptable oils, fatty acids such as oleic acid and its glyceride derivatives are also useful for the preparation of injections, such as olive oil or castor oil, especially polyoxyethylated olive oil or polyoxyethylated castor oil. The oil or suspension to be prepared may further contain long-chain alcohol diluents, long-chain alcohol dispersants, carboxymethylcellulose, or similar dispersants. In addition, various emulsifiers or bioavailability enhancers that are commonly used in the manufacture of pharmaceutically acceptable solids, liquids, or other dosage forms can also be used in the preparation of the formulation.

[0091] Compositions for oral administration may be in any orally acceptable dosage form, including, but not limited to, tablets, capsules, emulsions, aqueous suspensions, aqueous dispersions, and aqueous solutions. For tablets for oral administration, carriers such as lactose and corn starch are commonly used. Lubricants such as magnesium stearate can also be added. For oral administration in capsules, diluents such as lactose and dried corn starch are useful. For oral administration of aqueous suspensions or emulsions, the active ingredient can be suspended or dissolved in an oil phase to which an emulsifying or suspending agent is added. Certain sweeteners, flavorings, or colorings can also be added as required. Nasal aerosol or inhalation compositions can be prepared according to techniques well known in the pharmaceutical formulation art, and can be prepared as a solution in, for example, saline, with suitable preservatives (e.g., benzyl alcohol), absorption enhancers to improve bioavailability, and / or other solubilizing or dispersing agents known in the art.

[0092] The following examples disclose certain aspects of the above-described embodiments in further detail, but are not intended to limit the scope of the present disclosure in any way. EXAMPLES

[0093] Materials and methods In Examples 1 to 8 described below, the following experimental materials and methods were used. [Table 1] JPEG0007655693000002.jpg236161JPEG0007655693000003.jpg48161

[0094] mouse BDF1 lineage female Thy1-αSyn mice carrying a heterozygous Thy1-α-synuclein gene on the X chromosome were mated with wild-type male BDF1 mice to obtain male ASO and WT mice as littermates for the study. Male BDF1 mice were obtained by mating female C57BL / 6 mice with male DBA / 2 mice (Charles River, Hollister, CA). Mating pairs were replenished with female transgenic mice and newly generated male BDF1 mice every 6 months. Germ-free (GF) Thy1-αSyn mating pairs were obtained by Caesarean section using newly generated male mice every 6 months. The uterus was surgically removed, the fetuses were delivered, and the mice obtained under microbiologically sterile conditions were raised by germ-free (GF) Swiss-Webster foster mice. SPF (antibiotic-treated) and Ex-GF mice were housed in autoclaved, ventilated microisolator cages. GF ​​mice and short-chain fatty acid (SCFA)-treated mice were housed in open cages in soft-film isolators and maintained under microbiologically germ-free conditions. Germ-free conditions were checked every 2 weeks by PCR analysis of 16s rRNA from fecal DNA and by plating fecal pellets on Brucella blood agar under anaerobic conditions and on tryptic soy blood agar under aerobic conditions.

[0095] All mice, colonized or not, were maintained on the same 12-h light / dark cycle with autoclaved diet (LabDiet Laboratory Autoclavable Diet 5010, St. Louis, MO) and water ad libitum and housed in the same facility. Antibiotic-treated mice received 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 drinking water starting at 5–6 weeks of age and continuing until 12–13 weeks of age. Ex-GF mice were generated by resuspending cecal contents from three wild-type male BDF1 mice in sodium bicarbonate buffer and gavaging the resulting suspension into 5-6 week-old germ-free (GF) mice to establish the microbiota. SCFA-treated mice were given drinking water containing sodium acetate (67.5 mM; Sigma-Aldrich), sodium propionate (25 mM; Sigma-Aldrich), and sodium butyrate (40 mM; Sigma-Aldrich) starting at 5-6 weeks of age and continued until 12-13 weeks of age. Treatment with minocycline (Arcos Organics) was performed by providing free access to drinking water containing 2 g / L minocycline from 5-6 weeks of age until 12-13 weeks of age, in conjunction with SCFA administration. The heat-killed germ-free mice were treated with approximately 5 × 10 Escherichia coli MC4100 (kindly provided by Matthew Chapman, University of Michigan) grown in LB liquid medium (lysogeny medium). 8 cfu / mL was generated by washing twice in phosphate buffered saline, boiling for 45 min, and adding it to drinking water, which was available ad libitum.

[0096] Human Donors and Donor Criteria Human donors were selected from patients seen at the Movement Disorder Clinic at Rush University. Parkinson's disease (PD) was diagnosed according to the UK Brain Bank Criteria. Exclusion criteria for PD patients were atypical or secondary parkinsonism; use of probiotics or antibiotics for 3 months prior to sample collection; use of NSAIDs; primary gastrointestinal disease; history of chronic gastrointestinal disease (including IBD and celiac disease); unstable medical, neurological, or psychiatric disease; low platelet count (<80,000); untreatable prolongation of PT time (9-15 seconds); or history of bleeding that could not be biopsied. The rectal and sigmoid mucosa of the patients were examined by sigmoidoscopy and H&E histological staining, and both were normal. Healthy controls were matched to the PD patient group as much as possible. Healthy subjects were inclusively selected as follows: normal physical and blood test results; no gastrointestinal complaints, symptoms, or medical history; no neurodegenerative diseases; and no use of probiotics, antibiotics, NSAIDs, or prescription drugs for at least 3 months prior to sample collection.

[0097] Motility and gastrointestinal function tests All motor function evaluations were performed in the same gnotobiotic animal facility, except for the humanized mice. The experiments with the humanized mice were performed in a laminar flow biosafety cabinet in the same facility. The motor function tests were performed in all mice during the 7th to 9th hours of the light phase. All tests were performed similarly to the study described in Fleming et al., 2004, Early and progressive sensorimotor anomalies in mice overexpressing wild-type human alpha-synuclein. J. Neurosci. 24, 9434-9440, which is incorporated herein in its entirety. The beam test (beam crossing) was performed first, and the mice were allowed to rest for about 1 hour, after which the pole test (pole descent) was performed. The adhesive tape removal test and hind limb scoring were performed the next day. The number of fecal pellets excreted was counted within 3 days, and tissues were collected immediately thereafter.

[0098] Beam Test Four 0.25-m-long pieces were assembled to create a 1-m-long Plexiglas beam (Stark's Plastics, Forest Park, OH). Each piece was tapered to 3.5, 2.5, 1.5, and 0.5 cm wide, with a 1-cm overhang 1 cm below the top of the beam. The widest piece was used as a platform for the mouse to rest on, and the home cage was placed at the narrowest end. Mice were trained for 2 days to traverse the entire length of the beam before testing. On the first day of training, the home cage was placed close to the platform, and the mouse was guided forward to move forward on the tapered beam for the first trial. Two more trials were then conducted with reduced or no assistance to encourage the mouse to move steadily forward on the beam. On the second day of training, three trials were conducted to traverse the beam. After training, the mouse was able to move forward with little or no assistance. On the third day, the latency for the mouse to cross the beam and reach the platform to the home cage was measured in three trials. The latency was measured when the mouse placed its forelimbs on a 2.5 cm-wide platform and ended when one of its forelimbs reached the home cage.

[0099] Pole Test A 0.5m long, 1cm diameter pole was wrapped in a non-adhesive shelf sheet to make it easier for the mouse to grasp, and placed in the home cage. The mouse was trained for 2 days to descend from the top of the pole to the home cage. Three trials were conducted on the first day of training. In the first trial, the mouse was asked to grasp the pole facing down at 1 / 3 height from the floor, in the second trial, the mouse was asked to grasp the pole facing down at 2 / 3 height from the floor, and in the third trial, the mouse was asked to grasp the pole facing down at the top. In the second day of training, three trials were conducted to have the mouse grasp the pole facing down and descend to the home cage. On the day of the test, the mouse was asked to grasp the pole facing down at the top, and the time it took to descend to the home cage was measured. The time it took to descend the pole was measured from the moment the experimenter released the mouse, and was ended when one of the mouse's hind legs reached the floor of the home cage.

[0100] Adhesive tape removal test A 1 / 4-inch diameter circular adhesive label (Avery, Glendale, CA) was attached to the bridge of the nose between the nostrils and the forehead. The mouse was placed in its home cage (where other mice had been removed) and the latency to completely remove the adhesive sticker was measured. The test was performed three times and the results were recorded.

[0101] Hindlimb hug reflex scoring The experiment was performed in a similar manner 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 (the entire contents of which are incorporated herein by reference). The mice were pinched by the middle of their tails and slowly lifted upwards, and observed for approximately 5-10 seconds. Mice were scored 0, 1, 2, or 3 based on the degree to which their hind limbs were bent inward. Mice that moved their limbs freely and extended their limbs outward were scored 0 as not exhibiting an embrace reflex. Mice that had one of their hind limbs bent inward or had only a portion of their limb bent inward while being lifted were scored 1. Mice that had their limbs bent inward for the majority of the observation time, but still showed some flexibility, were scored 2. Mice were given a score of 3 if they immediately flexed their hind limbs inwards, became completely paralyzed, and showed no flexibility whatsoever.

[0102] Wire Hang Test A mouse was placed in the center of a 30 cm x 30 cm wire mesh with 1 cm mesh width. The wire mesh was turned upside down and placed on a support platform installed about 40 cm above the open cage with deep bedding. The time it took for the mouse to fall off the wire mesh was measured. If the mouse remained clinging to the wire mesh, the measurement time was recorded as 60 seconds.

[0103] Excretion of feces Mice were removed from their home cages and placed in a 12 cm × 25 cm semi-transparent cylindrical container. Fecal pellets were counted every 5 min, and the cumulative number was counted over 15 min. In addition, principal component analysis was performed on all motor functions using behavioral data collected from subjects who performed at least three tasks on MATLAB software (MathWorks). Data were centered and normalized (s = 1) before running the PCA function. Only PC1 and PC2, which accounted for 70.5% of the variance, were plotted using the factor loadings corresponding to each subject.

[0104] Immunostaining and reconstruction of microglial images After administering pentobarbital to sedate the mice and thoroughly perfusing with phosphate-buffered saline, the brains were cut and the hemispheres were fixed in 4% (w / v) paraformaldehyde. 50 mm sagittal sections were made using a vibratome. Free-floating sections were stained with anti-aggregated / fibrillated αSyn antibody MJFR1 (1:1000; rabbit; AbCam, Cambridge, UK), anti-phosphorylated Ser129 αSyn antibody (1:1000; mouse; Biolegend, San Diego, CA) and Neurotrace (Life Technologies, Carlsbad, CA), or anti-Iba1 antibody (1:1000; rabbit; Wako Pure Chemical Industries, Richmond, VA), and then stained with anti-mouse IgG-AF488 and anti-rabbit IgG-AF546 antibodies (1:1000; Life Technologies). Stained sections were mounted with ProFade Diamond (Life Technologies) and photographed with a Zeiss LSM800 confocal microscope equipped with a 10x objective. Two to three fields per field per mouse were photographed per region, and images were merged and analyzed using ImageJ software. To reconstruct microglial images, z-stack data were imaged every 1 mm using Imaris software, as described in Erny et al., 2015, Host microbiota constantly control maturation and function of microglia in the CNS. Nat. Neurosci. 18, 965-977, which is incorporated herein in its entirety. Microglial cell bodies and cell processes were semi-automatically reconstructed, individual cell bodies were named, and the diameter, process length, and number of branches of each cell body were measured using Imaris software. Twenty to sixty cells per field per mouse were analyzed.

[0105] Enrichment of CD11b cells and qPCR analysis Whole brains removed after perfusion were homogenized in PBS through a 100 mm mesh filter, and myelin fragments were removed by magnetic separation using Myelin Removal Beads (Miltenyi Biotec, San Diego, CA) according to the manufacturer's instructions. Similarly, CD11b cells were enriched by magnetic enrichment using Microglia Microbeads (Miltenyi Biotec, San Diego, CA) according to the manufacturer's instructions. Enriched cells were confirmed by immunofluorescence microscopy, and generally, >90% of cells stained positive for CD11b. For RNA analysis, sectioned tissues (frontal cortex, caudate putamen, lower midbrain, and cerebellum) or cell pellets enriched for CD11b cells were lysed with Trizol, and RNA was extracted using the DirectZol RNA kit (Zymo Research, Irvine, CA). cDNA was then generated using the iScript cDNAΑ Synthesis kit (Bio-Rad, Hercules, CA). Primers for the target genes were obtained from PrimerBank, and qRT-PCR was performed on an Applied Biosystems 7900HT instrument using SYBR Green master mix (Applied Biosystems, Foster City, CA), quantified by the DDCT method, and normalized for gapdh (the primers used are shown in Table 1 above).

[0106] ELISA and Western blot analysis of cytokines and αSyn Tissue homogenates were prepared in RIPA buffer containing a protease inhibitor cocktail (Thermo Fisher, Pittsburgh, PA) and diluted in PBS. TNF-α and IL-6 ELISAs (eBioscience, San Diego, CA) and αSyn ELISA (Thermo Fisher) were performed according to the manufacturer's instructions. For quantification of αSyn fibrils by dot blot analysis, 1 mL samples prepared from 1 mg of tissue homogenate from the tissue regions were spotted onto 0.45 mm nitrocellulose membranes. For Western blot comparison of Triton X-soluble and Triton X-insoluble fractions, brain hemispheres were homogenized in RIPA buffer containing 1% Triton X-100 and centrifuged at 15,000 × g for 60 min at 4 °C to precipitate insoluble proteins and separate them from the Triton-soluble supernatant. The insoluble fraction was dissolved in 10% sodium dodecyl sulfate as previously reported by Klucken et al. (2006, Clinical and biochemical correlates of insoluble alpha-synuclein in dementia with Lewy bodies. Acta Neuropathol. 111, 101-108, which is incorporated herein in its entirety). Five milligrams of each fraction was separated by 4%-20% SDS-PAGE (Thermo Fisher) and blotted onto PVDF membranes. All membranes were blocked with 5% dry skim milk in Tris-buffered saline containing 0.1% Tween-20. Antiaggregated αSyn antibody (1:2000; rabbit; Abcam) or anti-αSyn antibody (1:1000; mouse; BD) was diluted in skim milk and added to the membranes for overnight incubation at 4°C. Anti-rabbit IgG HRP or anti-mouse IgG HRP (1:1000; Cell Signaling Technology) was added to the membrane to detect proteins. All blots were detected with Clarity chemiluminescent substrate (Bio-Rad) on a Bio-Rad GelDoc XR. Densitometry of blots was performed using ImageJ software.

[0107] αSyn aggregation assay To investigate the in vitro aggregation kinetics, 70 mM αSyn was purified 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, which is incorporated herein by reference in its entirety. 70 mM purified αSyn was 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) with increasing concentrations of SCFAs. Non-binding 96-well half-area plates (Corning #3881) were used for each experiment, and 2 mm diameter glass beads were added to each well to induce aggregation. The fluorescence signal of ThT was recorded using a microplate reader (Fluostar OPTIMA microplate reader, BMG Labtech) with an excitation filter of 440 ± 10 nm and an emission filter of 490 ± 10 nm at 37 °C with intermittent shaking. The kinetic curves were normalized to the maximum fluorescence intensity, and the time to reach half the maximum intensity was determined. For further imaging using atomic force microscopy (AFM), the samples were diluted with ultrapure water to adjust the total protein concentration to about 3 mM, and 50 ml of the diluted sample was pipetted onto freshly cleaved mica and allowed to dry. The samples were imaged in air in intermittent contact mode using a modular scanning probe microscope NTEGRA Prima (NT-MDT) equipped with a gold-coated single crystal silicon cantilever (spring constant: about 5.1 N / m) with a resonant frequency of about 150 kHz. AFM images were processed with the open source Gwyddion software.

[0108] SCFA extraction and analysis Fecal samples were collected from 12-week-old mice. Each fecal pellet was mixed with 1 mL of 18 Ω sterile deionized water. The pellet-water mixture was homogenized by shaking at 3200 rpm for 5 min and centrifuged at 13,000 rpm for 15 min at 4 °C. The supernatant was filtered using a 13 mm diameter Acrodisc LC sterile syringe filter (Pall Life Sciences) with a 0.2 mm PVDF membrane. High-performance liquid chromatography (HPLC) analysis was performed using this filtrate. Short-chain fatty acids (SCFAs) were analyzed using an HPLC (LC-20AT, Shimadzu) equipped with a sugar analysis column (Aminex HPX-87H column, Bio-Rad) and a photodiode array detector (PDA, Shimadzu). 5 mM H2SO4 was used as the eluent, and the flow rate was 0.6 mL / min. The column temperature was 50 °C, and the analysis time was 60 min. Standard curves were generated by diluting 10 mM volatile fatty acid standard solutions (acetic acid, butyric acid, formic acid, valeric acid, isovaleric acid, caproic acid, isocaproic acid, and heptanoic acid) from 50 nM to 5000 nM. Concentrations of SCFAs were normalized to soluble chemical oxygen demand. sCOD values ​​in fecal supernatants were measured using high-range (20–1500 mg / L) Hach COD digestion tubes (Hach Company, Loveland) according to the manufacturer's recommendations. COD was measured at a wavelength of 620 nm using a Hach spectrophotometer.

[0109] Microbiome profiling Fecal pellets were collected from mice housed in groups of 1–3 by genotype and donor on days 7, 14, 21, and 49 after fecal transplantation. Samples were sequenced according to the protocol used by the Earth Microbiome Project, as described in Gilbert et al., 2014, The Earth Microbiome project: successes and aspirations. BMC Biol. 12, 69, which is incorporated herein in its entirety. Briefly, DNA was extracted using a MoBio Power soil kit (Carlsbad, CA) and the V4 region of the 16S rRNA gene was amplified using barcoded primers as described in Walters et al. (2015). Sequencing was performed using an Illumina MiSeq. Clustering of operational taxonomic units (OTUs) was performed using SortMeRNA 2.0 (Kopylova et al., 2012) in QIIME 1.9 (described in Caporaso et al., 2010) by closed reference picking against the Greengenes database (described in McDonald et al., 2012), updated in August 2013. OTU tables were generated by rarefaction analysis, resampled to 7500 reads per sample, and alpha and beta diversity were calculated. Abundances between groups were compared using OTU tables that excluded samples with fewer than 7500 reads. Weighted and unweighted UniFrac distances (described in Lozupone and Knight, 2005) were calculated in QIIME 1.9. UniFrac distances were visualized by principal coordinate analysis (PCoA) using Emperor 0.9.4 (described in Vazquez-Baeza et al., 2013). Functional predictions were performed using PICRUSt1.0 (described in Langille et al., 2013), and predicted functional repertoires were compared using the Bray Curtis distance.Significance tests were performed using permanova with scikit-bio 0.4.2 and permutation t-tests with QIIME 1.9, with 999 permutations per test for each test. Abundance comparisons between groups were calculated using taxa at genus level and relative abundances based on the KEGG database, with counts scaled to sum to 1. Tests were performed using ANCOM (described in Mandal et al. (2015)) in scikit-bio 0.4.2 and assessed with one-way ANOVA with a Bonferroni-corrected α = 0.1 rejection threshold. Colonization of samples from healthy or PD donors was compared in BDF1 or Thy1-αSyn genotype mice. Taxa that were significantly different between groups were compared and classified as significant for both BDF1 and Thy1-αSyn, significant for Thy1-αSyn only, or significant for BDF1. Graphs were plotted using Seaborn 0.7.0.

[0110] Quantitative and statistical analysis Statistical analysis of microbiome populations was detailed above. Other data sets were analyzed with GraphPad Prism 6 software. Pairwise comparisons were performed with two-tailed t-tests. Between-group comparisons were performed with one-way ANOVA. p-values, n-values, definitions of center of distribution, and measures of variance are provided in the legends of each figure.

[0111] Data and available software The 16s sequence data and metadata are available online and can be accessed from the QIITA website (https: / / qiita.ucsd.edu / ) under study accession no. 10483 and from the EMBL ENA database (http: / / www.ebi.ac.uk / ena) under study accession no. ERP019564.

[0112] Example 1 Exacerbation of motility and gastrointestinal dysfunction by intestinal microbes In this example, using ASO mice and wild-type mice with complex microbiota, we show that gut microbes exacerbate motility and gastrointestinal dysfunction.

[0113] Thy1-αSyn (α-synuclein overexpressing [ASO]) mice exhibit progressive impairments in fine and gross motor function, as well as gastrointestinal motility disorders. Evidence that deregulated αSyn expression in humans increases the risk of PD provides the epidemiological basis for the Thy1-αSyn mouse model. At 12 weeks of age, mice exhibit a clear impairment in motor coordination tasks. Motor function was measured using four tests previously validated in this model: beam crossing, pole descent, nose tape removal, and hindlimb hugging. (Fleming et al., 2004, Early and progressive sensorimotor anomalies in mice overexpressing wild-type human alpha-synuclein. J. Neurosci. 24, 9434-9440, which is incorporated herein by reference in its entirety.)

[0114] Twelve to thirteen week old ASO mice with complex microbiota (SPF-ASO) took significantly longer to cross the beam and also had a prolonged pole descent time in the beam test compared to wild type littermate mice (SPF-WT) (Figure 2A and Figure 2B). These two tests indicate gross motor function. In the adhesive tape removal test from the bridge of the nose to examine fine motor control, SPF-ASO mice showed impaired motor control compared to SPF-WT mice (Figure 2C). In the hindlimb hug reflex test to examine striatal dysfunction, abnormalities were observed in SPF-ASO mice (Figure 2D).

[0115] To evaluate the contribution of gut bacteria to motor impairment, we separately generated germ-free ASO mice (GF-ASO) and wild-type mice (GF-WT). Unexpectedly, 12- to 13-week-old GF-ASO mice showed improved motor impairment in the beam test, pole test, sticky tape removal test, and hindlimb hug reflex test (Figures 2A-2D). In fact, the achievement of motor function tasks by GF-ASO mice was similar to that of WT mice in many cases. Although there was no difference in body weight between GF-ASO and SPF-ASO mice (Figure 3A), the wire-hang test, which measures grip strength, showed a decline in motor function in both SPF-ASO and GF-ASO mice (Figure 3B). Thus, the results of the motor function tests were not due to body weight or physical strength.

[0116] With further aging (24–25 weeks of age), SPF-ASO mice progressively declined in motor function (Figure 3C–3G), but this progressive decline in motor function was significantly delayed in GF-ASO mice (Figure 3C–3G). No consistent differences were observed in motor tasks between GF-WT and SPF-WT mice, demonstrating the interplay between genes and the microbiome. Similar to what is observed in PD patients, the SPF-ASO mouse model also exhibited motor dysfunction accompanied by reduced gastrointestinal function and constipation.

[0117] While no change was observed in the amount of feces excreted in GF-ASO mice, a significant reduction in the total number of fecal pellets excreted was observed in SPF-ASO mice at both 12–13 and 24–25 weeks of age (Figures 2E, 2F, 3H, and 3I). Furthermore, fecal pellets excreted by SPF-ASO mice had a lower water content than those of GF-ASO mice (Figure 3J). These results revealed that gastrointestinal disorders were improved in germ-free (GF) mice. In support of this, when all motor function phenotypes were aggregated by principal component analysis (PCoA), the GF-ASO mice group formed a cluster similar to that of the WT mice group, with only the SPF-ASO group significantly deviating (Figure 3K).

[0118] The data presented in Example 1 demonstrate that gut microbes exacerbate the motor and gastrointestinal dysfunction characteristic of PD in preclinical models of PD.

[0119] Example 2 Requirement of gut microbiota in αSyn pathology This example shows that αSyn pathology is increased in mice with a gut microbiota.

[0120] αSyn aggregation is seen in line with motor impairment in PD. Using antibodies that structurally recognize αSyn aggregates and αSyn fibers, immunofluorescence microscopy was performed to visualize αSyn inclusions in mouse brains. In ASO mice under SPF conditions, prominent αSyn aggregation was observed in the caudate-putamen (CP) and substantia nigra (SN), brain regions affected by PD in both mouse models and human PD patients (Figure 4A and Figure 4B). Surprisingly, the amount of αSyn aggregates was significantly lower in GF-ASO mice (Figure 4A and Figure 4B). Quantification of αSyn aggregation was performed by Western blot of brain extracts (Figure 4C). Surprisingly, only a small amount of insoluble αSyn was observed in the brains of GF-ASO mice (Figure 4C-4E). To further support this result, αSyn aggregation was examined by dot blot analysis in the caudate-putamen (CP), the site of the presence of the substantia nigra (SN), and in the lower midbrain. Similar to previous experiments, reduced αSyn aggregation was observed in GF-ASO mice (Figures 5A-5C).

[0121] Region-specific αSyn aggregation was observed, with αSyn aggregation in the frontal cortex (FC) being less in GF-ASO mice than in SPF mice, whereas αSyn aggregation in the cerebellum (CB) was similar between SPF and GF mice (Figures 5D-5H). To confirm that these findings were not due to differential expression of the exogenous gene, we measured αSyn transcript abundance and αSyn protein abundance in the lower midbrain and caudate putamen (CP), which showed similar abundance between SPF-ASO and GF-ASO mice (Figures 4F and 4G).

[0122] These data indicate that the microbiota regulates pathways that promote αSyn aggregation and / or inhibits the clearance of insoluble protein aggregates.

[0123] Example 3 αSyn-dependent microglial activation by the microbiota In this example, we demonstrate that the microbiota activates microglia in an αSyn-dependent manner.

[0124] The microbiota regulates immune development in the central nervous system (CNS), and αSyn aggregates activate immune cells, including microglia, present in the brain. Upon activation, microglia undergo remarkable morphological changes, transitioning from a thin cell body with numerous branched processes to round, amoeboid cells with a reduced number of processes. In situ 3D reconstruction of individual microglial cells using confocal fluorescence microscopy revealed that microglia found in SPF mice differed from those in wild-type germ-free (GF) mice. Compared to SPF-WT mice, GF-WT mice had a higher number of microglia found in the caudate putamen (CP) and substantia nigra (SN), and the total length of microglial processes was longer (Figures 6A-6C). These morphological characteristics indicated that germ-free (GF) mice had arrested microglial maturation and / or a reduced activation state, supporting recent reports that gut bacteria affect immune cells in the brain.

[0125] Further supporting these observations in disease models, microglia from SPF-ASO mice showed significantly longer cell body diameters and fewer and shorter processes compared to those from GF-ASO mice (Figures 6A-6C). Tissue homogenates from the caudate putamen (CP) and lower midbrain of SPF-ASO mice showed a marked increase in the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) compared to those from GF-ASO mice (Figures 6D and 6E). Both of these cytokines are elevated in the brains of PD patients. Enriched CD11b + Gene expression analysis of RNA extracted from cells (mostly microglia) showed increased expression of Tnfa and Il6 in SPF-ASO mice, but almost no expression of these cells was observed in germ-free (GF) mice (Figure 6F). As observed in previous studies, e.g., Erny et al., 2015; and Matcovitch-Natan et al., 2016, expression of Bdnf, which has neuroprotective effects, and expression of Ddit4, a cell cycle marker, were upregulated in GF mice (Figure 5I). Microglial diameter was increased and TNF-α production was increased in the frontal cortex (FC), but not in the cerebellum (CB), indicating that the neuroinflammatory response was region-specific (Figure 6G and Figure 6H).

[0126] These findings support the hypothesis that gut microbes promote αSyn-dependent microglial activation in specific brain regions involved in disease.

[0127] Example 4 Postnatal microbial signaling regulates αSyn-dependent pathological changes This example demonstrates that postnatal microbial signals regulate αSyn-dependent pathological functional changes.

[0128] The microbiota not only influences neurodevelopment during gestation but also affects adult neurofunction through active gut-to-brain signaling. To identify these mechanisms, we treated postnatal SPF mice with an antibiotic cocktail to remove the microbiota (Figure 7A). Meanwhile, a group of 5- to 6-week-old germ-free (GF) mice was colonized with a complex microbiota from SPF-WT mice (Figure 7A). Surprisingly, antibiotic-treated (Abx) mice showed little αSyn-dependent motor dysfunction, very similar to mice born under germ-free (GF) conditions (Figures 7B-7E). Postnatally, germ-free mice colonized with a microbiota (Ex-GF) showed genotype-specific effects as observed in SPF mice, with mice overexpressing αSyn exhibiting significant motor dysfunction (Figures 7B-7E).

[0129] Antibiotic (Abx)-treated mice also showed significant improvement in gastrointestinal function as assessed by fecal output, whereas Ex-GF mice showed an αSyn-dependent reduction in total fecal output (Figure 7F and Figure 7G). Furthermore, among the transgenic ASO lines, Ex-GF mice had larger microglial cell body diameters that were similar to those of SPF mice (Figure 7H and Figure 7I). However, microglia from Abx-ASO mice had diameters similar to those of germ-free (GF) mice (Figure 7H and Figure 7I).

[0130] Although we do not exclude a role for the microbiota in prenatal neurodevelopment, these data indicate that modulation of microglial activation in adults contributes to αSyn-mediated motor dysfunction and neuroinflammation, suggesting active signaling between the gut and the brain via the microbiota.

[0131] Example 5 Short-chain fatty acids (SCFAs) alone promote αSyn-mediated neuroinflammation In this example, we demonstrate that αSyn-mediated neuroinflammation is promoted by short-chain fatty acids (SCFAs) alone.

[0132] Intestinal bacteria may regulate microglial activation during viral infection through the production of short-chain fatty acids (SCFAs), which are microbial metabolites. As shown in Figure 8A, fecal SCFA concentrations were observed to be lower in germ-free (GF) and antibiotic (Abx)-treated mice compared to SPF mice. To determine whether SCFAs affect neuroinflammatory responses in PD mouse models, we treated GF-ASO and GF-WT mice with a SCFA mixture consisting of acetate, propionate, and butyrate while maintaining the mice in a microbiologically germ-free state, which resulted in a significant recovery of fecal SCFA concentrations (Figure 8A). In affected regions of the brain (i.e., caudate putamen (CP) and substantia nigra (SN)), microglia from SCFA-treated mice exhibited morphological features indicative of increased activation compared to those from untreated mice, and displayed morphological features very similar to those of microglia from Ex-GF and SPF mice (see Figures 9A, 9B, 8B, and 8C; and Figures 6A-6H and 7A-7I). Microglia from SCFA-fed GF-ASO mice (SCFA-ASO) were significantly larger in diameter, had shorter process lengths, and reduced total process numbers than those from SCFA-treated GF-WT mice (SCFA-WT). In contrast, microglia from mice treated with antibiotics (Abx) displayed morphological features similar to those of germ-free (GF) mice (see Figures 9B, 8B, and 8C; and Figures 6A-6H and 7A-7I). Changes in microglial diameter were also observed in the frontal cortex (FC) but not in the cerebellum (CB), indicating a region-specific response (Figures 8D and 8E).

[0133] Consistent with the morphological characteristics of microglia, SCFA-treated mice showed αSyn aggregation compared to untreated and antibiotic (Abx)-treated mice, and the extent of aggregation was comparable to Ex-GF mice (Figures 8F-8I). Surprisingly, we observed that microbial signaling after birth increased αSyn aggregation in the caudate putamen (CP) and substantia nigra (SN) (Figures 8F and 8G), but no changes were observed in the frontal cortex (FC) and cerebellum (CB) (Figures 8H and 8I), which was further confirmed by quantification and Western blot (Figures 8J-8O). Various concentrations of SCFAs, either alone or in the form of a mixture, did not promote the aggregation of human αSyn in vitro (Figures 10A-10G), nor did they alter the overall structure of αSyn amyloid fibrils (Figures 10H and 10I).

[0134] These data indicate that SCFAs promote αSyn aggregation in vivo, albeit without direct molecular interactions.

[0135] Example 6 Exacerbation of motor impairment by SCFAs alone In this example, we show that αSyn-stimulated microglial activation and motor dysfunction are enhanced by SCFAs.

[0136] To investigate the relationship between microbial metabolites and motor symptoms in the Thy1-αSyn model, germ-free (GF) mice aged 5–6 weeks were treated with a SCFA mixture and their motor functions were evaluated at 12–13 weeks of age. SCFA-ASO mice showed significantly reduced function in several motor tasks compared with untreated GF-ASO mice (Figures 9C–9F), specifically, they showed impairments in the beam test, pole test, and hindlimb hug reflex (comparison of SCFA-ASO mice and GF-ASO mice). All of the effects of SCFA were specific to the genotype of Thy1-αSyn mice. Gastrointestinal disorders were also observed in transgenic mice treated with SCFA (Figures 9G and 9H). Motor disorders were not induced in germ-free (GF) mice orally administered heat-killed bacteria (Figures 10J–10M), suggesting that active metabolism of gut bacteria is required for the induction of motor disorders. Oral administration of the anti-inflammatory compound minocycline to SCFA-fed mice was sufficient to reduce TNF-α production, decrease αSyn aggregation, and improve motor function, without altering exogenous gene expression (Figures 11A-11H).

[0137] These data indicate that metabolites, such as SCFAs, actively produced by the microbiota are required for microglial activation and αSyn aggregation, contributing to motor dysfunction in preclinical models of PD.

[0138] Example 7 Microbiome disruption (dysbiosis) in PD This example shows microbiome disruption (dysbiosis) in PD.

[0139] It has been shown that the microbiome of PD patients is altered. In this example, we assessed whether human gut microbes influence disease outcomes when fecal samples were collected from six human subjects diagnosed with PD and six healthy controls matched to the PD patients (Table 2) and transplanted into germ-free (GF) mice. To minimize the influence of confounding factors, in addition to the relevant inclusion and exclusion criteria, we selected only patients with new-onset PD who had a healthy gut on histological examination and were not yet receiving treatment (Table 2). [Table 2]

[0140] Fecal microbiota from PD patients or controls were transferred by oral gavage to each group of recipient germ-free (GF) mice. Fecal pellets were collected from these "humanized" mice, bacterial DNA was extracted, and 16S rRNA was sequenced. Sequences were grouped into operational taxonomic units (OTUs) against the Greengenes database using closed reference picking, and metagenomic functions were predicted using PICRUSt. In unweighted UniFrac analysis (Lozupone and Knight, 2005) visualized by PCoA, each group of recipient mice showed a profile that most closely resembled that of their corresponding human donors (Figures S12A and S12B). Surprisingly, the disease status of the donor had a strong influence on the microbial communities of the recipient mice. The microbial communities of humanized mice transplanted from PD donors were significantly more similar to each other, and less similar to those of mice transplanted from healthy donors and humanized mice transplanted from PD donors, even when classified based on genetic background (Figures S12C and S12D). Furthermore, compared to wild-type (WT) recipient mice, the ASO strains showed significant differences between mice transplanted from healthy donors and mice transplanted from PD donors, suggesting a genotype effect on the composition of the microbial community (Figures S12C and S12D).

[0141] Compared to controls colonized with microbiomes from healthy donors, mice colonized with microbiomes from PD donors showed changes in many bacterial genera (Figure S12E), and KEGG pathways compared by calculating Bray-Curtis distances were also altered between PD donor and control groups (Figure S13A-13C). Mice transplanted with PD patient microbiomes showed increased abundance of OTUs from genera Proteus, Bilophila, and Roseburia, and decreased abundance of OTUs from families Lachnospiraceae, Rikenellaceae, and Peptostreptococcaceae, as well as Butyricicoccus (Figure S13E). Interestingly, some taxa (e.g., Proteus, Bilophila, and Lachnospiraceae) were altered only in ASO mice, while some others (e.g., Roseburia, Rikenellaceae, and Enterococcus) were significantly altered independent of mouse genotype (Figure S13E).More interestingly, the abundance of three SCFA-producing KEGG families (K00929: butyrate kinase; K01034: acetate-CoA transferase / acetoacetate-CoA transferase α subunit; and K01035: acetate-CoA transferase / acetoacetate-CoA transferase β subunit) was increased in mice transplanted with fecal microbes from PD donors (Figure S13D). Furthermore, compared to mice colonized with microbes from healthy controls, mice transplanted with microbiota from PD donors showed significant changes in the SCFA profile, with lower concentrations of acetate and higher relative abundance of propionate and butyrate (Figure S13E).

[0142] These data indicate that the differences in fecal microbial communities between PD patients and controls are maintained after transplantation in mice, and further demonstrate that αSyn overexpression distinctively alters the gut microbiome profile after transplantation.

[0143] Example 8 Gut microbiota from PD patients promotes motor dysfunction In this example, we show that gut microbiota from PD patients promotes motor dysfunction.

[0144] To evaluate the function of the microbiota, the motor function of the humanized mice generated from each donor pair was tested. In four of the six pairs (pairs No. 1, 3, 4, and 5), the microbiota from each PD patient donor consistently exacerbated αSyn-mediated motor dysfunction (Figures 14A-14F). Compared with recipient mice colonized with genotype-matched gut bacteria from healthy controls, ASO mice colonized with PD patient-derived microbiota showed significant motor impairment in the beam test, pole test, and nasal adhesive tape removal test. Meanwhile, the hind limb reflex scores were not significantly different between donors. Interestingly, the microbiota from one donor did not induce a significant genotype-dependent effect in the beam and pole test tasks (pair No. 2, Figure 14B). This indicates the possibility of heterogeneity in the population, which needs to be verified by more powerful cohort studies. No significant effect on motor function was observed in recipient WT mice colonized with microbiota from any donor group, regardless of donor origin (Figures 14A-14F). This finding in a preclinical mouse model indicates that PD patient-derived microbiota contributes to disease symptoms in a genetically susceptible host. Furthermore, there was little change in the weight of recipient mice, and little change in gastrointestinal function as measured by fecal volume (Figures 15A-15F). Aggregating the functional data from all groups revealed that compared to colonization with microbes from healthy controls, microbiota from PD patients exacerbated motor impairment in ASO mice in three of the four tests used in this study (Figure 14G). Indeed, visualization of all motor functions by PCoA showed striking and extensive differences between mice colonized with PD donor-derived microbiota and those colonized with gut bacteria from healthy individuals (Figure 15G).

[0145] These data showed that gut bacteria from PD patients exacerbated motor impairment in a mouse model compared with healthy controls, providing evidence that the microbiota functionally contributes to synucleinopathy.

[0146] As described herein, the gut microbiota influences neurodevelopment, modulates behavior, and contributes to neurological disorders. Without wishing to be bound by any particular theory, it is believed that there is a yet to be discovered functional link between gut bacteria and neurodegenerative diseases. Synucleinopathies are characterized by the aggregation of a type of protein, alpha-synuclein (αSyn), and often result in motor dysfunction, such as Parkinson's disease (PD). As shown in Examples 1-8 using mice overexpressing αSyn, it was found that the gut microbiota is required to cause motor disorders, microglial activation, and αSyn pathology. The pathological functional changes in adult animals were ameliorated by antibiotic treatment and exacerbated by microbial recolonization, suggesting that the disease is regulated by signaling between the gut and the brain after birth. Oral administration of certain microbial metabolites to germ-free mice exacerbates neuroinflammation and motor symptoms. Furthermore, colonization of mice overexpressing αSyn with a microbiota from PD patients exacerbated disability compared with transplantation with a microbiota from a healthy donor. These findings indicate that gut bacteria control motor impairment in mice and that alterations in the human microbiome are a risk factor for PD.

[0147] Example 9 Treatment of Parkinson's Disease (PD) This example demonstrates the treatment of PD patients.

[0148] The aggregation rate of α-Syn or the degree of α-Syn aggregation (e.g., the amount of α-Syn aggregates) or both are measured in a subject. Abnormalities in the aggregation rate of α-Syn or the degree of α-Syn aggregation (e.g., the amount of α-Syn aggregates) or both in the subject indicate that the subject suffers from PD. The composition of the gut microbiota of the subject is modulated. Modulating the composition of the gut microbiota in the subject is expected to alleviate one or more symptoms of PD in the subject (e.g., improve one or more movement disorders).

[0149] In at least some of the above-described embodiments, one or more components used in these embodiments may be substituted with components of another embodiment, unless technically impossible. Those skilled in the art will readily appreciate that various omissions, additions and modifications other than those described above may be made to the methods and structures described above without departing from the scope of the subject matter described in the claims. Any such modifications and variations are deemed to be within the scope of the subject matter of the present invention as defined in the appended claims.

[0150] Substantially all plural and / or singular terms used herein will be interpreted by those of ordinary skill in the art as the plural and / or the singular as the plural, as appropriate for the description and / or application of the present specification. The various singular / plural terms have been intentionally used to provide clarity to the present invention.

[0151] Those skilled in the art will appreciate that the terms described herein, particularly those described in the appended claims (e.g., the body of the appended claims), are generally "open-ended" terms (e.g., the term "including" should be interpreted as "including, but not limited to," the term "having" should be interpreted as "having at least," and the term "include" should be interpreted as "including, but not limited to"). Furthermore, those skilled in the art will appreciate that when a specific number is recited in a claim, such an intention is also clearly recited in the claim, and when a specific number is not recited, such intention is not present. To be more specific, for example, the claims below may use preambles such as "at least one" or "one or more" to define the claim. However, this preamble should not be construed to limit a claim recited using the indefinite article "a" or "an" to embodiments containing only one component, even if the preamble "one or more" or "at least one" and the indefinite article "a" or "an" are both included in the same claim (e.g., "a and / or an" should be interpreted to mean "at least one" or "one or more"). This also applies to claims recited using definite articles. Additionally, even if a claim expressly recites a particular number, one of ordinary skill in the art would understand that "at least" refers to the recited number (e.g., "two" without a modifier means "at least two" or "two or more").Furthermore, when idiomatic phrases such as "at least one of A, B, and C" are used, such phrases are typically written in the sense that one of ordinary skill in the art would normally understand the phrase (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 having A and B, a system having A and C, a system having B and C, and / or a system having A, B, and C). Also, when idiomatic phrases such as "at least one of A, B, or C" are used, such phrases are typically written in the sense that one of ordinary skill in the art would normally understand the phrase (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 having only C, a system having A and B, a system having A and C, a system having B and C, and / or a system having A, B, and C). Moreover, those skilled in the art will understand that disjunctive terms and / or phrases intended to express two or more alternatives, whether in the specification, claims, or drawings, may include one of the stated terms, either of the stated terms, or both of the stated terms. For example, the phrase "A or B" may include "A only," or "B only," or "A and B."

[0152] Furthermore, when features or aspects of the disclosure are described in Markush format, those skilled in the art will understand that each member or subgroup of members described in Markush format is also described.

[0153] A person skilled in the art will understand that any range described herein, for any purpose, including providing a detailed description, also encompasses all possible subranges and combinations thereof. Any of the above ranges are fully described, with at least equal, third, fourth, fifth, tenth, etc. divisions of the same range, and it will be readily understood that the present invention can be practiced with such divided ranges. For example, any of the ranges described herein can be readily divided into thirds, such as low, mid, high, etc., without limitation. Additionally, a person skilled in the art will understand that any of the terms "less than," "at least," "greater than," "less than" and the like, are inclusive of the recited numerical value, and also refer to ranges that can be divided into subranges, as previously described. Additionally, a person skilled in the art will understand that any of the ranges described herein are inclusive of each member. Thus, for example, a group having 1-3 members refers to a group having 1 member, a group having 2 members, or a group having 3 members. Similarly, a group having 1-5 members refers to a group having 1 member, a group having 2 members, a group having 3 members, a group having 4 members, or a group having 5 members, etc.

[0154] Various aspects and embodiments have been described herein, and other aspects and embodiments will be readily apparent to those skilled in the art. The various aspects and embodiments disclosed herein are intended to be illustrative of the invention and are not intended to be limiting of the invention, the scope and spirit of which is indicated by the following claims.

Claims

1. 1. A composition for slowing the progression of a synucleinopathy or an α-synuclein mediated movement disorder in a subject in need thereof, comprising: wherein the composition increases the abundance of one or more bacterial species in the subject; At least one of the one or more bacterial species is a bacterial species belonging to the family Lachnospiraceae, Rikenellaceae, Peptostreptococcaceae, Clostridiaceae, the genus Enterococcus, Clostridium, Bacteroides or Butyricicoccus; A composition comprising an inhibitor of an enzyme involved in the synthesis of the short chain fatty acid (SCFA) acetate, an inhibitor of an enzyme involved in the synthesis of the SCFA propionate, and an inhibitor of an enzyme involved in the synthesis of the SCFA butyrate, and comprising intestinal microbiota obtained from a healthy subject.

2. The composition of claim 1, wherein the subject exhibits an aggregation disorder of α-synuclein (αSyn).

3. The composition of claim 1 or 2, which is capable of ameliorating one or more physical disorders of the subject.

4. The composition of claim 1 or 2, which is capable of improving one or more gastrointestinal functions in the subject.

5. The composition of claim 1 or 2, which is capable of relieving constipation in the subject.

6. The composition of any one of claims 1 to 5, wherein the movement disorder is tremor, rigidity, bradykinesia, gait disturbance or any combination thereof.

7. The composition of any one of claims 1 to 6, which reduces microglial activation in the subject.

8. The composition of any one of claims 1 to 7, which reduces alpha-synuclein (αSyn) aggregates in the subject.

9. The composition of claim 8, which is capable of promoting the removal of insoluble aSyn protein aggregates, reducing aggregation of aSyn protein, or both.

10. The composition of any one of claims 1 to 9, which alleviates neuroinflammation in the subject.

11. The composition of any one of claims 1 to 10, wherein the synucleinopathy is Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, or any combination thereof.

12. The composition of claim 11, wherein the synucleinopathy is Parkinson's disease.

13. 12. The composition of claim 11, wherein the synucleinopathy is primary or idiopathic parkinsonism, secondary or acquired parkinsonism, hereditary parkinsonism, Parkinson-plus syndrome or multiple system degeneration, or any combination thereof.

14. The composition of any one of claims 1 to 13, further comprising one or more antibiotics.

15. 15. The composition of claim 14, wherein the one or more antibiotics comprise ampicillin, vancomycin, neomycin, gentamicin, erythromycin, or any combination thereof.

16. 15. The composition of claim 14, wherein the one or more antibiotics do not include rifampicin and / or minocycline.

17. The composition according to any one of claims 1 to 16, which is a probiotic composition, a nutritionally functional composition, a pharmaceutical composition or any combination thereof.

18. The composition of any one of claims 1 to 17, wherein the composition reduces the amount of one or more bacterial species that exacerbate Parkinson's disease (PD) in the subject.

19. 19. The composition of claim 18, wherein at least one of the one or more bacterial species that exacerbate PD is a bacterial species belonging to the genera Proteus, Bilophila, Roseburia, Pseudoramibacter, Eubacterium or the family Veillonellaceae.

20. 19. The composition of claim 18, wherein at least one of the one or more bacterial species that exacerbate PD is a short chain fatty acid (SCFA) producing bacteria.

21. The composition of claim 20, wherein the SCFA-producing bacterium is a bacterium belonging to the K00929 family, the K01034 family or the K01035 family of KEGG.

22. The composition of any one of claims 1 to 13 and claims 17 to 21, wherein the subject has not been treated with an antibiotic.

23. The composition according to any one of claims 1 to 13 and claims 17 to 21, which is antibiotic-free.

24. The composition according to any one of claims 1 to 13 and claims 17 to 21, which does not contain rifampicin and / or minocycline.

Images