Compositions containing bacterial strains of the genus *Macrococcus* and their uses

Abstract

The present invention provides compositions comprising bacterial strains for the treatment and prevention of neurodegenerative disorders.

Description

Technical Field

[0001] This invention relates to the field of compositions comprising bacterial strains isolated from the digestive tract of mammals and the use of such compositions in the treatment of diseases. Background Technology

[0002] The human gut is thought to be sterile in the womb, but immediately after birth it is exposed to a wide variety of maternal and environmental microbes. Then, a dynamic period of microbial colonization and proliferation occurs, influenced by factors such as mode of delivery, environment, diet, and host genotype, all of which affect the composition of the gut microbiota, especially during early life. Subsequently, the microbiota stabilizes and matures [1]. The human gut microbiota contains more than 500–1000 different phyla, which are essentially classified into two major bacterial groups: Bacteroidetes and Firmicutes [2]. Successful symbiotic relationships resulting from bacterial colonization in the human gut have generated a wide variety of metabolic, structural, protective, and other beneficial functions. The enhanced metabolic activity of the colonized gut ensures that dietary components that are otherwise difficult to digest are degraded with the release of byproducts, thus providing an important source of nutrients for the host. Similarly, the immunological importance of the gut microbiota is well recognized and exemplified in germ-free animals with impaired immune systems that have been functionally remodeled following the introduction of symbiotic bacteria [3–5].

[0003] Dynamic changes in the composition of the gut microbiota have been documented in gastrointestinal disorders such as inflammatory bowel disease (IBD). For example, Clostridium (… Clostridium The levels of cluster XIVa bacteria were reduced in IBD patients, while Escherichia coli ( E. coli The increase in the number of ) indicates a change in the balance between symbiotic and pathogenic organisms in the gut [6-9].

[0004] Following the recognition of the potential positive effects of certain bacterial strains on the animal gut, various strains have been proposed for the treatment of various diseases (see, for example, [10-13]). In addition, certain strains have been proposed, primarily including Lactobacillus (…). Lactobacillus ) and Bifidobacteria ( Bifidobacterium ) strains are used to treat a variety of inflammatory and autoimmune diseases not directly related to the gut (see reviews

[14] and

[15] for more information). However, the relationship between different diseases and different bacterial strains, as well as the precise role of specific bacterial strains in the gut and at the systemic level and in any particular type of disease, has not been well characterized, particularly for neurodegenerative disorders.

[0005] Recently, there has been increasing interest in the field of alterations to the gut microbiota that may play a pathophysiological role in human brain disorders

[16] . Preclinical and clinical evidence strongly suggests a link between brain development and the microbiota

[17] . A large body of preclinical literature suggests a bidirectional signaling pathway between the brain and the gut microbiota, involving multiple neurosecretory and endocrine signaling systems. Indeed, increased levels of Clostridium species in the microbiota have been associated with brain disorders

[18] , while imbalances in Bacteroidetes and Firmicutes have also been associated with brain development disorders

[19] . It has been shown that gut commensal bacteria (including Bifidobacterium spp.) Bifidobacterium Lactobacillus () Lactobacillus ), Sartella spp. Sutterella ), Prevotella spp. Prevotella ) and Rumenococcus spp. Ruminococcus Those symbiotic bacteria) and Alcaligenes family ( Alcaligenaceae Alterations in the microbiome (family) level are involved in immune-mediated central nervous system (CNS) disorders, but this has been challenged by studies showing no alteration in the microbiome between patients and healthy subjects

[19] . The administration of probiotics has also been shown to potentially be beneficial in the treatment of neurodegenerative disorders. However, these studies have failed to conclude that probiotic compositions alone can provide therapeutic benefits in the treatment of neurodegenerative diseases, and have not shown any useful role for any specific bacteria [20, 21]. This suggests that the actual role of the link between the microbiome and human brain diseases is not yet well characterized. Therefore, more direct analytical studies are needed to identify the therapeutic effects of altered microbiome on neurodegenerative disorders.

[0006] There is a need in this field for novel approaches to treating neurodegenerative disorders. There is also a need to characterize the potential role of gut bacteria so that new therapies using gut bacteria can be developed. Summary of the Invention

[0007] The inventors have developed novel therapies for the treatment and prevention of neurodegenerative disorders. The inventors have recognized that bacterial strains from the genus *Macrococcus* can effectively treat neurodegenerative diseases. As described in the examples, administration of a treatment containing *Macrococcus masculinii* (… Megasphaera massiliensis The composition can prevent reactive oxygen species and inflammation, thus acting as a neuroprotective agent. The inventors have also determined that treatment with *Macrococcus masculinii* can reduce the activity of pro-inflammatory molecules such as NFκB and IL-6 by LPS and mutants. - Synuclein activation. The inventors have determined that treatment with *Megacoccus maltii* in vitro reduces histone deacetylation activity and lipid peroxidation, which can help reduce cell death and apoptosis. The inventors have also determined that *Megacoccus maltii* can produce indole, which can alleviate inflammation and oxidative stress. Furthermore, the inventors have demonstrated that treatment with *Megacoccus maltii* can increase kynurenine levels.

[0008] The inventors have also determined that *M. martensii* produces certain organic acids, including hexanoic acid, valeric acid, and 4-hydroxyphenylacetic acid. They have also found that *M. martensii* can increase the activation of the pro-inflammatory cytokine IL-8, which can help promote neuronal myelination. Furthermore, they have determined that treatment with a combination of *M. martensii* and retinoic acid can increase the secretion of brain-derived neurotrophic factor (BDNF), which can help promote neurogenesis and axonogenesis and / or prevent cell death. They have also determined that treatment with *M. martensii* that produces valeric acid can reduce histone deacetylation, which can help reduce cell death and apoptosis. In addition, they have found that *M. martensii* can produce hexanoic acid, which can play a neuroprotective or neuroreparative role, for example, by promoting neurite growth. They have also found that *M. martensii* that produces hexanoic acid increases the expression of MAP2 (microtubule-associated protein 2), which is considered crucial for microtubule formation in axonogenesis. Therefore, the inventors discovered that *Macrococcus martensii*, which produces hexanoic acid, can be used to promote neurite growth. Thus, *Macrococcus martensii*, which produces organic acids such as hexanoic acid, valeric acid, and 4-hydroxyphenylacetic acid, and other bacteria, can be used to treat neurodegenerative disorders.

[0009] In a first embodiment, the present invention provides a composition comprising a bacterial strain of the genus *Macrococcus* for use in treatment, such as methods for treating or preventing neurodegenerative disorders.

[0010] In a particular embodiment, the present invention provides a composition comprising a bacterial strain of the genus *Macrococcus* for the treatment or prevention of diseases or symptoms selected from the group consisting of: Parkinson's disease, including progressive supranuclear palsy, Steele-Richardson-Olszewski syndrome, normal pressure hydrocephalus, vascular or arteriosclerotic Parkinson's disease, and drug-induced Parkinson's disease; Alzheimer's disease, including Benson's syndrome; multiple sclerosis; Huntington's disease; amyotrophic lateral sclerosis; Lou Gehrig's disease; motor neuron disease; prions; 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.

[0011] In a preferred embodiment, the present invention provides a composition comprising a bacterial strain of the genus *Macrococcus*, used in a method for treating or preventing Parkinson's disease, such as environmental, familial, or other Parkinson's diseases associated with a general inflammatory state. The inventors have determined that treatment with *Macrococcus* strains can reduce the inflammatory activity of pro-inflammatory molecules such as NFκB and IL-6 by LPS and mutants in in vitro models of environmental and familial Parkinson's disease. - Synuclein activation. In a preferred embodiment, the present invention provides a composition comprising a bacterial strain of *Macrococcus martini* for the treatment of Parkinson's disease. Compositions using *Macrococcus martini* can be particularly effective in treating Parkinson's disease.

[0012] In some embodiments, the compositions of the present invention are used in methods for treating or preventing early-onset neurodegenerative diseases. In some embodiments, the compositions of the present invention are used in methods for preventing or delaying the onset or progression of neurodegenerative disorders.

[0013] In a preferred embodiment of the invention, the bacterial strain in the composition is *Macrococcus masculinus*. Closely related strains, such as bacterial strains having a 16S rRNA sequence that has at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with the 16S rRNA sequence of a bacterial strain of *Macrococcus masculinus*, may also be used. Preferably, the bacterial strain has a 16S rRNA sequence having at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with SEQ ID NO:1 or 2. Preferably, the sequence identity is with respect to SEQ ID NO:2. Preferably, the bacterial strain used in the present invention has the 16S rRNA sequence represented by SEQ ID NO:2.

[0014] In some embodiments, the compositions of the present invention are for oral administration. Oral administration of the strains of the present invention can be effective against neurodegenerative disorders. Furthermore, oral administration is convenient for patients and practitioners and allows delivery to the intestine and / or partial or complete colonization of the intestine.

[0015] In some embodiments, the compositions of the present invention comprise one or more pharmaceutically acceptable excipients or carriers.

[0016] In some embodiments, the compositions of the present invention comprise lyophilized bacterial strains. Lyophilization is an efficient and convenient technique for preparing stable compositions that allow for the delivery of bacteria.

[0017] In some embodiments, the present invention provides a vaccine composition comprising the composition described above.

[0018] In addition, the present invention provides a method for treating or preventing neurodegenerative disorders, comprising administering a composition containing a bacterial strain of the genus *Macrococcus*.

[0019] In developing the above invention, the inventors have identified and characterized bacterial strains that are particularly useful for treatment. The *Macrococcus flavonoids* strain of the present invention has been shown to be effective in treating the diseases described herein, such as neurodegenerative diseases. Therefore, in another aspect, the present invention provides cells of a *Macrococcus flavonoids* strain or a derivative thereof deposited under accession number NCIMB 42787. The present invention also provides compositions comprising such cells or biologically pure cultures of such cells. The present invention further provides cells of a *Macrococcus flavonoids* strain or a derivative thereof deposited under accession number NCIMB 42787 for treatment, particularly for the treatment of the diseases described herein.

[0020] In some embodiments of the invention, the compositions are used to treat brain injury. The neuroprotective activity of the compositions of the invention and their ability to reduce histone deacetylase activity (HDAC) levels make them suitable for treating brain injury. In a preferred embodiment, the compositions of the invention are used to treat stroke, such as brain injury resulting from stroke. Attached Figure Description

[0021] Figure 1 Cell viability of neuroblastoma cells

[0022] Figure 2 Downregulation of IL-6 secretion

[0023] Figure 3 IL-8 secretion

[0024] Figure 4: -Inhibition of synuclein IL-6 and IL-8 secretion

[0025] Figure 5 : -Inhibition of synuclein-induced NFκB promoter activation

[0026] Figure 6 Inhibition of LPS-induced NFκB promoter activation

[0027] Figure 7 Changes in antioxidant capacity

[0028] Figure 8 Changes in total antioxidant capacity (lipid oxidation)

[0029] Figure 9 Changes in histone deacetylase (HDAC) activity

[0030] Figure 10 Indole production level

[0031] Figure 11 kynurenine production level

[0032] Figure 12: Mean dopamine (DA) level in the striatum ( Figure 12A ), DOPAC level ( Figure 12B ) and HVA level ( Figure 12C The data is presented as average + SEM.

[0033] Figure 13: Promoting neurite growth: optical microscopy and MAP2 gene expression ( Figure 13A ), Phalloidin immunofluorescence microscopy ( Figure 13B )

[0034] Figure 14: (a) Changes in ROS levels in U373 cells and (b) SHSY-5Y cells.

[0035] Figure 15 Neuroprotection – Cell Vitality. Figure 15 It shows the relationship with Figure 1 Same data.

[0036] Figure 16. Changes in histone deacetylase activity induced by the strain in whole cells and cell lysates ( Figure 16A Changes in acid-induced histone deacetylase activity ( Figure 16B The strain produces metabolites ( Figure 16C )

[0037] Figure 17 HDAC1 suppression ( Figure 17A HDAC2 suppression ( Figure 17B HDAC3 suppression ( Figure 17C )

[0038] Figure 18 Suppression of Class I HDACs ( Figure 18A HDAC1 suppression (); Figure 18B HDAC2 suppression Figure 18C HDAC3 suppression (); Figure 18D )

[0039] Figure 19 BDNF generation level

[0040] Figure 20 Metabolite production levels – neurotransmitters in the brain

[0041] Figure 21 : Metabolite production level – organic acids in the supernatant

[0042] Figure 22: Effects on intestinal barrier function.

[0043] Figure 23The production of neurotransmitters in the brain

[0044] Figure 24 Changes in hippocampal receptor expression – A) oxytocin receptor, B) vasopressin receptor, C) glucocorticoid receptor, and D) mineralocorticoid receptor

[0045] Figure 25 A) Changes in corticotropin-releasing hormone (CRH), B) BDNF expression, and C) TLR4 expression in the hippocampus.

[0046] Figure 26 A) Changes in hippocampal corticotropin-releasing hormone receptor 1 (CRFR1) expression and B) Corticotropin-releasing hormone receptor 2 (CRFR2) expression.

[0047] Figure 27 Changes in hippocampal expression of A) tumor necrosis factor, B) interleukin-1β, and C) IL-6.

[0048] Figure 28 A) Changes in hippocampal integrin αM (CD11b) expression and B) Changes in hippocampal serotonin 1A receptor (5-HT1A receptor) expression.

[0049] Figure 29 A) Changes in hippocampal NMDA subunit 2A (Grin2A) of the glutamate ionotropic receptor and B) Expression of NMDA subunit 2B (Grin2B) of the glutamate ionotropic receptor.

[0050] Figure 30 Changes in hippocampal expression of A) GABA A receptor 2 (GABA A2), B) GABA B receptor 1 (GABA BR1), and C) dopamine receptor 1 (DRD1).

[0051] Figure 31 Changes in amygdala receptor expression – A) oxytocin receptor, B) vasopressin receptor, C) glucocorticoid receptor, and D) mineralocorticoid receptor.

[0052] 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).

[0053] Figure 33Changes in amygdala expression of: A) integrin αM (CD11b), B) interleukin-6 (IL-6), C) glutamate ionotropic receptor NMDA subunit 2A (Grin2A), and D) glutamate ionotropic receptor NMDA subunit 2B (Grin2B).

[0054] Figure 34 Changes in amygdala expression of A) GABA-A receptor α2 subunit (GABRA2), B) GABA-A type B receptor 1 subunit (GABBR1), and C) dopamine receptor 1 (DRD1).

[0055] Figure 35 Changes in the expression of A) oxytocin receptor, B) brain-derived neurotrophic factor (BDNF), C) mineralocorticoid receptor, and D) glucocorticoid receptor in the prefrontal cortex.

[0056] Figure 36 Changes in the expression of the following receptors in the prefrontal cortex: A) Toll-like receptor 4 (TLR-4), B) corticotropin-releasing hormone receptor 1 (CRFR1), C) corticotropin-releasing hormone receptor 2 (CRFR2), and D) integrin αM (CD11b).

[0057] Figure 37 Changes in the expression of: A) interleukin-6 (IL-6), B) glutamate ionotropic receptor NMDA subunit 2A (Grin2A), C) glutamate ionotropic receptor NMDA subunit 2B (Grin2B), and D) GABA-A receptor α2 subunit (GABRA2) in the prefrontal cortex.

[0058] Figure 38 Changes in the expression of A) GABA-A receptor type B receptor subunit 1 (GABBR1) and B) dopamine receptor 1 (DRD1) in the prefrontal cortex.

[0059] Figure 39 Changes in colonic expression of A) tryptophan hydroxylase-1 (Tph1) and B) indoleamine 2,3-dioxygenase-1 (IDO1).

[0060] Figure 40 Changes in the ileum expression of A) tryptophan hydroxylase-1 (Tph1) and B) indoleamine 2,3-dioxygenase-1 (IDO1).

[0061] Figure 41 Changes in circulating tryptophan metabolite levels: A) kynurenine, B) tryptophan, and C) kynurenine / tryptophan metabolic index.

[0062] Figure 42Effects on interferon-γ production in mouse spleen cells of mice fed MRx0029

[0063] Figure 43 Effects on interleukin-1β production in spleen cells

[0064] Figure 44 Effects on interleukin-6 production in spleen cells

[0065] Figure 45 Effects on tumor necrosis factor production in spleen cells

[0066] Figure 46 Effects on interleukin-10 production in spleen cells

[0067] Figure 47 Effects of the chemical inducer CXCL1 on spleen cells

[0068] Figure 48 Changes in cecal short-chain fatty acid levels

[0069] Figure 49 Changes in gene expression levels of actin, villin, closure proteins TJP1, TJP2, MAP2, DRD2, GABRB3, SYP, PINK1, PARK7, and NSE induced by MRx0029 and MRX005.

[0070] Figure 50 SHSY5Y cell differentiation induced by MRx0005 and MRx0029. (AC) Representative image of cells immunolabeled with phalloidin and MAP2. (D-F) Image merged with DAPI images. (G-I) Cells immunolabeled with β3 tubulin. (JL) Image merged with DAPI images. Magnification x630. Western blot analysis of the effects of MRx0005 and MRx0029 treatments on SHSY5Y cells. Western blot membranes were detected with antibodies against MAP2 (M) and β3 tubulin (N). Actin was used as a loading control. Bottom panel: Representative blot from one of six independent experiments; Top panel: Relative optical density intensity. Detailed Implementation

[0071] bacterial strains

[0072] The compositions of this invention comprise bacterial strains of the genus *Macrococcus*. Examples demonstrate that bacteria of this genus can be used to treat or prevent neurodegenerative disorders. Preferred bacterial strains are *Macrococcus martensii*.

[0073] Examples of macrococcal strains used in this invention include *Megacoccus eherii* (…). Megasphaera elsdenii ), Saccharitococcus brewer Megasphaera cerevisiae), giant coccidia maseri, Megasphaera indica Oligophalae macrococcus ( Megasphaera paucivorans ), Megasphaera sueciensis and Megasphaera micronuciformis Another example of the *Macrococcus* strain used in this invention is... Megasphaera hexanoica 。 Macrococci are obligate anaerobic lactic acid fermenting gastrointestinal microorganisms of ruminant and non-ruminant mammals (including humans).

[0074] The typical strain of *Macrococcus martensii* is NP3 (=CSUR P245=DSM 26228)

[22] . The GenBank accession number for the 16S rRNA gene sequence of *Macrococcus martensii* strain NP3 is JX424772.1 (disclosed in this paper as SEQ ID NO:1).

[0075] The *Macrococcus masculinus* bacteria tested in this embodiment are referred to herein as strain MRx0029. The 16S rRNA sequence of the tested MRx0029 strain is provided in SEQ ID NO:2.

[0076] On July 13, 2017, strain MRx0029 was deposited by 4D Pharma Research Ltd (Life Sciences Innovation Building, Cornhill Road, Aberdeen, AB25 2ZS, Scotland) with NCIMB Ltd (Ferguson Building, Aberdeen, AB21 9YA, Scotland) under the designation NCIMB 42787.

[0077] It is also anticipated that bacterial strains closely related to the strains tested in the examples will be effective in treating or preventing neurodegenerative disorders. In some embodiments, the bacterial strains used in the present invention have a 16S rRNA sequence having at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with the 16S rRNA sequence of *Macrococcus maseri* bacterial strains. Preferably, the bacterial strains used in the present invention have a 16S rRNA sequence having at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with SEQ ID NO:1 or 2. Preferably, sequence identity is for SEQ ID NO:2. Preferably, the bacterial strains used in the present invention have the 16S rRNA sequence represented by SEQ ID NO:2.

[0078] Bacterial strains of biotype MRx0029 or NP3 are also expected to be effective in treating or preventing neurodegenerative disorders. Biotypes are closely related strains with the same or very similar physiological and biochemical characteristics.

[0079] Biotypes of strains MRx0029 or NP3 and suitable strains for use in this invention can be identified by sequencing other nucleotide sequences of strains MRx0029 or NP3. For example, essentially the whole genome can be sequenced, and the biotype strains used in this invention can have at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity across at least 80% (e.g., across at least 85%, 90%, 95%, or 99%, or across the whole genome) of the entire genome. Other suitable sequences for identifying biotype strains may include hsp60 or repetitive sequences such as BOX, ERIC, (GTG)5, or REP or

[23] . Biotype strains can have sequences having at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity with the corresponding sequences of strains MRx0029 or NP3.

[0080] Alternatively, the biotype of strain MRx0029 or NP3 and suitable for use in this invention can be identified by using strain MRx0029 or NP3 and restriction fragment analysis and / or PCR analysis, such as by using fluorescently amplified fragment length polymorphism (FAFLP) and repeat DNA element (rep)-PCR fingerprinting or protein analysis or partial 16S or 23S rDNA sequencing. In a preferred embodiment, such techniques can be used to identify other *Macrococcus malathi* strains.

[0081] In some embodiments, the biotype of strain MRx0029 or NP3 and suitable for use in this invention is a strain that provides the same pattern as strain MRx0029 or NP3 when subjected to amplified ribosomal DNA restriction analysis (ARDRA), for example when using Sau3AI restriction enzyme analysis (see, for example

[24] , for exemplary methods and guidelines). Alternatively, the biotype strain is identified as a strain having the same carbohydrate fermentation pattern as strain MRx0029 or NP3.

[0082] Other giant cocci strains that can be used in the compositions and methods of the present invention, such as biotypes of strains MRx0029 or NP3, can be identified using any suitable method or strategy, including the assays described in the examples. For example, strains used in the present invention can be identified by co-culturing with neuroblastoma cells and then assessing cytokine levels and neuroprotective or neuroproliferative levels. Specifically, bacterial strains having similar growth patterns, metabolic types, and / or surface antigens to strains MRx0029 or NP3 can be used in the present invention. The available strains have comparable immunomodulatory activity to strains MRx0029 or NP3. Specifically, biotype strains will evoke comparable effects in neurodegenerative disease models and at comparable cytokine levels compared to the effects shown in the examples, which can be identified by using the culture and administration protocols described in the examples.

[0083] A particularly preferred strain of the present invention is *Megacoccus vallis* strain MRx0029. This is an exemplary strain tested in the examples and has shown efficacy in treating diseases. Therefore, the present invention provides cells such as isolated cells of *Megacoccus vallis* strain MRx0029 or derivatives thereof. The present invention also provides a composition comprising cells of *Megacoccus vallis* strain MRx0029 or derivatives thereof. The present invention also provides a biologically pure culture of *Megacoccus vallis* strain MRx0029. The present invention further provides cells of *Megacoccus vallis* strain MRx0029 or derivatives thereof for treatment, particularly for the treatment of the diseases described herein.

[0084] A particularly preferred strain of the present invention is the *Megacoccus masculinus* strain deposited with accession number NCIMB 42787. This is the exemplary MRx0029 strain tested in the examples and showed efficacy in treating the disease. Therefore, the present invention provides cells, such as isolated cells, of the *Megacoccus masculinus* strain or derivative thereof deposited with accession number NCIMB 42787. The present invention also provides a composition comprising cells of the *Megacoccus masculinus* strain or derivative thereof deposited with accession number NCIMB 42787. The present invention also provides a biologically pure culture of the *Megacoccus masculinus* strain deposited with accession number NCIMB 42787. The present invention also provides cells of the *Megacoccus masculinus* strain or derivative thereof deposited with accession number NCIMB 42787 for treatment, particularly for the treatment of the disease described herein.

[0085] The derivatives of the strains of this invention can be progeny strains (offspring) or strains cultured from the original organism (subclones). The derivatives of the strains of this invention can be modified, for example, at the gene level without loss of biological activity. Specifically, the derivative strains of this invention are therapeutically effective. The derivative strains will have therapeutic activity comparable to that of the MRx0029 strain. Specifically, compared to the effects shown in the examples, the derivative strains will induce comparable effects in neurodegenerative disease models and at the cytokine level, which can be identified by using the culture and administration protocols described in the examples. The derivatives of the MRx0029 strain are typically biotypes of the MRx0029 strain.

[0086] References to cells of strain MRx0029 of *Macrococcus maseri* cover any cells that have the same safety and therapeutic efficacy characteristics as strain MRx0029, and such cells are covered in this invention.

[0087] In a preferred embodiment, the bacterial strains in the composition of the present invention are viable and capable of partially or completely colonizing the intestine.

[0088] The inventors have discovered that strains of *Macrococcus masculinus* reduce the activation of inflammatory cytokines such as IL-6 and increase the activation of the inflammatory cytokine IL-8. IL-8 is associated with myelination

[25] . Chronic inflammation induced by IL-6 ultimately leads to cell death. Therefore, the bacterial strains of the present invention are particularly useful in the treatment or prevention of neurodegenerative disorders. In some embodiments, the bacterial strains can be used to treat symptoms characterized by enhanced IL-6 activation. In some embodiments, the compositions of the present invention are used to treat or prevent neurodegenerative diseases characterized by demyelination. Many neurodegenerative diseases are characterized by demyelination. Demyelination impairs the propagation of action potentials within neurons, thereby impairing effective communication within the nervous system. IL-8 has been shown to have a positive effect on myelination and repair. Therefore, the compositions of the present invention are particularly beneficial for the treatment or prevention of neurodegenerative disorders characterized by demyelination, such as multiple sclerosis.

[0089] The inventors have discovered that the *Megacoccus masculinus* strain of the present invention alleviates symptoms of neurodegenerative diseases in disease models. For example, the inventors have found that the *Megacoccus masculinus* strain promotes neurite growth in vitro and can therefore be used to promote neuronal recovery for the treatment or prevention of neurodegenerative diseases. Therefore, the bacterial strain of the present invention is intended for the treatment or prevention of neurodegenerative diseases.

[0090] The inventors also discovered that the bacterial strains of the present invention increase BDNF activation. BDNF is a neurotrophic growth factor that has been shown to enhance neuronal differentiation and survival. Therefore, the compositions of the present invention can be used in methods for enhancing nerve cell survival to treat or prevent neurodegenerative diseases.

[0091] Another bacterium that can be used in the compositions of the present invention is *Pseudomonas diffusa*. Examples have shown that both *Pseudomonas diffusa* and *Macrococcus martensii* possess neuroprotective activity, but produce different metabolites and may have different mechanisms of action and specific neuroprotective activities. Therefore, these strains can be particularly effective when used in combination. In a preferred embodiment, the composition comprises strains of *Pseudomonas diffusa* and strains of *Macrococcus martensii*.

[0092] In this embodiment, *Parabacterium difficile*, deposited under accession number NCIMB 42382 and also referred to herein as strain MRx0005, was tested. MRX0005, MRX005, MRx005, and MRx0005 are used interchangeably herein. The 16S rRNA sequence of the tested strain MRx0005 is provided in SEQ ID NO:17. Strain MRx0005 was deposited on March 12, 2015, under the name “*Parabacterium* 755” by GT Biologics Ltd (Life Sciences Innovation Building, Aberdeen, AB25 2ZS, Scotland) with the international depository NCIMB Ltd (Ferguson Building, Aberdeen, AB21 9YA, Scotland) under accession number NCIMB 42382. GT Biologics Ltd was subsequently renamed 4DPharma Research Ltd.

[0093] In a preferred embodiment, the present invention provides a composition comprising a strain, or a derivative or biotype thereof, deposited in NCIMB with accession number NCIMB 42787, and a strain, or a derivative or biotype thereof, deposited in NCIMB with accession number NCIMB 42382, preferably for therapeutic purposes, and more preferably for treating neurodegenerative diseases such as Parkinson's disease.

[0094] Therapeutic uses

[0095] As demonstrated by the examples, the bacterial compositions of the present invention are effective in treating neurodegenerative disorders. Specifically, treatment with the compositions of the present invention increases neurogenesis and acts as a neuroprotective agent against agents that destroy dopaminergic neurons. Therefore, the compositions of the present invention can be used to treat or prevent neurodegenerative disorders caused by neuronal death.

[0096] The compositions of the present invention can reduce the activation of the NFκB promoter, which induces the production of cytokines such as IL-1β and IL-1. IL-18, TNF And IL-6. Using mutants - Synuclein processing of cells is a model of familial Parkinson's disease. A point mutation at position 53, changing from adenine to threonine, leads to... - Synaptic nuclein misfolding. Incorrectly folded - Synuclein then aggregates into insoluble fibrils, which form Lewy bodies. Therefore, the compositions of the present invention can be used to treat or prevent neurodegenerative disorders caused by neuroinflammation, protein misfolding and / or environmental exposure. The compositions of the present invention can be used to treat familial Parkinson's disease. Activation of the NFκB promoter is mediated by the TLR4 ligand. TLR4 is known to mediate cell death in the MPTP mouse model of Parkinson's disease. The compositions of the present invention can be used to inhibit the ability of TLR4 signaling to activate the NFκB promoter. Particularly relevant to PD is that both TLR2 and TLR4 are upregulated in the brains of PD patients

[26] . Furthermore, α-synuclein has been described as a ligand of TLR2

[27] , and we have demonstrated that α-synuclein also acts as a ligand of TLR4 using HEK-TLR4 cells

[28] .

[0097] The compositions of the present invention reduce the secretion of pro-inflammatory cytokines such as IL-6, which can be induced by lipopolysaccharide (LPS). Treatment of cells with LPS can mimic environmentally induced Parkinson's disease. The compositions of the present invention can be used to reduce IL-6 secretion. The compositions of the present invention can be used to treat environmental Parkinson's disease.

[0098] Examples of neurodegenerative diseases treated with the compositions of the present invention include: Parkinson's disease, including progressive supranuclear palsy, Steele-Richardson-Olszewski syndrome, normal pressure hydrocephalus, vascular or arteriosclerotic Parkinson's disease, and drug-induced Parkinson's disease; Alzheimer's disease, including Benson's syndrome; multiple sclerosis; Huntington's disease; amyotrophic lateral sclerosis; Lou Gehrig's disease; motor neuron disease; prions; 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. Other diseases treated with the compositions of the present invention are progressive inflammatory neuropathies.

[0099] In some embodiments, the compositions of the present invention are used to reduce neuronal death, particularly for treating neurodegenerative disorders. In some embodiments, the compositions of the present invention are used to protect neurons, particularly for treating neurodegenerative disorders.

[0100] In some embodiments, the compositions of the present invention are used to reduce or prevent the loss of dopaminergic cells in the substantia nigra. In some embodiments, the compositions of the present invention are used to reduce or prevent the degeneration of dopaminergic neurons in the pars compacta of the substantia nigra. In some embodiments, the compositions of the present invention are used to reduce or prevent... melanin Degeneration of dopaminergic neurons in the pars compacta and the resulting loss of their protruding nerve fibers in the striatum. In some embodiments, the compositions of the present invention are used to reduce or prevent the loss of dopaminergic neurons in the substantia nigra striatum.

[0101] In some embodiments, the compositions of the present invention are used to increase dopamine levels. In some embodiments, the compositions of the present invention are used to increase DOPAC (3,4-dihydroxyphenylacetic acid) levels. In some embodiments, the compositions of the present invention are used to increase both dopamine and DOPAC levels. In some embodiments, dopamine and / or DOPAC levels are increased in the striatum. The levels of dopamine and DOPAC can be measured using any suitable method known in the art, such as radioenzymatic methods, for example in plasma or CSF (e.g., as described in

[29] ), or reversed-phase HPLC, possibly in conjunction with electrochemical detection, for example in plasma or CSF (e.g., as described in

[30] ).

[0102] As illustrated in the embodiments, the neuroprotective effects of the compositions of the present invention mean that the compositions can be particularly effective in preventing or delaying the onset or progression of neurodegenerative disorders. In some embodiments, the compositions of the present invention are used to delay the onset or progression of neurodegenerative disorders.

[0103] The compositions of the present invention can increase the secretion of IL-8. IL-8 has been shown to play a role in neuronal myelination. In some embodiments, the compositions of the present invention can be used to increase IL-8 secretion.

[0104] The therapeutic compositions of the present invention can increase the activation of BDNF. BDNF acts on certain neurons in the central nervous system to support the survival of existing neurons and to aid in the growth and development of new neurons and synapses. BDNF is active in the hippocampus, cortex, and basal forebrain and is important for long-term memory. Therefore, the compositions of the present invention can be used to increase the secretion of BDNF. Therefore, the compositions can be used to treat neurodegenerative diseases associated with long-term memory impairment. The compositions of the present invention can be used to improve long-term memory, particularly for improving long-term memory impaired by neurodegenerative diseases.

[0105] In some embodiments, the compositions of the present invention increase mitochondrial metabolic activity in neuronal cells.

[0106] Regulation of the gut-microbiome axis

[0107] Communication between the gut and the brain (microbiota-gut-brain axis) occurs through a bidirectional neurohumoral communication system. Recent evidence suggests that the gut microbiota can regulate brain development and produce behavioral phenotypes through the microbiota-gut-brain axis. Indeed, many reviews have suggested the role of the microbiota-gut-brain axis in maintaining central nervous system function and have suggested its dysfunction in the development of central nervous system disorders and symptoms.

[16] ,

[19] ,

[31]

[0108] The bidirectional communication between the brain and the gut (i.e., the gut-brain axis) involves the central nervous system, neuroendocrine and neuroimmune systems, including the hypothalamus-pituitary-adrenal (HPA) axis, the sympathetic and parasympathetic arms of the autonomic nervous system (ANS), including the enteric nervous system (ENS) and the vagus nerve, and the gut microbiota.

[0109] As demonstrated in the examples, the compositions of the present invention can modulate the gut-microbiota axis and reduce cell death associated with neurodegenerative disorders. Therefore, the compositions of the present invention can be used to treat or prevent neurodegenerative disorders, particularly those disorders and symptoms associated with dysfunction of the gut-microbiota axis.

[0110] In certain embodiments, the compositions of the present invention can be used to treat or prevent diseases or conditions selected from the group consisting of: Parkinson's disease, including progressive supranuclear palsy, Steele-Richardson-Olszewski syndrome, normal pressure hydrocephalus, vascular or arteriosclerotic Parkinson's disease, and drug-induced Parkinson's disease; Alzheimer's disease, including Benson's syndrome; multiple sclerosis; Huntington's disease; amyotrophic lateral sclerosis; Lou Gehrig's disease; motor neuron disease; prions; spinocerebellar ataxia; spinal muscular atrophy; dementia, including Lewy bodies; vascular and frontotemporal dementia; primary progressive aphasia; mild cognitive impairment; HIV-related cognitive impairment; and corticobasal degeneration.

[0111] The compositions of the present invention can be used specifically for the treatment or prevention of chronic diseases, for the treatment or prevention of diseases in patients who are unresponsive to other therapies (such as treatment with levodopa, dopamine agonists, MAO-B inhibitors, COMT inhibitors, glutamate antagonists, and / or anticholinergic drugs), and / or for the treatment or prevention of tissue damage and symptoms associated with dysfunction of the gut-microbiota axis.

[0112] In some embodiments, the compositions of the present invention modulate the CNS. In some embodiments, the compositions of the present invention modulate the autonomic nervous system (ANS). In some embodiments, the compositions of the present invention modulate the enteric nervous system (ENS). In some embodiments, the compositions of the present invention modulate the hypothalamic-pituitary-adrenal (HPA) axis. In some embodiments, the compositions of the present invention modulate neuroendocrine pathways. In some embodiments, the compositions of the present invention modulate neuroimmune pathways. In some embodiments, the compositions of the present invention modulate the CNS, ANS, ENS, HPA axis and / or neuroendocrine and neuroimmune pathways. In some embodiments, the compositions of the present invention modulate the levels of symbiotic metabolites and / or gastrointestinal permeability of a subject.

[0113] Signal transduction along the gut-microbiota axis is regulated by the nervous system. Therefore, in some embodiments, the compositions of the present invention regulate signal transduction in the nervous system. In some embodiments, the compositions of the present invention regulate signal transduction in the central nervous system. In some embodiments, the compositions of the present invention regulate signal transduction in sensory neurons. In other embodiments, the compositions of the present invention regulate signal transduction in motor neurons. In some embodiments, the compositions of the present invention regulate signal transduction in the enteric nervous system (ANS). In some embodiments, the ANS is the parasympathetic nervous system. In a preferred embodiment, the compositions of the present invention regulate vagal nerve signal transduction. In other embodiments, the ANS is the sympathetic nervous system. In other embodiments, the compositions of the present invention regulate signal transduction in the enteric nervous system. In some embodiments, signal transduction in ANS and ENS neurons responds directly to the contents of the gastrointestinal tract. In other embodiments, signal transduction in ANS and ENS neurons responds indirectly to neurochemicals produced by luminal bacteria. In other embodiments, signal transduction in ANS and ENS neurons responds to neurochemicals produced by luminal bacteria or enteroendocrine cells. In some preferred embodiments, neuronal activation of the ENS affects vagal afferentation in CNS function. In some embodiments, the compositions of the present invention modulate the activity of enterochromaffin cells.

[0114] Neurodegenerative diseases

[0115] Parkinson's disease

[0116] Parkinson's disease is a common neurodegenerative disease characterized neuropathologically by the degeneration of heterogeneous populations of nerve cells (dopamine-producing cells). Clinical diagnosis of Parkinson's disease requires bradykinesia and at least one of the following core symptoms: resting tremor; muscle rigidity; and postural reflex disturbances. Other signs and symptoms that may appear or develop during disease progression include autonomic dysfunction (drooling, seborrhea, constipation, urinary dysfunction, sexual dysfunction, orthostatic hypotension, hyperhidrosis), sleep disturbances, and disturbances in olfactory or temperature sensation. Parkinson's disease is a neurodegenerative disease that may develop or persist due to dysfunction of the gut-microbiota axis. Therefore, in a preferred embodiment, the compositions of the present invention are used to treat or prevent Parkinson's disease in a subject.

[0117] In another preferred embodiment, the present invention provides a composition comprising a bacterial strain of the genus *Macrococcus*, used in a method for treating or preventing Parkinson's disease. The composition comprising a bacterial strain of the genus *Macrococcus* can improve motor and cognitive function in a Parkinson's disease model. Treatment with *Macrococcus* strains can modulate signal transduction in the central, autonomic, and enteric nervous systems; modulate the activity of the HPA axis pathway; modulate neuroendocrine and / or neuroimmune pathways; and modulate the levels of symbiotic metabolites, inflammatory markers, and / or gastrointestinal permeability in the subject, all of which are related to the neuropathology of Parkinson's disease. In a preferred embodiment, the present invention provides a composition comprising a bacterial strain of the species *Macrococcus vallis*, used in a method for treating or preventing Parkinson's disease. The composition using *Macrococcus vallis* can be particularly effective in treating Parkinson's disease.

[0118] In preferred embodiments, the compositions of the present invention prevent, reduce, or alleviate one or more symptoms of Parkinson's disease in a subject. In preferred embodiments, the compositions of the present invention prevent, reduce, or alleviate one or more core symptoms of Parkinson's disease in a subject. In some embodiments, the compositions of the present invention prevent, reduce, or alleviate bradykinesia in a subject. In some embodiments, the compositions of the present invention prevent, reduce, or alleviate resting tremor, muscle rigidity, and / or postural reflex disorders in a subject. In some embodiments, the compositions of the present invention prevent, reduce, or alleviate one or more symptoms associated with the progression of Parkinson's disease, said symptoms being selected from autonomic disorders (drooling, seborrhea, constipation, urinary disorders, sexual dysfunction, orthostatic hypotension, hyperhidrosis), sleep disorders, and disturbances of smell or temperature sensation.

[0119] In a preferred embodiment, the compositions of the present invention prevent, reduce, or alleviate depressive symptoms coexisting with Parkinson's disease. In some embodiments, the compositions of the present invention improve verbal memory and / or executive function. In some embodiments, the compositions of the present invention improve attention, working memory, verbal fluency, and / or anxiety.

[0120] In other preferred embodiments, the compositions of the present invention prevent, reduce, or alleviate cognitive impairment coexisting with Parkinson's disease.

[0121] In some embodiments, the compositions of the present invention prevent, reduce, or alleviate the progression of Parkinson's disease. In some embodiments, the compositions of the present invention prevent, reduce, or alleviate later motor complications. In some embodiments, the compositions of the present invention prevent, reduce, or alleviate later motor fluctuations. In some embodiments, the compositions of the present invention prevent, reduce, or alleviate neuronal loss. In some embodiments, the compositions of the present invention improve symptoms of Parkinson's disease dementia (PDD). In some embodiments, the compositions of the present invention prevent, reduce, or alleviate impairments in executive function, attention, and / or working memory. In some embodiments, the compositions of the present invention improve dopaminergic neurotransmission. In some embodiments, the compositions of the present invention prevent, reduce, or alleviate impaired dopaminergic neurotransmission.

[0122] In some embodiments, the compositions of the present invention improve symptoms of Parkinson's disease according to a symptom or diagnostic scale. In some embodiments, the test used to assess the improvement in motor function symptoms in Parkinson's disease is the Unified Parkinson's Disease Rating Scale (UPDRS). Specifically, UPDRS II takes into account activities of daily living, and UPDRS III takes into account motor examinations.

[0123] In some embodiments, the compositions of the present invention improve symptoms associated with PDD based on symptom or diagnostic tests and / or scales. In some embodiments, the tests or scales are selected from the Hopkins Language Learning Test-Revised (HVLT-R); the Delis-Kaplan Executive Function System (D-KEFS) Color-Word Interference Test; the Hamilton Depression Rating Scale (HAM-D 17; Depression); the Hamilton Anxiety Rating Scale (HAM-A; Anxiety); and the Unified Parkinson's Disease Rating Scale (UPDRS; PD Symptom Severity).

[0124] In some embodiments, the compositions of the present invention improve the Clinical Global Impression – Overall Improvement (CGI-I) scale used to assess mental and neurological disorders. In some embodiments, the compositions of the present invention show a positive effect on overall social and occupational impairment in subjects with Parkinson's disease.

[0125] In some embodiments, the compositions of the present invention are used to treat or prevent neurological disorders such as Parkinson's disease in a subject, wherein said use includes reducing or preventing the loss of dopaminergic cells in the substantia nigra. In some embodiments, the compositions of the present invention are used to treat or prevent neurological disorders such as Parkinson's disease in a subject, wherein said use includes reducing or preventing the degeneration of dopaminergic neurons in the pars compacta of the substantia nigra. In some embodiments, the compositions of the present invention are used to treat or prevent neurological disorders such as Parkinson's disease in a subject, wherein said use includes reducing or preventing the degeneration of dopaminergic neurons in the pars compacta of the substantia nigra and the resulting loss of their protruding nerve fibers in the striatum. In some embodiments, the compositions of the present invention are used to treat or prevent neurological disorders such as Parkinson's disease in a subject, wherein said use includes reducing or preventing the loss of dopaminergic neurons in the substantia nigra striatum.

[0126] In some embodiments, the compositions of the present invention are used to treat or prevent neurological disorders such as Parkinson's disease in a subject, wherein said use includes increasing dopamine levels. In some embodiments, the compositions of the present invention are used to treat or prevent neurological disorders such as Parkinson's disease in a subject, wherein said use includes increasing DOPAC levels. In some embodiments, the compositions of the present invention are used to treat or prevent neurological disorders such as Parkinson's disease in a subject, wherein said use includes increasing dopamine and DOPAC levels. In some embodiments, dopamine and / or DOPAC levels are increased in the striatum.

[0127] Alzheimer's disease and dementia

[0128] In DSM-5, the term dementia is replaced by the terms severe neurocognitive impairment and mild neurocognitive impairment. Neurocognitive impairment is a heterogeneous class of mental illnesses. The most common neurocognitive impairment is Alzheimer's disease, followed by vascular dementia or a combination of both. 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 associated with dementia.

[0129] Alzheimer's disease and dementia are also characterized by neuronal loss; therefore, the neuroprotective and neuroproliferative effects shown in the embodiments of the compositions of the present invention suggest that they may be used to treat or prevent these conditions.

[0130] The criteria for dementia under DSM-5 are evidence of a significant decline in cognition from previous performance levels in one or more of the following cognitive domains: learning and memory; language; executive function; complex attention; perceptual-motor and social cognition. Cognitive deficits must interfere with the independence of daily activities. Furthermore, cognitive deficits do not occur only in cases of delirium and cannot be better explained by another mental disorder (such as MDD or schizophrenia).

[0131] In addition to the main symptoms, subjects with neurodegenerative disorders also exhibited behavioral and psychiatric symptoms, including agitation, aggression, depression, anxiety, apathy, psychosis, and sleep-wake cycle disturbances.

[0132] Neurodegenerative disorders may develop or persist due to dysfunction of the gut-microbiota axis. Therefore, in a preferred embodiment, the compositions of the present invention are used to treat or prevent neurodegenerative disorders in a subject. In a preferred embodiment, the neurodegenerative disorder is Alzheimer's disease. In other embodiments, the neurodegenerative disorder is selected from vascular dementia; mixed Alzheimer's disease and vascular dementia; Lewy body disease; frontotemporal dementia; Parkinson's dementia; Creutzfeldt-Jakob disease; Huntington's disease; and Weil-Codd syndrome.

[0133] In preferred embodiments, the compositions of the present invention prevent, reduce, or alleviate one or more symptoms of neurodegenerative disorders in a subject. In some embodiments, the compositions of the present invention prevent, reduce, or alleviate the occurrence of cognitive decline in a subject. In some embodiments, the compositions of the present invention improve the performance level of a subject with neurodegenerative disorders in one or more cognitive domains selected from: learning and memory; language; executive function; complex attention; perceptual-motor and social cognition. In some embodiments, the compositions of the present invention prevent, reduce, or alleviate the occurrence of one or more behavioral and psychiatric symptoms associated with neurodegenerative disorders selected from: agitation, aggression, depression, anxiety, apathy, psychosis, and sleep-wake cycle disorders.

[0134] In some embodiments, the compositions of the present invention prevent, reduce, or alleviate symptomatic disease by intervening in suspected pathogenic mechanisms in the preclinical stage. In some embodiments, the compositions of the present invention improve disease correction by slowing or halting symptom progression. In some embodiments, the slowing or halting of symptom progression is associated with evidence of delaying underlying neuropathological processes. In a preferred embodiment, the compositions of the present invention improve symptoms of neurodegenerative disorders, including enhanced cognitive and functional improvements. In a preferred embodiment, the compositions of the present invention improve behavioral and psychiatric symptoms of dementia (BPSD). In a preferred embodiment, the compositions of the present invention improve the ability of subjects with neurodegenerative disorders to perform daily activities.

[0135] In a preferred embodiment, the compositions of the present invention improve both cognition and function in subjects with Alzheimer's disease. In some embodiments, the compositions of the present invention improve cognitive endpoints in subjects with Alzheimer's disease. In some embodiments, the compositions of the present invention improve functional endpoints in subjects with Alzheimer's disease. In a preferred embodiment, the compositions of the present invention improve both cognitive and functional endpoints in subjects with Alzheimer's disease. In another preferred embodiment, the compositions of the present invention improve overall clinical response (overall endpoint) in subjects with Alzheimer's disease.

[0136] In some embodiments, the compositions of the present invention improve symptoms of neurodegenerative disorders based on symptom or diagnostic tests. In some embodiments, tests for assessing improvement in symptoms of Alzheimer's disease (and other neurodegenerative disorders) are selected from objective cognition, activities of daily living, overall assessments of changes, health-related quality of life tests, and tests assessing behavioral and psychiatric symptoms of neurodegenerative disorders.

[0137] In some implementations, objective cognitive tests used to assess symptom improvement employ the Alzheimer's Disease Rating Scale Cognitive Subscale (ADAS-cog) and the classic ADAS scale. In some implementations, the Neurophysiological Tests for Alzheimer's Disease (NTB) suite is used to assess cognitive symptom improvement.

[0138] In some implementations, the overall assessment of change uses the Clinical Global Impression – Overall Improvement (CGI-I) scale to assess mental and neurological disorders. In some implementations, the overall scale is the Clinician Interview-Based Change Impression Plus (CIBIC-plus). In some implementations, the overall scale is the Alzheimer's Disease Collaborative Research Unit Clinician Global Change Impression (ADCS-CGIC).

[0139] In some implementations, health-related quality of life measures are Alzheimer's disease-related QOL (ADRQL) and QOL-Alzheimer's disease (QOL-AD).

[0140] In some implementations, the tests for assessing the behavioral and psychiatric symptoms of neurodegenerative disorders are selected from behavioral pathologies including: the Alzheimer's Disease Rating Scale (BEHAVE-AD); the Behavioral Rating Scale for Dementia (BRSD); the Neuropsychiatric Inventory (NPI); and the Cohen-Mansfield Agitation Inventory (CMAI).

[0141] In some embodiments, the compositions of the present invention are particularly effective in preventing, reducing, or alleviating neurodegenerative disorders when used in combination with another therapy for treating neurodegenerative disorders. In some embodiments, such a therapy includes acetylcholinesterase inhibitors, including donepezil (Aricept®), galantamine (Razadyne®), rivastigmine (Exelon®), and memantine.

[0142] Multiple sclerosis

[0143] Multiple sclerosis (MS) is a demyelinating disease in which the myelin sheath surrounding neurons in the brain and spinal cord is destroyed. The exact underlying cause of MS is unknown, but it is thought to vary from person to person. Some forms of MS are hereditary. Environmental factors are also considered a cause of MS. In some individuals, a combination of both genetic and environmental factors may trigger the onset of MS.

[0144] Symptoms associated with MS are diverse. Subjects may exhibit almost any neurological symptom related to autonomic, visual, motor, or sensory control disturbances. The exact symptoms will vary depending on the location of neuronal damage / demyelination.

[0145] IL-8 is involved in myelin formation. Therefore, the compositions of the present invention can be used for myelin regeneration of neurons in subjects suffering from MS. The compositions of the present invention can also be used to protect neurons from demyelination. In other words, the compositions of the present invention can be used in methods of treating or preventing multiple sclerosis by restoring or preventing the loss of neuronal myelin sheaths.

[0146] In some embodiments, the compositions of the present invention prevent, reduce, or alleviate one or more symptoms of MS in a subject. In some embodiments, the compositions of the present invention prevent, reduce, or alleviate fatigue in a subject. In some embodiments, the compositions of the present invention prevent, reduce, or alleviate resting tremor, muscle weakness, muscle spasms, muscle rigidity, paresthesia, and / or ataxia in a subject. In some embodiments, the compositions of the present invention prevent, reduce, or alleviate one or more symptoms associated with the progression of MS, said symptoms being selected from the list of: autonomic disorders: constipation, urinary disorders, sexual dysfunction, dysphagia, dysarthria, syncope, vertigo, and / or dizziness; sleep disorders; and disturbances of smell or temperature sensation. In some embodiments, the compositions of the present invention prevent, reduce, or alleviate one or more ocular symptoms associated with MS. In some embodiments, ocular symptoms are selected from the list of: vision loss, eye pain, color blindness, diplopia, and / or involuntary eye movements in a subject.

[0147] In some embodiments, the compositions of the present invention prevent, reduce or alleviate dizziness, vertigo, neuropathic pain, musculoskeletal pain, cognitive impairment, fecal incontinence, dysphagia, dysarthria, or any combination thereof.

[0148] In some embodiments, the compositions of the present invention prevent, reduce, or alleviate depressive symptoms or anxiety symptoms coexisting with MS.

[0149] In some implementations, symptom improvement is determined using the 2017 McDonald guidelines for diagnosing MS.

[0150] In some embodiments, treatment with the compositions of the present invention results in a reduction in the incidence or severity of MS. In some embodiments, the compositions of the present invention are used to reduce the relapse rate or severity of relapse. In some embodiments, treatment with the compositions of the present invention can prevent a decline in motor function or cause an improvement in MS-related motor function. In some embodiments, the compositions of the present invention are used to prevent a decline in motor function or to improve motor function to treat MS. In some embodiments, treatment with the compositions of the present invention can prevent the development of paralysis in MS. In some embodiments, the compositions of the present invention are used to prevent paralysis to treat MS.

[0151] In some embodiments, the compositions of the present invention are used to prevent multiple sclerosis (MS) in patients identified as having a risk of MS or diagnosed with early-stage or relapsing-remitting MS. The compositions of the present invention can be used to prevent the development of MS. The compositions of the present invention can be used to prevent the progression of MS. In some embodiments, the compositions of the present invention are used in patients identified as having a genetic predisposition to MS, such as the major histocompatibility complex (MHC) class II phenotype, human leukocyte antigen (HLA)-DR2, or HLA-DR4.

[0152] The compositions of the present invention can be used to control or alleviate multiple sclerosis. The compositions of the present invention can be particularly useful in reducing symptoms associated with multiple sclerosis. Treatment or prevention of multiple sclerosis can refer to, for example, a reduction in the severity of symptoms or a decrease in the frequency or extent of exacerbations that are triggers for the patient. In some embodiments, the compositions of the present invention slow or stop the progression of the disease.

[0153] In some embodiments, the compositions of the present invention are used to treat relapsing-remitting MS. In alternative embodiments, the compositions of the present invention are used to treat progressive MS, such as secondary progressive MS (SPMS), which develops over time after a diagnosis of RRMS; primary progressive MS (PPMS), which exhibits gradual, continuous neurological deterioration; and progressive relapsing MS (PRMS), which is similar to PPMS but has relapse overlap.

[0154] In some embodiments, the compositions of the present invention are used to treat one or more symptoms of MS, said MS symptoms being selected from the group consisting of: fatigue, vision problems, numbness, tingling, muscle spasms, muscle stiffness, muscle weakness, mobility problems, pain, thinking, learning and planning problems, depression and anxiety, sexual problems, bladder problems, bowel problems, and speech and swallowing difficulties.

[0155] Neurochemical factors, neuropeptides and neurotransmitters, and the gut-brain axis

[0156] As mentioned above, the gut-microbiota axis is regulated by many different physiological systems. This axis is modulated by numerous signaling molecules. Alterations in the levels of these signaling molecules can lead to neurodegenerative diseases. Experiments conducted by the inventors have shown that administration of macrococcal strains, particularly *Megacoccus masculinii*, can regulate the levels of indole and kynurenine. Dysregulation of these metabolites may contribute to neurodegenerative diseases such as Parkinson's disease.

[0157] In some embodiments, the compositions of the present invention regulate the levels of brain monoamines and their metabolites. In a preferred embodiment, the metabolite is kynurenine. In some embodiments, the compositions of the present invention regulate kynurenine, which is a major pathway of tryptophan metabolism that acts as a pathway for the production of nicotinamide adenine dinucleotide (NAD+). Kynurenine can be metabolized to neuroactive compounds such as kynurenine quinolinic acid (KYNA) and 3-hydroxy-1-kynurenine (3-OH-1-KYN), and further metabolized to quinolinic acid (QUIN) in a further step. Dysregulation of the kynurenine pathway may lead to activation of the immune system and accumulation of potentially neurotoxic compounds. Alterations in kynurenine metabolism may be involved in the development of Parkinson's disease. Kynurenine levels have been shown to be reduced in the frontal cortex, putamen, and substantia nigra pars compacta of PD patients

[32] . Therefore, in some embodiments, the compositions of the present invention are used to increase kynurenine levels to treat Parkinson's disease.

[0158] In some embodiments of the invention, the compositions of the invention can increase the level of kynurenine. Increased kynurenine levels have been shown to alleviate MPP+-induced neuronal cell death in vitro in human dopaminergic neuroblastoma cell lines

[33] . In some embodiments, kynurenine and kynurenine quinolinate can activate the GI aryl hydrocarbon receptor (Ahr) and the GPR35 receptor. Activation of the Ahr receptor induces the production of IL-22, which can suppress local inflammation. Activation of GPR35 induces the production of inositol triphosphate and Ca2+ mobilization.

[0159] In some embodiments, the compositions of the present invention regulate indole levels. In a preferred embodiment, the metabolite is kynurenine. In some embodiments, the compositions of the present invention regulate kynurenine, which is a major pathway in tryptophan metabolism.

[0160] Signal transduction along the gut-brain axis is regulated by the levels of neurochemicals, neuropeptides, and neurotransmitters. Therefore, in some embodiments, the compositions of the present invention modulate the levels of neurochemicals, neuropeptides, and neurotransmitters. Thus, in some preferred embodiments, the compositions of the present invention directly alter CNS biochemistry.

[0161] Signal transduction along the gut-microbiota axis is regulated by gamma-aminobutyric acid (GABA) levels. Therefore, in a preferred embodiment, the compositions of the present invention regulate GABA levels. GABA is an inhibitory neurotransmitter that reduces neuronal excitability. In some embodiments, the compositions of the present invention increase GABA levels. In some embodiments, the compositions of the present invention decrease GABA levels. In some embodiments, the compositions of the present invention alter GABAergic neurotransmission. In some embodiments, the compositions of the present invention regulate GABA transcription levels in different regions of the central nervous system. In some embodiments, symbiotically derived GABA crosses the blood-brain barrier and directly affects neurotransmission. In some embodiments, the compositions of the present invention result in a reduction of GABA in the hippocampus, amygdala, and / or locus coeruleus. In some embodiments, the compositions of the present invention result in an increase of GABA in cortical regions.

[0162] Immune response

[0163] Signal transduction along the gut-microbiota axis is modulated through immune responses and alterations in inflammatory factors and markers. Therefore, in some embodiments, the compositions of the present invention can modulate immune responses. In some embodiments, the compositions of the present invention modulate systemic levels of circulating neuroimmune signaling molecules. In some preferred embodiments, the compositions of the present invention modulate the production of pro-inflammatory cytokines and inflammation. In some embodiments, the compositions of the present invention modulate inflammatory states. In some embodiments, the compositions of the present invention reduce the production and secretion of IL-6. In some embodiments, the compositions of the present invention reduce the activation of the NFκB promoter. In some embodiments, the compositions of the present invention are capable of modulating the activation of IL-6 production via potent pro-inflammatory endotoxin lipopolysaccharide (LPS). In some embodiments, the compositions of the present invention are capable of modulating the activation of IL-6 production via LPS and - Activation of the NFκB promoter by mutant synuclein proteins such as A53T. Increased circulating cytokine levels are closely associated with various neurodegenerative disorders, including Parkinson's disease, dementia, and Alzheimer's disease. In some embodiments, the compositions of the present invention are used to reduce IL-6 and / or NFκB levels to treat neurodegenerative disorders.

[0164] In some embodiments, the compositions of the present invention increase the secretion of IL-8. IL-8 has been shown to induce myelination and restore or maintain effective neuronal communication. Therefore, in some embodiments, the compositions of the present invention are used to induce myelination to treat neurodegenerative diseases. In some embodiments, the compositions of the present invention are used to restore neuronal communication. In some embodiments, the compositions of the present invention are used to maintain neuronal communication.

[0165] Signaling along the gut-microbiota axis is regulated by the levels of symbiotic metabolites. Therefore, in some embodiments, the compositions of the present invention regulate systemic levels of microbiota metabolites. In some preferred embodiments, the compositions of the present invention regulate the levels of short-chain fatty acids (SCFAs). In some embodiments, SCFA levels are increased or decreased. In some embodiments, the SCFA is butyrate (BA). In some embodiments, the SCFA is propionic acid (PPA). In some embodiments, the SCFA is acetic acid. In some embodiments, the compositions of the present invention regulate the ability of SCFAs to cross the blood-brain barrier.

[0166] Histone acetylation and deacetylation are important epigenetic regulators of gene expression. Imbalances in histone acetylation and deacetylation can lead to apoptosis. Dysregulation of such histone acetyltransferases is involved in the pathogenesis of age-related neurodegenerative diseases such as Parkinson's disease, Huntington's disease, Alzheimer's disease, amyotrophic lateral sclerosis, and cognitive decline

[34] . Therefore, in some embodiments, the compositions of the present invention can modulate histone deacetylase activity. In some embodiments, the compositions of the present invention can reduce histone deacetylase activity. In some embodiments, the compositions of the present invention can reduce histone acetylase activity.

[0167] Patients with neurodegenerative diseases (including Parkinson's disease, Huntington's disease, Alzheimer's disease, and amyotrophic lateral sclerosis) exhibit high levels of lipid peroxidation. Lipids are readily oxidized by reactive oxygen species, and the brain is rich in polyunsaturated fatty acids. Therefore, in some embodiments, the compositions of the present invention can modulate lipid peroxidation. In some embodiments, the compositions of the present invention can reduce lipid peroxidation. Reducing oxidative damage caused by reactive oxygen species can be used to target early stages of neurodegenerative diseases. Therefore, in some embodiments, the compositions of the present invention are used to treat early neurodegeneration. Similarly, therefore, in some embodiments, the compositions of the present invention are used to prevent the development of neurodegenerative disorders. In such embodiments, the compositions of the present invention can be used in patients who have been identified as being at risk of developing neurodegenerative disorders.

[0168] Signal transduction along the gut-microbiota axis is regulated by gastrointestinal permeability levels. Therefore, in some embodiments, the compositions of the present invention alter the integrity of the gastrointestinal epithelium. In some embodiments, the compositions of the present invention modulate gastrointestinal permeability. In some embodiments, the compositions of the present invention modulate the barrier function and integrity of the gastrointestinal tract. In some embodiments, the compositions of the present invention modulate gastrointestinal motility. In some embodiments, the compositions of the present invention modulate the transport of symbiotic metabolites and inflammatory signaling molecules from the gastrointestinal lumen into the bloodstream.

[0169] Signal transduction along the gut-microbiota axis is regulated by the composition of the gut microbiome. Therefore, in some embodiments, the compositions of the present invention regulate the gut microbiome composition. In some embodiments, the compositions of the present invention prevent microbiome dysbiosis and the increase of associated toxic metabolites (e.g., LPS). In some embodiments, the compositions of the present invention regulate the level of Clostridium in the gut. In a preferred embodiment, the compositions of the present invention reduce the level of Clostridium in the gut. In some embodiments, the compositions of the present invention reduce the level of Campylobacter jejuni. In some embodiments, the compositions of the present invention regulate the proliferation of harmful anaerobic bacteria and the production of neurotoxins by these bacteria. In some embodiments, the compositions of the present invention regulate the microbiome levels of Lactobacillus and / or Bifidobacterium. In some embodiments, the compositions of the present invention regulate the microbiome levels of Sartella, Prevotella, Ruminococcus, and / or Alcaligenes. In some embodiments, the compositions of the present invention increase Lactobacillus plantarum and / or Saccharomyces boulardii (…). Saccharomyces boulardii ) level.

[0170] Brain injury

[0171] Examples demonstrate that the compositions of the present invention have neuroprotective effects and HDAC inhibitory activity. HDAC2 is a key target for functional recovery from stroke

[35] , and HDAC inhibition can prevent white matter damage

[36] , therefore the compositions of the present invention can be used to treat brain injury.

[0172] In some embodiments, the compositions of the present invention are used to treat brain injury. In some embodiments, the brain injury is traumatic brain injury. In some embodiments, the brain injury is acquired brain injury. In some embodiments, the compositions of the present invention are used to treat brain injury caused by trauma. In some embodiments, the compositions of the present invention are used to treat brain injury caused by a tumor. In some embodiments, the compositions of the present invention are used to treat brain injury caused by a stroke. In some embodiments, the compositions of the present invention are used to treat brain injury caused by cerebral hemorrhage. In some embodiments, the compositions of the present invention are used to treat brain injury caused by encephalitis. In some embodiments, the compositions of the present invention are used to treat brain injury caused by cerebral hypoxia. In some embodiments, the compositions of the present invention are used to treat brain injury caused by cerebral hypoxia.

[0173] In a preferred embodiment, the composition of the present invention is used to treat stroke. The effects shown in the examples are particularly relevant to the treatment of stroke. A stroke occurs when blood flow to at least a portion of the brain is interrupted. If there is insufficient blood supply to provide oxygen and nutrients to brain tissue and to remove waste from it, brain cells rapidly begin to die. Stroke symptoms depend on the brain region affected by insufficient blood flow. Symptoms include paralysis, muscle numbness or weakness, loss of balance, dizziness, sudden severe headache, speech impairment, memory loss, loss of reasoning ability, sudden confusion, visual disturbances, coma, or even death. Stroke is also known as a brain attack or cerebrovascular accident (CVA). If sufficient blood flow is restored within a short period, stroke symptoms may be transient. However, if insufficient blood flow persists for a long time, symptoms may be permanent.

[0174] In some embodiments, a stroke is cerebral ischemia. Cerebral ischemia occurs when there is insufficient blood flow to brain tissue to meet metabolic needs. In some embodiments, cerebral ischemia is focal cerebral ischemia, i.e., limited to a specific area of ​​the brain. In some embodiments, cerebral ischemia is global cerebral ischemia, i.e., encompassing a wide area of ​​brain tissue. Focal cerebral ischemia typically occurs when cerebral blood vessels are partially or completely blocked, thereby reducing blood flow to a specific area of ​​the brain. In some embodiments, focal cerebral ischemia is ischemic stroke. In some embodiments, ischemic stroke is thrombotic, i.e. caused by a thrombus or blood clot that develops in cerebral blood vessels and restricts or obstructs blood flow. In some embodiments, ischemic stroke is thrombotic stroke. In some embodiments, ischemic stroke is embolic, i.e. caused by an embolus or an unattached mass traveling through the bloodstream and restricting or obstructing blood flow at a location remote from its point of origin. In some embodiments, ischemic stroke is embolic stroke. Global cerebral ischemia typically occurs when blood flow to the brain is generally obstructed or reduced. In some implementations, global cerebral ischemia is caused by insufficient perfusion, i.e., due to shock. In other implementations, global cerebral ischemia is a result of cardiac arrest.

[0175] In some embodiments, the subject diagnosed with brain injury suffers from cerebral ischemia. In some embodiments, the subject diagnosed with brain injury suffers from focal cerebral ischemia. In some embodiments, the subject diagnosed with brain injury suffers from ischemic stroke. In some embodiments, the subject diagnosed with brain injury suffers from thrombotic stroke. In some embodiments, the subject diagnosed with brain injury suffers from embolic stroke. In some embodiments, the subject diagnosed with brain injury suffers from global cerebral ischemia. In some embodiments, the subject diagnosed with brain injury suffers from hypoperfusion. In some embodiments, the subject diagnosed with brain injury suffers from cardiac arrest.

[0176] In some embodiments, the compositions of the present invention are used to treat cerebral ischemia. In some embodiments, the compositions of the present invention are used to treat focal cerebral ischemia. In some embodiments, the compositions of the present invention are used to treat ischemic stroke. In some embodiments, the compositions of the present invention are used to treat thrombotic stroke. In some embodiments, the compositions of the present invention are used to treat embolic stroke. In some embodiments, the compositions of the present invention are used to treat global cerebral ischemia. In some embodiments, the compositions of the present invention are used to treat hypoperfusion.

[0177] In some embodiments, the stroke is a hemorrhagic stroke. A hemorrhagic stroke is caused by bleeding within or around the brain, leading to swelling, pressure, and damage to brain cells and tissues. Hemorrhagic strokes are often the result of a weakened blood vessel rupture and seeping into the surrounding brain. In some embodiments, a hemorrhagic stroke is a cerebral hemorrhage, which is caused by bleeding within the brain tissue itself. In some embodiments, a cerebral hemorrhage is caused by bleeding in the brain parenchyma. In some embodiments, a cerebral hemorrhage is caused by intraventricular hemorrhage. In some embodiments, a hemorrhagic stroke is a subarachnoid hemorrhage, which is bleeding occurring outside the brain tissue but still within the skull. In some embodiments, a hemorrhagic stroke is the result of cerebral amyloid angiopathy. In some embodiments, a hemorrhagic stroke is the result of a cerebral aneurysm. In some embodiments, a hemorrhagic stroke is the result of a cerebral arteriovenous malformation (AVM).

[0178] In some embodiments, the subject diagnosed with brain injury has hemorrhagic stroke. In some embodiments, the subject diagnosed with brain injury has cerebral hemorrhage. In some embodiments, the subject diagnosed with brain injury has parenchymal hemorrhage. In some embodiments, the subject diagnosed with brain injury has intraventricular hemorrhage. In some embodiments, the subject diagnosed with brain injury has subarachnoid hemorrhage. In some embodiments, the subject diagnosed with brain injury has cerebral amyloid angiopathy. In some embodiments, the subject diagnosed with brain injury has cerebral aneurysm. In some embodiments, the subject diagnosed with brain injury has cerebral AVM.

[0179] In some embodiments, the compositions of the present invention are used to treat hemorrhagic stroke. In some embodiments, the compositions of the present invention are used to treat cerebral hemorrhage. In some embodiments, the compositions of the present invention are used to treat parenchymal hemorrhage. In some embodiments, the compositions of the present invention are used to treat intraventricular hemorrhage. In some embodiments, the compositions of the present invention are used to treat subarachnoid hemorrhage. In some embodiments, the compositions of the present invention are used to treat cerebral amyloid angiopathy. In some embodiments, the compositions of the present invention are used to treat cerebral aneurysm. In some embodiments, the compositions of the present invention are used to treat cerebral AVM.

[0180] While restoring sufficient blood flow to the brain after a period of interruption can effectively alleviate stroke-related symptoms, it can also lead to further damage to brain tissue. During the interruption, affected tissues lack oxygen and nutrients, and the sudden restoration of blood flow can cause inflammation and oxidative damage by inducing oxidative stress. This is known as reperfusion injury, and it is well-documented not only after a stroke but also after a period of ischemia or hypoxia when blood supply returns to tissues, after a heart attack, or after other tissue injuries. In some embodiments, subjects diagnosed with brain injury have suffered reperfusion injury due to stroke. In some embodiments, the compositions of the present invention are used to treat stroke-induced reperfusion injury.

[0181] Transient ischemic attack (TIA) (commonly known as mini-stroke) is a recognized warning sign of a more serious stroke. Therefore, subjects with one or more TIAs have a higher risk of stroke. In some embodiments, subjects diagnosed with brain injury have TIA. In some embodiments, the compositions of the present invention are used to treat TIA. In some embodiments, the compositions of the present invention are used to treat brain injury in subjects with TIA.

[0182] Hypertension, hyperlipidemia, family history of stroke, heart disease, diabetes, cerebral aneurysm, arteriovenous malformation, sickle cell disease, vasculitis, hemorrhagic conditions, use of nonsteroidal anti-inflammatory drugs (NSAIDs), smoking, heavy alcohol consumption, illicit drug use, obesity, lack of physical activity, and unhealthy diet are all considered risk factors for stroke. Specifically, lowering blood pressure has been shown to prevent both ischemic and hemorrhagic strokes [37, 38]. In some embodiments, the compositions of the present invention are used to treat brain injury in a subject with at least one stroke risk factor. In some embodiments, the subject has two stroke risk factors. In some embodiments, the subject has three stroke risk factors. In some embodiments, the subject has four stroke risk factors. In some embodiments, the subject has more than four stroke risk factors. In some embodiments, the subject has hypertension. In some embodiments, the subject has high cholesterol. In some embodiments, the subject has a family history of stroke. In some embodiments, the subject has heart disease. In some embodiments, the subject has diabetes. In some embodiments, the subject has a cerebral aneurysm. In some embodiments, the subject has arteriovenous malformation. In some embodiments, the subject has vasculitis. In some implementations, the subject has sickle cell disease. In some implementations, the subject has a bleeding disorder. In some implementations, the subject has a history of using nonsteroidal anti-inflammatory drugs (NSAIDs). In some implementations, the subject smokes. In some implementations, the subject drinks alcohol heavily. In some implementations, the subject uses drugs illegally. In some implementations, the subject is obese. In some implementations, the subject is overweight. In some implementations, the subject lacks physical activity. In some implementations, the subject has an unhealthy diet.

[0183] Examples demonstrate that the compositions of the present invention, when administered before the occurrence of an injury event, can be used to treat brain injury and aid recovery. Therefore, the compositions of the present invention can be particularly effective in treating brain injury when administered to subjects at risk of brain injury (such as stroke).

[0184] In some embodiments, the compositions of the present invention are used to reduce damage caused by potential brain injury, preferably stroke. When the compositions are administered before potential brain injury occurs, particularly to patients identified as being at risk of brain injury, the resulting damage can be reduced.

[0185] Examples demonstrate that the compositions of the present invention, when administered after an injury event, can be used to treat brain injury and aid recovery. Therefore, when administered to subjects after a brain injury such as a stroke, the compositions of the present invention can be particularly used to treat brain injury.

[0186] In some embodiments, the compositions of the present invention treat brain injury by reducing motor impairment. In some embodiments, the compositions of the present invention treat brain injury by improving motor function. In some embodiments, the compositions of the present invention treat brain injury by improving muscle strength. In some embodiments, the compositions of the present invention treat brain injury by improving memory. In some embodiments, the compositions of the present invention treat brain injury by improving social acceptance. In some embodiments, the compositions of the present invention treat brain injury by improving neurological function.

[0187] Treatment of brain injury can refer to, for example, reducing the severity of symptoms. Treatment of brain injury can also refer to reducing neurological impairment following a stroke. The compositions of the present invention for treating stroke can be provided to subjects before a stroke occurs, for example, in patients identified as having a stroke risk. The compositions of the present invention for treating stroke can be provided after a stroke, for example, during the recovery period. The compositions of the present invention for treating stroke can be provided during the acute recovery period (i.e., up to one week after the stroke). The compositions of the present invention for treating stroke can be provided during the subacute recovery period (i.e., one week to three months after the stroke). The compositions of the present invention for treating stroke can be provided during the chronic recovery period (three months after the stroke).

[0188] In some embodiments, the compositions of the present invention are used in combination with a second active agent. In some embodiments, the compositions of the present invention are used in combination with aspirin or tissue plasminogen activator (tPA). Other second agents include other antiplatelet drugs (such as clopidogrel), anticoagulants (such as heparin, warfarin, apixaban, dabigatran, edoxaban, or rivaroxaban), antihypertensive drugs (such as diuretics, ACE inhibitors, calcium channel blockers, beta-blockers, or alpha-blockers), or statins. The compositions of the present invention can improve patient response to a second active agent.

[0189] In some embodiments, the compositions of the present invention reduce the effects of ischemia on tissues. In some embodiments, the compositions of the present invention reduce the amount of tissue damage caused by ischemia. In some embodiments, the tissue damaged by ischemia is brain tissue. In some embodiments, the compositions of the present invention reduce the number of necrotic or dead cells. In some embodiments, the compositions of the present invention reduce the number of apoptotic or dead cells. In some embodiments, the compositions of the present invention reduce the number of both necrotic and apoptotic cells. In some embodiments, the compositions of the present invention prevent cell death due to necrosis and / or apoptosis. In some embodiments, the compositions of the present invention prevent cell death due to ischemia-induced necrosis and / or apoptosis. In some embodiments, the compositions of the present invention improve the recovery of tissues damaged by ischemia. In some embodiments, the compositions of the present invention increase the clearance rate of necrotic and / or apoptotic cells. In some embodiments, the compositions of the present invention improve the clearance efficiency of necrotic and / or apoptotic cells. In some embodiments, the compositions of the present invention improve the replacement and / or regeneration of cells within tissues. In some embodiments, the compositions of the present invention improve the replacement and / or regeneration of cells within tissues damaged by ischemia. In some embodiments, the compositions of the present invention improve the overall histology of the tissue (e.g., during biopsy).

[0190] Application method

[0191] Preferably, the compositions of the present invention are administered to the gastrointestinal tract to allow the bacterial strains of the present invention to be delivered into the intestine and / or to partially or completely colonize the intestine. Typically, the compositions of the present invention are administered orally, or they may be administered rectally, intranasally, or via the oral or sublingual route.

[0192] In some embodiments, the compositions of the present invention can be applied as foam, spray, or gel.

[0193] In some embodiments, the compositions of the present invention can be administered as suppositories such as rectal suppositories, for example in the form of cocoa butter, synthetic stearin (e.g., suppocire, witepsol), glycerin-gelatin, polyethylene glycol, or soap-glycerin compositions.

[0194] In some embodiments, the compositions of the present invention are administered to the gastrointestinal tract via tubes (such as nasogastric tubes, orogastric tubes, gastric tubes, jejunostomy tubes (J tubes), percutaneous endoscopic gastrostomy (PEG)) or ports (such as chest wall ports providing a pathway to the stomach, jejunal or other suitable pathway ports).

[0195] The compositions of the present invention may be administered once, or they may be administered sequentially as part of a treatment regimen. In some embodiments, the compositions of the present invention are administered daily.

[0196] In some embodiments of the invention, treatment according to the invention is accompanied by an assessment of the patient's gut microbiota. If the delivery of the strains of the invention is not achieved and / or partial or complete colonization of the strains of the invention is not achieved, resulting in no observed efficacy, treatment may be repeated; or if delivery and / or partial or complete colonization is successful and efficacy is observed, treatment may be discontinued.

[0197] In some embodiments, the compositions of the present invention may be applied to pregnant animals, such as mammals, such as humans, to prevent inflammatory or autoimmune diseases in the womb and / or in the offspring after birth.

[0198] The compositions of the present invention can be administered to patients who have been diagnosed with or identified as being at risk of developing neurodegenerative diseases. The compositions can also be administered as a preventative measure to prevent the development of neurodegenerative diseases in healthy patients.

[0199] The compositions of the present invention can be administered to patients identified as having an abnormal gut microbiota. For example, the patients may have reduced or absent colonization of macrococci, particularly *Macrococcus masculinii*.

[0200] Typically, the compositions of the present invention are used to treat humans, although they can be used to treat animals, including monogastric mammals such as poultry, pigs, cats, dogs, horses, or rabbits. The compositions of the present invention can be used to enhance the growth and performance of animals. If administered to animals, oral tube feeding can be used.

[0201] Composition

[0202] Typically, the compositions of the present invention contain bacteria. In a preferred embodiment of the invention, the compositions are formulated in lyophilized form. For example, the compositions of the present invention may comprise granules or gelatin capsules, such as hard gelatin capsules, containing the bacterial strains of the present invention.

[0203] Preferably, the composition of the present invention comprises lyophilized bacteria. The lyophilization of bacteria is a well-established procedure, and relevant instructions can be found, for example, in references

[39] , [],

[41] ).

[0204] Alternatively, the compositions of the present invention may comprise live, effective bacterial cultures.

[0205] In some embodiments, the bacterial strains in the compositions of the present invention are not inactivated, for example, not heat-inactivated. In some embodiments, the bacterial strains in the compositions of the present invention are not killed, for example, not heat-killed. In some embodiments, the bacterial strains in the compositions of the present invention are not attenuated, for example, not heat-attenuated. For example, in some embodiments, the bacterial strains in the compositions of the present invention are not killed, inactivated, and / or attenuated. For example, in some embodiments, the bacterial strains in the compositions of the present invention are live. For example, in some embodiments, the bacterial strains in the compositions of the present invention are viable. For example, in some embodiments, the bacterial strains in the compositions of the present invention are capable of partially or completely colonizing the intestine. For example, in some embodiments, the bacterial strains in the compositions of the present invention are viable and capable of partially or completely colonizing the intestine.

[0206] In some embodiments, the composition comprises a mixture of live bacterial strains and killed bacterial strains.

[0207] In a preferred embodiment, the compositions of the present invention are encapsulated to allow delivery of bacterial strains to the intestine. Encapsulation protects the compositions from degradation until delivery to the target site by rupture, for example, by chemical or physical stimulation such as pressure, enzyme activity, or physical disintegration that can be triggered by pH changes. Any suitable encapsulation method may be used. Exemplary encapsulation techniques include embedding within a porous matrix, attaching or adsorbing onto the surface of a solid carrier, agglomerating by flocculation or with a crosslinking agent, and mechanically encapsulating after a microporous membrane or microcapsule. Guidance on encapsulation that can be used to prepare the compositions of the present invention can be found, for example, in references

[42] and

[43] .

[0208] The composition can be administered orally and may be in the form of tablets, capsules, or powder. Encapsulated products are preferred because *Macrococcus* is an anaerobic bacterium. Other ingredients (such as vitamin C) may be included as oxygen scavengers and probiotic substrates to improve in vivo delivery and / or partial or complete colonization and survival.

[0209] The composition can be formulated as probiotics.

[0210] The compositions of the present invention comprise a therapeutically effective amount of the bacterial strain of the present invention. A therapeutically effective amount of the bacterial strain is sufficient to exert a beneficial effect on the patient. A therapeutically effective amount of the bacterial strain may be sufficient to produce delivery to the patient's intestine and / or partial or complete colonization of the subject's intestine.

[0211] For example, for adults, a suitable daily dose of bacteria could be approximately 1 x 10⁻⁶. 3 To approximately 1 x 10 11 One colony-forming unit (CFU); for example, approximately 1 x 10⁻⁶. 7 To approximately 1 x 1010 One CFU; in another instance, approximately 1 x 10 6 To approximately 1 x 10 10 One CFU.

[0212] In some embodiments, the composition comprises about 1 x 10⁻⁶ units relative to the weight of the composition. 6 To approximately 1 x 10 11 A bacterial strain in the amount of CFU / g; for example, about 1 x 10⁻⁶ CFU / g. 8 To approximately 1 x 10 10 One CFU / g. The dosage can be, for example, 1g, 3g, 5g, and 10g.

[0213] Typically, probiotics (such as the compositions of the present invention) are optionally combined with at least one suitable probiotic compound. The probiotic compound is typically a poorly digestible carbohydrate, such as oligosaccharides or polysaccharides, or sugar alcohols, which are not degraded or absorbed in the upper digestive tract. Known probiotics include commercial products such as inulin and galactose-oligosaccharides.

[0214] In some embodiments, the probiotic composition of the present invention comprises a probiotic compound in an amount of about 1 to about 30% by weight (e.g., about 5 to 20% by weight) relative to the total weight composition. The carbohydrate may be selected from the group consisting of: fructose-oligosaccharides (or FOS), short-chain fructose-oligosaccharides, inulin, isomaltitol-oligosaccharides, pectin, xylooligosaccharides (or XOS), chitosan-oligosaccharides (or COS), β-glucan, modified gum arabic and resistant starch, polydextrin, D-tagatose, acacia fiber, carob, oats, and citrus fiber. In one aspect, the probiotic is a short-chain fructose-oligosaccharide (hereinafter referred to herein as FOS-cc for simplicity); said FOS-cc is a poorly digestible carbohydrate, typically obtained by the conversion of beet sugar and comprising a sucrose molecule bound with three glucose molecules.

[0215] In some embodiments, the compositions of the present invention are used in combination with another therapeutic compound to treat or prevent neurodegenerative disorders. In some embodiments, the compositions of the present invention are administered together with a nutritional supplement that modulates neuroprotection or neuroproliferation. In preferred embodiments, the nutritional supplement comprises or consists of nutritional vitamins. In some embodiments, the vitamins are vitamin B6, magnesium, dimethylglycine (vitamin B16), and vitamin C. In some embodiments, the compositions of the present invention are administered in combination with another probiotic.

[0216] In some embodiments, the compositions of the present invention are used to enhance the effect of a second agent on neurodegenerative diseases. The immunomodulatory effects of the compositions of the present invention can make the brain more sensitive to conventional therapies such as levodopa, dopamine agonists, MAO-B inhibitors, COMT inhibitors, glutamate antagonists, or anticholinergic drugs, which are exemplary second agents administered in combination with the compositions of the present invention (sequentially or simultaneously).

[0217] The compositions of the present invention may contain pharmaceutically acceptable excipients or carriers. Examples of such suitable excipients can be found in reference

[44] . Acceptable carriers or diluents for therapeutic purposes are well known in the pharmaceutical industry and are described, for example, in reference

[45] . Examples of suitable carriers include lactose, starch, glucose, methylcellulose, magnesium stearate, mannitol, sorbitol, etc. Examples of suitable diluents include ethanol, glycerol, and water. The choice of pharmaceutical carrier, excipient, or diluent may be made with consideration of the intended route of administration and standard pharmaceutical practice. The pharmaceutical composition may contain any suitable binder, lubricant, suspending agent, coating agent, or solubilizer in addition to the carrier, excipient, or diluent. Examples of suitable binders include starch; gelatin; natural sugars such as glucose, anhydrous lactose, free-flowing lactose, β-lactose; corn sweeteners; natural and synthetic gums such as gum arabic, tragacanth, or sodium alginate, carboxymethyl cellulose, and polyethylene glycol. Examples of suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, etc. Preservatives, stabilizers, dyes, and even flavoring 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 also be used.

[0218] In some embodiments, the compositions of the present invention comprise one or more strains of bacteria from the genus *Macrococcus* and do not contain bacteria from any other genus, or contain only trace or biologically irrelevant amounts of bacteria from another genus. Therefore, in some embodiments, the present invention provides a composition for treatment comprising one or more strains of bacteria from the genus *Macrococcus*, which does not contain bacteria from any other genus, or contains only trace or biologically irrelevant amounts of bacteria from another genus.

[0219] In some embodiments, the compositions of the present invention comprise bacterial strains of one or more species of *Macrococcus maseri* and do not contain bacteria from any other species, or contain only trace or biologically irrelevant amounts of bacteria from another species. Therefore, in some embodiments, the present invention provides a composition for treatment comprising bacterial strains of one or more species of *Macrococcus maseri*, which does not contain bacteria from any other species, or contains only trace or biologically irrelevant amounts of bacteria from another species.

[0220] In some embodiments, the compositions of the present invention comprise bacterial strains of one or more species of *Macrococcus masculinus* and do not contain bacteria from any other species of *Macrococcus*, or contain only trace or biologically irrelevant amounts of bacteria from another species of *Macrococcus*. Therefore, in some embodiments, the present invention provides a composition for treatment comprising bacterial strains of one or more species of *Macrococcus masculinus*, which does not contain bacteria from any other species of *Macrococcus*, or contains only trace or biologically irrelevant amounts of bacteria from another species of *Macrococcus*.

[0221] In some embodiments, the compositions of the present invention comprise a single bacterial strain or species and contain no other bacterial strains or species. Such compositions may contain only trace or biologically unrelated amounts of other bacterial strains or species. Such compositions may be cultures substantially free of other kinds of organisms.

[0222] In some embodiments, the present invention provides a composition for treatment comprising a single bacterial strain of the genus *Macrococcus*, which does not contain bacteria from any other strain, or contains only trace or biologically unrelated amounts of bacteria from another strain.

[0223] In some embodiments, the present invention provides a composition for treatment comprising a single bacterial strain of *Macrococcus maseri*, which does not contain bacteria from any other strain, or contains only trace or biologically unrelated amounts of bacteria from another strain.

[0224] In some embodiments, the compositions of the present invention comprise more than one bacterial strain. For example, in some embodiments, the compositions of the present invention comprise more than one strain from 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 contain no bacteria from any other species. In some embodiments, the compositions of the present invention comprise fewer than 50 strains from the same species (e.g., fewer than 45, 40, 35, 30, 25, 20, 15, 12, 10, 9, 8, 7, 6, 5, 4, or 3 strains), and optionally contain no bacteria from any other species. In some embodiments, the compositions of the present 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 bacterial strains from the same species, and optionally do not contain bacteria from any other species. The present invention includes any combination of the foregoing.

[0225] In some embodiments, the composition comprises a microbial aggregate. For example, in some embodiments, the composition comprises a *Macrococcus* bacterial strain as part of the microbial aggregate. For example, in some embodiments, the *Macrococcus* bacterial strain exists in combination with one or more (e.g., at least 2, 3, 4, 5, 10, 15, or 20) other bacterial strains from other genera, and the *Macrococcus* bacterial strain can survive symbiotically with said other bacterial strains in vivo in the intestine. For example, in some embodiments, the composition comprises a *Macrococcus* bacterial strain combined with bacterial strains from different genera. In some embodiments, the microbial aggregate comprises two or more bacterial strains obtained from a fecal sample from a single organism, such as a human. In some embodiments, the microbial aggregate is not found together in nature. For example, in some embodiments, the microbial aggregate comprises bacterial strains obtained from fecal samples from at least two different organisms. In some embodiments, the two different organisms are from the same species, such as 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 human and non-human mammals.

[0226] In some embodiments, the compositions of the present invention further comprise a bacterial strain having the same safety and therapeutic efficacy characteristics as strain MRx0029, but which is not MRx0029 or not *Macrococcus martensii*.

[0227] In some embodiments of the present invention, where the composition comprises more than one bacterial strain, species, or genus, each bacterial strain, species, or genus can be used individually, simultaneously, or sequentially. For example, the composition may comprise all of the more than one bacterial strain, species, or genus, or the bacterial strain, species, or genus may be stored separately and applied individually, simultaneously, or sequentially. In some embodiments, the more than one bacterial strain, species, or genus is stored separately but mixed together before use.

[0228] In some embodiments, the bacterial strains used in this invention are obtained from adult feces. In some embodiments where the compositions of this invention comprise more than one bacterial strain, all bacterial strains are obtained from adult feces, or, if other bacterial strains are present, they are present in only trace amounts. The bacteria may be cultured after being obtained from adult feces and used in the compositions of this invention.

[0229] As described above, in some embodiments, one or more strains of *Macrococcus* are the sole therapeutic agent in the composition of the present invention. In some embodiments, the bacterial strain in the composition is the sole therapeutic agent in the composition of the present invention.

[0230] The compositions used in accordance with the present invention may or may not require market approval.

[0231] In some embodiments, the present invention provides the above-described pharmaceutical composition, wherein the bacterial strain is lyophilized. In some embodiments, the present invention provides the above-described pharmaceutical composition, wherein the bacterial strain is spray-dried. In some embodiments, the present invention provides the above-described pharmaceutical composition, wherein the bacterial strain is lyophilized or spray-dried and is viable. In some embodiments, the present invention provides the above-described pharmaceutical composition, wherein the bacterial strain is lyophilized or spray-dried and is viable. In some embodiments, the present invention provides the above-described pharmaceutical composition, wherein the bacterial strain is lyophilized or spray-dried and is capable of partially or completely colonizing the intestine. In some embodiments, the present invention provides the above-described pharmaceutical composition, wherein the bacterial strain is lyophilized or spray-dried and is viable and capable of partially or completely colonizing the intestine.

[0232] In some cases, the lyophilized bacterial strains are reconstituted prior to application. In some cases, this reconstitution is performed using the diluents described herein.

[0233] The compositions of the present invention may contain pharmaceutically acceptable excipients, diluents or carriers.

[0234] In some embodiments, the present invention provides a pharmaceutical composition comprising: the bacterial strain of the present invention; and a pharmaceutically acceptable excipient, carrier, or diluent; wherein the amount of said bacterial strain is sufficient to treat neurodegenerative disorders when administered to a subject in need.

[0235] In some embodiments, the present invention provides a pharmaceutical composition comprising: the bacterial strain of the present invention; and a pharmaceutically acceptable excipient, carrier, or diluent; wherein the amount of said bacterial strain is sufficient to treat or prevent neurodegenerative disorders.

[0236] In some embodiments, the present invention provides the above-described pharmaceutical composition, wherein the amount of the bacterial strain is about 1 × 10⁻⁶ per unit weight of the composition. 3 From approximately 1 × 10 11 Colony forming units / gram.

[0237] In some embodiments, the present invention provides the above pharmaceutical compositions, wherein the compositions are administered at doses of 1 g, 3 g, 5 g, or 10 g.

[0238] In some embodiments, the present invention provides the above-described pharmaceutical compositions, wherein the compositions are administered by methods selected from the group consisting of: oral, rectal, subcutaneous, nasal, oral, and sublingual administration.

[0239] In some embodiments, the present invention provides the above pharmaceutical composition comprising a carrier selected from the group consisting of lactose, starch, glucose, methylcellulose, magnesium stearate, mannitol, and sorbitol.

[0240] In some embodiments, the present invention provides the above pharmaceutical composition comprising a diluent selected from the group consisting of ethanol, glycerol, and water.

[0241] In some embodiments, the present invention provides the above pharmaceutical composition comprising excipients selected from the group consisting of: starch, gelatin, glucose, anhydrous lactose, free-flowing lactose, β-lactose, corn sweetener, gum arabic, tragacanth, sodium alginate, carboxymethyl cellulose, polyethylene glycol, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, and sodium chloride.

[0242] In some embodiments, the present invention provides the above-described pharmaceutical composition, which further comprises at least one of a preservative, an antioxidant, and a stabilizer.

[0243] In some embodiments, the present invention provides the above pharmaceutical composition comprising a preservative selected from the group consisting of sodium benzoate, sorbic acid, and esters of p-hydroxybenzoic acid.

[0244] In some embodiments, the present invention provides the above-described pharmaceutical composition, wherein the bacterial strain is lyophilized.

[0245] In some embodiments, the present 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 atmosphere with 50% relative humidity, at least 80% of the bacterial strains, as measured in colony-forming units, remain for at least the following time periods: 1 month, 3 months, 6 months, 1 year, 1.5 years, 2 years, 2.5 years, or 3 years.

[0246] In some embodiments, the compositions of the present invention are provided in a sealed container containing the composition as described herein. In some embodiments, the sealed container is a pouch or bottle. In some embodiments, the compositions of the present invention are provided in a syringe containing the composition as described herein.

[0247] In some embodiments, the compositions of the present invention are provided as pharmaceutical preparations. For example, the compositions may be provided as tablets or capsules. In some embodiments, the capsules are gelatin capsules (“gel sleeves”).

[0248] In some embodiments, the compositions of the present invention are administered orally. Oral administration may involve swallowing, allowing the compound to enter the gastrointestinal tract, and / or may involve oral, tongue, or sublingual administration, through which the compound enters the bloodstream directly from the mouth.

[0249] Pharmaceutical formulations suitable for oral administration include solid fillers, solid microparticles, semi-solids, and liquids (including multiphase or dispersion systems), such as tablets; soft or hard capsules containing multiple particles or nanoparticles, liquids (e.g., aqueous solutions), emulsions, or powders; sugar tablets (including liquid fillers); chewable tablets; gels; rapidly dispersible dosage forms; films; oval bodies; sprays; and oral / mucosal adhesive patches.

[0250] In some embodiments, the pharmaceutical formulation is an enteric-coated formulation, i.e., a gastric-tolerant formulation (e.g., tolerant to gastric pH) suitable for delivering the composition of the invention to the intestine via oral administration. Enteric-coated formulations can be particularly useful when the bacteria or another component of the composition are acid-sensitive, for example, readily degraded under acidic conditions.

[0251] In some embodiments, the enteric-coated formulation comprises an enteric coating. In some embodiments, the formulation is an enteric-coated dosage form. For example, the formulation may be an enteric-coated tablet or an enteric-coated capsule, etc. The enteric coating may be a conventional enteric coating, such as the conventional coating for tablets, capsules, etc., for oral delivery. The formulation may include a film coating, such as a thin film layer of an enteric polymer, such as an acid-insoluble polymer.

[0252] In some embodiments, the enteric-coated formulation is inherently enteric-coated, for example, gastric-tolerant and does not require an enteric coating. Therefore, in some embodiments, the formulation is an enteric-coated formulation that does not include an enteric coating. In some embodiments, the formulation is a capsule made of a thermogelatinizing material. In some embodiments, the thermogelatinizing material is a cellulose material, such as methylcellulose, hydroxymethylcellulose, or hydroxypropyl methylcellulose (HPMC). In some embodiments, the capsule comprises a shell free of any film-forming polymers. In some embodiments, the capsule comprises a shell and the shell contains hydroxypropyl methylcellulose and does not contain any film-forming polymers (e.g., see

[46] ). In some embodiments, the formulation is an inherently enteric-coated capsule (e.g., Vcaps® from Capsugel).

[0253] In some embodiments, the formulation is a soft capsule. A soft capsule is a capsule that possesses a certain elasticity and softness due to the addition of softening agents such as glycerin, sorbitol, maltitol, and polyethylene glycol present in the capsule shell. Soft capsules can be produced, for example, based on gelatin or starch. Gelatin-based soft capsules are commercially available from various suppliers. Depending on the method of administration, such as oral or rectal, soft capsules can have various shapes; they can be, for example, round, oval, elliptical, or torpedo-shaped. Soft capsules can be produced by conventional methods such as the Scherer process, Accogel process, or microdrop or blow-drip methods.

[0254] Cultivation methods

[0255] The bacterial strains used in this invention can be cultured using standard microbiological techniques as detailed in, for example, references

[47] , [] and

[49] .

[0256] The solid or liquid culture medium used for cultivation can be YCFA agar or YCFA medium. YCFA medium may contain (approximately per 100 ml): tyrophonium (1.0 g), yeast extract (0.25 g), NaHCO3 (0.4 g), cysteine ​​(0.1 g), K2HPO4 (0.045 g), KH2PO4 (0.045 g), NaCl (0.09 g), (NH4)2SO4 (0.09 g), MgSO4·7H2O (0.009 g), CaCl2 (0.009 g), resazurin (0.1 mg), heme chloride (1 mg), biotin (1 μg), cobalamin (1 μg), para-aminobenzoic acid (3 μg), folic acid (5 μg), and pyridoxine (15 μg).

[0257] Bacterial strains used in vaccine compositions

[0258] The inventors have determined that the bacterial strains of the present invention can be used to treat or prevent neurodegenerative disorders. This is likely a result of the effects of the bacterial strains of the present invention on the host's immune system. Therefore, the compositions of the present invention can also be used to prevent neurodegenerative disorders when administered as a vaccine composition. In some such embodiments, the bacterial strains of the present invention can be killed, inactivated, or attenuated. In some such embodiments, the composition may contain a vaccine adjuvant. In some embodiments, the composition is intended for administration by injection, such as subcutaneous injection.

[0259] General Principles

[0260] Unless otherwise indicated, the practice of this invention will be carried out using conventional chemical, biochemical, molecular biological, immunological, and pharmaceutical methods within the art. Such techniques are well described in the literature. See, for example, references

[50] and [51, 57], etc.

[0261] The term “comprising” encompasses both “including” and “consisting of”, such as a composition that “comprising” X may consist of only X or may include other things, such as X+Y.

[0262] With numerical values x The relevant term "about" is optional and refers to an average value, for example... x + 10%.

[0263] The word "substantially" does not exclude, for example, "completely," meaning that a composition "substantially free of" Y can be completely free of Y. If necessary, the word "substantially" can be omitted from the definition of this invention.

[0264] The reference to percentage sequence identity between two nucleotide sequences means that, when aligned, the percentage of nucleotides in the two sequences being compared is the same. This alignment and percentage homology or sequence identity can be determined using software programs known in the art, such as those described in section 7.7.18 of reference

[58] . Preferred alignments are determined using the Smith-Waterman homology search algorithm with an affine gap search having a gap open penalty of 12, a gap extension penalty of 2, and a BLOSUM matrix of 62. The Smith-Waterman homology search algorithm is disclosed in reference

[59] .

[0265] Unless expressly stated otherwise, a process or method comprising multiple steps may include additional steps at the beginning or end of the method, or may include additional inserted steps. Furthermore, where appropriate, steps may be combined, omitted, or performed in an alternative order.

[0266] Various embodiments of the invention are described herein. It should be understood that the features specified in each embodiment can be combined with other specified features to provide further embodiments. Specifically, embodiments highlighted herein as suitable, typical, or preferred can be combined with each other (except where they are mutually exclusive).

[0267] Modes for implementing the present invention

[0268] Example 1 – Efficacy of bacterial inoculum as a neuroprotective agent

[0269] Overview

[0270] Neuroblastoma cells were treated with a composition comprising the bacterial strain according to the invention. The SH-SY5Y neuroblastoma cells used are dopamine-producing and have been established as an in vitro model for studying neurodegenerative diseases. The ability of the bacterial strain to increase neurogenesis was observed. Neuroblastoma cells were also treated with the dopaminergic neurotoxin 1-methyl-4-phenylpyridinium (MPP), which induces permanent symptoms of Parkinson's disease in neuroblastoma cells. The ability of the bacterial strain to act as a neuroprotective agent against MPP was investigated.

[0271] Materials and methods

[0272] bacterial strains

[0273] Macrococcus martensii MRx0029 ； Parabacterium dilatatum MRX0005

[0274] cell lines

[0275] SH-SY5Y neuroblastoma cells were purchased from ECCACC (catalog number: 94030304) and grown in MEM (Sigma Aldrich, catalog number M2279) supplemented with nutrient mixture F-12 Ham (Sigma Aldrich, catalog number N4888).

[0276] method

[0277] Once growth began, SH-SY5Y neuroblastoma cells were seeded at 11,000 cells / well in 96-well plates and incubated for 2 days. Cells were then transferred to differentiation medium (containing 1% FBS) and 10 μM retinoic acid (Sigma Aldrich, catalog number R2625-100MG). The differentiation medium was changed every other day, and cells were harvested on day 7 of differentiation. Cells were pretreated with MPP (Sigma Aldrich, catalog number D048-1G) for 8 hours, with or without. Subsequently, cells were treated with 10% bacterial supernatant and incubated overnight. Cell viability was measured using the CCK-8 reagent (Sigma Aldrich, Cell Counting Kit – 8, catalog number 96992-3000TESTS-F) and read at 450 nm.

[0278] result

[0279] The results of these experiments are Figure 1 As shown in the figure, treatment of neuroblastoma cells with MRx0029 or MRX0005 resulted in increased neuronal proliferation. Neuroblastoma cells treated with MPP and a bacterial strain together exhibited increased cell viability compared to cells treated with MPP alone (which had reduced viability). These data suggest that the bacterial strain can act as a neuroprotective agent. For cells treated with MRX0029, the protective effect was greater, salvaging more viability than the positive control cells treated with quercetin. These data suggest that the bacterial strain can act as a neuroprotective agent.

[0280] Example 2 – Efficacy of bacterial inoculum in reducing IL-6 secretion.

[0281] Overview

[0282] Activation of pro-inflammatory cytokines is associated with neuronal damage in neurodegenerative diseases. Lipopolysaccharide (LPS) is a known stimulant of the pro-inflammatory cytokine IL-6. Human glioblastoma astrocytomas were treated with a composition comprising a bacterial strain according to the invention and LPS to observe its ability to regulate IL-6 levels.

[0283] Materials and methods

[0284] bacterial strains

[0285] Macrococcus martensii MRx0029

[0286] cell lines

[0287] MG U373 is a human glioblastoma astrocytoma derived from a malignant tumor, acquired from Sigma-Aldrich (catalog number 08061901-1VL). MG U373 human glioblastoma astrocytoma cells were grown in MEM (Sigma-Aldrich, catalog number M-2279) supplemented with 10% FBS, 1% penicillin-streptomycin, 4 mM L-Glut, 1X MEM non-essential amino acid solution, and 1X sodium pyruvate.

[0288] method

[0289] Once growth was complete, MG U373 cells were seeded at 100,000 cells / well in 24-well plates. Cells were treated alone with LPS (1 μg / mL) or with 10% MRx0029 bacterial supernatant for 24 h. A control was also performed in which cells were incubated in untreated medium. Afterward, the cell-free supernatant was collected and centrifuged at 10,000 g at 4°C for 3 min. IL-6 was measured using the Peprotech Human IL-6 ELISA Kit (catalog number #900-K16) according to the manufacturer's instructions.

[0290] result

[0291] The results of these experiments are Figure 2 As shown in the figure, treatment of neuroblastoma cells with LPS and bacterial strains resulted in a decrease in secreted IL-6 levels.

[0292] Example 2b – The effect of bacterial inoculum on regulating IL-8 secretion.

[0293] Overview

[0294] Since neuroinflammation plays a key role in neurodegenerative diseases, and IL-8 has been shown to have positive neuroactive effects, the effect of a composition comprising the bacterial strain of the present invention and LPS on IL-8 activation was evaluated. Human glioblastoma astrocytoma cells were treated with a composition comprising the bacterial strain of the present invention and LPS to observe its ability to regulate IL-8 levels.

[0295] Materials and methods

[0296] bacterial strains

[0297] Macrococcus martensii MRX0029; Bacteroides dysenteriae MRX0005

[0298] cell lines

[0299] MG U373 is a human glioblastoma astrocytoma derived from a malignant tumor, acquired from Sigma-Aldrich (catalog number 08061901-1VL). MG U373 human glioblastoma astrocytoma cells were grown in MEM (Sigma-Aldrich, catalog number M-2279) supplemented with 10% FBS, 1% penicillin-streptomycin, 4 mM L-Glut, 1X MEM non-essential amino acid solution, and 1X sodium pyruvate.

[0300] method

[0301] Once growth was complete, MG U373 cells were seeded at 100,000 cells / well in 24-well plates. Cells were treated alone with LPS (1 μg / mL) or with 10% MRX0029 bacterial supernatant for 24 h. Afterward, the cell-free supernatant was collected and centrifuged at 10,000 g at 4°C for 3 min. IL-8 levels were measured using the Peprotech Human IL-8 ELISA Kit (catalog number #900-K18) according to the manufacturer's instructions.

[0302] result

[0303] The results of these experiments are Figure 3 As shown in the figure, treatment of neuroblastoma cells with bacterial strains resulted in an increase in IL-8 secretion independent of LPS.

[0304] Example 2C – The efficacy of bacterial inoculum in reducing α-synuclein-induced inflammation.

[0305] Overview

[0306] Neuroinflammation plays a crucial role in Parkinson's disease, and α-synuclein has been shown to induce neuroinflammation in vivo. Therefore, the ability of the bacterial strain of the present invention to inhibit α-synuclein-induced neuroinflammation was evaluated. Co-cultures of human glioblastoma astrocytoma cells and neuroblastoma cells were exposed to wild-type α-synuclein and mutant isoforms E46K and A53T, and treated with a composition containing the bacterial strain according to the present invention. The ability of the bacterial strain to inhibit α-synuclein-induced IL-6 secretion was then tested.

[0307] Materials and methods

[0308] bacterial strains

[0309] Macrococcus martini MRX0029 ； Parabacterium dilatatum MRX0005

[0310] cell lines

[0311] MG U373 is a human glioblastoma astrocytoma derived from a malignant tumor, acquired from Sigma-Aldrich (catalog number 08061901-1VL). MG U373 human glioblastoma astrocytoma cells were grown in MEM (Sigma-Aldrich, catalog number M-2279) supplemented with 10% FBS, 1% penicillin-streptomycin, 4 mM L-Glut, 1X MEM non-essential amino acid solution, and 1X sodium pyruvate.

[0312] SH-SY5Y is a human neuroblastoma cell line derived from malignant neuroblastoma and is available from Sigma-Aldrich (catalog number 94030304-1VL). Cells were grown in 50% MEM and 50% F-12 Ham medium supplemented with 2 mM L-glutamine, 10% heat-inactivated FBS, 100 U / ml penicillin, and 100 µg / ml streptomycin. Cells from the growth medium were seeded at 11,000 cells / well in 96-well plates and incubated. After 2 days, the medium was replaced with differentiation medium (growth medium containing 1% FBS) and 10 µM retinoic acid. The differentiation medium was changed every other day, and cells were used after 7 days of differentiation.

[0313] method

[0314] SHSY5Y cells were seeded at a density of 50,000 cells / well in 12-well plates. Cells were grown in 50% MEM and 50% F-12 Ham medium supplemented with 2 mM glutamine, 10% heat-inactivated FBS, 100 U / ml penicillin, and 100 µg / ml streptomycin. Cells from the growth medium were then seeded at 11,000 cells / well in 96-well plates and incubated. After 2 days, the medium was replaced with differentiation medium (growth medium containing 1% FBS) and 10 µM retinoic acid. Differentiation medium was changed every other day, and cells were used after 7 days of differentiation. U373 cells were seeded at a density of 50,000 cells / well in 12-transwell plates (0.4 µm polyester membrane, Costa) for 72 hours. Cells were co-cultured together for 24 hours before treatment with differentiation medium (growth medium containing 1% FBS but without retinoic acid).

[0315] Subsequently, cells were treated with 25 μg / ml α-synuclein (Wt, A53T, E46K) for 48 hours, with or without 10% bacterial supernatant. Cell-free supernatants were collected, centrifuged at 10,000 g for 3 min at 4°C, aliquoted, and stored at -80°C. Human IL-6 and IL-8 were measured as described above.

[0316] result

[0317] The results of these experiments are shown in Figure 4. Treatment of cells with wild-type α-synuclein and mutant isoforms E46K and A53T induced moderate secretion of IL-6. Figure 4A In cells treated with the bacterial strain, α-synuclein-induced IL-6 secretion was inhibited. Figure 4A The reduction in IL-6 secretion was greatest when MRX0029 was administered.

[0318] Example 3 – Efficacy of bacterial inoculum in reducing NFκB activation

[0319] Overview

[0320] Activation of the NFκB promoter leads to the production of pro-inflammatory cytokines, including IL-1β and IL-1. IL-18, TNF And IL-6. The NFκB promoter can be activated by stimulating TLR4 ligands. - Synuclein and LPS activation. - Mutations in synuclein (such as...) Synuclein A53T is associated with familial Parkinson's disease. Treatment of neuronal cells with LPS simulated environmentally induced Parkinson's disease. The ability of compositions containing the bacterial strain according to the invention to inhibit NFκB promoter activation was investigated.

[0321] Materials and methods

[0322] bacterial strains

[0323] Macrococcus martensii MRx0029

[0324] cell lines

[0325] Human Hek blue TLR4 was purchased from InvivoGen (catalog number hkb-htlr4). Human Hek blue TLR4 was grown in DMEM high glucose (Sigma Aldrich, catalog number D-6171) supplemented with 10% FBS, 1% penicillin-streptomycin, 4 mM L-Glut, Normocin and 1X HEK Blue selectant.

[0326] method

[0327] Once growth begins, human Hek blue cells are seeded at 25,000 cells / well in 96-well plates, repeated four times. One group of cells is then used alone... - Synuclein A53T (1 μg / mL) or 10% MRx0029 bacterial supernatant was treated for 22 h. A second group of cells was treated alone with LPS (10 ng / mL, derived from Salmonella Typhimurium serotype, Sigma Aldrich, catalog number L6143) or 10% MR029 bacterial supernatant for 22 h. Cells were then centrifuged, and 20 μL of the supernatant was mixed with 200 μL of Quanti Blue reagent (InvivoGen, catalog number rep-qb2), incubated for 2 h, and the absorbance was read at 655 nm.

[0328] result

[0329] The results of these experiments are Figure 5 and Figure 6 As shown in the image. Figure 5 The results show that MRx0029 inhibits... - Activation of the NFκB promoter by synuclein. Figure 6 The results showed that MRx0029 inhibited the activation of the NFκB promoter by LPS.

[0330] Example 4 – The effect of bacterial inoculum on altering antioxidant capacity

[0331] Overview

[0332] Compositions containing bacterial strains according to the invention have the ability to alter antioxidant capacity. The antioxidant capacity of the bacterial strains was determined using the well-known ABTS (2,2'-azinyl-bis(3-ethylbenzothiazoline-6-sulfonic acid)) assay.

[0333] bacterial strains

[0334] Macrococcus martensii MRx0029

[0335] method

[0336] Collect bacterial cells (10) 6 (One or more) and centrifuge. Resuspend them in assay buffer (using three times the precipitate volume). Sonicate the suspension on ice for 5 minutes, then centrifuge at 12,000 xg for 10 minutes. Remove the supernatant according to the manufacturer's instructions and measure using the ABTS assay kit (code CS0790) manufactured by Sigma Aldrich.

[0337] result

[0338] The results of these experiments are Figure 7 As shown in the image. Figure 7 This indicates that MRx0029 has an antioxidant capacity of approximately 2 mM compared to Trolox.

[0339] Example 5 – Efficacy of bacterial inoculum in altering lipid peroxidation levels

[0340] Overview

[0341] The ability of compositions containing bacterial strains according to the invention to alter lipid peroxidation levels was investigated. The thiobarbituric acid reactive assay (TBAR) was used to measure byproducts of lipid peroxidation.

[0342] Materials and methods

[0343] bacterial strains

[0344] Macrococcus martensii MRx0029

[0345] method

[0346] Collect bacterial cells (10) 6 (One or more) and centrifuged, washed with isotonic saline, and then resuspended the precipitate in potassium chloride assay buffer. The suspension was sonicated on ice for 10 min, and then centrifuged at 10,000 x g for 10 min. The supernatant was removed, and the level of lipid peroxidation was assessed using the thiobarbituric acid reactant assay.

[0347] result

[0348] Experimental results in Figure 8 As shown in the image. Figure 8 The results showed that MRx029 was able to inhibit lipid peroxidation by approximately 20%, which is a higher antioxidant capacity than the positive control butylated hydroxytoluene (1% w / v).

[0349] Example 6 – Effect of bacterial inoculum on histone deacetylase activity

[0350] Overview

[0351] The ability of compositions containing bacterial strains according to the present invention to alter histone deacetylase activity was investigated. Dysregulation of histone deacetylases is involved in the pathogenesis of age-related neurodegenerative diseases.

[0352] Materials and methods

[0353] bacterial strains

[0354] Macrococcus martensii MRx0029

[0355] cell lines

[0356] The HT-29 cell line was used because it contains histone deacetylase.

[0357] method

[0358] Cell-free supernatant from the stationary phase bacterial culture was separated by centrifugation and filtration through a 0.22 uM filter. HT-29 cells were used 3 days after confluence, with 1 mL of DTS added 24 hours before the start of the experiment. HT-29 cells were challenged with 10% cell-free supernatant diluted in DTS and incubated for 48 hours. Nuclease proteins were then extracted using a Sigma Aldrich nuclease extraction kit, and the samples were flash-frozen before HDAC activity measurement. HDAC activity was assessed for fluorescence using a Sigma Aldrich (UK) kit.

[0359] result

[0360] Experimental results in Figure 9 As shown in the image. Figure 9 This indicates that MRx0029 can reduce the activity level of histone deacetylase.

[0361] Example 7 – Indole Production Levels in Bacteria

[0362] Overview

[0363] The ability of the bacteria of this invention to produce indole was investigated. Indole is associated with reducing inflammation and oxidative stress.

[0364] Materials and methods

[0365] bacterial strains

[0366] Macrococcus martensii MRx0029

[0367] ATCC 11775 is the reference strain of bacteria known to produce indole.

[0368] method

[0369] Intact bacterial cells in the stationary phase were incubated with 6 mM tryptophan for 48 hours. Bacterial strains possessing tryptophanase utilize tryptophan as a substrate to produce indole. After 48 hours of incubation, the supernatant was removed and added to Kovac reagent to quantify indole. Standards, stock solutions, and reagents were prepared using internally validated standardized methods.

[0370] result

[0371] Experimental results in Figure 10 As shown in the image. Figure 10 This indicates that MRx0029 has the ability to generate indole from tryptophan at a concentration of approximately 0.2 mM.

[0372] Example 8 – Production Level of Kynurenine in Bacteria

[0373] Overview

[0374] The ability of the bacteria of this invention to produce kynurenine was investigated. Dysregulation of the kynurenine pathway may lead to activation of the immune system and accumulation of potentially neurotoxic compounds. Alterations in kynurenine metabolism may be involved in the development of Parkinson's disease.

[0375] bacterial strains

[0376] Macrococcus martensii MRx0029

[0377] DSM 17136 is known to produce kynurenine. Bacteroides copricola strains.

[0378] method

[0379] Cell-free supernatant from the stationary phase bacterial culture was separated by centrifugation and filtration through a 0.22 μM filter and frozen until use. Kynuronic acid standards, stock solutions, and reagents were prepared using an internally validated standardized method. Samples were treated with trichloroacetic acid and centrifuged at 10,000 x g at 4 °C for 10 min. The supernatant was collected and aliquoted into 96-well plates. Ehrlich reagent was used for kynuronic acid detection, added at a 1:1 ratio.

[0380] result

[0381] Experimental results in Figure 11 As shown in the image. Figure 11The results showed that MRx0029 has the ability to produce kynurenine at a concentration of approximately 40 µM.

[0382] Example 9 – Levels of dopamine, DOPAC, and HVA in the striatum of bacterial-treated MPTP mice

[0383] Parkinson's disease is a common neurodegenerative disorder characterized by tremor, bradykinesia, rigidity, and postural instability. These symptoms are primarily attributed to the degeneration of dopaminergic neurons in the substantia nigra pars compacta and the resulting loss of prominent nerve fibers in the striatum

[60] . Mice treated with MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) have selectively lost large numbers of dopaminergic neurons in the substantia nigra striatum

[61] . The loss of MPTP-induced dopaminergic cells in the substantia nigra mimics the clinical condition of Parkinson's disease and is therefore a useful model for testing anti-Parkinson's drugs.

[0384] The aim of this study was to evaluate the role of the MRX0029 anaerobic bacteria using MPTP-damaged mice.

[0385] Forty-eight male mice were divided into four distinct treatment groups (A, B, E, and I, n=12 animals per group). The treatment groups are shown in Table 1 below, and the time course of the events is outlined below.

[0386] Table 1: Treatment Groups

[0387] Groups A, B, E, and I were treated daily via tube feeding with either bacteria (MRx0029–E group) or a carrier (PBS) for 18 days. Oral treatment began 14 days prior to MPTP injury. Group I animals received daily oral administration of the carrier (PBS) and intraperitoneal injection of the reference drug 30 minutes before and 90 minutes after the first MPTP on day 0. The administration volume for both oral and carrier treatments was 200 μl per mouse. The bacterial strain for group E was derived from stock glycerol (gly). For oral treatment, the tubes used for administration were stored in vials containing 70% ethanol and rinsed with distilled water before and after each use. Each treatment group had its own tube, ethanol vial, and distilled water vial. There were no changes to the tubes or tubes used between groups. Each syringe was rinsed directly with N2 prior to treatment.

[0388] On day 0, animals in groups B, E, and I received intraperitoneal injections of MPTP (20 mg / kg body weight (bw), 4 times, at 2-hour intervals). A sham injury was created in group A by intraperitoneal administration of the MPTP medium (0.9% saline). The administration volume was 10 μl per gram of body weight. Animals were weighed prior to MPTP treatment to ensure administration based on their actual body weight. Subsequently, animals received po treatment daily.

[0389] Formulation of preparations for drug administration and preparation of stock glycerol solutions for drug administration.

[0390] For processing group E (MRx0029)

[0391] 1.) Take one portion of glycerol stock solution from the -80℃ freezer and place it under anaerobic conditions at 37℃ (anaerobic container with sac) to thaw (requires 30-40 min).

[0392] 2.) Centrifuge the completely thawed glycerol stock solution at 6000 xg for 10 min at room temperature.

[0393] 3.) Discard the supernatant without disturbing the precipitation (e.g., using a pipette).

[0394] 4.) Add 4.22 mL of sterile preheated (37°C) 1 x PBS and mix gently with a pipette.

[0395] 5.) Administer 200 µL of bacterial solution to mice. Resuspend the precipitate in PBS and administer the solution to the animals within 15 min.

[0396] Reference drug group preparation

[0397] Dissolve an appropriate amount of 7-nitroindazole in peanut oil to achieve a final concentration of 50 mg / kg.

[0398] Materials and methods

[0399] animal

[0400] Special treatment and randomization of animals

[0401] Change gloves between each treatment group and spray gloves with a 70% ethanol solution between each cage in the same group to minimize the risk of contamination when handling animals (e.g., handling, behavioral testing, cleaning, and tissue sampling).

[0402] Treatment is randomized and alternates daily to prevent the same group from being treated at the same time each day. During the sampling process, animals in each cage are randomized.

[0403] Organization collection and processing

[0404] On day 4, all animals in all groups were sacrificed and their brains were collected. Mice were thus deeply anesthetized with pentobarbital injection (600 mg / kg).

[0405] Blood (approximately 500 µl) was collected via cardiac puncture. The mice were then perfused with 0.9% saline via the heart, and the brain was removed and halved. The left hemisphere was subdivided into striatum (for HPLC), substantia nigra, and residual brain tissue, weighed, and immediately frozen and stored at -80°C. Instruments and surfaces that came into contact with the animals must be cleaned with 70% ethanol before dissecting the next animal.

[0406] Biochemical analysis of dopamine, DOPAC, and HVA levels in the striatum was performed using HPLC.

[0407] Striatal samples (n=6 per treatment group; 24 samples in total) were mixed with 0.2 M perchloric acid containing 100 μM EDTA-2Na at a ratio of 1:10 (w / v) and homogenized in a glass vise microhomogenizer at 0 °C. After incubation on ice for 30 min, the homogenate was centrifuged at 10,000 RPM for 10 min in a refrigerated centrifuge (Heraeus Instruments, Germany). The supernatant was carefully aspirated and mixed with 0.4 M sodium acetate buffer pH 3 at a ratio of 1:2 (v / v) and filtered through a 0.22 μm centrifuge filter (Merck Millipore, Germany) at 14,000 g at 4 °C for 4 min. The filtrate was stored at -80 °C prior to HPLC analysis.

[0408] HPLC analysis

[0409] The concentrations of DA, DOPAC, and HVA in the striatal samples were determined by column HPLC-electrochemical detection [62; 63]. The HPLC system used (HTEC-500, Eicom Corp., Kyoto, Japan) included a pulseless microflow pump, a degasser, and an amperometric detector equipped with a glassy carbon electrode operating at +0.45 V relative to an Ag / AgCl reference electrode. Samples were injected using a CMA / 200 cryogenic microsyringe (CMA / Microdialysis, Stockholm, Sweden). Chromatograms were recorded and integrated using a computerized data acquisition system (DataApex, Prague, Czech Republic). DA, DOPAC, and HVA were separated on a 150 x 2.1 id mm column (CA5-ODS, Eicom Corp., Kyoto, Japan). The mobile phase consisted of 0.1 M phosphate buffer (pH 6.0), 0.13 mM EDTA, 2.3 mM sodium 1-octanesulfonate, and 20% (v / v) methanol. The limit of detection (LOD) for DA (signal-to-noise ratio = 3) was estimated to be 0.5 fmol in 15 μL (0.03 nM) injected into the column.

[0410] result

[0411] Animals tolerated the bacterial strain well. On the day of MPTP injury, heat the animals with a red lamp the following day if necessary. If the animals are in poor condition (feeling cold, dehydrated, behaving abnormally), provide them with wet food and administer subcutaneous saline treatment if necessary.

[0412] To analyze dopamine, DOPAC, and HVA levels, striatal tissue from six animals in each treatment group was used. Data were analyzed using the Kruskal-Wallis test, followed by Dunn multiple comparison post-hoc tests or one-way ANOVA, and then Bonferroni post-hoc tests (A = all...). B is relative to all, I is relative to all (#)) / # = p<0.05 = p<0.01 = p<0.001.

[0413] Healthy animals in group A had high levels of dopamine, DOPAC, and HVA, while MPTP treatment in group B reduced these levels, and the positive control (group I) partially recovered production (Figure 12). Group I animals tended to have higher dopamine levels compared to the bacterial treatment groups and group B. DOPAC (dopamine metabolite) levels in group B animals were generally significantly lower than those in the undamaged animals of group A. Figure 12B ).

[0414] Notably, treatment with MRx0029 (Group E) restored the production of dopamine and DOPAC (respectively). Figure 12A and Figure 12B Therefore, treatment with MRx0029 can be used to treat or prevent neurodegenerative disorders.

[0415] Example 10 – The effect of bacteria on altering neurite growth

[0416] Overview

[0417] Neuronal spur growth is a crucial process in the development of connections between neurons. Therefore, the ability of bacterial strains and organic acids to induce neurite spur growth was tested by measuring the transcriptional level of microtubule-associated protein MAP2 (a specific marker of neuronal differentiation).

[0418] bacterial strains

[0419] Giant cellulococcus martensii MRX0029.

[0420] method

[0421] SHSY5Y was inoculated into 10 cm Piper dishes at a density of 2 x 10⁻⁶ cm. 6 Cells were collected. After 24 h, cells were treated for 17 h in differentiation medium (containing 1% FBS but without RA) containing 10% bacterial supernatant or YCFA+, 10 μM RA, 200 μM hexanoic acid, or 200 μM valproic acid. Representative images were then captured at 40X / 0.65 magnification using a phase-contrast EVOS XL core microscope. Cells were collected, and total RNA was isolated according to the RNeasy microkit protocol (Qiagen). cDNA was prepared using a high-capacity cDNA reverse transcription kit (Applied Biosystems). Gene expression was measured using qPCR. GAPDH was used as an internal control. (Based on 2...) (-ΔΔct) The method calculates the multiple change.

[0422] Immunofluorescence and confocal microscopy

[0423] Cells were spaced at 5x10 4Cells / well were seeded onto 8-well chamber slides (Marienfeld Laboratory Glassware) and incubated overnight, followed by treatment with 10% bacterial supernatant for 24 hours. For differentiation, cells were treated with 10 nM retinoic acid for 5 days, then treated with bacterial supernatant. Cells were then fixed for 20 minutes at room temperature (RT) with 4% paraformaldehyde in PBS. The fixed cells were washed with PBS and permeabilized with 1% Triton X-100 in PBS for 10 minutes. After washing with PBS, the slides were incubated with blocking buffer (4% BSA / PBS) at RT for 1 hour, followed by incubation at 4°C for 12 hours with anti-MAP2 antibody (sc-74421, Santa Cruz Biotechnology Inc.) diluted in 1% BSA / PBS. They were then washed twice with PBS and incubated for 1 hour at RT with Alexa Flour 488-conjugated anti-mouse (Molecular Probes Inc.) and Alexa Flour 594-conjugated phalloidin (ab176757, Abcam). After washing three times with PBS, the slides were fixed with a Vectorshield containing DAPI (Sigma, Aldrich). The slides were observed using a Zeiss Axioscope microscope equipped with a 63x / 1.2 W Korr objective and a filter set suitable for detecting the fluorescent dyes used. The manual exposure time for digitally acquiring images immunolabeled with MAP-2 was kept constant, allowing comparisons between different wells and treatments. The exposure times for phalloidin (F-actin) and DAPI were varied to suit the field of view. Random fields of view were acquired using a QImaging camera controlled by Image Pro Plus software. Save the images as TIF and open them in Adobe Photoshop CC 2015.1.2, then overlay and merge the overlays of the MAP-2, DAPI, and phalloidin images. Select representative images to illustrate the differences in abundance and location of the examined proteins.

[0424] result

[0425] The results are shown in Figure 13. Figure 13A Representative microscopic images of undifferentiated SHSY-5Y cells incubated with each acid and bacterial supernatant are shown. Treatment with MRX0029 induced a neuron-like phenotype, exhibiting characteristics similar to cells treated with retinoic acid (used for terminal differentiation of neuroblastoma cells), with larger, cone-shaped cell bodies, neurites, and processed branching into networks with neighboring cells. Figure 13BThe results showed that MRx0029 significantly upregulated MAP2 in undifferentiated neuroblastoma cells. Phalloidin (an actin cytoskeleton binder) staining further demonstrated that the cytoskeleton arrangement differed in cells treated with MRx0029, further supporting the neuronal differentiation hypothesis of MRx0029. Figure 13B ).

[0426] Example 11 – Efficacy of bacterial inoculants in reducing cellular oxidative levels

[0427] background

[0428] The production of reactive oxygen species (ROS) contributes to the pathology of neurodegenerative diseases. The ability of bacterial strains to protect differentiated SHSY-5Y and U373 cells from ROS generated by treatment with tert-butyl hydroperoxide (TBHP) was investigated.

[0429] Materials and methods

[0430] bacterial strains

[0431] Macrococcus martini MRX0029

[0432] method

[0433] SHSY-5Y cells were seeded at a density of 5000 cells / well in black flat-bottomed 96-well plates and incubated in a CO2 incubator. After 24 h, the culture medium was replaced with differentiation medium (growth medium containing 1% FBS) and 10 µM retinoic acid. The differentiation medium was changed every other day. On day 10, the differentiation medium was removed, and the cells were washed with pre-warmed PBS and stained with 10 µM DCFDA molecular probes in growth medium containing 1% FBS for 20 min. Then, the cells were washed again with pre-warmed PBS and treated with 100 µM TBHP for 2 h, with or without 10% bacterial supernatant. Fluorescence intensity was measured at Ex / Em 485 / 530 nm using a TECAN plate reader.

[0434] result

[0435] The experimental results are shown in Figure 14. Figure 14b shows that MRX0029 can inhibit ROS production in differentiated SHSY-5Y neuroblastoma cells. MRX0029 has no effect on ROS production in U373 glioblastoma cells (Figure 14a). This indicates that this aspect of the antioxidant effect is neuron-specific.

[0436] Example 12 – Neuroprotection

[0437] RA-differentiated SHSY-5Y cells were treated with MPP+ (the active metabolite of MPTP), a chemical widely used in vitro and in vivo to mimic some pathological features of PD. Cell viability was measured using mitochondrial respiration rate. Figure 15 Both MRx0005 and MRx0029 showed significant effects and directly promoted increased mitochondrial metabolic activity in SHSY-5Y cells. MRx0029 showed complete resistance to MPP+, restoring cell viability almost to the same level as untreated cells and exceeding that of the quercetin-positive control. The protective effect of MRx0005 was approximately 20% compared to YCFA-MPP+ treated samples, roughly the same as the protective effect observed in the quercetin-positive control. Figure 15 ).

[0438] Example 13 – Further Analysis of the Histone Deacetylation Inhibition Mechanism

[0439] introduce

[0440] The gut microbiota possesses immense diversity and metabolic capacity, representing a vast metabolic pool capable of generating a variety of molecules that may influence HDAC activity. Few studies have evaluated the HDAC-inhibiting activity of microbial-derived metabolites other than butyrate, which has been shown to inhibit HDAC and is associated with improved motor function in Huntington's disease

[64] . Therefore, the inventors sought to identify which metabolites are responsible for HDAC inhibition and further elucidate the mechanisms by which this inhibition is achieved.

[0441] Materials and methods

[0442] Bacterial culture and cell-free supernatant collection

[0443] Pure bacterial cultures were anaerobically grown in YCFA broth until a stable growth phase was reached. The cultures were centrifuged at 5,000 xg for 5 minutes, and the cell-free supernatant (CFS) was filtered using a 0.2 µM filter (Millipore, UK). One-mL aliquots of the CFS were stored at -80°C until use. Sodium butyrate, hexanoic acid, and valeric acid were obtained from Sigma Aldrich (UK) and suspensions were prepared in YCFA broth.

[0444] Quantitative analysis of SCFA and MCFA in bacterial supernatant

[0445] Short-chain fatty acids (SCFA) and medium-chain fatty acids (MCFA) in bacterial supernatants were analyzed and quantified by MS Omics APS as follows. Samples were acidified with hydrochloric acid and a deuterium-labeled internal standard was added. All samples were analyzed in random order. Analyses were performed using a high-polarity column (Zebron™ ZB-FFAP, GC capillary column 30 m x 0.25 mm x 0.25 µm) in a GC (7890B, Agilent) coupled to a quadrupole detector (59977B, Agilent). The system was controlled by ChemStation (Agilent). Raw data were converted to netCDF format using ChemStation (Agilent) and then imported and processed in Matlab R2014b (Mathworks, Inc.) using PARADISe software as described in

[65] .

[0446] Specific HDAC activity analysis

[0447] Specific HDAC inhibitory activity for HDAC1, 2, 3, 4, 5, 6, and 9 was analyzed using a fluorescence assay kit (BPS Bioscience, CA) for each type of HDAC. Assays were performed according to the manufacturer's instructions, with each sample repeated. Cell-free supernatants were diluted to one-tenth of their original volume and exposed to the specific HDAC protein provided in the kit to maintain consistency between methods.

[0448] result

[0449] The intestinal symbiotic microbial metabolites that inhibit histone deacetylases are butyrate and valerate.

[0450] The supernatant of MRx0029 cells showed strong HDAC inhibition in both whole HT29 cells and HT29 cell lysates, producing average concentrations of valerate and hexanoate of 5.08 mM and 1.60 mM, respectively. Figure 16A and Figure 16C ).

[0451] To investigate which metabolites are responsible for strain-induced HDAC inhibition, the HDAC inhibitory effects of different concentrations of hexanoic acid, valeric acid, and sodium butyrate on whole HT-29 cells and HT-29 cell lysates were measured. Figure 16B The results showed that sodium butyrate significantly inhibited HDAC activity in whole cells and cell lysates (P < 0.05), while hexanoic acid did not show significant inhibitory activity. Valeric acid inhibited total HDAC activity. (p<0.05) (p<0.005) (P<0.001) (p<0.0001)).

[0452] The study found that effective total HDAC inhibitors target class I HDACs.

[0453] The specific HDAC inhibition of the tested bacterial strains was investigated. Specific HDAC inhibition assays were performed for class I and class II HDACs (BPS Bioscience, CA). The ability of the bacterial strains to inhibit HDAC enzymes was compared with that of butyrate, hexanoate, and valerate. Our results indicate that MRX0029 is a very potent inhibitor of class I HDAC enzymes (HDAC1, 2, and 3). Inhibition of class II HDACs was less significant (data not shown).

[0454] Discussion

[0455] The strains exhibiting HDAC inhibitory activity produced large amounts of valeric acid and hexanoic acid, as well as large amounts of sodium butyrate (…). Figure 16C When tested as pure substances, valeric acid and sodium butyrate cause significant HDAC inhibition (p<0.0001).

[0456] Interestingly, the results of specific HDAC activity indicated that the tested strains were effective inhibitors of class I HDACs, especially HDAC2 (Figs. 17 and 18). Class I HDACs (HDAC1, 2, 3, and 8) are located in the cell nucleus and are universally expressed in several human cell types. HDACs 1–3 share more than 50% homology but have unique structures and cellular functions

[66] . They are primarily involved in cell survival, proliferation, and differentiation, so their inhibition could be useful for a variety of diseases

[67] ;

[68] ;

[69] ;

[70] ;

[71] .

[0457] Example 14 – Level of BDNF secretion in SHSY-5Y cells

[0458] background

[0459] Brain-derived neurotrophic factor (BDNF) is a ubiquitous molecule in the brain associated with neurodevelopment, neuroprotection, and neuroregeneration. BDNF not only prevents neurodegeneration but also mental disorders such as depression and anxiety, which are common in patients diagnosed with Parkinson's disease (PD) or Alzheimer's disease (AD).

[0460] method

[0461] SH-SY5-SY cells were seeded at a density of 60,000 cells / well in 24-well plates and incubated. After 24 h, the medium was replaced with differentiation medium (growth medium containing 1% FBS) and 10 µM retinoic acid. The differentiation medium was changed every other day, and the cells were used on day 10 of differentiation. For treatment, the differentiation medium was removed and replaced with 450 µL of complete growth medium, and 50 µL of bacterial SN was added to the treated wells, or YCFA+ was added as a negative control.

[0462] result

[0463] The result is Figure 19 The study showed that administration of MRX0029 in combination with retinoic acid increased the secretion of BDNF from differentiated neuroblastoma cells. Therefore, compositions containing symbiotic bacteria and organic acids may be used for treatment.

[0464] Example 15 – Production of Metabolites – Metabolites in the Brain

[0465] background

[0466] Metabolites present in bacterial supernatants can directly influence the host's response to oxidative stress, intercellular communication, and neuroprotection. During in vitro screening, metabolites that play key roles in nervous system processes were measured in brain tissue from mice fed MRx0005 and MRx0029.

[0467] method

[0468] animal

[0469] Adult male BALBc (Envigo, UK) mice were housed in groups under a 12-hour light-dark cycle; standard rodent food and water were provided freely. All experiments were conducted in accordance with European guidelines, with approval from the University College Cork Animal Ethics Experimentation Committee. Animals were 8 weeks old at the start of the experiments.

[0470] Research Design

[0471] After entering the animal unit, the animals are allowed to acclimatize in their enclosures for one week. Between 3:00 PM and 5:00 PM, they are fed at a 1 x 10 ratio. 9 One dose of CFU was administered orally via tube feeding (200 µL) for 6 consecutive days as part of a live biotherapy regimen. On day 7, the animals were decapitated and tissues were collected for experiments.

[0472] Organization Collection

[0473] For treatment and testing conditions, animals were euthanized randomly; sampling was performed between 9:00 AM and 1:00 PM. Blood from the trunk was collected in EDTA (ethylenediaminetetraacetic acid) tubes and centrifuged at 4000 g for 15 min. Plasma was separated and stored at -80°C for further analysis. The brain was rapidly removed, dissected, and each brain region was rapidly frozen on dry ice and stored at -80°C for further analysis. The spleen was removed and immediately processed for in vitro immunostimulation. Intestinal tissue (removing the 2 cm ileum and colon closest to the cecum and using the 1 cm tissue furthest from the cecum) was fitted into the using chamber for intestinal permeability testing. The cecum was removed, weighed, and stored at -80°C for SCFA analysis.

[0474] Monoamine analysis

[0475] Neurotransmitter concentrations in brainstem samples were analyzed by HPLC. Briefly, brainstem tissue was sonicated in a 500 μl frozen mobile phase containing 4 ng / 40 μl N-methyl 5-HT (Sigma Chemical Co., UK) as an internal standard. The mobile phase contained 0.1 M citric acid, 5.6 mM octane-1-sulfonic acid (Sigma), 0.1 M sodium dihydrogen phosphate, 0.01 mM EDTA (Alkem / Reagecon, Cork), and 9% (v / v) methanol (Alkem / Reagecon), and was adjusted to pH 2.8 using 4 N sodium hydroxide (Alkem / Reagecon). The homogenate was then centrifuged at 22,000 × g for 15 min at 4 °C, and 40 μl of the supernatant was injected into an HPLC system consisting of an SCL 10-Avp system controller, a LECD 6A electrochemical detector (Shimadzu), an LC-10AS pump, a CTO-10A oven, a SIL-10A autosampler (with the sample cooler maintained at 40 °C), and an online Gastorr degasser (ISS, UK). Separation was performed using a reversed-phase column (Kinetex 2.6 u C18100 × 4.6 mm, Phenomenex) maintained at 30 °C (flow rate 0.9 ml / min). A glassy carbon working electrode was used in combination with an Ag / AgCl reference electrode (Shimdazu) at an operating voltage of +0.8 V, and the resulting chromatograms were analyzed using Class-VP 5 software (Shimadzu). Neurotransmitters were identified by characteristic retention times determined by standard injections performed at regular intervals during sample analysis. The ratio of the peak height of the analyte to that of the internal standard was measured and compared with that of the standard injection. The results are expressed as ng neurotransmitter / g fresh weight tissue.

[0476] Metabolite analysis

[0477] For GC metabolite analysis, bacterial supernatant samples were derivatized with methyl chloroformate using a slightly modified version of the protocol described in

[72] . All samples were analyzed in a randomized order. Analyses were performed using a GC (7890B, Agilent) coupled to a quadrupole detector (59977B, Agilent). The system was controlled by ChemStation (Agilent). Raw data were converted to netCDF format using ChemStation (Agilent) and then imported and processed in Matlab R2014b (Mathworks, Inc.) using PARADISe software described in

[65] .

[0478] For fatty acid analysis, samples were acidified with hydrochloric acid and a deuterium-labeled internal standard was added. All samples were analyzed in a randomized order. Analyses were performed using a high-polarity column (Zebron™ ZB-FFAP, GC capillary column 30 m x 0.25 mm x 0.25 µm) mounted in a GC (7890B, Agilent) coupled to a quadrupole detector (59977B, Agilent). The system was controlled by ChemStation (Agilent). Raw data were converted to netCDF format using ChemStation (Agilent) and then imported and processed in Matlab R2014b (Mathworks, Inc.) using the PARADISe software described in

[65] .

[0479] Result – Production of neurotransmitters

[0480] The result is Figure 20The figure shows that norepinephrine levels were elevated (p=0.0507) in the brains of mice fed MRx0029, while serotonin and 5-HIAA were slightly elevated. These data support the metabolite analysis listed below, which suggests that MRx00029 is a major producer of 4-hydroxyphenylacetic acid (a known antioxidant)

[73] . More importantly, 4-hydroxyphenylacetic acid is a synthetic intermediate for dopamine and norepinephrine and is an important bioactive molecule

[74] . Indeed, in PD, denaturation changes extend beyond the dopaminergic system, also affecting the serotonergic and norepinephrine systems, leading to decreased serotonin (5-hydroxytryptamine, 5-HT) and norepinephrine (norepinephrine) levels in the striatum and extrastriatal structures

[75] . L-DOPA primarily targets the dopamine-related features of PD, but it does not address the decreases in both 5-HT and norepinephrine. Furthermore, the longer the duration of L-DOPA treatment, the more pronounced a range of motor and non-motor complications (e.g., movement disorders, psychiatric symptoms) became

[76] . Thus, these data demonstrate that bacteria that produce organic acids (such as 4-hydroxyphenylacetic acid) can be used for treatment, particularly for neurodegenerative diseases.

[0481] Result – Production of metabolites

[0482] Metabolites present in bacterial supernatants can specifically and directly affect the host's response to oxidative stress, intercellular communication, and neuroprotection. Metabolites in the supernatants of MRX0029 and MRX0005 cultures were analyzed, and the results showed... Figure 21 As shown in the image.

[0483] Several metabolites revealed significant differences between the two analyzed strains. Succinic acid concentration was particularly elevated in MRx0005. Interestingly, the sample / medium ratio of 4-hydroxyphenylacetic acid was significantly higher in MRx0029. Figure 21 A).

[0484] Fatty acid analysis of the supernatant revealed an interesting divergence between the two strains: MRx0005 primarily produced acetic acid and propionic acid, while MRx0029 produced butyric acid, valeric acid, and hexanoic acid in both linear and branched forms. Figure 21 B). The two strains appear very different, and specifically, notably, MRx0005 and MRx0029 produce succinic acid and 4-hydroxyphenylacetic acid, respectively. Figure 21 A). In addition, MRx0005 appears to produce more C2 and C3 short-chain fatty acids, while MRx00029 produces more C4 (butyrate) and straight-chain and branched medium-chain fatty acids, including hexanoic acid.

[0485] Succinate is a metabolite of the Krebs cycle involved in oxidative phosphorylation. Oxidative phosphorylation complexes are key steps in transporting proteins and vesicle synapses to proximal and distal regions

[77] . Its dysfunction has been reported in neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease, and spinocerebellar ataxia type 1

[78] . These findings are particularly interesting because succinate can enhance mitochondrial activity and support vulnerable neurons in neurodegenerative diseases involving misfolded proteins, including PD

[79] . BDNF and succinate have similar protective activities not only in neurodegeneration but also in mental disorders such as depression and anxiety, which are very common in patients diagnosed with PD or AD.

[0486] Figure 21 B also demonstrated that MRX0029 is a producer of butyrate (butyric acid). This may be important because butyrate has known effects, such as reducing the impermeability of the blood-brain barrier, and has neuroprotective effects

[80] . This property of MRx0029 (and other neuroprotective bacteria) may contribute to its efficacy.

[0487] Example 16 - Regulation of tight junction protein mRNA expression by MRx0029

[0488] Since recent evidence suggests that intestinal dysfunction and inflammation are nonmotor symptoms associated with PD, the ability of the bacterial strains of this invention to induce any intestinal barrier dysfunction was investigated. A monolayer of mucin-producing cells from HT29-mtx epithelium

[81] was used as an in vitro model to assess intestinal barrier disruption and immunostimulation following treatment with MRx0005 and MRx0029. HT29-mtx cells differentiated from phorbol 12-myristate-13-acetate (PMA) secreted large amounts of IL-8; in contrast, treatment with MRx005 and MRx0029 bacterial supernatant for 24 h induced even lower IL-8 secretion compared to untreated and YCFA-treated cells. Figure 22A ).

[0489] The ability of MRx0005 and MRx0029 to regulate epithelial permeability by modifying intracellular signal transduction involved in the expression and localization of proteins involved in intestinal barrier formation was then investigated.

[0490] RNA was isolated and quantitative RT-PCR (qRT-PCR) was performed to characterize changes in the gene expression of tight junction proteins during incubation with MRx0005 and MRx0029. After 2 h of incubation, administration of MRx0029 enhanced the expression of closure protein, villi protein, and tight junction proteins 1 and 2 (TJP1 and TJP2, respectively) mRNA. Figure 22BIn contrast, exposure to MRx0005 did not alter the gene expression of tight junction proteins, suggesting that the two strains have different effects on the intestinal barrier.

[0491] In vitro results were compared with parallel in vitro data from mouse intestines fed with MRx0005 and MRx0029. Gene expression of TJP2 and closure protein in the colon and ileum was quantified. Since MRx0029 significantly upregulated TJP1 and closure protein in the mouse colonic region (p=0.073), the in vitro data fully reflected the in vitro data. Figure 22C +22D). MRx0029 also reduced colonic permeability in the same mouse colon ( Figure 22E +22F).

[0492] Materials and Methods - RNA Extraction and qPCR Analysis

[0493] Total RNA was extracted using the RNeasy microkit (Qiagen, Manchester, JUK) according to the manufacturer's instructions, and RNA concentration was determined by absorbance at 260 / 280 nm using a spectrophotometer (nano-Drop ND-1000; Thermo Scientific, Wilmington, DE). For mRNA expression analysis, cDNA was prepared from total RNA using a high-capacity cDNA reverse transcription kit (Applied Biosystems, UK) according to the manufacturer's instructions. The reverse transcription reaction was performed in a thermal cycler (Biometra, Germany) at 25°C for 10 min, 37°C for 120 min, 85°C for 5 min, and maintained at 4°C. cDNA was amplified in duplicate using the SYBR-Green PCR assay, and the products were detected on a QuantStudio 6 flex real-time PCR instrument (Applied Biosystems, UK) using a normalized spectrum (95°C initial denaturation for 10 min, followed by 95°C denaturation for 15 s, annealing / extension at 60 / 65°C for 60 s, 40 cycles, depending on primers). A dissociation phase was added after 40 cycles to generate melting curves. Analysis was performed using Applied Biosystems QuantStudio real-time PCR software v1.2. Primer sequences for actin, villin, and the closing proteins TJP1 and TJP2 are provided in the sequence listing.

[0494] Example 16 – Stability Test

[0495] The compositions described herein, containing at least one bacterial strain described herein, are stored in sealed containers at 25°C or 4°C, and said containers are placed in an atmosphere with a relative humidity of 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, or 95%. After 1 month, 2 months, 3 months, 6 months, 1 year, 1.5 years, 2 years, 2.5 years, or 3 years, at least 50%, 60%, 70%, 80%, or 90% of the bacterial strain remains, as measured in colony-forming units using a standard protocol.

[0496] Example 17

[0497] method

[0498] animal

[0499] The animals and research design used were the same as in Example 15.

[0500] bacterial strains 755: Parabacterium dilatatum (MRX005) Giant cellulococcus martensii (MRX0029)

[0501] Organization Collection

[0502] For treatment and testing conditions, animals were euthanized randomly; sampling was performed between 9:00 AM and 2:30 PM. Trunk blood was collected in EDTA (ethylenediaminetetraacetic acid) tubes and centrifuged at 4000 g for 15 min. Plasma was separated and stored at -80°C for further analysis. The brain was rapidly excised, dissected, and each brain region was rapidly frozen on dry ice and stored at -80°C for further analysis. The spleen was removed and collected in 5 mL of RPMI medium (containing L-glutamine and sodium bicarbonate, R8758 Sigma + 10% FBS (F7524, Sigma) + 1% penicillin-streptomycin (P4333, Sigma)) and processed immediately after removal for in vitro immunostimulation. Intestinal tissue (removing the 2-3 cm ileum and colon closest to the cecum, and using the 1-2 cm tissue furthest from the cecum) was fitted into the using chamber for intestinal permeability assays. The cecum was removed, weighed, and stored at -80°C for SCFA analysis.

[0503] Monoamine analysis

[0504] Neurotransmitter concentration was analyzed as described in Example 10.

[0505] Spleen cytokine assay

[0506] Immediately after sacrifice, spleens were collected in 5 mL of RPMI medium and cultured. Splenic cells were first homogenized in this RPMI medium and then incubated for 5 min with 1 mL of RBC lysis buffer (11814389001 ROCHE, Sigma). Another 10 mL of RPMI medium was added, followed by centrifugation at 200 G for 5 min. The supernatant was then filtered through a 40 μm filter. Cells were counted and seeded (4,000,000 / mL medium). After acclimation for 2.5 h, cells were stimulated for 24 h with either lipopolysaccharide (LPS - 2 μg / mL) or concanavalin A (ConA - 2.5 μg / mL). Following stimulation, the supernatant was harvested using the Pro-inflammatory Plate 1 (Mouse) V-PLEX kit for TNFα, IL-10, IL-1β, interferon γ, CXCL2, and IL6 (Meso Scale Discovery, MD, USA) to assess cytokine release. Analysis was performed using MESO QuickPlex SQ 120, SECTOR Imager 2400, SECTOR Imager 6000, and SECTOR S 600.

[0507] Gene expression analysis

[0508] Total RNA was extracted using the mirVana™ miRNA Isolation Kit (Ambion / Llife Technologies, Paisley, UK) and treated with DNase (Turbo DNA-free, Ambion / life technologies) according to the manufacturer's recommendations. RNA was quantified using a NanoDrop™ spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, Delaware, USA) according to the manufacturer's instructions. RNA quality was assessed and RNA integrity count (RIN) was calculated using an Agilent Bioanalyzer (Agilent, Stockport, UK) according to the manufacturer's procedures. RNA with an RIN value >7 was used for subsequent experiments. RNA was reverse transcribed into cDNA using the Applied Biosystems High-Capacity cDNA Kit (Applied Biosystems, Warrington, UK) according to the manufacturer's instructions. In short, Multiscribe reverse transcriptase (50 U / µL) (1)(2)(1)(10) was added as part of the RT master mixture and incubated at 25°C for 10 min, 37°C for 2 h, 85°C for 5 min, and stored at 4°C. Quantitative PCR was performed using a mouse-specific target gene probe (6-carboxyfluorescein-FAM) designed by Applied Biosystems, with β-actin as an endogenous control. The amplification reaction contained 1 µl cDNA, 5 µl 2X PCR master mixture (Roche), 900 nM of each primer, and RNase-free water to a total of 10 µl. All reactions were performed in triplicate on a LightCycler® 480 system using 96-well plates. Thermal cycling conditions were followed for 55 cycles as recommended by the manufacturer (Roche). To check for amplicon contamination, each run was performed in triplicate without a template control for each probe used. The cycle threshold (Ct) was recorded. Data were standardized using β-actin and transformed using the 2-ΔΔCT method, and displayed as fold changes relative to the control group.

[0509] Analysis of short-chain fatty acids in cecal contents

[0510] The cecal contents were mixed and vortexed with MilliQ water, then incubated at room temperature for 10 min. Bacteria and other solids were precipitated by centrifugation (10000 g, 5 min, 4 °C), and the supernatant was obtained by filtration through a 0.2 μm filter. This supernatant was transferred to clear GC vials, and 2-ethylbutyric acid (Sigma) was used as an internal standard. The concentration of SCFA was analyzed using a Varian 3500 GC flame ionization system equipped with a ZB-FFAP column (30 m x 0.32 mm x 0.25 mm; Phenomenex). Standard curves were established using a mixture of standards containing acetate, propionate, isobutyrate, n-butyrate, isovalerate, and valerate (Sigma). Peaks were integrated using Varian Star Chromatography Workstation version 6.0 software. All SCFA data are expressed in µmol / g.

[0511] Statistical analysis

[0512] Normally distributed data were represented as mean ± SEM; nonparametric datasets were represented as the median of the interquartile range. Unpaired two-tailed t-tests were used to analyze parametric data, and the Mann-Whitney test was used for nonparametric data. Spearman's rank correlation coefficient was used for correlation analysis in the pooled datasets. In all cases, a p-value < 0.05 was considered significant.

[0513] Result – Production of neurotransmitters

[0514] Figure 23 The results showed the effect of MRx005 treatment on neurotransmitter concentrations in the mouse brain. Most notably, treatment with MRx005 resulted in a decrease in dopamine.

[0515] Results – Gene Expression

[0516] The gene expression of neurotransmitter receptors [serotonin receptor 1a (5-HTR1a), dopamine D1 receptor, GABA receptor subunit B1, GABAA receptor, NMDA2A (Grin2A) and NMDA2B (Grin2b) receptors], inflammatory markers [IL-1β, IL6, CD11b, TNFα and TLR4], and endocrine markers [corticosterone-releasing factor (CRF), corticosterone-releasing factor receptors 1 and 2 (CRFR1, CRFR2), brain-derived neurotrophic factor (BDNF), angiotensin receptor, oxytocin receptor, glucocorticoid receptor and mineralocorticoid receptor] in brain tissues of the hippocampus, amygdala and prefrontal cortex was analyzed.

[0517] Figures 24 to 38 The changes in gene expression in the hippocampus, amygdala, and prefrontal cortex after treatment with MRX005 or MRX0029 are shown. Treatment with MRx0029 resulted in increased expression of glucocorticoid receptors in the amygdala. Figure 31 C). Figure 32 A showed that MRx005 significantly increased BDNF expression in the amygdala, while treatment with MRx0029 significantly increased TLR4 expression in the amygdala. Figure 32 ).

[0518] Both MRx005 and MRx0029 can increase the expression of CD11b in the amygdala. Figure 33 A), while MRx005 treatment reduced the expression of IL-6, Grin2a, and Grin2b. Figure 33 B to Figure 33 D). In addition, MRx005 and MRx0029 significantly increased the expression of GABRA2 and GABBR1 in the amygdala.

[0519] Treatment with MRx005 resulted in a significant increase in BDNF expression in the prefrontal cortex. Figure 35 B).

[0520] Discussion

[0521] Administration of MRx005 and MRx0029 caused changes in gene expression, particularly in the amygdala.

[0522] Results – Effects on Tph1 and IDO-1 expression

[0523] Figure 39 The results showed that MRx0029 significantly increased the expression of tryptophan hydroxylase-1 (Tph1) in the colon, and MRX005 treatment increased the expression of IDO-1 in the colon. Treatment with MRX005 increased the expression of Tph1 and IDO1 in the ileum. Figure 40 ).

[0524] Indoleamine-pyrrole 2,3-dioxygenase-1 (IDO-1) is the first rate-limiting enzyme in the tryptophan / kynurenine pathway, while tryptophan hydroxylase 1 (Tph1) is an isoform of tryptophan hydroxylase responsible for serotonin synthesis. These data suggest that MRx0029 and MRx005 may affect serotonin levels and the tryptophan / kynurenine pathway.

[0525] Results – Effects on tryptophan metabolite levels

[0526] Figure 41The effects of treatment with MRx005 on circulating kynurenine and tryptophan levels are shown.

[0527] Results – Effects on cytokine expression in spleen cells

[0528] In vitro spleen cell assays involve attacking spleen cells (cells isolated from the spleen—a major organ involved in immune defense) with bacteria or viruses to simulate an attack.

[0529] After LPS attack, MRX005 significantly reduced the level of interferon-γ in splenocytes. Figure 42 Furthermore, after LPS attack, MRX005 reduced the levels of interleukin-6 and tumor necrosis factor (respectively). Figure 44 and Figure 45 Following LPS attack, treatment with MRx0029 resulted in a decrease in interferon-γ, interleukin-1β, and interleukin-6 (respectively). Figure 42 , Figure 43 and Figure 44 ).

[0530] Treatment with MRx005 and MRx0029 resulted in an increase in the level of the chemical attractant CXCL1. Figure 47 ).

[0531] Results – Effect on cecal short-chain fatty acid levels

[0532] When indigestible fiber in the diet is fermented by gut bacteria, short-chain fatty acids (SCFAs) are produced. The effect of MRX005 administration is... Figure 48 As shown in the image.

[0533] Example 18 – Further analysis of changes in gene expression levels induced by MRX029 and MRX005

[0534] method

[0535] cell lines

[0536] SH-SY5Y cells

[0537] bacterial strains 755: Parabacterium dilatatum (MRX005) Giant cellulococcus martensii (MRX0029)

[0538] qPCR

[0539] SHSY5Y was inoculated into 10 cm Piper dishes at a density of 2 x 10⁻⁶ cm. 6Cells were collected. After 24 h, cells were treated for 17 h in differentiation medium (containing 1% FBS but without RA) containing 10% bacterial supernatant or YCFA+, 10 μM RA, 200 μM hexanoic acid, or 200 μM valproic acid. Representative images were then captured at 40X / 0.65 magnification using a phase-contrast EVOS XL core microscope. Cells were collected, and total RNA was isolated according to the RNeasy microkit protocol (Qiagen). cDNA was prepared using a high-capacity cDNA reverse transcription kit (Applied Biosystems). Gene expression was measured using qPCR. GAPDH was used as an internal control. (Based on 2...) (-ΔΔct) Methods were used to calculate fold changes. Primer sequences for MAP2, DRD2, GABRB3, SYP, PINK1, PARK7, and NSE are provided in the sequence listing.

[0540] Immunolabeling and cell imaging

[0541] Cells were spaced at 5x10 4Cells / well were seeded onto 8-well chamber slides (Marienfeld Laboratory Glassware) and incubated overnight, followed by treatment with 10% bacterial supernatant for 24 h. For differentiation, cells were treated with 10 nM RA for 5 days, followed by treatment with cell-free bacterial supernatant for 24 h. Cells were then fixed for 20 min at room temperature (RT) with 4% paraformaldehyde in PBS. The fixed cells were washed with PBS and permeabilized with 1% Triton X-100 in PBS for 10 min. After washing with PBS, the slides were incubated with blocking buffer (4% BSA / PBS) at RT for 1 h, followed by the addition of anti-MAP2 antibody or β3-tubulin diluted in 1% BSA / PBS (sc-74421 and sc-80005, respectively, Santa Cruz Biotechnology Inc.), and incubated at 4°C for 12 h. They were then washed twice with PBS and incubated for 1 h at RT with Alexa Flour 488-conjugated anti-mouse (Molecular Probes Inc.) and Alexa Flour 594-conjugated phalloidin (ab176757, Abcam). After washing three times with PBS, the slides were stained with DAPI and fixed with Vectashield® (VectorLaboratories). The slides were observed using an Axioskop 50 microscope (Zeiss) equipped with a 63x / 1.2 W Korr objective and a filter set suitable for detecting the fluorescent dyes used. The manual exposure time for digitally acquiring images immunolabeled with MAP-2 was kept constant, allowing comparisons between different wells and treatments. The exposure times for phalloidin (F-actin) and DAPI were varied to suit the field of view. Random fields of view were acquired using a QImaging camera controlled by Image Pro Plus software. Images were saved as TIFF files and opened in Adobe Photoshop CC 2015.1.2. The images of MAP-2, DAPI, and phalloidin were then overlaid and merged. Representative images were selected to illustrate differences in the abundance and location of the proteins examined.

[0542] Immunoblotting

[0543] SH-SY5Y cells were cultured under the above-described conditions, treated with MRx0005 and MRx0029 for 24 h, and then lysed in RIPA buffer containing a mixture of protease inhibitors (Roche Diagnostics, UK). Protein concentrations were estimated using a BCA protein assay kit (Pierce Biotechnology, Rockford, Illinois), separated by SDS-PAGE, and transferred to PVDF membranes. The membranes were then blocked with 5% skim milk powder or 5% BSA and incubated overnight at 4°C with primary antibodies (MAP2 and β3-tubulin, respectively). The blots were then incubated with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies, and protein was detected by a chemiluminescence assay kit (Pierce Biotechnology, Rockford, Illinois). For both MAP2 and β3-tubulin, β-actin served as a control to monitor changes in protein loading in the samples.

[0544] Results and discussion

[0545] Gene expression

[0546] Figure 13a (illustration) and Figure 49 The changes in expression levels of actin, chorionic villi, closure proteins TJP1, TJP2, MAP2, DRD2, GABRB3, SYP, PINK1, PARK7, and NSE induced by MRx0029 and MRX005 were shown.

[0547] Microscopy and immunoblotting

[0548] Figure 50 The changes in MAP2 expression levels in SHSY5Y cells, as determined by confocal microscopy, are shown. The expression levels of MAP2 and B3-tubulin were also quantified by Western blotting analysis. Figure 50 M and Figure 50 The results shown in N indicate that MRX029 induces an increase in MAP2 expression levels.

[0549] sequence

[0550] SEQ ID NO:1 (Partial sequence of 16S ribosomal RNA gene from *Megacoccus martensii*, strain: NP3-JX424772.1)

[0551] SEQ ID NO:2 (Common 16S rRNA sequence of *Macrococcus masculinii* strain MRx0029)

[0552] Primers used for qPCR (SEQ ID NO in parentheses)

[0553] SEQ ID NO:17 (Common 16S rRNA sequence of Parabacterium digile strain MRX0005)

[0554] Primers and probes for in vitro qPCR (SEQ ID NO in parentheses)

[0555] Additional primers for qPCR (SEQ ID NO in parentheses)

[0556] References sequence list <110> 4D Pharmaceutical Research Limited <120> Composition containing bacterial strains <130> P077072 <141> 2018-06-13 <150> 1709468.1 <151> 2017-06-14 <150> 1709534.0 <151> 2017-06-15 <150> 1712851.3 <151> 2017-08-10 <150> 1803826.5 <151> 2018-03-09 <150> 1805989.9 <151> 2018-04-11 <150> 1805991.5 <151> 2018-04-11 <150> 1805990.7 <151> 2018-04-11 <150> 1806780.1 <151> 2018-04-25 <150> 1806779.3 <151> 2018-04-25 <160> 37 <170> SeqWin2010, version 1.0 <210> 1 <211> 1519 <212> DNA <213> NP3 (Megasphaera massiliensis strain NP3) <400> 1 agagtttgat cctggctcag gacgaacgct ggcggcgtgc ttaacacatg caagtcgaac 60 gagaagagat gagaagcttg cttcttatca atcgagtgg caaacgggtg agtaacgcgt 120 aagcaacctg cccttcagat ggggacaaca gctggaaacg gctgctaata ccgaatacgt 180 tctttccgcc gcatgacggg aagaagaaag ggaggccttc gggctttcgc tggaggaggg 240 gcttgcgtct gattagctag ttggaggggt aacggcccac caaggcgacg atcagtagcc 300 ggtctgagag gatgaacggc cacattggga ctgagacacg gcccagactc ctacgggagg 360 cagcagtggg gaatcttccg caatggacga aagtctgacg gagcaacgcc gcgtgaacga 420 tgacggcctt cgggttgtaa agttctgtta tatgggacga acagggcatc ggttaatacc 480 cggtgtcttt gacggtaccg taagaagaag ccacggctaa ctacgtgcca gcagccgcgg 540 taatacgtag gtggcaagcg ttgtccggaa ttattgggcg taaagggcgc gcaggcggca 600 tcgcaagtcg gtcttaaaag tgcggggctt aaccccgtga ggggaccgaa actgtgaagc 660 720 aggaacacca gtggcgaaag cggctttctg gacgaacact gacgctgagg cgcgaaagcc 780 agggagcaa acgggattag ataccccggt agtcctggcc gtaaacgatg gatactaggt 840 gtaggaggta tcgactcctt ctgtgccgga gttaacgcaa tagtatccc gcctggggag 900 tacggccgca aggctgaaac tcaaaggaat tgacggggc ccgcacaagc ggtggagtat 960 gtggtttaat tcgacgcaac gcgaagaacc ttaccaagcc ttgacattga ttgctacgga 1020 aagagatttc cggttcttct tcggagaca agaaaacagg tggtgcacgg ctgtcgtcag 1080 ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg caacccctat cttctgttgc 1140 cagcacctcg ggtggggact cagaagagac tgccgcagac aatgcggagg aaggcggggga 1200 tgacgtcaag tcatcatgcc ccttatggct tgggctacac acgtactaca atggctctta 1260 atagagggac gcgaaggagc gatccggagc aaaccccaaa aacagagtcc cagttcggat 1320 tgcaggctgc aactcgcctg catgaagcag gaatcgctag taatcgcagg tcagcatact 1380 gcggtgaata cgttcccggg ccttgtacac accgcccgtc acaccacgaa agtcattcac 1440 acccgaagcc ggtgaggcaa ccgcaaggaa ccagccgtcg aaggtggggg cgatgattgg 1500 ggtgaagtcg taacaaggt 1519 <210> 2 <211> 1398 <212> DNA <213> Megasphaera massiliensis strain MRX0029 <400> 2 tgagaagctt gcttcttatc gattctagtg gcaaacgggt gagtaacgcg taagcaacct 60 gcccttcaga tggggacaac agctggaaac ggctgctaat accgaatacg ttctttccgc 120 cgcatgacgg gaagaagaaa gggaggcctt cgggctttcg ctggaggagg ggcttgcgtc 180 tgattagcta gttggagggg taacggccca ccaaggcgac gatcagtagc cggtctgaga 240 ggatgaacgg ccacattggg actgagacac ggcccagact cctacgggag gcagcagtgg 300 ggaatcttcc gcaatggacg aaagtctgac ggagcaacgc cgcgtgacg atgacggcct 360 tcggttgta aagttctgtt atatgggacg aacaggacat cggttaatac ccggtgtctt 420 tgacggtacc gtaagagaaa gccacggcta actacgtgcc agcagccgcg gtaatacgta 480 ggtggcaagc gttgtccgga attattgggc gtaagggcg cgcaggcggc atcgcaagtc 540 ggtcttaaaa gtgcggggct taaccccgtg agggaccga aactgtgaag ctcgagtgtc 600 ggagaggaaa gcggaatttcc tagtgtagcg gtgaaatgcg tagattag gaggaacacc 660 agtggcgaaa gcggctttct ggacgacaac tgacgctgag gcgcgaaagc caggggagca 720 aacgggatta gataccccgg tagtcctggc cgtaaacgat ggatactagg tgtaggaggt 780 atcgactcct tctgtgccgg agttaacgca ataagtatcc cgcctgggga gtacggccgc 840 aaggctgaaa ctcaaggaa ttgacgggg cccgcacaag cggtggagta tgtggttaa 900 ttcgacgcaa cgcgaagaac cttaccaagc cttgacattg attgctacgg aaagagattt 960 ccggttcttc ttcggaagac aagaaaacag gtggtgcacg gctgtcgtca gctcgtgtcg 1020 tgagatgttg ggttaagtcc cgcaacgagc gcaaccccta tcttctgttg ccagcacctc 1080 gggtggggac tcagaagaga ctgccgcaga caatgcggag gaaggcgggg atgacgtcaa 1140 gtcatcatgc cccttatggc ttgggctaca cacgtactac aatggctctt aatagaggga 1200 agcgaaggag cgatccggag caaaccccaa aaacagagtc ccagttcgga ttgcaggctg 1260 caactcgcct gcatgaagca ggaatcgcta gtaatcgcag gtcagcatac tgcggtgaat 1320 acgttcccgg gccttgtaca caccgcccgt cacaccacga aagtcattca cacccgaagc 1380 cggtgaggca accgcaag 1398 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> <400> 3 gatcaagatc attgctcctc 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> <400> 4 ttgtcaagaa agggtgtaac 20 <210> 5 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> <400> 5 ggtatcgtgg aaggactcat g 21 <210> 6 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> <400> 6 atgccagtga gcttcccgtt c 21 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> <400> 7 ctcagcaccg ctaacagagg 20 <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> <400> 8 cattggcgct tctctcctc 19 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> <400> 9 aagaggaatt ttgacactgg 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> <400> 10 gccatgtact cttcactttc 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> <400> 11 aagtcacact ggtgaaatcc 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> <400> 12 ctcttgctgc caaactatct 20 <210> 13 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> <400> 13 ccctcccctg gatcaggat 19 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> <400> 14 gccatcaaac tcgtccatca 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> <400> 15 cattacctgc tctacgtttg 20 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> <400> 16 agatggacat aagatgaggt g 21 <210> 17 <211> 1403 <212> DNA <213> Parabacteroides distasonis strain MRX0005 <400> 17 acccgggtgg cgaccggcgc acgggtgagt aacgcgtatg caacttgcct atcagagggg 60 gataacccgg cgaaagtcgg actaataccg catgaagcag ggatcccgca tgggaatatt 120 tgctaaagat tcatcgctga tagataggca tgcgttccat taggcagttg gcggggtaac 180 ggcccaccaa accgacgatg gataggggtt ctgagaggaa ggtcccccac attggtactg 240 agacacggac caaactccta cgggaggcag cagtgaggaa tattggtcaa tgggcgtgag 300 cctgaaccag ccaagtcgcg tgagggatga aggttctatg gatcgtaaac ctcttttata 360 agggaataaa gtgcgggacg tgtcccgttt tgtatgtacc ttatgaataa ggatcggcta 420 actccgtgcc agcagccgcg gtaatacgga ggatccgagc gttatccgga tttattgggt 480 ttaaagggtg cgtaggcggc cttttaagtc agcggtgaaa gtctgtggct caaccataga 540 attgccgttg aaactgggag gcttgagtat gtttgaggca ggcggaatgc gtggtgtagc 600 ggtgaaatgc atagatatca cgcagaaccc cgattgcgaa ggcagcctgc caagccatta 660 ctgacgctga tgcacgaaag cgtggggatc aaacaggatt agataccctg gtagtccacg 720 cagtaaacga tgatcactag ctgtttgcga tacactgtaa gcggcacagc gaaagcgtta 780 agtgatccac ctggggagta cgccggcaac ggtgaaactc aaaggaattg acgggggccc 840 gcacaagcgg aggaacatgt ggtttaattc gatgatacgc gaggaacctt acccgggttt 900 gaacgcattc ggacmgakgt ggaaacacat tttctagcaa tagccatttg cgaggtgctg 960 catggttgtc gtcagctcgt gccgtgaggt gtcggcttaa gtgccataac gagcgcaacc 1020 cttgccacta gttactaaca ggtaaagctg aggactctgg tgggactgcc agcgtaagct 1080 gcgaggaagg cggggatgac gtcaaatcag cacggccctt acatccgggg cgacacacgt 1140 gttacaatgg cgtggacaaa gggaagccac ctggcgacag ggagcgaatc cccaaaccac 1200 gtctcagttc ggatcggagt ctgcaacccg actccgtgaa gctggattcg ctagtaatcg 1260 cgcatcagcc atggcgcggt gaatacgttc ccgggccttg tacacaccgc ccgtcaagcc 1320 atgggagccg ggggtacctg aagtccgtaa ccgcgaggat cggcctaggg taaaactggt 1380 gactggggct aagtcgtacg ggg 1403 <210> 18 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> <400> 18 gattactgct ctggctccta g 21 <210> 19 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> <400> 19 gactcatcgt actcctgctt g 21 <210> 20 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> <400> 20 ctggcctcac tgtccacctt cc 22 <210> twenty one <211> 19 <212> DNA <213> Artificial Sequence <220> <223> <400> twenty one aatggtgaag gtcggtgtg 19 <210> twenty two <211> twenty four <212> DNA <213> Artificial Sequence <220> <223> <400> twenty two gtggagtcat actggaacat gtag 24 <210> twenty three <211> 20 <212> DNA <213> Artificial Sequence <220> <223> <400> twenty three tgcaaatggc agccctggtg 20 <210> twenty four <211> twenty three <212> DNA <213> Artificial Sequence <220> <223> <400> twenty four gctgccttga tgtttacttt gac 23 <210> 25 <211> twenty three <212> DNA <213> Artificial Sequence <220> <223> <400> 25 gcaaccgaag tatgaaataa cca 23 <210> 26 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> <400> 26 accaggtgag aagagtgatg accatcc 27 <210> 27 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> <400> 27 agccagagtc cttcagaga 19 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> <400> 28 tccttagcca ctccttctgt 20 <210> 29 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> <400> 29 cctaccccaa tttccaatgc tctcct 26 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> <400> 30 ccctgtatcg taagaacggt 20 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> <400> 31 gccaccattg atcacgttga 20 <210> 32 <211> 19 <212> DNA <213> <400> 32 cccaagcaac tagcccctc 19 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> <400> 33 ggcagcacat cagggtagtc 20 <210> 34 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> <400> 34 gtagccgtga tgtggtcatt t 21 <210> 35 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> <400> 35 ctgtgcgccc agattacct 19 <210> 36 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> <400> 36 ctcggctttg tgaaggtgct 20 <210> 37 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> <400> 37 ggcttcatgg catcaacttc a 21

Claims

1. Contains giant cocci ( Megasphaera The use of a composition of bacterial strains of the genus ( ) in the preparation of a medicament for the treatment or prevention of neurodegenerative disorders, wherein the bacterial strain is *Macrococcus martensii* MRx0029, deposited with accession number NCIMB 42787.

2. The use as described in claim 1, wherein the drug is used to treat or prevent neurodegenerative disorders selected from the group consisting of: Parkinson's disease; Alzheimer's disease; multiple sclerosis; Huntington's disease; amyotrophic lateral sclerosis; prions; spinocerebellar ataxia; spinal muscular atrophy; primary progressive aphasia; mild cognitive impairment; HIV-related cognitive impairment; and corticobasal degeneration.

3. The use as described in claim 1, wherein the drug is used to treat or prevent motor neuron disease.

4. The use as described in claim 1, wherein the drug is used to treat or prevent dementia.

5. The use as claimed in claim 1, wherein the neurodegenerative disorder is progressive supranuclear palsy, i.e., Steele-Richardson-Olszewski syndrome, normal pressure hydrocephalus, vascular or arteriosclerotic Parkinson's disease, or drug-induced Parkinson's disease.

6. The use as claimed in claim 1, wherein the neurodegenerative disorder is Benson's syndrome.

7. The use as claimed in claim 4, wherein the dementia is Lewy body, vascular, or frontotemporal dementia.

8. The use as described in claim 2, wherein the drug is used to treat or prevent Parkinson's disease.

9. The use as described in any one of claims 1-8, wherein the drug is used to treat or prevent early-onset neurodegenerative diseases, and / or wherein the drug is used to prevent or delay the onset or progression of neurodegenerative disorders.

10. The use as described in claim 1, wherein the drug is used to treat or prevent Parkinson's disease.

11. The use according to any one of claims 1-8, wherein the drug is for oral administration, and / or wherein the drug comprises one or more pharmaceutically acceptable excipients or carriers, and / or wherein the bacterial strain is lyophilized.

12. Contains giant cocci ( Megasphaera The use of a composition of bacterial strains of the genus ( ) in the preparation of a medicament for the treatment of brain injury, wherein the bacterial strain is *Macrococcus martensii* MRx0029, deposited under accession number NCIMB 42787.

13. The use as described in claim 12, wherein the brain injury is a stroke.

14. The use as described in claim 13, wherein the stroke is an ischemic stroke or a hemorrhagic stroke.

15. The use as described in claim 13, wherein the stroke is cerebral ischemia.

16. The use as described in claim 13, wherein the stroke is focal cerebral ischemia.

17. The use as described in claim 12, wherein the drug is used to treat brain injury caused by stroke.

18. The use as claimed in any one of claims 12-16, wherein the drug is for oral administration, and / or wherein the drug comprises one or more pharmaceutically acceptable excipients or carriers, and / or wherein the bacterial strain is lyophilized.

19. Cells of a strain of *Macrococcus martensii* deposited under accession number NCIMB 42787.

20. A composition comprising the cells as described in claim 19.

21. The composition of claim 20, wherein it comprises a pharmaceutically acceptable carrier or excipient.

22. A biologically pure culture of a strain of *Macrococcus martensii* deposited under accession number NCIMB 42787.

Images