NZ760605B2

NZ760605B2 - Compositions comprising bacterial strains

Info

Publication number: NZ760605B2
Application number: NZ760605A
Authority: NZ
Inventors: Suaad Ahmed; Anna Ettorre; Parthena Fotiadou; Imke Elisabeth MULDER; Helene Savignac; Joseph Roby Iringan Urcia; Samantha Yuille
Original assignee: D Pharma Research Limited
Priority date: 2017-06-14
Filing date: 2018-06-14
Publication date: 2022-06-28

Abstract

The invention provides compositions comprising commensal bacteria, such as Megasphaera massiliensis strains (e.g., strain NCIMB 42787), capable of producing valeric acid that are for treating neurodegenerative disorders or brain injuries in a subject.

Description

COMPOSITIONS COMPRISING BACTERIAL STRAINS TECHNICAL FIELD This invention is in the field of compositions comprising bacterial strains isolated from the ian digestive tract and the use of such compositions in the treatment of e.

BACKGROUND TO THE INVENTION The human ine is thought to be sterile in utero, but it is exposed to a large variety of maternal and environmental microbes immediately after birth. fter, a dynamic period of microbial colonization and succession occurs, which is influenced by factors such as ry mode, environment, diet and host pe, all of which impact upon the composition of the gut microbiota, particularly during early life. Subsequently, the microbiota izes and becomes adult-like [1], The human gut microbiota contains more than 500-1000 different phylotypes belonging essentially to two major bacterial divisions, the Bacteroidetes and the Firmicutes [2], The successful symbiotic relationships arising from bacterial zation of the human gut have yielded a wide variety of metabolic, structural, protective and other beneficial functions. The enhanced metabolic activities of the colonized gut ensure that otherwise indigestible dietary components are degraded with release of by-products providing an ant nutrient source for the host. Similarly, the immunological importance of the gut microbiota is well-recognized and is exemplified in germfree animals which have an impaired immune system that is functionally reconstituted following the introduction of commensal bacteria [3-5], Dramatic changes in microbiota composition have been documented in gastrointestinal disorders such as inflammatory bowel disease (IBD). For example, the levels of Clostridium cluster XIYa bacteria are reduced in IBD patients whilst numbers of E. coli are sed, suggesting a shift in the e of nts and pathobionts within the gut [6-9], In recognition of the potential positive effect that n bacterial strains may have on the animal gut, various s have been proposed for use in the ent of various diseases (see, for example, [10- 13]). Also, certain strains, including mostly Lactobacillus and Bifidobacterium strains, have been proposed for use in treating various inflammatory and autoimmune diseases that are not directly linked to the intestines (see [14] and [15] for reviews). However, the relationship between different diseases and different bacterial s, and the precise effects of particular bacterial strains on the gut and at a systemic level and on any ular types of diseases are poorly characterised, particularly for neurodegenerative disorders.

Recently, there has been increased interest in the art regarding alterations in the gut microbiome that may play a pathophysiological role in human brain diseases [16], Preclinical and clinical evidence are strongly suggesting a link between brain development and microbiota [17], A growing body of nical literature has demonstrated bidirectional signalling between the brain and the gut microbiome, involving multiple neurocrine and endocrine signalling systems. Indeed, increased levels [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM of Clostridium species in the microbiome have been linked to brain disorders [18], and an imbalance of the oidetes and Firmicutes phyla has also been implicated in brain development disorders . Suggestions that altered levels of gut commensals, including those of Bifidobacterium, Lactobacillus, Sutterella, Prevotella and Ruminococcus genera and of the Alcaligenaceae family are involved in immune-mediated central nervous system (CNS) disorders, are questioned by s suggesting a lack of alteration in the microbiota between patients and healthy subjects [19]. There have also been suggestions that the administration of probiotics may be beneficial in the treatment of neurological disorders. However, these studies failed to conclude that probiotic compositions per se can achieve therapeutic benefits with t to the treatment of neurodegeneration and did not show any useful effects for any particular bacteria [20,21], This indicates that, at present, the practical effect of the link between the microbiome and human brain diseases is poorly characterised. Accordingly, more direct analytical studies are required to identify the therapeutic impact of altering the iome on neurodegenerative disorders.

There is a requirement in the art for new methods of treating neurodegenerative ers. There is also a requirement for the potential effects of gut bacteria to be characterised so that new therapies using gut ia can be developed.

SUMMARY OF THE ION The inventors have developed new therapies for treating and preventing neurodegenerative disorders.

In particular, the inventors have fied that commensal bacteria that produce certain organic acids have neuroprotective activities and may be effective for treating neurodegenerative disease. Related to this, the inventors have also fied that adding c acids to compositions sing commensal bacteria, or using organic acids in ation with commensal bacteria, may provide enhanced therapeutic s, in particular in the treatment of neurodegenerative disorders.

The inventors have identified that commensal bacterial strains that produce one or more organic acids with the formula Rx-COOH, wherein Rx ses an alkyl group comprising in the range of 4 to 11 carbons, or wherein Rx ses a phenyl group with a substituent hydroxyl group, wherein optionally the hydroxyl group is at position 4, maybe effective for treating egenerative diseases. In another aspect of the invention, the inventors have found that a combination of a commensal bacterial strain and an organic acid may be effective for ng or ting neurodegenerative diseases. As used herein, the terms "the combination of the invention", "the therapeutic ation of the invention" and "the therapeutic combination" may be used interchangeably and refer to a therapeutic combination of: (a) a composition comprising a commensal bacterial strain; and (b) a composition comprising an organic acid. It is to be understood that the term "combination" in the context of the therapeutic ation does not refer to components (a) and (b) of the combination necessarily being in the same composition and/or administered at the same time.

As described in the examples, stration of itions comprising Megasphaera massiliensis can protect against reactive oxygen s and t inflammation, thus actingas a neuroprotectant. The inventors have identified that Megasphaera massiliensis produces certain organic acids including hexanoic acid, valeric acid and oxyphenylacetic acid. The inventors have also identified that treatment with Megasphaera massiliensis can reduce the activation of proinflammatory molecules, such as NFκB and IL-6, by LPS and mutant α-synuclein. The inventors have also found that Megasphaera massiliensis can increase the activation of the proinflammatory cytokine IL-8, which can help to promote neuron myelination. The inventors have also identified that treatment with a combination of Megasphaera massiliensis and retinoic acid can increase the secretion of brain-derived neurotrophic factor (BDNF), which can help e neurogenesis and neuritogenesis and/or prevent cell death. The inventors have identified that treatment with Megasphaera massiliensis can attenuate oxidative stress, reduce histone deacetylation activity and lipid peroxidation in vitro, which can help to reduce cell death and sis. In particular, the ors have identified that treatment with Megasphaera massiliensis, which can produce c acid, can reduce histone deacetylation, which can help to reduce cell death and apoptosis. The inventors have also identified that Megasphaera massiliensis can produce indole that can attenuate inflammation and oxidative stress. The inventors have also demonstrated that treatment with Megasphaera massiliensis can increase kynurenine levels.

Furthermore, the inventors have also found that Megasphaera iensis can produce hexanoic acid, which can be neuroprotective or neurorestorative, for example by promoting neurite outgrowth. The inventors have found that Megasphaera massiliensis that can produce hexanoic acid increase the sion of MAP2 (Microtubule –associated protein 2), which is thought to be essential for microtubule formation in neuritogenesis. Therefore, the inventors have found that haera massiliensis that can produce hexanoic acid can be used to promote neurite outgrowth. Megasphaera iensis and other bacteria that produce organic acids like hexanoic acid, valeric acid and 4-hydroxyphenylacetic acid may therefore be useful for treating egenerative disorders.

Accordingly, in one aspect, the present disclosure provides for the use of a composition comprising a strain of a commensal bacteria in the manufacture of a ment for treating or ting a neurodegenerative disorder in a subject, wherein the strain produces valeric acid and wherein the strain is not a bacterial strain of the genus Megasphaera.

In a related aspect, the present disclosure es for the use of a composition comprising a strain of a commensal bacteria in the manufacture of a medicament for treating brain injury in a subject, wherein the strain es c acid and wherein the strain is not a bacterial strain of the genus Megasphaera.

Thus, in a first aspect, the invention describes a composition comprising a commensal bacterial strain for use in a method of treating or preventing a neurodegenerative disorder, wherein the strain 3A followed by page 4 produces one or more organic acids each having the following formula: Rx-COOH, wherein Rx comprises an alkyl group comprising at least 4 carbons, or wherein Rx ses a phenyl group with a substituent hydroxyl group, wherein optionally the hydroxyl group is at position 4.

In a second aspect, the invention provides a composition comprising a commensal bacterial strain and one or more organic acids having the formula H or ceutically acceptable salts or esters thereof. In preferred embodiments, the composition comprises one or more of the organic acids valproic acid, valeric acid, hexanoic acid, retinoic acid, and 4-hydroxyphenylacetic acid or pharmaceutically acceptable salts or esters thereof. The examples demonstrate that such combinations have therapeutic benefits.

[Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM In some embodiments, the invention describes a composition comprising a commensal ial strain for use in a method of treating or preventing a disease in a t, wherein the composition is to be administered in combination with one or more organic acids or pharmaceutically acceptable salts or esters thereof. The examples demonstrate that such combinations may be particularly effective.

In some ments, the invention describes a composition comprising one or more organic acids or pharmaceutically acceptable salts or esters thereof for use in a method of treating or preventing a disease in a subject, wherein the composition is to be administered in combination with a commensal bacterial strain. The examples demonstrate that such combinations may be ularly effective.

Preferred embodiments of both aspects of the invention are discussed below and throughout the application.

In some embodiments, the commensal bacterial strain produces a short chain fatty acid. Preferably, the short chain fatty acid is c acid. Butyrate may contribute to reducing impermeability of the blood brain barrier, which has a neuroprotective .

In some embodiments, the composition is for use in a method of treating or preventing e.

In some embodiments, the compositions are for use in a method of ng or preventing a egenerative disorder.

In particular embodiments, the compositions are for use in a method of treating or preventing a disease or condition selected from the group consisting of: Parkinson’s e, including ssive supranuclear palsy, ssive supranuclear palsy, Steele-Richardson-Olszewski syndrome, normal pressure hydrocephalus, vascular or arteriosclerotic parkinsonism and drug-induced parkinsonism; Alzheimer’s disease, ing Benson's syndrome; multiple sclerosis; gton’s disease; amyotrophic lateral sclerosis; Lou Gehrig's disease; motor neurone disease; prion disease; spinocerebellar ataxia; spinal muscular atrophy; dementia, including Lewy body, vascular and frontotemporal dementia; primary progressive aphasia; mild cognitive impairment; HIV-related cognitive impairment, progressive inflammatory neuropathy, and obasal ration.

In preferred embodiments, the compositions of the invention are for use in a method of treating or preventing Parkinson’s disease, such as environmental, familial or Parkinson’s associated with general inflammatory status. The inventors have identified that treatment with the compositions of the invention can reduce the activation of proinflammatory molecules, such as NFkB and IL-6, by LPS and mutant a-synuclein in in vitro models of environmental and familial Parkinson’s.

In some embodiments, the compositions of the invention are for use in a method of treating or preventing early-onset neurodegenerative disease. In some embodiments, the compositions of the ion are for use in a method of preventing or delaying onset or progression of a egenerative disorder.

[Annotation] KJM None set by KJM [Annotation] KJM ionNone set by KJM [Annotation] KJM Unmarked set by KJM ation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM In preferred embodiments, the invention provides a combination comprising a bacterial strain of the s Megasphaera massiliensis, and an organic acid selected from the list consisting of hexanoic acid, valeric acid, valproic acid, retinoic acid or 4-hydroxyphenylacetic acid for use in the treatment of Parkinson’s disease. Combinations using Megasphaera massiliensis and hexanoic acid may be particularly effective for use in a method of treating neurodegenerative diseases, in particular Parkinson’s. red compositions of the invention comprise a commensal bacteria and retinoic acid.

Further preferred compositions of the invention comprise a commensal bacteria and hexanoic acid.

Further preferred compositions of the invention comprise a commensal bacteria and valeric acid In some embodiments of the invention, the commensal bacterial strain is from the genus selected from the list consisting of oides, Enterococcus, Escherichia, Klebsiella, Bifidobacterium, Staphylococcus, Lactobacillus, Megasphaera, Clostridium, Proteus, Pseudomonas, Salmonella, Faecalibacterium, Peptostreptococcus or Peptococcus, or a combination thereof. In some embodiments, the commensal bacterial strain is from the genus Megasphaera. In some red embodiments the strain is of the species Megasphaera massiliensis. Closely related strains may also be used, such as bacterial strains that have a 16S rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to the 16S rRNA sequence of a bacterial strain of Megasphaera massiliensis. Preferably, the bacterial strain has a 16S rRNA ce that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to SEQ ID NO: 2. Preferably, the bacterial strain for use in the invention has the 16S rRNA sequence represented by SEQ ID NO:2.

In preferred embodiments, the strain for use in the ion es one or more organic acids each having the following formula: Rx-COOH, wherein Rx comprises an alkyl group comprising at least 4 carbons, or n Rx comprises a phenyl group with a substituent hydroxyl group, wherein optionally the hydroxyl group is at position 4, wherein the organic acid is effective in the ent or prevention of a neurodegenerative disorder. In some embodiments, the strain for use in the invention produces an organic acid selected from the list consisting of hexanoic acid, valeric acid, valproic acid, ic acid or 4-hydroxyphenylacetic acid. Preferably, strains of the invention are e of increasing the tion of IL-8 and reducing the tion of IL-6.

In some embodiments of the invention, the commensal ial strain of the composition is engineered to produce the organic acid of the invention.

In some embodiments, the compositions of the invention are for oral administration. Oral administration of the strains of the invention and where applicable the organic acid of the ion can be effective for neurodegenerative disorders. Also, oral administration is convenient for ts and practitioners and allows delivery to and / or partial or total sation of the intestine.

[Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM In certain embodiments, the composition of the invention comprises one or more pharmaceutically acceptable excipients or carriers.

In certain embodiments, the composition of the invention comprises a commensal bacterial strain that has been lyophilised. Lyophilisation is an effective and convenient technique for preparing stable compositions that allow delivery of bacteria.

In certain embodiments, the invention provides a food product comprising any of the compositions as described above.

In certain embodiments, the invention provides a vaccine composition comprising the composition as described above.

Additionally, the ion provides a method of treating or preventing neurodegenerative diseases, comprising administering a composition comprising a commensal bacterial strain that produces one or more organic acids each having the following a: Rx-COOH, wherein Rx comprises an alkyl group comprising at least 4 s, or wherein Rx ses a phenyl group with a substituent hydroxyl group, wherein optionally the hydroxyl group is at position 4.

The invention also provides a method of treating or prevention egenerative diseases comprising administering a combination of a commensal bacterial strain and an organic acid or a pharmaceutically acceptable salt or ester thereof.

In ping the above invention, the inventors have identified and characterised a bacterial strain that is particularly useful for therapy. The Megasphaera massiliensis strain of the invention is shown to be effective for treating the es described herein, such as neurodegenerative diseases.

Therefore, in another aspect, the invention es a cell of the Megasphaera massiliensis strain deposited under ion number NCIMB 42787, or a derivative f. The invention also provides compositions sing such cells, or biologically pure cultures of such cells. The invention also provides a cell of the Megasphaera massiliensis strain ted under accession number NCIMB 42787, or a derivative thereof, for use in y, in particular for the diseases described herein.

In certain embodiments of either aspect of the invention, the composition is for use in ng brain injury. The neuroprotective activity of the compositions of the invention and their ability to reduce levels of histone deacetylase activity (HDAC) may make them useful for treating brain injury. In preferred embodiments, the compositions of the invention are for use in treating stroke, such as treating brain injury resulting from a stroke.

BRIEF DESCRIPTION OF DRAWINGS Figure 1: Cell viability of neuroblastoma cells Figure 2: Downregulation of IL-6 secretion Figure 3: Secretion of IL-8 [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM ation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM Figure 4: Inhibition of a- synuclein IL-6 and IL-8 secretion Figure 5: Inhibition of a- synuclein induced NFkB promoter activation Figure 6: Inhibition of LPS d NFkB promoter activation Figure 7: Promoting neurite outgrowth: light microscopy and MAP2 gene expression (Figure 7A), Phalloidin immunofluorescence copy (Figure 7B) Figure 8: Change in antioxidant capacity Figure 9: Change in total anti-oxidant capacity (lipid oxidation) Figure 10: Change in ROS levels in (a) U373 cells and (b) Y cells Figure 11: Neuroprotection - cell viability. Figure 11 shows the same data as Figure 1.

Figure 12: Change in histone deacetylase (HDAC) activity Figure 13 Strain-induced changes in whole cell and cell lysate e deacetylase activity (Figure 13A), acid-induced s in histone deacetylase activity (Figure 13B), metabolite production by strains (Figuure 13C) Figure 14 HDAC1 inhibition (Figure 14A), HDAC2 inhibition (Figure 14B), HDAC3 inhibition (Figure 14C) Figure 15 Inhibition of Class I HDACs (Figure 15A); inhibition of HDAC 1 (Figure 15B); inhibition ofHDAC2 (Figure 15C); inhibition of HDAC3 (Figure 15D) Figure 16: Level of Indole production Figure 17: Level of Kyrunenine production Figure 18: Level of BDNF production Figure 19: Levels of metabolite production - neurotransmitters in the brain Figure 20: Levels of lite production - c acids in the supernatant Figure 21: Mean Dopamine (DA) levels (Figure 21 A), DOPAC levels (Figure 2IB) and HVA levels (Figure 21C) in striatum. Data is displayed as Mean + SEM.

Figure 22: Effect on intestinal barrier function.

Figure 23: Production of neurotransmitters in the brain Figure 24: Changes in ampal Receptor Expression - A) Oxytocin Receptor, B) Vasopressin Receptor, C) Glucocorticoid Receptor and D) Mineralocorticoid Receptor [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM Figure 25: Changes in Hippocampal Expression of A) Corticotropin-Releasing e (CRH), B) BDNF Expression and C) TLR4 Figure 26: A) Changes in Hippocampal Corticotropin Releasing Hormone or 1 ) Expression and B) Corticotropin Releasing Hormone or 2 ) Expression Figure 27: Changes in Hippocampal Expression of A) Tumour Necrosis Factor, B) Interleukin lb and C) IL-6 Figure 28: A) Changes in Hippocampal Integrin Alpha M (CD1 lb) Expression and B) Changes in Hippocampal Serotonin 1A Receptor (5-HT1A receptor) Expression Figure 29: A) Changes in Hippocampal Glutamate Ionotropic Receptor NMDA Type Subunit 2A (Grin2A) and B) Glutamate Ionotropic Receptor NMDA Type Subunit 2B (Grin2B) expression Figure 30: s in Hippocampal Expression of A) Gamma-Aminobutyric Acid A Receptor 2 (GABA A2), B) Gamma-Aminobutyric Acid B Receptor 1 (GABA BR1) and C) Dopamine Receptor 1 (DRD1) Figure 31: Changes in Amygdala Receptor Expression - A) Oxytocin Receptor, B) Vasopressin Receptor, C) Glucocorticoid or and D) Mineralocorticoid or Figure 32: Changes in la Expression of A) Brain Derived Neurotrophic Factor , B) Toll-like or 4 (TLR-4), C) Corticotropin Releasing Hormone Receptor 1 (CRFR1) and D) Corticotropin Releasing e Receptor 2 (CRFR2) Figure 33: Changes in Amygdala Expression of A) Integrin Alpha M (CD1 lb), B) Interleukin-6 (IL- 6), C) Glutamate Ionotropic Receptor NMDA Type Subunit 2A (Grin2A) and D) Glutamate Ionotropic Receptor NMDA Type Subunit 2B (Grin2B) Figure 34: Changes in la Expression of A) GABA-A Receptor Alpha 2 Subunit (GABRA2), B) GABA-A Type B Receptor 1 Subunit (GABBR1) and C) Dopamine Receptor 1 (DRD1) Figure 35: Changes in ntal Cortex Expression of A) Oxytocin Receptor, B) Brain Derived Neurotrophic Factor (BDNF), C) Mineralocorticoid Receptor and D) Glucocorticoid Receptor Figure 36: Changes in ntal Cortex Expression of A) Toll-like Receptor 4 (TLR-4), B) Corticotropin Releasing Hormone Receptor 1 (CRFR1), C) Corticotropin Releasing Hormone Receptor 2 (CRFR2) and D) Integrin Alpha M (CD1 lb) Figure 37: Changes in Prefrontal Cortex sion of A) Interleukin-6 (IL-6), B) Glutamate Ionotropic Receptor NMDA Type Subunit 2A (Grin2A), C) Glutamate Ionotropic Receptor NMDA Type Subunit 2B (Grin2B) and D) GABA-A Receptor Alpha 2 Subunit (GABRA2) [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM Figure 38: Changes in ntal Cortex Expression of A) GABA-A Receptor Type B Receptor Subunit 1 1) and B) Dopamine Receptor 1 (DRD1) Figure 39: Changes in Colon Expression of A) Tryptophan Hydroxylase-1 (Tphl) and B) Indoleamine2,3-Dioxygenase-1 (IDO 1) Figure 40: Changes in Ileum Expression of A) Tryptophan Hydroxylase-1 (Tphl) and B) Indoleamine2,3-Dioxygenase-1 (IDO 1) Figure 41: Changes in Circulating Tryptophan Metabolite Levels A) Kynurenine, B) Tryptophan and C) Kynurenine/ Tryptophan Index of metabolism Figure 42: Effect on eron-y Production from from mouse Splenocytes from mice fed with MRx0029 Figure 43: Effect on Interleukin-ip Production from Splenocytes Figure 44: Effect on Interleukin-6 Production from Splenocytes Figure 45: Effect on Tumour Necrosis Factor Production from Splenocytes Figure 46: Effect on Interleukin-10 Production from cytes Figure 47: Effect on Chemoattractant CXCL1 Production from Splenocytes Figure 48: s in Caecal Short Chain Fatty Acid Levels Figure 49: MRx0029 and MRXOOS-induced changes in gene expression levels of Actin, Villin, Occludin TJP1, TJP2, MAP2, DRD2, GABRB3, SYP, PINK1, PARK? .

Figure 50: SHSY5Y cell differentiation induced by MRxOOOS and MRx0029. (A-C) Representative images of immuno labelled cells with Phalloidin and MAP2. (D—F) images of A-C merged with DAPI images. (G—I) P3 tubulin immunolabelled cells. (J-L) merged with DAPI images. ication x630. Western blot analysis of effects of MRxOOOS and MRx0029 treatment on SHSY5Y cells. Western blot membranes were probed with antibodies to MAP2 (M) and b3 tubulin (N). Actin was used as a loading control. Lower panels: representative blots from one of six separate experiments; upper panels: relative densitometric intensity.

DISCLOSURE OF THE INVENTION Bacterial strains The compositions of the invention comprise a sal ial strain useful for treating or preventing es. In a first aspect, the commensal bacterial strains of the invention produce one or more organic acids each having the following formula: Rx-COOH, wherein Rx ses an alkyl group comprising at least 4 carbons, or wherein Rx comprises a phenyl group with a substituent [Annotation] KJM None set by KJM [Annotation] KJM ionNone set by KJM [Annotation] KJM ed set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM hydroxyl group, n optionally the hydroxyl group is at position 4. In a second aspect, the invention provides compositions comprising a commensal bacterial strain and one or more organic acids having the formula R-COOH, or pharmaceutically acceptable salts or esters thereof. The commensal bacterial strain of the second aspect may or may not produce an organic acid according to the invention.

The ors have found that certain sal bacterial strains reduce the activation of matory cytokines such as IL-6 and increase the activation of the inflammatory cytokine IL-8.

IL-8 has been implicated in myelin sheath formation. Chronic mation induced by IL-6 can ultimately lead to cell death. Therefore, the commensal bacterial strains of the invention are particularly useful in the treatment or prevention of egenerative disorders. In some embodiments, the commensal bacterial species are useful in the treatment of conditions characterised by the enhanced activation of IL-6. In some embodiments, the compositions of the ion are for use in the treatment or tion of neurodegenerative diseases characterised by demyelination. Many neurodegenerative es are characterised by demyelination. Demyelination impedes the propagation of action potentials within neurons, impairing effective communication within the nervous system. IL-8 has been shown to contribute positively to myelin sheath formation and repair [22], Therefore, the compositions of the ion are particularly beneficial in the treatment or prevention of neurodegenerative disorders characterised by demyelination, such as Multiple Sclerosis.

The inventors have found that the sal bacteria of the invention alleviate symptoms of neurodegenerative diseases in models of the disease. For example, the inventors have found that the particular commensal bacteria strains promote neurite outgrowth in vitro, and may therefore be used in promoting neuron restoration for the treatment or tion of neurodegenerative diseases. Thus, commensal bacterial strains of the invention are for use in the treatment or prevention of neurodegenerative diseases.

The commensal bacterial strain of the first aspect of the invention may produce one or more of the organic acids of the invention and be administered in ation with one or more of the c acids of the invention.

Commensal bacterial strains of the second aspect that are beneficial for the treatment or prevention of neurodegenerative diseases may be administered in combination with an organic acid that has the neurorestorative or neuroprotective properties described herein.

The commensal bacterial strains of the first and second aspect of the invention comprise those that exhibit the beneficial effects across the IL-6/IL-8 axis, whether or not the commensal bacterial strain produces one or more of the organic acids according to the invention.

The inventors have also found that the commensal bacterial s of ion increase the activation of BDNF. BDNF is a neurotrophic growth factor that has been shown to enhance neuron differentiation and survival. The effect is most prominently observed when a commensal bacterial strain is [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM ation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM administered in combination with the c acid of the invention. Thus, the compositions of the invention can be used in a method of enhancing nerve cell survival in the treatment or prevention of neurodegenerative diseases.

As used herein, "increasing" or "decreasing" "activation" refers to enhancing or reducing the concentration of bio-active biomolecules available for performing physiological functions, preferably following administration to the gastrointestinal tract. In other words, "increasing" or "decreasing" refers to the modulating the level of a biomolecule such that a physiological response is appropriately modified. This may be achieved, for e, by modulating the expression or secretion of the biomolecule.

The compositions of the invention comprise a commensal ial strain. A commensal bacterial strain is a symbiont derived from the gastrointestinal tract of a mammal, such as a human. Examples of genera from which the commensal ial strain may be d e Bacteroides, Enterococcus, Escherichia, Klebsiella, Bifidobacterium, Staphylococcus, Lactobacillus, Megasphaera, Clostridium, Proteus, Pseudomonas, Salmonella, Faecalibacterium, Peptostreptococcus or Peptococcus. In some embodiments, the commensal bacterial strain is of the genus Megasphaera. Preferrably, Megasphaera species for use in the invention include Megasphaera elsdenii, Megasphaera cerevisiae, Megasphaera massiliensis, Megasphaera indica, Megasphaera paucivorans, Megasphaera sueciensis and haera micronuciformis. A further example of a Megasphaera species for use in the invention is Megasphaera hexanoica. The Megasphaera are obligately anaerobic, lactate-fermenting, intestinal microbe of nt and non-ruminant s, including humans. Preferably, the ial strain is derived from the species to which the composition is intended to be administered.

The type strain ofM massiliensis is NP3 (=CSUR P245=DSM 26228) [23], The GenBank accession number for the 16S rRNA gene sequences of M massiliensis strain NP3 is JX424772.1 (disclosed herein as SEQ ID NOT).

The Megasphaera massiliensis bacterium tested in the Examples is referred to herein as strain MRx0029. MRX0029 and MRx0029 are used herein hangeably. A 16S rRNA sequence for the MRx0029 strain that was tested is provided in SEQ ID NO:2. Preferably, the ia for use in the invention is of the species Megasphaera massiliensis, in particular the strain MRx0029.

Strain MRx0029 was deposited with the ational tary authority NCIMB, Ltd. (Ferguson Building, Aberdeen, AB21 9YA, Scotland) by 4D Pharma Research Ltd. (Life es Innovation Building, Cornhill Road, Aberdeen, AB25 2ZS, Scotland) on 13th July 2017 as "Megasphaera massiliensis MRx0029" and was assigned accession number NCIMB 42787. sal bacterial strains closely related to the strain tested in the examples are also ed to be effective for treating or preventing neurodegenerative diseases. In certain embodiments, the bacterial strain for use in the invention has a 16S rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM ation] KJM Unmarked set by KJM 99.5% or 99.9% identical to the 16S rRNA sequence of a bacterial strain of Megasphaera massiliensis.

Preferably, the ial strain for use in the invention has a 16S rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to SEQ ID NO: 2. Preferably, the ial strain for use in the invention has the 16S rRNA sequence represented by SEQ ID NO:2.

Commensal bacterial strains that are biotypes of strains MRx0029 are also expected to be effective for treating or preventing neurodegenerative disorders. A biotype is a closely related strain that has the same or very similar physiological and biochemical characteristics.

Strains that are biotypes of MRx0029 and that are suitable for use in the invention may be identified by sequencing other nucleotide ces for strains MRx0029. For example, substantially the whole genome may be sequenced and a biotype strain for use in the invention may have at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity across at least 80% of its whole genome (e.g. across at least 85%, 90%, 95% or 99%, or across its whole genome). Other suitable sequences for use in identifying biotype strains may include hsp60 or repetitive sequences such as BOX, ERIC, (GTG)5, or REP [24], Biotype strains may have sequences with at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity to the corresponding sequence of the strain MRx0029.

In some embodiments, a commensal bacterial strain useful in the invention is one that produces organic acids of the invention. Such biotypes may be identified by profiling the tion of metabolites of a bacterial strain. lite ing can be used to identity ate bacterial s that produce the timulatory organic acids of the ion. Metabolite profiling can be performed by mass spectrometry. Candidate strains that e the organic acid and that are le for the uses bed herein can then be identified by performing the methods and assays as set out in the examples.

Alternatively, suitable biotypes e of producing an organic acid of the invention are those that contain metabolic pathways that produce the organic acids. Such strains can be identified by genomic is, for example by determining whether the bacterial strain encodes for enzymes required for the biosynthesis of the desired organic acids. For example, hexanoic acid production is a multistep catalytic process requiring the presence of multiple enzymes. A crucial step in the production of hexanoic acid is the condensation of acetyl-CoA and butyric-CoA to form a C6 acyl-CoA intermediate, such as 3- ketohexanoyl-CoA. Enyzmes required for the production of C4 and C2-CoA sors are well known to a person skilled in the art. An example of a suitable enzyme for the condensation of C2 and C4 precursors is Beta-ketothiolase. Enzymatic pathways for the ion of C4 CoA are known to have activity with C6 substrates. Therefore, once the C6 intermediate has been formed, enzymes that catalyse the production C4 butryrl-CoA are also suitable for the generation of hexanoic acid. As such, suitable biotypes are those that encode C4-CoA and C2-CoA production- enzymes and an enzyme with beta-ketothiolase activity. le strains may be identified using homology sequence searching [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM databases such as BLASTP or UNIPROT KB by using query sequences of well known enzymes involved in the production of hexanoic acid.

Listed below are bacteria that are known to e organic acids of use according to the invention, together with supporting references. Using these lists and referenced documents, further strains and species useful for ng neurodegenerative disorders may be identified.

The following species and strains are known to produce hexanoic acid and so may be useful in the compositions of the ion. Preferably, the itions of the invention comprise a bacterial strain of a species selected from: Megasphaera hexanoica [25], in particular strain Megasphaera hexanoica KFCC11466P 745084B1); haera sp. MH [26]; Megasphaera elsdenii [27] and [28], in particular sixain haera elsdenii NCIMB 702410 [29]; Clostridium kluyveri [30]; Clostridium sp. BS-1 [31], CPB6 {Ruminococcaceae bacterium, Clostridium cluster IV) [32]; Lactobacillus sanfranciscensis CB1 [33]; Pediococcus acidilactici [34]; Lysinibacillus spp. Y20 [35] or Eubacterium pyruvativorans (1-6) [36], Bacillus cereus is known to produce retinoic acid and so may be useful in the compositions of the invention [37], In certain embodiments, the composition of the invention ses a strain of the species Bacillus cereus.

The ing genera, species and strains are known to produce (4)-hydroxyphenylacetic acid and so may be useful in the compositions of the invention. ably, the itions of the invention comprise a bacterial strain of the genus Clostridia [38] and [39], preferably a bacterial strain of the species C. difficile [40] and [41], in particular strains C. difficile CDC A567 [42], C. difficile DSM 101085 [43], C. ile DSM 102978 [43], C. difficile DSM 102860 [43], and C. difficile DSM 28666 ; C. putrefaciens [44]); C. stricklandii [44] and Great Plains Lab, Organic Acid Test, Nutritional and Metabolic Profile, Clostridia Bacterial Markers); C. lituseburense [44] and Great Plains Lab, Organic Acid Test, Nutritional and Metabolic Profile, idia Bacterial Markers; C. subterminale id="p-45"

[45], [46] and [44]; C. propionicum [44] [46]; C. clostridiforme [46]; C. cochlearium [46]; C. glycolicum [46]; C. hast forme [46]; C. irregularis [46]; C. perfringens [47]; or C. botulinum Type G and [49], Preferably, the compositions of the invention comprise a bacterial strain of a species selected from: Bacteroides fragilis [50] and [51]); Bacteroides thetaiotaomicron [51]; Bacteroides eggerthii [51]; Bacteroides ovatus [51]; Parabacteroides distasonis [51]; Eubacterium hallii [51]; idium bartlettii [51]; Porphyromonas alis [52]; Flavobacterium sp. [53]; Desulfitobacterium dehalogenans [54] and [55]; Desulfitobacterium hafniense DCB-2 [54]; or Streptomyces sentonii [56], Commensal bacteria that have neuroprotective properties but do not produce organic acids in ance with the first aspect of the ion may be useful in the second aspect of the invention, in compositions in combination with an organic acid. Bacteria which may be employed in the compositions of the second aspect of the present invention include those belonging to the genera [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM ation] KJM None set by KJM [Annotation] KJM ionNone set by KJM [Annotation] KJM Unmarked set by KJM Roseburia (e.g. Roseburia s), Bacteroides, Parabacteroides (e.g. Parabacteroides distasonis), Blautia (e.g. Blautia hydrogenotrophica, Blautia stercoris, Blautia wexlerae), Ruminococcus (e.g.

Ruminococcus albus [57]), Clostridium (e.g. Clostridium butyricum [58]), Lactococcus (e.g.

Lactococcus , Lactococcus rhamnosus). Bifidobacterium (e.g. Bifidobacterium breve, Bifidobacterium , Bifidobacterium adolescentis, Bifidobacterium lactis, Bifidobacterium infantis), Lactobacillus (e.g. Lactobacillusparacasei, Lactobacillus delbrueckii, Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus acidophilus) or Streptococcus (e.g. ococcus thermophilus) ([59], [60]), Alternatively, strains that are biotypes of strains MRX0029 and that are suitable for use in the invention may be identified by using strains MRX0029 and restriction fragment analysis and/or PCR analysis, for example by using fluorescent amplified fragment length polymorphism (FAFLP) and repetitive DNA element (rep)-PCR fingerprinting, or n profiling, or partial 16S or 23S rDNA sequencing.

In preferred embodiments, such techniques may be used to identify other Megasphaera iensis strains.

In certain embodiments, strains that are biotypes of strains MRX0029 and that are suitable for use in the invention are strains that provide the same pattern as strains MRX0029 when analysed by amplified ribosomal DNA restriction is (ARDRA), for example when using Sau3Al ction enzyme (for exemplary methods and guidance see, for example, [61]). Alternatively, biotype s are identified as strains that have the same carbohydrate tation patterns as strains MRX0029.

Other commensal bacterial strains that are useful in the itions and methods of the invention, such as biotypes of MRX0029, may be identified using any appropriate method or strategy, including the assays bed in the examples. For instance, strains for use in the invention may be identified by culturing with lastoma cells and then ing cytokine levels and levels of neuroprotection or neuroproliferation. In particular, ial strains that have similar growth patterns, lic type and/or surface antigens to MRX0029 may be useful in the invention. A useful strain will have comparable immune modulatory activity to MRX0029. In particular, a biotype strain will elicit comparable effects on the neurodegenerative disease models and comparable effects on cytokine levels to the effects shown in the es, which may be identified by using the culturing and administration protocols described in the Examples.

A particularly red strain of the invention is the Megasphaera massiliensis MRX0029 strain. This is the exemplary strain tested in the examples and shown to be effective for treating disease. Therefore, the invention provides a cell, such as an isolated cell, of the Megasphaera massiliensis strain MRX0029, or a derivative thereof. The invention also provides a composition comprising a cell of the Megasphaera massiliensis strain MRX0029, or a derivative thereof. The invention also provides a biologically pure culture of the Megasphaera massiliensis strain MRX0029. The invention also [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM ation] KJM None set by KJM ation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM es a cell of the Megasphaera massiliensis strain MRX0029, or a derivative thereof, for use in therapy, in particular for the diseases bed herein.

A ularly preferred strain of the invention is the Megasphaera massiliensis strain deposited under accession number NCIMB 42787. This is the exemplary MRx0029 strain tested in the examples and shown to be effective for treating disease. Therefore, the invention provides a cell, such as an isolated cell, of the Megasphaera massiliensis strain deposited under accession number NCIMB 42787, or a derivative thereof. The invention also provides a composition comprising a cell of the Megasphaera massiliensis strain ted under accession number NCIMB 42787, or a derivative thereof. The invention also provides a biologically pure culture of the Megasphaera massiliensis strain deposited under accession number NCIMB 42787. The invention also provides a cell of the Megasphaera iensis strain deposited under accession number NCIMB 42787, or a derivative f, for use in therapy, in particular for the es described herein.

A derivative of the strain of the invention may be a er strain (progeny) or a strain cultured (subcloned) from the original. A derivative of a strain of the ion may be modified, for example at the c level, without ablating the ical activity. In particular, a derivative strain of the invention is therapeutically active. A derivative strain will have comparable therapeutic activity to the MRX0029 strain. In particular, a derivative strain will elicit comparable effects on the neurodegenerative disease models and comparable effects on cytokine levels to the effects shown in the Examples, which may be identified by using the culturing and stration protocols described in the Examples. A derivative of the MRX0029 strain will generally be a biotype of the MRX0029 strain.

References to cells of the Megasphaera massiliensis MRX0029 strain encompass any cells that have the same safety and therapeutic efficacy characteristics as the strain 9, and such cells are encompassed by the invention.

In red embodiments, the bacterial strains in the compositions of the invention are viable and capable of partially or totally colonising the intestine.

A further commensal bacteria that may be used in the compositions of the invention is the species Parabacteroides distasonis. The examples demonstrate that Parabacteroides distasonis and Megasphaera massiliensis both have neuroprotective activities, but produce different metabolites and may have different mechanisms of action and specific neuroprotective activities. Therefore, these s may be particularly effective when used in combination. In red embodiments, the composition comprises a strain of the species Parabacteroides distasonis and a strain of the species Megasphaera massiliensis. The composition may also include an organic acid, as defined herein.

The Parabacteroides distasonis ium deposited under accession number NCIMB 42382 was tested in the Examples and is also referred to herein as strain MRxOOOS. MRX0005, MRX005, MRx005 and MRxOOOS are used herein interchangeably. A 16S rRNA sequence for the MRxOOOS [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM strain that was tested is provided in SEQ ID NO: 17. Strain MRxOOOS was deposited with the international depositary authority NCIMB, Ltd. (Ferguson Building, Aberdeen, AB21 9YA, Scotland) by GT Biologies Ltd. (Life Sciences Innovation Building, Aberdeen, AB25 2ZS, Scotland) on 12th March 2015 as "Parabacteroides sp 755" and was assigned accession number NCIMB 42382. GT Biologies Ltd. Subsequently changed its name to 4D Pharma Research Limited.

In preferred embodiments, the invention provides a ition comprising the strain deposited at NCIMB under accession number NCIMB 42787, or a derivative or biotype thereof, and the strain deposited at NCIMB under accession number NCIMB 42382, or a derivative or biotype thereof, preferably for use in therapy, preferably for use in treating a neurodegenerative disease such as Parkinson’s disease.

In certain embodiments, the composition of the invention does not comprise a bacterial strain of the genus Megasphaera. In n embodiments, the composition of the invention does not comprise a bacterial strain of the species Megasphaera massiliensis. In certain embodiments, the composition of the ion does not comprise the haera iensis strain deposited under accession number NCIMB 42787.

Organic acids As demonstrated in the Examples, one or more organic acids described herein have neuroprotective and/or neurorestorative properties. For e, the c acids of the invention may promote neurite outgrowth. Neurite outgrowth is the process of neuritogenesis, which refers to the formation of axonal or dendritic projections from the cell body of a neuron. Axons and dendrites are responsible for receiving and itting action potentials. Thus, increasing neurite outgrowth can restore or preserve effective communication within neural networks within the s system. Therefore, in some embodiments, organic acids suitable for use in the invention are those capable of promoting neurite outgrowth. Such c acids may be for use in a method of preventing disease. In some embodiments, the organic acids are for use in a method of treating or ting a neurodegenerative disease.

The one or more organic acids se the following formula: R-COOH.

In some embodiments, R comprises an alkyl group. As referred to herein, an alkyl group is a linear, ed, or cyclic saturated hydrocarbon chain or any tuent derived therefrom. In some embodiments, R comprises an l group. As referred to herein, an alkenyl group is a , branched, or cyclic unsaturated hydrocarbon sing at least one carbon-to-carbon double bond, or any substituent derived therefrom. In some embodiments, R comprises an l group. As referred to herein, an alkynyl group is a linear, branched, or cyclic unsaturated arbon comprising at least one carbon-carbon triple bond, or any substituent derived therefrom. In some embodiments, R comprises an aryl group. As referred to herein, an aryl group is a linear, branched or cyclic aromatic hydrocarbon, or any substituent derived therefrom.

[Annotation] KJM None set by KJM ation] KJM MigrationNone set by KJM [Annotation] KJM ed set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM As referred to herein, a substituent is an atom or group of atoms that replaces one or more hydrogens of the parent hydrocarbon chain. In some embodiments, R comprises one or more substituents. In some embodiments, the one or more substituents are selected from the list consisting of yl, carbonyl, aldehyde, haloformyl, ate ester, carboxylate, carboxyl, ester, methoxy, hydroperoxy, peroxy, ether, hemiacetal, hemiketal, acetal, ketal, orthoester, methylenedioxy, carboxamide, amine, imine, nitrate, nitrile, nitrite, pyridyl, sulfhydryl, sulphide, disulphide, sulfinyl, yl, sulfino, sulfo, carbonothioyl, catechol or a combination thereof.

In some embodiments, R comprises at least 4 carbon atoms. In some embodiments, R ses at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 carbon atoms. In some embodiments, R comprises in the range of 4 to 19 carbon atoms.

In some embodiments, R comprises in the range of 4 to 11 carbons. In some ments, R comprises an alkyl group consisting of 5 carbon atoms. In some embodiments, the one or more organic acids comprise hexanoic acid. The inventors have found that hexanoic acid and ial strains bed herein that produce hexanoic acid enhance e outgrowth. As such, these organic acids can be used to promote neurite outgrowth. In some embodiments, these organic acids can be used to promote neurite outgrowth in the treatment or prevention of a neurodegenerative disease. Exemplary bacterial s of the invention that produce hexanoic acid se those from the genus Megasphaera.

In some embodiments, R comprises an alkyl group consisting of 4 carbon atoms. In some ments, the one or more organic acids comprise valeric acid. Valeric acid and pentanoic acid are herein used interchangeably. The inventors have found that valeric acid and commensal bacterial strains described herein that produce valeric acid can reduce histone deacetylation. In particular the inventors have found that valeric acid histone deacetylase inhibitor. Histone deacetylases (HDACs) regulate gene expression by modulating chromatin acetylation. The pression of ent isoforms of HDACs has been found in several types of cancer cells as well as in neurological and inflammatory pathologies. In humans, there are a total of 13 HDACs, which are categorised into four main classes - Class I (HDACs 1, 2, 3 and 8), Class Ha (HDACs 4,5,7 and 9) and Class lib (HDACs 6 and 10), Class III (sirtl-sirt7) and class IV (HDAC 11). The inventors have found that valeric acid is a class 1 HDAC inhibitor.

In some embodiments, R comprises a branched alkyl group. In some embodiments, R comprises at least 5 carbon atoms. In some embodiments, R comprises in the range of 5 to 9 carbon atoms. In some embodiments, R comprises a butyric group. In some embodiments, R consists of a butyl group and a propyl group. In some ments, the one or more organic acids se valproic acid. The inventors have shown increases neurite outgrowth and thus can be for use in a method of neurorestoration. Valproic acid has also been shown to aid neurorestoration in a rat model of PD [62], although not in combination with a bacterial therapy. Valproic acid has been shown to increase MAP2 expression.

[Annotation] KJM None set by KJM ation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM In some embodiments, R comprises an aryl group. In some embodiments, the aryl group is a phenyl group. In some embodiments, the phenyl group comprises a hydroxyl group. In some embodiments, the hydroxyl group is at ring position 2, 3, 4, 5 or 6. In some embodiments, the hydroxyl group is at ring position 4. In some embodiments, R consists of a 4-hydroxyphenylacetyl group. In some embodiments, the one or more organic acids comprise 4-hydroxyphenylacetic acid. The inventors have shown that this organic acid or bacterial strains bed herein that produce this organic acid can be used to promote e wth. oxyphenylacetic acid is also an antioxidant. In some embodiments, the one or more organic acids comprising 4-hydroxyphenylacetic acid can be used in the treatment or prevention of egenerative disorders to reduce oxidative stress. 4- hydroxyphenylacetic acid is an intermediate of dopamine and norepinephrine. Dopamine and norepinephrine (also known as noradrenaline) are neurotransmitters acting in the sympathetic nervous system. Levels of dopamine and norepinephrine are reduced in subjects suffering from neurodegenerative diseases, for example Parkinson’s disease. The ors have found that commensal bacterial s described herein that produce 4-hydroxyphenylacetic acid increase the levels of noradrenaline, nin or 5-hydroxytryptamine (5-HT) in the brain. Therefore, 4- hydroxyphenylacetic acid may be useful in the treatment or prevention of egenerative diseases characterised by lower levels of norepheniphrine, dopamine, serotonin or 5- hydroxytryptamine. In some embodiments, 4- hydroxyphenylacetic acid may be useful in the treatment or prevention of neurodegenerative diseases.

In some embodiments, R comprises an alkenyl group. In some embodiments, the alkenyl group is at least partially cyclical. As referred to herein, a "partially cyclical" group is a hydrocarbon chain comprising a linear chain n and a cyclical chain portion. In some embodiments, the partially cyclical alkenyl group comprises 19 carbon atoms. In some embodiments, R comprises in the range of 1 to 8 methyl substituent . In some embodiments, R ses in the range of 1 to 8 carbon- to- carbon double bonds. In some embodiments, the organic acid is retinoic acid or a derivative thereof. The inventors have found that these organic acids when administered in combination with the bacterial strains of the ion increase the activation of BDNF. As such, in some embodiments, these organic acids may be used to increase the activation of BDNF. In some embodiments, these organic acids can be used to increase the activation of BDNF in the treatment or prevention of neurodegenerative diseases.

The organic acids for use in the invention may be identified by using the culturing and administration protocols described in the Examples. For ce, c acids for use in the invention can be fied by culturing with neuroblastoma cells and then ing cytokine levels and levels of neuroprotection or neuroproliferation and neuritogenesis. A useful organic acid may enhance neurite outgrowth activity at least comparable to valproic acid, hexanoic acid or retinoic acid, as shown in the Examples. In ular, a useful organic acid may elicit comparable effects on the neurodegenerative disease models and comparable effects on cytokine and growth factor levels, to the effects shown in [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM the Examples, when the organic acid is administered in combination with a sal bacterial strain of the invention. For example, suitable organic acids will increase the activation of BDNF comparable with retinoic acid when stered in combination with a bacterial strain of the invention. In the first aspect, the organic acid is produced by the commensal bacterial strain of the invention.

In some embodiments, le organic acids of the invention as those that act together with the commensal bacterial strain of the invention to enhance a eutic response. For instance, the Examples show that administration of valproic acid together with MRX0029 increases the secretion of BDNF. BDNF can help support the survival of existing neurons and helps the growth and development of new neurons and synapses. ore, n organic acids of the invention may be for use in methods of neuropreservation, or in other words, to t neuron death.

The compositions of the invention may further comprise short chain fatty acids (SCFAs). SCFAs are organic acids comprising a carboxylic acid group bonded to a linear or branched hydrocarbon alkyl group consisting of 0 to 4 carbon atoms. SCFAs have been shown to regulate the permeability of the blood brain barrier. In ular, SCFAs have been shown to redress defective BBB permeability, thus restoring permeability to physiologically appropriate levels. In some embodiments, the SCFA is selected from the list consisting of formic acid, ethanoic acid, propanoic acid, butyric acid, 2- methylpropanoic acid isovaleric acid, or a salts or esters thereof. In some embodiments, the SCFA is part of the therapeutic combination. In some embodiments, the SCFA is produced by the commensal bacterial strain of the invention.

In some embodiments, the SCFA for use in the invention is butyric acid or a salt thereof. In some embodiments, the commensal bacterial strain produces butyric acid or a salt thereof. In some embodiments, the commensal bacterial strain that produces butyric acid or a salt thereof is the M.massiliensis strain referred to herein as strain MRX0029. Suitable biotypes of the bacterial strain that e the SCFA can be identified using any of the other methods described in the previous n, for example, metabolite profiling, genome analysis, or amplified ribosomal DNA restriction analysis.

In some embodiments, the itions of the ion may further comprise succinic acid or a salt thereof. In some embodiments, the commensal bacterial strain produce succinic acid or a salt f.

In some embodiments, the commensal bacterial strain that produces succinic acid is the strain referred to herein as strain MRX0005. Succinic acid is a Krebs cycle metabolite involved in oxidative phosphorylation. Dysfunction of oxidative phosphorylation has been reported in neurodegenerative disorders including Alzheimer’s disease, Parkinson’s disease and Spinocerebellar ataxia type 1 [63,64]). Therefore increasing the bility of ic acid may reverse defects in oxidative phosphorylation and increase mitochondrial ty to e the health of neurons t to neurodegenerative disease, such as PD [65], [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM ation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM ation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM Therapeutic uses As demonstrated in the es, the bacterial itions of the invention are effective for treating neurodegenerative disorders. In particular, treatment with compositions of the invention increase neuroproliferation or neuritogenesis or act as a neuroprotectant against agents that y neurons, such as dopaminergic neurons. Therefore, the compositions of the invention may be useful for treating or preventing neurodegenerative diseases that are the result of neuron death.

Compositions of the invention can decrease the activation of the NFkB promoter, which activates cytokine production, for example IL-ip, IL-la, IL-18, TNFa and IL-6. Treating cells with mutant asynuclein is a model for familial Parkinson’s. A point mutation at position 53 from adenine to ine leads to a-synuclein mis-folding. The incorrectly folded a-synuclein subsequently aggregates into insoluble fibrils which form Lewy bodies. Therefore, the compositions of the invention may be useful for treating or preventing neurodegenerative disorders that are the result of neuroinflammation, protein misfolding and/or environmental exposure. Compositions of the invention can be used for treatment of familial Parkinson’s. tion of the NFkB promoter is mediated through the TLR4 ligand. TLR4 is known to mediate cell death in the mouse model MPTP, which simulates of Parkinson’s disease.

Compositions of the ion can be used to inhibit the ability of TLR4 to activate the NFkB promoter.

Of particular relevance for PD, both TLR2 and TLR4 were found to be lated in brains of PD patients [66], Moreover a-syn has been described as a ligand for TLR2 [67] and we have demonstrated that a-syn is also a ligand for TLR4 using R4 cells [68], Compositions of the invention decrease the secretion of pro-inflammatory cytokines such as IL-6, which can be induced by lipopolysaccharide (LPS). Treatment of cells with LPS simulates Parkinson’s caused by environmental factors. Compositions of the invention can be used to decrease IL-6 ion.

Compositions of the ion can be used for treatment of environmental Parkinson’s.

Compositions of the ion can increase the secretion of IL-8. IL-8 has been shown to play a role in neuron myelination. In some embodiments, compositions of the invention can be used to increase IL-8 secretion.

The therapeutic compositions of the invention can increase the activation of BDNF. BDNF acts on certain neurons of the central nervous system to support the survival of existing neurons and help the growth and pment of new s and es. BDNF is active in the hippocampus, cortex and basal forebrain, and is important for long-term . The compositions of the invention can therefore be used to se the secretion of BDNF. The compositions may therefore be used in the treatment of egenerative diseases associated with the impairment of long-term memory. The compositions of the invention may be used for improving long-term memory, in particular for improving long-term memory that is impaired by a neurodegenerative disease.

In certain embodiments, the compositions of the invention increase the mitochondria metabolic activity in neuronal cells.

[Annotation] KJM None set by KJM [Annotation] KJM ionNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM ation] KJM Unmarked set by KJM Examples of neurodegenerative diseases to be treated by compositions of the invention include: Parkinson’s disease, including ssive supranuclear palsy, progressive supranuclear palsy, - Richardson-Olszewski syndrome, normal pressure hydrocephalus, vascular or arteriosclerotic parkinsonism and drug-induced parkinsonism; Alzheimer’s disease, including Benson's syndrome; multiple sclerosis; gton’s e; amyotrophic lateral sclerosis; Lou Gehrig's disease; motor e disease; prion disease; spinocerebellar ataxia; spinal muscular y; dementia, including Lewy body, vascular and frontotemporal dementia; primary progressive aphasia; mild cognitive impairment; HIV-related ive impairment, progressive inflammatory neuropathy, and corticobasal degeneration.

In certain embodiments, the compositions of the invention are for use in reducing neuron death, in particular, in the treatment of neurodegenerative disorders. In certain ments, the compositions of the invention are for use in protecting neurons, in particular in the treatment of neurodegenerative disorders.

In n embodiments, the compositions of the invention are for use in reducing or preventing loss of dopaminergic cells in the substantia nigra. In certain embodiments, the compositions of the invention are for use in reducing or preventing the degeneration of dopaminergic neurons in the substantia nigra pars compacta. In certain embodiments, the compositions of the invention are for use in reducing or preventing the degeneration of dopaminergic neurons in the substantia nigra pars compacta and the consequent loss of their projecting nerve fibers in the striatum. In certain ments, the compositions of the invention are for use in reducing or preventing loss of triatal dopaminergic neurons.

In certain embodiments, the compositions of the invention are for use in increasing dopamine levels.

In n embodiments, the compositions of the invention are for use in increasing DOPAC (3,4- Dihydroxyphenylacetic acid) levels. In certain embodiments, the compositions of the invention are for use in increasing dopamine and DOPAC levels. In n embodiments, the dopamine and/or DOPAC levels are increased in the striatum. Dopamine and DOPAC levels may be measured using any riate method known in the art, such as a radio enzymatic method, for e in plasma or CSF (for example as described in [69]), or a reverse-phase HPLC method, perhaps with electrochemical detection, for example in plasma or CSF (for example as described in [70]).

The neuroprotective properties of the compositions of the invention, as shown in the Examples, mean that the compositions may be particularly effective for preventing or delaying onset or progression of neurodegenerative disorders. In certain embodiments, the compositions of the invention are for use in delaying onset or progression of neurodegenerative ers.

Modulation of the microbiota-gut-brain axis Communication between the gut and the brain (the microbiota-gut-brain axis) occurs via a bidirectional neurohumoral ication system. Recent evidence shows that the microbiota that resides in the [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM gut can modulate brain development and produce behavioural phenotypes via the microbiota-gut-brain axis. Indeed, a number of reviews suggest a role of the microbiota-gut-brain axis in maintaining central nervous system functionality and implicate dysfunction of the microbiota-gut-brain axis in the development of central nervous system disorders and conditions [16],[19],[71], The bidirectional communication between the brain and the gut (i. e. the-gut-brain axis) includes the central nervous system, neuroendocrine and neuroimmune systems, including the hypothalamuspituitary-adrenal (HPA) axis, sympathetic and parasympathetic arms of the autonomic nervous system (ANS), including the enteric nervous system (ENS) and the vagus nerve, and the gut microbiota.

As demonstrated in the examples, the compositions of the present invention can modulate the microbiota-gut-brain axis and reduce cell death associated with neurodegenerative disorders.

Accordingly, the compositions of the invention may be useful for treating or preventing neurodegenerative disorders, in particular those disorders and conditions associated with dysfunction of the iota-gut-brain axis.

In particular embodiments, the compositions of the invention may be useful for treating or ting a disease or condition selected from the group consisting of: Parkinson’s disease, including progressive supranuclear palsy, progressive uclear palsy, Steele-Richardson-Olszewski syndrome, normal pressure hydrocephalus, vascular or arteriosclerotic parkinsonism and drug-induced sonism; Alzheimer’s disease, including Benson's syndrome; multiple sclerosis; Huntington’s e; amyotrophic l sclerosis; Lou Gehrig's disease; motor neurone disease; prion disease; erebellar ataxia; spinal muscular atrophy; ia; including Lewy body; vascular and temporal ia; primary progressive aphasia; mild cognitive impairment; lated cognitive impairment, progressive inflammatory neuropathy, and corticobasal degeneration.

The compositions of the invention may be ularly useful for treating or preventing chronic disease, treating or preventing disease in patients that have not responded to other therapies (such as treatment with Levodopa, dopamine ts, MAO-B inhibitors, COMT inhibitors, ate antagonists, and/or anticholinergics), and/or treating or ting the tissue damage and symptoms associated with dysfunction of the iota-gut-brain axis.

In certain embodiments, the compositions of the invention modulate the CNS. In some embodiments, the compositions of the invention modulate the autonomic nervous system (ANS). In some embodiments, the compositions of the invention modulate the enteric nervous system (ENS). In some embodiments, the itions of the invention modulate the hypothalamic, pituitary, adrenal (HPA) axis. In some embodiments, the compositions of the ion modulate the neuroendocrine pathway.

In some embodiments, the compositions of the invention modulate the neuroimmune pathway. In some embodiments, the compositions of the invention modulate the CNS, the ANS, the ENS, the HPA axis and/or the neuroendocrine and neuroimmune pathways. In n ments, the compositions of [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM the invention module the levels of commensal metabolites and/or the gastrointestinal permeability of a subject.

The signalling of the microbiota-gut-brain axis is modulated by neural systems. Accordingly, in some embodiments, the compositions of the invention modulate signalling in neural systems. In certain embodiments, the compositions of the invention modulate the signalling of the l s system.

In some ments, the compositions of the invention modulate signalling in sensory neurons. In other embodiments, the itions of the invention modulate signalling in motor neurons. In some embodiments, the compositions of the invention modulate the signalling in the ANS. In some embodiments, the ANS is the parasympathetic nervous . In preferred embodiments, the compositions of the invention modulate the ling of the vagus nerve. In other embodiments, the ANS is the sympathetic nervous system. In other embodiments, the compositions of the invention modulate the signalling in the enteric nervous system. In certain embodiments, the signalling of ANS and ENS neurons responds directly to luminal contents of the gastrointestinal tract. In other ments, the signalling of ANS and ENS neurons responds indirectly to neurochemicals produced by luminal bacteria. In other embodiments, the signalling of ANS and ENS neurons responds to neurochemicals produced by luminal bacteria or enteroendocrine cells. In certain preferred embodiments, the neurons of the ENS activate vagal afferents that influence the functions of the CNS.

In some embodiments, the compositions of the invention regulate the activity of enterochromaffin cells.

Neurodegenerative diseases Parkinson’s disease son’s disease is a common neurodegenerative disease neuropathologically characterised by degeneration of geneous populations of neural cells ine-producing cells). The clinical diagnosis of son’s disease requires bradykinesia and at least one of the following core ms: resting tremor; muscle rigidity and postural reflex impairment. Other signs and symptoms that may be t or develop during the progression of the disease are autonomic disturbances (sialorrhoea, seborrhoea, constipation, micturition disturbances, sexual functioning, orthostatic hypotension, hyperhydrosis), sleep disturbances and disturbances in the sense of smell or sense of temperature.

Parkinson’s disease is a neurodegenerative diseases that may develop or persist due to dysfunction of the microbiota-gut-brain axis. Therefore, in preferred embodiments, the compositions of the invention are for use in treating or preventing Parkinson’s disease in a t.

In further preferred ments, compositions of the ion are for use in a method of treating or ting Parkinson’s disease. Compositions of the invention may e motor and cognitive functions in models of Parkinson’s disease. Treatment with the compositions may modulate ling in the central, autonomic and enteric nervous systems; may modulate the activity of the HPA axis y; may modulate neuroendocrine and/or mmune pathways; and may modulate the levels [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM of commensal metabolites, inflammatory markers and/or gastrointestinal permeability of a subject, all of which are implicated in the neuropathology of son’s disease. In preferred embodiments, the invention provides a composition comprising a bacterial strain of the species Megasphaera massiliensis for use in a method of treating or preventing Parkinson’s disease. Compositions using Megasphaera massiliensis may be particularly effective for treating son’s disease. The composition may further comprise an organic acid.

In preferred embodiments, the itions of the invention prevent, reduce or ate one or more of the symptoms of Parkinson’s disease in a subject. In preferred embodiments, the compositions of the invention prevent, reduce or alleviate one or more core symptoms of son’s disease in a t. In certain embodiments, the compositions of the invention prevent, reduce or ate bradykinesia in a subject. In n ments, the compositions of the invention prevent, reduce or alleviate resting tremor; muscle rigidity and/or postural reflex impairment in a subject. In certain ments, the compositions of the invention prevent, reduce or alleviate one or more symptoms associated with Parkinson’s disease progression selected from autonomic disturbances (sialorrhoea, seborrhoea, constipation, micturition disturbances, sexual functioning, orthostatic hypotension, hyperhydrosis), sleep disturbances and bances in the sense of smell or sense of temperature.

In preferred embodiments, the compositions of the invention prevent, reduce or alleviate depressive symptoms id with Parkinson’s disease. In certain ments, the compositions of the invention e verbal memory and/or executive functions. In certain embodiments, the compositions of the invention improve attention, g memory, verbal fluency and/or anxiety.

In other preferred embodiments, the compositions of the invention prevent, reduce or alleviate cognitive dysfunctions comorbid with Parkinson’s disease.

In certain ments, the compositions of the invention prevent, reduce or alleviate son’s disease progression. In certain embodiments, the compositions of the invention prevent, reduce or alleviate later motor complications. In certain embodiments, the compositions of the invention prevent, reduce or alleviate late motor fluctuations. In certain embodiments, the compositions of the invention prevent, reduce or alleviate neuronal loss. In certain embodiments, the compositions of the invention improve ms of Parkinson’s disease ia (PDD). In certain embodiments, the compositions of the invention prevent, reduce or alleviate impairment of executive function, attention and/or working memory. In n embodiments, the compositions of the invention improve dopaminergic neurotransmission. In certain embodiments, the compositions of the invention prevent, reduce or alleviate ed dopaminergic neurotransmission.

In some embodiments, the compositions of the invention e the symptoms of Parkinson’s disease according to a symptomatic or diagnostic scale. In certain embodiments, the tests for ing symptomatic improvement of motor function in Parkinson’s disease is the Unified Parkinson’s Disease [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM Rating Scale. In particular, UPDRS II considers the activity of daily life and UPDRS III considers motor-examination.

In some embodiments, the compositions of the invention e the symptoms associated with PDD according to a symptomatic or diagnostic test and/or scale. In certain embodiments, the test or scale is ed from the s Verbal Learning Test - Revised (HVLT-R); the Delis-Kaplan Executive on System S) Color-Word Interference Test; the Hamilton Depression Rating Scale (HAM-D 17; depression); the Hamilton Anxiety Rating Scale (HAM-A; anxiety) and the Unified Parkinson’s Disease Rating Scale (UPDRS; PD symptom severity).

In some embodiments, the compositions of the invention improve the Clinical Global Impression - Global Improvement (CGI-I) scale for assessing psychiatric and neurological disorders. In some embodiments, the compositions of the invention display a positive effect on global social and tional ment of the subject with Parkinson’s disease.

In certain embodiments, the compositions of the invention are for use in treating or preventing neurological ers such as Parkinson’s disease in a subject wherein said use es reducing or preventing loss of dopaminergic cells in the substantia nigra. In certain embodiments, the compositions of the invention are for use in treating or preventing ogical disorders such as Parkinson’s disease in a subject wherein said use involves reducing or preventing the degeneration of dopaminergic neurons in the substantia nigra pars compacta. In certain ments, the compositions of the invention are for use in treating or preventing neurological ers such as Parkinson’s disease in a subject wherein said use involves reducing or preventing the degeneration of dopaminergic neurons in the ntia nigra pars compacta and the consequent loss of their projecting nerve fibers in the striatum. In certain embodiments, the itions of the invention are for use in ng or preventing neurological disorders such as Parkinson’s disease in a t wherein said use involves reducing or preventing loss of nigrostriatal dopaminergic neurons.

In certain embodiments, the compositions of the invention are for use in treating or preventing neurological ers such as Parkinson’s disease in a subject wherein said use involves increasing dopamine levels. In certain embodiments, the compositions of the invention are for use in treating or preventing neurological disorders such as Parkinson’s disease in a t wherein said use involves increasing DOPAC levels. In certain embodiments, the compositions of the invention are for use in treating or preventing neurological disorders such as Parkinson’s disease in a subject n said use involves increasing dopamine and DOPAC levels. In certain embodiments, the dopamine and/or DOPAC levels are increased in the striatum.

Alzheimer’s disease and dementia In DSM-5, the term dementia was replaced with the terms major neurocognitive disorder and mild neurocognitive disorder. Neurocognitive disorder is a heterogeneous class of psychiatric diseases. The most common neurocognitive disorder is mer’s e, followed by vascular dementias or [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM ation] KJM ed set by KJM mixed forms of the two. Other forms of egenerative disorders (e.g. Lewy body disease, frontotemporal dementia, Parkinson’s dementia, Creutzfeldt-Jakob disease, Huntington’s disease, and Wernicke-Korsakoff syndrome) are accompanied by dementia.

Alzheimer’s disease and dementia are also characterised by neuronal loss, so the neuroprotective and neuroproliferative effects shown in the examples for the compositions of the invention indicate that they may be useful for treating or preventing these conditions.

The symptomatic criteria for ia under DSM-5 are ce of significant cognitive decline from a previous level of performance in one or more cognitive domains selected from: learning and memory; language; executive function; complex attention; perceptual-motor and social cognition. The cognitive ts must interfere with independence in everyday activities. In addition, the cognitive deficits do not occur exclusively in the context of a delirium and are not better explained by another mental disorder (for example MDD or schizophrenia).

In addition to the primary symptom, subjects with neurodegenerative ers display behavioural and psychiatric symptoms including agitation, aggression, depression, y, apathy, psychosis and sleep-wake cycle disturbances.

Neurodegenerative ers may develop or t due to dysfunction of the microbiota-gut-brain axis. Therefore, in preferred embodiments, the compositions of the invention are for use in treating or preventing neurodegenerative disorders in a subject. In preferred embodiments, the neurodegenerative disorder is Alzheimer’s disease. In other embodiments, the egenerative disorder is selected from vascular dementias; mixed form Alzheimer’s disease and vascular dementia; Lewy body disease; frontotemporal dementia; Parkinson’s dementia; Creutzfeldt-Jakob disease; gton’s disease; and Wernicke-Korsakoff me.

In red embodiments, the compositions of the ion prevent, reduce or alleviate one or more of the symptoms of neurodegenerative disorders in a subject. In certain ments, the compositions of the invention prevent, reduce or alleviate the occurrence of cognitive decline in a subject. In certain embodiments, the compositions of the invention improve the level of performance of a subject with neurodegenerative disorders in one or more cognitive domains selected from: learning and memory; language; executive function; complex attention; perceptual-motor and social cognition. In some embodiments, the compositions of the invention prevent, reduce or alleviate the occurrence of one or more behavioural and atric symptoms ated with neurodegenerative disorders selected from agitation, aggression, depression, anxiety, apathy, sis and sleep-wake cycle disturbances.

In certain embodiments, the compositions of the invention prevent, reduce or alleviate symptomatic disease by intervention in suspected pathogenic mechanisms at a preclinical stage. In certain embodiments, the compositions of the ion improve disease modification, with slowing or arrest of symptom ssion. In some embodiments, the slowing or arrest of symptom progression correlates with evidence in ng the ying neuropathological process. In preferred [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM ation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM ation] KJM Unmarked set by KJM ments, the compositions of the invention e symptoms of neurodegenerative disorders comprising enhanced cognitive and functional improvement. In preferred embodiments, the compositions of the invention improve the behavioural and psychiatric symptoms of dementia (BPSD).

In preferred embodiments, the compositions of the invention improve the ability of a t with neurodegenerative er to undertake everyday activities.

In preferred embodiments, the compositions of the invention e both ion and functioning in a subject with Alzheimer’s e. In some embodiments, the composition of the invention es the cognitive endpoint in a subject with Alzheimer’s disease. In some embodiments, the compositions of the invention improve the functional endpoint in a subject with Alzheimer’s disease.

In preferred embodiments, the compositions of the invention improve the cognitive and functional endpoint in a subject with Alzheimer’s disease. In yet further preferred embodiments, the compositions of the invention improve the overall clinical response (the global endpoint) in a subject with mer’s disease.

In some embodiments, the compositions of the invention improve the symptoms of neurodegenerative disorders according to a symptomatic or diagnostic test. In certain embodiments, the tests for assessing symptomatic improvement of Alzheimer’s disease (and other neurodegenerative disorders) are selected from objective cognitive, activities of daily living, global assessment of change, health related quality of life tests and tests assessing behavioural and psychiatric symptoms of neurodegenerative disorders.

In certain embodiments, the objective cognitive tests for assessment of symptomatic improvement use the Alzheimer’s disease Assessment Scale cognitive subscale (ADAS-cog) and the classic ADAS scale. In certain embodiments, symptomatic improvement of cognition is assessed using the Neurophysiological Test Battery for Use in Alzheimer’s Disease (NTB).

In some embodiments, the global assessment of change test uses the Clinical Global sion - Global Improvement (CGI-I) scale for assessing psychiatric and neurological ers. In some embodiments, the global scale is the Clinician's Interview Based Impression of Change plus (CIBIC-plus). In some embodiments, the global scale is the Alzheimer’s Disease Cooperative Study Unit Clinician’s Global sion of Change (ADCS-CGIC).

In n embodiments, the health related quality of life measures are the Alzheimer’s e-Related QOL (ADRQL) and the QOL-Alzheimer’s Disease (QOL-AD).

In n embodiments, the tests assessing behavioural and atric symptoms of neurodegenerative disorders are selected from the Behavioural pathology in Alzheimer’s Disease Rating Scale (BEHAVE-AD); the Behavioural Rating Scale for Dementia (BRSD); the Neuropsychiatric ory (NPI); and the Mansfield Agitation Inventory (CMAI).

[Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM In some embodiments, the compositions of the invention are particularly effective at preventing, reducing or alleviating neurodegenerative disorders when used in ation with another y for treating neurodegenerative disorders. In certain embodiments, such therapies include acetylcholinesterase inhibitors including donepezil pt®), galantamine (Razadyne®) and rivastigmine n ®), and memantine.

Multiple Sclerosis Multiple sclerosis (MS) is a demyelinating disease in which the myelin sheath surrounding neurons in the brain and spinal cord are damaged. The exact underlying causes of MS are unknown, but are thought to vary between individuals. Certain forms of MS are hereditary. Environmental factors are also thought to bute to MS. In some individuals, a combination of both genetic and environmental factors may trigger the onset of MS.

There are a wide variety of symptoms associated with MS. Subjects may exhibit almost any neurological symptom associated with the impairment of autonomic, visual, motor or sensory control.

The exact symptoms will vary ing on the site of neuronal damage/demyelination.

IL-8 has been implicated in the formation of myelin sheaths. The compositions of the invention may therefore be for use in the remyelination of neurons in subjects with MS. The compositions of the ion may also be used to t neurons from demyelination. In other words, the compositions of the invention may be for use in a method of treating or preventing multiple sclerosis by restoring or ting loss of neuron myelin sheaths.

In some embodiments, the compositions of the invention prevent, reduce or alleviate one or more symptoms of MS in a subject. In some embodiments, the compositions of the invention t, reduce or alleviate fatigue in a subject. In certain embodiments, the compositions of the ion prevent, reduce or alleviate resting tremor, muscle weakness, muscle spasms, muscle stiffness, paraesthesia and/or ataxia in a subject. In certain embodiments, the itions of the ion prevent, reduce or alleviate one or more symptoms ated with MS progression selected from the list consisting of autonomic disturbances: constipation, micturition disturbances, sexual functioning, dysphagia, dysarthria, syncope, o and/or dizziness; sleep disturbances; and disturbances in the sense of smell or sense of temperature. In some embodiments, the compositions of the invention prevent, reduce or alleviate one or more ocular symptoms associated with MS. In some embodiments, the ocular symptom is selected from the list ting of loss of vision, eye pain, colour blindness, double vision and/or involuntary eye movements in a subject.

In some embodiments, the compositions of the invention t, reduce or alleviate dizziness, vertigo, neuropathic pain, musculoskeletal pain, cognitive ction, bowel incontinence, dysphagia, dysarthria, or any ation thereof.

[Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM ed set by KJM In some embodiments, the compositions of the invention prevent, reduce or alleviate sive symptoms or anxiety comorbid with MS.

In some ments, the compositions of the invention the improvement of ms are determined using the 2017 McDonald criteria for sing MS.

In certain embodiments, treatment with the compositions of the invention results in a reduction in MS incidence or MS severity. In certain embodiments, the compositions of the invention are for use in reducing relapse incidence or relapse severity. In certain embodiments, treatment with the compositions of the invention prevents a decline in motor function or results in improved motor function ated with MS. In n embodiments, the compositions of the invention are for use in preventing a decline in motor function or for use in improving motor function in the treatment of MS.

In certain embodiments, treatment with the compositions of the invention prevents the pment of sis in MS. In certain embodiments, the itions of the invention are for use in preventing sis in the treatment of MS.

In certain embodiments the itions of the invention are for use in preventing multiple sclerosis in a patient that has been identified as at risk of multiple sclerosis, or that has been diagnosed with stage multiple sclerosis or "relapsing-remitting" multiple sclerosis. The compositions of the invention may be useful for preventing the development of MS. The compositions of the invention may be useful for preventing the progression of MS. In n ments, the compositions of the invention are for use in a patient identified as having a genetic predisposition to MS, such as major histocompatibility complex (MHC) class II phenotype, human leukocyte antigen (HLA)-DR2 or HLADR4.

The compositions of the invention may be useful for ng or alleviating multiple sclerosis. The compositions of the invention may be particularly useful for reducing symptoms associated with multiple sclerosis. Treatment or prevention of multiple sclerosis may refer to, for example, an alleviation of the severity of ms or a reduction in the frequency of exacerbations or the range of triggers that are a problem for the patient. In certain embodiments, the compositions of the invention slow or stop ssion of the disease.

In certain embodiments, the compositions of the invention are for use in treating relapsing-remitting MS. In alternative embodiments, the compositions of the invention are for use in treating progressive MS, such as secondary progressive MS (SPMS), which develops over time ing diagnosis of RRMS, primary progressive MS (PPMS) which ts gradual continuous neurologic deterioration and progressive relapsing MS (PRMS), which is similar to PPMS but with overlapping relapses.

In certain embodiments, the compositions of the invention are for use in treating one or more of symptoms of MS selected from the group consisting of: fatigue, vision problems, numbness, tingling, muscle spasms, muscle stiffness, muscle weakness, mobility problems, pain, problems with thinking, [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM learning and planning, depression and anxiety, sexual problems, bladder problems, bowel problems, speech and swallowing difficulties.

Neurochemical factors, eptides and neurotransmitters and the iota-gut-brain axis As outlined above, the microbiota-gut-brain axis is modulated by a number of different physiological systems. The microbiota-gut-brain axis is modulated by a number of signalling molecules. Alterations in the levels of these signalling molecules s in neurodegenerative diseases. The experiments med by the inventors te that administration of haera commensal bacterial strains, and in particular Megasphaera massiliensis, can modulate levels of indole and kynurenine.

Dysregulation of these lites can lead to neurodegenerative es, such as Parkinson’s disease.

In certain embodiments, the compositions of the ion modulate the levels of brain monoamines and metabolites thereof. In preferred embodiments the metabolite is kynurenine. In certain embodiments, the compositions of the invention modulate nine, which is the main route of tryptophan metabolism, which serves as a route to nicotinamide e dinucleotide (NAD+) production. Kynurenine can be metabolized to neuroactive compounds such as kynurenic acid (KYNA) and 3-hydroxykynurenine (3-OH-l-KYN), and in further steps to quinolinic acid (QUIN).

Dysregulation of the kynurenine y can lead to activation of the immune system and the accumulation of potentially neurotoxic compounds. Alterations in the kynurenine metabolism may be involved in the development of Parkinson’s diseases. Kynurenine levels have been demonstrated to be decreased in the frontal cortex, putamen and ntia nigra pars compacta of patients with PD (Parkinson’s e) [72], Therefore, in certain ments the compositions of the invention are for use in increasing the levels of kynurenine in the treatment of Parkinson’s disease.

In certain embodiments of the invention the compositions of the invention can increase the levels kynurenin. Increased levels of kynurenine have been shown to attenuate MPP+-induced neuronal cell death in vitro in a human dopaminergic neuroblastoma cell line [73], In certain embodiments nine and kynurenic acid, can activate GI aryl hydrocarbon receptor (Ahr) and GPR35 receptors.

Activation of Ahr receptor s IL-22 production, which can t local mation. Activation of GPR35 inducing the production of inositol triphosphate and Ca2+ mobilization.

In certain embodiments, the compositions of the invention modulate the levels of indole. In preferred embodiments the metabolite is kynurenine. In certain embodiments, the compositions of the invention modulate kynurenine, which it the main route of tryptophan metabolism.

The signalling of the microbiota-gut-brain axis is modulated by levels of neurochemical factors, neuropeptides and neurotransmitters. Accordingly, in certain embodiments, the compositions of the invention modulates levels of neurochemical factors, neuropeptides and neurotransmitters.

Accordingly, in certain preferred embodiments, the compositions of the invention directly alter CNS biochemistry.

[Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM ionNone set by KJM [Annotation] KJM Unmarked set by KJM The signalling of the microbiota-gut-brain axis is modulated by levels of y-aminobutyric acid (GABA).

Accordingly, in preferred embodiments, the compositions of the invention modulate the levels of GABA. GABA is an inhibitory neurotransmitter that reduces neuronal excitability. In n embodiments, the compositions of the ion increase the levels of GABA. In certain embodiments, the compositions of the invention decrease the levels of GABA. In certain embodiments, the itions of the invention alter GABAergic neurotransmission. In certain embodiments, the compositions of the invention modulate the level of GABA transcription in different regions of the l nervous system. In certain embodiments, the commensal derived GABA crosses the bloodbrain barrier and affects neurotransmission directly. In certain embodiments, the compositions of the invention lead to a reduction of GABA in the hippocampus, amygdala and/or locus coeruleus. In n embodiments, the compositions of the invention lead to an increase of GABA in cortical regions.

Immune response The signalling of the microbiota-gut-brain axis is modulated by alterations in the immune response and inflammatory factors and markers. Accordingly, in certain embodiments, the compositions of the invention may modulate the immune response. In certain embodiments, the compositions of the invention te the systemic levels of circulating neuroimmune signalling les. In certain red embodiments, the itions of the invention modulate pro-inflammatory ne production and inflammation. In certain embodiments, the compositions of the invention modulate the inflammatory state. In n embodiments, the compositions of the ion decrease IL-6 production and secretion. In certain ments, the compositions of the invention decrease the activation of the NFkB er. In certain embodiments, the compositions of the invention are able to modulate the activation of IL-6 production by the potent pro-inflammatory xin lipopolysaccharide (LPS). In certain embodiments, the compositions of the invention are able to modulate the activation of the NFkB promoter by LPS and a-synuclein mutant proteins such as A53T.

Increased circulating levels of cytokines are closely associated with various neurodegenerative disorders, including Parkinson’s, dementia and Alzheimer’s. In certain ments, the compositions of the invention are for use in reducing IL-6 levels and/or NFkB levels in the treatment of a neurodegenerative disorder. In some embodiments, the compositions of the ion increase the secretion of IL-8. IL-8 has been shown to induce myelin sheath formation and e or preserve effective neuronal communication. Thus, in some embodiments, the compositions of the invention are for use in ng myelin sheath formation in the treatment of neurodegenerative diseases. In some embodiments, the compositions of the invention are for use in restoring neuronal communication. In some embodiments, the compositions of the invention are for use in preserving neuronal communication.

The signalling of the iota-gut-brain axis is modulated by levels of commensal metabolites.

Accordingly, in certain embodiments, the compositions of the invention modulate the systemic levels [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM ation] KJM Unmarked set by KJM of microbiota metabolites. In certain preferred embodiments, the compositions of the invention te the level of short chain fatty acids (SCFAs). In n embodiments the level of SCFAs is sed or decreased. In some embodiments, the level of SCFAs is increased. In some embodiments, the SCFA is butyric acid (BA) (or butyrate). In some embodiments, the SCFA is propionic acid (PPA).

In some embodiments, the SCFA is acetic acid. In certain ments, the compositions of the invention modulate the ability of SCFAs to cross the blood-brain barrier.

Histone acetylation and deacetylation are important epigenetic regulators of gene expression. An imbalance in histone acetylation and deacetylation can result in apoptosis. Dysregulation of such histone acetyltransferases has been implicated in the pathogenesis associated with age-associated neurodegenerative diseases, such as Parkinson’s disease, Huntington's disease, Alzheimer's disease, amyotrophic lateral sclerosis and cognitive e [74], Accordingly, in certain ments, the compositions of the invention can modulate histone deacetylase activity. In certain ments, the compositions of the invention can reduce histone deacetylase ty. In certain ments, the compositions of the invention can reduce histone acetylase activity. In certain embodiments, the compositions of the invention can reduce class 1 histone deacetylase activity. ts with neurodegenerative diseases, including Parkinson’s e, Huntington's e, Alzheimer's disease and amyotrophic lateral sclerosis, exhibit high levels of lipid peroxidation. Lipid are vulnerable to oxidation by reactive oxygen species, and the brain is rich in saturated fatty acids. Accordingly, in certain embodiments, the compositions of the invention can modulate lipid peroxidation. In certain embodiments, the compositions of the invention can reduce lipid peroxidation.

Reducing the oxidative damage caused by reactive oxygen species can be used to target early the stages neurodegenerative diseases. Accordingly, in certain embodiments, the compositions of the invention are for use in treating early stage egeneration. Also accordingly, in certain embodiments, the compositions of the invention are for use in ting the development of a neurodegenerative disorder. In such embodiments, the compositions of the invention may be for use in a patient that has been identified as at risk of developing a neurodegenerative disorder.

The signalling of the microbiota-gut-brain axis is modulated by levels of gastrointestinal permeability. ingly, in some embodiments, the compositions of the invention alter the integrity of the gastrointestinal tract epithelium. In certain embodiments, the compositions of the invention modulate the permeability of the gastrointestinal tract. In certain embodiments, the compositions of the invention te the barrier on and integrity of the gastrointestinal tract. In certain ments, the compositions of the invention modulate gastrointestinal tract motility. In n embodiments, the compositions of the invention modulate the translocation of commensal metabolites and inflammatory signalling molecules into the tream from the gastrointestinal tract lumen.

The signalling of the microbiota-gut-brain axis is modulated by microbiome composition in the gastrointestinal tract. Accordingly, in certain embodiments, the compositions of the invention [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM ation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM modulates the microbiome composition of the gastrointestinal tract. In certain embodiments, the compositions of the invention prevents microbiome dysbiosis and associated increases in toxic metabolites (e.g. LPS). In certain embodiments, the compositions of the invention modulate the levels of Clostridium in the gastrointestinal tract. In preferred embodiments, the compositions of the invention reduce the level of idium in the gastrointestinal tract. In certain ments, the compositions of the invention reduce the levels of Campylobacter jejuni. In certain embodiments, the itions of the invention modulate the proliferation of l anaerobic bacteria and the production of oxins produced by these bacteria. In certain embodiments, the compositions of the invention modulate the microbiome levels of Lactobacillus and/or Bifidobacterium. In certain embodiments, the compositions of the invention modulate the microbiome levels of Sutterella, Prevotella, Rumincoccus genera and/or the genaceae family. In certain embodiments, the itions of the invention increase the level of Lactobacillus plantarum and/or Saccharomyces boulardii.

Brain injury The examples demonstrate that the compositions of the invention are neuroprotective and have HD AC inhibitory activity. HDAC2 is a crucial target for functional recovery from stroke [75] and HDAC tion can prevent white matter injury [76], so the compositions of the invention may be useful in the treatment of brain injury.

In certain embodiments, the compositions of the invention are for use in treating brain injury. In some embodiments, the brain injury is a traumatic brain . In some embodiments, the brain injury is an acquired brain injury. In some embodiments, the compositions of the invention are for use in ng brain injury resulting from trauma. In some ments, the compositions of the invention are for use in treating brain injury resulting from a . In some embodiments, the compositions of the invention are for use in treating brain injury resulting from a stroke. In some embodiments, the compositions of the invention are for use in treating brain injury resulting from a brain haemorrhage.

In some embodiments, the compositions of the invention are for use in treating brain injury resulting from encephalitis. In some embodiments, the compositions of the invention are for use in treating brain injury resulting from cerebral hypoxia. In some embodiments, the compositions of the invention are for use in treating brain injury resulting from cerebral anoxia.

In preferred embodiments, the compositions of the invention are for use in treating stroke. The effects shown in the examples are particularly relevant to the treatment of stroke. Stroke occurs when blood flow to at least a part of the brain is interrupted. Without an adequate supply of blood to provide oxygen and nutrients to the brain tissue and to remove waste products from the brain tissue, brain cells rapidly begin to die. The symptoms of stroke are ent on the region of the brain which is affected by the uate blood flow. ms include paralysis, numbness or weakness of the muscles, loss of balance, dizziness, sudden severe headaches, speech impairment, loss of memory, loss of reasoning ability, sudden confusion, vision impairment, coma or even death. A stroke is also referred [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM ed set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM to as a brain attack or a ovascular accident (CVA). The symptoms of stroke may be brief if adequate blood flow is restored within a short period of time. However, if inadequate blood flow continues for a significant period of time, the symptoms can be ent.

In some embodiments, the stroke is cerebral ischemia. Cerebral ischemia results when there is insufficient blood flow to the tissues of the brain to meet metabolic demand. In some embodiments, the cerebral ischemia is focal cerebral ischemia, i.e. confined to a specific region of the brain. In some embodiments the al ischemia is global cerebral ischemia, i.e. encompassing a wide area of the brain tissue. Focal cerebral ischemia commonly occurs when a cerebral vessel has become d, either lly or tely, reducing the flow of blood to a specific region of the brain. In some embodiments the focal cerebral ischemia is ischemic stroke. In some embodiments, the ischemic stroke is thrombotic, i.e. caused by a thrombus or blood clot, which develops in a cerebral vessel and restricts or blocks blood flow. In some embodiments the ischemic stroke is a thrombotic stroke. In some embodiments, the ischemic stroke is embolic, i.e. caused by an embolus, or an unattached mass that travels through the bloodstream and restricts or blocks blood flow at a site distant from its point of origin. In some ments the ischemic stroke is an embolic stroke. Global cerebral ischemia ly occurs when blood flow to the brain as a whole is blocked or reduced. In some embodiments the global cerebral ischemia is caused by hypoperfusion, i.e. due to shock. In some embodiments the global cerebral ia is a result of a cardiac arrest.

In some embodiments the subject diagnosed with brain injury has suffered cerebral ia. In some embodiments, the subject diagnosed with brain injury has suffered focal cerebral ischemia. In some embodiments, the subject diagnosed with brain injury has suffered an ischemic stroke. In some embodiments, the subject diagnosed with brain injury has suffered a otic stroke. In some embodiments, the subject diagnosed with brain injury has suffered an embolic stroke. In some embodiments, the subject diagnosed with brain injury has suffered global cerebral ischemia. In some embodiments, the subject diagnosed with brain injury has suffered hypoperfusion. In some embodiments, the subject sed with brain injury has suffered a cardiac arrest.

In some embodiments, the compositions of the invention are for use in treating cerebral ischemia. In some embodiments, the compositions of the invention are for use in treating focal cerebral ischemia.

In some ments, the itions of the ion are for use treating ischemic stroke. In some embodiments, the compositions of the invention are for use in treating thrombotic stroke. In some embodiments, the compositions of the invention are for use in treating embolic . In some embodiments, the compositions of the invention are for use in treating global cerebral ischemia. In some embodiments, the compositions of the invention are for use in ng hypoperfusion.

In some embodiments, the stroke is hemorrhagic stroke. Hemorrhagic stroke is caused by bleeding into or around the brain resulting in swelling, pressure and damage to the cells and tissues of the brain.

Hemorrhagic stroke is commonly a result of a ed blood vessel that ruptures and bleeds into the [Annotation] KJM None set by KJM ation] KJM ionNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM ed set by KJM surrounding brain. In some embodiments, the hemorrhagic stroke is an erebral hemorrhage, i.e. caused by bleeding within the brain tissue itself. In some embodiments the intracerebral hemorrhage is caused by an intraparenchymal hemorrhage. In some embodiments the intracerebral hage is caused by an intraventricular hemorrhage. In some embodiments the hemorrhagic stroke is a subarachnoid hemorrhage i.e. bleeding that occurs outside of the brain tissue but still within the skull.

In some embodiments, the hemorrhagic stroke is a result of cerebral amyloid angiopathy. In some embodiments, the hemorrhagic stroke is a result of a brain aneurysm. In some embodiments, the hemorrhagic stroke is a result of cerebral arteriovenous malformation (AVM).

In some embodiments the subject diagnosed with brain injury has suffered hemorrhagic stroke. In some embodiments, the subject diagnosed with brain injury has suffered an intracerebral hemorrhage.

In some embodiments, the subject sed with brain injury has suffered an intraparenchymal hemorrhage. In some embodiments, the subject diagnosed with brain injury has suffered an intraventricular hemorrhage. In some embodiments, the subject diagnosed with brain injury has suffered a subarachnoid hemorrhage. In some embodiments, the subject diagnosed with brain injury has suffered cerebral amyloid angiopathy. In some embodiments, the subject diagnosed with brain injury has suffered a brain aneurysm. In some embodiments, the subject diagnosed with brain injury has suffered cerebral AVM.

In some embodiments, the compositions of the invention are for use in treating hemorrhagic stroke. In some embodiments, the itions of the invention are for use in treating an intracerebral hemorrhage. In some embodiments, the compositions of the invention are for use in treating an intraparenchymal hemorrhage. In some embodiments, the compositions of the invention are for use in treating an intraventricular hemorrhage. In some embodiments, the compositions of the invention are for use in treating a subarachnoid hage. In some ments, the compositions of the invention are for use in treating a cerebral amyloid athy. In some embodiments, the compositions of the invention are for use in treating a brain sm. In some embodiments, the itions of the invention are for use in treating al AVM. ation of adequate blood flow to the brain after a period of interruption, though effective in alleviating the symptoms ated with stroke, can paradoxically result in r damage to the brain tissue. During the period of interruption, the ed tissue suffers from a lack of oxygen and nutrients, and the sudden restoration of blood flow can result in inflammation and oxidative damage through the induction of oxidative stress. This is known as reperfusion injury, and is well documented not only following stroke, but also following a heart attack or other tissue damage when blood supply returns to the tissue after a period of ischemia or lack of oxygen. In some embodiments the subject diagnosed with brain injury has suffered from reperfusion injury as a result of stroke. In some ments, the compositions of the invention are for use in treating reperfusion injury as a result of stroke.

[Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM A transient ischemic attack (TIA), often referred to as a mini-stroke, is a recognised warning sign for a more serious stroke. Subjects who have suffered one or more TIAs are therefore at greater risk of stroke. In some embodiments the t diagnosed with brain injury has suffered a TIA. In some embodiments, the compositions of the ion are for use in treating a TIA. In some embodiments, the compositions of the invention are for use in treating brain injury in a subject who has suffered a High blood pressure, high blood cholesterol, a familial history of stroke, heart disease, es, brain aneurysms, arteriovenous malformations, sickle cell disease, vasculitis, bleeding disorders, use of nonsteroidal anti-inflammatory drugs s), smoking tobacco, drinking large amounts of alcohol, illegal drug use, obesity, lack of physical activity and an unhealthy diet are all ered to be risk s for stroke. In particular, lowering blood pressure has been conclusively shown to prevent both ischemic and hemorrhagic strokes [77], [78], In some embodiments, the compositions of the invention are for use in treating brain injury in a subject who has at least one risk factor for stroke. In some embodiments the subject has two risk factors for stroke. In some embodiments the subject has three risk factors for stroke. In some embodiments the subject has four risk factors for . In some embodiments the subject has more than four risk factors for stroke. In some embodiments the subject has high blood pressure. In some embodiments the subject has high blood cholesterol. In some embodiments the subject has a familial history of stroke. In some embodiments the t has heart disease. In some embodiments the subject has diabetes. In some embodiments the subject has a brain aneurysm. In some embodiments the subject has arteriovenous malformations. In some embodiments the subject has vasculitis. In some embodiments the subject has sickle cell disease. In some ments the subject has a bleeding disorder. In some ments the subject has a history of use of nonsteroidal anti-inflammatory drugs (NSAIDs). In some embodiments the subject smokes tobacco. In some embodiments the subject drinks large amounts of l. In some ments the subject uses illegal drugs. In some ments the t is obese. In some embodiments the subject is overweight. In some embodiments the subject has a lack of physical ty. In some embodiments the subject has an unhealthy diet.

The es indicate that the compositions of the invention may be useful for treating brain injury and aiding recovery when administered before the injury event occurs. Therefore, the compositions of the invention may be particularly useful for ng brain injury when administered to subjects at risk of brain injury, such as stroke.

In certain embodiments, the compositions of the invention are for use in reducing the damage caused by a potential brain injury, preferably a stroke. The compositions may reduce the damage caused when they are administered before the potential brain injury occurs, in particular when administered to a patient fied as at risk of a brain injury. ation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM The examples indicate that the compositions of the invention may be useful for treating brain injury and aiding ry when administered after the injury event . Therefore, the compositions of the invention may be particularly useful for treating brain injury when administered to subjects following a brain injury, such as stroke.

In some embodiments, the compositions of the invention treat brain injury by ng motoric damage. In some embodiments, the compositions of the invention treat brain injury by improving motor function. In some embodiments, the compositions of the invention treat brain injury by improving muscle strength. In some embodiments, the compositions of the invention treat brain injury by improving memory. In some embodiments, the compositions of the invention treat brain injury by improving social recognition. In some embodiments, the compositions of the invention treat brain injury by improving neurological function. ent of brain injury may refer to, for example, an ation of the severity of symptoms.

Treatment of brain injury may also refer to reducing the neurological impairments following stroke.

Compositions of the invention for use in treating stroke maybe provided to the subject in advance of the onset of stroke, for e in a patient identified as being at risk of stroke. Compositions of the ion for use in treating stroke may be provided after a stroke has occurred, for e, during recovery. Compositions of the invention for use in treating stroke may be provided during the acute phase of ry (i.e. up to one week after stroke). Compositions of the invention for use in treating stroke may be provided during the subacute phase of recovery (i.e. from one week up to three months after stroke). Compositions of the invention for use in treating stroke may be provided during the chronic phase of recovery (from three months after stroke).

In certain embodiments, the compositions of the invention are for use in combination with a secondary active agent. In certain embodiments, the itions of the invention are for use in combination with aspirin or tissue plasminogen activator (tPA). Other secondary agents include other antiplatelets (such as clopidogrel), anticoagulants (such as heparins, warfarin, apixaban, tran, edoxaban or rivaroxaban), antihypertensives (such as diuretics, ACE inhibitors, calcium channel blockers, betablockers or alpha-blockers) or s. The compositions of the invention may improve the patient’s se to the secondary active agent.

In certain ments, the compositions of the invention reduce the effect of ischemia on tissues. In certain embodiments, the compositions of the invention reduce the amount of damage to tissues caused by ischemia. In certain embodiments, the tissues damaged by ischemia are the cerebral tissues. In certain embodiments, the compositions of the invention reduce necrosis or the number of necrotic cells.

In certain embodiments, the compositions of the invention reduce sis or the number of apoptotic cells. In n ments, the compositions of the invention reduce the number of necrotic and apoptotic cells. In certain embodiments, the compositions of the invention prevent cell death by necrosis and/or apoptosis. In certain embodiments, the compositions of the invention prevent cell death [Annotation] KJM None set by KJM ation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM by necrosis and/or apoptosis caused by ischemia. In certain embodiments, the compositions of the invention improve the recovery of the tissue damaged by ischemia. In n embodiments, the compositions of the invention improve the speed of clearance of necrotic cells and/or apoptotic cells.

In certain embodiments, the compositions of the invention improve the cy of the clearance of necrotic cells and/or apoptotic cells. In n embodiments, the compositions of the invention improve the replacement and/or regeneration of cells within tissues. In certain embodiments, the compositions of the invention improve the replacement and/or regeneration of cells within tissues damaged by ischemia. In certain embodiments, the compositions of the invention improve the overall histology of the tissue (for example upon a biopsy).

Modes of administration Preferably, the compositions of the invention are to be administered to the gastrointestinal tract in order to enable delivery to and / or partial or total colonisation of the intestine with the commensal bacterial strain of the invention. Generally, the compositions of the invention are administered orally, but they may be administered rectally, intranasally, or via buccal or sublingual routes. In some embodiments, a ition comprises the strain and the c acid of the invention. In some embodiments, separate compositions of the organic acid and the commensal bacterial strain are ed.

In n embodiments, the compositions comprising the commensal bacterial strain of the invention may be administered as a foam, as a spray or a gel.

In certain embodiments, the compositions of the invention may be stered as a suppository, such as a rectal suppository, for example in the form of a theobroma oil (cocoa butter), synthetic hard fat (e.g. suppocire, witepsol), glycero-gelatin, polyethylene glycol, or soap glycerin composition.

In certain embodiments, the composition of the ion is administered to the gastrointestinal tract via a tube, such as a nasogastric tube, orogastric tube, gastric tube, jejunostomy tube (J tube), percutaneous endoscopic gastrostomy (PEG), or a port, such as a chest wall port that provides access to the stomach, jejunum and other le access ports.

The compositions of the invention may be stered once, or they may be administered sequentially as part of a treatment n. In n embodiments, the compositions of the invention are to be administered daily.

In certain embodiments of the invention, treatment according to the invention is accompanied by assessment of the patient’s gut microbiota. Treatment may be ed if delivery of and / or partial or total colonisation with the strain of the invention is not ed such that efficacy is not ed, or treatment may be ceased if delivery and / or partial or total colonisation is successful and efficacy is observed.

[Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM ation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM ed set by KJM In certain embodiments, the composition of the invention may be administered to a pregnant animal, for example a mammal such as a human in order to prevent an inflammatory or autoimmune disease developing in her child in utero and / or after it is born.

The compositions of the invention may be administered to a t that has been sed with a neurodegenerative disease, or that has been fied as being at risk of a neurodegenerative disease.

The compositions may also be administered as a prophylactic measure to prevent the development of neurodegenerative disease in a healthy patient.

The compositions of the invention may be administered to a patient that has been fied as having an al gut microbiota. For example, the patient may have d or absent colonisation by Megasphaera , and in particular Megasphaera massiliensis.

The compositions comprising the bacteria of the invention may be administered as a food product, such as a nutritional supplement.

In some embodiments, a composition comprising a pharmaceutically acceptable salt or ester of the organic acid may be administered intravenously. In some embodiments, the organic acid is lyophilised and administered orally.

In some embodiments, the composition comprising the commensal bacterial strain and the composition comprising the organic acid are to be administered simultaneously, separately or sequentially. Each of the different compositions may be stered by any combination of the modes of administration described herein. Generally, the compositions of the invention are for the treatment of humans, although they may be used to treat animals including monogastric mammals such as poultry, pigs, cats, dogs, horses or s. The compositions of the invention may be useful for enhancing the growth and performance of animals. If administered to s, oral gavage may be used.

Compositions lly, the composition of the invention comprises bacteria. In some embodiments, the composition further comprises an organic acid or a pharmaceutically acceptable salt or ester thereof. In red embodiments of the invention, the composition is formulated in freeze-dried form. For example, the ition of the invention may se granules or gelatin capsules, for example hard n capsules, comprising a bacterial strain of the invention and/or an organic acid of the invention. In some embodiments, the organic acid of the invention and the bacteria of the invention and ated as separate compositions. In some embodiments, each of the separate compositions are formulated in a freeze-dried form. General guidance on the formulation of the compositions of the invention can be found for example, in Aulton's Pharmaceutics: The Design and Manufacture of Medicines.

Preferably, the composition of the invention comprises lyophilised bacteria. Lyophilisation of ia is a well-established procedure and relevant guidance is available in, for example, references [79], [], id="p-81"

[81].

[Annotation] KJM None set by KJM ation] KJM MigrationNone set by KJM [Annotation] KJM ed set by KJM [Annotation] KJM None set by KJM ation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM Alternatively, the composition of the invention may comprise a live, active bacterial culture.

In some embodiments, the bacterial strain in the composition of the invention has not been inactivated, for example, has not been nactivated. In some embodiments, the ial strain in the composition of the invention has not been killed, for example, has not been heat-killed. In some embodiments, the bacterial strain in the composition of the invention has not been attenuated, for example, has not been heat-attenuated. For example, in some embodiments, the bacterial strain in the composition of the ion has not been killed, inactivated and/or attenuated. For example, in some embodiments, the bacterial strain in the composition of the invention is live. For e, in some embodiments, the bacterial strain in the composition of the invention is viable. For example, in some embodiments, the bacterial strain in the composition of the invention is capable of partially or y colonising the intestine. For example, in some embodiments, the bacterial strain in the composition of the invention is viable and capable of partially or totally colonising the intestine.

In some embodiments, the composition comprises a mixture of live bacterial strains and bacterial strains that have been killed.

In preferred embodiments, the composition of the invention is encapsulated to enable delivery of the ial strain and/or the organic acid or a pharmaceutically acceptable salt or ester f to the intestine. Encapsulation protects the composition from degradation until ry at the target location through, for example, ing with chemical or physical stimuli such as pressure, enzymatic activity, or physical disintegration, which may be triggered by changes in pH. Any appropriate encapsulation method may be used. Exemplary encapsulation techniques include entrapment within a porous matrix, ment or adsorption on solid r surfaces, self-aggregation by flocculation or with crosslinking agents, and mechanical nment behind a microporous membrane or a microcapsule.

Guidance on encapsulation that may be useful for preparing compositions of the invention is available in, for example, nces [82] and [83], The composition may be administered orally and may be in the form of a tablet, capsule or powder.

Encapsulated products are preferred because Megasphaera are anaerobes. Other ingredients (such as vitamin C, for example), may be included as oxygen scavengers and prebiotic substrates to improve the delivery and / or partial or total colonisation and survival in vivo. atively, the probiotic composition of the invention may be stered orally as a food or nutritional product, such as milk or whey based fermented dairy product, or as a pharmaceutical t.

The composition may be formulated as a probiotic.

A composition of the invention includes a therapeutically effective amount of a bacterial strain of the invention and/or a eutically effective amount an organic acid or a pharmaceutically acceptable salt or ester thereof of the invention. A therapeutically effective amount of a bacterial strain and/or an organic acid of the invention is sufficient to exert a beneficial effect upon a patient. A therapeutically [Annotation] KJM None set by KJM ation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM effective amount of a bacterial strain may be sufficient to result in delivery to and / or partial or total colonisation of the patient’s intestine.

A suitable daily dose of the bacteria, for example for an adult human, may be from about 1 x 103 to about 1 x 1011 colony forming units (CFU); for example, from about 1 x 107 to about 1 x 1010 CFU; in another e from about 1 x 106 to about 1 x 1010 CFU.

In certain embodiments, the composition contains the bacterial strain in an amount of from about 1 x 106to about 1 x 1011 CFU/g, respect to the weight of the composition; for example, from about 1 x 108 to about 1 x 1010 CFU/g. The dose maybe, for e, 1 g, 3g, 5g, and lOg.Typically, a probiotic, such as the composition of the ion, is optionally combined with at least one suitable prebiotic compound. A prebiotic nd is y a non-digestible ydrate such as an oligo- or polysaccharide, or a sugar alcohol, which is not degraded or absorbed in the upper digestive tract.

Known prebiotics include commercial products such as inulin and transgalacto-oligosaccharides.

In certain ments, the probiotic composition of the present ion includes a prebiotic compound in an amount of from about 1 to about 30% by weight, t to the total weight composition, (e.g. from 5 to 20% by weight). Carbohydrates may be selected from the group consisting of: - oligosaccharides (or FOS), short-chain fructo-oligosaccharides, inulin, isomalt- oligosaccharides, pectins, xylo-oligosaccharides (or XOS), chitosan-oligosaccharides (or COS), betaglucans , arable gum modified and resistant starches, polydextrose, D-tagatose, acacia fibers, carob, oats, and citrus fibers. In one aspect, the prebiotics are the short-chain fructo-oligosaccharides (for simplicity shown herein below as FOSs-c.c); said FOSs-c.c. are not ible carbohydrates, generally obtained by the conversion of the beet sugar and including a saccharose molecule to which three glucose molecules are bonded.

In some embodiments, the organic acid is provided as a lyophilised salt or ester thereof. The lyophilised organic acid product may be formulated in a single dosage form. The lyophilised organic acid may be formulated in a repeat dosage form. The lyophilised organic acid product may be formulated with the lyophilised bacteria in a single dosage form. atively, the organic acid product may be ated with the lyophilised bacteria in a repeat dosage form.

In some embodiments, the organic acid t may be formulated as a "slow e" or "fast release" composition. "Slow e" compositions result in lower initial increases in serum plasma concentrations of the organic acid product but have a more sustained effect, with elevated plasma levels of the organic acid remaining longer. By contrast, "fast release" compositions are formulated to provide a high initial dose of the organic acid product, resulting in higher initial plasma concentration but with a shorter duration effect.

In some embodiments, the c acid is administered intravenously. For example, the organic acid may be sodium valproate and may be administered to a subject in a 60 min infusion at a rate of no more than 20 mg/min. A typical daily dose of the organic acid or a pharmaceutically acceptable salt or [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM ed set by KJM ester thereof will vary depending on the physical characteristics of the t, the route of administration and/or the desired duration of the effect of the composition. In certain embodiments, the compositions of the invention are used in combination with another therapeutic compound for treating or preventing the neurodegenerative disorder.

In some embodiments, the compositions of the invention are administered with nutritional supplements that modulate neuroprotection or neuroproliferation. In preferred embodiments, the nutritional supplements comprise or consist of nutritional vitamins. In n embodiments, the vitamins are n B6, magnesium, dimethylglycine (vitamin B16) and vitamin C. In certain embodiments, the compositions of the invention are administered in combination with another probiotic.

In certain embodiments, the itions of the invention are for use in enhancing the effect of a second agent on a neurodegenerative disease. The immune modulatory effects of the compositions of the invention may make the brain more susceptible to conventional therapies such as Levodopa, dopamine agonists, MAO-B tors, COMT inhibitors, Glutamate antagonists, or anticholinergics, which are exemplary secondary agents to be administered in ation (sequentially or contemporaneously) with the compositions of the invention.

The itions of the invention may comprise pharmaceutically acceptable excipients or rs.

Examples of such suitable excipients may be found in the reference [84], Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art and are bed, for example, in reference [85], Examples of suitable carriers e lactose, starch, e, methyl cellulose, magnesium stearate, mannitol, sorbitol and the like. es of suitable diluents include ethanol, glycerol and water. The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as, or in addition to, the carrier, ent or diluent any suitable (s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s). Examples of suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flow lactose, beta-lactose, com sweeteners, l and tic gums, such as acacia, tragacanth or sodium alginate, carboxymethyl cellulose and hylene glycol. Examples of suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium te, sodium acetate, sodium chloride and the like. Preservatives, stabilizers, dyes and even flavouring agents may be provided in the pharmaceutical composition. Examples of preservatives include sodium te, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may be also used.

The compositions of the invention may be formulated as a food product. For example, a food product may provide nutritional benefit in addition to the therapeutic effect of the invention, such as in a ional supplement. Similarly, a food product may be formulated to enhance the taste of the composition of the invention or to make the composition more attractive to e by being more similar to a common food item, rather than to a pharmaceutical composition. In certain embodiments, [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM the composition of the invention is formulated as a milk-based product. The term "milk-based product" means any liquid or semi-solid milk- or whey- based product having a varying fat content. The milkbased product can be, e.g., cow's milk, goafs milk, sheep's milk, skimmed milk, whole milk, milk recombined from powdered milk and whey without any processing, or a processed product, such as yoghurt, curdled milk, curd, sour milk, sour whole milk, butter milk and other sour milk products.

Another important group includes milk beverages, such as whey beverages, fermented milks, condensed milks, infant or baby milks; flavoured milks, ice cream; milk-containing food such as sweets.

In some embodiments, the compositions of the ion comprise one or more bacterial strains of the genus Megasphaera and do not contain bacteria from any other genera, or which se only c/e minimis or biologically irrelevant amounts of bacteria from another genera. Thus, in some embodiments, the invention provides a composition comprising one or more ial strains of the genus haera, which does not contain ia from any other genera or which comprises only de minimis or ically irrelevant s of bacteria from another , for use in therapy.

In some embodiments, the compositions of the invention comprise one or more bacterial strains of the species Megasphaera massiliensis and do not contain bacteria from any other species, or which se only de s or biologically irrelevant amounts of bacteria from another species. Thus, in some embodiments, the invention provides a composition comprising one or more bacterial strains of the species Megasphaera massiliensis, which does not contain bacteria from any other species or which comprises only de s or biologically vant amounts of bacteria from another species, for use in therapy.

In some embodiments, the compositions of the invention comprise one or more bacterial strains of the species haera massiliensis and do not contain bacteria from any other Megasphaera species, or which comprise only de minimis or biologically irrelevant amounts of bacteria from another Megasphaera species. Thus, in some embodiments, the invention provides a ition comprising one or more bacterial strains of the species Megasphaera iensis, which does not contain bacteria from any other Megasphaera species or which comprises only de minimis or biologically irrelevant amounts of bacteria from another Megasphaera species, for use in therapy.

In certain embodiments, the compositions of the invention contain a single bacterial strain or species and do not n any other bacterial strains or s. Such compositions may comprise only de s or biologically irrelevant amounts of other bacterial strains or species. Such compositions may be a culture that is substantially free from other species of sm.

In some embodiments, the invention provides a composition comprising a single bacterial strain of the genus Megasphaera, which does not contain bacteria from any other strains or which comprises only de minimis or biologically irrelevant amounts of bacteria from r strain for use in therapy.

[Annotation] KJM None set by KJM ation] KJM ionNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM In some embodiments, the invention provides a composition sing a single bacterial strain of the species Megasphaera massiliensis, which does not contain bacteria from any other strains or which comprises only de s or biologically irrelevant amounts of bacteria from another strain for use in In some embodiments, the compositions of the invention comprise more than one ial strain. For example, in some embodiments, the compositions of the invention comprise more than one strain from within the same species (e.g. more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40 or 45 strains), and, optionally, do not contain bacteria from any other species, In some embodiments, the itions of the invention se less than 50 strains from within the same species (e.g. less than 45, 40, 35, 30, 25, 20, 15, 12, 10, 9, 8, 7, 6, 5, 4 or 3 strains), and, optionally, do not contain bacteria from any other species. In some embodiments, the compositions of the invention comprise 1-40, 1-30, 1- 20, 1-19, 1-18, 1-15, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-50, 2-40, 2-30, 2-20, 2-15, 2-10, 2- 5, 6-30, 6-15, 16-25, or 31-50 strains from within the same s and, optionally, do not contain bacteria from any other species. The invention comprises any combination of the foregoing.

In some embodiments, the composition ses a microbial consortium. For example, in some embodiments, the composition comprises the Megasphaera commensal bacterial strain as part of a microbial consortium. For example, in some embodiments, the Megasphaera commensal bacterial strain is present in combination with one or more (e.g. at least 2, 3, 4, 5, 10, 15 or 20) other bacterial strains from other genera with which it can live symbiotically in vivo in the ine. For example, in some embodiments, the composition comprises a commensal bacterial strain of Megasphaera the invention in combination with a bacterial strain from a different genus. In some embodiments, the microbial consortium comprises two or more bacterial s obtained from a faeces sample of a single organism, e.g. a human. In some embodiments, the microbial consortium is not found together in nature. For example, in some embodiments, the microbial consortium ses bacterial strains obtained from faeces samples of at least two different organisms. In some embodiments, the two different sms are from the same species, e.g. two different humans. In some embodiments, the two ent organisms are an infant human and an adult human. In some embodiments, the two ent organisms are a human and a non-human mammal.

In some embodiments, the composition of the invention additionally comprises a ial strain that has the same safety and eutic efficacy characteristics as strain MRX0029, but which is not MRX0029, or which is not a Megasphaera massiliensis.

In some embodiments in which the composition of the invention comprises more than one bacterial strain, species or genus, the individual bacterial strains, species or genera may be for separate, simultaneous or sequential administration. For example, the composition may comprise all of the more than one bacterial strain, species or genera, or the bacterial strains, species or genera may be stored separately and be administered separately, aneously or sequentially. In some embodiments, the [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM ed set by KJM ation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM more than one bacterial strains, species or genera are stored separately but are mixed together prior to In some embodiments, the bacterial strain for use in the invention is obtained from human adult .

In some embodiments in which the composition of the invention comprises more than one bacterial strain, all of the bacterial strains are ed from human adult faeces or if other bacterial strains are present they are present only in tie minimis s. The bacteria may have been cultured subsequent to being ed from the human adult faeces and being used in a composition of the invention.

As mentioned above, in some embodiments, the one or more Megasphaera commensal bacterial strains is/are the only therapeutically active agent(s) in a composition of the invention. In some embodiments, the bacterial strain(s) in the composition is/are the only eutically active agent(s) in a composition of the invention.

The compositions for use in accordance with the invention may or may not e marketing approval.

In certain embodiments, the ion provides the above pharmaceutical composition, wherein said bacterial strain is lyophilised. In certain embodiments, the invention provides the above pharmaceutical composition, wherein said bacterial strain is spray dried. In certain embodiments, the invention provides the above pharmaceutical composition, wherein the bacterial strain is lyophilised or spray dried and wherein it is live. In certain embodiments, the invention provides the above pharmaceutical composition, wherein the bacterial strain is lyophilised or spray dried and wherein it is viable. In n embodiments, the invention provides the above ceutical composition, wherein the ial strain is lyophilised or spray dried and wherein it is capable of partially or totally colonising the intestine. In n embodiments, the invention es the above pharmaceutical composition, wherein the bacterial strain is lised or spray dried and wherein it is viable and capable of partially or totally sing the intestine.

In some cases, the lyophilised bacterial strain is reconstituted prior to administration. In some cases, the reconstitution is by use of a diluent described herein.

The compositions of the invention can comprise ceutically able excipients, diluents or carriers.

In certain embodiments, the invention provides a pharmaceutical composition comprising: a bacterial strain of the invention; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the bacterial strain is in an amount sufficient to treat a neurodegenerative disorder when administered to a subject in need thereof.

In certain embodiments, the invention provides pharmaceutical composition comprising: a bacterial strain of the invention; and a pharmaceutically able excipient, carrier or diluent; wherein the bacterial strain is in an amount sufficient to treat or prevent a neurodegenerative disorder.

[Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM In certain embodiments, the invention provides the above pharmaceutical composition, wherein the amount of the bacterial strain is from about 1 x 103 to about 1 x 1011 colony forming units per gram with t to a weight of the ition.

In certain embodiments, the invention es the above pharmaceutical composition, n the composition is administered at a dose of 1 g, 3 g, 5 g or 10 g.

In n embodiments, the invention es the above pharmaceutical composition, wherein the composition is administered by a method selected from the group consisting of oral, rectal, subcutaneous, nasal, buccal, and sublingual.

In certain embodiments, the invention provides the above pharmaceutical composition, comprising a r ed from the group consisting of lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol and ol.

In certain embodiments, the invention provides the above pharmaceutical composition, comprising a diluent selected from the group consisting of ethanol, glycerol and water.

In certain embodiments, the ion provides the above pharmaceutical composition, comprising an excipient selected from the group consisting of starch, n, glucose, anhydrous lactose, free-flow lactose, beta-lactose, corn sweetener, acacia, tragacanth, sodium alginate, carboxymethyl cellulose, polyethylene glycol, sodium oleate, sodium stearate, magnesium te, sodium benzoate, sodium acetate and sodium chloride.

In n embodiments, the invention provides the above pharmaceutical composition, further comprising at least one of a preservative, an antioxidant and a stabilizer.

In n embodiments, the invention provides the above pharmaceutical composition, comprising a preservative selected from the group consisting of sodium benzoate, sorbic acid and esters of phydroxybenzoic acid.

In certain embodiments, the invention provides the above pharmaceutical composition, wherein said bacterial strain is lyophilised.

In certain embodiments, the invention provides the above pharmaceutical composition, n when the composition is stored in a sealed container at about 4°C or about 25°C and the container is placed in an atmosphere having 50% relative humidity, at least 80% of the bacterial strain as measured in colony forming units, remains after a period of at least about: 1 month, 3 , 6 months, 1 year, 1.5 years, 2 years, 2.5 years or 3 years.

In some embodiments, the ition of the invention is provided in a sealed container comprising a ition as described herein. In some embodiments, the sealed container is a sachet or bottle. In some embodiments, the composition of the invention is provided in a syringe comprising a composition as described herein.

[Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM The composition of the present ion may, in some embodiments, be provided as a pharmaceutical formulation. For e, the composition may be provided as a tablet or capsule. In some embodiments, the e is a gelatine capsule ("gel-cap").

In some embodiments, the compositions of the invention are administered . Oral administration may e swallowing, so that the compound enters the gastrointestinal tract, and/or buccal, lingual, or sublingual administration by which the compound enters the blood stream directly from the mouth.

Pharmaceutical ations suitable for oral administration include solid plugs, solid microparticulates, semi-solid and liquid (including multiple phases or sed systems) such as tablets; soft or hard capsules containing multi- or nano-particulates, liquids (e.g. aqueous solutions), ons or powders; lozenges (including liquid-filled); chews; gels; fast dispersing dosage forms; films; ovules; sprays; and buccal/mucoadhesive s.

In some embodiments the pharmaceutical ation is an enteric formulation, i.e. a -resistant ation (for example, resistant to gastric pH) that is suitable for delivery of the composition of the invention to the intestine by oral administration. Enteric formulations may be particularly useful when the bacteria or another component of the composition is acid-sensitive, e.g. prone to degradation under gastric conditions.

In some embodiments, the enteric formulation comprises an enteric coating. In some ments, the formulation is an enteric-coated dosage form. For example, the formulation may be an entericcoated tablet or an enteric-coated capsule, or the like. The enteric coating may be a conventional enteric coating, for example, a conventional coating for a tablet, capsule, or the like for oral delivery. The formulation may se a film coating, for example, a thin film layer of an enteric polymer, e.g. an nsoluble r.

In some embodiments, the enteric formulation is intrinsically enteric, for example, gastro-resistant without the need for an c g. Thus, in some embodiments, the formulation is an enteric formulation that does not comprise an enteric coating. In some embodiments, the formulation is a capsule made from a thermogelling material. In some embodiments, the thermogelling material is a cellulosic al, such as methylcellulose, hydroxymethylcellulose or hydroxypropylmethylcellulose (HPMC). In some embodiments, the capsule comprises a shell that does not contain any film forming polymer. In some embodiments, the capsule comprises a shell and the shell comprises hydroxypropylmethylcellulose and does not comprise any film forming polymer (e.g. see [86 ]). In some ments, the formulation is an intrinsically enteric capsule (for example, Vcaps® from Capsugel).

In some embodiments, the formulation is a soft capsule. Soft capsules are capsules which may, owing to additions of softeners, such as, for example, glycerol, sorbitol, maltitol and polyethylene glycols, present in the capsule shell, have a certain elasticity and softness. Soft capsules can be produced, for example, on the basis of gelatine or starch. Gelatine-based soft capsules are commercially available [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM ation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM from various suppliers. Depending on the method of administration, such as, for example, orally or rectally, soft capsules can have various shapes, they can be, for example, round, oval, oblong or torpedo-shaped. Soft capsules can be produced by conventional processes, such as, for example, by the Scherer process, the l process or the droplet or blowing process.

Culturing s The bacterial s for use in the present invention can be ed using standard microbiology techniques as detailed in, for example, references [87], [] and [89], The solid or liquid medium used for culture may be YCFA agar or YCFA medium. YCFA medium may include (per 100ml, approximate values): Casitone (1.0 g), yeast extract (0.25 g), NaHCCT (0.4 g), cysteine (0.1 g), K2HP04 (0.045 g), KH2P04 (0.045 g), NaCl (0.09 g), S04 (0.09 g), MgS04 • 7H20 (0.009 g), CaCl2 (0.009 g), rin (0.1 mg), hemin (1 mg), biotin (1 pg), cobalamin (1 pg), /;-aminobcnzoic acid (3 pg), folic acid (5 pg), and pyridoxamine (15 pg).

Bacterial strains for use in vaccine compositions The inventors have identified that the bacterial strains of the invention are useful for treating or preventing neurodegenerative disorders. This is likely to be a result of the effect that the bacterial s of the invention have on the host immune system. Therefore, the compositions of the invention may also be useful for preventing egenerative ers, when administered as vaccine compositions. In n such embodiments, the ial strains of the invention may be killed, inactivated or attenuated. In certain such embodiments, the compositions may comprise a vaccine adjuvant. In certain embodiments, the compositions are for stration via injection, such as via subcutaneous injection.

General The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, immunology and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., references [90] and [91, 97], etc.

The term "comprising" encompasses "including" as well as "consisting" e.g. a composition "comprising" X may consist exclusively of X or may include something onal e.g. X + Y.

The term "about" in relation to a numerical value x is optional and means, for example, x+10%.

The word "substantially" does not exclude etely" e.g. a composition which is antially free" from Y may be completely free from Y. Where necessary, the word "substantially" may be omitted from the definition of the invention.

References to a percentage sequence identity between two nucleotide sequences means that, when aligned, that percentage of nucleotides are the same in comparing the two sequences. This alignment and the percent homology or ce identity can be determined using software programs known in [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM the art, for example those bed in section 7.7.18 of ref. [98], A preferred alignment is determined by the Smith-Waterman gy search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 2, BLOSUM matrix of 62. The Smith-Waterman homology search algorithm is disclosed in ref. [99], Unless ically stated, a process or method sing numerous steps may comprise additional steps at the beginning or end of the method, or may se additional intervening steps. Also, steps may be combined, d or performed in an alternative order, if appropriate.

Various embodiments of the invention are described herein, ft will be appreciated that the features specified in each embodiment may be combined with other specified features, to provide further embodiments. In particular, embodiments ghted herein as being suitable, typical or preferred may be combined with each other (except when they are mutually exclusive).

MODES FOR CARRYING OUT THE ION Example 1 - Efficacy of bacterial inocula to act as a neuroprotectant Summary Neuroblastoma cells were treated with compositions comprising ial strains according to the invention. The SH-SY5Y neuroblastoma cells used are dopamine ing and well-established as an in vitro model for studying neurodegenerative diseases. The ability of the bacterial strains to increase neuroproliferation was observed. The lastoma cells were treated with dopaminergic neurotoxin 1-methylphenylpyridinium (MPP), which induces permanent ms of Parkinson’s disease in neuroblastoma cells. The ability of the bacterial s to act as a rotectant against MPP was investigated.

Material and Methods Bacterial strains Megasphaera massiliensis MRX0029; Parabacteroides distasonis MRX0005 Cell line SH-SY5Y neuroblastoma cells were purchased from ECCACC (Cat. no: 94030304) and were grown in MEM (Sigma Aldrich, cat n. M2279) supplemented with Nutrient Mixture F-12 Ham (Sigma Aldrich, cat n. N4888).

Method Once grown the SH-SY5Y neuroblastoma cells were plated on 96-well plate at 11,000 cells/well and incubated for 2 days. The cells were then transferred to differentiation medium (which contains FBS at 1%) and 10 uM retinoic acid (Sigma Aldrich, cat. n. R2625-100MG). Differentiation medium was replaced every other day and cells were harvested at 7 day of differentiation. Cells were pre-treated [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM ed set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM with or without MPP (Sigma Aldrich, cat. n. D048-1G) for 8 hours. Subsequently, cells were treated with 10% bacterial supernatant and incubated overnight. Cell viability was measured by using CCK-8 reagent (Sigma Aldrich, Cell Counting Kit - 8, cat. n. 96992-3000TESTS-F) and read at 450nm wavelength.

Results The results of these experiments are shown in Figure 1. Treatment of neuroblastoma cells with MRX0029 or MRX0005 led to an se in the proliferation of neurons. Neuroblastoma cells that were treated with MPP together with the bacterial strain had increased cell ity compared to the cells treated with MPP alone (which had decreased viability). The protective effect was greater for 9-treated cells, which d viability more than the positive control cells treated with Quercetin. These data show that the bacterial strains can act as a neuroprotectant.

Example 2a - Efficacy of bacterial inocula to reduce IL-6 secretion.

Summary Activation of proinflammatory cytokines has been associated with neuron damage in neurodegenerative disease. Lipopolysaccharide (LPS) is a known stimulator of the proinflammatory cytokine IL-6. Human astoma astrocytoma cells were treated with compositions sing bacterial strains according to the invention in combination with LPS to observe their ability to modulate the levels of IL-6.

Material and Methods Bacterial s Megasphaera massiliensis MRX0029; Parabacteraides distasonis MRX0005 Cell line MG U373 is a human glioblastoma astrocytoma d from a malignant tumour and were purchased from Sigma-Aldrich (cat n. 08061901-1VL). MG U373 human glioblastoma astrocytoma cells were grown in MEM (Sigma Aldrich, cat n. M-2279) supplemented with 10% FBS, 1% Pen Strep, 4mM L-Glut, IX MEM Non ial Amino Acid solution and IX Sodium Piruvate.

Method Once grown the MG U373 cells were plated on 24-well plate at 100,000 cells/well. The cells were treated with LPS (lug/mL) alone or with 10% of bacteria supernatant from MRX0029 or MRX0005 for 24h. A control was also med where the cells were incubated in untreated media.. Afterwards the cell free atants were collected, centrifuged at 10,000g for 3min at 4°C. IL-6 was measured using the Human IL-6 ELISA Kit from Peprotech (cat n.#900-K16) according to manufacturer instructions.

[Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM Results The results of these experiments are shown in Figure 2. Treatment of neuroblastoma cells with LPS and the bacteria strains led to a decrease in the level of IL-6 secreted.

Example 2b - cy of bacterial inocula to modulate IL-8 secretion.

Summary As neuro-inflammation plays a pivotal role in neurodegenerative diseases and IL-8 has been shown to have neuro-positive effects, the effect of compositions comprising bacterial strains of the invention and LPS on the activation of IL-8 were assessed. Human glioblastoma astrocytoma cells were treated with compositions comprising bacterial strains according to the ion in combination with LPS to observe their ability to modulate the levels of IL-8.

Material and Methods Bacterial strains haera massiliensis 9; Parabacteroides distasonis MRX0005 Cell line MG U373 is a human glioblastoma astrocytoma derived from a malignant tumour and were purchased from Sigma-Aldrich (cat n. 08061901-1VL). MG U373 human glioblastoma astrocytoma cells were grown in MEM (Sigma Aldrich, cat n. M-2279) supplemented with 10% FBS, 1% Pen Strep, 4mM L-Glut, IX MEM Non ial Amino Acid on and IX Sodium Piruvate.

Method Once grown the MG U373 cells were plated on 24-well plate at 0 cells/well. The cells were treated with LPS (lug/mL) alone or with 10% of bacteria supernatant from MRX0029 for 24h.

Afterwards the cell free supernatants were ted, centrifuged at 10,000g for 3min at 4°C. IL-8 was measured using Human IL-8 ELISA Kit from ech (cat n.#900-K18) according to manufacturer instruction.

Results The results of these experiments are shown in Figure 3. ent of neuroblastoma cells with the bacteria strains lead to an increase in IL-8 secretion independently of the presence of LPS.

Example 2C - Efficacy of bacterial inocula to reduce a-synuclein-induced inflammation.

Summary Neuroinflammation plays a l role in Parkinson’s disease and a-synuclein has been shown to induce neuroinflammation in vivo. Therefore, the ability of the bacteria strains of the invention to inhibit a-synuclein-induced neuroinflammation was assessed. A ture of human glioblastoma [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM astrocytoma cells and neuroblastoma cells were exposed to wild-type a-synuclein and the mutant isoforms E46K and A53T and treated with compositions comprising bacterial strains according to the invention. The ability of the bacteria strains to inhibit a-synuclein-induced secretion of IL-6 was then Material and Methods Bacterial strains Megasphaera massiliensis MRX0029; Parabacteraides distasonis MRX0005 Cell line MG U373 is a human glioblastoma astrocytoma derived from a malignant tumour and were purchased from Sigma-Aldrich (cat n. 08061901-1VL). MG U373 human glioblastoma astrocytoma cells were grown in MEM (Sigma Aldrich, cat n. M-2279) supplemented with 10% FBS, 1% Pen Strep, 4mM L-Glut, IX MEM Non essential Amino Acid on and IX Sodium Pimvate.

SH-SY5Y is a human neurobastoma cell line derived from a malignant neuroblastoma and can be purchased from Sigma-Aldrich (cat n. 94030304-1VL). The cells were grown in 50 % MEM and 50% Nutrient Mixture F-12 Ham media supplemented with 2mM L-Glutamine, 10% heat inactivated FBS, 100 U/ml penicillin, 100 pg/ml streptomycin. Cells on growth medium were plated on 96-well plate at 11,000 cells/well and placed in the incubator. After 2 days, media were replaced with entiation medium (growth medium containing 1% FBS) and 10 uM retinoic acid. Differentiation medium was replaced every other day and cells were used after 7 days of differentiation.

Method SHSY5Y cells were plated on 12 well plates at a density of 50,000 well. The cells were grown in 50 % MEM and 50% nt Mixture F-12 Ham media supplemented with 2mM L-Glutamine, 10% heat inactivated FBS, 100 U/ml penicillin, 100 pg/ml streptomycin. Cells on growth medium were plated on 96-well plate at 11,000 cells/well and placed in the incubator. After 2 days, media were replaced with differentiation medium h medium containing 1% FBS) and 10 pM ic acid.

Differentiation medium was replaced every other day and cells were used after 7 days of differentiation. U373 were plated on 12 transwell plates (0.4pm polyester membrane. Costar) at a density of cells/well for 72 hrs. Cells were co-cultured together for 24hrs before treatment in entiation medium (growth medium ning 1% FBS without ic acid). fter cells were treated with 25pg/ml a-synuclein (Wt, A53T, E46K) in the presence or absence of 10% bacteria supernatant for 48 hrs. Cell free Supernatants were collected, spun-dwon at lOOOOg for 3 min at 4°C, aliquoted and stored at -80 0C. Human IL-6 and IL-8 were measured as described above.

[Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM ation] KJM Unmarked set by KJM Results The results of these ments are shown in Figure 4. ent of cells with wild-type a-synuclein and the mutant isoforms E46K and A53T d moderate secretion of IL-6 (Figure 4A). The nduced secretion of IL-6 was inhibited in cells treated with the ia strains (Figure 4A). The reduction in IL-6 secretion was greatest on administration of MRX0029.

Example 3 - Efficacy of bacterial a to reduce NFtcB activation Summary Activation of the NFkB promoter leads to the production of proinflammatory cytokines including IL- ip, IL-la, IL-18, TNFa and IL-6. The NFkB promoter can be ted by a-synuclein and LPS by stimulating the TLR4 ligand. Mutations in a-synuclein, such as a-synuclein A53T, are implicated in familial Parkinson’s. Treatment of neuronal cells with LPS tes son’s caused by environmental factors. The ability of compositions comprising bacterial strains according to the invention to inhibit the activation of the NFkB promoter was investigated.

Material and Methods Bacterial strain Megasphaera massiliensis MRX0029 Cell line Human Hek blue TLR4 were purchased from InvivoGen (cat n. hkb-htlr4). Human Hek blue TLR4 were grown in DMEM high glucose (Sigma Aldrich, cat n. D-6171) supplemented with 10% FBS, 1% Pen Strep, 4mM L-Glut, Normocin and IX HEK Blue selection solution.

Method Once grown the Human Hek blue cells were plated in 96 well plates at 25,000 well in 4 replicates.

One set of cells were treated with a-synuclein A53T (lug/mL) alone or with 10% of bacteria supernatant from MRX0029 for 22h. The second set of cells were d with LPS (10 ng/mL, from Salmonella enterica serotype Typhimurium, Sigma Aldrich, cat n. L6143) alone or with 10% of bacteria supernatant from MR029 for 22h. The cells were subsequently spun down and 20ul of the supernatant was mixed with 200ul of Quanti Blue reagent (InvivoGen, cat n. rep-qb2), incubated for 2 h and absorbance read at 655nm.

Results The results of these experiments are shown in Figure 5 and 6. Figure 5 shows that the activation of the NFkB promoter by a-synuclein is not inhibited by MRX0029. Figure 6 shows that the activation of the NFkB promoter by LPS is inhibited by MRX0029.

[Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM Example 4 - Efficacy of bacteria to alter neurite outgrowth Summary Neurite outgrowth is an important process for the development of connections between neurons. The ability of bacterial strains and organic acids to induce neurite outgrowth was therefore tested by ing transcriptional levels of microtubule associated protein MAP2, a specific neuronal differentiation marker.

Bacterial strain Megasphaera massiliensis 9.

Method SHSY5Y were plated in 10cm petri dishes a density of 2xl06 cells. After 24h cells were treated in differentiation medium (growth medium containing 1% FBS without RA) with 10% bacteria atants or YCFA+, lOuM RA, 200uM hexanoic acid or 200uM valproic acid, for 17 hrs. There after representative images were taken using phase contrast EVOS XL core microscope at 40X/0.65 magnification. Cells were collected, and total RNA was isolated according to RNeasy mini kit protocol (Qiagen). cDNAs were made using the high capacity cDNA reverse transcription kit (Applied Biosystems). Gene expression was measured using qPCR. GAPDH was used as internal control. Fold change was calculated according to the 2( AAct) method.

Immunofluorescence and Confocal microscopy Cells were seeded onto 8 well chamber slides (Marienfeld Laboratory are) at 5xl04 cells/well overnight and were treated with 10% ial atant for 24 hrs. For differentiation, cells were treated with lOnM Retinoic acid for 5 days before treating with bacterial supernatant. Cells were then fixed with 4% paraformaldehyde in PBS for 20 minutes at room temperature (RT). Fixed cells were washed with PBS, and permeabilized with 1% Triton X-100 in PBS for 10 minutes. After washing with PBS, the slides were incubated with blocking buffer (4% BSA/PBS) for Ihr at RT before adding anti-MAP2 antibody (sc-74421, Santa Cruz Biotechnology Inc) diluted in 1% BSA/PBS for 12hr at 4°C. They were then washed twice with PBS, followed by tion with Alexa Flour 488 conjugated anti-mouse (Molecular Probes Inc) and Alexa Flour 594 conjugated Phalloidin (ab 176757, Abeam) for Ihr at RT. After washing 3X with PBS, the slides were d with Vcctorshicld ning DAP1 (Sigma, Aldrich). Slides were viewed using a Zeiss Axioscope cope equipped with a 63x/l .2 W Korr objective and filter sets suitable for detection of the fluorochromes used. Manual exposure times for the digital acquisition of images immuno-labelled with MAP-2 were kept constant allowing comparison between ent wells and ents. Phalloidin in) and DAP1 exposure times varied to suit the field of view. Randomised fields of view were acquired using a Qlmaging camera lled by Image Pro Plus software. Images were saved as TIFs and opened in Adobe Photoshop CC 2015.1.2 and overlays of the MAP-2, DAP1 and idion images overlaid and merged.

[Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM ation] KJM MigrationNone set by KJM [Annotation] KJM ed set by KJM Representative images were selected to illustrate the ences in abundance and on of the proteins examined Results The results are shown in Figure 7. Figure 7A shows representative microscopy images of undifferentiated SHSY-5Y cells ted with each of the acids and bacteria supernatants. Treatment of cells with MRX0029 induced a neuron-like phenotype, showing similar features to cells treated with retinoic acid (which is used for terminal differentiation of neuroblastoma cells), where cell bodies are bigger and pyramidal-shaped, with neurites and sed branching out to network with neighbour cells. Figure 7B shows that MRx0029 significantly upregulates MAP2 in undifferentiated neuroblastoma cells. Phalloidin (an actin cytoskeleton-binding agent) staining further proved a ent arrangement of cytoskeletal structure in cells treated with MRx0029, further supporting the neuronal differentiation hypothesis for MRx0029 (Fig. 7B) Example 5 - Efficacy of bacterial inocula to alter antioxidant capacity Summary The ability of compositions comprising ial strains according to the invention to alter the antioxidant capacity. The antioxidant capacity of the bacterial strain was established using the own ABTS (2,2'-azino-bis(3-ethylbenzothiazolinesulphonic acid)) assay.

Bacterial strain Megasphaera massiliensis MRX0029 Method Bacterial cells (106 or greater) were collected and centrifuged. They were resuspended in assay buffer (using three times the pellet volume). The suspension was sonicated on ice for 5 minutes and then spun down at 12,000 x g for 10 minutes. The atant was removed and measured using the ABTS assay kit produced by Sigma Aldrich (code CS0790), in accordance with manufacturer’s instructions.

Results The s of these experiments are shown in Figure 8. Figure 8 shows that the MRX0029 has an antioxidant capacity of approximately 2mM compared to .

Example 6 - Efficacy of bacterial a to alter lipid peroxidation levels Summary The y of compositions comprising bacterial strains according to the invention to alter lipid peroxidation levels was investigated. The thiobarbituric reactive substances assay (TBARs) was used to measure the by-products of lipid peroxidation.

[Annotation] KJM None set by KJM [Annotation] KJM ionNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM Material and Methods ial strain Megasphaera massiliensis MRX0029 Method Bacterial cells (106 or greater) were collected and centrifuged, a wash step was performed with isotonic saline before the pellet was re-suspended in potassium chloride assay . The suspension was sonicated on ice for 10 minutes and then spun down at 10,000 x g for 10 minutes. The supernatant was removed and the level of lipid peroxidation evaluated using the thiobarbituric reactive substances assay.

Results The results of the experiments are shown in Figure 9. Figure 9 shows that 9 is able to inhibit lipid peroxidation by approximately 20 %, which is a higher antioxidant capacity than the positive control, ted hydroxytoluene (1% w/v).

Example 7a - Efficacy of bacterial inocula to reduce oxidative levels in cells Background The generation of reactive oxygen species contributes to the pathology of neurodegenerative diseases.

The ability of bacterial strains to protect differentiated SHSY-5Y and U373 cells from reactive oxygen species (ROS) generated by treatment with Tert-Butyl Hydrogen Peroxide (TBHP) was igated.

Material and Methods ial strain Megasphaera massiliensis MRX0029 Method SHSY-5Y cells were plated in black flat bottom 96 well plate at density of 5000 cells/well and placed in the C02 incubator. After 24 h, media were replaced with differentiation medium (growth medium containing 1% FBS) and 10 uM retinoic acid. Differentiation medium was replaced every other day.

On Day 10 the entiation medium was removed and cells were washed with pre-warmed PBS and stained with lOuM DCFDA molecular probe for 20 mins in growth medium containing 1% FBS. Then cells were washed with pre-warmed PBS again and treated with lOOuM TBHP in the presence or absence of 10% bacteria supernatant for 2h. Fluorescence intensity was ed using TECAN plate reader at Ex/Em 0 nm.

Results The results of the experiments are shown in Figure 10. Figure 10b shows that MRX0029 is able to inhibit ROS production in entiated SHSY-5Y neuroblastoma cells. MRX0029 did not have an [Annotation] KJM None set by KJM ation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM ation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM ation] KJM Unmarked set by KJM effect on the generation of ROS in U373 astroglioblastoma cells (Figure 10a). This shows that this aspect of the antioxidant effect is neuron-specific.

Example 7b - neuroprotection RA-differentiated SHSY-5Y cells were treated with MPP+, the active metabolite of MPTP, a chemical widely used to mimic in vitro and in vivo some of the features of PD pathology. Cell viability was measured as the rate of mitochondria respiration e 11). Both MRxOOOS and MRx0029 showed significant effects and promote per se an increase of the mitochondria metabolic activity in SHSY-5Y cells. MRX0029 showed complete protection from MPP+, ing cell viability nearly to the same level of ted cells and higher than quercetin positive control. MRxOOOS protection was about 20% compared to YCFA-MPP+ d sample, about the same observed for the quercetin positive control (Fig. 11).

Example 8 - Efficacy of ial inocula on e deacetylase activity Summary The ability of compositions comprising bacterial strains according to the invention to alter histone deacetylase activity was investigated. Dysregulation of histone deacetylase has been implicated in the pathogenesis associated with age-associated neurodegenerative diseases.

Material and Methods Bacterial strain Megasphaera massiliensis MRX0029 Cell line The cell line HT-29 was used because histone deacetylase is present.

Method Cell free supernatants of stationary phase bacterial cultures were isolated by centrifugation and filtering in a 0.22 uM filter. HT-29 cells were used 3 days’ post confluence and stepped down in 1 mL DTS 24 hours prior to commencement of the experiment. The HT-29 cells were challenged with 10 % cell free supernatant diluted in DTS and this was left to incubate for 48 hours. Nuclease ns were then extracted using the Sigma Aldrich se extraction kit and samples were snap frozen prior to HDAC activity measurement. HDAC ty kit was assessed fluorometrically using the Sigma Aldrich (UK) kit.

Results The results of the experiments are shown in Figure 12. Figure 12 shows that MRX0029 is able reduce the levels of histone ylase activity.

[Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM ation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM e 8a - r analysis of the mechanism of histone deacetylation inhibition Introduction The gut microbiota, with its immense diversity and metabolic capacity, represents a huge metabolic reservoir for production of a vast variety of molecules with potential to influence HD AC activity. Few studies have assessed the HDAC inhibitory activity of microbially-derived metabolites other than butyrate, which has been shown to inhibit HDAC and is associated with improvement of motor function in Huntington’s disease [100], The inventors therefore sought to determine which metabolites are responsible for HDAC inhibition and r elucidate the mechanisms by which inhibition is al and s Bacterial culture and cell-free supernatant collection Pure cultures of bacteria were grown anaerobically in YCFA broth until they d their stationary growth phase. es were centrifuged at 5,000 x g for 5 minutes and the cell-free supernatant (CFS) was filtered using a 0.2 uM filter (Millipore, UK). 1 mL aliquots of the CFS were stored at -80 °C until use. Sodium butyrate, hexanoic and valeric acid were obtained from Sigma Aldrich (UK) and suspensions were prepared in YCFA broth.

SCFA and MCFA quantification of bacterial supernatants Short chain fatty acids (SCFAs) and medium chain fatty acids (MCFAs) from bacterial supernatants were analysed and quantified by MS Omics APS as follows. Samples were acidified using hydrochloride acid, and deuterium labelled internal rds where added. All samples were analyzed in a randomized order. is was performed using a high polarity column (Zebron™ P, GC Cap. Column 30 m x 0.25 mm x 0.25 jam) installed in a GC (7890B, Agilent) coupled with a quadropole detector (59977B, Agilent). The system was controlled by ChemStation (Agilent). Raw data was converted to netCDF format using Chemstation (Agilent), before the data was imported and processed in Matlab R2014b (Mathworks, Inc.) using the PARADlSe software described in [101].

Specific HDAC activity analysis Specific HDAC inhibition ty was analysed for HDAC1, 2, 3, 4, 5, 6, 9 using genic assay kits for each type of HDAC (BPS Bioscience, CA). Assays were conducted according to manufacturer’s instructions and each sample were performed in replicates. Cell free supernatants were diluted 1 in 10 and exposed to specific HDAC proteins provided in the kit to maintain consistency between methods.

Results Histone deacetylase-inhibiting gut commensal microbial metabolites are butyrate and valeric acid ation] KJM None set by KJM [Annotation] KJM ionNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM MRx0029, whose supernatant showed strong HDAC inhibition in assays with either HT29 whole cell or HT29 cell lysate, produced valeric acid and hexanoic acid at mean concentrations of 5.08 mM and 1.60 mM, respectively (Figure 13A and C).

To investigate which metabolites were responsible for the strain-induced HDAC inhibition, different concentrations of hexanoic acid, valeric acid and sodium butyrate were measured for their HDAC inhibition on whole HT-29 cells and on HT-29 cell lysate. The results in Fig. 13B show significant (P<0.05) inhibition of HDAC activity by sodium butyrate on whole cells as well as on the cell lysate, while hexanoic acid did not show significant inhibitory ty. Valeric acid inhibited total HDAC activity (* (p<0.05), ** (p<0.005), *** (P<0.001), **** (pO.OOOl)).

Potent total HDAC inhibitors investigated target class IHDACs.

The specific HDAC tion profile of the test bacteria strain was investigated. Specific HDAC inhibition assays (BPS Bioscience, CA) were d out for Class f and Class ff HDACs. The ability of the bacterial strain to inhibit HDAC s was compared to butyrate, hexanoic and valeric acid (Figures 14 and 15). Our results demonstrate that MRX0029, is a very potent inhibitor of Class 1 HDAC enzymes (HDAC1, 2 and 3). tion of class ff HDACs was not as significant (data not shown).

The strain with HDAC tory activity produced significant amounts of valeric acid and hexanoic acid as well as significant amounts of sodium butyrate (Figure 13C). When tested as pure substances, valeric acid and sodium butyrate resulted in significant HDAC inhibition (p<0.0001). interestingly, the results for specific HDAC activity show that the tested strain is a potent inhibitor of Class f HDACs, and particularly HDAC2. Class f HDACs (HDAC1, 2, 3 and 8) reside in the nucleus and are ubiquitously expressed in several human cell types. HDACs 1-3 share more than 50% homology, but have distinct structures and cellular ons [ 102]. They are primarily involved in cell survival, proliferation and differentiation, and thus there inhibition may be useful is wide array of diseases [103]; [104]; [105]; [106]; [107], Example 9 - Level of indole production in ia Summary The ability of the bacteria of the invention to produce indole was igated, fndole has been implicated in attenuating inflammation and oxidative stress.

Material and Methods Bacterial strain haera iensis MRX0029 ATCC 11775 is a bacterial reference strain that is known to produce indole.

[Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM Method Intact bacterial cells in stationary phase were incubated with 6mM Tryptophan for 48 hours. Bacterial s which possess the enzyme phanase will utilise tryptophan as a substrate to produce indole. ing the 48 hour tion period, the supernatant was removed and added to Kovac's reagent for quantification of indole. Standards, stock solutions and reagents were prepared using standardised methods validated in-house.

Results The s of the experiments are shown in Figure 16. Figure 16 shows that 9 has the capacity to produce indole from tryptophan, at concentrations of approximately 0.2mM.

Example 10 - Level ofkynurenineproduction in bacteria Summary The ability of the bacteria of the invention to produce kynurenine was investigated. Dysregulation of the kynurenine pathway can lead to activation of the immune system and the accumulation of potentially neurotoxic compounds. Alterations in the kynurenine metabolism may be involved in the development of Parkinson’s diseases.

Bacterial strain Megasphaera massiliensis 9 DSM 17136 is a strain of oides copricola that is known to produce kynurenine.

Method Cell free supernatants of nary phase bacterial cultures were isolated by centrifugation and ing in a 0.22 uM filter and frozen until use. Kynurenine standards, stock solutions and reagents were prepared using standardised methods validated in-house. Sample were treated with trichloroacetic acid and centrifuged at 10,000xg for 10 minutes at 4°C. The supernatant was ted and dispensed into a 96 well plate. Ehrlich’s reagent was used for kynurenine detection and added at a ratio of 1:1.

Results The s of the experiments are shown re 17. Figure 17 shows that MRX0029 has the capacity to produce kynurenine at a concentration of approximately 40 uM.

Example 11 - Levels of Dopamine, DOPAC and HVA in striatum in bacteria-treated MPTP mice Parkinson's disease is a common neurodegenerative disorder whose cardinal clinical features include tremor, slowness of movement, ess, and postural instability. These symptoms are primarily attributable to the degeneration of dopaminergic neurons in the substantia nigra pars compacta and the consequent loss of their projecting nerve fibers in the striatum [108], Mice treated with MPTP (I- phenyl-f,2,3,6-tetrahydropyridine) selectively lose significant numbers of nigrostriatal [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM dopaminergic neurons [ 109]. MPTP induced loss of dopaminergic cells in substantia nigra mimics the clinical condition in Parkinson’s disease and is therefore a useful model to test anti-parkinsonian drugs.

The aim of this study was to te the effects of MRX0029 anaerobic bacteria using MPTP lesioned mice. 48 male mice were allocated to 4 different treatment groups s A, B, E and I, with n=12 s in each group). The treatment groups are shown in Table 1 below and the project time course is outlined below.

Table 1: Treatment groups Treatment Lesion Group n Safety level Substance Dose Route Schedule Substance Dose Route Schedule A 12 Vehicle (PBS) p.o. 18 days: day(- Vehicle (0.9% i-P- dayO 14) - dav3 saline) B 12 Vehicle (PBS) p.o. 18 days: day(- MPTP 4x20 i-P- dayO 141 - dav3 mq/kq MRx0029 E 12 Megasphaera S1/S2 2x10A8 p.o. 18 days: day(- MPTP 4x20 i-P- dayO sp. bacteria 14) - day3 mg/kg tolvl Vehicle (PBS) p.o. 18 days: day(- I 12 14) - dayS MPTP 4x20 i-P- dayO mg/kg 7-nitroindazole 50 mg/kg i-P- dayO (2x i.p.) Groups A, B, E and I were treated daily for 18 days via oral gavage with either bacteria (MRx0029 - group E), or vehicle (PBS). Oral treatment started 14 days before MPTP lesion. Group I animals received a daily vehicle (PBS) p.o. (per oral) treatment and were injected i.p. (intraperitoneal) with the nce drug 30 min before and 90 min after first MPTP on day 0. The application volume for p.o. and vehicle treatment was 200 pi per mouse. Bacteria strain of group E was from glycerol stocks (gly).

For oral treatment, gavages for applications were stored in vial containing 70% l and were flushed before and after each use with distilled water. Every treatment group had its own gavage and l vial and distilled water vial. The tubes and gavages were not changed between the groups.

Directly before treatment each syringe was flushed with N2.

On day 0 MPTP (20 mg/kg bodyweight (b.w.) 4 times, 2h inter-treatment interval) was injected i.p. in s of groups B, E and I. One group of animals (A) was sham lesioned by i.p. administration of the MPTP vehicle (0.9% saline). The application volume was 10 pi per g body weight. Weighing of the animals was performed before the MPTP ent to dose the animals ing to their actual body weight. ards animals ed the daily p.o. treatment.

Formulation of preparations for dosing and preparation of glycerol stocks for dosing [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM MRx0029 haera sp.

Name of the Bacteria strain: e condition/stability: -80 °C Vehicle: 1 x PBS Treatment dosages: 2 x 10A8 bacteria Administration: 200nl Lot Number: n/a For treatment group E 29) 1.) 1 glycerol stock was taken from the -80 °C freezer and placed under anaerobic conditions obic jar with sachet) at 37 °C in order to thaw (this took 30-40 mins). 2.) The completely thawed glycerol stock was centrifuged at 6000 x g for 10 min at room temperature. 3.) The atant was discarded without disturbing the pellet (e.g. using a pipette). 4.) 4.22 mL of sterile pre-warmed (37 °C) 1 x PBS was added and gently mixed using a pipette.

.) The mice were dosed with 200 jaL of the bacterial solution. The animals were dosed within 15 mins after resuspension of the pellet with PBS.

Reference drug group formulation Storage condition/stability: -20°C Vehicle: Peanut oil Treatment dosages: 50 mg/kg stration: i.p. (30 min before and 90 min after 1stMPTP treatment) Batch Number: MKBS6671V The appropriate amount of 7-Nitroindazole was dissolved in peanut oil to reach the final concentration of 50 mg/kg.

[Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM ionNone set by KJM [Annotation] KJM Unmarked set by KJM Materials and Methods Animals C57BL/6J (JAX™ Mice ) Mouse line: JAX™ Mice Stock Number 000664 Provider: Charles River Laboratories Age at start: ~ 10 weeks Sex: Male Number of s: 48 Specific handling of Animals and Randomization Gloves were changed between each treatment group and sprayed with 70% l solution between each cage of the same group to minimize the risk of contamination whenever animals were handled (e.g.: treatment, behavioural testing, cleaning and tissue sampling).

The treatment was at random and alternated daily so as to t the same groups being treated at the same time each day. Animals were randomized per cage at the tissue sampling.

Tissue sampling and sing On day 4 animals of all groups were sacrificed and brains were collected. Therefore, mice were deeply anesthetized by Pentobarbital injection /kg).

Blood (approximately 500 pi) was collected by heart puncture. Mice were then transcardially perfused with 0.9% saline and brains were removed and hemisected. The left hemisphere was subdivided into striatal tissue (for HPLC), substantia nigra tissue as well as residual brain, weighed and immediately frozen and stored at -80°C. Instruments and surfaces which were in contact with the animals had to be cleaned with 70% ethanol before the next animal was dissected.

Biochemical Analysis of Dopamine, DOPAC and HVA levels with HPLC in striatum The striatal s (n=6 from each treatment group; total 24 samples) were mixed at a ratio of 1:10 (w/v) with 0.2 M perchloric acid including 100 pM EDTA-2Na and homogenized at 0 °C in a glass- pestlemicro-homogenizer. Following standing for 30 min on ice, the nates were centrifuged at ,000 RPM for 10 minutes in a refrigerated centrifuge Biofuge Fresco (Heraeus Instruments, Germany). The supernatants were carefully aspirated and mixed with 0.4 M Na-acetate buffer, pH 3 at a ratio 1:2 (v/v) and filtered through a 0.22 pm centrifugal filter (Merck ore, Germany) for 4 min at 14 000 g at 4 °C. The filtrates were stored at -80 °C before HPLC analysis.

[Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM ionNone set by KJM [Annotation] KJM Unmarked set by KJM HPLC analysis Concentrations of DA, DOPAC and HVA in the striatal samples were determined by column liquid chromatography with electrochemical detection [110;111], The HPLC system (HTEC-500, Eicom Corp., Kyoto, Japan) including a pulse-free microflow pump, a degasser and an amperometric or equipped with a glassy-carbon electrode operating at +0.45 V vs. an Ag/AgCl ref. electrode was used.

Samples were injected by use of a CMA/200 Refrigerated Microsampler (CMA/Microdialysis, Stockholm, Sweden). The chromatograms were recorded and integrated by use of a computerized data acquisition system (DataApex, , Czech ic). DA, DOPAC and HVA were separated on a 150 x 2.1 i.d. mm column (CA5-ODS, Eicom Corp., Kyoto, . The mobile phase consisted of 0.1 M phosphate buffer at pH 6.0, 0.13 mM EDTA, 2.3 mM sodiumoctanesulfonate and 20 % (v/v) methanol. The detection limit (signal-to-noise ratio 3) for DA was estimated to 0.5 fmol in 15 pi (0.03 nM) injected onto the column.

Results Administration of bacteria strains was well tolerated by the animals. On the MPTP lesion day and if ary on the day afterwards a red light was used to warm the animals. If animals were in bad conditions (felt cold, dehydrated, abnormal behaviour), they were supplied with wet food and subcutaneous saline treatment if necessary.

For analysis of Dopamine, DOPAC and HVA levels, striatal tissue of 6 animals per treatment group were used. Data were analyzed by using Kruskal-Wallis test ed by Dunn’s multiple comparison post hoc test or One-way analysis of variance ed by Bonferroni post hoc test (A vs. all(*), B vs. all, I vs. all (#)). */# = p<0.05; ** = p<0.01; *>!<>!< — p<0.001.

The y animals in group A had high levels of Dopamine, DOPAC and HVA whereas MPTP treatment in group B reduced this and the positive control (group I) recovered the production to some degree (Figure 21). Animals of group I tended to have higher Dopamine levels than the bacteria treated group and group B (Figure 21 A). DOPAC (a ne metabolite) levels in general were significantly lower in animals of group B compared to DOPAC levels of unlesioned animals of group A e 21B).

Significantly, treatment with MRx0029 (group E) was found to recover tion of Dopamine and DOPAC (Figures 21A and 21B, respectively). Treatment with MRx0029 may therefore be useful for treating or preventing neurodegenerative ers.

Example 12 - Stability testing A composition described herein containing at least one bacterial strain described herein is stored in a sealed container at 25°C or 4°C and the container is placed in an atmosphere having 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90% or 95% ve ty. After 1 month, 2 months, 3 months, 6 months, 1 year, 1.5 years, 2 years, 2.5 years or 3 years, at least 50%, 60%, 70%, 80% or 90% of the bacterial [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM ed set by KJM strain shall remain as measured in colony forming units determined by standard protocols.

Example 13 - Level of BDNF secretion in SHSY-5Y cells Background derived neurotrophic factor (BDNF) is a ubiquitous molecule in the brain associated with neural development, neuro-protection and neuro-regeneration. BDNF not only protects against neurodegeneration but also mental disorders like sion and anxiety, which are quite common amongst patients diagnosed with PD or AD.

Methods SH-SY5-SY were plated in 24 wells plate at density of 60,000 cells/well and placed in the tor.

After 24 h, media were replaced with differentiation medium (growth medium containing 1% FBS) and 10 uM retinoic acid. Differentiation medium was ed every other day and cells were used on day 10 of differentiation. F or the ent differentiation medium was removed and replaced with 450ul of full growth media and 50 jal of bacteria SN was added to the treated wells or YCFA+ was added as ve Control.

Results The results are shown in Figure 18, which shows that administration of MRX0029 in combination with retinoic acid increases the secretion of BDNF from differentiated neuroblasoma cells. Compositions comprising commensal ia and organic acids may therefore be useful in therapy.

Example 14 - Metabolite production - metabolites in the brain Background Metabolites present in bacteria supernatants can directly influence the host response to oxidative stress, cell-to-cell communication and rotection. Metabolites that play a key role in neurological processes were measured during the ex vivo screening in brain tissue of mice fed with and MRx0029.

Methods Animals BALBc (Envigo, UK) adult male mice were group housed under a 12 h light-dark cycle; standard rodent chow and water were available ad libitum. All experiments were performed in accordance with European guidelines following approval by University College Cork Animal Ethics Experimentation Committee. Animals were 8 weeks old at the start of the experiment.

Study Design [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM Animals were allowed to ate to their holding room for one week after arrival into the animal unit. They receive oral gavage (200jaL dose) of live biotherapeutics at a dose of 1 X 109 CPU for 6 consecutive days between 15:00 and 17:00. On day 7, the animals are decapitated, and tissues are harvested for experimentation.

Tissue Collection Animals were sacrificed in a random fashion regarding treatment and testing condition; sampling occurred between 9.00 a.m. and 1:00 p.m. Trunk blood was collected in potassium EDTA (Ethylene Diamine Tetra Acetic Acid) tubes and spun for 15 min at 4000 g. Plasma was isolated and stored at -80 °C for further is. The brain was quickly excised, dissected and each brain region was snap- frozen on dry ice and stored at -80 °C for further analysis. Spleen was d and sed ately after culls for ex-vivo immune stimulation. Intestinal tissue (2 cm segments of ileum and colon t to the caecum were excised, and the furthest 1 cm of tissue from the caecum were used) were mounted into the Ussing rs for intestinal permeability assay. The caecum was removed, weighted and stored at -80 °C for SCFAs analysis. ine Analysis Neurotransmitter concentration was analysed by HPLC on samples from the brainstem. Briefly, brainstem tissue was sonicated in 500 pi of chilled mobile phase spiked with 4 ng/40 pi of yl -HT (Sigma Chemical Co., UK) as internal standard. The mobile phase contained 0.1 M citric acid, .6 mM octanesulphonic acid (Sigma), 0.1 M sodium dihydrogen phosphate, 0.01 mM EDTA (Alkem/Reagecon, Cork) and 9% (v/v) methanol (Alkem/Reagecon) and was adjusted to pH 2.8 using 4 N sodium hydroxide (Alkem/Reagecon). Homogenates were then centrifuged for 15 min at 22,000 x g at 4 °C and 40 pi of the supernatant injected onto the HPLC system which consisted of a SCL 10- Avp system controller, LECD 6A electrochemical detector (Shimadzu), a LC-10AS pump, a CTO- 10A oven, a SIL-10A autoinjector (with sample cooler maintained at 40 C) and an online Gastorr er (ISS, UK). A reverse-phase column (Kinetex 2.6 u Cl8 100 4.6 mm, Phenomenex) maintained at 30 °C was employed in the separation (Flow rate 0.9 ml/min). The glassy carbon working electrode combined with an Ag/AgCl reference electrode (Shimdazu) operated a +0.8 V and the chromatograms generated were analyzed using Class-VP 5 software dzu). The ransmitters were identified by their characteristic ion times as ined by standard injections, which run at regular intervals during the sample analysis. The ratios of peak heights of analyte versus internal standard were measured and compared with standard injection. Results were expressed as ng of neurotransmitter per g fresh weight of tissue.

Metabolite analysis For GC-metabolite analysis, samples of bacterial supernatants were derivatized with methyl chloroformate using a slightly modified version of the protocol described in [112], All samples were analyzed in a randomized order. Analysis was performed using GC (7890B, Agilent) coupled with a [Annotation] KJM None set by KJM ation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM ation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM quadropole detector (59977B, Agilent). The system was lled by ChemStation (Agilent). Raw data was converted to netCDF format using Chemstation (Agilent), before the data was imported and processed in Matlab R2014b orks, Inc.) using the PARADISe software described in [101].

For fatty acid analysis samples were acidified using hloride acid, and deuterium labelled internal standards where added. All samples were analyzed in a randomized order. Analysis was performed using a high polarity column (Zebron™ ZB-FFAP, GC Cap. Column 30 m x 0.25 mm x 0.25 pm) installed in a GC (7890B, Agilent) coupled with a quadropole detector (59977B, Agilent). The system was controlled by ChemStation nt). Raw data was converted to netCDF format using Chemstation (Agilent), before the data was imported and processed in Matlab R2014b (Mathworks, Inc.) using the PARADISe software described by [101], Results - neurotransmitter tion The results are shown in Figure 19, which shows that in brains of mice fed with MRx0029, noradrenaline levels are increased (p=0.0507), accompanied with a slight increase of serotonin and 5- HIAA. These data support the metabolite analysis set out below, suggesting that MRx00029 is a major producer of 4-hydroxyphenylacetic acid, a known antioxidant [113]. More importanly, 4- hydroxyphenylacetic acid is a synthetic intermediate of ne and norepinephrine and an important bio-active molecule [114], In fact, in PD, degenerative changes extend beyond the dopaminergic system, affecting equally the serotonergic and noradrenergic systems, which in turn leads to decreased levels of serotonin (5-hydroxytryptamine, 5-HT) and noradrenaline (norepinephrine) in both striatal and extra-striatal structures [115]. L-DOPA targets mainly the dopamine-related features of PD, however it does not address the decreases in both 5-HT and noradrenaline. Adding to this is that the longer is the duration of L-DOPA treatment, the more visible are a range of motor and nonmotor complications (e.g. dyskinesia, psychiatric symptoms) [116]. Therefore, these data demonstrate that bacteria that produce organic acids, such as 4-hydroxyphenylacetic acid, may be useful in therapy, in particular in the treatment of neurodegenerative es.

Results - metabolite production Metabolites t in bacteria atants can directly influence the host response to oxidative , cell-to-cell communication and rotection in the specific. Metabolites in the supernatant of cultures of MRX0029 and 5 were analysed and the results are shown in Figure 20.

A few metabolites showed a striking difference between the two strains analysed. The concentration of succinic acid was ularly elevated in MRxOOOS. Interestingly, the ratio sample/media for 4- hydroxyphenylacetic acid was significantly higher in 9 (Fig. 20A).

Fatty acid analysis in the supernatants revealed an interesting omy in the two strains: MRxOOOS produced mainly acetic and propanoic acid, while MRx0029 ed butanoic, pentanoic and hexanoic acid, both in the linear and branched forms (Fig. 20B). The two strains looked very different [Annotation] KJM None set by KJM ation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM and in particular, the production of succinic acid and 4-hydroxyphenylacetic acid by S and MRx0029 respectively was notable (Figure 20A). Furthermore, MRxOOOS seems to produce more C2 and C3 short chain fatty acids, while MRx00029 produced more C4 (butyrate) and both linear and branched medium chain fatty acids, including hexanoic acid.

Succinic acid is a Krebs cycle metabolite involved in oxidative phosphorylation. Oxidative orylation x is a key step for synaptic trafficking of proteins and vesicles to proximal and distal regions [117]. Its dysfunction has been reported in neurodegenerative disorders including Alzheimer’s disease, Parkinson’s disease and Spinocerebellar ataxia type 1 [63], These findings are particularly interesting as succinic acid can augment ondrial ty and support vulnerable s in neurodegenerative disease related to misfolded proteins including PD [65], BDNF and succinic acid have both a similar protective activity not only in neuro-degeneration but also in mental disorders like depression and anxiety, which are quite common amongst ts diagnosed with PD or AD.

Figure 20B also demonstrates that MRX0029 is a butyrate (butanoic acid) producer. This may be significant e butyrate has a known role is reducing impermeability of the blood brain barrier, which has a rotective effect [118]. This property of MRx0029 (and other neuroprotective bacteria) may contribute to its effacy.

Example 15 - Modulation of the mRNA expression of tight junction proteins by MRx0029 Since recent evidence suggests that intestinal dysfunction and inflammation is a non-motor symptom ated with PD, the y of the ial s of the invention to cause any intestinal barrier dysfunction was investigated. HT29-mtx epithelial, mucin-producing cell monolayers [119] were used as an in vitro model to evaluate gut barrier disruption and immune stimulation following treatment with S and MRx0029. Differentiated HT29-mtx cells exposed to phorbol 12-myristate acetate (PMA) secreted a significant amount of IL-8; in contrast treatment for 24h with MRxOOS and 9 bacterial supernatants, induced an even lower secretion of IL-8 compared than both untreated and YCFA-treated cells (Fig. 22A).

The ability of MRxOOOS and MRx0029 to regulate epithelial bility by modifying intracellular signal transduction involved in the expression and localization of proteins involved in the gut barrier formation was then investigated.

RNA was isolated and Quantitative RT-PCR (qRT-PCR) analysis was performed to characterize the s in gene expression of tight junction proteins during incubation with MRxOOOS and MRx0029.

The administration of MRx0029 enhanced Occludin, Vlillin, Tight Junction Protein 1 and 2 (respectively TJP1 and TJP2) mRNA expression after 2h incubation (Fig. 22B). In contrast, exposure [Annotation] KJM None set by KJM [Annotation] KJM ionNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM ionNone set by KJM [Annotation] KJM Unmarked set by KJM to MRxOOOS did not alter the gene expression of tight junction proteins indicating that the two strains act entially on the intestinal barrier.

The in vitro results were compared with data from the ex vivo parallel analysis on the gut of mice fed with MRxOOOS and MRx0029. Gene expression of TJP2 and occludin was quantified in the colon and ileum. The ex vivo data perfectly mirror the in vitro data as 9 was able to significantly upregulate TJP1 and Occludin (p=0.073) in the colon region of the murine intestine (Fig. 22C+22D).

MRx0029 was also able to decrease the permeability function in the colon of the same mice (Fig. 22E+22F).

Materials and methods - RNA extraction and qPCR analysis Total RNA was extracted using the RNeasy mini kit (Qiagen, ster, JUK) according to the manufacturer's ctions, and the RNA tration determined by absorbance at 260/280nm using a spectrophotometer (nano-Drop ND-1000; Thermo Scientific, Wilmington, DE). For mRNA expression analysis, cDNA was ed from total RNA using the High-Capacity cDNA reverse transcription kit (Applied Biosystems, UK) according to the manufacturer's instructions. The reverse transcription ons were performed in a Thermo cycler (Biometra, Germany) at 25°C for lOmin, 37°C for 120min, and 85°C for 5 min, hold on at 4°C. Resulting cDNA was amplified in duplicates by the SYBR-Green PCR assay, and products were detected on QuantStudio 6 flex real-time PCR machine ed Biosystems, UK) using a standardised profile (initial denaturation of 95°C for 10 minutes, followed by 40 cycles of 15 seconds of denaturation at 95°C and 60 seconds of annealing/extension at 60/65°C, depending on the primers. A dissociation stage was added after the 40 cycles to generate a melting curve. Analysis was performed using the Applied Biosystems tudio Real-Time PCR Software vl.2. The primer sequences for Actin, , Occludin TJP1 and TJP2 are provided in the sequence listing.

Example 16 Methods Animals The animals and study design used were the same as for Example 14.

Bacterial strains • 755: cteroides distasonis (MRX005) • Megasphaera massiliensis (MRX0029) Tissue Collection

Claims

1. Use of a composition comprising a strain of a commensal bacteria in the manufacture of a medicament for treating or preventing a neurodegenerative disorder in a subject, wherein the strain produces valeric acid and wherein the strain is not a bacterial strain of the genus Megasphaera.

2. Use of a composition comprising a strain of a commensal bacteria in the manufacture of a medicament for treating brain injury in a subject, wherein the strain produces valeric acid and wherein the strain is not a bacterial strain of the genus Megasphaera.

3. The use of claim 1 or claim 2, wherein the strain also produces a short chain fatty acid.

4. The use of claim 3, wherein the short chain fatty acid is butyric acid.

5. The use of any one of claims 1-4, wherein the strain also produces succinic acid.

6. The use of any one of claims 1-5, wherein the medicament is formulated to increase the activation of IL-8 when administered to the gastrointestinal tract.

7. The use of any one of claims 1-6, wherein the medicament comprises two or more strains of commensal bacteria.

8. The use of any one of claims 1-7, wherein the medicament comprises a strain of the species Parabacteroides distasonis.

9. The use of claim 8, wherein the strain of the species Parabacteroides distasonis has a 16s rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to SEQ ID NO:17, or wherein the strain of the species Parabacteroides distasonis has the 16s rRNA sequence represented by SEQ ID NO:17.

10. The use of claim 9, comprising the strain deposited at NCIMB under accession number NCIMB 42382.

11. The use of any one of claims 1 or 3-10, wherein the neurodegenerative disorder is selected from the group consisting of Parkinson’s disease, including progressive supranuclear palsy, Steele-