OA21082A - Compositions comprising a bacterial strain of the genus megasphera and uses thereof. - Google Patents

Abstract

The invention provides compositions comprising bacterial strains for treating and preventing neurodegenerative disorders.

Description

COMPOSITIONS COMPRISING BACTERIAL STRAINS

TECHNICAL FIELD

This invention is in the field of compositions comprising bacteria] strains isolated from the mammalian digestive tract and the use of such compositions in the treatment of disease.

BACKGROUND TO THE INVENTION

The human intestine is thought to be stérile in utero, but it is exposed to a large variety of maternai and environmental microbes immédiately after birth. Thereafter, a dynamic period of microbial colonization and succession occurs, which is influenced by factors such as delivery mode, environment, diet and host génotype, ail of which impact upon the composition of the gut microbiota, particularly during early life. Subsequently, the microbiota stabilizes and becomes adult-Iike [l], The human gut microbiota contains more than 500-1000 different phylotypes belonging essentially to two majoi bacteria! divisions, the Bacteroîdetes and the Firmicutes [2], The successful symbiotic relationships arising from bacterial colonization of the human gut hâve yielded a wide variety of metabolic, structural, protective and other bénéficiai functions. The enhanced metabolic activîties of the colonized gut ensure that otherwise indigestible dietary components are degraded with release of by-products ptoviding an important nutrient source for the host. Similarly, the immunological importance of the gut microbiota is well-recognized and is exemplified in germfree animais which hâve an impaired immune System that is functionally reconstituted following the introduction of commensal bacteria [3-5].

Diamatic changes in microbiota composition bave been documented in gastrointestinal disorders such as inilammatory bowel disease (IBD). For example, the levels of Clostridium cluster XIVa bacteria aie reduced în IBD patients whilst numbers of E. coli are increased, suggesting a shift in the balance of symbionts and pathobionts within the gut [6-9].

In récognition of the potential positive effect that certain bacterial strains may hâve on the animal gut, various strains hâve been proposed for use in the treatment of various diseases (see, for example, [ 10-13J). Also, certain strains, including mostly Lactobacillus and Bifidobacterium strains, hâve been proposed for use in treating various inilammatory and autoimmune diseases that are not directly hnked to the intestines (see [14] and [15] for reviews). However, the relationship between different diseases and different bacterial strains, and the précisé effects of particular bacterial strains on the gut and at a systemic level and on any particular 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 rôle in human brain diseases [16], Preclinical and clinical evidence are strongly suggesting a link between brain development and microbiota [l 7]. A growing body of preclinieal literature has demonstrated bidirectional signalling between the brain and the gut microbiome, involving multiple neurocrme and endocrine signalling Systems. Indeed, increased levels of Clostridium species in the microbiome hâve been linked to brain disorders [18], and an imbalance of the Bacteroidetes and Firmieûtes phyla has also been implicated in brain development disorders [19], Suggestions that altered levels of gut commensals, including those of Bifidobacterium, Lactobacillus, Sutterella, Prevotella and Ruminococcus généra and of the Alcaligenaceae family are involved in immune-mediated central nervous system (CNS) disorders, are questioned by studies suggesting a lack of alteration in the microbiota between patients and healthy subjects [ 19]. There hâve also been suggestions that the administration of probiotics may be bénéficiai in the treatment of neurological disorders. However, these studies failed to conclude that probiotic compositions per se can achieve therapeutic benefits with respect 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 îdentify the therapeutic impact of altering the microbiome on neurodegenerative disorders.

There is a requirement in the ait for new methods of treating neurodegenerative disorders. There is also a requirement for the potential effects of gut bacteria to be characterised so that new thérapies using gut bacteria can be developed.

SUMMARY OF THE INVENTION

The inventors hâve developed new thérapies for treating and preventing neurodegenerative disorders. The inventors hâve identified that bacterial strains from the genus Megasphaera may be effective for treating neurodegenerative diseases. As described in the examples, administration of compositions comprising Megasphaera massiliensis can protect against reactive oxygen species and prevent inflammation, thus acting as a neuroprotectant. The inventors hâve also identified that treatment with Megasphaera massiliensis can reduce the activation of proinflammatory molécules, such as NFkB and IL-6, by LPS and mutant α-synuclein. The inventors hâve identified that treatment with Megasphaera massiliensis can reduce histone deacetylation activity and lipid peroxidation in vitro, which can help to reduce cell death and apoptosis. The inventors hâve also identified that Megasphaera massiliensis can produce indole that can attenuate inflammation and oxidative stress. Furthermore, the inventors hâve demonstrated that treatment with Megasphaera massiliensis can increase kynurenine levels.

The inventors hâve also identified that Megasphaera massiliensis produces certain organic acids including hexanoic acid, valeric acid and 4-hydroxyphenylacetic acid. The inventors hâve also found that Megasphaera massiliensis can increase the activation of the pro-inflammatory cytokine IL-8, which can help to promote neuron myelination. The inventors hâve also identified that treatment with a combination of Megasphaera massiliensis and retinoic acid can increase the sécrétion of brainderived neurotrophic factor (BDNF), which can help promote neurogenesis and neuritogenesîs and/or prevent cell death. The inventors hâve also identified that treatment with Megasphaera massiliensis, which can produce valeric acid, can reduce histone deacetylation, which can help to reduce cell death and apoptosis. Furthermore, the inventors hâve also found that Megasphaera massiliensis can produce hexanoic acid, which can be neuroprotectîve or neurorestorative, for example by promoting neurite outgrowth. The inventors hâve found that Megasphaera massiliensis that can produce hexanoic acid increase the expression of MAP2 (Microtubule -associated protein 2), which is thought to be essential for microtubule formation in neuritogenesîs. Therefore, the inventors hâve found that Megasphaera massiliensis that can produce hexanoic acid can be used to promote neurite outgrowth. Megasphaera massiliensis and other bacteria that produce organic acids like hexanoic acid, valeric acid and 4-hydroxyphenylacetic acid may therefore be usefiil for treating neurodegenerative disorders.

In a first embodiment, the invention provides a composition comprising a bacterial strain of the genus Megasphaera, for use in therapy, such as for use in a method of treating or preventing a neurodegenerative disorder.

In particular embodiments, the invention provides a composition comprising a bacterial strain of the genus Megasphaera, for use in a method of treating or preventing a disease or condition selected from the group consisting of: Parkinson’s disease, includîng progressive supranuclear palsy, progressive supranuclear palsy, Steele-Richardson-Olszewski syndrome, normal pressure hydrocephalus, vascular or arteriosclerotic parkinsonîsm and drug-induced parkinsonism; Aizheimer’s disease, includîng Benson's syndrome; multiple sclerosis; Huntington’s disease; amyotrophie latéral sclerosis; Lou Gehrig's disease; motor neurone disease; prion disease; spinocerebellar ataxia; spinal muscular atrophy; dementia, includîng Lewy body, vascular and frontotemporal dementia; primary progressive aphasia; mild cognitive împairment; HIV-related cognitive împairment and corticobasal degeneration.

In preferred embodiments, the invention provides a composition comprising a bacterial strain of the genus Megasphaera, 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 hâve identified that treatment with Megasphaera strains can reduce the activation of proinflammatory molécules, such as NFkB and IL-6, by LPS and mutant α-synuclein in in vitro models of environmental and familial Parkinson’s. In preferred embodiments, the invention provides a composition comprising a bacterial strain of the species Megasphaera massiliensis, for use în the treatment of Parkinson’s disease. Compositions using Megasphaera massiliensis may be particularly effective for treating 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 invention are for use in a method of preventing or delayîng onset or progression of a neurodegenerative disorder.

In preferred embodiments of the invention, the bacterial strain in the composition is of Megasphaera massiliensis. Closely related strains may also be used, such as bacterial strains that hâve 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 sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to SEQ ID NO: l or 2. Preferably, the sequence identity is to SEQ ID NO:2. Preferably, the bacterial strain for use in the invention has the I6S rRNA sequence represented by SEQ ID NO:2.

In certain embodiments, the composition of the invention is for oral administration. Oral administration ot the strains of the invention can be effective for neurodegenerative disorders. Also, oral administration is convenient for patients and practitioners and allows delivery to and / or partial or total colonisation of the intestine.

In certain embodiments, the composition oi the invention comprises one or more pharmaceutically acceptable excipients or carriers.

In certain embodiments, the composition of the invention comprises a 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 the composition as described above.

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

Additionally, the invention provides a method of treating or preventing neurodegenerative disorders, comprising administering a composition comprising a bacterial strain ofthe genus Megasphaera.

In developing the above invention, the inventors hâve identified and characterised a bacterial strain that îs particularly useiul for therapy. The Megasphaera massiliensis strain ofthe invention is shown to be effective for treating the diseases described herein, such as neurodegenerative diseases.

Therefore, in another aspect, the invention provides a cell of the Megasphaera massiliensis strain deposited under accession number NCIMB 42787, or a dérivative thereof The invention also provides compositions comprising such cells, or biologically pure cultures of such cells. The invention also provides a cell of the Megasphaera massiliensis strain deposited under accession number NCIMB 42787, or a dérivative thereof, for use in therapy, in particular for the diseases described herein.

In certain embodiments of the invention, the composition is for use in treating brain injury. The neuroprotective activity of the compositions of the invention and their ability to reduce levels of hîstone 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: Down-regulation of IL-6 sécrétion

Figure 3 Sécrétion of IL-8

Figure 4: Inhibition of a- synuclein IL-6 and IL-8 sécrétion

Figure 5: Inhibition of a- synuclein induced NFkB promoter activation

Figure 6: Inhibition of LPS induced NFkB promoter activation

Figure 7: Change in antioxidant capacity

Figure 8: Change in total anti-oxidant capacity (lipid oxidation)

Figure 9: Change in histone deacetylase (HDAC) activity

Figure 10: Level of Indole production

Figure II: Level of Kyrunenine production

Figure 12: Mean Dopamine (DA) levels (Figure 12A), DOPAC levels (Figure 12B) and HVA levels (Figure 12C) in striatum. Data is displayed as Mean + SEM.

Figure 13: Piomoting neurite outgrowth: light microscopy and MAP2 gene expression (Figure 13A), Phalioidin immunofluorescence microscopy (Figure 13B)

Figure 14: Change in ROS levels in (a) U373 cells and (b) SHSY-5Y cells

Figure 15: Neuroprotection - cell viability. Figure 15 shows the same data as Figure 1.

Figure 16 Strain-induced changes in whole cell and cell lysate histone deacetylase activity (Figure 16A), acid-induced changes in histone deacetylase activity (Figure 16B), métabolite production by strains (Figure 16C)

Figure 17 HDAC1 inhibition (Figure 17A), HDAC2 inhibition (Figure 17B), HDAC3 inhibition (Figure 17C)

Figure 18 Inhibition of Class I HDACs (Figure ISA); inhibition of HDAC1 (Figure 18B); inhibition of HDAC2 (Figure 18C); inhibition of HDAC3 (Figure 18D)

Figure 19: Level of BDNF production

Figure 20: Levels of métabolite production - neurotransmitters in the brain

Figure 21: Levels of métabolite production - organic acids in the supernatant

Figure 22: Effect on intestinal barrier function.

Figure 23: Production of neurotransmitters in the brain

Figure 24: Changes in Hippocampal Receptor Expression - A) Oxytocin Receptor, B) Vasopressin Receptor, C) Glucocorticoid Receptor and D) Mineralocorticoid Receptor

Figure 25: Changes in Hippocampal Expression of A) Corticotropin-Releasing Hormone (CRH), B) BDNF Expression and C) TLR4

Figure 26: A) Changes in Hippocampal Corticotropin Releasing Hormone Receptor 1 (CRFR1 ) Expression and B) Corticotropin Releasing Hormone Receptor 2 (CRFR2) Expression

Figure 27: Changes in Hippocampal Expression of A) Tumour Necrosis Factor, B) Interleukin Ib and C) IL-6

Figure 28: A) Changes in Hippocampal Integrin Alpha M (CDi 1b) Expression and B) Changes in

Hippocampal Serotonin 1A Receptor (5-HT1A receptor) Expression

Figure 29: A) Changes în Hippocampal Glutamate lonotropîc Receptor NMDA Type Subunit 2A (Grin2A) and B) Glutamate lonotropîc Receptor NMDA Type Subunit 2B (Grin2B) expression

Figure 30: Changes in Hippocampal Expression of A) Gamma-Aminobutyric Acid A Receptor 2 (GABA A2), B) Gamma-Aminobutyric Acid B Receptor 1 (GABA BRI) and C) Dopamine Receptor 1 (DRD1)

Figure 31: Changes in Amygdala Receptor Expression - A) Oxytocin Receptor, B) Vasopressin Receptor, C) Glucocorticoid Receptor and D) Mineralocorticoid Receptor

Figure 32: Changes in Amygdala Expression of A) Brain Derived Neurotrophic Factor (BDNF), B) Toll-like Receptor 4 (TLR-4), C) Corticotropin Releasing Hormone Receptor 1 (CRFR1) and D) Corticotropin Releasing Hormone Receptor 2 (CRFR2)

Figure 33: Changes in Amygdala Expression of A) Integrin Alpha M (CD11 b), B) Interleukin-6 (IL6), C) Glutamate lonotropic Receptor NMDA Type Subunit 2A (Grîn2A) and D) Glutamate lonotropic Receptor NMDA Type Subunît 2B (Grin2B)

Figure 34: Changes in Amygdala Expression of A) G AB A-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 Prefrontal Cortex Expression of A) Oxytocin Receptor, B) Brain Derived Neurotrophic Factor (BDNF), C) Mineralocorticoid Receptor and D) Glucocorticoid Receptor

Figure 36: Changes in Prefrontal 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 (CD11b)

Figure 37: Changes in Prefrontal Cortex Expression of A) Interleukin-6 (IL-6), B) Glutamate lonotropic Receptor NMDA Type Subunit 2A (Grin2A), C) Glutamate lonotropic Receptor NMDA Type Subunit 2B (Grin2B) and D) GABA-A Receptor Alpha 2 Subunit (GABRA2)

Figure 38: Changes in Prefrontal Cortex Expression of A) GABA-A Receptor Type B Receptor Subunit I (GABBR1) and B) Dopamine Receptor 1 (DRD1)

Figure 39: Changes in Colon Expression of A) Tryptophan Hydroxylase-1 (Tphl) and B) Indoleamine2,3-Dioxygenase-1 (IDO1)

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 Métabolite Levels A) Kynurenine, B) Tryptophan and C) Kynurenine/ Tryptophan Index of metabolism

Figure 42: Effect on Interferon-γ Production from mouse Splénocytes from mice fed with MRx0029

Figure 43: Effect on Interleukin-ΐβ Production from Splénocytes

Figure 44: Effect on lnterleukin-6 Production from Splénocytes

Figure 45: Effect on Tumour Necrosîs Factor Production from Splénocytes

Figure 46: Effect on Interleukin-lO Production from Splénocytes

Figure 47: Effect on Chemoattractant CXCLl Production from Splénocytes

Figure 48: Changes in Caecal Short Chain Fatty Acid Levels

Figure 49: MRx0029 and MRXOO5-induced changes in gene expression levels of Actin, Villin, Occludin TJPI, TJP2, MAP2, DRD2, GABRB3, SYP, PINKI, PARK7 and NSE.

Figure 50: SHSY5Y cell différentiation induced by MRx0005 and MRxÛ029. (A-C) Représentative images of immuno labelled cells with Phalloidin and MAP2. (D—F) images of A-C merged with DAPI images. (G—I) β3 tubulîn immunolabelled cells. (J-L) merged with DAPI images.

Magnification x630. Western blot analysis of effects of MRx0005 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: représentative blots from one of six separate experiments; upper panels: relative densitométrie intensity.

DISCLOSURE OF THE INVENTION

Bacterial strains

The compositions of the invention comprise a bacterial strain of the genus Megasphaera. The examples demonstrate that bacteria of this genus are usefui for treating or preventing neurodegenerative disorders. The preferred bacterial strains are of the species Megasphaera massiliensis.

Examples of Megasphaera species for use in the invention include Megasphaera elsdenii, Megasphaera cerevisiae, Megasphaera massiliensis, Megasphaera indîca, Megasphaera paucivorans, Megasphaera sueciensis and Megasphaera micronttciformis. A further example of a Megasphaera species for use in the invention îs Megasphaera hexanoica^ The Megasphaera are obligately anaérobie, lactate-fermenting, gastrointeslinal microbe of ruminant and non-ruminant mammals, including humans.

The type strain of M. massiliensis is NP3 (=CSUR P245=DSM 26228)[22], The GenBank accession number for the 16S rRNA gene sequences of M. massiliensis strain NP3 is JX424772.1 (disclosed herein as SEQ ID NO: l ).

The Megasphaera massiliensis bacterium tested in the Examples is referred to herein as strain MRx0029. A 16S rRNA sequence for the MRx0029 strain that was tested is provided in SEQ ID NO:2.

Strain MRx0029 was deposited with the international depositary authority NCIMB, Ltd, (Ferguson Building, Aberdeen, AB2l 9YA, Scotland) by 4D Pharma Research Ltd. (Life Sciences Innovation Building, Comhill Road, Aberdeen, AB25 2ZS, Scotland) on 13th July 2017 as “Megasphaera massiliensis MRx0029” and was assigned accession number NCIMB 42787.

Bacterial strains closely related to the strain tested in the examples are also expected to be effective for treating or preventing neurodegenerative disorders. 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%, 99.5% or 99.9% identical to the 16S rRNA sequence of a bacterial strain of Megasphaera massiliensis. Preferably, the bacterial 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: 1 or 2. Preferably, the sequence identity is 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.

Bacterial strains that are biotypes of strains MRx0029 or NP3 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 strains MRx0029 or NP3 and that are suitable for use in the invention may be identified by sequencing other nucléotide sequences for strains MRx0029 or NP3. For example, substantially the whole genome may be sequenced and a biotype strain for use in the invention may hâve 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 répétitive sequences such as BOX, ERIC, (GTG)₅, or REP or [23], Biotype strains may hâve sequences with at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity to the corresponding sequence of the strains MRxOO29 or NP3.

Alternatively, strains that are biotypes of strains MRx0029 or NP3 and that are suitable for use in the invention may be identified by using strains MRxOO29 or NP3 and restriction fragment analysis and/or PCR analysis, for example by using fluorescent amplified fragment length polymorphism (FAFLP) and repedtive DNA element (rep)-PCR fïngerprinting, or protein profiling, or partial I6S or 23S rDNA sequencing. In preferred embodiments, such techniques may be used to identify other Megasphaera massiliensis strains.

ΙΟ ln certain embodiments, strains that are biotypes of strains MRx0029 or NP3 and that are suitable for use în the invention are strains that provide the same pattern as strains MRx0029 or NP3 when analysed by amplified ribosomal DNA restriction analysis (ARDRA), for example when using Sau3AI restriction enzyme (for exemplary methods and guidance see, for example,[24]). Alternatively, biotype strains are îdentified as strains that hâve the same carbohydrate fermentation patterns as strains MRx0029 or NP3.

Other Megasphaera strains that are useful in the compositions and methods of the invention, such as biotypes of strains MRx0029 or NP3, may be îdentified using any appropriate method or strategy, including the assays described in the examples. For instance, strains for use in the invention may be îdentified by culturing with neuroblastoma cells and then assessing cytokine levels and levels of neuroprotection or neuroproliferation. In particular, bacterial strains that hâve similar growth patterns, metabolic type and/or surface antigens to strains MRx0029 or NP3 may be useful in the invention. A useful strain will hâve comparable immune modulâtory activity to strains MRx0029 or NP3. 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 Examples, which may be îdentified by using the culturing and administration protocols described in the Examples.

A particularly preferred strain of the invention îs 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 dérivative thereof. The invention also provides a composition comprising a cell of the Megasphaera massiliensis strain MRx0029, or a dérivative thereof. The invention also provides a biologically pure culture of the Megasphaera massiliensis strain MRx0029. The invention also provides a cell of the Megasphaera massiliensis strain MRx0029, or a dérivative thereof, for use in therapy, în particular for the diseases described herein.

A particularly 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 dérivative thereof. The invention also provîdes a composition comprising a cell of the Megasphaera massiliensis strain deposited under accession number NCIMB 42787, or a dérivative thereof. The invention also provides a biologically pure culture of the Megasphaera massiliensis strain deposited under accession number NCIMB 42787. The invention also provîdes a cell of the Megasphaera massiliensis strain deposited under accession number NCIMB 42787, or a dérivative thereof, for use in therapy, in particular for the diseases described herein.

i i

A dérivative of the strain of the invention may be a daughter strain (progeny) or a strain cultured (subcloned) from the original. A dérivative of a strain ofthe invention may be modified, for example at the genetic level, without ablating the biological activity. In particular, a dérivative strain of the invention is therapeutically active. A dérivative strain will hâve comparable therapeutic activity to the MRx0029 strain. In particular, a dérivative strain will elicît comparable effects on the neurodegenerative disease models and comparable effects on cytokine levels to the effects shown in the Examples, which may be îdentifîed by using the culturing and administration protocols described in the Examples. A dérivative of the MRxOO29 strain will generally be a biotype ofthe MRx0029 strain.

Référencés to cells of the Megasphaera massiliensis MRx0029 strain encompass any cells that hâve the same safety and therapeutic efficacy characteristics as the strain MRx0029, and such cells are encompassed by the invention.

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

The inventors hâve found that Megasphaera massiliensis strains reduce the activation of inflammatory cytokines such as IL-6 and increase the activation of the inflammatory cytokine IL-8. IL-8 has been implicated in myelin sheath formation [25]. Chronic inflammation induced by IL-6 can ultimately lead to cell dealh. Therefore, the bacterial strains of the invention are particularly useful in the treatment or prévention of neurodegenerative disorders. In some embodiments, the bacterial strains are useful in the treatment of conditions characterised by the enhanced activation of IL-6. In some embodiments, the compositions of the invention are for use in the treatment or prévention of neutodegenerative diseases characterised by demyelination. Many neurodegenerative diseases 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. Therefore, the compositions of the invention are particulaily bénéficiai in the treatment or prévention of neurodegenerative disorders characterised by demyelination, such as Multiple Sclerosis.

The inventors hâve found that the Megasphaera massiliensis strains of the invention alleviate symptoms of neurodegenerative diseases in models oi the disease. For example, the inventors hâve found that the Megasphaera massiliensis strains promote neurite outgrowth in vitro, and may therefore be used in promoting neuron restoration for the treatment or prévention of neurodegenerative diseases. Thus, bacterial strains of the invention are for use in the treatment or prévention of neurodegenerative diseases.

The inventors hâve also found that the bacterial strains of invention increase the activation of BDNF. BDNF is a neurotrophic growth factor that has been shown to enhance neuron différentiation and survival. Thus, the compositions of the invention can be used in a method of enhancing nerve cell survival in the treatment or prévention of neurodegenerative diseases.

A further 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 hâve neuroprotective activities, but produce different métabolites and may hâve different mechanisms of action and spécifie neuroprotective activities. Therefore, these species may be particularly effective when used in combination. In preferred embodiments, the composition comprises a strain of the species Parabacteroides distasonis and a strain of the species Megasphaera mass il iensis,

The Parabacteroides distasonis bacterium deposited under accession number NCIMB 42382 was tested în the Examples and is also referred to herein as strain MRxOOOS. MRX0005, MRX005, MRx005 and MRx0005 are used herein interchangeably. A 16S rRNA sequence for the MRx0005 strain that was tested is provided in SEQ ID NO: 17. Strain MRx0005 was deposited with the international depositary authority NCIMB, Ltd. (Ferguson Building, Aberdeen, AB21 9 Y A, Scotland) by GT Biologics 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 Biologics Ltd. Subsequently changed its name to 4D Phanna Research Limited.

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

Therapeutic uses

As demonstrated in the examples, the bacterial compositions of the invention are effective for treating neuiodegeneiative disorders. In particular, treatment with compositions of the invention increase neuro-proliferation and act as a neuroprotectant against agents that destroy dopaminergic neuions. Therefore, the compositions ot the invention may be useful for treating or preventing neurodegenerative disorders that are the resuit of neuron death.

Compositions of the invention can decrease the activation of the NFkB promoter, which activâtes cytokine production, for example IL-1 β, IL-la, IL-18, TNFa and IL-6. Treating cells with mutant asynuclein îs a model for familial Parkinson’s. A point mutation at position 53 from adenine to threonine leads to a-synuclein mis-folding. The incorrectly folded α-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 resuit of neuro in fl amination, protein misfoldîng and/or environmental exposure. Compositions of the invention can be used for treatment of familial Parkinson’s. Activation of the NFkB promoter is mediated through the TLR4 ligand. TLR4 is known to médiate cell death in the mouse model MPTP, which simulâtes Parkinson’s disease. Compositions of the invention can be used to inhibit the ability of TLR4 sîgnalling to activate the NFkB promoter. Of particular relevance for PD, both TLR2 and TLR4 were found to be upregulated in brains of PD patients [26], Moreover ct-syn has been described as a ligand for TLR2 [27] and we hâve demonstrated that α-syn is also a ligand for TLR4 using HEK-TLR4 cells [28].

Compositions of the invention decrease the sécrétion of pro-inilammatory cytokines such as IL-6, which can be induced by lipopolysaccharide (LPS). Treatment of cells with LPS simulâtes Parkinson’s caused by environmental factors. Compositions of the invention can be used to decrease IL-6 sécrétion. Compositions of the invention can be used for treatment of environmental Parkinson’s.

Examples of neurodegenerative diseases to be treated by compositions of the invention include: Parkinson’s disease, including progressive supranuclear palsy, progressive supranuclear palsy, Steele-Richardson-Olszewski syndrome, normal pressure hydrocephalus, vascular or arteriosclerotic parkinsonism and drug-induced parkinsonism; Alzheimer’s disease, including Benson's syndrome; multiple sclerosis; Huntington’s disease; amyotrophie latéral 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, , and corticobasal degeneration. A further disease to be treated by compositions of the invention is progressive inilammatory neuropathy.

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

In certain embodiments, the compositions of the invention are for use în 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 în the substantiel nigra pars compacta and the conséquent loss of their projecting nerve fibers in the striatum. In certain embodiments, the compositions of the invention are for use in reducing or preventing loss of nigrostriatal dopaminergic neurons.

In certain embodiments, the compositions of the invention are for use in increasing dopamine levels. In certain embodiments, the compositions of the invention are for use in increasing DOPAC (3,4Dihydroxyphenylacetic acid) levels. In certain embodiments, the compositions of the invention are for use in increasing dopamine and DOPAC levels. In certain embodiments, the dopamine and/or DOPAC levels are increased in the striatum. Dopamine and DOPAC levels may be measured using any approprîate method known in the art, such as a radioenzymatic method, for example in plasma or CSF (for example as described in [29]), or a reverse-phase FIPLC method, perhaps with electrochemical détection, for example in plasma or CSF (for example as described in [30]).

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 delayîng onset or progression of neurodegenerative disorders. In certain embodiments, the compositions ofthe invention are for use in delayîng onset or progression of neurodegenerative disorders.

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

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 development of new neurons and synapses. BDNF is active in the hippocampus, cortex and basal forebrain, and is important for long-tenn memory. The compositions of the invention can therefore be used to increase the sécrétion of BDNF. The compositions may therefore be used in the treatment of neurodegenerative 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.

Modulation of the microbiota-gut-brain axis

Communication between the gut and the brain (the microbiota-gut-brain axis) occurs via a bidirectional neurohumoral communication System. Recent evidence shows that the mîcrobiota that résides in the gut can moduiate brain development and produce behavioural phenotypes via the mîcrobiota-gut-brain axis. Indeed, a number of reviews suggest a rôle of the microbiota-gut-brain axis in inaintaining central nervous System functionality and implicate dysfonction ofthe microbiotagut-brain axis in the development of central nervous system disorders and conditions [l6],[l9],[31],

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 hypothalamuspi tuitary-adrenal (HPA) axis, sympathetic and parasympathetic amis ol the autonomie 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 moduiate the mîciobiota-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 dysfonction of the microbiota-gut-brain axis.

In particulai embodiments, the compositions of the invention may be useful for treating or preventing a disease or condition selected from the group consisting of: Parkinson’s disease, including progressive supranuclear palsy, progressive supranuclear palsy, Steele-Richardson-Olszewski syndrome, normal pressure hydrocephalus, vascular or arteriosclerotic parkinsonism and druginduced parkinsonism; Alzheimer’s disease, including Benson's syndrome; multiple sclerosis; Huntington s disease, amyotrophie latéral sclerosis; Lou Gehrig's disease; motor neurone disease; prion disease; spinocerebellar ataxia; spinal muscular atrophy; denientia; including Lewy body; vascular and frontotemporal denientia; primary progressive aphasia; mild cognitive impairment; HIV-related cognitive impairment and corticobasal degeneration.

The compositions of the invention may be particularly useful for treating or preventing chronic disease, treating or preventing disease in patients that hâve not responded to other thérapies (such as treatment with Levodopa, dopamine agonists, MAO-B inhibitors, COMT inhibitors, Glutamate antagonists, and/or anticholinergics), and/or treating or preventing the tissue damage and symptoms associated with dysfonction of the microbiota-gut-brain axis.

In certain embodiments, the compositions ofthe invention moduiate the CNS. In some embodiments, the compositions of the invention moduiate the autonomie nervous System (ANS). In some embodiments, the compositions of the invention moduiate the enteric nervous system (ENS). In some embodiments, the compositions of the invention moduiate the hypothalamic, pituitary, adrenal (HPA) axis. In some embodiments, the compositions of the invention moduiate 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 certain embodiments, the compositions of the invention module the levels of commensal métabolites and/or the gastrointestinal permeability of a subject.

The signalling of the miciobiota-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 central nervous system. In some embodiments, the compositions of the invention modulate signalling in sensory neurons. In other embodiments, the compositions 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 system. In preferred embodiments, the compositions of the invention modulate the signalling ofthe 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 embodiments, 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 ofthe 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

Parkinson’s disease îs a common neurodegenerative disease neuropathologically characterised by degeneration of heterogeneous populations of neural cells (dopamine-producing cells). The clinical diagnosis of Parkinson’s disease requires bradykinesia and at least one of the following core symptoms: resting tremor; muscle rigidity and postural reflex impairment. Other signs and symptoms that may be present or develop during the progression of the disease are autonomie disturbances (sialorrhoea, seborrhoea, constipation, micturîtion disturbances, sexual functioning, orthostatic hypotension, hyperhydrosis), sleep disturbances and disturbances in the sense of smell or sense of température. Parkinson’s disease is a neurodegenerative disease that may develop or persist due to dysfonction of the microbiota-gut-brain axis. Therefore, in preferred embodiments, the compositions ofthe invention are for use in treating or preventing Parkinson’s disease in a subject.

In further preferred embodiments, the invention provides a composition comprising a bacterial strain of the genus Megasphaera, for use in a method of treating or preventing Parkinson’s disease. Compositions comprising a bacterial strain of the genus Megasphaera may improve mot or and cognitive functions în models of Parkinson’s disease. Treatment with Megasphaera strains may modulate signalling in the central, autonomie and enteric nervous Systems; may modulate the activity of the HPA axis pathway; may modulate neuroendocrine and/or neuroimmune pathways; and may modulate the levels of commensal métabolites, inflainmatory markers and/or gastrointestinal permeability of a subject, ail of which are implicated in the neuropathology of Parkinson’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 Parkinson’s disease.

In preferred embodiments, the compositions of the invention prevent, reduce or allevîate 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 Parkinson’s disease in a subject. In certain embodiments, the compositions of the invention prevent, reduce or alleviate bradykinesia in a subject. In certain embodiments, the compositions of the invention prevent, reduce or alleviate resting tremor; muscle rigidîty and/or postural reflex impairment m a subject. In certain embodiments, the compositions of the invention prevent, reduce or alleviate one or more symptoms associated with Parkinson’s disease progression selected from autonomie disturbances (sialorrhoea, seborrhoea, constipation, micturîtion disturbances, sexual functîoning, orthostatic hypotension, hyperhydrosis), sleep disturbances and disturbances in the sense of smell or sense of température.

In preferred embodiments, the compositions of the invention prevent, reduce or alleviate dépressive symptoms coniorbid with Parkinson’s disease. In certain embodiments, the compositions of the invention improve verbal memory and/or executive functions. In certain embodiments, the compositions of the invention improve attention, working memory, verbal fluency and/or anxiety.

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

In certain embodiments, the compositions of the invention prevent, reduce or alleviate Parkinson’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 ofthe invention impiove symptoms of Parkinson’s disease dementia (PDD). In certain embodiments, the compositions of the invention prevent, reduce or alleviate impairment of executive function, attention and/or working memory. In certain embodiments, the compositions of the invention improve dopaminergic neurotransmission. In certain embodiments, the compositions of the invention prevent, reduce or alleviate impaired dopaminergic neurotransmission.

In some embodiments, the compositions of the invention improve the symptoms of Parkinson’s disease according to a symptomatic or diagnostic scale. In certain embodiments, the tests for assessing symptomatic improvement of motor function in Parkinson’s disease is the Unified Parkinson’s Disease 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 improve the symptoms associated with PDD according to a symptomatic or diagnostic test and/or scale. In certain embodiments, the test or scale is selected from the Hopkins Verbal Leaming Test - Revised (HVLT-R); the Delis-Kaplan Executive Function System (D-KEFS) Color-Word Interférence Test; the Hamilton Dépréssion Rating Scale (HAM-D 17; dépréssion); the Hamilton Anxîety 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 psychiatrie and neurological disorders. In some embodiments, the compositions of the invention display a positive effect on global social and occupational impairment of the subject with Parkinson’s disease.

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 wherein said use involves 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 neurological disorders such as Parkinson’s disease in a subject wherein said use invoives 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 treating or preventing neurological 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 and the conséquent loss of their projecting nerve fibers in the striatum. 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 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 disorders 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 pieventing neurological disorders such as Parkinson’s disease in a subject wherein said use involves încreasing 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 wherein said use involves încreasing 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 ternis major neurocognitive disorder and mild neurocognîtive disorder. Neurocognitive disorder is a heterogeneous class of psychiatrie diseases. fhe most common neutocogmtive disorder îs Alzheimer’s disease, followed by vascular dementias or mixed forms of the two, Other forms of neurodegenerative 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.

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

The symptomatic ciiteria for dementia under DSM-5 are evidence of signifîcant cognitive décliné from a préviens level of performance in one or more cognitive domains selected from: leaming and memory; language; executive function; complex attention; perceptual-motor and social cognition. The cognitive déficits must interfère with independence in everyday activities. In addition, the cognitive déficits 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 disorders display behavioural and psychiatrie symptoms includîng agitation, aggression, dépréssion, anxiety, apathy, psychosis and sleep-wake cycle disturbances.

Neurodegenerative disorders may develop or persist due to dysfonction ofthe microbiota-gut-brain axis. Therefore, in preferred embodiments, the compositions ofthe invention are for use in treating or preventing neurodegenerative disorders in a subject. In preferred embodiments, the neuiodegeneiative disorder is Alzheimer’s disease. In other embodiments, the neurodegenerative 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; Huntington’s disease; and Wernicke-Korsakoff syndrome.

lu preferred embodiments, the compositions of the invention prevent, reduce or alleviate one or more of the symptoms of neurodegenerative disorders in a subject. In certain embodiments, the compositions of the invention prevent, reduce or alleviate the occurrence of cognitive décliné 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: leaming 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 psychiatrie symptoms associated with neurodegenerative disorders selected from agitation, aggression, dépréssion, anxiety, apathy, psychosis and sleep-wake cycle disturbances.

In certain embodiments, the compositions of the invention prevent, reduce or alleviate syinptomatic disease by intervention in suspected pathogenic mechanisms at a preclinical stage. In certain embodiments, the compositions of the invention improve disease modification, with slowing or arrest of symptom progression. In some embodiments, the slowing or arrest of symptom progression correlates with evidence in delaying the underlying neuropathological process. In preferred embodiments, the compositions of the invention improve symptoms of neurodegenerative disorders comprising enhanced cognitive and functional improvement. In preferred embodiments, the compositions of the invention improve the behavioural and psychiatrie symptoms of dementia (BPSD). In preferred embodiments, the compositions of the invention improve the ability of a subject with neurodegenerative disorder to undertake everyday activities.

In preferred embodiments, the compositions of the invention improve both cognition and functioning in a subject with Alzheimer’s disease. In some embodiments, the composition of the invention improves 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 clînical response (the global endpoint) in a subject with Alzheimer’s disease.

In some embodiments, the compositions of the invention improve the symptoms of neurodegenerative disorders according to a syinptomatic 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 psychiatrie symptoms of neurodegenerative disorders.

[n certain embodiments, the objective cognitive tests for assessment of symptomatic improvement use the Alzheimer’s disease Assessment Scale cognitive subscaie (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 Impression Global Improvement (CGI-I) scale for assessing psychiatrie and neurological disorders. 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 Impression of Change (ADCS-CGIC).

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

In certain embodiments, the tests assessing behavioural and psychiatrie symptoms of neurodegenerative disorders are selected from the Behavioural pathology in Alzheimer’s Disease Ratmg Scale (BEHAVE-AD); the Behavioural Rating Scale for Dementia (BRSD); the

Neuropsychiatrie Inventory (NPI); and the Cohen-Mansfield Agitation Inventory (CMAI).

In some embodiments, the compositions of the invention are particularly effective at preventing, reducing or aHeviating neurodegenerative disorders when used in combination with another therapy for treating neurodegenerative disorders. In certain embodiments, such thérapies include acetylcholinesteiase inhibitors including donepezil (Aricept®), gaiantamine (Razadyne®) and 20 rivastigmine (Exelon ®), and memantîne.

Multiple Sclerosis

Multiple sclerosis (MS) is a demyelînating 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 25 also thought to contribute 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 autonomie, visual, motor or sensory control. The exact symptoms will vary depending 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 invention may also be used to protect neurons from demyelination. In other words, the compositions ofthe invention may be for use in a method of treating or preventing multiple sclerosis by restoring or preventing loss of neuron myelin sheaths.

In some embodiments, the compositions of the invention prevent, reduce or allevîate one or more symptoms of MS in a subject. In some embodiments, the compositions of the invention prevent, reduce or allevîate fatigue in a subject. In certain embodiments, the compositions of the invention prevent, reduce or allevîate restîng tremor, muscle weakness, muscle spasms, muscle stiffness, paraesthesia and/or ataxia in a subject. in certain embodiments, the compositions of the invention prevent, reduce or allevîate one or more symptoms associated with MS progression selected from the list consisting of autonomie disturbances: constipation, micturition disturbances, sexual functioning, dysphagîa, dysarthria, syncope, vertigo and/or dizziness; sleep disturbances; and disturbances in the sense of smell or sense of température. In some embodiments, the compositions of the invention prevent, reduce or allevîate one or more ocular symptoms associated with MS. In some embodiments, the ocular symptom is selected from the list consisting 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 prevent, reduce or allevîate dizziness, vertigo, neuropathie pain, musculoskelétal pain, cognitive dysfunction, bowel incontinence, dysphagîa, dysarthria, or any combination thereof.

In some embodiments, the compositions of the invention prevent, reduce or allevîate dépressive symptoms or anxiety comorbid with MS.

In some embodiments, the improvement of symptoms are determined using the 2017 McDonald criteria for diagnosing MS.

In certain embodiments, treatment with the compositions of the invention results in a réduction 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 pre vents a décliné in motor function or results in improved motor function associated with MS. In certain embodiments, the compositions of the invention are for use in preventing a décliné 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 development oi paralysis in MS. In certain embodiments, the compositions of the invention are for use in preventing paralysis in the treatment of MS.

In certain embodiments the compositions 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 early-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 certain embodiments, the compositions of the invention are for use in a patient identified as having a genetic prédisposition to MS, such as major histocompatibility complex (MHC) class II phenotype, human leukocyte antigen (HLA)-DR2 or HLA-DR4.

The compositions of the invention may be useful for managing or alleviating multiple sclerosis. The compositions of the invention may be particularly useful for reducing symptoms associated with multiple sclerosis. Treatment or prévention of multiple sclerosis may refer to, for example, an alleviation of the severity of symptoms or a réduction 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 progression 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 following dîagnosis of RRMS, primary progressive MS (PPMS) which exhibîts graduai contînuous neurologie détérioration 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 consistîng of: fatigue, vision probiems, numbness, tingling, muscle spasms, muscle stiffness, muscle weakness, mobîlity probiems, pain, probiems with thinking, learning and planning, dépréssion and anxîety, sexual probiems, bladder probiems, bowel probiems, speech and swallowing diffîculties.

Neurochemical factors, neuropeptides and neurotransmitters and the microbiota-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 molécules. Alterations in the levels of these signalling molécules results in neurodegenerative diseases. The experiments performed by the inventors indicate that administration of Megasphaera species, and in particular Megasphaera massiliensis, can modulate levels of indole and kynurenine. Dysrégulation of these métabolites can lead to neurodegenerative diseases, such as Parkinson’s disease.

In certain embodiments, the compositions of the invention modulate the levels of brain monoamines and métabolites thereof. In preferred embodiments the metaboiite is kynurenine. In certain embodiments, the compositions of the invention modulate kynurenine, which is the main route of tryptophan metabolism, which serves as a route to nicotinamide adenine dinucleotîde (NAD+) production. Kynurenine can be metabolized to neuroactive compounds such as kynurenic acid (KYNA) and 3-hydroxy-l-kynurenine (3-OH-I-KYN), and in further steps to quinolinic acid (QUIN). Dysrégulation of the kynurenine pathway can lead to activation of the immune system and the accumulation ofpotentially neurotoxic compounds. Alterations in the kynurenine metabolism may be involved in the development of Parkinson’s diseases. Kynurenine levels hâve been demonstrated to be decreased in the frontal cortex, putamen and substantia nigra pars compacta of patients with PD [32], Therefore, in certain embodiments the compositions of the invention are for use in increasing the levels of kynurenine în 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 hâve been shown to attenuated MPP+-înduced neuronal cell death in vitro in a human dopaminergîc neuroblastoma cell line [33], In certain embodiments kynurenine and kynurenic acid, can activa te GI aryl hydrocarbon receptor (Ahr) and GPRS 5 receptors. Activation of Ahr receptor induces IL-22 production, which can inhibit local inflammation. Activation of GPR35 inducing the production of inositol triphosphate and Ca2+ mobilizatîon.

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

The signal]ing of the microbiota-gut-brain axis is modulâted by levels of neurochemical factors, neuropeptides and neurotransmitters. Accordingly, in certain embodiments, the compositions ofthe invention modulâtes levels of neurochemical factors, neuropeptides and neurotransmitters. Accordingly, in certain preferred embodiments, the compositions ofthe invention directly alter CNS biochemistry.

The signalling of the microbiota-gut-brain axis is modulated by levels of γ-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 certain embodiments, the compositions of the invention increase the levels of GABA. In certain embodiments, the compositions of the invention decrease the levels of GABA. In certain embodiments, the compositions of the invention alter GABAergic neurotransmission. In certain embodiments, the compositions of the invention modulate the level of GABA transcription in different régions of the central nervous system. In certain embodiments, the commensal derived GABA crosses the blood-brain barrier and affects neurotransmission directly. In certain embodiments, the compositions of the invention lead to a réduction of GABA in the hippocampus, amygdala and/or locus coeruleus. In certain embodiments, the compositions of the invention lead to an increase of GABA in cortical régions.

Immune response

The signalling of the microbiota-gut-brain axis is modulated by alterations in the immune response and inflammaîory factors and markers. Accordingly, in certain embodiments, the compositions ofthe invention may modulate the immune response. In certain embodiments, the compositions of the invention modulate the systemic levels of circuiating neuroimmune signalling molécules. In certain preferred embodiments, the compositions of the invention modulate piO-inflammatory cytokine production and inflammation. In certain embodiments, the compositions of the invention modulate the inilanimatory State. In certain embodiments, the compositions of the invention decrease IL-6 production and sécrétion. In certain embodiments, the compositions of the invention decrease the activation of the NFkB promoter. In certain embodiments, the compositions of the invention are able to modulate the activation of IL-6 production by the potent pro-inflammatory endotoxin lipopolysaccharide (LPS). In certain embodiments, the compositions of the invention are able to modulate the activation of the NFkB promoter by LPS and ct-synuclein mutant proteins such as A53T. Increased circuiating levels of cytokines are closely associated with various neurodegenerative disorders, including Parkinson’s, dementia and Alzheimer’s. In certain embodiments, 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 invention increase the sécrétion of IL-8. IL-8 has been shown to induce myelin sheath formation and restore or preserve effective neuronal communication. Thus, in some embodiments, the compositions of the invention are for use in inducing 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 microbiota-gut-brain axis is modulated by levels of commensal métabolites. Accordingly, in certain embodiments, the compositions of the invention modulate the systemic levels of microbiota métabolites. In certain preferred embodiments, the compositions of the invention modulate the level of short chain fatty acids (SCFAs). In certain embodiments the level of SCFAs is increased or decreased. 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 embodiments, the compositions of the invention modulate the ability of SCFAs to cross the blood-brain barrier.

Histone acétylation and deacetylation are important epigenetic regulators of gene expression. An imbalance in histone acétylation and deacetylation can resuit in apoptosis. Dysrégulation of such histone acetyltransferases has been implicated in the pathogenesis associated with age-assocîated neurodegenerative diseases, such as Parkinson’s disease, Huntington's disease, Alzheimer's disease, amyotrophie latéral sclerosis and cognitive décliné [34]. Accordingly, in certain embodiments, the compositions of the invention can modulate histone deacetylase activity. In certain embodiments, the compositions of the invention can reduce histone deacetylase activity. In certain embodiments, the compositions of the invention can reduce histone acetylase activity.

Patients with neurodegenerative diseases, including Parkinson’s disease, Huntington's disease, Alzheimer's disease and amyotrophie latéral sclerosis, exhibit high levels of lipid peroxidation. Lipid are vulnérable to oxidation by reactive oxygen species, and the brain is rich in polyunsaturated 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 eariy stage neurodegeneration. Also accordingly, in certain embodiments, the compositions of the invention are for use in preventing 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. Accordingly, 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 modulate the barrier function and integrity of the gastrointestinal tract. In certain embodiments, the compositions of the invention modulate gastrointestinal tract motility. In certain embodiments, the compositions of the invention modulate the translocation of commensal métabolites and inflammatory signalling molécules into the bloodstream 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 modulâtes the microbiome composition of the gastrointestinal tract. In certain embodiments, the compositions of the invention prevents microbiome dysbiosis and associated increases in toxic metabohtes (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 Clostridium in the gastrointestinal tract. In certain embodiments, the compositions of the invention reduce the levels of Campylobacter jejuni. In certain embodiments, the compositions of the invention modulate the prolifération of harmful anaérobie bacteria and the production of neurotoxins 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, Ruminococcus généra and/or the Alcaligenaceae family. In certain embodiments, the compositions of the invention increase the level of Lactobacillus plantarum and/or Saccharomyces boulardii.

Brain injurv

The examples demonstrate that the compositions of the invention are neuroprotective and hâve HDAC inhibitory activity. HDAC2 is a crucial target for functional recovery from stroke [35] and HDAC inhibition can prevent white matter înjury [36], 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 injury. In some embodiments, the brain injury is an acquired brain injury. In some embodiments, the compositions of the invention are for use in treating brain injury resulting from trauma. In some embodiments, the compositions of the invention are for use in treating brain injury resulting from a tumour. 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 cérébral hypoxia. In some embodiments, the compositions of the invention are for use in treating brain injury resulting from cérébral 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 adéquate 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 dépendent on the région of the brain which is affected by the inadéquate blood flow. Symptoms 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 to as a brain attack or a cerebrovascular accident (CVA). The symptoms of stroke may be brief if adéquate blood flow is restored within a short period of time. However, if inadéquate blood flow continues for a significant period of time, the symptoms can be permanent.

In some embodiments, the stroke îs cérébral ischemia. Cérébral ischemia results when there is insuffic ient blood flow to the tissues of the brain to meet metabolic demand. In some embodiments, the cérébral ischemia is focal cérébral ischemia, i.e. confined to a spécifie région of the brain. In some embodiments the cérébral ischemia is global cérébral ischemia, Le, encompassing a wide area of the brain tissue. Focal cérébral ischemia commonly occurs when a cérébral vessel has become blocked, either partially or completely, reducing the flow of blood to a spécifie région of the brain. In some embodiments the focal cérébral ischemia is ischémie stroke. In some embodiments, the ischémie stroke is thrombotic, i.e. caused by a thrombus or blood dot, which develops in a cérébral vessel and restricts or blocks blood flow. In some embodiments the ischémie stroke is a thrombotic stroke. In some embodiments, the ischémie stroke is embolie, 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 embodiments the ischémie stroke is an embolie stroke. Global cérébral ischemia commonly occurs when blood flow to the brain as a whole is blocked or reduced. In some embodiments the global cérébral ischemia is caused by hypoperfusion, i.e. due to shock. In some embodiments the global cérébral ischemia is a resuit of a cardîac arrest.

In some embodiments the subject diagnosed with brain injury has suffered cérébral ischemia. In some embodiments, the subject diagnosed with brain injury has suffered focal cérébral ischemia. In some embodiments, the subject diagnosed with brain injury has suffered an ischémie stroke. In some embodiments, the subject diagnosed with brain injury has suffered a thrombotic stroke. In some embodiments, the subject diagnosed with brain injury has suffered an embolie stroke. In some embodiments, the subject diagnosed with brain înjury has suffered global cérébral ischemia. In some embodiments, the subject diagnosed with brain injury has suffered hypoperfusion. In some embodiments, the subject diagnosed with brain injury has suffered a cardiac amest.

In some embodiments, the compositions ofthe invention are for use în treating cérébral ischemia. In some embodiments, the compositions of the invention are for use in treating focal cérébral ischemia. In some embodiments, the compositions of the invention are for use treating ischémie stroke. In some embodiments, the compositions of the invention are for use în treating thrombotic stroke. In some embodiments, the compositions of the invention are for use in treating embolie stroke. In some embodiments, the compositions of the invention are for use in treating global cérébral ischemia. In some embodiments, the compositions of the invention are for use in treating hypoperfusion.

In some embodiments, the stroke îs hémorrhagie stroke. Hémorrhagie stroke is caused by bleeding înto or around the brain resulting in swelling, pressure and damage to the cells and tissues of the brain. Hémorrhagie stroke is commonly a resuit of a weakened blood vessel that ruptures and bleeds into the surrounding brain. In some embodiments, the hémorrhagie stroke is an intracérébral hemorrhage, i.e. caused by bleeding within the brain tissue itself. In some embodiments the intracérébral hemorrhage is caused by an intraparenchymal hemorrhage. In some embodiments the intracérébral hemorrhage îs caused by an intraventricular hemorrhage. In some embodiments the hémorrhagie stroke is a subarachnoid hemonhage i.e. bleeding that occurs outside of the brain tissue but still within the skull. In some embodiments, the hémorrhagie stroke is a resuit of cérébral amyloid angiopathy. In some embodiments, the hémorrhagie stroke is a resuit of a brain aneurysm. In some embodiments, the hémorrhagie stroke is a resuit of cérébral arteriovenous malformation (AVM).

In some embodiments the subject diagnosed with brain injury has suffered hémorrhagie stroke. In some embodiments, the subject diagnosed with brain injury has suffered an intracérébral hemorrhage. In some embodiments, the subject diagnosed 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 cérébral 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 cérébral AVM,

In some embodiments, the compositions of the invention are for use in treating hémorrhagie stroke. In some embodiments, the compositions of the invention are for use in treating an intracérébral hemorrhage. In some embodiments, the compositions of the invention are for use in treating an intraparenchymal hemorrhage. In some embodiments, the compositions ofthe invention are for use in treating an intraventricular hemorrhage. In some embodiments, the compositions of the invention are for use in treating a subarachnoid hemorrhage. In some embodiments, the compositions of the invention are for use in treating a cérébral amyloid angiopathy. In some embodiments, the compositions of the invention are for use in treating a brain aneurysm. In some embodiments, the compositions of the invention are for use in treating cérébral AVM.

Restoration of adéquate blood flow to the brain after a period of interruption, though effective in allevîating the symptoms associated with stroke, can paradoxically resuit in further damage to the brain tissue. During the period of interruption, the affected tissue suffers from a lack of oxygen and nutrients, and the sudden restoration of blood flow can resuit in inflammation and oxidative damage thiough the induction oi 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 retums 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 resuit of stroke. In some embodiments, the compositions of the invention are for use in treating reperfusion injury as a resuit of stroke.

A transient ischémie attack (TIA), often refened to as a mini-stroke, is a recognised waming sign for a more serious stroke. Subjects who hâve suffered one or more TIAs are therefore at greater risk of stroke. In some embodiments the subject diagnosed with brain injury has suffered a TIA. In some embodiments, the compositions of the invention are for use in treating a TIA. In some embodiments, the compositions ofthe invention are for use in treating brain injury in a subject who has suffered a TIA.

High blood pressure, high blood cholestérol, a familial history of stroke, heart disease, diabètes, brain aneurysms, arteriovenous malfonnations, sickle cell disease, vasculitis, bleeding disorders, use of nonsteroidal anti-inflammatory drugs (NSAIDs), smoking tobacco, drinking large amounts of alcohol, illégal drug use, obesity, lack of physical activity and an unhealthy diet are ail considered to be risk factors for stroke. In particular, lowering blood pressure has been conciusively shown to prevent both ischémie and hémorrhagie strokes [37, 38], 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 stroke. 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 cholestérol. In some embodiments the subject has a familial histoiy of stroke. In some embodiments the subject has heart disease. In some embodiments the subject has diabètes. 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 dîsease. In some embodiments the subject has a bleeding disorder. In some embodiments 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 alcohol. In some embodiments the subject uses illégal drugs. In some embodiments the subject is obese. In some embodiments the subject îs overweight. In some embodiments the subject has a lack of physical activity. In some embodiments the subject has an unhealthy diet.

The examples 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 treating brain injury when administered to subjects at risk of brain injuiy, such as stroke.

In certain embodiments, the compositions of the invention are for use in reducing the damage caused by a potentîal 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 identified as at risk of a brain injury,

The examples indicate that the compositions of the invention may be useful for treating brain injury and aiding recovery when administered after the injury event occurs, 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 injuiy by reducing motoric damage. In some embodiments, the compositions of the invention treat brain injury by împroving motor function. In some embodiments, the compositions of the invention treat brain injury by împroving muscle strength. In some embodiments, the compositions of the invention treat brain injury by împroving memory. In some embodiments, the compositions of the invention treat brain injury by împroving social récognition. In some embodiments, the compositions of the invention treat brain injuiy by împroving neurological function.

Treatment of brain injuiy may refer to, for example, an alleviation 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 may be provided to the subject in advance of the onset ofstroke, for example in a patient identified as being at risk ofstroke. Compositions ofthe invention for use in treating stroke may be provided after a stroke has occurred, for example, during recovery. Compositions of the invention for use in treating stroke may be provided during the acute phase of recoveiy (i.e. up to one week after stroke). Compositions ofthe 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 tn 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 widi a secondary active agent. In certain embodiments, the compositions 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, apîxaban, dabigatran, edoxaban or rivaroxaban), antihypertensives (such as diuretics, ACE inhibitors, calcium channel blockeis, beta-blockers or alpha-blockers) or statins. The compositions of the invention may improve the patient’s response to the secondary active agent.

In certain embodiments, 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 cérébral tissues. In certain embodiments, the compositions of the invention reduce necrosis or the number of 5 necrotic cells. In certain embodiments, the compositions of the invention reduce apoptosis or the number of apoptotic cells. In certain embodiments, 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 by necrosis and/or apoptosis caused by ischemia. In certain I0 embodiments, the compositions of the invention improve the recovery of the tissue damaged by ischemia. In certain 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 efficacy of the clearance of necrotic cells and/or apoptotic cells. In certain embodiments, the compositions of the invention improve the replacement and/or régénération of cells within 15 tissues. In certain embodiments, the compositions of the invention improve the replacement and/or régénération 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 adminîstered to the gastrointestinal tract in 20 order to enable delivery to and / or partial or total colonisation of the intestine with the bacterial strain of the invention. Generally, the compositions of the invention are adminîstered orally, but they may be adminîstered rectally, intranasally, or via buccal or sublingual routes.

in certain embodiments, the compositions of the invention may be adminîstered as a foam, as a spray or a gel.

In certain embodiments, the compositions of the invention may be adminîstered 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 invention is adminîstered to the gastrointestinal tract via a tube, such as a nasogastric tube, orogastric tube, gastric tube, jejunostomy tube (J tube), 30 percutaneous endoscopie gastrostomy (PEG), or a port, such as a chest wall port that provides access to the stomach, jéjunum and other suitable access ports.

The compositions of the invention may be administered once, or they may be administered sequentially as part of a treatment regimen. In certain embodiments, the compositions of the invention are to be administered daîly.

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

In certain embodiments, the composition of the invention may be administered to a prégnant 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 bom.

The compositions of the invention may be administered to a patient that has been diagnosed with a neurodegenerative disease, or that has been identified 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 identified as having an abnormal gut microbiota. For example, the patient may hâve reduced or absent colonisation by Megasphaera, and in particular Megasphaera massiliensis.

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

Generally, the compositions of the invention are for the treatment of humans, although they may be used to treat animais including monogastric mammals such as poultry, pigs, cats, dogs, horses or rabbits. The compositions of the invention may be useful for enhancing the growth and performance of animais. If administered to animais, oral gavage may be used.

Compositions

Generally, the composition of the invention comprises bacteria. In preferred embodiments of the invention, the composition is formulated in freeze-dried form. For example, the composition of the invention may comprise granules or gelatin capsules, for example hard gelatin capsules, comprising a bacterial strain of the invention.

Preferably, the composition of the invention comprises lyophilised bacteria. Lyophilisation of bacteria is a well-established procedure and relevant guidance is available in, for example, references [39], [], [41]],

Altematively, 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 heat-inactivated. In some embodiments, the bacterial 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 invention 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 example, in some embodiments, the bacterial strain în the composition of the invention is viable. For example, în some embodiments, the bacterial strain in the composition of the invention is capable of partially or totally colonising the intestine. For example, in some embodiments, the bacterial strain in the composition ofthe 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 hâve been killed.

In preferred embodiments, the composition of the invention is encapsulated to enable delivery of the bacterial strain to the intestine. Encapsulation protects the composition from dégradation until delivery at the target location through, for example, rupturing 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, attachment or adsorption on solid carrier surfaces, selfaggregation by flocculation or with cross-linking agents, and mechanical containment 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, references [42] and [43],

The composition may be administered orally and may be in the form of a tablet, capsule or powder. Encapsulated products are preferred bec au se Megasphaera are anaerobes. Other ingrédients (such as vitamin C, for example), may be included as oxygen scavengers and prebiotîc substrates to improve the delivery and / or partial or total colonisation and survival in vivo. Altematively, the probîotic composition of the invention may be administered orally as a food or nutritional product, such as milk or whey based femiented dairy product, or as a pharmaceutical product.

The composition may be formulated as a probiotic.

A composition of the invention includes a therapeutically effective amount of a bacterial strain ofthe invention. A therapeutically effective amount of a bacterial strain is sufficient to exert a bénéficiai effect upon a patient. A therapeutically effective amount of a bacterial strain may be sufficient to resuit in delivery to and / or partial or total colonisation of the patient’s intestine.

A suitabie daily dose of the bacteria, for example for an adult human, may be from about l x 10³ to about l x l O¹¹ colony forming units (CFU); for example, from about 1 x 10⁷ to about 1 x 10¹⁰ CFU; in another example 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 I x 10⁶ to about 1 x 10¹¹ CFU/g, respect to the weight of the composition; for example, from about 1 x 10^s to about 1 x 10¹⁰ CFU/g. The dose may be, for example, 1 g, 3g, 5g, and 10g.

Typically, a probiotic, such as the composition of the invention, is optionally combined with at least one suitabie prebiotic compound. A prebiotic compound is usually a non-digestible carbohydrate 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 transgalactooligosaccharides.

In certain embodiments, the probiotic composition of the présent invention includes a prebiotic compound in an amount of from about 1 to about 30% by weight, respect to the total weight composition, (e.g. from 5 to 20% by weight). Carbohydrates may be selected from the group consisting of: fructo- oligosaccharides (or FOS), short-chain fructo-oligosaccharides, inulin, isomaltoligosaccharides, pectins, xylo-oligosaccharides (or XOS), chitosan-oligosaccharides (or COS), betaglucans, arable gum modified and résistant 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 digestible carbohydrates, generally obtained by the conversion ofthe beet sugar and includîng a saccharose molécule to which three glucose molécules are bonded.

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 nutritîonal suppléments that modulate neuroprotection or neuroproliferation. In preferred embodiments, the nutritîonal suppléments comprise or consist of nutritîonal vitamins. in certain embodiments, the vitamins are vitamin B6, magnésium, dimethyiglycine (vitamin B16) and vitamin C. In certain embodiments, the compositions of the invention are administered in combination with another probiotic.

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

The compositions ofthe invention may comprise pharmaceutically acceptable excipients or carriers.

Examples of such suitable excipients may be found in the reference [44]. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art and are described, for example, in reference [45]. Examples of suitable carriers include lactose, starch, glucose, methyl cellulose, magnésium stéarate, mannitol, sorbitol and the like. Examples of suitable diluents include éthanol, glycerol and water. The choice of pharmaceutical carrier, excipient or diluent can be selected with 10 regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as, or in addition to, the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s). Examples of suitable binders include starch, gelatîn, natural sugars such as glucose, anhydrous lactose, freeflow lactose, b eta-lactose, corn sweeteners, natural and synthetic gums, such as acacia, tragacanth or 15 sodium alginate, carboxymethyl cellulose and polyethylene glycol. Examples of suitable lubricants include sodium oleate, sodium stéarate, magnésium stéarate, sodium benzoate, 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 benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may be also 20 used.

The compositions ofthe invention may be formulated as a food product. For example, a food product may provide nutritîonal benefit in addition to the therapeutic effect of the invention, such as in a nutritional supplément. 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 consume by being more 25 similar to a common food item, rather than to a pharmaceutical composition. In certain embodiments, 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 milk-based product can be, e.g., cow's milk, goat’s milk, sheep's milk, skimmed milk, whole milk, milk recombined from powdered milk and whey without any processing, or a processed 30 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-contaîning food such as sweets.

In some embodiments, the compositions of the invention comprise one or more bacterial strains of the genus Megasphaera and do not contain bacteria from any other généra, or which comprise only de minimis or biologically irrelevant amounts of bacteria from another généra. Thus, in some embodiments, the invention provides a composition comprising one or more bacterial strains of the genus Megasphaera, which does not contain bacteria from any other généra or which comprises only de minimis or biologically irrelevant amounts of bacteria from another généra, 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 comprise only de minimis 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 minimis or biologically irrelevant 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 Megasphaera 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 composition comprising one or more bacterial strains of the species Megasphaera massiliensis, 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 contain any other bacterial strains or species. Such compositions may comprise only de minimis or biologically irrelevant amounts of other bacterial strains or species. Such compositions may be a culture that is substantially free from other species of organism.

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 another strain for use in therapy.

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

In some embodiments, the compositions of the invention comprise more than one bacterial 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 compositions of the invention comprise 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 species and, optionally, do not contain bacteria from any other species. The invention comprises any combination of the foregoing.

In some embodiments, the composition comprises a microbial consortium. For example, in some embodiments, the composition comprises the Megasphaera bacterial strain as part of a microbial consortium. For example, in some embodiments, the Megasphaera bacterial strain is present în combination with one or more (e.g. at least 2, 3, 4, 5, 10, 15 or 20) other bacterial strains from other généra with which it can live symbiotically in vivo in the intestine. For example, in some embodiments, the composition comprises a bacterial strain of Megasphaera in combination with a bacterial strain from a different genus. In some embodiments, the microbial consortium comprises two or more bacterial strains obtaîned 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 comprises bacterial strains obtaîned from faeces samples of at least two different organisms. In some embodiments, the two different organisme are from the same species, e.g. two different humans. In some embodiments, the two different organisms are an infant human and an adult human. In some embodiments, the two different organisms are a human and a non-human mammal.

In some embodiments, the composition of the invention additionally comprises a bacterial strain that has the same safety and therapeutic 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 généra may be for separate, simultaneous or sequential administration. For example, the composition may comprise ail of the more than one bacterial strain, species or généra, or the bacterial strains, species or généra may be stored separate ly and be adminîstered separately, simultaneously or sequential ly. In some embodiments, the more than one bacterial strains, species or généra are stored separately but are mixed together prior to use.

In some embodiments, the bacterial strain for use in the invention is obtained from human adult faeces. In some embodiments in which the composition of the invention comprises more than one bacterial strain, ail of the bacterial strains are obtained from human adult faeces or if other bacterial strains are present they are present only in de mini mis amounts. The bacteria may hâve been cultured subséquent to being obtained 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 bacterial strains is/are the only therapeutically active agent(s) in a composition of the invention. In some embodiments, the bacterial strain(s) în the composition is/are the only therapeutically active agent(s) in a composition of the invention.

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

In certain embodiments, the invention provides the above phannaceuticai composition, wherein said bacterial strain is lyophilised. In certain embodiments, the invention provides the above phannaceuticai composition, wherein said bacterial strain is spray dried. In certain embodiments, the invention provides the above phamiaceutical 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 certain embodiments, the invention provides the above pharmaceutical composition, wherein the bacterial strain is lyophilised or spray dried and wherein it is capable of partially or totally colonising the intestine. In certain embodiments, the invention provides the above pharmaceutical composition, wherein the bacterial strain is lyophilised or spray dried and wherein it is viable and capable of partially or totally colonising 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 pharmaceutically acceptable 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 acceptable excipient, carrier or diluent; wherein the bacterial strain is in an amount sufficient to treat or prevent a neurodegenerative disorder.

In certain embodiments, the invention provides the above phannaceutical composition, wherein the amount of the bacterial strain is from about I x 10³ to about l x I0 colony fonning units per gram with respect to a weight ofthe composition.

In certain embodiments, the invention provides the above phannaceutical composition, wherein the composition is administered at a dose of l g, 3 g, 5 g or 10 g.

In certain embodiments, the invention provides 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 carrier selected from the group consisting of lactose, starch, glucose, methyl cellulose, magnésium stéarate, mannîtol and sorbitol.

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

In certain embodiments, the invention provides the above pharmaceutical composition, comprising an excipient selected from the group consisting of starch, gelatin, glucose, anhydrous lactose, freeflow lactose, beta-lactose, corn sweetener, acacia, tragacanth, sodium alginate, carboxymethyl cellulose, polyethylene glycol, sodium oleate, sodium stéarate, magnésium stéarate, sodium benzoate, sodium acetate and sodium chloride.

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

In certain 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 phannaceutical composition, wherein said bacterial strain is lyophilised.

In certain embodiments, the invention provides the above pharmaceutical composition, wherein when the composition is stored in a sealed container at about 4°C or about 25°C and the container is placed in an atmosphère having 50% relative humidity, at least 80% of the bacterial strain as measured in colony form ing units, remains after a period of at least about: l month, 3 months, 6 months, l year, l .5 years, 2 years, 2.5 years or 3 years.

In some embodiments, the composition ofthe invention is provided in a sealed container comprising a composition as described herein. In some embodiments, the sealed container is a sachet or bottie.

In some embodiments, the composition of the invention is provided in a syringe comprising a composition as described herein.

The composition of the présent invention may, in some embodiments, be provided as a pharmaceutical formulation. For example, the composition may be provided as a tablet or capsule. In some embodiments, the capsule is a gélatine capsule (“gel-cap”).

In some embodiments, the compositions of the invention are adminîstered orally. Oral administration may involve 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 formulations suitable for oral administration include solid plugs, solid 15 microparticulates, semi-solid and liquid (including multiple phases or dispersed Systems) such as tablets; soft or hard capsules containing multi- or nano-particulates, liquids (e.g. aqueous solutions), émulsions or powders; lozenges (including liquid-filled); chews; gels; fast dispersing dosage forms; films; ovules; sprays; and buccal/mucoadhesive patches.

In some embodiments the pharmaceutical formulation is an enteric formulation, i.e. a gastro-résistant 20 formulation (for example, résistant to gastric pH) that îs 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. prône to dégradation under gastric conditions.

In some embodiments, the enteric formulation comprises an enteric coating. In some embodiments, 25 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 comprise a film coating, for example, a thin film layer of an enteric polymer, e.g. an acid-insoluble polymer.

In some embodiments, the enteric formulation is întrinsically enteric, for example, gastro-résistant without the need for an enteric coating. Thus, in some embodiments, the formulation îs an enteric formulation that does not comprise an enteric coating. In some embodiments, the formulation is a capsule made from a thermogelLing materiai. In some embodiments, the thermogelling materiai is a cellulosic materiai, such as methylcellulose, hydroxymethylcelhilose 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 5 the shell comprises hydroxypropylmethylcellulose and does not comprise any film forming polymer (e.g. see [46 ]). In some embodiments, 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 10 glycols, present in the capsule shell, hâve a certain elasticity and softness. Soft capsules can be produced, for example, on the basis of gélatine or starch. Gelatine-based soft capsules are commercially available from various suppliers. Depending on the method of administration, such as, for example, orally or rectally, soft capsules can hâve various shapes, they can be, for example, round, oval, oblong or torpedo-shaped. Soft capsules can be produced by conventional processes, 15 such as, for example, by the Scherer process, the Accogel process or the droplet or blowing process.

Culturing methods

The bacterial strains for use in the present invention can be cultured using standard microbiology techniques as detailed in, for example, references [47], [] and [49].

The solid or liquid medium used for culture may be YCFA agar or YCFA medium, YCFA medium 20 may include (per lOOml, approximate values): Casitone (l.O g), yeast extract (0.25 g), NaHCO₃ (0.4 g), cysteine (O.l g), K₂HPO₄ (0.045 g), KH₂PO₄ (0.045 g), NaCi (0.09 g), (NH₄)₂SO₄ (0.09 g), MgSO₄ 7HiO (0.009 g), CaCl₂ (0.009 g), resazurin (O.l mg), hemin (I mg), biotin (l gg), cobalamin ( I gg), p-aminobenzoic acid (3 gg), folie acid (5 gg), and pyridoxamine ( 15 gg).

Bacterial strains for use in vaccine compositions

The inventors hâve identified that the bacterial strains of the invention are useful for treating or preventing neurodegenerative disorders. This is likely to be a resuit of the effect that the bacterial strains of the invention hâve on the host immune system. Therefore, the compositions of the invention may also be useful for preventing neurodegenerative disorders, when administered as vaccine compositions. In certain such embodiments, the bacterial strains of the invention may be 30 killed, inactivated or attenuated. In certain such embodiments, the compositions may comprise a vaccine adjuvant. In certain embodiments, the compositions are for administration via injection, such as via subeutaneous 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 fuliy in the literature. See, e.g., references [50] and [51,57], 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 additional e.g. X + Y.

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

The word “substantially” does not exclude “completely” e.g. a composition which is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the définition of the invention.

References to a percentage sequence identity between two nucléotide sequences means that, when aligned, that percentage of nucléotides are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in section 7.7.18 of ref [58]. A preferred alignment is determined by the Smith-Waterman homology 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 SmithWaterman homology search algorithm is disclosed in ref. [59],

Unless specifically stated, a process or method comprising numéro us steps may comprise additional steps at the beginning or end of the method, or may comprise additional intervening steps. Also, steps may be combined, omitted or performed in an alternative order, if appropriate.

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

MODES FOR CARRYING OUT THE INVENTION

Example 1 - Efficacy of bacterial inocula to act as a neuroprotectant

Sutnmarv

Neuroblastoma cells were treated with compositions comprising bacterial strains according to the invention. The SH-SY5Y neuroblastoma cells used are dopamine producing and well-established as an in vitro model for studying neurodegenerative diseases. The ability of the bacterial strains to increase neuroproliferation was observed. The neuroblastoma cells were also treated with dopaminergic neurotoxin l -methyl-4-phenylpyridinium (MPP), which induces permanent symptoms of Parkinson’s disease in neuroblastoma cells. The ability of the bacterial strains to act as a neuroprotectant against MPP was investigated.

Material and Methods

Bacterial strain

Megasphaera massiliensis MRx0029; Parabacteroides distasonis MRX0005

Cell line

SH-SY5Y neuroblastoma cells were purchased from ECCACC (Cat. no: 94030304) and were grown în MEM (Sigma Aldrich, cat η. M2279) supplemented with Nutrîent Mixture F-12 Ham (Sigma Aldrich, cat η. 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 différentiation medium (which contains FBS at 1%) and 10 uM retinoic acid (Sigma Aldrich, cat. n. R2625-100MG). Différentiation medium was replaced every other day and cells were harvested al 7 day of différentiation. Cells were pre-treated with or without MPP (Sigma Aldrich, cat. n. D048-1G) for 8 hours. Subsequently, cells were treated with 10% bacterial supernatant and incubated ovemight. Cell viability was measured by using CCK8 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 increase in the prolifération of neurons. Neuroblastoma cells that were treated with MPP together with the bacterial strain had increased cell viability compared to the cells treated with MPP alone (which had decreased viability).These data show that the bacterial strain can act as a neuroprotectant. The protective effect was greater for MRX0029-treated cells, which rescued viability more than the positive control cells treated with Quercetin. These data show that the bacterial strains can act as a neuroprotectant

Example 2 - Efficacy af bacterial inocula to redit ce IL-6 sécrétion.

Summarv

Activation of proinflammatory cytokines has been associated with neuron damage in neurodegenerative disease. Lipopolysaccharide (LPS) is a known stîmuiator of the proinflammatory cytokine IL-6. Human glioblastoma astrocytoma cells were treated with compositions comprising 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 strain

Megasphaera massiliensis MRx0029

Cell Une

MG U373 is a human glioblastoma astrocytoma derived from a ma lignant tumour and were purchased from Sigma-Aldrich (cat n. 08061901-1VL). MG U373 human glioblastoma astrocytoma cells were grown in MEM (Sigma Aldrîch, cat η. M-2279) supplemented with 10% FBS, 1% Pen Strep, 4mM L-GIut, IX MEM Non essential 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 ίθ% of bacteria supernatant from MRx0029 for 24h. A control was also performed where the cells were incubated în untreated media. Afterwards the cell free supematants 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.

Results

The results of these experiments are shown in Figure 2. Treatment of neuroblastoma cells with LPS and the bacteria strain led to a decrease in the level of IL-6 secreted.

Example 2b - Efficacy of bacterial inocula to modulate IL-8 sécrétion.

S uni mary

As neuro-inflammation plays a pivotai rôle in neurodegenerative diseases and IL-8 has been shown to hâve neuro-positive effects, the effect ofcompositions comprising bacterial strains ofthe 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 invention in combination with LPS to observe their ability to modulate the levels of IL-8.

Material and Methods

Bacterial strains

Megasphaera massiliensis MRX0029; Parabacteroides distasonis MRX0005

Cell line

MG U373 is a human glioblastoma astrocytoma derived from a malignant tumeur and were purchased from Sigma-Aldrîch (cat n. 0806190l-l VL). MG U373 human glioblastoma astrocytoma cells were grown in MEM (Sigma Aldrich, cat η. M-2279) supplemented with I0% FBS, l% Pen Strep, 4mM L-Glut, IX MEM Non essential Amino Acid solution and IX Sodium Piruvate.

Method

Once grown the MG U373 cells were piated on 24-weli plate at 100,000 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 supematants were collected, centrifuged at 10,000g for 3min at 4°C. IL-8 was measured using Human IL-8 ELISA Kit from Peprotech (cat n.#900-K18) according to manufacturer instruction.

Results

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

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

Summary

Neuroinflammation plays a pivotai rôle in Parkinson’s disease and α-synuclein has been shown to induce neuroinflammation in vivo. Therefore, the ability of the bacteria strains of the invention to inhibit α-synuclein-induced neuroinflammation was assessed. A co-culture of human glioblastoma astrocytoma cells and neuroblastoma cells were exposed to wild-type α-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 α-synuclein-induced sécrétion of IL-6 was then tested.

Material and Methods

Bacterial strains

Megasphaera massiliensis MRX0029; Parabacteroides distasonis MRX0005

Cell line

MG U373 is a human gliobiastoma astrocytoma derived from a malignant tumour and were purchased from Sigma-Aldrich (cat n. 0806190I-1VL). MG U373 human gliobiastoma astrocytoma cells were grown in MEM (Sigma Aldrich, cat η, M-2279) supplemented with 10% FBS, l% Pen Strep, 4mM L-Glut, IX MEM Non-essentîal Amino Acid solution and IX Sodium Piruvate.

SH-SY5Y is a human neurobastoma cell line derived from a malignant neuroblastoma and can be purchased from Sigma-Aldrich (cat n. 94030304-1 VL). 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 în the incubator. After 2 days, media were replaced with différentiation medium (growth medium containing 1% FBS) and 10 μΜ retinoic acid. Différentiation medium was replaced eveiy other day and cells were used after 7 days of différentiation.

Method

SHSY5Y cells were plated on 12 well plates at a density of 50,000 cells/well. 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 différentiation medium (growth medium containing 1% FBS) and 10 μΜ retinoic acid. Différentiation medium was replaced every other day and cells were used after 7 days of différentiation. U373 were plated on 12 transwell plates (0.4pm polyester membrane, Costar) at a density of 50,000cells/well for 72 hrs. Cells were co-cultured together for 24hrs before treatment in différentiation medium (growth medium containing 1% FBS without retinoic acid).

Thereafter cells were treated with 25pg/ml α-synuclein (Wt, A53T, E46K) in the presence or absence of 10% bacteria supernatant for 48 hrs, Cell free Supernatants were collected, spun-dwon at 10000g for 3 min at 4C, aiiquoted and stored at -80 0C. Human IL-6 and IL-8 were measured as described above.

Results

The results of these experiments are shown in Figure 4. Treatment of cells with wild-type asynuclein and the mutant isoforms E46K and A53T induced moderate sécrétion of IL-6 (Figure 4A). The α-syn-induced sécrétion of IL-6 was inliibited in cells treated with the bacteria strains (Figure 4A). The réduction in IL-6 sécrétion was greatest on administration of MRX0029.

Example 3 - Efficacy of bacterial inocula to reduce NFkB activation

Summary

Activation of the NFkB promoter leads to the production of proinflammatory cytokines including ILΙβ, IL-la, IL-18, TNFa and IL-6. The NFkB promoter can be activated by α-synuclein and LPS by stimulating the TLR4 ligand. Mutations in α-synuclein, such as α-synuclein A53T, are implicated in familial Parkinson’s. Treatment of neuronal cells with LPS simulâtes Parkinson’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 investîgated.

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-6I71) supplemented with 10% FBS, 1% Pen Strep, 4mM L-Glut, Normocin and IX HEK Blue sélection solution.

Method

Once grown the Human Hek blue cells were plated in 96 well plates at 25,000 cells/well in 4 replicates. One set of cells were treated with α-synuclein A53T (lug/mL) alone or with 10% of bacteria supematant from MRx0029 for 22h. The second set of cells were treated with LPS (10 ng/mL, from Salmonella enterica serotype Typhimurium, Sigma Aldrich, cat n. L6143) alone or with 10% of bacteria supematant from MR029 for 22h. The cells were subsequently spun down and 20ul of the supematant 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 ct-synuclein is inhibited by MRx0029. Figure 6 shows that the activation of the NFkB promoter by LPS is inhibited by MRx0029.

Example 4 — Efficacy of bacterial inocula to alter antioxidant capacity

Summary

The ability of compositions comprising bacterial strains according to the invention to alter the antioxidant capacity. The antioxidant capacity of the bacterial strain was established using the wellknown ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic 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 peliet volume). The suspension was sonicated on ice for 5 minutes and then spun down at 12,000 x g for 10 minutes. The supematant was removed and measured using the ABTS assay kit produced by Sigma Aidrich (code CS0790), in accordance with manufacturer’s instructions.

Results

The results of these experiments are shown in Figure 7. Figure 7 shows that MRx0029 has an antioxidant capacity of approximately 2mM compared to Trolox.

Example 5 - Efficacy of bacterial inocula to alter lipid peroxidation levels

Summary

The ability 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.

Material and Methods

Bacterial strain

Megasphaera massiliensis MRx0029

Method

Bacterial cells (10⁶ or greater) were collected and centrifuged, a wash step was performed with isotonie saline before the pellet was re-suspensed in potassium chloride assay buffer. The suspension was sonicated on ice for I0 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 8. Figure 8 shows that MRxO29 îs able to inhibit lipid peroxidation by approximately 20 %, which is a higher antioxidant capacity than the positive control, butylated hydroxytoluene (1% w/v).

Exemple 6 - Efficacy of bacterial inocula on histone deacetylase activity

Summary

The ability of compositions comprising bacterial strains according to the invention to alter histone deacetylase activity was investigated. Dysrégulation 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 supematants of stationary phase bacterial cultures were isolated by centrifùgation and Altering 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 ofthe experiment. The HT-29 cells were challenged with 10 % cell free supernatant diluted in DTS and was is left to incubate for 48 hours. Nuclease proteins were then extracted using the Sigma Aldrich Nuclease extraction kit and samples were snap frozen prior to HDAC activity measurement. HDAC activity was assessed fluorometrically using the Sigma Aldrich (UK) kit.

Results

The results of the experiments are shown in Figure 9. Figure 9 shows that MRxOO29 is able reduce the levels of histone deacetylase activity.

Example 7 - Level of indole production in bacteria

Summary

The ability of the bacteria of the invention to produce indole was investigated, Indole has been implicated in attenuating inflammation and oxidative stress.

Material and Methods

Bacterial strain

Megasphaera massiliensis MRxOO29

ATCC 11775 is a bacterial reference strain that is known to produce indole.

Method

Intact bacterial cells in stationary phase were incubated with 6mM Tryptophan for 48 hours. Bacterial species which possess the enzyme tryptophanase will utilise tryptophan as a substrate to produce indole. Following the 48 hour incubation 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 results of the experiments are shown in Figure I0. Figure 10 shows that MRxOO29 has the capacity to produce indole from tryptophan, at concentrations of approximately 0.2mM.

Example 8 — Level of kynurenîne production în bacteria

Summary

The ability of the bacteria of the invention to produce kynurenîne was investigated. Dysrégulation of the kynurenîne pathway can lead to activation of the immune system and the accumulation of potentially neurotoxic compounds. Alterations in the kynurenîne metabolism may be involved in the development of Parkinson’s diseases.

Bacterial strain

Megasphaera massiliensis MRx0029

DSM 17136 is a strain of Bacteroides copricola that is known to produce kynurenîne.

Method

Cell free supematants of stationary phase bacterial cultures were isolated b y centrifugation and filtering in a 0.22 uM filter and frozen until use. Kynurenîne standards, stock solutions and reagents were prepared using standardised methods validated in-house. Sample were treated with trichloroacetic acid and centrifuged at I0,000xg for 10 minutes at 4°C. The supernatant was collected and dispensed into a 96 well plate. Ehrlich’s reagent was used for kynurenîne détection and added at a ratio of l ; l.

Results

The results of the experiments are shown in Figure H. Figure 11 shows that MRx0029 has the capacity to produce kynurenîne at a concentration of approximately 40 μΜ.

Example 9 - Levels of Dopamine, DOPAC and H VA in striatum in bacteria-treated MPTP mice

Parkinsoris disease is a commun neurodegenerative disorder whose cardinal clinical features include tremor, slowness of movement, stiffness, and postural instability. These symptoms are primarily attributable to the degeneratîon of dopaminergic neurons in the substantia nigra pars compacta and the conséquent loss of their projecting nerve fibers in the striatum [60], Mice treated with MPTP (lmethyl-4-phenyl-l,2,3,6-tetrahydropyridine) selectîvely lose significant numbers of nigrostriatal dopaminergic neurons [61]. 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 evaluate the effects of MRX0029 anaérobie bacteria using MPTP lesioned mice.

male mice were allocated to 4 different treatment groups (groups A, B, E and I, with n=l2 animais in each group). The treatment groups are shown in Table l below and the project time course îs outlined below.

Table 1: Treatment groups

Group	n	Treatment	Lésion
Substance	Safety level	Dose	Route	Schedule	Substance	Dose	Route	Schedule
A	12	Vehicle (PBS)		*	P-0-	18 days: day(14}-day3	Vehicle (0.9% saline)		Lp.	dayO

B	12	Vehicle (PBS)	-	-	p.o.	18 days: day(14)-day3	MPTP	4x 20 mg/kg	i.p.	dayO
e	12	MRx0029 Mepasphaerâ sp inlv)	S1/S2	2 x 10Λ8 bacteria	po	18 days: day(14)-day3	MPTP	4x 20 mg/kg	i.p.	dayO
1	12	Vehicle (PBS)	-		ρ,ο.	18 days; day(-	MPTP	4x20 mg/kg	i.p.	dayO
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 lésion. Group I animais received a daily vehicle (PBS) p.o. (per oral) treatment and were înjected i.p. (intraperitoneal) with 5 the reference drug 30 min before and 90 min after first MPTP on day 0. The application volume for p.o. and vehicle treatment was 200 μΙ per mouse. Bacteria strain of group E was from glycerol stocks (gly). For oral treatment, gavages for applications were stored in vial containing 70% Ethanol and were flushed before and after each use with distilled water. Every treatment group had its own gavage and éthanol vial and distilled water vial. The tubes and gavages were not changed between 10 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 animais of groups B, E and I. One group of animais (A) was sham lesioned by i.p. administration of the MPTP vehicle (0.9% saline). The application volume was 10 μΙ per g body weight. Weighing of the animais was performed before the MPTP treatment to dose the animais according to their 15 actual body weight. Afterwards animais received the daily p.o. treatment.

Formulation of préparations for closing and préparation of glycerol stocks for dosing

Name of the Bacteria strain:	MRxÛ029 Megasphaera sp.
Storage condition/stability:	-80 eC
Vehicle:	1xPBS
Treatment dosages:	2 x 10Λ8 bacteria
Administration:	200μΙ
Lot Number:	n/a

For treatment group E (MRx0029)

.) 1 glycerol stock was taken from the -80 °C freezer and placed under anaérobie conditions (anaérobie jar with sachet) at 37 °C in order to thaw (this took 30-40 mins).

.) The completely thawed glycerol stock was centrifuged at 6000 x g for 10 min at room 5 température.

.) The supernatant was discarded without disturbing the peliet (e.g. using a pipette).

.) 4.22 mL of stérile pre-waimed (37 °C) 1 x PBS was added and gently mixed using a pipette.

.) The mice were dosed with 200 pL of the bacterial solution. The animais were dosed within 15 mins after resuspension of the peliet with PBS.

Reference drug group formulation

Name of the Reference item:	7-Nitroindazole
Storage condition/stability:	-20°C
Vehicle:	Peanut oil
Treatment dosages:	50 mg/kg
Administration:	i.p. (30 min before and 90 min after 1S,MPTP treatment)
Batch Number:	MKBS6671V

The appropriate amount of 7-Nitroindazole was dissolved in peanut oil to reach the final concentration of 50 mg/kg.

Materials and Methods

Animais

Mouse fine:	C57BL/6J (JAX™ Mice Strain) JAX™ Mice Stock Number ________________________000664___________
Provider:	Charles River Laboratories
Age at start:	~10 weeks
Sex:	Maie
Number of animais:	48

Spécifie handling of Animais and Randomisation

Gloves were changée! between each treatment group and sprayed with 70% éthanol solution between each cage of the same group to minimize the risk of contamination whenever animais were handled (e.g.: treatment, behavioural testing, cleaning and tissue sampling).

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

Tissue sampling and processing

On day 4 animais of ali groups were sacrificed and brains were collected. Therefore, mice were deeply anesthetized by Pentobarbital injection (600mg/kg).

Blood (approximately 500 μΐ) 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 braîn, weighed and immedîately frozen and stored at -80°C. Instruments and surfaces which were in contact with the animais had to be cleaned with 70% éthanol before the next animal was dissected.

Biochemical Analysis of Dopamine, DOPAC and HVA levels with HPLC in striatum

The striatal samples (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 μΜ EDTA-2Na and homogenized at 0 °C in a glasspestlemicro-homogenizer. Following standing for 30 min on ice, the homogenates were centrifuged at 10,000 RPM for 10 minutes in a refrigerated centrifuge Bîofuge Fresco (Heraeus Instruments, Germany). The supematants 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 centrîfugal filter (Merck Millipore, Germany) for 4 min at 14 000 g at 4 °C. The filtrâtes were stored at -80 °C before HPLC analysis.

HPLC analysis

Concentrations of DA, DOPAC and HVA in the striatal samples were determined by column liquid chromatography with electrochemical détection [62;63]. The HPLC system (HTECaOO, Eicom Corp., Kyoto, Japan) including a puise-free microflow pump, a degasser and an amperometric detector 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, Prague, Czech Republic). DA, DOPAC and HVA were separated on a 150 x 2.1 i.d. mm column (CA5-ODS, Eicom Corp., Kyoto, Japan). The mobile phase consisted of 0.1 M phosphate buffer at pH 6.0, 0.13 mM EDTA, 2.3 mM sodium-1 octanesulfonate and 20 % (v/v) methanol. The détection limit (signal-to-noise ratio = 3) for DA was estimated to 0.5 fmol in 15 μΙ (0.03 nM) injected onto the column.

Results

Administration of bacteria strains was well tolerated by the animais. On the MPTP lésion day and if necessary on the day afterwards a red light was used to wann the animais. If animais 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 animais per treatment group were used. Data were analyzed by using Kruskal-Wallis test followed by Dunn’s multiple comparison post hoc test or One-way analysis of variance followed by Bonferronî post hoc test (A vs. all(*), B vs. ail, I vs. ail (#)). */# = p<0.05; ** = p<0.0l; *** = p<0.00l.

The healthy animais in group A had high levels of Dopamine, DOPAC and HVA whereas MPTP treatment în group B reduced this and the positive control (group I) recovered the production to some degree (Figure 12). Animais of group I tended to hâve higher Dopamine levels than the bacteria treated group and group B. DOPAC (a Dopamine métabolite) levels in general were significantly lower in animais of group B compared to DOPAC levels of unlesioned animais of group A (Figure 12B).

Significantly, treatment with MRx0029 (group E) was found to recover production of Dopamine and DOPAC (Figures ! 2A and 12B, respectively). Treatment with MRx0029 may therefore be useful for treating or preventing neurodegenerative disorders.

Example 10 - 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 measuring transcriptional levels of microtubule associated protein MAP2, a spécifie neuronal différentiation marker.

Bacterial strain

Megasphaera massiliensis MRX0029,

Method

SHSY5Y were plated in lOcin pétri dishes a density of 2xl0⁶ cells. After 24h cells were treated in différentiation medium (growth medium containing 1% FBS without RA) with 10% bacteria supematants or YCFA+, lOuM RA, 200uM hexanoic acid or 200uM valproic acid, for 17 hrs, There after représentative 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 internai control. Fold change was calculated according to the 2^{( àict)} method.

Immunofluorescence and Confocal microscopy

Cells were seeded onto 8 well chamber slides (Marienfeld Laboratory Glassware) at 5xl0⁴ cells/well ovemight and were treated with 10% bacterial supernatant for 24 hrs. For différentiation, cells were treated with lOnM Retinoic acid for 5 days before treating with bacterial supernatant. Cells were then fixed with 4% paraformaldéhyde in PBS for 20 minutes at room température (RT). Fixed cells were washed with PBS, and permeabilized with l% 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 antî-MAP2 antibody (sc-7442l, Santa Cruz Biotechnology Inc) diluted in l% BSA/PBS for 12hr at 4°C. They were then washed twice with PBS, followed by incubation with Alexa Flour 488 conjugated anti-mouse (Molecular Probes Inc) and Alexa Flour 594 conjugated Phalloidin (abl76757, Abcam) for Ihr at RT, After washing 3X with PBS, the slides were mounted with VectorshieldD containing DAPI (Sigma, Aldrich). Slides were viewed using a Zeiss Axioscope microscope equipped with a 63x/l .2 W Korr objective and filter sets suitable for détection of the fluorochromes used. Manual exposure times for the digital acquisition of images immuno-labelled with MAP-2 were kept constant allowing comparison between different wells and treatments. Phalloidin (F-actin) and DAPI exposure times varied to suit the field of view. Randomised fields of view were acquired using a Qlmaging caméra controlled 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, DAPI and Phalloidion images overlaid and merged. Représentative images were selected to il lustrale the différences în abundance and location of the proteins examined

Results

The results are shown in Figure 13. Figure 13A shows représentative microscopy images of undifferentiated S HS Y-5 Y cells incubated with each of the acids and bacteria supematants. Treatment of cells with MRX0029 induced a neuron-like phenotype, showing similar features to cells treated with retinoic acid (which is used for terminal différentiation of neuroblastoma cells), where cell bodies are bigger and pyramidal-shaped, with neurites and processed branching out to network with neighbour cells. Figure 13B shows that MRx0029 significantly upregulates MAP2 in undifferentiated neuroblastoma cells, Phalloidin (an actin cytoskeleton-binding agent) staining further proved a different arrangement of cytoskeletal structure in cells treated with MRx0029, further supporting the neuronal différentiation hypothesis for MRx0029 (Fig. 13B).

Example 11 - Efficacy of bacterial inocula to reduce oxidative levels in cells

Background

The génération of réactivé 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 (TB HP) was investigated.

Material and Methods

Bacterial strain

Megasphaera massiliensis MRX0029

Method

SHSY-5Y cells were plated in black fiat bottom 96 well plate at density of 5000 cells/well and placed in the CO2 incubator. After 24 h, media were replaced with différentiation medium (growth medium containing 1% FBS) and 10 μΜ retinoic acid. Différentiation medium was replaced every other day. On Day 10 the différentiation medium was removed and cells were washed with prewarmed 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 întensity was measured using TEC AN plate reader at Ex/Em 485/530 nm.

Results

The results of the experiments are shown in Figure 14. Figure I4b shows that MRX0029 is able to inhibît ROS production in differentiated SHSY-5Y neuroblastoma cells. MRX0029 did not hâve an effect on the génération of ROS in U373 astroglioblastoma cells (Figure 14a). This shows that this aspect of the antioxîdant effect is neuron-specific.

Example 12 - neuroprotection

RA-differentiated SHSY-5Y cells were treated with MPP+, the active métabolite 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 (Figure 15). Both MRx0005 and MRx0029 showed significant effects and promote per se an increase of the mitochondria metabolic activity in SHSY-5Y cells. MRX0029 showed complété protection from MPP+, restoring cell viability nearly to the same level of untreated cells and higher than quercetin positive control. MRx0005 protection was about 20% compared to YCFA-MPP+ treated sample, about the same observed for the quercetin positive control (Fig. 15).

Example 13 - Further analysis of the mechanism of histone deacetylation inhibition

Introduction

The gut microbiota, with its immense diversîty and metabolic capacity, represents a huge metabolic réservoir for production of a vast variety of molécules with potenlial to influence HDAC activity. Few studies hâve assessed the HDAC inhibitory activity of microbially-derived métabolites other than butyrate, which has been shown to inhibit HDAC and is associated with improvement of motor fonction in Huntington’s disease [64], The inventors therefore sought to determine which métabolites are responsible for HDAC inhibition and forther elucidate the mechanisms by which inhibition is achieved.

Material and Methods

Bacterial culture and cell-free supernatant collection

Pure cultures of bacteria were grown anaerobically in YCFA broth until they reached their stationary growth phase. Cultures were centrifoged at 5,000 x g for 5 minutes and the celi-free supernatant (CFS) was fîltered using a 0.2 μΜ filter (Millipore, UK). 1 mL aliquots ofthe 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 MC FA 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 internai standards where added. Ail 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 (7890Β, 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 R20l4b (Mathworks, Inc.) using the PARADISe software described in [65],

Spécifie HDAC activity analysis

Spécifie HDAC inhibition activity was analysed for HDACl, 2, 3, 4, 5, 6, 9 using fluorogenic 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 supematants were diluted l in 10 and exposed to spécifie HDAC proteins provided in the kit to maintaîn consistency between methods.

Results

Historié deacetylase-inhibiting gut commensal microbial métabolites are butyrate and valeric acid

MRx0029, whose supernatant showed strong HDAC inhibition in both HT29 whole cells and HT29 cell lysâtes, produced valeric acid and hexanoic acid at mean concentrations of 5.08 mM and l .60 mM, respectively (Figure 16A and C).

To investigate which métabolites 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. 16B 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 activity. Valérie acid inhibited total HDAC activity (* (p<0.05), ** (p<0.005), *** (P<0.001), **** (p<0.000l)).

Potent total HDAC inhibitors investigated target class IHDACs,

The spécifie HDAC inhibition profile of the test bacteria strain was investigated. Spécifie HDAC inhibition assays (BPS Bioscience, CA) were carried out for Class I and Class II HDACs. The ability of the bacterial strain to inhibit HDAC enzymes was compared to butyrate, hexanoic and valeric acid. Our results demonstrate that MRX0029, is a very potent inhibitor of Class l HDAC enzymes (HDACl, 2 and 3). Inhibition of class II HDACs was not as significant (data not shown).

Discussion

The strain with HDAC inhibitory activity produced significant amounts of valeric acid and hexanoic acid as well as significant amounts of sodium butyrate (Figure 16C). When tested as pure substances, valeric acid and sodium butyrate resulted in significant HDAC inhibition (p<0.000l).

Interestîngly, the results for spécifie HDAC activity show that the tested strain is a potent inhibitor of Class l HDACs, and particularly HDAC2 (Figure 17 and 18). Class I HDACs (HDACl, 2, 3 and 8) résidé in the nucléus and are ubiquitously expressed in several human cell types. HDACs l-3 share more than 50% homology, but hâve distinct structures and cellular functions [66], They are primarily involved in cell survival, prolifération and différentiation, and thus their inhibition may be useful is wide arrayof diseases [67]; [68]; [69]; [70]; [71].

Example 14- Level of BDNF sécrétion in SHSY-5Y cells

Baçkground

Brain-derived neurotrophic factor (BDNF) is a ubiquitous molécule in the brain associated with neural development, neuro-protection and neuro-regeneration. BDNF not only protects against neurodegeneration but also mental disorders like dépréssion and anxiety, which are quite common amongst patients diagnosed with PD or AD.

Methods

SH-SY5-SY were plated în 24 wells plate at density of 60,000 cells/well and placed in the incubator. After 24 h, media were replaced with différentiation medium (growth medium containing 1% FBS) and 10 pM retinoic acid. Différentiation medium was replaced eveiy other day and cells were used on day 10 of différentiation. For the treatment différentiation medium was removed and replaced with 450ul of full growth media and 50 pl of bacteria SN was added to the treated wells or YCFA+ was added as négative Control,

Results

The results are shown in Figure 19, which shows that administration of MRX0029 in combination with retinoic acid increases the sécrétion of BDNF from differentiated neuroblasoma cells. Compositions comprising commensal bacteria and organic acids may therefore be useful in therapy.

Example 15 - Métabolite production - métabolites in the brain

Background

Métabolites present in bacteria supematants can directly influence the host response to oxidative stress, cell-to-cell communication and neuroprotection. Métabolites that play a key rôle in neurological processes were measured during the ex vivo screening in brain tissue of mice fed with MRxOOO5 and MRx0029.

Methods

Animais

BALBc (Envîgo, UK) adult male mice were group housed under a 12 h light-dark cycle; standard rodent chow and water were available ad libitum. Ail experiments were perfonned in accordance with Europcan guidelines following approval by University College Cork Animal Ethics Expérimentation Committee. Animais were 8 weeks old at the start ofthe experiment.

Study Design

Animais were allowed to habituate to their holding room for one week after arrivai into the animal unit. They receive oral gavage (200pL dose) of live biotherapeutics at a dose of l X I0⁹ CFU for 6 consecutive days between 15:00 and 17:00. On day 7, the animais are decapitated, and tissues are harvested for expérimentation.

Tissue Collection

Animais 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 analysis. The brain was quickly excised, dissected and each brain région was snap10 frozen on dry ice and stored at -80 °C for further analysis. Spleen was removed and processed immediately after culls for ex-vivo immune stimulation. Intestinal tissue (2 cm segments of ileum and colon closest to the caecum were excised, and the furthest 1cm of tissue from the caecum were used) were mounted into the Using chambers for intestinal permeability assay. The caecum was removed, weighted and stored at -80 °C for SCFAs analysis.

Monoamine Analysis

Neurotransmitter concentration was analysed by HPLC on samples from the braînstem. Briefly, brainstem tissue was sonîcated in 500 μΐ of chilied mobile phase spiked with 4 ng/40 μΐ ofN-Methyl 5-HT (Sigma Chemical Co., UK) as internai standard. The mobile phase contained 0.1 M citric acid, 5.6 mM octane-1-sulphonic acid (Sigma), 0.1 M sodium dihydrogen phosphate, 0.01 mM EDTA 20 (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 μΐ 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 S1L-10A autoinjector (with sample cooler maintained at 40 C) and an online 25 Gastorr Degasser (ISS, UK). A reverse-phase column (Kinetex 2.6 u Cl8 100 ^x 4.6 mm, Phenomenex) maintained at 30 °C was employed in the séparation (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 (Shimadzu). The neurotransmitters were identified by their characteristic rétention times as 30 determined by standard injections, which run at regular intervais during the sample analysis. The ratios of peak heights of analyte versus internai standard were measured and compared with standard injection. Results were expressed as ng of neurotransmitter per g fresh weight of tissue.

Métabolite analysis

For GC-métabolite analysis, samples of bacterial supematants were derivatized with methyl chloroformate using a slightly modified version of the protocol described in [72], Ali samples were analyzed in a randomized order. Analysis was performed using GC (7890B, Agitent) coupled with a quadropole detector (59977B, Agitent). The system was controlled by ChemStation (Agitent). Raw data was converted to netCDF format using Chemstation (Agitent), before the data was imported and processed in Matlab R20l4b (Mathworks, Inc.) using the PARADISe software described in [65],

For fatty acid analysis samples were acidified using hydrochloride acid, and deuterium labelled internai standards where added. Ail 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 qm) installed in a GC (7890B, Agitent) coupled with a quadropole detector (59977B, Agitent). The system was controlled by ChemSlation (Agitent). Raw data was converted to netCDF format using Chemstation (Agitent), before the data was imported and processed in Matlab R20l4b (Mathworks, Inc.) using the PARADISe software described in [65],

Results - neurotransmitter production

The results are shown in Figure 20, which shows that in brains of mice fed with MRx0029, noradrenalîne levels are increased (p=0.0507), accompanied with a slight increase of serotonin and 5HIAA. These data support the métabolite analysis set out below, suggesting that MRx00029 is a major producer of 4-hydroxyphenylacetic acid, a known antioxidant [73], More importanlly, 4hydroxyphenyl acetic acid is a synthetic intermediate of dopamine and norepinephrine and an important bio-active molécule [74]. In fact, in PD, degenerative changes extend beyond the dopaminergic system, affecting equally the serotonergic and noradrenergic Systems, which in tum leads to decreased levels of serotonin (5-hydroxytryptamine, 5-HT) and noradrenalîne (norepinephrine) in both striatal and extra-striatal structures [75], L-DOPA targets mainly the dopamine-related features of PD, however ît does not address the decreases in both 5-HT and noradrenalîne. 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, psychiatrie symptoms) [76], Therefore, these data demonstrate that bacteria that produce organic acids, such as 4hydroxyphenyl acetic acid, may be useful in therapy, in particular in the treatment of neurodegenerative diseases.

Results - metaholite production

Métabolites present in bacteria supematants can directly influence the host response to oxidative stress, cell-to-ceil communication and neuroprotection in the spécifie. Métabolites in the supernatant of cultures of MRX0029 and MRX0005 were analysed and the results are shown in Figure 21.

A few métabolites showed a striking différence between the two strains analysed, The concentration of succinic acid was particularly elevated in MRx0005. Interestingly, the ratio sample/media for 4hydroxyphenylacetic acid was significantly higher in MRx0029 (Fig. 21 A).

Fatty acid analysis in the supematants revealed an interestîng dichotomy in the two strains: MRxOOO5 produced mainly acetic and propanoic acid, while MRxOO29 produced butanoic, pentanoic and hexanoic acid, both in the linear and branched forms (Fig. 2lB). The two strains looked very different and in particular, the production of succinic acid and 4-hydroxyphenylacetic acid by MRx0005 and MRx0029 respectively was notable (Figure 2lA). Furthennore, MRx0005 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 métabolite involved in oxidative phosphorylation. Oxidative phosphorylation complex is a key step for synaptic trafficking of proteins and vesicles to proximal and distal régions [77], Its dysfunction has been reported in neurodegenerative disorders including Alzheimer’s disease, Parkinson’s disease and Spinocerebellar ataxia type l [78], These findings are particularly interestîng as succinic acid can augment mitochondrial activity and support vulnérable neurons in neurodegenerative disease related to misfolded proteins including PD [79], BDNF and succinic acid hâve both a similar protective activity not only in neuro-degeneration but also în mental disorders like dépréssion and anxiety, which are quite common amongst patients diagnosed with PD or AD.

Figure 2lB also demonstrates that MRX0029 is a butyrate (butanoic acid) producer. This may be significant because butyrate has a known rôle is reducing impermeability of the blood brain barrier, which has a neuroprotective effect [80], This property of MRx0029 (and other neuroprotective bacteria) may contribute to its efficacy.

Example 16- Modulation of the mRNA expression of tightfunction proteins by MRx0029

Since recent evidence suggests that intestinal dysfonction and inflammation is a non-motor symptom associated with PD, the ability of the bacterial strains of the invention to cause any intestinal barrier dysfonction was investigated. HT29-mtx épithélial, mucin-producing cell monolayers [81] were used as an in vitro model to evaluate gut barrier disruption and immune stimulation following treatment with MRx0005 and MRxOO29, Differentiated HT29-mtx cells exposed to phorbol l2-myristate-13acetate (PMA) secreted a significant amount of IL-8; în contrast treatment for 24h with MRx005 and MRx0029 bacterial supematants, induced an even lower sécrétion of IL-8 compared than both untreated and YCFA-treated cells (Fig. 22A).

The ability of MRx0005 and MRx0029 to regulate épithélial permeability by modifying intraceilular signal transduction involved in the expression and localization of proteins involved in the gut bamer formation was then investigated.

RNA was isolated and Quantitative RT-PCR (qRT-PCR) analysis was performed to characterize the 5 changes in gene expression of tight junction proteins during incubation with MRx0005 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 to MRx0005 did not alter the gene expression of tight junction proteins îndicating that the two strains act differentially 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 MRx0005 and MRx0029. Gene expression of TJP2 and occludin was quantified in the colon and ileum. The ex vivo data perfectly miiTor the in vitro data as MRxOO29 was able to significantly up-regulate TJP1 and Occludin (p=0.073) in the colon région of the murine intestine (Fig. 22C+22D). MRx0029 was also able to decrease the permeability function in the colon of the same 15 mice (Fig. 22E+22F).

Materials and methods - RNA extraction and a PC R analysis

Total RNA was extracted using the RNeasy mini kit (Qiagen, Manchester, JUK) according to the manufacturer's instructions, and the RNA concentration determined by absorbance at 260/280nm using a spectrophotometer (nano-Drop ND-1000; Thermo Scientific, Wilmington, DE). For mRNA 20 expression analysis, cDNA was prepared from total RNA using the High-Capacity cDNA reverse transcription kit (Applied Biosystems, UK) according to the manufacturées instructions. The reverse transcription reactions were performed in a Thermo cycler (Biometra, Germany) at 25°C for 10min, 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 QuantStudîo 6 flex real-time PCR 25 machine (Applied Biosystems, UK) using a standardiscd profile (initial dénaturation of 95°C for 10 minutes, followed by 40 cycles of 15 seconds of dénaturation 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 QuantStudîo Real-Time PCR Software vl.2. The primer sequences for Actin, Villin, Occludin TJP1 30 and TJP2 are provided in the sequence listing.

Example 16 — Stability testing

A composition described herein containing at least one bacterial strain described herein is stored in a sealed container at 25G or 4°C and the container is placed in an atmosphère having 30%, 40%, 50%,

60%, 70%, 75%, 80%, 90% or 95% relative humidity. After l month, 2 months, 3 months, 6 months, l year, 1.5 years, 2 years, 2.5 years or 3 years, at least 50%, 60%, 70%, 80% or 90% of the bacterial strain shall remain as measured in colony forming units determined by standard protocols.

Exemple 17

Methods

Animais

The animais and study design used were the same as for Example 15.

Bacterial strains • 755: Parabacteroides distasonis (MRX005) • Megasphaera massiliensis (MRX0029)

Tissue Collection

Animais were sacrificed in a random fashion regarding treatment and testing condition; sampling occurred between 9.00 a.m. and 2:30 p.m. Trunk blood was collected in potassium EDTA (Ethyiene Diamine Tetra Acetic Acid) tubes and spun for 15 min at 4000 g. Plasma was isolated and stored at -80 °C for further analysis. The brain was quickly excised, dissected and each brain région was snapfrozen on dry ice and stored at -8Û °C for further analysis. Spleen was removed, collected in 5 mL RPMI media (with L-glutamine and sodium bicarbonate, R8758 Sigma + 10 % FBS (F7524, Sigma) + 1% Pen/Strep (P4333, Sigma)) and processed immediately after culls for ex-vivo immune stimulation. Intestinal tissue (2 3cm segments of ileum and colon closest to the caecum were excised, and the furthest 1cm 2cm of tissue from the caecum were used) were mounted into the Ussing chambers for intestinal permeability assay. The caecum was removed, weighted and stored at -80 °C for SCFAs analysis.

Monoamine Analysis

The neurotransmitter concentration was analysed as described in Example 10

Spleen Cytokine Assay

Spleens were collected immediately in 5mL RPMI media following sacrifice and cultured immediately. Spleen cells were first homogenised in this RPMI media, followed by 5 mins incubation with 1ml of RBC lysis buffer (11814389001 ROCHE, Sigma). A further 10 ml of RPMI media was added, followed by 200G centrifugation for 5 mins. The supernatant was then fîltered through 40um strainer. Cells were counted and seeded (4,000,000/mL media). After 2.5 h of adaptation, cells were stimulated with lipopolysaccharide (LPS-2 pg/ml) or coneanavalin A (ConA21082

2.5 pg/ml) for 24 h. Following stimulation, the supematants were harvested to assess the cytokine release using Proinflammatory Panel l (mouse) V-PLEX Kit (Meso Scale Discovery, Maryland, USA) for TNFot, IL-IO, IL-1β, Interferon γ, CXCL2 and IL6. The analyses were performed using MESO QuickPlex SQ 120, SECTOR Imager 2400, SECTOR Imager 6000, SECTOR S 600.

Gene Expression Analysis

Total RNA was extracted using the mirVana™ miRNA Isolation kit (Ambion/Llife technologies, Paisley, UK) and DNase treated (Turbo DNA-free, Ambion/life technologies) according to the manufacturers recommendations. RNA was quantified using NanoDrop™ spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, Delaware, USA) according to the manufacturer's instructions. RNA quality was assessed using the Agilent Bioanalyzer (Agilent, Stockport, UK) according to the manufacturer's procedure and an RNA integrity number (RIN) was calculated. RNA with RIN value >7 was used for subséquent experiments. RNA was reverse transcribed to cDNA using the Applied Bîosystems High Capacity cDNA kit (Applied Biosystems, Warrington, UK) according to manufacturer's instructions. Briefly, Multiscribe Reverse Transcriptase (50 U/pL) (I )(2)(1)(10) was added as part of RT master mix, incubated for 25°C for 10 min, 37°C for 2 h, 85°C for 5 min and stored at 4°C. Quantitative PCR was carried out using probes (6 carboxy fluorescein FAM) desîgned by Applied Biosystems to mouse spécifie targeted genes, while using β-actin as an endogenous control. Amplification reactions contained 1 pl cDNA, 5 pl of the 2X PCR Master mix (Roche), 900 nM of each primer and were brought to a total of 10 pl by the addition of RNase-free water. Ail reactions were performed in triplicate using 96-well plates on the LightCycler®480 System. Thermal cycling conditions were as recommended by the manufacturer (Roche) for 55 cycles. To check for amplicon contamination, each run contained no template Controls in triplicate for each probe used. Cycle threshold (Ct) values were recorded. Data was normalized using β-actin and transformed using the 2-AACT method and presented as a fold change vs. control group.

Short Chain Fatty Acids Analysis in the Caecal Content

Caecum content was mixed and vortexed with MilliQ water and incubated at room température for 10 min. Supematants were obtained by centrifugation (10000 g, 5 min, 4 °C) to pellet bacteria and other solids and filiation by 0.2pm. It was transferred to a clear GC vial and 2-Ethylbutyric acid (Sigma) was used as the internai standard. The concentration of SCFA was analyzed using a Varian 3500 GC flame-ionization System, fitted with a with a ZB-FFAP column (30 m x 0.32 mm x 0.25 mm; Phenomenex). A standard curve was built with different concentrations of a standard mix containing acetate, propionate, iso-butyrate, n-butyrate, isovalerate and valerate (Sigma). Peaks were integrated by using the Varian Star Chromatography Workstation version 6.0 software. Ail SCFA data are expressed as pmol/g.

Statistical Analysis

Nonnally distributed data are presented as mean ± SEM; Non-parametric datasets are presented as médian with inter-quartile range. Unpaired two-tailed t-test were applied to analyse parametric data and Mann-Whitney test was used for non-parametric. Speannan's rank corrélation coefficient was employed for the corrélation analysis in the pooied datasets. A p value < 0.05 was deemed significant in ail cases.

Results - Neurotransmitter production

The results în Figure 23 show the effect of MRx005 treatment on the concentration of neurotransmitters in the brain of mice. Most notably, treatment with MRx005 leads to a decrease in dopamine.

Results — Gene expression

Expression of genes for neurotransmitter receptors [serotonin receptor la(5-HTRla), dopamine Dl receptor, GABA receptor subunit Bl, GABAA receptor, NMDA2A (Grin2A) and NMDA2B (Grin2b) receptor], inilammatory markers [IL-Ιβ, IL6, CDllb, TNFa and TLR4], and endocrine markers [corticosterone releasing factor (CRF), corticosterone reieasing factor receptors l and 2 (CRFRl, CRFR2), brain-derived neurotrophin factor (BDNF), vasopressin receptor, oxytocin receptor, glucocorticoîd receptor and mineralocorticoid receptor] were analysed în brain tissue from the hippocampus, amygdala and prefrontal cortex.

Figures 24-38 show the changes in gene expression after MRX005 or MRX0029 treatment in the hippocampal, amygdala and prefrontal cortex. Treatment with MRxOO29 led to an increase in glucocorticoîd receptor expression in the amygdala (Figure 3lC). Figure 32A shows that MRx005 significantly increased the expression of BDNF in the amygdala, while treatment with MRx0029 significantly increased the expression of TLR4 in the amygdala (Figure 32).

Both MRx005 and MRx0029 can increase expression of CDl Ib in the amygdala (Figure 33A), while the expression of IL-6, Grin2a and Grin2b is reduced after MRx005 treatment (Figures 33B-D). In addition, MRx005 and MRx0029 significantly increased the expression of GABRA2 and increased the expression of GABBRl in the amygdala.

Treatment with MRx005 led to a significant increase in the expression of BDNF in the prefrontal cortex (Figure 35B).

Discussion

MRx005 and MRx0029 administration caused changes in gene expression, especially in the amygdala.

Results - Effect on Tphl and IDO-l expression

Figure 39 shows that MRx0029 can significantly increase the expression tryptophan hydroxylase- 1 (Tphl) in the colon and that MRX005 treatment can increase IDO-l expression in the colon. Treatment with MRX005 increased the expression of Tphl and IDO1 in the ileum (Figure 40).

Indoleamine-pyiTole 2,3-dioxygenase-l (IDO-l) the first and rate-limiting enzyme in the tryplophan/kynurenine pathway while tryptophan hydroxylase 1 (Tphl), an isoform ofthe enzyme tryptophan hydroxylase, responsible for the synthesis of serotonin. These data suggest that MRx0029 and MRx005 may affect serotonin levels and the tryptophan/kynurenine pathway.

Results - Effect on tryptophan métabolite levels

Figure 41 shows the effect of treatment with MRx005 on the levels of circulating kynurenine and tryptophan.

Results - Effect on cytokine expression from splénocytes

The ex-vivo splénocyte assay involves challenging the splénocytes (cells isolated from the spleen - a main organ învolved in immune defence), with a bacterio- or viral-mimetic challenge.

MRX005 significantly reduced the levels of interferon-γ in splénocytes following a challenge with LPS (Figure 42). In addition, MRX0Ü5 reduced the levels of interleukin-6 and tumour necrosis factor after a challenge with LPS (Figures 44 and 45, respectively). Treatment with MRx0029 led to a réduction in interferon-γ, interleukin-ΐβ and interleukin-6 following a challenge with LPS (Figures 42, 43 and 44, respectively).

Treatment with MRx005 and MRxOO29 led to an increase in the levels of the chemoattractant CXCL1 (Figure 47).

Results — Effect on Caecal Short Chain Fatty AcidLevels

Short chain fatty acids (SCFAs) are produced when non-digestible fibres from the diet are fermented by bacteria in the gut. The effects of MRX005 administration are shown in Figure 48.

Example 18— Further analysis of MRX029 and MRXOOS-induced changes in gene expression levels

Methods

Cell line

SH-SY5Y cells

Bacterial strains • 755: Parabacteroides distasonis (MRX005) • Megasphaera massiliensis (MRX0029) qPCR

SHSY5Y were plated in 10cm pétri dishes a density of2xlO⁶ cells. After 24h cells were treated in différentiation medium (growth medium containing l% FBS without RA) with 10% bacteria supernatants or YCFA+, lOuM RA, 200uM hexanoic acid or 200uM valproic acid, for 17 his. There after représentative 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 internai control. Fold change was calculated according to the 2⁽·^ΔΔα) method. The primer sequences for MAP2, DRD2, GABRB3, S Y P, PINKl, PARK7 and NSE are provided in the sequence listing.

Immuno-labelling and cell imaging

Cells were seeded onto 8-well chamber slîdes (Marienfeld Laboratory Glassware) at 5xl0⁴ cells/well overnight and were treated with 10% bacterial supernatant for 24 h. For différentiation, cells were treated with 10 nM RA for 5 days before treating with cell-free bacterial supernatant for 24 h. Afterwards, the cells were fixed with 4% paraformaldéhyde in PBS for 20 minutes at room température (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 blockîng buffer (4% BSA/PBS) for 1 h at RT before adding anti-MAP2 antibody or p3-tubulin (sc-74421 and sc-8OOO5 respectively, Santa Cruz Biotechnology Inc) diluted in 1% BSA/PBS for 12 h at 4C. They were then washed twice with PBS, followed by incubation with Alexa Flour 488 conjugated anti-mouse (Molecular Probes Inc) and Alexa Flour 594 conjugated Phalloidin (ab 176757, Abcam) for 1 h at RT, After washing 3X with PBS, the slides were staining with DAPI and mounted with Vectashield® (Vector Laboratories). Slides were viewed using a Axioskop 50 microscope (Zeiss) equipped with a 63x/1.2 W Korr objective and filter sets suitable for détection of the fluorochromes used. Manuai exposure times for the digital acquisition of images immunodabelled with MAP-2 were kept constant allowing comparison between different wells and treatments. Phalloidin (F-actin) and DAPI exposure times varied to suit the field of view. Randomised fields of view were acquired using a Qlmaging caméra controlled by Image Pro Plus software. Images were saved as TIFF files and opened in Adobe Photoshop CC 2015.1.2. Images of the MAP-2, DAPI and Phalloidin images were then overlaid and merged. Représentative images were selected to illustrate the différences in abundance and location of the proteins examined,

Immunoblotting

SH-SY5Y cells cultured under the indicated conditions described above, treated with MRx0005 and MRx0029 for 24h and then lysed in RIPA buffer containing cocktail of protease inhibitors (Roche Diagnostics, UK). Protein concentration was estimated using the BCA protein assay kit (Pierce Biotechnology, Rockford, IL), separated by SDS-PAGE and transferred to a PVDF membrane. Membranes were then blocked with 5% non-fat dry milk or 5% BSA and incubated ovemight at 4°C with the primary antibodies (respectiveîy MAP2 and P3-tubulin). The blots were then incubated with the approprîate horseradish peroxidase (HRP)-conjugated secondary antibody, and proteins were detected by chemiluminescence détection kit (Pierce Biotechnology, Rockford, IL). For both MAP2 and p3-tubulin, β-actin served as a control to monitor protein loading variability amongst samples,

Results and Discussion

Gene expression

Figures 13a (graph insert) and 49 show the MRxOO29 and MRX005-induced changes in expression levels of Actin, Villin, Occludin TJP1, TJP2, MAP2, DRD2, GABRB3, SYP, PINK1, PARK7 and NSE.

Microscopy and Immunoblotting

Figure 50 shows the change in the level of expression of MAP2 in SHSY5Y cells as determined by confocal microscopy. The expression levels of MAP2 and B3-tubulin were also quanti fied by immunoblot analysis. The results shown in Figure 50M and N indicate that MRX029 induces an increase în the level expression of MAP2

Sequences

SEQ ID NO:1 (Megasphaera massiliensis gene for 16S ribosomal RNA, partial sequence, strain:

NP3 - JX424772.1) agagtttgat cctggctcag gacgaacgct ggcggcgtgc ttaacacatg caagtcgaac gagaagagat gagaagcttg cttcttatca attcgagtgg caaacgggtg agtaacgcgt 121 aagcaacctg cccttcagat ggggacaaca gctggaaacg gctgctaata ccgaatacgt 181 tctttccgcc gcatgacggg aagaagaaag ggaggccttc gggctttcgc tggaggaggg 241 gcttgcgtct gattagctag ttggaggggt aacggcccac caaggcgacg atcagtagcc 301 ggtctgagag gatgaacggc cacattggga ctgagacacg gcccagactc ctacgggagg

	361	cagcagtggg	gaatcttccg	caatggacga	aagtctgacg	gagcaacgcc	gcgtgaacga
	421	tgacggcct t	cgggttgtaa	agttctgtta	tatgggacga	acagggcatc	ggttaatacc
	481	cggtgtcttt	gacggtaccg	taagagaaag	ccacggctaa	ctacgtgcca	gcagccgcgg
	541	taatacgtag	gtggcaagcg	ttgtccggaa	ttat tgggcg	taaagggcgc	gcaggcggca
5	601	tcgcaagtcg	gtcttaaaag	tgcggggct t	aaccccgtga	ggggaccgaa	actgtgaagc
	661	tcgagtgtcg	gagaggaaag	cggaattcct	agtgtagcgg	tgaaatgcgt	agatattagg
	721	aggaacacca	gtggcgaaag	cggct ttctg	gacgacaact	gacgctgagg	cgcgaaagcc
	781	aggggaqcaa	acgggattag	ataccccggL	agtcctggcc	gtaaacgatg	gatactaggt
	841	gtaggaggta	tcgactcctt	ctgtgccgga	gttaacgcaa	taagtatccc	gcctggggag
10	901	tacggccgca	aggctgaaac	tcaaaggaat	tgacgggggc	ccgcacaagc	ggtggagtat
	961	g tggtttaat	tcgacgcaac	gcgaagaacc	ttaccaagcc	ttgacattga	ttgctacgga
	1021	aagagatttc	cggttcttct	tcggaagaca	agaaaacagg	tggtgcacgg	ctgtcgtcag
	1081	ctcgtgtcgt	gagatgttgg	gttaagtccc	gcaacgagcg	caacccctat	cttctgttgc
	1141	cagcacctcg	ggtggggact	cagaagagac	tgccgcagac	aatgcggagg	aaggcgggga
15	1201	tgacgtcaag	tcatcatgcc	cct tatggct	tgggctacac	acgtactaca	atggctctta
	1261	atagagggac	gcgaaggagc	gatccggagc	aaaccccaaa	aacagagtcc	cagttcggat
	1321	tgcaggctgc	aactcgcctg	catgaagcag	gaatcgctag	taatcgcagg	tcagcatact
	1381	gcggtgaata	cgttcccggg	ccttgtacac	accgcccgtc	acaccacgaa	agtcat tcac
	1441	acccgaagcc	ggtgaggcaa	ccgcaaggaa	ccagccgtcg	aaggtggggg	cgatgattgg
20	1501	ggtgaagtcg	taacaaggt

SEQ ID NO:2 (consensus 16S rRNA sequence for Megasphaera massiliensis strain MRx0029)

TGAGAAGCTTGCTTCTTATCGATTCTAGTGGCAAACGGGTGAGTAACGCGTAAGCAACCTGCCCTTCAGATGGGGAC AACAGCTGGAAACGGCTGCTAATACCGAATACGTTCTTTCCGCCGCATGACGGGAAGAAGAAAGGGAGGCGTTCGGG 25 CTTTCGCTGGAGGAGGGGCTTGCGTCTGATTAGCTAGTTGGAGGGGTAACGGCCCACCAAGGCGACGATCAGTAGCC

GGTCTGAGAGGATGAACGGCCACATTGGGACTGAGACACGGCCCAGACTCGTACGGGAGGCAGCAGTGGGGAATCTT CCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAACGATGACGGCCTTCGGGTTGTAAAGTTCTGTTATATG GGACGAACAGGACATCGGTTAATACCCGGTGTCTTTGACGGTACCGTAAGAGAAAGCCACGGCTAACTACGTGCCAG CAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCGCGCAGGCGGCATCGCAAGT 30 CGGTCTTAAAAGTGCGGGGCTTAACCCCGTGAGGGGACCGAAACTGTGAAGCTCGAGTGTCGGAGAGGAAAGCGGAA

TTCCTAGTGTAGCGGTGAAATGCGTAGATATTAGGAGGAACACCAGTGGCGAAAGCGGCTTTCTGGACGACAACTGA CGCTGAGGCGCGAAAGCCAGGGGAGCAAACGGGATTAGATACCCCGGTAGTCCTGGCCGTAAACGATGGATACTAGG TGTAGGAGGTATCGACTCCTTCTGTGCCGGAGTTAACGCAATAAGTATCCCGCCTGGGGAGTACGGCCGCAAGGCTG AAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGTATGTGGTTTAATTCGACGCAACGCGAAGAACCTTA

CCAAGCCTTGACATTGATTGCTACGGAAAGAGATTTCCGGTTCTTCTTCGGAAGACAAGAAAACAGGTGGTGCACGG GTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATCTTCTGTTGCCAGCACC

TCGGGTGGGGACTCAGAAGAGACTGCCGCAGACAATGCGGAGGAAGGCGGGGATGACGTCAAGTCATCATGCCCCTT ATGGCTTGGGCTACACACGTACTACAATGGCTCTTAATAGAGGGAAGCGAAGGAGCGATCCGGAGCAAACCCCAAAA ACAGAGTCCCAGTTCGGATTGCAGGCTGCAACTCGCCTGCATGAAGCAGGAATCGCTAGTAATCGCAGGTCAGCATA

CTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAAAGTCATTCACACCCGAAGCCGGTGA GGCAACCGCAAG

Primers used for qPCR (with SEQ ID NO in brackets)

Name			Forward sequence		Reverse sequence	l|
ACTB	i	GATCAAGATCATTGCTCCTC	(3)	TTGTCAAGAAAGGGTGTAAC	(4 )
GAPDH	i	GGTATCGTGGAAGGACTCATG	(5)	ATGCCAGTGAGCTTCCCGTTC	(6)
MAP2 Occludin		CTCAGCACCGCTAACAGAGG AAGAG GAATT T T GACAC TGG	(7) (9)	CATTGGCGCTTCTCTCCTC (8) GCCATGTACTCTTCACTTTC (10)
lIJi	L	AAGTCACACTGGTGAAATCC	(11)	CTCTTGCTGCCAAACTATCT	(12)
ÎTJP2		CCCTCCCCTGGATCAGGAT (	13)	GCCATCAAACTCGTCCATCA	(14)
jvillin		CATTACCTGCTCTACGTTTG	(15)	AGAT GGACATAAGAT GAG GT G	(16)

SEQ ID NO:l7 (consensus 16S rRNA sequence for Parabacteroides distasonis strain MRX0005)

AMCCGGGTGGCGACCGGCGCACGGGTGAGTAACGCGTATGCAACTTGCCTATCAGAGGGGGATAACCCGGCGAAAGT CGGACTAATACCGCATGAAGCAGGGATCCCGCATGGGAATATTTGCTAAAGATTCATCGCTGATAGATAGGCATGCG TTCCATTAGGCAGTTGGCGGGGTAACGGCCCACCAAACCGACGATGGATAGGGGTTCTGAGAGGAAGGTCCCCCACA TTGGTACTGAGACACGGACCAAACTCCTACGGGAGGCAGCAGTGAGGAATATTGGTCAATGGGCGTGAGCCTGAACC AGCCAAGTCGCGTGAGGGATGAAGGTTCTATGGATCGTAAACCTCTTTTATAAGGGAATAAAGTGCGGGACGTGTCC CGTTTTGTATGTACCTTATGAATAAGGATCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGATCCGAGCGT TATCCGGATTTATTGGGTTTAAAGGGTGCGTAGGCGGCCTTTTAAGTCAGCGGTGAAAGTCTGTGGCTCAACCATAG AATTGCCGTTGAAACTGGGAGGCTTGAGTATGTTTGAGGCAGGCGGAATGCGTGGTGTAGCGGTGAAATGCATAGAT ATCACGCAGAACCCCGATTGCGAAGGCAGCCTGCCAAGCCATTACTGACGCTGATGCACGAAAGCGTGGGGATCAAA CAGGATTAGATACCCTGGTAGTCCACGCAGTAAACGATGATCACTAGCTGTTTGCGATACACTGTAAGCGGCACAGC GAAAGCGTTAAGTGATCCACCTGGGGAGTACGCCGGCAACGGTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAG CGGAGGAACATGTGGTTTAATTCGATGATACGCGAGGAACCTTACœGGGTTTGAACGCATTCGGACMGAKGTGGAA ACACATTTTCTAGCAATAGCCATTTGCGAGGTGCTGCATGGTTGTCGTCAGCTCGTGCCGTGAGGTGTCGGCTTAAG TGCCATAACGAGCGCAACCCTTGCCACTAGTTACTAACAGGTAAAGCTGAGGACTCTGGTGGGACTGCCAGCGTAAG CTGCGAGGAAGGCGGGGATGACGTCAAATCAGCACGGCCCTTACATCCGGGGCGACACACGTGTTACAATGGCGTGG ACAAAGGGAAGCCACCTGGCGACAGGGAGCGAATCCCCAAACCACGTCTCAGTTCGGATCGGAGTCTGCAACCCGAC TCCGTGAAGCTGGATTCGCTAGTAATCGCGCATCAGCCATGGCGCGGTGAATACGTTCCCGGGCCTTGTACACACCG CCCGTCAAGCCATGGGAGCCGGGGGTACCTGAAGTCCGTAACCGCGAGGATCGGCCTAGGGTAAAACTGGTGACTGG GGCTAAGTCGTACGGGG

Primers and probes used for ex vivo qPCR (with SEQ ID NO in brackets)

Ex vivo:

^tfViafâSi <rse sggtrenc& ί

GAT TAC TGC TCT GGC TCC TAG (10)

AAT GGT GAA GGT CGG TGT G

GCT GCC TTG ATG TTT ACT TTG AC (24)

AGC CAG AGT CCT TC A GAG A _i27)

GAC TCATCG TAC TCC TGC TTG _{10)

GTG GAG TCA TAC TGG AAC ATG TAG (22)

GCA ACC GAA GTA TGA AAT AAC CA (25)

TCC TTA GCC ACT CCT TCT GT (23)

Æ6-FAM/CTG GCC TCA /ZEN/CTG TCC ACC TTC C'SfABkFQ/ (²⁰) /56-FAM/TGC AAA TGG /ZEN/CAG CCC

TGQTG/3IABkFQ/ (23) /56-FAM/ACC AGG TGA /ZEN/GAA GAG TGA TGA CCA TCC /3IASkFQ/ (^2e>

/56-FAM/CCT ACC CCA /ZEN/ATT TCC AAT GCT CTC CT/SIABkFQ/ (23)

Additional primers used in qPCR (with SEQ ID NO in brackets)

Gene ID	Forward sequence	Reverse sequence
NSE	CCCTGTATCGTAAGAACGGT (30)	GCCACCATTGATCACGTTGA (31)
PINK1	CCCAAGCAACTAGCCCCTC (32)	GGCAGCACATCAGGGTAGTC (33)
PARK7	GTAGCCGTGATGTGGTCATTT (34)	CTGTGCGCCCAGATTACCT (35)

GGCTTCATGGCATCAACTTCA

CTCGGCTTTGTGAAGGTGCT (36) (37)

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Claims (18)

1. l. A composition comprising a bacterial strain of the genus Megasphaera, for use in a method of treating or preventing a neurodegenerative disorder.
2. 2. The composition of claim l, wherein the composition is for use in a method of treating or preventing a disease or condition selected from the group consisting of Parkinson’s disease, includîng progressive supranuclear palsy, progressive supranuclear palsy, Steele-RichardsonOlszewski syndrome, normal pressure hydrocephalus, vascular or arteriosclerotic parkinsonism and drug-înduced parkinsonism; Alzheimer’s disease, includîng Benson's syndrome; multiple sclerosis; Huntington’s disease; amyotrophie latéral sclerosis; Loti Gehrîg's disease; motor neurone disease; prion disease; spinocerebellar ataxia; spinal muscular atrophy; dementia, includîng Lewy body, vascular and frontotemporal dementia; primary progressive aphasia; mild cognitive împairment; HIV-related cognitive împairment and corticobasal degeneration.
3. 3. The composition of claim 2, wherein the composition is for use in a method of treating or preventing Parkinson’s disease.
4. 4. The composition of any of claims I-3, wherein the composition is for use in a method of treating or preventing early-onset neurodegenerative disease.
5. 5. The composition of any preceding claim, wherein the composition is for use in reducing neuron death or protecting neurons.
6. 6. The composition of any preceding claim, wherein the composition is for use in a method of preventing or delaying onset or progression of a neurodegenerative disorder.
7. 7. A composition comprising a bacterial strain of the genus Megasphaera, for use in a method of treating brain injury.
8. 8. The composition of claim 7, wherein the brain injury is stroke, includîng cérébral ischemia, focal cérébral ischemia, ischémie stroke or hémorrhagie stroke.
9. 9. The composition of any preceding daim, wherein the bacterial strain is of Megasphaera massiliensis.
10. 10. The composition of any of claims l -9, wherein the bacterial strain has a I6s rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identîcal to SEQ ID NO: l or 2.
11. 11. The composition of claim 10, wherein the bacterial strain has a 16s rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identîcal to SEQ ID NO:2, or wherein the bacterial strain has the 16s rRNA sequence represented by SEQ ID NO:2.
12. 12. The composition of claim l or 2, wherein the composition comprises a bacterial strain of the species Megasphaera massiliensis, for use in a method of treating or preventing Parkinson’s disease.

    I3. The composition of claim 7, wherein the composition comprises a bacterial strain of the species Megasphaera massiliensis, for use in a method of treating brain injury resulting from a stroke.
13. 14. The composition of any preceding claim, wherein the bacterial strain is lyophilised.

    5
14. 15. A food product or vaccine composition comprising the composition of any preceding claim, for the use of any preceding claim.
15. 16. A cell of the Megasphaera massiliensis strain deposited under accession number NCIMB 42787, or a dérivative thereof.
16. 17. A biologically pure culture of the Megasphaera massiliensis strain deposited under accession 10 number NCIMB 42787, or a dérivative thereof.
17. 18. A cell of the Megasphaera massiliensis strain deposited under accession number NCIMB 42787, or a dérivative thereof, for use in therapy.
18. 19. The cell of claim 18, wherein the cell is for use in a method defined in any of daims l -9.