Compound for dopaminergic neuron culture and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a compound capable of being used for culturing dopaminergic neurons and application of the compound. The glycopeptide fiber is constructed on the basis of natural amino acid and natural saccharide molecules through a simple chemical reaction, and the glycopeptide fiber is clear in preparation structure and simple in step, has excellent biological safety and good biological activity, and has an obvious supporting effect on the growth of dopaminergic neuronal cells.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a compound that can be used for culturing dopaminergic neurons and an application thereof. Background Art

[0002] The extracellular matrix (ECM) is an important biological scaffold, mainly composed of collagen, fibronectin and elastin as fibril components, and proteoglycans and glycoproteins as non-fibril components (SharmaA, SharmaP, Roy S. Elastin-inspired supramolecular hydrogels: a multifaceted extracellular matrix protein in biomedical engineering [J]. Soft Matter, 2021, 17 (12): 3266-3290.). At present, researchers have designed a variety of compounds to simulate the natural cell environment in vitro and regulate the in vitro cell process to a certain extent. For example, traditional synthetic polymers, including polyethylene glycol hydrogels (Zhang X, BattigMR, Chen N, et al. Chimeric Aptamer-Gelatin Hydrogels as an Extracellular Matrix Mimic for Loading Cells and Growth Factors[J]. Biomacromolecules, 2016, 17(3): 778-787.), poly(ε-caprolactone) (PCL) fiber scaffolds (Formica FA, E, Hess SC, et al. A Bioinspired Ultraporous Nanofiber-Hydrogel Mimic of the Cartilage Extracellular Matrix [J]. Advanced Healthcare Materials, 2016, 5 (24): 3129-3138.), polyallylguanidine (PAG) (Ji YR, Homaeigohar S, Wang YH, et al. Selective Regulation of Neurons, Glial Cells, and Neural Stem / Precursor Cells by Poly (allylguanidine)-Coated Surfaces [J]. ACS Applied Materials & Interfaces, 2019, 11 (51): 48381-48392.), etc.; modifying naturally extracted polysaccharides, such as hyaluronic acid (Chantre CO, Gonzalez GM, Ahn S, et al. Porous Biomimetic Hyaluronic Acid and Extracellular Matrix Protein Nanofiber Scaffolds for Accelerated Cutaneous Tissue Repair[J].ACS Applied Materials&Interfaces,2019,11(49):45498-45510.) and chondroitin sulfate (Menezes R, Hashemi S, Vincent R,et al.Investigation of glycosaminoglycanmimetic scaffolds for neurite growth[J].Acta Biomaterialia,2019,90:169-178.); and hydrogels formed by self-assembly of amphiphilic molecules (Jain R,Roy S.Controlling Neuronal Cell Growth through Composite Laminin Supramolecular Hydrogels[J].ACS Biomaterials Science&Engineering,2020,6(5):2832-2846.), etc.

[0003] Parkinson's disease (PD) is the second largest neurodegenerative disease, and its pathological feature is the loss of dopaminergic neurons in the substantia nigra of the midbrain, which leads to motor symptoms such as resting tremor. Although the currently used dopaminergic drugs and deep brain stimulation can improve symptoms, they have certain side effects and cannot completely prevent the progression of the disease. With the development of stem cell technology, cell therapy has become a promising new method for PD treatment in recent years. Directed differentiation of human pluripotent stem cells (hPSC) into dopaminergic progenitors (DAPs) and then transplantation into the brain of PD patients can effectively improve the symptoms of patients. In order to prepare cell products for clinical application, all reagents for culturing DAPs should meet the GMP (Good manufactor practice) level. During the DAP culture process, the surface of the culture dish needs to be coated with a neuronal culture matrix to promote cell adhesion and growth. In neural tissue engineering, damaged nerve cells are usually difficult to regenerate. The growth matrices for culturing nerve cells currently used in mainstream laboratories mainly include poly-L-Ornithine, fibronectin and laminin co-coating (P / F / L) (Song B, Cha Y, Ko S, et al. Human autologous iPSC–derived dopaminergic progenitors restore motor function in Parkinson's disease models[J]. The Journal of Clinical Investigation, 2020, 130(2):904-920.); Geltrex (Tofoli F A, Semeano ATS, Oliveira-Giacomelli et al.: Midbrain Dopaminergic NeuronsDifferentiated from Human-Induced Pluripotent Stem Cells, Daadi MM, editor, Neural Stem Cells: Methods and Protocols, New York, NY: Springer New York, 2019:97-118.), Laminin111 (Kirkeby A, Nolbrant S, Tiklova K, et al. Predictive MarkersGuide Differentiation to Improve Graft Outcome in Clinical Translation of hESC-Based Therapy for Parkinson's Disease[J]. Cell Stem Cell, 2017, 20(1):135-148.).

[0004] At present, the internationally recognized neuronal culture matrix mainly includes poly-L-Ornithine (PLO), fibronectin and laminin co-coating (denoted as P / F / L) as well as GelTrex and Laminin111. However, Laminin, GelTrex and Laminin111 are not GMP grade, especially Laminin and GelTrex are purified and extracted from the basement membrane of mouse sarcoma (Engelbreth-Holm-Swarm tumor), which does not meet the requirements of clinical transformation. This will also become a huge hidden danger in the process of culturing neural cells in vitro for disease treatment. Therefore, finding a neuronal culture matrix that meets the GMP grade is a key link in the preparation of clinical-grade DAP and the clinical application of Parkinson's cell therapy. Summary of the invention

[0005] The present invention is based on the construction of glycopeptide fibers through simple chemical reactions of natural amino acids and natural sugar molecules. The glycopeptide fibers have a clear structure, simple steps, excellent biosafety, and obvious growth-supporting effects on dopaminergic neuron cells. The present invention is completed based on this.

[0006] In a first aspect, the present invention provides a glycopeptide fiber, wherein the glycopeptide fiber is composed of a glycopeptide molecule and a class of hydrophobic polypeptides through an acylation reaction to form a hydrazide bond or a click reaction, and the structural formula of the glycopeptide fiber is shown in formula (I):

[0007]

[0008] Wherein, n=1-10000, the oxygen-containing ring is a glycopeptide molecule, oligopeptide is a hydrophobic polypeptide, Linker is a connecting bond between the glycopeptide molecule and the polypeptide, and R is a substituent.

[0009] Furthermore, the glycopeptide molecule includes one or more of sialyllactose (SL), lactose (Lac) and / or chondroitin sulfate hexasaccharide (CS-H).

[0010] Furthermore, the linker includes but is not limited to a hydrazide bond, a triazole bond, an O bond, an N bond or an S bond.

[0011] Furthermore, the substituent is selected from one or more of OH, COOH, NHAc and / or Sia.

[0012] In one embodiment of the present invention, when the glycopeptide molecule is sialyllactose (SL), the glycopeptide fiber is preferably of the following structural formula:

[0013]

[0014] In one embodiment of the present invention, when the glycopeptide molecule is lactose (Lac), the glycopeptide fiber is preferably of the following structural formula:

[0015]

[0016] In one embodiment of the present invention, when the glycopeptide molecule is chondroitin sulfate hexasaccharide (CS-H), the glycopeptide fiber is preferably of the following structural formula:

[0017]

[0018] In a second aspect, the present invention provides a pharmaceutical composition comprising the glycopeptide fiber according to the first aspect.

[0019] Furthermore, the pharmaceutical composition may also contain a stereoisomer of the glycopeptide fiber described in the first aspect, or a pharmaceutically acceptable salt thereof, or a tautomer thereof, or an isotope-labeled compound thereof.

[0020] Furthermore, the pharmaceutical composition can be prepared into a variety of dosage forms, including but not limited to one or more of tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories and / or lyophilized powder injections.

[0021] Furthermore, the various preparations may also add colorants, preservatives, spices, flavoring agents, sweeteners or other materials to the pharmaceutical preparations as needed.

[0022] Furthermore, the pharmaceutical composition can be administered by injection, cavity administration, respiratory tract administration or mucosal administration.

[0023] Furthermore, the injection administration includes subcutaneous injection, intravenous injection, intramuscular injection and intracavitary injection, etc.; the cavity administration includes rectal or vaginal administration; and the respiratory tract administration is nasal administration.

[0024] In a third aspect, the present invention provides use of the glycopeptide fiber described in the first aspect in preparing a matrix for culturing dopaminergic neurons.

[0025] In a fourth aspect, the present invention provides use of the glycopeptide fiber described in the first aspect in the preparation of a drug for treating neurodegenerative diseases.

[0026] Furthermore, the neurodegenerative diseases include, but are not limited to, Alzheimer's disease, Parkinson's disease, primary tauopathy, frontotemporal dementia, amyotrophic lateral sclerosis, synucleinopathy, Huntington's disease and related polyglutamine diseases, prion disease, traumatic brain injury, chronic traumatic encephalopathy, stroke, spinal cord injury, and multiple sclerosis.

[0027] Preferably, the neurodegenerative disease is Parkinson's disease.

[0028] Beneficial Effects

[0029] 1. The structural design of the glycopeptide fiber in the present invention is mainly based on the construction of natural amino acids and natural sugar molecules through simple chemical reactions. Compared with other products with similar functions, the preparation structure is clear and the steps are simple.

[0030] 2. The glycopeptide fiber of the present invention has excellent biosafety and has the potential for further development.

[0031] 3. The glycopeptide fiber of the present invention has a significant effect on supporting the growth of dopaminergic neuron cells and has good biological activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Characterization of SL(2,3)-2F sialic acid glycopeptide.

[0033] Figure 2 Characterization of SL(2,3)-3F sialic acid glycopeptide.

[0034] Figure 3 Characterization of SL(2,3)-4F sialic acid glycopeptide.

[0035] Figure 4Characterization of Lac-4F lactose glycopeptide.

[0036] Figure 5 Characterization of CS-H-3F chondroitin sulfate hexasaccharide glycopeptide.

[0037] Figure 6 This is a cytotoxicity test for SL(2,3)-3F sialic acid glycopeptide.

[0038] Figure 7 The effect of PLO, Fibronectin and SL-4F co-encapsulation on the growth of dopaminergic neurons. Note: A is the experimental flow chart; B is PLO, Fibronectin and SL-4F (0.5 mg / mL) co-encapsulation; C is PLO, Fibronectin and SL-4F (1 mg / mL) co-encapsulation.

[0039] Figure 8 The effect of combined coating of PLO, Fibronectin and Lac-4F or CS-H-3F on the growth of dopaminergic neurons.

[0040] Fig. 9 This is an in vitro safety and efficacy test of PLO, Fibronectin and SL-4F co-encapsulation on the growth of dopaminergic neurons. Note: A and B are images taken with a confocal microscope after immunofluorescence staining; C and D are statistical graphs of the percentage of positive cells.

[0041] Fig.10 This is an in vivo safety and efficacy test of PLO, Fibronectin and SL-4F co-encapsulation on the growth of dopaminergic neurons. Note: Figure A is a brain slice after cells were implanted into the mouse brain after improved culture; Figure B is a brain slice after HE staining; Figure C is a confocal fluorescence image of brain slices after immunofluorescence staining; Figure D is the corresponding positive molecule statistical graph. DETAILED DESCRIPTION

[0042] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.

[0043] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0044] Explanation of terms

[0045] GMP: Good manufacturing practice, which means "good manufacturing practice" or "production quality management practice" in Chinese. The World Health Organization defines GMP as a regulation guiding the production and quality management of food, drugs, and medical products, aiming to ensure the safety, effectiveness, and controllability of the quality and production process of food, drugs, and medical products. GMP requires that manufacturers of food, drugs, and medical products should have good production equipment, reasonable production processes, perfect quality management, and strict testing systems to ensure that the quality of the final product (including food safety and hygiene) meets regulatory requirements.

[0046] Ki67: It is a cell cycle-related nuclear protein of proliferating cells, encoded by the MKI67 gene, expressed at various stages of the cell cycle, and expressed at a higher level in actively proliferating cells. There is a positive correlation between the expression level of Ki67 protein and cell proliferation activity, that is, high expression of Ki67 usually means active cell proliferation. In clinical medicine, Ki67 protein is widely used in the diagnosis, treatment and prognosis evaluation of tumors. Ki67 can accurately reflect the proliferation activity of tumor cells and is closely related to the development, metastasis and prognosis of various tumors.

[0047] SOX1: Also known as sex-determining region Y-box protein 1 (SRY-box 1) protein, it is a transcription factor. It belongs to the SOX transcription factor family, whose members are widely expressed in embryonic development and nervous system development and regulate a variety of developmental processes. During the development of the nervous system, SOX1 protein can promote the differentiation and connection of neurons. In adults, SOX1 protein is still actively expressed in a few parts (such as cerebellar Bergmann glial cells), while its expression in other parts is downregulated.

[0048] PAX6: is a transcription factor that plays a key role in the process of neural development. It belongs to the paired box gene family, whose members play an important role in the development of the nervous system, such as the eyes, brain, and olfactory bulb. PAX6 protein is a key marker of early neural ectoderm and is involved in regulating the differentiation and development of nerve cells. If NSC (neural stem cell) continues to express PAX6, it may be restricted from differentiating into specific neuronal subtypes.

[0049] PLO: Poly-L-ornithine, is a polymer composed of ornithine monomers connected by peptide bonds. In aqueous solution, PLO is cationic and can interact with negatively charged substances. PLO has good biocompatibility and low toxicity to organisms, and can be used as a carrier for drug delivery systems. In addition, PLO can reverse the inhibitory effect of fibronectin on oligodendrocyte differentiation and promote the regeneration of myelin.

[0050] Fibronectin: Fibronectin, referred to as FN, is a high molecular weight glycoprotein widely present in animal tissues and tissue fluids. It mainly exists in three forms: plasma fibronectin (PFN), cell surface fibronectin (CFN) and matrix fibronectin (such as fetal fibronectin). It has many important functions in the body, such as cell adhesion and migration, wound repair and healing, immune regulation and promotion of cell metabolism.

[0051] Example 1 Synthesis of oligopeptide segments

[0052]

[0053] Synthetic route of oligopeptide segments

[0054] Synthesis of 1: Weigh Boc-L-phenylalanine (10.0 g, 38 mmol), 1-hydroxybenzotriazole (6.00 g, 45 mmol) and EDC (8.67 g, 45 mmol), add DCM (200 mL) under ice bath, stir and dissolve completely, slowly add DIPEA (14.6 g, 113 mmol) to the solution, stir for about 10 minutes, then slowly add L-phenylalanine methyl ester hydrochloride, and react at room temperature for 24 hours. After the reaction is completed, vacuum distillation is performed to remove most of the solvent, and equal volumes of HCl solution, NaOH solution and saturated NaCl solution are added for liquid extraction. After the organic phase is combined, it is dried, filtered and spin-dried with anhydrous MgSO4, and separated by silica gel column chromatography. The mobile phase is DCM / EA (v / v) = 20 / 1, and the yield is 72%.

[0055] Synthesis of 2: Weigh 1 (510 mg, 0.89 mmol) and dissolve it completely with as little methanol or a mixed solvent of methanol and dichloromethane as possible, then add hydrazine hydrate (862 μL, 17.8 mmol), stir and react at room temperature for one hour, a large amount of white precipitate appears, and the white precipitate is collected after washing with water, and freeze-dried to obtain a white powder with a yield of 93%.

[0056] Synthesis of 3: Weigh 1 (2.00 g, 4.7 mmol) and dissolve it in an appropriate amount of methanol, add 1 M aqueous solution of NaOH (0.3 g, 7 mmol), react at room temperature, and point the plate to determine the end of the reaction. Dilute with ethyl acetate and extract with potassium carbonate solution and saturated brine in sequence, dry the organic phase with anhydrous magnesium sulfate, and spin dry to obtain a white solid product 3 with a yield of 95%.

[0057] Synthesis of 6: Weigh 1 (2.00 g, 4.7 mmol) and add it to DCM (40 mL) and stir to dissolve. Add trifluoroacetic acid (10.5 mL, 141 mmol) under stirring and react at room temperature for 3 hours. After the reaction is completed, spin dry to obtain a white solid product 6 with a yield of 98%.

[0058] Synthesis of 4 and 7: refer to the synthesis of 1.

[0059] Synthesis of 5 and 8: refer to the synthesis of 2.

[0060] Example 2 Synthesis of glycopeptide molecules

[0061]

[0062] Synthetic route of SL(2,3)-2F sialic acid glycopeptide

[0063] Synthesis of SL(2,3)-2F: 3 (181 mg, 0.32 mmol) and SL(2,3) (200 mg, 0.32 mmol) were weighed and dissolved in anhydrous methanol (4 mL), and yttrium trifluoromethanesulfonate (51 mg, 0.095 mmol) was added. The reaction was carried out at 50°C under argon protection for 48 hours. After the reaction, the solvent was dried and separated by silica gel column chromatography. The mobile phase was DCM / MeOH (v / v) = 2 / 3. The yield was 59%. Characterization of SL(2,3)-2F sialic acid glycopeptide Figure 1 shown.

[0064] The synthesis of SL(2,3)-3F and SL(2,3)-4F was based on the synthesis of SL(2,3)-2F. Figure 2 As shown; SL (2,3) -4F sialic acid glycopeptide characterization as shown Figure 3 shown.

[0065]

[0066] Synthesis route of lactose glycopeptide (Lac-4F)

[0067] The synthesis of Lac-4F lactose glycopeptide was based on the synthesis of SL(2,3)-2F. The characterization of Lac-4F lactose glycopeptide was as follows: Figure 4 shown.

[0068]

[0069] Synthesis route of chondroitin sulfate hexaglycopeptide (CS-H-3F)

[0070] Synthesis of CS-H-3F: Weigh CS-H-N3 (50 mg, 0.04 mmol) and 3F (21 mg, 0.04 mmol) and dissolve them in a mixed solution of water and DMF, add copper sulfate pentahydrate (2 mg, 0.008 mmol) and sodium ascorbate (16 mg, 0.08 mmol), and react at 50°C under argon protection for 48 hours. After the reaction, separate using a C18 column. Characterization of CS-H-3F chondroitin sulfate hexasaccharide glycopeptide Figure 5 shown.

[0071] Example 3 Cytotoxicity test of glycopeptide fibers on neural cells

[0072] SL(2,3)-3F was co-incubated with HepG2 cells and RAW264.7 cells for 24 h, and the cell viability was determined by the CCK8 method.

[0073] from Figure 6 It can be observed that at a concentration of 250 μg / mL, there is still no obvious cytotoxicity.

[0074] Example 4 Detection of the biological activity of glycopeptide fibers on nerve cells

[0075] SL-4F, Lac-4F, and CS-H-3F were used in combination with PLO and Fibronectin. First, human embryonic stem cells were plated on a culture dish, and induction medium was added for culture on days 1-5. Neural progenitor cell induction medium was used on days 6-7, and neural progenitor cell induction medium without β-mercaptoethanol was used on days 8-11. Quercetin was added starting on day 9 to remove residual undifferentiated hPSCs. On day 12 of differentiation, 0.0015% PLO was added to the culture medium, and on day 13, the PLO solution was aspirated and replaced with 2μg / mL Fibronectin, and on day 14, it was replaced with 2μg / mL SL-4F, Lac-4F, and CS-H-3F. On day 15, the cells were dissociated, and the single-cell suspension was re-plated on a new plate and cultured. The cell growth status of SL-4F (4 μg / mL and 2 μg / mL) combined with PLO and Fibronectin and the positive control was observed on days 16, 17, 19 and 21.

[0076] like Figure 7 As shown, SL-4F combined with PLO and Fibronectin can effectively support DAP culture, and the cell morphology is similar to that of the positive control group P / F / L.

[0077] After the neural cells cultured to day 19 were digested and replated, they were coated with PLO, Fibronectin and Lac-4F or CS-H-3F, and the cells were observed to grow successfully on the wall.

[0078] like Figure 8 As shown, it was proved that Lac-4F, CS-H-3F combined with PLO and Fibronectin can also effectively support DAP culture.

[0079] Example 4 In vitro safety and efficacy testing of PLO, Fibronectin and SL-4F combined coating on DAP growth

[0080] The safety and efficacy of DAP cultured with SL-4F and PLO, Fibronectin combined coating groups were tested by immunofluorescence. Cells were fixed with cold paraformaldehyde (PFA) and then rinsed. Cells were permeabilized with 0.3% Triton-X100 in 0.3% PBS (0.3% PBST) and blocked for 1 hour with 3% normal donkey serum and 3% bovine serum albumin (BSA) in 0.3% PBST, and then incubated with primary antibodies overnight. After rinsing the primary antibody with 0.3% PBST, cells were incubated with fluorescent conjugated secondary antibodies and Hoechst at room temperature for 1 hour. After rinsing the secondary antibody with 0.3% PBST, images were collected using a confocal microscope.

[0081] like Fig. 9 As shown, compared with the control group, DAPs cultured in the SL-4F and PLO, Fibronectin combined coating groups all expressed the same proportion of markers (FOXA2, LMX1A and TH), and no stem cell markers (OCT4 and SSEA4) were detected.

[0082] Example 5 In vivo safety and efficacy of PLO, Fibronectin and SL-4F combined coating on DAP growth

[0083] SL-4F and PLO, Fibronectin-coated DAP were stereotaxically injected into NODSCID mice, and brain slices were analyzed 3 months after transplantation.

[0084] like Fig.10 As shown, compared with the control group, DAPs cultured in the SL-4F and PLO, Fibronectin co-coated groups could express the same proportion of TH-positive cells. H&E staining revealed that no tumor formation or other abnormal structures were observed in the grafts. Further staining of brain slices revealed that Ki67 / SOX1 / PAX6 co-labeled cells were not detected in either the experimental or control groups, indicating the long-term safety of PLO, Fibronectin and SL-4F co-coated DAPs in mice.

Claims

1. A glycopeptide fiber, wherein the glycopeptide fiber is composed of a glycopeptide molecule and a class of hydrophobic polypeptides through an acylation reaction to form a hydrazide bond or a click reaction, and the structural formula of the glycopeptide fiber is shown in formula (I): in, n=1-10000, the oxygen-containing ring is a glycopeptide molecule, oligopeptide is a hydrophobic polypeptide, Linker is a linker connecting the glycopeptide molecule and the polypeptide, and R is a substituent.

2. The glycopeptide fiber according to claim 1, wherein the glycopeptide molecule comprises one or more of sialyllactose (SL), lactose (Lac) and / or chondroitin sulfate hexasaccharide (CS-H).

3. The glycopeptide fiber according to any one of claims 1 or 2, wherein the linker includes but is not limited to a hydrazide bond, a triazole connection, an O connection, an N connection or an S connection.

4. The glycopeptide fiber according to any one of claims 1 to 3, wherein the substituent is selected from one or more of OH, COOH, NHAc and / or Sia.

5. The glycopeptide fiber according to any one of claims 1 to 4, when the glycopeptide molecule is sialyllactose (SL), the glycopeptide fiber is preferably of the following structural formula: When the glycopeptide molecule is lactose (Lac), the glycopeptide fiber is preferably of the following structural formula: When the glycopeptide molecule is chondroitin sulfate hexasaccharide (CS-H), the glycopeptide fiber is preferably of the following structural formula:

6. A pharmaceutical composition comprising the glycopeptide fiber according to claim 1 or its stereoisomer, or its pharmaceutically acceptable salt, or its tautomer, or its isotope-labeled compound.

7. The pharmaceutical composition according to claim 6, which can be prepared into various dosage forms, including but not limited to one or more of tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories and / or lyophilized powder injections.

8. Use of the glycopeptide fiber as claimed in claim 1 in preparing a matrix for culturing dopaminergic neurons.

9. Use of the glycopeptide fiber according to claim 1 in the preparation of a drug for treating neurodegenerative diseases.

10. The use as claimed in claim 9, wherein the neurodegenerative disease includes but is not limited to Alzheimer's disease, Parkinson's disease, primary tau disease, frontotemporal dementia, amyotrophic lateral sclerosis, synuclein disease, Huntington's disease and related polyglutamine diseases, prion disease, traumatic brain injury, chronic traumatic encephalopathy, stroke, spinal cord injury and multiple sclerosis; preferably Parkinson's disease.

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