Method for regulating neurotoxic reactive astrocytes, regulator and application

Abstract

TMEM164 and the like can prevent and inhibit the induced activation of neurotoxic reactive astrocytes and neuron death in neurodegenerative diseases. The present invention relates to the field of neuroscience, in particular the medical treatment of neurodegenerative diseases. TMEM164, SOC18, CAPN15 and the like are identified as early reaction regulatory factors of neurotoxic astrocyte reactivity, and it is found that for example, TMEM164 overexpression can prevent induced activation of neurotoxic reactive astrocytes and neuron death in mouse and human astrocytes, as well as in an inflammation-induced PD mouse model and a 5XFAD mouse model; therefore, a basis is provided for treatment of neurodegenerative diseases related to neurotoxic reactive astrocytes.

Description

Technical Field

[0001] The present invention relates to the field of neuroscience, and specifically provides a method capable of regulating neurotoxic reactive astrocytes and a regulator of neurotoxic reactive astrocyte activation, and also provides the use of the method or the regulator for preventing or treating nerve damage diseases and / or protecting nerves. Background Art

[0002] Astrocytes are the most abundant cell type in the mammalian central nervous system, playing a protective role in the brain, performing important functions such as synapse formation, neuronal trophic support, regulation of neuronal metabolite supply, and propagation of action potentials. However, in brain injury, stroke, infection, or neurodegenerative diseases (such as Alzheimer's disease, Parkinson's disease, and multiple sclerosis), astrocytes undergo significant morphological and gene expression changes.

[0003] Jennifer L Zamanian et al. (J Neurosci. 2012 May 2; 32(18): 6391-410) and Shane A Liddelow et al. (Nature. 2017 Jan 26; 541(7638): 481-487.) performed genetic analysis of reactive astrocytes from inflammatory and ischemic mouse models and showed that neuroinflammation and ischemia induce the activation of quiescent astrocytes into neurotoxic reactive astrocytes (also known as A1 astrocytes) and neuroprotective reactive astrocytes (also known as A2 astrocytes). During this process, neuroinflammatory microglia release interleukin-1α (IL-1α), tumor necrosis factor (TNF) and complement component subunit 1q (C1q). These three cytokines induce the transformation of astrocytes into neurotoxic reactive astrocytes. Neurotoxic reactive astrocytes not only lose the normal functions of resting astrocytes, such as the function of promoting neuronal survival, growth, synaptogenesis and phagocytosis, but also induce neuronal and oligodendrocyte death through saturated lipids.

[0004] Although there have been some previous studies on reactive astrocytes, there is still a lack of effective means to regulate the activation of neurotoxic reactive astrocytes. Summary of the invention

[0005] The purpose of the present invention is to provide a method and a regulator for regulating neurotoxic reactive astrocytes, and the use of the regulator for treating nervous system diseases.

[0006] In order to achieve the above object, the technical solution provided by the present invention is as follows:

[0007] In a first aspect of the present invention, a method for regulating neurotoxic reactive astrocytes is provided, the method comprising: administering to an individual in need a TMEM164 regulator capable of upregulating the expression level of TMEM164, or administering to an individual in need a SOX18 regulator capable of upregulating the expression level of SOX18, or administering to an individual in need a CAPN15 regulator capable of downregulating the expression level of CAPN15; or administering a combination of at least two.

[0008] In the second aspect of the present invention, a method for preventing or treating nerve injury diseases is provided, comprising: administering to an individual in need a TMEM164 regulator capable of upregulating the expression level of TMEM164, or administering to an individual in need a SOX18 regulator capable of upregulating the expression level of SOX18, or administering to an individual in need a CAPN15 regulator capable of downregulating the expression level of CAPN15; or administering a combination of at least two of the above; preferably, the nerve injury disease is a disease related to neuronal death.

[0009] In some specific embodiments, in the methods described in the first aspect and the second aspect of the present invention, the TMEM164 regulator can upregulate the expression level of TMEM164 in glial cells, the SOX18 regulator can upregulate the expression level of SOX18 in glial cells; or the CAPN15 regulator can downregulate the expression level of CAPN15 in glial cells; preferably, the glial cells are from the central nervous system, the visual nervous system, or the auditory nervous system.

[0010] In some specific embodiments, the glial cells described in any of the above items include astrocytes, Muller glial cells, or cochlear glial cells (satellite cells); preferably, the astrocytes include GFAP-positive astrocytes, fibrous astrocytes or protoplasmic astrocytes.

[0011] In some specific embodiments, the astrocytes described in any of the foregoing are non-reactive astrocytes.

[0012] In some specific embodiments, the modulator described in any of the above items can regulate the activation of non-reactive astrocytes into reactive astrocytes; preferably, the regulation is to inhibit the transformation of non-reactive astrocytes into neurotoxic reactive astrocytes; and / or to enhance the transformation of non-reactive resting astrocytes into neuroprotective reactive astrocytes.

[0013] In some specific embodiments, in any of the methods described above, compared with cells not given the modulator, cells given any of the above modulators have a reduced expression of at least one marker of neurotoxic reactive astrocytes after administration of the modulator; preferably, the marker is selected from C3, H2-T23, H2-D1, Iigp1, Gbp2, Fkbp5, Psmb8, Srgn, Serping1, or Amigo2, or the expression of at least two markers is reduced.

[0014] In some specific embodiments, in any of the methods described above, compared with cells not administered with any of the aforementioned modulators, the amount of resting astrocytes converted into neurotoxic reactive astrocytes after administration of the modulator is reduced.

[0015] In some specific embodiments, in any of the methods described above, compared with cells not given the modulator, the amount of resting astrocytes converted into neurotoxic reactive astrocytes after being stimulated by the modulator is reduced in cells given the modulator.

[0016] In some specific embodiments, the stimulus received includes ischemia, trauma, inflammation, neurodegeneration and aging.

[0017] In some specific embodiments, in any of the methods described above, compared with cells not given the modulator, cells given any of the above modulators have an increased level of at least one marker of neuroprotective reactive astrocytes after administration of the modulator; preferably, the marker is selected from Clcf1, S100a10, Sphk1, Ptgs2, Emp1 or Tm4sf1.

[0018] In some specific embodiments, in any of the methods described above, compared with cells not given the modulator, the amount of resting astrocytes converted into neuroprotective reactive astrocytes after being stimulated by the modulator is increased in cells given the modulator.

[0019] In some specific embodiments, the stimulus received includes ischemia, trauma, inflammation, neurodegeneration and aging.

[0020] In some specific embodiments, in any of the methods described above, the TMEM164 regulator is an epigenetic regulation tool, a TMEM164 gene activator, a TMEM164 gene expression activator, a TMEM164 protein degradation inhibitor, a TMEM164 mRNA stabilizer, an exogenous TMEM164 or a functional fragment of TMEM164.

[0021] In a preferred embodiment, the exogenous TMEM164 or functional fragment of TMEM164 is a polypeptide of TMEM164 or a biologically active fragment thereof, or a nucleic acid encoding a polypeptide of TMEM164 or a biologically active fragment thereof; TMEM164 is preferably human TMEM164.

[0022] In a preferred embodiment, the exogenous TMEM164 or functional fragment of TMEM164 is a mutant of the TMEM164 polypeptide or a biologically active fragment thereof that retains the function of TMEM164; preferably, the mutant has at least 80%, 85%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% homology with the TMEM164 polypeptide or a biologically active fragment thereof.

[0023] In a preferred embodiment, the TMEM164 polypeptide comprises a sequence as shown in SEQ ID NO: 31, 32, 33, 34, 35, 36, 37, or 38, or a sequence having at least 80%, 85%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% homology to the sequence as shown in SEQ ID NO: 31, 32, 33, 34, 35, 36, 37, or 38.

[0024] In some specific embodiments, in any of the methods described above, the Sox18 regulator is an epigenetic regulation tool, a Sox18 gene expression activator, a Sox18 protein degradation inhibitor, a Sox18 mRNA stabilizer, exogenous Sox18 or a functional fragment of Sox18.

[0025] In a preferred embodiment, the exogenous Sox18 or the functional fragment of Sox18 is a Sox18 polypeptide or a biologically active fragment thereof, or a nucleic acid encoding a Sox18 polypeptide or a biologically active fragment thereof; Sox18 is preferably human Sox18.

[0026] In a preferred embodiment, the exogenous Sox18 or functional fragment of Sox18 is a mutant of the Sox18 polypeptide or its biologically active fragment that retains the Sox18 function; preferably, the mutant has at least 80%, 85%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% homology with the Sox18 polypeptide or its biologically active fragment.

[0027] In a preferred embodiment, the Sox18 refers to the gene with the gene number 54345 in NCBI.

[0028] In some specific embodiments, in any of the methods described above, the Capn15 regulator is an epigenetic regulation tool, a Capn15 gene expression inhibitor, a Capn15 activity inhibitor, a Capn15 antibody, a Capn15 protein degradation activator, a gene editing tool targeting Capn15, PROTAC, ASO, miRNA, RNAi, shRNA, Capn15 binding protein or protein domain.

[0029] In a preferred embodiment, the Capn15 refers to the gene with a gene number of 6650 in NCBI.

[0030] In some specific embodiments, in any of the aforementioned methods, the regulator is a small molecule compound, an antibody, a protein, a polypeptide, a gene fragment, or a polysaccharide.

[0031] In a preferred embodiment, the TMEM164 gene expression activator is a modifier of endogenous TMEM164, an agonist that enhances the expression of the TMEM164 gene or protein, or an inhibitor of a TMEM164 competitive gene.

[0032] In a preferred embodiment, the Sox18 gene expression activator is a modifier of endogenous Sox18, an agonist that enhances the expression of Sox18 gene or protein, or an inhibitor of Sox18 competitive gene.

[0033] In a preferred embodiment, the Capn15 gene expression inhibitor is a modifier of endogenous Capn15, an inhibitor that reduces the expression of Capn15 gene or protein, or an agonist of Capn15 competitive gene.

[0034] In a preferred embodiment, the enhancer for enhancing TMEM164 gene or protein expression is selected from: agonists of positive regulatory factors that enhance TMEM164 expression, inhibitors of negative regulatory factors that reduce TMEM164 expression, and agonists that increase TMEM164 protein expression.

[0035] In a preferred embodiment, the enhancer for enhancing Sox18 gene or protein expression is selected from: agonists of positive regulatory factors that enhance Sox18 expression, inhibitors of negative regulatory factors that reduce Sox18 expression, and agonists that increase Sox18 protein expression.

[0036] In a preferred embodiment, the inhibitor that reduces the expression of Capn15 gene or protein is selected from: an inhibitor of a positive regulatory factor that reduces the expression of Capn15, an agonist of a negative regulatory factor that enhances the expression of Capn15, and an inhibitor that reduces the expression of Sox18 protein.

[0037] In a preferred embodiment, the inhibitor of TMEM164 competitive gene is a small molecule inhibitor, a ligand of TMEM164 competitive gene, a gene silencing tool, or an antibody of TMEM164 competitive gene.

[0038] In a preferred embodiment, the inhibitor of the Sox18 competitive gene is a small molecule inhibitor, a ligand of the Sox18 competitive gene, a gene silencing tool, or an antibody of the Sox18 competitive gene;

[0039] In a preferred embodiment, the agonist of the Capn15 competitive gene is a small molecule agonist, a ligand of the Sox18 competitive gene, or an antibody of the Sox18 competitive gene.

[0040] In some specific embodiments, in any of the aforementioned methods, the nerve damage disease is brain nerve damage, spinal cord damage, or optic nerve damage.

[0041] In some specific embodiments, in any of the methods described above, the diseases causing brain nerve damage include: brain trauma, stroke, cerebral ischemia, physical injury, tumor, inflammation, infection, ischemia caused by cardiac arrest or severe hypotension (shock), hypoxic-ischemic encephalopathy caused by hypoxia, hypoglycemia or anemia, meningitis, dehydration, subarachnoid hemorrhage, intracranial hematoma, weak cerebral blood vessels, cerebral vascular malformations, hypertension, or neurodegenerative diseases, or a combination of at least two diseases.

[0042] In a preferred embodiment, the disease of brain nerve damage includes Alzheimer's disease (AD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), multiple sclerosis (MS), or frontotemporal dementia (FTLD).

[0043] In a preferred embodiment, the disease causing optic nerve damage includes glaucoma, retinal detachment, optic nerve inflammation, vision damage caused by RGC cell death, photoreceptor cell damage, age-related macular degeneration (AMD), diabetic retinopathy, retinal ischemia or hemorrhage, Leber hereditary optic neuropathy, or ocular trauma, or a combination of at least two diseases.

[0044] In some specific embodiments, in any of the aforementioned methods, the modulator is administered to an individual in need thereof via a carrier.

[0045] In a preferred embodiment, the carrier is selected from: viral vectors, lipid nanoparticles (LNP), liposomes, cationic polymers (such as PEI), nanoparticles, exosomes; more preferably, the carrier is an AAV vector or lipid nanoparticles, wherein AAV vectors or lipid nanoparticles are preferred.

[0046] In a preferred embodiment, the carrier is selected from viral vectors, lipid nanoparticles (LNP), liposomes, cationic polymers (such as PEI), nanoparticles, exosomes, liposomes, microvesicles, and gene guns.

[0047] In a preferred embodiment, the viral vector is adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, herpes simplex virus, or oncolytic virus.

[0048] In a preferred embodiment, the vector further comprises a regulatory sequence for regulating the expression of the coding sequence; preferably, the regulatory sequence comprises expression elements including promoters, introns, Poly (A) sequences, WPRE, etc. In a specific embodiment, the coding sequence may refer to a nucleic acid encoding a TMEM164 regulator, a SOX18 regulator, or a Capn15 regulator.

[0049] In specific embodiments, the nucleic acid can be DNA and RNA.

[0050] In some specific embodiments, in any of the methods described above, the astrocytes are from the retina, vitreous cavity, striatum, substantia nigra, inner ear, spinal cord, prefrontal cortex, motor cortex, thalamus, ventral tegmental area (VTA), hippocampus, cerebellum, brainstem, or inner ear cochlea or vestibule, more preferably, the astrocytes are from the striatum, substantia nigra, retina, or vitreous cavity.

[0051] In some specific embodiments, in any of the methods described above, the regulator is administered to an individual in need by direct targeted administration to the affected site of the neurological injury disease.

[0052] In some specific embodiments, in any of the methods described above, the modulator is administered to an individual in need thereof by cell transfection, cell infection, cell endocytosis, injection, intracranial administration, spinal administration, inhalation, parenteral administration, intravenous administration, intramuscular administration, intradermal administration, topical administration, local administration, targeted administration, subretinal injection, intravitreal injection, or oral administration.

[0053] In some specific embodiments, in any of the aforementioned methods, the nerve injury disease is a central nervous system injury disease, and the regulator is injected into the striatum, hippocampus or substantia nigra.

[0054] In some specific embodiments, in any of the aforementioned methods, the modulator is capable of increasing the proportion of neuroprotective astrocytes.

[0055] In some specific embodiments, in any of the aforementioned methods, the TMEM164 modulator is co-administered with a SOX18 modulator, or the TMEM164 modulator is co-administered with a Capn15 modulator.

[0056] In the third aspect of the present invention, provided is the use of a TMEM164 modulator that upregulates the expression level of TMEM164, a SOX18 modulator that upregulates the expression level of SOX18, or a Capn15 modulator that downregulates the expression level of Capn15 for preparing a drug for preventing, delaying, or treating diseases related to neural injury or neuronal aging.

[0057] In the fourth aspect of the present invention, provided is the use of an AAV vector carrying a TMEM164 modulator that upregulates the expression level of TMEM164, a SOX18 modulator that upregulates the expression level of SOX18, or a Capn15 modulator that downregulates the expression level of Capn15 for preparing a drug for preventing, delaying, or treating diseases related to neural injury or neuronal aging, or alleviating the symptoms of neural injury or neuronal aging.

[0058] In some specific embodiments, in any of the aforementioned uses, the neurological injury disease is brain injury.

[0059] In some specific embodiments, in the aforementioned use, the brain injury includes Alzheimer's disease (AD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), multiple sclerosis (MS), and FTLD.

[0060] In a fifth aspect of the present invention, provided is the use of a TMEM164 modulator that upregulates the expression level of TMEM164, a SOX18 modulator that upregulates the expression level of SOX18, or a Capn15 modulator that downregulates the expression level of Capn15 for preparing a neuroprotective agent.

[0061] In the sixth aspect of the present invention, a method for regulating the CAPN15-ELOVL1 pathway is provided, wherein the use of a TMEM164 modulator that upregulates the expression level of TMEM164, a SOX18 modulator that upregulates the expression level of SOX18, or a Capn15 modulator that downregulates the expression level of Capn15 is provided; preferably, the regulation is to reduce the expression level of the neurotoxic saturated lipid synthase Elovl1.

[0062] In the seventh aspect of the present invention, a TMEM164 modulator that upregulates the expression level of TMEM164, a SOX18 modulator that upregulates the expression level of SOX18, or a Capn15 modulator that downregulates the expression level of Capn15 is provided for downregulating the expression of Capn15, Kidinis220, Zfp1, or Zfp626.

[0063] In some specific embodiments, the use described in the seventh aspect of the present invention is used to down-regulate the expression of Capn15.

[0064] In some specific embodiments, in the uses described in the third, fourth, fifth, sixth and seventh aspects of the present invention, the TMEM164 regulator can upregulate the expression level of TMEM164 in glial cells; preferably, the glial cells are from the central nervous system, visual nervous system, or auditory nervous system.

[0065] In some specific embodiments, in the uses described in the third, fourth, fifth, sixth and seventh aspects of the present invention, the SOX18 regulator can upregulate the expression level of SOX18 in glial cells; preferably, the glial cells are from the central nervous system, visual nervous system, or auditory nervous system.

[0066] In some specific embodiments, in the uses described in the third, fourth, fifth, sixth and seventh aspects of the present invention, the Capn15 regulator can downregulate the expression level of Capn15 in glial cells; preferably, the glial cells are from the central nervous system, visual nervous system, or auditory nervous system.

[0067] Furthermore, in any of the aforementioned uses, the glial cells include astrocytes, Müller glial cells, or cochlear glial cells (satellite cells); preferably, the astrocytes include GFAP-positive astrocytes, fibrous astrocytes or protoplasmic astrocytes.

[0068] Furthermore, in any of the aforementioned uses, the astrocytes are non-reactive astrocytes.

[0069] Furthermore, in any of the aforementioned uses, the regulator can regulate the activation of non-reactive astrocytes into reactive astrocytes; preferably, the regulation is to inhibit the activation of non-reactive astrocytes into neurotoxic reactive astrocytes; and / or to enhance the transformation of non-reactive resting astrocytes into neuroprotective reactive astrocytes.

[0070] Furthermore, in any of the uses described above, the expression of markers C3, H2-T23, H2-D1, Iigp1, Gbp2, Fkbp5, Psmb8, Srgn, Serping1, or Amigo2, or at least two of the markers is reduced in the cells administered with the regulator compared to the cells not administered with the regulator.

[0071] Furthermore, in any of the aforementioned uses, the TMEM164 regulator is an epigenetic regulation tool, a TMEM164 gene activator, a TMEM164 gene expression activator, a TMEM164 protein degradation inhibitor, an mRNA stabilizer, exogenous TMEM164 or a functional fragment of TMEM164.

[0072] Furthermore, the exogenous TMEM164 or the functional fragment of TMEM164 is a polypeptide of TMEM164 or a biologically active fragment thereof, or a nucleic acid encoding a polypeptide of TMEM164 or a biologically active fragment thereof; TMEM164 is preferably human TMEM164.

[0073] Furthermore, in a preferred embodiment, the exogenous TMEM164 or functional fragment of TMEM164 is a mutant of the TMEM164 polypeptide or its biologically active fragment that retains the function of TMEM164; preferably, the mutant has at least 80%, 85%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% homology with the TMEM164 polypeptide or its biologically active fragment.

[0074] Furthermore, in a preferred embodiment, the TMEM164 polypeptide has a sequence as shown in SEQ ID NO: 31 or 32.

[0075] In some specific embodiments, in any of the aforementioned uses, the Sox18 regulator is an epigenetic regulation tool, a Sox18 gene expression activator, a Sox18 protein degradation inhibitor, a Sox18 mRNA stabilizer, exogenous Sox18 or a functional fragment of Sox18.

[0076] Furthermore, the exogenous Sox18 or the functional fragment of Sox18 is a Sox18 polypeptide or a biologically active fragment thereof, or a nucleic acid encoding a Sox18 polypeptide or a biologically active fragment thereof; Sox18 is preferably human Sox18.

[0077] Furthermore, the exogenous Sox18 or functional fragment of Sox18 is a mutant of the Sox18 polypeptide or its biologically active fragment that retains the Sox18 function; preferably, the mutant has at least 80%, 85%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% homology with the Sox18 polypeptide or its biologically active fragment.

[0078] In a preferred embodiment, the Sox18 refers to the gene with the gene number 54345 in NCBI.

[0079] In some specific embodiments, in any of the aforementioned uses, the Capn15 regulator is an epigenetic regulation tool, a Capn15 gene expression inhibitor, a Capn15 activity inhibitor, a Capn15 antibody, a Capn15 protein degradation activator, a gene editing tool targeting Capn15, PROTAC, ASO, miRNA, RNAi, shRNA, Capn15 binding protein or protein domain.

[0080] Furthermore, the Capn15 refers to the gene with the gene number 6650 in NCBI.

[0081] In some specific embodiments, in any of the aforementioned uses, the regulator is a small molecule compound, an antibody, a protein, a polypeptide, a gene fragment, or a polysaccharide.

[0082] Furthermore, the TMEM164 gene expression activator is a modifier of endogenous TMEM164, an agonist that enhances the expression of the TMEM164 gene or protein, or an inhibitor of a TMEM164 competitive gene.

[0083] Furthermore, the Sox18 gene expression activator is a modifier of endogenous Sox18, an agonist that enhances the expression of Sox18 gene or protein, or an inhibitor of Sox18 competitive gene.

[0084] Furthermore, the Capn15 gene expression inhibitor is a modifier of endogenous Capn15, an inhibitor that reduces the expression of Capn15 gene or protein, or an agonist of Capn15 competitive gene.

[0085] Furthermore, the enhancer for enhancing TMEM164 gene or protein expression is selected from: agonists of positive regulatory factors that enhance TMEM164 expression, inhibitors of negative regulatory factors that reduce TMEM164 expression, and agonists that increase TMEM164 protein expression.

[0086] Furthermore, the enhancer for enhancing the expression of Sox18 gene or protein is selected from: an agonist of a positive regulatory factor that enhances Sox18 expression, an inhibitor of a negative regulatory factor that reduces Sox18 expression, and an agonist that increases Sox18 protein expression.

[0087] Furthermore, the inhibitor that reduces the expression of Capn15 gene or protein is selected from: an inhibitor of a positive regulatory factor that reduces the expression of Capn15, an agonist of a negative regulatory factor that enhances the expression of Capn15, and an inhibitor that reduces the expression of Sox18 protein.

[0088] Furthermore, the inhibitor of TMEM164 competitive gene is a small molecule inhibitor, a ligand of TMEM164 competitive gene, a gene silencing tool, or an antibody of TMEM164 competitive gene.

[0089] Further, the inhibitor of the Sox18 competitive gene is a small molecule inhibitor, a ligand of the Sox18 competitive gene, a gene silencing tool, or an antibody of the Sox18 competitive gene;

[0090] Furthermore, the agonist of the Capn15 competitive gene is a small molecule agonist, a ligand of the Sox18 competitive gene, or an antibody of the Sox18 competitive gene.

[0091] In some specific embodiments, in any of the aforementioned uses, the nerve damage disease is brain nerve damage, spinal cord damage, or optic nerve damage.

[0092] In some specific embodiments, in any of the aforementioned uses, the diseases causing brain nerve damage include: brain trauma, stroke, cerebral ischemia, physical injury, tumor, inflammation, infection, ischemia caused by cardiac arrest or severe hypotension (shock), hypoxic-ischemic encephalopathy caused by hypoxia, hypoglycemia or anemia, meningitis, dehydration, subarachnoid hemorrhage, intracranial hematoma, weak cerebral blood vessels, cerebral vascular malformations, hypertension, or neurodegenerative diseases, or a combination of at least two diseases.

[0093] Furthermore, the diseases of brain nerve damage include Alzheimer's disease (AD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), multiple sclerosis (MS), or frontotemporal dementia (FTLD).

[0094] Furthermore, the diseases causing optic nerve damage include glaucoma, retinal detachment, optic nerve inflammation, vision damage caused by RGC cell death, photoreceptor cell damage, age-related macular degeneration (AMD), diabetic retinopathy, retinal ischemia or hemorrhage, Leber hereditary optic neuropathy, or ocular trauma, or a combination of at least two diseases.

[0095] In some specific embodiments, in any of the aforementioned uses, the modulator is administered to an individual in need thereof via a carrier.

[0096] Furthermore, the carrier is selected from: viral vectors, lipid nanoparticles (LNP), liposomes, cationic polymers (such as PEI), nanoparticles, exosomes; more preferably, the carrier is an AAV vector or lipid nanoparticles, wherein AAV vectors or lipid nanoparticles are preferred.

[0097] Furthermore, the carrier is selected from viral vectors, lipid nanoparticles (LNP), liposomes, cationic polymers (such as PEI), nanoparticles, exosomes, liposomes, microvesicles, and gene guns.

[0098] Furthermore, the viral vector is adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, herpes simplex virus, or oncolytic virus.

[0099] Furthermore, the vector also includes a regulatory sequence for regulating the expression of the coding sequence; preferably, the regulatory sequence includes expression elements such as promoter, intron, Poly (A) sequence, WPRE, etc. In a specific embodiment, the coding sequence may refer to a nucleic acid encoding a TMEM164 regulator, a SOX18 regulator, or a Capn15 regulator.

[0100] In specific embodiments, the nucleic acid can be DNA and RNA.

[0101] Furthermore, in any of the aforementioned uses, the astrocytes are from the retina, vitreous cavity, striatum, substantia nigra, inner ear, spinal cord, prefrontal cortex, motor cortex, thalamus, ventral tegmental area (VTA), hippocampus, cerebellum, brainstem, or inner ear cochlea or vestibule, more preferably, the astrocytes are from the striatum, substantia nigra, retina, or vitreous cavity.

[0102] Furthermore, in any of the aforementioned uses, the regulator is administered to an individual in need by direct targeted administration to the affected site of the nerve damage disease.

[0103] Furthermore, in any of the uses described above, the regulator is administered to an individual in need by cell transfection, cell infection, cellular endocytosis, injection, intracranial administration, spinal administration, inhalation, parenteral administration, intravenous administration, intramuscular administration, intradermal administration, topical administration, local administration, targeted administration, subretinal injection, intravitreal injection, or oral administration.

[0104] Furthermore, in any of the aforementioned uses, the nerve injury disease is a central nervous system injury disease, and the regulator is injected into the striatum, hippocampus or substantia nigra.

[0105] Furthermore, in any of the aforementioned uses, the regulator can increase the proportion of neuroprotective astrocytes.

[0106] In the eighth aspect of the present invention, a composition is provided, comprising the regulator for upregulating the expression level of TMEM164 as described in any one of the first to seventh aspects.

[0107] Furthermore, the regulator is a gene.

[0108] Furthermore, the regulator is a gene carried by a vector, and the vector is selected from viral vectors, lipid nanoparticles (LNP), liposomes, cationic polymers (such as PEI), nanoparticles, exosomes, liposomes, microvesicles, and gene guns.

[0109] Furthermore, the viral vector is adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, herpes simplex virus, or oncolytic virus.

[0110] Furthermore, the adeno-associated virus is: AAV vector serotype 1 (AAV1), AAV vector serotype 2 (AAV2), AAV vector serotype 3 (AAV3), AAV vector serotype 4 (AAV4), AAV vector serotype 5 (AAV5), AAV vector serotype 6 (AAV6), AAV vector serotype 7 (AAV7), AAV vector serotype 8 (AAV8), AAV vector serotype 9 (AAV9), AAV vector serotype 10 (AAV10), AAV vector serotype 11 (AAV11) or AAV vector serotype 12 (AAV12); preferably AAV9.

[0111] The beneficial effects of the present invention are:

[0112] 1. This application first discovered that TMEM164, SOX18 and Capn15 are related to the occurrence of nerve damage diseases. In particular, this application first discovered that TMEM164, SOX18 and Capn15 are related to the occurrence of multiple neurodegenerative diseases.

[0113] 2. This application discovered for the first time that TMEM164, SOX18 and Capn15 are early response regulators of neurotoxic reactive astrocytes. By increasing the expression of TMEM164 or SOX18, or reducing the expression of Capn15, neurotoxic reactive astrocytes can be reduced, or the activation of non-reactive astrocytes into neurotoxic reactive astrocytes can be inhibited.

[0114] 3. The present application overexpresses TMEM164, SOX18 or Capn15 in mouse astrocytes and human astrocytes, and finds that increasing the expression of TMEM164 and SOX18, or decreasing the expression of Capn15, can prevent the induction of neurotoxic reactive astrocytes and neuronal death, thereby reducing the production of neurotoxic reactive astrocytes, protecting the normal function of astrocytes, and also protecting neurons. This indicates that increasing the expression of TMEM164 can prevent or treat diseases related to the induction of neurotoxic reactive astrocytes.

[0115] 4. This application found that TMEM164 can inhibit the activation of neurotoxic reactive astrocytes in vivo and reduce the toxic effects of neurotoxic reactive astrocytes on neurons by administering TMEM164 to inflammation-induced PD mouse models and AD mouse models, thereby playing a therapeutic role in nerve injury diseases.

[0116] 5. The present application found that upregulating TMEM164 in astrocytes and downregulating the expression level of CAPN15 can synergistically inhibit the generation of neurotoxic reactive astrocytes.

[0117] 6. The present application found that upregulating TMEM164 in astrocytes and simultaneously upregulating the expression of SOX18 can synergistically inhibit the generation of neurotoxic reactive astrocytes.

[0118] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0119] Figure 1 It is the qPCR primer sequence of mouse gene and human gene and the sequence map of single guide RNA of mouse gene.

[0120] Figure 2 is a heat map of transcriptional levels of reactive genes in non-reactive, reactive, EGFP-ITC, and TMEM164-ITC astrocytes.

[0121] FIG3 is the regulatory effect of Tmem164 expression level on the non-reactive astrocyte state. Figure 3A The transcript levels of A1 neurotoxicity-specific markers in TMEM164-EGFP or EGFP-overexpressing astrocytes without stimulation. Figure 3BThe transcription levels of A1 neurotoxicity-specific markers in control or TMEM164 knockdown astrocytes without stimulation. All data are expressed as mean ± SEM, n = 3 replicates / group. *p < 0.05, ***p < 0.001, unpaired two-tailed student t test.

[0122] FIG4 shows the effect of overexpression of TMEM164 on the phagocytosis and synapse promotion of neurotoxic reactive astrocytes. Figure 4A Representative images of EGFP-PBS, EGFP-ITC, and TMEM164-ITC astrocytes engulfing pHrodo-bound synaptosomes. EGFP-ITC astrocytes showed reduced phagocytic capacity, but TMEM164-ITC astrocytes maintained normal phagocytic capacity. Figure 4B Figure 3 is the time-dependent change in the extent of phagocytosis of pHrodo-bound synaptosomes by EGFP-PBS, EGFP-ITC, and TMEM164-ITC astrocytes. Figure 4C The transcription levels of genes encoding phagocytic receptors (Megf10 and Mertk) and genes encoding bridging molecules (Gas6) in EGFP-PBS, EGFP-ITC and TMEM164-ITC astrocytes.

[0123] Figure 5A The transcription levels of related genes encoding synaptogenic factors (Thbs1, Gpc6, and Sparcl1) in EGFP-PBS, EGFP-ITC, and TMEM164-ITC astrocytes are shown. Figure 5B This is the result of the effect of EGFP-PBS, EGFP-ITC and TMEM164-ITC on neuronal synaptic growth after co-culture with mouse cortical neurons.

[0124] FIG. 6 shows the results of the inhibition of neurotoxicity of reactive astrocytes by overexpression of TMEM164. Fig. 6A Representative images of ACM-induced cortical neuron death analyzed by Calcein AM / homoethidium-1 (live / dead) staining assay. Scale bar = 100 μm. Figure 6B Quantitative analysis of neuronal death after 48 hours of ACM treatment.

[0125] FIG. 7 shows the effect of TMEM164 overexpression on Elovl1 expression. Fig. 7A Comparison of Elovl1 transcription levels in EGFP-PBS, EGFP-ITC and TMEM164-ITC astrocytes, with 3 replicates in each group. Figure 7B Immunoblot staining of TMEM164, C3 and ELOVL1. Figure 7C For the quantitative analysis of C3 and ELOVL1, three replicates were performed in each group.

[0126] Figure 8 The concentration of saturated fatty acids in astrocyte conditioned medium was measured by gas chromatography-mass spectrometry (GC-MS). There were 4-6 replicates in each group.

[0127] Figure 9 shows the differentially expressed genes in EGFP-ITC and TMEM164-ITC astrocytes RNA sequencing ( Fig. 9A ) and differentially expressed transcription factors ( Fig. 9B ) analysis results, showing the top 5 up-regulated and down-regulated genes and transcription factors, with 3 replicates in each group.

[0128] FIG. 10 shows the expression of downstream transcription factors of TMEM164. Fig. 10A Transcription levels of A1 neurotoxicity-specific markers in EGFP-PBS, EGFP-ITC, TMEM164-ITC, and TMEM164+CAPN15 (TMEM164 and CAPN15 co-overexpressed)-ITC astrocytes are shown, with 3 replicates per group. Fig. 10B Transcription levels of C3 in astrocytes overexpressing TMEM164 together with individual transcription factors are shown, with three replicates per group.

[0129] FIG. 11 is an analysis testing whether Elovl1 is a downstream target of Capn15. Fig.11A Transcription levels of Tmem164, Capn15, and Elovl1 in primary mouse astrocytes treated with PBS or ITC, with 3 replicates in each group. Fig. 11B Effects of knocking down Capn15 transcription level on Elovl1 transcription level in non-reactive astrocytes and reactive astrocytes, respectively, with 5 replicates in each group.

[0130] FIG. 12 shows that TMEM164 overexpression inhibits the activation of human neurotoxic reactive astrocytes. Fig. 12A Representative images of C3 and TMEM164 immunostaining in human non-reactive and neurotoxic reactive astrocytes, scale bar = 50 μm. Fig. 12B Quantitative analysis results of the protein expression levels of C3 and TMEM164 in human non-reactive and neurotoxic reactive astrocytes. Fig. 12CRepresentative images of TMEM164, ZO1 and α-tubulin immunostaining of human astrocytes treated with IL-1α, TNF and C1q (ITC) over time. The results show that the protein level of TMEM164 decreased with the induction time during the induction of human neurotoxic reactive astrocytes. Scale bar = 10 μm. Fig.12D Transcription levels of human A1 neurotoxicity-specific markers in EGFP-PBS, EGFP-ITC, and TMEM164-ITC human astrocyte groups are shown.

[0131] FIG. 13 shows the effects of overexpression of human TMEM164 on phagocytosis, promotion of synaptic growth and neurotoxicity of human neurotoxic reactive astrocytes. Fig.13A Representative images of EGFP-PBS, EGFP-ITC, and TMEM164-ITC human astrocytes engulfing pHrodo-bound synaptosomes. Scale bar = 100 μm. Fig. 13B Quantitative analysis of phagocytosis of pHrodo-bound synaptosomes in EGFP-PBS, EGFP-ITC, and TMEM164-ITC human astrocyte groups for 12 hours. Fig. 13C Representative images of death of neurons differentiated from human embryonic stem cells induced by human ACM analyzed by Calcein AM / homoethidium-1 live / dead cell staining assay, Scale bar = 100 μm. Fig.13D Quantitative analysis of cell death rate in neurons differentiated from human embryonic stem cells 48 hours after human ACM treatment. Fig.13E The staining results of EGFP-PBS, EGFP-ITC and TMEM164-ITC human astrocytes promoting synaptic growth of neurons differentiated from human embryonic stem cells. Scale bar = 20 μm. Fig.13F Statistical analysis results of neuronal synapses.

[0132] Fig.14 Schematic diagram of AAV plasmid elements for overexpressing Tmem164-P2A-mCherry or mCherry.

[0133] FIG. 15 shows the effect of TMEM164 overexpression on the treatment of Parkinson's disease mouse model. Fig.15A Representative images of colocalization of mCherry and astrocyte marker GFAP, microglia marker Iba1, or dopaminergic neuron marker TH are shown. Scale bar = 100 μm. Fig. 15B is a statistical analysis of the proportions of GFAP+mCherry+, Iba1+mCherry+, and TH+mCherry+ double-positive cells, indicating that AAV virus specifically infects astrocytes. Fig. 15CRepresentative images of TH staining in the SNc are shown. Scale bar = 100 μm. Fig.15D It is a quantitative analysis of the number of TH-positive neurons. Fig.15E These are the behavioral experimental results of mice injected with AAV in the rotarod test.

[0134] Fig.16A are representative images of C3-positive astrocytes in the SNc. Fig. 16B The quantitative results are for the proportion of C3 expression area in SNc. Fig. 16C As a result, AAV-mediated TMEM164 overexpression inhibited the induction of neurotoxic reactive astrocytes. Double staining of GFAP, a pan-reactive marker of astrocytes, and C3, a marker of A1 astrocytes, in the SNc of control and AAV-injected PD mice showed that astrocytes infected with TMEM164-overexpressing AAV showed downregulated GFAP and C3 signals (area below the white dashed line in the AAV-Tmem164+LPS group). Scale bar = 100 μm.

[0135] FIG. 17 shows the results of analysis of microglial activation in the SNc of control and AAV-injected PD mice. Fig.17A Representative images of Iba1 staining in the LPS-induced PD model are shown, scale bar = 50 μm. Fig. 17B Quantification results of the area covered by Iba1-positive microglia in the SNc, which showed no significant difference in the extent of inflammatory microglial activation between the AAV-mCherry group and the AAV-Tmem164 group (n=6 mice).

[0136] FIG. 18 shows infection status of whole brain AAV delivery in the 5XFAD Alzheimer's disease mouse model. Fig.18A Statistical analysis of the proportion of GFAP+mCherry+, Iba1+mCherry+, and NeuN+mCherry+ double-positive cells in the cerebral cortex and hippocampus of 5XFAD mice, showing that mCherry is specifically expressed in astrocytes, n=6 mice. Fig.18B Representative images of colocalization of mCherry with astrocyte marker GFAP, microglia marker Iba1, or neuronal marker NeuN in the cerebral cortex and hippocampus of 5XFAD mice are shown, scale bar = 50 μm. Fig. 18C Statistical analysis of the proportion of AAV-infected astrocytes in the cerebral cortex and hippocampus of 5XFAD mice, n = 6 mice.

[0137] Fig.19AThis is a quantitative analysis of the results of a novel object recognition test conducted on the 5XFAD Alzheimer's disease mouse model. Mice injected with AAV overexpressing TMEM164 showed better recognition ability than the control group without changing the mice's motor ability. Fig.19B This is a quantitative analysis of the Y-maze performed on the 5XFAD Alzheimer's disease mouse model. Mice injected with AAV overexpressing TMEM164 showed better spatial memory ability than the control group without changing the total movement distance of the mice.

[0138] FIG. 20 shows the results of immunostaining to evaluate the neurotoxicity level of astrocytes. Fig. 20A The double staining results of GFAP, a pan-reactive marker of astrocytes, and C3, a neurotoxic marker, in the cortex and hippocampus. Scale bar = 50 μm. Fig. 20B Quantification of the ratio of C3 expression areas in the cortex and hippocampus, n = 4 mice.

[0139] FIG. 21 shows the effect of TMEM164 overexpression on neuronal degeneration in the cortex and hippocampus. Fig.21A Representative images of degeneration of neurons labeled with Fluoro-Jade C in the cortex and hippocampus, scale bar = 50 μm. Fig.21B For quantitative analysis of degenerating neurons in the cortex and hippocampus in A, n = 4 mice.

[0140] Fig. 22 Whole-brain TMEM164 overexpression inhibits Aβ deposition in 9-month-old 5XFAD mice. Fig. 22 Left: Representative images of Aβ staining in the cerebral cortex and hippocampus of 5XFAD mice injected with AAV. Scale bar = 50 μm. Fig. 22 The right panel shows quantitative analysis of Aβ coverage in the cortex and hippocampus, n = 4 mice. Data are presented as mean ± standard error. Unpaired two-tailed student-t test.

[0141] Fig.23 The results show that whole-brain TMEM164 overexpression suppresses neuronal death in layer 5 of the cerebral cortex and the inferior peduncle of the hippocampus of 9-month-old 5XFAD mice. Fig.23 The left image shows the staining results of NeuN (green) and GFAP (gray) in the 5th layer of the cerebral cortex and the inferior peduncle of the hippocampus of 5XFAD mice 6 months after AAV injection. Scale bar = 100 μm. Fig.23 The right figure is a statistical diagram of the number of neurons in the inferior peduncle of the hippocampus (upper right) and the fifth layer of the cortex (lower right), n = 3 mice. Data are expressed as mean ± standard error. One-way ANOVA test.

[0142] FIG. 24 shows the results of RNA-seq analysis of astrocytes isolated from the brains of 9-month-old 5XFAD mice injected with control group and TMEM164 overexpression group AAV. Fig.24A This is a differential gene analysis diagram. Fig. 24B and Fig.24C The results of differential signaling pathway analysis.

[0143] FIG. 25 shows the results of specific expression analysis of TMEM164 overexpressing AAV in the brain and peripheral organs. Fig.25A The expression of TMEM164 overexpressing AAV in astrocytes in the cerebral cortex, hippocampus and thalamus. Fig.25B The expression of TMEM164 overexpressing AAV in peripheral organs such as liver, muscle, kidney, heart and lung. Fig.25C This is a graph showing the proportion of astrocytes infected by TMEM164-overexpressing AAV in different brain regions and organ cells infected in peripheral organs. DETAILED DESCRIPTION

[0144] In the present invention, we provide a method for regulating the activation of neurotoxic reactive astrocytes (A1 astrocytes) by regulating the expression of TMEM164. Further, we also provide a modulator for regulating the expression of TMEM164, such as overexpressing TMEM164 to increase the expression of TMEM164. Further, we also provide a method for treating neurological damage diseases (such as AD, PD, HD, MS, ALS, etc.) by regulating the activation of neurotoxic reactive astrocytes (A1 astrocytes).

[0145] We identified an endogenous early response regulator, TMEM164, and creatively discovered that TMEM164 is an important intrinsic regulator of the induction of neurotoxic reactive astrocytes. We found that TMEM164 can strongly prevent astrocytes from transitioning from a non-reactive state to a neurotoxic reactive state, theoretically inhibiting the release of neurotoxic factors from the source.

[0146] We also confirmed that TMEM164 overexpression maintained normal human astrocyte function, prevented human neurotoxic reactive astrocyte induction and human neurotoxic reactive astrocyte-induced human neuron death, which means that it has therapeutic prospects for treating a variety of human neurodegenerative diseases associated with neurotoxic reactive astrocytes. Since neurotoxic reactive astrocytes are abundant in a variety of neurodegenerative diseases including AD, PD, HD, MS and ALS, as well as brain injuries, through the technical solutions provided in this application, we can screen clinically significant candidates that are not only responsible for regulating neurotoxic reactive astrocytes in animals, but also involved in the pathophysiological conditions of human diseases.

[0147] In addition, we have developed an AAV-based therapeutic strategy to prevent the induction of neurotoxic reactive astrocytes in vivo by overexpressing TMEM164 specifically in astrocytes. AAV-mediated overexpression of TMEM164 not only produced a strong and sustained inhibition of the induction of neurotoxic reactive astrocytes, but also prevented neurotoxic reactive astrocyte-induced neuronal death in in vivo inflammatory PD models as well as AD models, which provides a new astrocyte-specific gene therapy for the prevention of neurodegenerative diseases.

[0148] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a modulator" includes a plurality of modulators and reference to "the modulator" includes reference to one or more modulators and equivalents thereof known to those skilled in the art, and so forth.

[0149] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those of ordinary skill in the art to which the present invention belongs. In the embodiments of the present application, all provided are exemplary methods and materials, and those skilled in the art may use any similar or equivalent methods and reagents in the art for the specific practice of the technical solutions disclosed in the present invention.

[0150] All publications, patents, and patent applications mentioned herein are incorporated by reference in their entirety for the purpose of describing and disclosing the methodologies described in the publications, which may be used in connection with the description herein, as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0151] To the extent that any terms appearing in one or more publications are similar or identical to terms expressly defined herein, the definitions of those terms expressly provided herein shall control in all respects.

[0152] Unless stated otherwise, the use of "and" may mean "and / or." Similarly, "including" and "comprising" are interchangeable and are not intended to be limiting.

[0153] It should be further understood that, while the description of various embodiments uses the term “comprising,” those skilled in the art will understand that, in some specific instances, the embodiments may alternatively be described using the language “consisting essentially of” or “consisting of.”

[0154] the term

[0155] "Astrocytes" are the most widely distributed type of cells in the mammalian brain. The classic metal immersion technique (silver staining) shows that this type of glial cell is star-shaped, with many long and branched protrusions extending from the cell body, which extend and fill between the cell body and protrusions of the nerve cells, playing a role in supporting and separating the nerve cells. The astrocytes of this application may refer to the characteristic astrocytes in the brain and spinal cord; they may also refer to some special types of astrocytes, such as Bergman glial cells in the cerebellum; Muller glial cells in the retina, also known as radial neuroglia cells; pituitary cells of the pituitary gland and tanycytes in the median eminence, etc.

[0156] In certain embodiments, astrocytes may refer to non-neuronal cells in the nervous system that express a transgene (eg, red fluorescent protein (RFP), Cre recombinase) driven by the glial fibrillary acidic protein (GFAP) promoter.

[0157] "TMEM164" is a seven-transmembrane protein predicted by AlphaFold2, also known as transmembrane protein 164, which is mainly distributed in the cytoplasm and cell membrane, especially in the cell junctions of astrocytes.

[0158] The term "contacting" refers to placing a composition (e.g., a compound, nucleic acid, viral vector, etc.) in a position that allows it to contact a cell to produce a "contacted" cell. Contacting can be accomplished using any suitable method. For example, in one embodiment, contacting is by adding the compound to a cell culture. Contacting can also be accomplished by injecting it or delivering the composition to a location in the body so that the composition "contacts" the targeted cell type.

[0159] The term "induce" refers to changing a default cell type ((genotype and / or phenotype)) to a non-default cell type ((genotype and / or phenotype)).

[0160] The term "function" refers to the ability to exhibit and / or maintain the ability to perform an action and / or task, such as a neuron or astrocyte.

[0161] The term "treatment" refers to reducing, inhibiting or improving symptoms or signs caused by neuroinflammatory reactive astrocyte activation, including but not limited to therapeutic and / or preventive treatment.

[0162] The term "therapeutically effective amount" refers to the amount of a modulator required to effectively reduce, improve or prevent the activation of astrocytes in the neuroinflammatory response to be treated.

[0163] The term "non-reactive astrocytes" refers to astrocytes that do not express or lowly express pan reactive, neurotoxic specific (A1 specific) and neuroprotective specific (A2 specific) markers.

[0164] The term "reactive astrocytes" refers to astrocytes that are in an activated state and highly express reactive markers relative to a non-reactive state.

[0165] The term "neuroinflammatory reactive astrocytes" refers to reactive astrocytes that highly express neurotoxic specific markers, are activated by neuroinflammatory responses, and have neurotoxic properties.

[0166] The term "neuroprotective reactive astrocytes" refers to reactive astrocytes that highly express neuroprotective specific markers and can secrete neurotrophic factors to promote neuronal survival.

[0167] The term "pan-reactive" refers to high expression in both neuroprotective reactive astrocytes and neuroinflammatory reactive astrocytes.

[0168] Unless otherwise specified, the term "astrocyte-conditioned medium (ACM)" refers to astrocyte culture medium obtained by stimulating astrocytes with PBS or IL-1α, TNF and C1q for 24 hours.

[0169] The term "neurological injury" may refer to a sudden injury, such as a closed head injury (CHI) due to an impact to the head, or a penetrating head injury. A non-limiting example of a penetrating head injury is an object piercing the skull and entering the brain, such as but not limited to a direct or indirect impact to the head from a fall, a car accident, a sports accident, being hit by an object, or an indirect impact such as a shock wave from an explosion.

[0170] The term "neurological damage disease" may also refer to hereditary neurological damage, such as hereditary glaucoma, hereditary mitochondrial encephalopathy, neurological Tourette syndrome, fibromatosis, etc.

[0171] The term "neurological damage" may also refer to damage caused by other medical causes, such as tumors.

[0172] The term "neurovascular disease" may also refer to some neurodegenerative diseases, such as Alzheimer's disease (AD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), or multiple sclerosis (MS).

[0173] The term "individual in need thereof" may be a human or a non-human mammal, or a non-mammal, or some in vitro cells, etc.

[0174] The term "subject" may refer to a human or a non-human mammal, including but not limited to non-human primates, cats, dogs, sheep, goats, horses, cattle, pigs, and rodents (including but not limited to mice and rats); non-mammals include but are not limited to birds, poultry, reptiles, and amphibians. In a preferred embodiment, the subject is a human.

[0175] The term "AAV vector" refers to the packaged capsid form of the AAV virus that delivers its nucleic acid genome into cells.

[0176] The term "recombinant" is used to indicate a nucleic acid construct in which two or more nucleic acids are linked and are not found linked in nature. Expression vectors include, but are not limited to, plasmids, viruses, BACs, and YACs. Specific viral expression vectors illustratively include viral expression vectors derived from adenoviruses, adeno-associated viruses, retroviruses, and lentiviruses.

[0177] The term "promoter" refers to a DNA sequence operably connected to a nucleic acid sequence (such as a nucleic acid sequence encoding TMEN164) waiting for transcription. The promoter is usually positioned upstream of the nucleic acid sequence to be transcribed, and is provided for sites specifically bound by RNA polymerase and other transcription factors. In a particular embodiment, the promoter is usually positioned upstream of the nucleic acid sequence transcribed to produce a desired molecule, and is provided for sites specifically bound by RNA polymerase and other transcription factors.

[0178] The term "nucleic acid" refers to any form of RNA or DNA molecule having more than one nucleotide including single-stranded, double-stranded, oligonucleotide or polynucleotide. The term "nucleotide sequence" refers to the order of nucleotides in an oligonucleotide or polynucleotide in a single-stranded form of a nucleic acid.

[0179] Those skilled in the art will appreciate that the 5' noncoding region of a gene can be separated and used as a promoter for driving the expression of an operably connected nucleic acid in its entirety. Alternatively, a portion of a 5' noncoding region can be separated and used to drive the expression of an operably connected nucleic acid. Typically, about 500-6000bp of the 5' noncoding region of a gene is used to drive the expression of an operably connected nucleic acid. Optionally, the portion of the 5' noncoding region containing the minimum amount of 5' noncoding region required to drive the expression of an operably connected nucleic acid of the 5' noncoding region of a gene is used. The determination of the ability of the specified portion of the 5' noncoding region of a gene to drive the expression of an operably connected nucleic acid is well known in the art.

[0180] In this application, the process of introducing genetic material into a recipient host cell, such as for transient or stable expression of a desired protein encoded by the genetic material in a host cell is referred to as "transfection" or "transduction". Transfection techniques are well known in the art and include, but are not limited to, electroporation, particle accelerated transformation (also known as "gene gun" technology), liposome-mediated transfection, calcium phosphate or calcium chloride coprecipitation-mediated transfection, DEAE-dextran-mediated transfection, microinjection, polyethylene glycol-mediated transfection, and heat shock-mediated transfection. Transduction refers to the viral-mediated introduction of genetic material into a recipient host cell.

[0181] Viral-mediated transfection can be accomplished using viral vectors, such as those derived from adenovirus, AAV, and lentivirus.

[0182] Optionally, the host cells are transfected or transduced ex vivo and then reintroduced into the host organism.For example, cells or tissues can be removed from a subject, transfected or transduced with an expression vector encoding NeuroD1, and then returned to the subject.

[0183] In some specific embodiments, the nerve damage disease is brain nerve damage, spinal cord damage, or optic nerve damage.

[0184] In some specific embodiments, the diseases causing brain nerve damage include: brain trauma, stroke, cerebral ischemia, physical injury, tumor, inflammation, infection, ischemia caused by cardiac arrest or severe hypotension (shock), hypoxic-ischemic encephalopathy caused by hypoxia, hypoglycemia or anemia, meningitis, dehydration, subarachnoid hemorrhage, intracranial hematoma, weak cerebral blood vessels, cerebral vascular malformation, hypertension, or neurodegenerative diseases, or a combination of at least two diseases.

[0185] In some embodiments, the disease causing brain nerve damage includes Alzheimer's disease (AD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), or multiple sclerosis (MS).

[0186] In some specific embodiments, the disease causing optic nerve damage includes glaucoma, retinal detachment, optic nerve inflammation, vision damage caused by RGC cell death, photoreceptor cell damage, age-related macular degeneration (AMD), diabetic retinopathy, retinal ischemia or hemorrhage, Leber hereditary optic neuropathy, or ocular trauma, or a combination of at least two diseases.

[0187] Signs and symptoms of neurological disorders, and methods for detecting and evaluating such disorders are well known in the art. Some signs and symptoms of neurological disorders include loss of consciousness, confusion, disorientation, headaches, fatigue, speech problems, sleep problems, dizziness, balance problems, sensory problems, sensitivity to light, blindness or changes in vision, loss or changes in smell, loss or changes in taste, tinnitus, loss or changes in hearing, memory problems, difficulty concentrating, depression, anxiety, agitation, mood swings, seizures, loss or impairment of coordination, movement problems, cognitive problems, including learning difficulties, negative changes in reasoning ability, negative changes in judgment, and negative changes in attention or concentration.

[0188] In some embodiments, the method for treating a nerve injury disease comprises administering a therapeutically effective amount of a TMEM164 modulator (such as TMEM164) to a subject in need thereof in a local area of ​​nerve damage, particularly in or near the area of ​​astrocytes.

[0189] According to various aspects of the present disclosure, administration of a therapeutically effective amount of a TMEM164 modulator can improve nerve damage, or inhibit the progression of nerve damage. According to various aspects of the present disclosure, administration of a TMEM164 modulator can regulate the activation of reactive astrocytes, for example, can reduce or block the activation of neurotoxic reactive astrocytes, and / or can increase the activation of neuroprotective reactive astrocytes.

[0190] In some embodiments, administration of a therapeutically effective amount of a TMEM164 modulator, or co-administration of a TMEM164 modulator with other agents, may result in treatments including but not limited to: repairing skull fractures, removing blood clots, relieving pressure within the skull, regulating speech, and / or regulating cognition.

[0191] In some embodiments, a TMEM164 modulator can be administered to an individual in need thereof via a carrier.

[0192] In some specific embodiments, the carrier can be selected from: viral vectors, lipid nanoparticles (LNP), liposomes, cationic polymers (such as PEI), nanoparticles, exosomes; more preferably, the carrier is an AAV vector or lipid nanoparticles, among which AAV vectors or lipid nanoparticles are preferred.

[0193] In some embodiments, the carrier further comprises a promoter. Specifically, an AAV vector comprising a nucleic acid encoding TMEM164 under the transcriptional control of a ubiquitous (constitutive) promoter or a neuron-specific promoter can be delivered to the subject's brain by stereotaxic injection together with an AAV encoding a site-specific recombinase, wherein the DNA sequence encoding TMEM164 is reversed and in the wrong orientation for TMEM164 expression and further comprises a site of recombinase activity of the site-specific recombinase until the site-specific recombinase reverses the reverse DNA sequence encoding TMEM164, thereby allowing the expression of TMEM164.

[0194] In the present application, adeno-associated virus (AAV) vectors or lentiviral vectors are used to carry TMEM164 regulators (such as TMEN164 gene). For example, by injecting the AAV vector carrying the TMEN164 gene in situ into the substantia nigra, striatum, etc., the activation of reactive astrocytes can be well regulated. Optionally, the site of directional injection can also be located in or near the glial scar caused by the interruption of normal blood flow in the CNS.

[0195] AAV is a ubiquitous, non-cytopathic, replication-incompetent member of the ssDNA animal virus family of Parvoviridae. As used herein, "AAV vector" refers to AAV packaged with a DNA vector construct. According to aspects of the present disclosure, the AAV vector can be selected from: AAV vector serotype 1, AAV vector serotype 2, AAV vector serotype 3, AAV vector serotype 4, AAV vector serotype 5, AAV vector serotype 6, AAV vector serotype 7, AAV vector serotype 8, AAV vector serotype 9, AAV vector serotype 10, AAV vector serotype 11, and AAV vector serotype 12.

[0196] In some embodiments, a concentration of 10 10 -10 14 AAV particles / ml, AAV particles in a volume of 1-1000 μl. According to various aspects of the present disclosure, the concentration of injection is between 10 10 AAV particles / mL and 10 11 AAV particles / mL, between 10 10 AAV particles / mL and 10 12 AAV particles / mL, between 10 10 AAV particles / mL and 10 13 AAV particles / mL, between 10 11AAV particles / mL and 10 12 AAV particles / mL, between 10 11 AAV particles / mL and 10 13 AAV particles / mL, between 10 11 AAV particles / mL and 10 14 AAV particles / mL, between 10 12 AAV particles / mL and 10 13 AAV particles / mL, between 10 12 AAV particles / mL and 10 14 AAV particles / mL, or between 10 13 AAV particles / mL and 10 14 According to aspects of the present disclosure, between 1 μL and 100 μL, between 1 μL and 200 μL, between 1 μL and 300 μL, between 100 μL and 200 μL, between 100 μL and 300 μL, between 100 μL and 400 μL, between 200 μL and 300 μL, between 200 μL and 400 μL, between 200 μL and 500 μL, between 300 μL and 400 μL, between 300 μL and 500 μL, between 300 μL and 600 μL, between 400 μL and 500 μL, between 400 μL and 600 μL, between 100 μL and 200 μL, between 100 μL and 300 μL, between 100 μL and 400 μL, between 200 μL and 500 μL, between 300 μL and 400 μL, between 300 μL and 500 μL, between 300 μL and 600 μL, between 100 μL and 2 ... The AAV particles are injected in a volume of between 400uL and 700uL, between 500uL and 600uL, between 500uL and 700uL, between 500uL and 800uL, between 600uL and 700uL, between 600uL and 800uL, between 600uL and 900uL, between 700uL and 800uL, between 700uL and 900uL, between 700uL and 1000uL, between 800uL and 900uL, between 800uL and 1000uL, or between 900uL and 1000uL.

[0197] Methods for regulating neurotoxic reactive astrocytes

[0198] The "method for regulating neurotoxic reactive astrocytes" provided in the present application is to administer to an individual in need a TMEM164 regulator that can upregulate the expression level of TMEM164, or to administer to an individual in need a SOX18 regulator that can upregulate the expression level of SOX18, or to administer to an individual in need a Capn15 regulator that can downregulate the expression level of Capn15; or to administer a combination of at least two.

[0199] In some embodiments, the regulator that can increase the expression level of TMEM164 can be a regulator that increases the TMEM164 gene, or increases the expression of TMEM164 protein. In some embodiments, some feasible regulators can be small molecule compounds. In some embodiments, some feasible regulators can also be antibodies, proteins, polypeptides, gene fragments, binding proteins or protein domains, polypeptides, nucleic acid aptamers, PROTACs, expression vectors containing promoters, protein analogs, or polysaccharides. In some embodiments, some feasible regulators can also be some gene regulation tools. Gene fragments can be mRNA, microRNA, siRNA, shRNA, antisense oligonucleotides, etc. Some gene regulation tools can be epigenetic regulation tools.

[0200] In some specific embodiments, the regulator capable of upregulating the expression level of TMEM164 can be a TMEM164 gene expression activator, a TMEM164 protein degradation inhibitor, an mRNA stabilizer, exogenous TMEM164 or a functional fragment of TMEM164.

[0201] In some embodiments, the TMEM164 protein degradation inhibitor may be some methods that intervene in the protein degradation of TMEM164, such as but not limited to, by adjusting the protein degradation pathway, such as inhibiting protease activity or using protease inhibitors, to slow down the degradation rate of TMEM164, thereby increasing the expression level. The methods may be, for example, small molecule inhibitors or macromolecular inhibitors.

[0202] In some embodiments, the TMEM164 gene expression activator is an endogenous TMEM164 modifier, an agonist that enhances TMEM164 gene or protein expression, or an inhibitor of TMEM164 competitive genes. In some embodiments, the endogenous TMEM164 modifier can be a modification of endogenous TMEM164 by genetic engineering technology (e.g., using ubiquitination) to prevent or slow down the degradation of TMEM164.

[0203] In some specific embodiments, the enhancer for enhancing the expression of the TMEM164 gene or protein is selected from: an agonist of a positive regulatory factor that enhances the expression of TMEM164, an inhibitor of a negative regulatory factor that reduces the expression of TMEM164, and an agonist that increases the expression of TMEM164 protein. In some embodiments, it can be, for example, some indirect ways of enhancing the expression of TMEM164, such as but not limited to: regulating the upstream positive or negative factors of TMEM164 expression, such as by overexpressing positive regulatory factors or knocking down / knocking out the expression of negative factors, thereby indirectly enhancing the expression level of TMEM164. In some embodiments, it can be to adjust the translation efficiency of the TMEM164 protein, such as but not limited to by optimizing the 5' untranslated region (5'UTR) of the TMEM164 gene to improve the translation efficiency of the TMEM164 protein.

[0204] In some embodiments, the inhibitor of TMEM164 competitive gene is a small molecule inhibitor, a ligand of TMEM164 competitive gene, a gene silencing tool, or an antibody of TMEM164 competitive gene. In some embodiments, the expression of endogenous proteins that compete with TMEM164 for resources can be reduced by silencing or knocking out competitive genes, thereby increasing the expression level of TMEM164 protein.

[0205] In some specific embodiments, the exogenous TMEM164 or functional fragment of TMEM164 is a polypeptide of TMEM164 or a biologically active fragment thereof, or a nucleic acid encoding a polypeptide of TMEM164 or a biologically active fragment thereof; TMEM164 is preferably human TMEM164.

[0206] In some specific embodiments, the exogenous TMEM164 or functional fragment of TMEM164 is a mutant of the TMEM164 polypeptide or its biologically active fragment that retains the function of TMEM164; preferably, the mutant has at least 80%, 85%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% homology with the TMEM164 polypeptide or its biologically active fragment.

[0207] In some specific embodiments, the TMEM164 polypeptide has a sequence shown in SEQ ID NO: 31 or 32.

[0208] In some specific embodiments, the modulator can upregulate the expression level of TMEM164 in glial cells; in some embodiments, the glial cells are preferably from the central nervous system, the visual nervous system, or the auditory nervous system.

[0209] In some specific embodiments, the glial cells include astrocytes, Muller glial cells, or cochlear glial cells (satellite cells); preferably, the astrocytes include GFAP-positive astrocytes, fibrous astrocytes or protoplasmic astrocytes.

[0210] In some embodiments, the astrocytes are non-reactive astrocytes.

[0211] In some embodiments, the modulator is capable of regulating the activation of non-reactive astrocytes into reactive astrocytes. In some preferred embodiments, the regulation is to inhibit the activation of non-reactive astrocytes into neurotoxic reactive astrocytes. In some preferred embodiments, the regulation is to increase the conversion of non-reactive resting astrocytes into neuroprotective reactive astrocytes. In some preferred embodiments, the regulation is to inhibit the activation of non-reactive astrocytes into neurotoxic reactive astrocytes and to increase the conversion of non-reactive resting astrocytes into neuroprotective reactive astrocytes.

[0212] In some specific embodiments, the astrocytes are from the retina, vitreous cavity, striatum, substantia nigra, inner ear, spinal cord, prefrontal cortex, motor cortex, thalamus, ventral tegmental area (VTA), hippocampus, cerebellum, brainstem, or inner ear cochlea or vestibule, more preferably, the astrocytes are from the striatum, substantia nigra, retina, or vitreous cavity.

[0213] In some embodiments, the modulator is administered to an individual in need via a carrier. In some embodiments, the carrier is selected from: a viral vector, a lipid nanoparticle (LNP), a liposome, a cationic polymer (such as PEI), a nanoparticle, an exosome; more preferably, the carrier is an AAV vector or a lipid nanoparticle, wherein an AAV vector or a lipid nanoparticle is preferred.

[0214] In some specific embodiments, the carrier further comprises a promoter. In some specific embodiments, the promoter may be a broad-spectrum promoter or a specific promoter. In some specific embodiments, the broad-spectrum promoter may be selected from CMV, CBH, CAG, PGK, SV40, EF1A, EFS, pGlobin promoters. In some specific embodiments, the specific promoter is a glial cell-specific promoter or a Muller glial cell (MG) cell-specific promoter, preferably, the glial cell-specific promoter is selected from GFAP promoter, ALDH1L1 promoter, EAAT1 / GLAST promoter, glutamine synthetase promoter, S100β promoter and EAAT2 / GLT-1 promoter, NG2 promoter, CD68 promoter, F4 / 80 promoter, or the MG cell-specific promoter is selected from GFAP promoter, ALDH1L1 promoter, Glast (also known as Slc1a3) promoter and Rlbp1 promoter.

[0215] Methods for preventing, delaying, or treating diseases related to nerve damage and neuronal aging, and methods for alleviating symptoms of diseases related to nerve damage and neuronal aging

[0216] The "methods for preventing, delaying, or treating nerve injury diseases and diseases related to neuronal aging" and "methods for alleviating symptoms of nerve injury and neuronal aging" provided in this application are: administering a TMEM164 modulator capable of upregulating the expression level of TMEM164 to an individual in need, or administering a SOX18 modulator capable of upregulating the expression level of SOX18 to an individual in need, or administering a Capn15 modulator capable of downregulating the expression level of Capn15 to an individual in need; or administering a combination of at least two. .

[0217] In some embodiments, the neurological damage disease refers to a disease associated with neuronal death.

[0218] In some specific embodiments, the nerve damage disease is brain nerve damage, spinal cord damage, or optic nerve damage.

[0219] In some specific embodiments, the diseases causing brain nerve damage include: brain trauma, stroke, cerebral ischemia, physical injury, tumor, inflammation, infection, ischemia caused by cardiac arrest or severe hypotension (shock), hypoxic-ischemic encephalopathy caused by hypoxia, hypoglycemia or anemia, meningitis, dehydration, subarachnoid hemorrhage, intracranial hematoma, weak cerebral blood vessels, cerebral vascular malformation, hypertension, or neurodegenerative diseases, or a combination of at least two diseases.

[0220] In some embodiments, the disease of brain nerve damage includes Alzheimer's disease (AD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), multiple sclerosis (MS), or frontotemporal dementia (FTLD).

[0221] In some specific embodiments, the disease causing optic nerve damage includes glaucoma, retinal detachment, optic nerve inflammation, vision damage caused by RGC cell death, photoreceptor cell damage, age-related macular degeneration (AMD), diabetic retinopathy, retinal ischemia or hemorrhage, Leber hereditary optic neuropathy, or ocular trauma, or a combination of at least two diseases.

[0222] In some specific embodiments, the modulator is administered to an individual in need by direct targeted administration to the affected site of a neurological injury disease.

[0223] In some embodiments, the modulator is administered to a subject in need thereof by cell transfection, cell infection, cell endocytosis, injection, intracranial administration, spinal administration, inhalation, parenteral administration, intravenous administration, intramuscular administration, intradermal administration, topical administration, local administration, targeted administration, subretinal injection, intravitreal injection, or oral administration.

[0224] In some specific embodiments, the nerve injury disease is a central nervous system injury disease, and the regulator is injected into the striatum, hippocampus or substantia nigra.

[0225] In some embodiments, the modulator is administered to an individual in need via a carrier. In some embodiments, the carrier is selected from: a viral vector, a lipid nanoparticle (LNP), a liposome, a cationic polymer (such as PEI), a nanoparticle, an exosome; more preferably, the carrier is an AAV vector or a lipid nanoparticle, wherein an AAV vector or a lipid nanoparticle is preferred.

[0226] In some specific embodiments, the carrier further comprises a promoter. In some specific embodiments, the promoter may be a broad-spectrum promoter or a specific promoter. In some specific embodiments, the broad-spectrum promoter may be selected from CMV, CBH, CAG, PGK, SV40, EF1A, EFS, pGlobin promoters. In some specific embodiments, the specific promoter is a glial cell-specific promoter or a Muller glial cell (MG) cell-specific promoter, preferably, the glial cell-specific promoter is selected from GFAP promoter, ALDH1L1 promoter, EAAT1 / GLAST promoter, glutamine synthetase promoter, S100β promoter and EAAT2 / GLT-1 promoter, NG2 promoter, CD68 promoter, F4 / 80 promoter, or the MG cell-specific promoter is selected from GFAP promoter, ALDH1L1 promoter, Glast (also known as Slc1a3) promoter and Rlbp1 promoter.

[0227] Composition and use

[0228] The present application provides a composition comprising a TMEM164 modulator that upregulates the expression level of TMEM164.

[0229] The present application provides a composition comprising a SOX18 regulator that upregulates the expression level of SOX18.

[0230] The present application provides a composition comprising a CAPN15 regulator that downregulates the expression level of CAPN15.

[0231] The present application provides a composition, comprising a TMEM164 regulator that upregulates the expression level of TMEM164 and a Capn15 regulator that downregulates the expression level of CAPN15.

[0232] The present application provides a composition, comprising a TMEM164 regulator that upregulates the expression level of TMEM164 and a SOX18 regulator that upregulates the expression level of SOX18.

[0233] In some embodiments, the regulator that can increase the expression level of TMEM164 can be a regulator that increases the TMEM164 gene, or increases the expression of TMEM164 protein. In some embodiments, some feasible regulators can be small molecule compounds. In some embodiments, some feasible regulators can also be antibodies, proteins, polypeptides, gene fragments, binding proteins or protein domains, polypeptides, nucleic acid aptamers, PROTACs, expression vectors containing promoters, protein analogs, or polysaccharides. In some embodiments, some feasible regulators can also be some gene regulation tools. Gene fragments can be mRNA, microRNA, siRNA, shRNA, antisense oligonucleotides, etc. Some gene regulation tools can be epigenetic regulation tools.

[0234] In some specific embodiments, the regulator capable of upregulating the expression level of TMEM164 can be a TMEM164 gene expression activator, a TMEM164 protein degradation inhibitor, an mRNA stabilizer, exogenous TMEM164 or a functional fragment of TMEM164.

[0235] In some embodiments, the TMEM164 protein degradation inhibitor may be some methods that intervene in the protein degradation of TMEM164, such as but not limited to, by adjusting the protein degradation pathway, such as inhibiting protease activity or using protease inhibitors, to slow down the degradation rate of TMEM164, thereby increasing the expression level. The methods may be, for example, small molecule inhibitors or macromolecular inhibitors.

[0236] In some embodiments, the TMEM164 gene expression activator is an endogenous TMEM164 modifier, an agonist that enhances TMEM164 gene or protein expression, or an inhibitor of TMEM164 competitive genes. In some embodiments, the endogenous TMEM164 modifier can be a modification of endogenous TMEM164 by genetic engineering technology (e.g., using ubiquitination) to prevent or slow down the degradation of TMEM164.

[0237] In some specific embodiments, the enhancer for enhancing the expression of the TMEM164 gene or protein is selected from: an agonist of a positive regulatory factor that enhances the expression of TMEM164, an inhibitor of a negative regulatory factor that reduces the expression of TMEM164, and an agonist that increases the expression of TMEM164 protein. In some embodiments, it can be, for example, some indirect ways of enhancing the expression of TMEM164, such as but not limited to: regulating the upstream positive or negative factors of TMEM164 expression, such as by overexpressing positive regulatory factors or knocking down / knocking out the expression of negative factors, thereby indirectly enhancing the expression level of TMEM164. In some embodiments, it can be to adjust the translation efficiency of the TMEM164 protein, such as but not limited to by optimizing the 5' untranslated region (5'UTR) of the Tmem164 gene to improve the translation efficiency of the TMEM164 protein.

[0238] In some embodiments, the inhibitor of TMEM164 competitive gene is a small molecule inhibitor, a ligand of TMEM164 competitive gene, a gene silencing tool, or an antibody of TMEM164 competitive gene. In some embodiments, the expression of endogenous proteins that compete with TMEM164 for resources can be reduced by silencing or knocking out competitive genes, thereby increasing the expression level of TMEM164 protein.

[0239] In some specific embodiments, the exogenous TMEM164 or functional fragment of TMEM164 is a polypeptide of TMEM164 or a biologically active fragment thereof, or a nucleic acid encoding a polypeptide of TMEM164 or a biologically active fragment thereof; TMEM164 is preferably human TMEM164.

[0240] In some specific embodiments, the exogenous TMEM164 or functional fragment of TMEM164 is a mutant of the TMEM164 polypeptide or its biologically active fragment that retains the function of TMEM164; preferably, the mutant has at least 80%, 85%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% homology with the TMEM164 polypeptide or its biologically active fragment.

[0241] In some specific embodiments, the TMEM164 polypeptide has a sequence shown in SEQ ID NO: 31 or 32.

[0242] In some specific embodiments, the modulator can upregulate the expression level of TMEM164 in glial cells; in some embodiments, the glial cells are preferably from the central nervous system, the visual nervous system, or the auditory nervous system.

[0243] In some specific embodiments, the glial cells include astrocytes, Muller glial cells, or cochlear glial cells (satellite cells); preferably, the astrocytes include GFAP-positive astrocytes, fibrous astrocytes or protoplasmic astrocytes.

[0244] In some embodiments, the astrocytes are non-reactive astrocytes.

[0245] In some embodiments, the modulator is capable of regulating the activation of non-reactive astrocytes into reactive astrocytes. In some preferred embodiments, the regulation is to inhibit the activation of non-reactive astrocytes into neurotoxic reactive astrocytes. In some preferred embodiments, the regulation is to increase the conversion of non-reactive resting astrocytes into neuroprotective reactive astrocytes. In some preferred embodiments, the regulation is to inhibit the activation of non-reactive astrocytes into neurotoxic reactive astrocytes and to increase the conversion of non-reactive resting astrocytes into neuroprotective reactive astrocytes.

[0246] In some specific embodiments, the astrocytes are from the retina, vitreous cavity, striatum, substantia nigra, inner ear, spinal cord, prefrontal cortex, motor cortex, thalamus, ventral tegmental area (VTA), hippocampus, cerebellum, brainstem, or inner ear cochlea or vestibule, more preferably, the astrocytes are from the striatum, substantia nigra, retina, or vitreous cavity.

[0247] In some specific embodiments, the composition provided includes: a first recombinant expression vector, which includes a glial cell-specific promoter operably linked to a nucleic acid encoding a site-specific recombinase; and a second recombinant expression vector, which includes a promoter operably linked to a nucleic acid sequence encoding TMEM164, a nucleic acid sequence encoding a reporter gene, an enhancer, and a regulatory element.

[0248] In some specific embodiments, the composition includes: a first recombinant AAV expression vector, which includes a glial cell-specific promoter operably linked to a nucleic acid encoding a site-specific recombinase; and a second recombinant AAV expression vector, which includes a promoter operably linked to a nucleic acid sequence encoding TMEM164, a nucleic acid sequence encoding a reporter gene, an enhancer, and a regulatory element.

[0249] In some specific embodiments, the site-specific recombinase is Cre recombinase, and the site of recombinase activity is the recognition site loxP and lox2272 site.

[0250] The present application also provides the use of the above-mentioned compounds for regulating neurotoxic reactive astrocytes, preventing, delaying, or treating nerve injury diseases, preventing, delaying, or treating diseases related to neuronal aging, or alleviating symptoms of nerve injury and neuronal aging.

[0251] Scientific and technical terms used herein are intended to have the meanings that are commonly understood by one of ordinary skill in the art. These terms are illustratively defined and used in the context of a variety of standard reference texts, including: J. Sambrook and D. W. Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 3rd Ed., 2001; F. M. Asubel, Ed., Short Protocols in Molecular Biology, Current Protocols; 5th Ed., 2002; B. Alberts et al., Molecular Biology of the Cell, 4th Ed., Garland, 2002; D. L. Nelson and M. M. Cox, Lehninger Principles of Biochemistry, 4th Ed., W. H. Freeman & Company, 2004; Engelke, D. R., RNA Interference (RNAi): Nuts and Bolts of RNAi Technology, DNA Press LLC, Eagleville, Pa., 2003; Herdewijn, p. (Ed.), Oligonucleotide Synthesis: Methods and Applications, Methods in Molecular Biology, Humana Press, 2004; A.Nagy, M.Gertsenstein, K.Vintersten, R.Behringer, Manipulating the MouseEmbryo: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 3rd Ed.; December 15, 2002, ISBN-10: 0879695919; Kursad Turksen (Ed.), Embryonic StemCells: Methods and Protocols in Methods in Molecular Biology, 2002; 185, Human Press: Current Protocols in Stem Cell Biology, ISBN: 9780470151808.

[0252] Details of some of the techniques mentioned in this application are known in the art, for example, as described in the following references: J. Sambrook and DW Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 3rd edition, 2001; FM Ausubel, ed., Molecular Biology: A Laboratory Manual; 5th edition, 2002; and Engelke, DR, RNA Interference (RNAi): Nuts and Bolts of RNAi Technology, DNA Press LLC, Eagleville, PA, 2003.

[0253] Some methods used by the present invention:

[0254] The animals used in the examples were mixed-sex C57BL / 6 and male 5XFAD mice.

[0255] Obtaining and culturing primary mouse astrocytes: The cortex of a mouse pup on the first day after birth (P1) was taken. After removing the meninges, the cortex was washed three times in DMEM / F12 medium supplemented with 1% penicillin / streptomycin (P / S), transferred to 0.25% trypsin-EDTA, and then gently stirred for 15 minutes. Trypsin digestion was terminated using DMEM / F12 medium supplemented with 10% fetal bovine serum and 1% P / S. The cortex was then washed three times in DMEM / F12 medium and ground in DMEM / F12 medium, then passed through a 70 μm nylon mesh and centrifuged at 1,000 rpm for 3 minutes to obtain a single cell suspension. The single cell suspension was resuspended in DMEM / F12 medium and cultured in a T75 flask for 7 days, with complete medium replacement on the second day. Astrocyte populations were isolated and trypsinized and plated in 6-well or 12-well plates for stimulation. Primary astrocytes were stimulated with 30 ng / ml TNF, 3 ng / ml IL-1α and 400 ng / ml C1q for 24 h for reactive phenotype induction.

[0256] Bulk RNA-seq analysis: For untransfected astrocytes, cells were stimulated with PBS, IL-1α+TNF+C1q or bFGF for a specified time, and RNA was subsequently isolated. For astrocytes transfected with plasmids, cells were stimulated with PBS or IL-1α+TNF+C1q for 24 hours, and then EGFP-positive cells were sorted using a MoFlo XDP cell sorter (Beckman) for RNA isolation. RNA-seq library construction and sequencing were performed by Novogene. For bulk RNA-seq analysis, bulk RNA-seq sequencing reads with poor quality were filtered using Trimmomatic (version -0.32) with default parameters. High-quality sequencing reads were then aligned with the mouse genome (GRCm38) (mm10) using the HISAT2 program (version hisat2-2.2.1) with default parameters. After this, the mapped Bam files were sorted by Samtools (version 1.7). Stringtie (version stringtie-2.1.4) was used to identify the read counts of each mRNA gene from the sorted RNA-seq bam files. The DESeq2 R package was used to determine the differentially expressed gene (DEG) analysis. Significant DEGs between the treatment and control groups were determined using the following combined criteria: |log2 fold change|>0 and p value <0.05. DEG Gene Ontology and KEGG pathway analysis were then performed using online web software (metascape (https: / / metascape.org) and DAVID (version v2022q2)) with default parameters.

[0257] Single-nucleus RNA-seq analysis: The analysis of the snRNA-seq dataset mainly includes two parts: de novo identification of differentially expressed genes (DEGs) in astrocytes (disease group vs. healthy group) and direct downloading of the supplementary table of DEGs in astrocytes (disease group vs. healthy group) from the corresponding literature. The former mainly uses the expression matrix provided by the author and converts it into a standard input file for Seurat. Then the downstream analysis uses Seurat v.4.1.3. In short, the disease group and the healthy group are combined into a single Seurat object, in which cells with low mitochondrial gene coverage (percent mt < 5%) are saved, and then normalization, identification of highly variable genes, principal component analysis, clustering (UMAP and t-SNE), identification of marker genes in different clusters, and identification of cell types by SingleR and CellMarker (http: / / xteam.xbio.top / CellMarker / ). By focusing on astrocytes, the DEGs of the disease group and the healthy group were obtained by the FindMarkers function.

[0258] Real-time quantitative PCR: Total RNA from astrocytes was extracted using RNA isolater total RNA extraction reagent (Vazyme, R401-01). Reverse transcription was performed using HiScript II Q RT SuperMix for qPCR Kit (Vazyme, R223-01). Quantitative PCR reactions were performed using AceQ qPCR SYBR Green Master Mix (Vazyme, Q111-02). All production was performed according to the manufacturer's instructions. The reaction was performed using a 480Instrument II (Roche Diagnostics) with a program consisting of 40 cycles, each cycle consisting of an initiation phase at 95°C for 5 s, an annealing phase at 60°C for 10 s, and an amplification phase at 95°C for 10 s, followed by 65°C for 60 s. Quantitative RT-PCR primers for mouse A1 or A2-specific markers, phagocytic mediators, and synaptogenic factors were used as described previously4, and other RT-PCR primers were as described Figure 1 shown.

[0259] Plasmid construction and transfection: To construct overexpression vectors of individual potential factors, cDNA of each candidate was prepared from the mRNA of astrocytes by reverse transcription. The individual cDNA fragments were cloned into a linearized lentiviral backbone vector, in which the cDNA was driven by a short CAG promoter and connected to the EGFP and puromycin N-acetyltransferase sequences through a 2A sequence. For transfection, 2 μg of plasmid was transfected into primary astrocytes seeded in one well of a 6-well plate using Lipofectamine 3000 transfection reagent (Invitrogen, L3000015). The culture medium was replaced 8 hours after transfection. After 48 hours, astrocytes were treated with IL-1α, TNF, and C1q to induce a reactive state.

[0260] Lentivirus production and infection: Lentivirus overexpression vector (22.5 μg), lentivirus packaging plasmid (16.9 μg, Addgene, #12259) and VSV-G envelope expression plasmid (5.6 μg, Addgene, #12260) were added to 1 ml of Opti-MEM (Gibco, 31985070) and incubated at room temperature for 5 minutes. EZ cell transfection reagent (90 μl) (Life-iLab, AC04L092) was added to 1 ml of Opti-MEM and incubated at room temperature for 5 minutes. The above solutions were mixed and further incubated at room temperature for 20 minutes. The transfection complex (2 ml) was added to HEK293T cells grown to 80% to 90% density in a 15 cm dish, and the culture medium was replaced with DMEM containing 5% FBS, 1% NEAA and 1% P / S 6 hours before transfection. 8 hours after transfection, the medium was replaced with fresh complete medium containing 10% FBS, 1% NEAA and 1% P / S. The viral supernatant was collected 72 hours after transfection and centrifuged at 4°C, 27,500rpm for 2 hours. The viral pellet was dissolved in 100 μl PBS and stored at -80°C for long-term storage. Primary astrocytes inoculated in 6-well plates were infected with concentrated lentivirus for 5 days in the presence of 10 μg / ml polybrene. The medium was replaced 24 hours after lentiviral infection.

[0261] Neuronal synapse formation assay: Sorted EGFP or TMEM164 overexpressing mouse primary astrocytes or human astrocytes were cultured on transmembrane well plates (Corning, #3470) and treated with PBS or ITC for 24 hours to reach a reactive state, and then co-cultured with mouse cortical neurons or human ES cell-derived neurons for 5 days. For quantitative analysis of structural synapses, mouse cortical neurons or human ES cell-derived neurons were fixed and stained with MAP2 (Abcam, ab5392) neuronal marker antibodies, presynaptic marker Bassoon (Novus, NB120-13249) and postsynaptic marker Homer1 (Abcam, ab184955). The number of synapses was quantified by measuring the number of spots where Bassoon and Homer1 signals colocalized using ImageJ.

[0262] Stereotaxic injection of AAV in LPS-induced PD model: Three-month-old C57BL / 6 male mice were deeply anesthetized. Then 1 μl of AAV-PHP.eB-sGFAP-Tmem164-P2A-mCherry-WPRE (4.00×1011vg / ml) or AAV-PHP.eB-sGFAP-mCherry-WPRE (2.58×1011vg / ml) (produced by Shanghai Genemagic Biosciences) was injected into the SNc (anterior / posterior: -2.90; medial: ±1.30; dorsal / ventral: -4.40). After three weeks, 1 μl of 0.5ug / ul LPS (Santa Cruz, sc-3535) was microinjected bilaterally into the SNc region of the mice to establish an inflammatory PD model. The mice were sacrificed one week later to harvest brain samples for subsequent immunofluorescence staining assays.

[0263] Immunofluorescence staining: After deep anesthesia of mice, PBS and 4% PFA were perfused. The brain was fixed in 4% PFA for 24 hours and then dehydrated in 30% sucrose. The dehydrated brain was sectioned coronally (30 μm) and stored in PBS at 4°C before staining. For staining on mouse and human astrocytes, cells were washed with PBS and fixed with 4% PFA for 10 minutes before staining. Mouse brain sections or cells were washed with PBS and incubated overnight at 4°C with the following primary antibodies diluted in primary antibody dilution buffer (Beyotime, P0262): anti-GFAP (Proteintech, 16825-1-AP), anti-GFAP (Proteintech, 60190-1-Ig), anti-Iba1 (Wako, 019-19741), anti-C3 (Abcam, ab11862), anti-C3 (Dako, A0063), anti-TH (Sigma-Aldrich, AB152), anti-TMEM164 (Novus, NBP1-93939). The sections were then incubated with the corresponding secondary antibodies diluted in secondary antibody dilution buffer (Beyotime, P0265) at room temperature for 2 hours and incubated with DAPI to stain the nuclei. The sections were mounted using fluorescent mounting medium and images were collected by FV3000 confocal microscope.

[0264] In the following examples, ITC treatment refers to adding 3 ng / ml IL-1α, 30 ng / ml TNF and 400 ng / ml C1q three cytokines to the culture medium to stimulate the cells, and PBS treatment refers to adding PBS in an equal volume to ITC to the culture medium.

[0265] Statistical analysis: Comparisons were analyzed by unpaired two-tailed student t-test, one-way ANOVA test, or two-way ANOVA test. Data were analyzed using GraphPad Prism 6 software. Differences were considered significant when p value < 0.05 (*), p value < 0.01 (**), and p value < 0.001 (***).

[0266] The embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention.

[0267] Example

[0268] Example 1. Effect of overexpression of TMEM164 on the activation of normal astrocytes and neurotoxic reactive astrocytes in mice

[0269] To determine the functional role of TMEM164 in neurotoxic astrocyte activation, we performed bulk RNA-seq analysis using mouse primary astrocytes. The transcription levels of specific markers of pan-reactive, A1 neurotoxic, and A2 neuroprotective astroglial cells were compared in mouse astrocytes treated with PBS for 24 h (non-reactive astrocytes), mouse astrocytes treated with ITC for 24 h (reactive astrocytes), EGFP-overexpressing (plasmid structure: Lenti-sCAG-EGFP-P2A-Puro-WPRE) astrocytes treated with ITC for 24 h (EGFP-ITC astrocytes), and TMEM164-EGFP-overexpressing (plasmid structure: Lenti-sCAG-Tmem164-P2A-EGFP-T2A-Puro-WPRE) astrocytes treated with ITC for 24 h (TMEM164-ITC astrocytes). ITC treatment refers to the addition of three cytokines, 3 ng / ml IL-1α, 30 ng / ml TNF, and 400 ng / ml C1q, to ​​the astrocyte culture medium to stimulate the cells.

[0270] Primary mouse astrocytes were obtained, and the primary mouse astrocytes were transfected with EGFP or TMEM164-P2A-EGFP overexpression plasmids for 48 hours using conventional biological methods in the art, and EGFP-positive cells were sorted to obtain EGFP-overexpressing astrocytes and astrocytes co-overexpressing TMEM164 and EGFP.

[0271] Mouse primary astrocytes, EGFP-overexpressing astrocytes, and TMEM164 and EGFP-co-overexpressing astrocytes were plated at a density of 100,000 cells / well in a 24-well plate pre-coated with 10 ng / ml poly-D-lysine (Sigma-Aldrich, P7886). After 12 hours, 30 ng / ml TNF, 3 ng / ml IL-1α, and 400 ng / ml C1q were added to the culture medium. After 24 hours of co-culture, the cells were harvested to obtain reactive astrocytes (IL-1α+TNF+C1q(Reactive)), EGFP-ITC astrocytes (EGFP overexpression IL-1α+TNF+C1q(EGFP-ITC)), TMEM164-ITC astrocytes (TMEM164 overexpression IL-1α+TNF+C1q(TMEM164-ITC)); primary mouse astrocytes were taken and co-cultured with PBS in the culture medium for 24 hours as a negative control, labeled as non-reactive astrocytes, namely: PBS (non-reactive).

[0272] The transcription levels of the specific markers of the four obtained astrocytes, pan-reactive astrocytes, A1 neurotoxic astrocytes, and A2 neuroprotective astrocytes, were detected respectively. Figure 2 As shown in the data, compared with EGFP-ITC astrocytes, the expression levels of neurotoxic astrocyte-specific markers such as C3, Serping1, Iigp1, Psmb8, Srgn and Amigo2 were decreased, while the expression levels of neuroprotective astrocyte-specific markers such as Clcf1, S100a10, Sphk1, Ptgs2, Emp1 and Tm4sf1 were increased in the TMEM164-ITC astrocyte group, indicating that the upregulation of TMEM164 can reduce the level of A1 glial cells and increase the level of A2 glial cells, which has a potential protective effect on neurons.

[0273] To evaluate whether the altered expression of Tmem164 affects the quiescent state of astrocytes, we overexpressed TMEM164 protein in primary mouse astrocytes or downregulated the mRNA expression level of Tmem164 using the CasRx / gRNA system. The sequence of sgRNA is shown in SEQ ID NO: 29. Figure 3A and Figure 3B As shown, neither increasing nor decreasing the expression of Tmem164 could significantly affect the expression levels of neurotoxicity-specific markers in non-reactive astrocytes.

[0274] These data suggest that TMEM164 overexpression prevents the activation of neurotoxic reactive astrocytes in response to inflammatory stimuli, whereas under resting conditions, modulation of Tmem164 expression fails to alter the state of non-reactive astrocytes.

[0275] Example 2: Effects of TMEM164 overexpression on phagocytosis and synapse formation in neurotoxic reactive astrocytes of mice

[0276] We tested whether overexpression of TMEM164 could maintain normal astrocyte function in the face of activating stimuli. The ability of astrocytes to phagocytose dead synaptosomes was determined by measuring the engulfment of purified synaptosomes.

[0277] Synaptosomes were purified from mouse brain and bound to pHrodo Red, succinimidyl ester (Invitrogen, P36600) in 0.1 M sodium carbonate (pH 9.0) by gentle incubation for 1 hour at room temperature. After incubation, unbound pHrodo Red was washed away by five centrifugations. Finally, pHrodo-bound synaptosomes were resuspended in PBS containing 5% DMSO and frozen at -80°C.

[0278] Mouse astrocytes were infected with lentivirus overexpressing EGFP or Tmem164-P2A-EGFP for 5 days to express EGFP. EGFP-positive cells were sorted to obtain EGFP-expressing astrocytes and TMEM164-EGFP co-expressing astrocytes. Astrocytes expressing EGFP and astrocytes co-expressing TMEM164-EGFP were plated at a cell density of 100,000 cells / well in 24-well plates pre-coated with 10ng / ml poly-D-lysine (Sigma-Aldrich, P7886). After 12 hours, they were treated with IL-1α+TNF+C1q for 24 hours and labeled as EGFP-ITC and TMEM164-ITC, respectively. Another astrocyte expressing EGFP was inoculated and treated with PBS 12 hours later as a control group, labeled as EGFP-PBS. The three groups of cells were then incubated with 5 μl of pHrodo Red-conjugated synaptosomes and imaged using a 20x objective at random areas at 12-hour intervals.

[0279] The results are as follows Figure 4A , Figure 4B ,and Figure 4CAs shown, TMEM164-ITC astrocytes engulfed more pHrodo-bound synaptosomes compared with EGFP-ITC astrocytes, which was consistent with the increased mRNA expression levels of the engulfment receptors Megf10 and Mertk and the bridging molecule Gas6 in TMEM164-ITC astrocytes.

[0280] We further examined the effect of TMEM164 overexpression on synapse formation in mouse cortical neurons. The results are shown in Figure 5. Figure 5A In the 3D images, genes encoding astrocyte-secreted synapse-forming factors, including Thbs1, Gpc6, and Sparcl1, were significantly upregulated in TMEM164-ITC astrocytes compared with EGFP-ITC astrocytes, indicating that TMEM164-ITC astrocytes retain the ability of normal astrocytes to promote synapse formation.

[0281] We co-cultured three groups of astrocytes, EGFP-ITC, TMEM164-ITC, and EGFP-PBS, with mouse cortical neurons for 5 days, stained them with MAP2 (Abcam, ab5392) neuronal marker antibody, presynaptic marker Bassoon (Novus, NB120-13249) and postsynaptic marker Homer1 (Abcam, ab184955), and used ImageJ to measure the number of spots where Bassoon and Homer1 signals co-localized to quantify the number of synapses. The results are shown in Figure 2. Figure 5B In the results, TMEM164-ITC astrocytes had a stronger ability to promote synaptic growth of mouse cortical neurons than EGFP-ITC astrocytes.

[0282] These data suggest that increased TMEM164 expression can maintain normal astrocyte function in astrocytes in response to inflammatory stimuli.

[0283] Example 3: Overexpression of TMEM164 can inhibit the neurotoxicity of reactive astrocytes

[0284] Neurotoxicity assay of primary cortical neurons of mice: Primary cortical neurons of mice were prepared from embryonic day 18 mice and cultured on tissue culture dishes coated with 100ng / ml poly-D-lysine in neurobasal medium supplemented with B-27, 0.5mM L-glutamine, 1% P / S. Neuronal culture was maintained by replacing half of the culture medium every 3 days. Cortical neurons were treated with conditioned medium collected from EGFP-PBS, TMEM164-PBS, EGFP-ITC, TMEM164-ITC astrocytes for 48 hours on the 7th day to induce neuronal death. Neuronal viability was measured by the calcein AM / EthD-1 method using the Viability / Cytotoxicity Assay Kit for Animal Live&DeadCells (Proteintech, PF00008) for animal live and dead cells.

[0285] The results are as follows Fig. 6A and 6B As shown, conditioned medium collected from TMEM164-ITC astrocytes induced less neuronal death compared with that from EGFP-ITC astrocyte controls, indicating that TMEM164 overexpression can inhibit cell death induced by neurotoxic reactive astrocytes.

[0286] Example 4: TMEM164 overexpression reduces the secretion of neurotoxic saturated lipids via the CAPN15-ELOVL1 pathway

[0287] To investigate the potential pathways underlying the protective effects of TMEM164, we first examined the effect of TMEM164 overexpression on the expression of Elovl1. Downregulation of Elovl1, a gene encoding a metabolic enzyme specifically responsible for the synthesis of neurotoxic saturated lipids, has been shown to mitigate the neurotoxic effects of reactive astrocytes.

[0288] We infected mouse astrocytes with lentiviruses that overexpress EGFP and TMEM164-EGFP for 5 days, sorted out two groups of EGFP-positive cells and continued to culture them. After stimulating the sorted astrocytes with ITC for 24 hours, we collected mRNA and detected the expression level of the Elovl1 gene by QPCR. Fig. 7AAs shown in Figure 2, compared with the EGFP-ITC astrocyte control group, the expression of Elovl1 at both the mRNA and protein levels in TMEM164-ITC astrocytes was reduced. To further prove that TMEM164 overexpression can inhibit the activation of the neurotoxic state of astrocytes and the expression of Elovl1, we detected the changes in the protein expression levels of ELOVL1 and C3, a typical marker of the neurotoxic astrocyte state. The results are shown in Figure 2. Figure 7B and 7C As shown, we found that after TMEM164 overexpression, the protein levels of C3 and ELOVL1 in the TMEM164-ITC group were significantly lower than those in the EGFP-ITC group (β-actin was the internal reference protein), indicating that TMEM164 overexpression significantly inhibited the activation of the neurotoxic state and suppressed the expression of ELOVL1.

[0289] To determine whether TMEM164 overexpression can reduce the secretion of neurotoxic saturated fatty acids, we measured the concentration of saturated fatty acids in the conditioned medium of astrocytes from three groups of EGFP-PBS, EGFP-ITC, and TMEM164-ITC by gas chromatography-mass spectrometry (GC-MS). Figure 8 As shown, we found that the concentrations of all detected neurotoxic saturated fatty acids (C16:0 and C18:0) were significantly reduced in the conditioned medium of TMEM164-ITC astrocytes compared with those of control EGFP-ITC astrocytes, suggesting that TMEM164 overexpression inhibits neuronal death induced by neurotoxic reactive astrocytes by reducing the expression of ELOVL1 and the subsequent secretion of neurotoxic saturated fatty acids.

[0290] To explore how TMEM164 overexpression affects Elovl1 expression, we performed bulk RNA-seq on EGFP-ITC and TMEM164-ITC astrocytes to screen for downstream targets of TMEM164. Fig. 9A and 9B As shown, most of the top-ranked down-regulated (left) differentially expressed genes induced by TMEM164 overexpression are transcription factors, including Capn15, Kidinis220, Zfp1, and Zfp626, suggesting that TMEM164 overexpression may reduce the expression of Elovl1 by affecting the expression levels of transcription factors.

[0291] To identify the downstream transcription factors of TMEM164, we overexpressed TMEM164 and each candidate transcription factor, including Capn15, Kidinis220, Sox18, Zfp1, and Zfp626, in resting astrocytes and then stimulated astrocytes with ITC. Fig. 10A and Fig. 10B As shown, it was found that overexpression of CAPN15 (Calpain 15) could counteract the inhibitory effect of TMEM164 overexpression on the induction of reactive astrocytes, and overexpression of SOX18 (SRY (sex determining region Y)-box18) could further enhance the inhibitory effect on the induction of reactive astrocytes, indicating that both CAPN15 and SOX18 are downstream transcription factors of TMEM164 in the activation of reactive astrocytes. The results suggest that upregulating or activating the expression level of SOX18 may inhibit neurotoxic reactive astrocytes, and SOX18 modulators that can upregulate the expression level of SOX18 have a synergistic effect with TMEM164 modulators that upregulate the expression level of TMEM164; inhibiting or reducing the expression level of CAPN15 may inhibit neurotoxic reactive astrocytes, and Capn15 modulators that downregulate the expression level of Capn15 have a synergistic effect with TMEM164 modulators that upregulate the expression level of TMEM164.

[0292] Since TMEM164 overexpression reduced the expression of Elovl1, a gene encoding a neurotoxic saturated lipid synthase, and CAPN15 is a downstream transcription factor of TMEM164 in astrocyte activation, we hypothesized that downregulating the expression of Capn15 could reduce the expression of Elovl1. To test our hypothesis, we first compared the expression levels of Tmem164, Capn15, and Elovl1 between non-reactive astrocytes (PBS-treated for 24 h) and reactive astrocytes (ITC-treated for 24 h) in mice. Fig.11A As shown in the figure, compared with non-reactive astrocytes, reactive astrocytes showed lower Tmem164 expression levels, but increased expression levels of Capn15 and Elovl1, indicating that there is a strong correlation between the expression levels of Tmem164, Capn15 and Elovl1. In order to test whether Elovl1 is a downstream target of Capn15, we first designed a gRNA sequence based on the CasRx knockdown system, and overexpressed CasRx and gRNA in astrocytes to screen the gRNA with the highest knockdown efficiency (i.e., SEQ ID No.30). Then we transfected a plasmid that can simultaneously express CasRx, gRNA targeting Capn15, and EGFP into astrocytes, stimulated them with ITC or PBS for 24 hours, and finally sorted out EGFP-positive cells for QPCR to detect the expression levels of Capn15 and Elovl1. The results are shown in the figure. Fig. 11BAs shown, reduced Capn15 expression levels downregulated Elovl1 expression under both non-responsive (PBS treatment) and responsive conditions (ITC treatment), indicating that Elovl1 is a downstream target of Capn15.

[0293] These results suggest that TMEM164 overexpression, CAPN15 underexpression, or SOX18 overexpression, or their combination, may reduce the neurotoxicity of reactive astrocytes through the TMEM164-CAPN15-ELOVL1 pathway.

[0294] Example 5: TMEM164 overexpression inhibits activation of the neurotoxic response state of human astrocytes

[0295] Because neurotoxic astrocytes are prevalent in multiple human neurodegenerative diseases, we investigated whether TMEM164 overexpression could prevent the activation of the neurotoxic reactive state in human astrocytes.

[0296] Human primary astrocytes were obtained from Hunan Fenghui Biotechnology Co., Ltd. and cultured in human astrocyte culture medium (purchased from Fenghui Biotechnology). They were incubated with ITC (30 ng / ml TNF, 3 ng / ml IL-1α, and 400 ng / ml C1q) and PBS for 24 h, and astrocytes were obtained for staining.

[0297] We performed immunofluorescence co-staining of the neurotoxic state markers C3 and TMEM164 in human astrocytes, and then we used ImageJ to quantitatively analyze the expression levels of C3 and TMEM164 in astrocytes of the PBS group and the ITC group. The results are shown in Fig. 12A and Fig. 12B As shown in the figure, it was found that the expression level of C3 in reactive astrocytes (ITC group) was increased compared with non-reactive astrocytes (PBS group), but the expression level of TMEM164 was decreased, indicating that the expression level of TMEM164 in astrocytes under neurotoxicity was lower. By immunostaining analysis, the changes of TMEM164, ZO1 and α-tubulin in astrocytes of the ITC group over time were detected. The results are shown in Fig. 12C As shown, the results showed that the protein level of TMEM164 decreased over time.

[0298] Human astrocytes were obtained and transfected with EGFP or TMEM164-P2A-EGFP overexpression plasmids for 48 hours using conventional biological methods in the art. EGFP-positive cells were sorted to obtain EGFP-overexpressing astrocytes and human astrocytes co-overexpressing TMEM164 and EGFP. EGFP-overexpressing astrocytes and TMEM164 and EGFP-co-overexpressing astrocytes were plated at a density of 100,000 cells / well in a 24-well plate pre-coated with 10ng / ml poly-D-lysine (Sigma-Aldrich, P7886). After 12 hours, 30ng / ml TNF, 3ng / ml IL-1α and 400ng / ml C1q were added, respectively, and co-cultured for 24 hours to obtain EGFP-ITC astrocytes and TMEM164-ITC astrocytes; EGFP-overexpressing astrocytes were also taken, and PBS was added to the culture medium and co-cultured for 24 hours as a negative control, labeled as EGFP-PBS. The transcription levels of A1 neurotoxicity-specific markers in the three groups of cells, EGFP-PBS, EGFP-ITC, and TMEM164-ITC, were detected. The results are shown in the figure. Fig.12D As shown, TMEM164 overexpression can significantly inhibit the expression of neurotoxic state markers such as C3, GBP2, SERPING1, HAL-E, SRGN, PSMB8, FBLN5 and FKBP5.

[0299] The above results indicate that overexpression of TMEM164 prevents the activation of human neurotoxic reactive astrocytes.

[0300] Example 6 Effects of TMEM164 overexpression on phagocytosis, neurotoxicity, and synaptosome growth ability of human neurotoxic reactive astrocytes

[0301] The phagocytosis of synaptosomes by human EGFP-PBS, EGFP-ITC, and TMEM164-ITC cells was detected. Fig.13A and Fig. 13B , overexpression of TMEM164 in human astrocytes also maintained phagocytic activity under ITC stimulation.

[0302] To investigate the effect of TMEM164 overexpression on the neurotoxic function of human astrocytes, we first differentiated human embryonic stem cells (hESCs) into neurons. TM -E8 TMCulture medium (STEMCELL, #05990) and transformed into neurons by overexpressing two doxycycline-inducible transcription factors NEUROG2 (pCDH-TRE3GV-Neurog2-EF1α-Tet3G-T2A-Puro), ASCL1 (pCDH-TRE3GV-Ascl1-EF1α-Tet3G-T2A-Neo) and TUBB3-driven GFP (pCDH-TUBB3(801)-copGFP) according to a previously published neuronal induction protocol. Human embryonic stem cells were cultured at 80% density in neural growth medium containing 50% DMEM / F12 medium, 50% neurobasal medium, 0.5% N2, 1% B27, 1% GlutaMAX, 1% NEAA and doxycycline (1 μg / ml). The next day, the neural growth medium was replaced with a neuronal medium (STEMCELL, #05795) containing doxycycline (1ug / ml) to allow embryonic stem cells to differentiate into neurons for 2 weeks, with complete replacement of the medium every 3 days. Embryonic stem cell-derived neurons were then treated with conditioned medium collected from EGFP-PBS, EGFP-ITC, human TMEM164-PBS, and human TMEM164-ITC astrocytes for 48 hours to induce cell death. Cell viability was measured by the calcein AM / EthD-1 method. The neurotoxicity of conditioned medium from human astrocytes overexpressing TMEM164 to neurons differentiated from human embryonic stem cells (ESC) was analyzed. The results are shown in Fig. 13C and Fig.13D As shown, the neurotoxicity of human astrocyte ACM overexpressing TMEM164 to neurons differentiated from human embryonic stem cells (ESC) was reduced.

[0303] The effect of human astrocytes expressing TMEM164 on the ability of neurons to grow synapses was analyzed. Fig.13E and Fig.13F TMEM164 overexpression can maintain the ability of astrocytes to promote neuronal synaptic growth, confirming that TMEM164 overexpression can maintain the function of normal astrocytes and inhibit the neurotoxicity of reactive astrocytes under ITC stimulation.

[0304] Example 7 AAV-mediated astrocyte-specific TMEM164 overexpression can prevent dopaminergic neuron death induced by neurotoxic reactive astrocytes in a Parkinson's disease mouse model

[0305] To determine whether TMEM164 overexpression can prevent the activation of neurotoxic reactive astrocytes and subsequent neuronal death in vivo, we examined the function of TMEM164 overexpression in an LPS-induced PD mouse model. LPS has no obvious direct toxic effects on neurons, but strongly activates classical neuroinflammatory microglia and induces the activation of neurotoxic reactive astrocytes.

[0306] In order to express TMEM164 specifically in astrocytes, we packaged AAV overexpressing TMEM164 (AAV-sGFAP-Tmem164-P2A-mCherry-WPRE), in which the expression of TMEM164 was driven by the astrocyte-specific promoter of short glial fibrillary acidic protein (sGFAP). AAV-sGFAP-mCherry-WPRE without TMEM164 was also constructed as a control. The construction diagram is shown in Fig.14 shown.

[0307] We injected AAV-sGFAP-Tmem164-P2A-mCherry-WPRE or AAV-sGFAP-mCherry-WPRE virus into the substantia nigra compacta (SNc) region of the brain of 3-month-old mice. Three weeks later, LPS was injected into the SNc region. One week later, an inflammatory PD model was established. Fig.15A and 15B As shown, almost all mCherry signals co-localized with the astrocyte marker GFAP, but not with the microglia marker Iba1 or the dopaminergic neuron marker tyrosine hydroxylase (TH), indicating that TMEM164 is specifically overexpressed in astrocytes.

[0308] To investigate whether TMEM164 overexpression can suppress the neuronal death phenotype in PD mouse models, we used immunofluorescence staining to count the number of TH-positive dopaminergic neurons in the SNc region. Fig. 15C and Fig.15D As shown, neuroinflammation-induced death of dopaminergic neurons was significantly inhibited in the SNc injected with AAV-sGFAP-Tmem164-P2A-mCherry-WPRE but not in the control group.

[0309] To investigate whether TMEM164 overexpression can improve the behavioral performance of PD mice, we compared the motor ability of mice using the rotarod test. Three-month-old mixed-sex C57BL / 6 mice were deeply anesthetized. Then 1 μl of AAV-PHP.eB-sGFAP-Tmem164-P2A-mCherry-WPRE or AAV-PHP.eB-sGFAP-mCherry-WPRE ( x 10 11 vg / ml) was injected into the SNc region (anterior / posterior: -2.90; medial / lateral: ±1.30; dorsal / ventral: -4.40). Three weeks later, the mice were microinjected with 2μl of LPS at a concentration of 5μg / ul in the right SNc region. The rotarod test was performed two weeks after the LPS injection. For the rotarod test, the control group and LPS-injected mice were trained on the rotarod apparatus for 5 minutes using a fixed speed of 4rpm on the first day, for a total of 4 times. On the second and third days, the mice were trained and tested at a speed accelerating from 4rpm to 40rpm, for a total of 4 times. The time the mouse stayed on the rotarod before falling was recorded as the latency. The average of the three longest latencies recorded on the third day was used for analysis. The results are shown in Fig.15E As shown, injection of AAV-sGFAP-Tmem164-P2A-mCherry-WPRE can improve the behavioral performance of mice in the rotarod test.

[0310] In addition, if Fig.16A , 16B As shown in Figures 16C, C3 signaling was significantly reduced in astrocytes infected with AAV-sGFAP-Tmem164-P2A-mCherry-WPRE, but not in astrocytes infected with control AAV, indicating that AAV-mediated overexpression of TMEM164 in astrocytes can prevent the activation of neurotoxic reactive astrocytes in vivo.

[0311] To exclude the possibility that the difference in C3 expression was caused by microglial activation, we stained Iba1, and the results are shown in Figure 17. No significant difference in microglial activation was observed between the two groups.

[0312] Collectively, these results demonstrate that TMEM164 overexpression prevents LPS-induced activation of reactive astrocytes and neurotoxicity in vivo, confirming the protective role of TMEM164 in models of neurodegenerative diseases.

[0313] Example 8. Whole-brain astrocyte-specific TMEM164 overexpression inhibits activation of neurotoxic reactive astrocytes, neuronal degeneration and memory deficits in 5XFAD Alzheimer's mice

[0314] We studied the inhibitory effect of TMEM164 overexpression on neurotoxic reactive astrocyte activation and neuronal death in 5XFAD mice. 5XFAD mice are a widely used mouse model of Alzheimer's disease. Aged 5XFAD mice are characterized by severe neuroinflammation, neuronal death, and behavioral defects. In order to analyze the whole-brain astrocyte-specific TMEM164 overexpression, the AAV9 variant PHP.eB that can penetrate the blood-brain barrier was used for packaging to obtain AAV-sGFAP-Tmem164-P2A-mCherry-WPRE or AAV-sGFAP-mCherry-WPRE. The obtained AAV was injected through the retro-orbital vein to infect the brains of 3-month-old 5XFAD mice. The results are shown in Fig.18A , 18B As shown in Figure 18C, whole-brain AAV delivery achieved astrocyte-specific infection. Fig. 18C As shown, whole-brain AAV delivery achieved high infection efficiency, with nearly 80% of astrocytes in the cortex and nearly 40% of astrocytes in the hippocampus being infected.

[0315] To determine the therapeutic effect of TMEM164 overexpression, we performed behavioral tests on 5XFAD mice to evaluate the memory performance of 5XFAD mice injected with AAV-sGFAP-Tmem164-P2A-mCherry-WPRE or AAV-sGFAP-mCherry-WPRE. Fig.19A and Fig.19B As shown, we found that 5XFAD mice injected with AAV-sGFAP-Tmem164-P2A-mCherry-WPRE performed better than control mice injected with AAV-sGFAP-mCherry-WPRE in recognition and spatial memory in the novel object recognition test and Y-maze spontaneous alternation test, indicating that overexpression of TMEM164 in whole-brain astrocytes prevented the decline in recognition and spatial memory abilities of 5XFAD mice.

[0316] To investigate the protective mechanism of cognitive performance in 5XFAD mice injected with AAV-sGFAP-Tmem164-P2A-mCherry-WPRE, we performed immunostaining to assess the extent of the neurotoxic response level of astrocytes. Fig. 20A and Fig. 20B As shown, we found that 5XFAD mice injected with AAV-sGFAP-Tmem164-P2A-mCherry-WPRE had reduced activation of neurotoxic reactive astrocytes in the cortex and hippocampus, brain regions associated with higher-order cognitive functions.

[0317] like Fig.21A and 21B As shown, whole-brain astrocyte TMEM164 overexpression also significantly reduced neuronal degeneration in the cortex and hippocampus, as determined by staining for Fluoro-Jade C, a marker for degenerative neurons.

[0318] like Fig. 22 As shown, AAV-mediated overexpression of TMEM164 in astrocytes reduced Aβ deposition in the brains of 9-month-old 5XFAD mice injected with AAV-sGFAP-Tmem164-P2A-mCherry-WPRE for 6 months.

[0319] like Fig.23 As shown, AAV-mediated overexpression of TMEM164 in astrocytes can significantly inhibit neuronal loss in the cortex and subiculum of the hippocampus in the brains of 9-month-old 5XFAD mice.

[0320] In order to study the specificity of AAV-sGFAP-Tmem164-P2A-mCherry-WPRE expression in organs in vivo, we sliced ​​and stained the brain, heart, liver, muscle, lung and kidney of 5XFAD mice injected with the virus, and counted the proportion of cells expressing mCherry fluorescence signals in different tissues. The results are shown in Figure 24, AAV-sGFAP-Tmem164-P2A-mCherry-WPRE is specifically expressed in astrocytes in the brain of 5XFAD mice, and there is little accumulation in peripheral organs such as the heart, liver, muscle, lung and kidney.

[0321] As shown in the results of differentially expressed genes and signaling pathway analysis in Figure 25 , overexpression of TMEM164 in astrocytes in vivo can inhibit the activation of astrocytes and change the function of astrocytes.

[0322] In conclusion, our data show that overexpression of TMEM164 in whole-brain astrocytes can inhibit the formation of neurotoxic reactive astrocytes and its mediated neuronal degeneration and memory decline in 5XFAD mice.

[0323] All documents mentioned in the invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

[0324] The sequence information involved in the present invention is as follows:

[0325]

[0326]

[0327]

Claims

1. A method for regulating neurotoxic reactive astrocytes, characterized in that: include: A TMEM164 modulator that can upregulate the expression level of TMEM164 is administered to an individual in need, or a SOX18 modulator that can upregulate the expression level of SOX18 is administered to an individual in need, or a CAPN15 modulator that can downregulate the expression level of CAPN15 is administered to an individual in need; or a combination of at least two.

2. A method for preventing or treating nerve damage diseases, characterized in that: include: Administering a TMEM164 modulator capable of upregulating the expression level of TMEM164 to an individual in need thereof, or administering a SOX18 modulator capable of upregulating the expression level of SOX18 to an individual in need thereof, or administering a CAPN15 modulator capable of downregulating the expression level of CAPN15 to an individual in need thereof; or administering a combination of at least two thereof; Preferably, the nerve damage disease is a disease related to neuronal death.

3. The method according to claim 1 or 2, characterized in that The TMEM164 regulator can upregulate the expression level of TMEM164 in glial cells, the SOX18 regulator can upregulate the expression level of SOX18 in glial cells; or the CAPN15 regulator can downregulate the expression level of CAPN15 in glial cells; Preferably, the glial cells are from the central nervous system, the visual nervous system, or the auditory nervous system.

4. The method according to claim 3, characterized in that The glial cells include astrocytes, Muller glial cells, or cochlear glial cells (satellite cells); Preferably, the astrocytes include GFAP-positive astrocytes, fibrous astrocytes or protoplasmic astrocytes.

5. The method according to claim 4, characterized in that The astrocytes are non-reactive astrocytes.

6. The method according to claim 1 or 2, characterized in that: The regulator can regulate the activation of non-reactive astrocytes into reactive astrocytes; Preferably, the regulation is to inhibit the transformation of non-reactive astrocytes into neurotoxic reactive astrocytes; and / or to enhance the transformation of non-reactive resting astrocytes into neuroprotective reactive astrocytes.

7. The method according to any one of claims 1 to 6, characterized in that: Cells given any one of the modulators showed reduced expression of at least one marker of neurotoxic reactive astrocytes after administration of the modulator compared to cells not given the modulator; Preferably, the marker is selected from C3, H2-T23, H2-D1, Igp1, Gbp2, Fkbp5, Psmb8, Srgn, Serping1, or Amigo2, or the expression of at least two markers is reduced.

8. The method according to any one of claims 1 to 6, characterized in that: The TMEM164 regulator is an epigenetic regulation tool, a TMEM164 gene expression activator, a TMEM164 protein degradation inhibitor, a TMEM164 mRNA stabilizer, an exogenous TMEM164 or a functional fragment of TMEM164; The Sox18 regulator is an epigenetic regulation tool, a Sox18 gene expression activator, a Sox18 protein degradation inhibitor, a Sox18 mRNA stabilizer, exogenous Sox18 or a functional fragment of Sox18; and / or The CAPN15 regulator is an epigenetic regulation tool, a CAPN15 gene expression inhibitor, a CAPN15 activity inhibitor, a CAPN15 antibody, a CAPN15 protein degradation activator, a gene editing tool targeting CAPN15, PROTAC, ASO, miRNA, RNAi, shRNA, a CAPN15 binding protein or a protein domain.

9. The method according to claim 8, characterized in that The exogenous TMEM164 or functional fragment of TMEM164 is a polypeptide of TMEM164 or a biologically active fragment thereof, or a nucleic acid encoding a polypeptide of TMEM164 or a biologically active fragment thereof; The exogenous Sox18 or the functional fragment of Sox18 is a Sox18 polypeptide or a biologically active fragment thereof, or a nucleic acid encoding a Sox18 polypeptide or a biologically active fragment thereof; TMEM164 is preferably human TMEM164, and Sox18 is preferably human Sox18.

10. The method according to claim 8, characterized in that The exogenous TMEM164 or functional fragment of TMEM164 is a mutant of the TMEM164 polypeptide or its biologically active fragment that retains the function of TMEM164; preferably, the mutant has at least 80%, 85%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% homology with the TMEM164 polypeptide or its biologically active fragment; or The exogenous Sox18 or functional fragment of Sox18 is a mutant of the Sox18 polypeptide or its biologically active fragment that retains the Sox18 function; preferably, the mutant has at least 80%, 85%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% homology with the Sox18 polypeptide or its biologically active fragment.

11. The method according to claim 9 or 10, characterized in that The TMEM164 polypeptide contains the sequence shown in SEQ ID NO: 31, 32, 33, 34, 35, 36, 37, or 38; or contains a sequence having at least 80%, 85%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% homology to the sequence shown in SEQ ID NO: 31, 32, 33, 34, 35, 36, 37, or 38.

12. The method according to claim 8, characterized in that The regulator is a small molecule compound, an antibody, a protein, a polypeptide, a gene fragment, or a polysaccharide.

13. The method according to claim 8, characterized in that The TMEM164 gene expression activator is a modifier of endogenous TMEM164, an agonist that enhances the expression of the TMEM164 gene or protein, or an inhibitor of a TMEM164 competitive gene; The Sox18 gene expression activator is a modifier of endogenous Sox18, an agonist that enhances Sox18 gene or protein expression, or an inhibitor of Sox18 competitive gene; or The CAPN15 gene expression inhibitor is a modifier of endogenous CAPN15, an inhibitor that reduces the expression of CAPN15 gene or protein, and an agonist of CAPN15 competitive gene.

14. The method according to claim 13, characterized in that The enhancer for enhancing the expression of TMEM164 gene or protein is selected from: an agonist of a positive regulatory factor that enhances the expression of TMEM164, an inhibitor of a negative regulatory factor that reduces the expression of TMEM164, and an agonist that increases the expression of TMEM164 protein; The enhancer for enhancing the expression of Sox18 gene or protein is selected from: an agonist of a positive regulatory factor that enhances Sox18 expression, an inhibitor of a negative regulatory factor that reduces Sox18 expression, and an agonist that increases Sox18 protein expression; or The inhibitor for reducing CAPN15 gene or protein expression is selected from: an inhibitor of a positive regulatory factor that reduces CAPN15 expression, an agonist of a negative regulatory factor that enhances CAPN15 expression, and an inhibitor for reducing Sox18 protein expression.

15. The method according to claim 13, characterized in that The inhibitor of the TMEM164 competitive gene is a small molecule inhibitor, a ligand of the TMEM164 competitive gene, a gene silencing tool, or an antibody of the TMEM164 competitive gene; The inhibitor of the Sox18 competitive gene is a small molecule inhibitor, a ligand of the Sox18 competitive gene, a gene silencing tool, or an antibody of the Sox18 competitive gene; The agonist of CAPN15 competitive gene is a small molecule agonist, a ligand of Sox18 competitive gene, or an antibody of Sox18 competitive gene.

16. The method according to claim 2, characterized in that The nerve injury disease is brain nerve injury, spinal cord injury, or optic nerve injury.

17. The method according to claim 16, characterized in that The diseases causing brain nerve damage include: brain trauma, stroke, cerebral ischemia, physical injury, tumor, inflammation, infection, ischemia caused by cardiac arrest or severe hypotension (shock), hypoxic-ischemic encephalopathy caused by lack of oxygen, hypoglycemia or anemia, meningitis, dehydration, subarachnoid hemorrhage, intracranial hematoma, weak cerebral blood vessels, cerebral vascular malformation, hypertension, or neurodegenerative diseases, or a combination of at least two diseases.

18. The method according to claim 17, characterized in that The diseases of brain nerve damage include Alzheimer's disease (AD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), multiple sclerosis (MS), or frontotemporal dementia (FTLD).

19. The method according to claim 16, characterized in that The diseases causing optic nerve damage include glaucoma, retinal detachment, optic nerve inflammation, vision damage caused by RGC cell death, photoreceptor cell damage, age-related macular degeneration (AMD), diabetic retinopathy, retinal ischemia or hemorrhage, Leber hereditary optic neuropathy, or ocular trauma, or a combination of at least two diseases.

20. The method according to any one of claims 1 to 19, characterized in that: The modulator is administered to a subject in need thereof via a carrier.

21. The method of claim 20, wherein: The carrier is selected from: viral vectors, lipid nanoparticles (LNP), liposomes, cationic polymers (such as PEI), nanoparticles, exosomes; more preferably, the carrier is an AAV vector or lipid nanoparticles, wherein AAV vectors or lipid nanoparticles are preferred.

22. The method according to claim 21, characterized in that The vector also includes a regulatory sequence for regulating the expression of the coding sequence; Preferably, the regulatory sequence includes expression elements including promoter, intron, Poly (A) sequence, WPRE and the like.

23. The method of claim 3, wherein: The astrocytes are from the retina, vitreous cavity, striatum, substantia nigra, inner ear, spinal cord, prefrontal cortex, motor cortex, thalamus, ventral tegmental area (VTA), hippocampus, cerebellum, brain stem, or inner ear cochlea or vestibule. More preferably, the astrocytes are from the striatum, substantia nigra, retina, or vitreous cavity.

24. The method according to any one of claims 1 to 23, characterized in that: The regulator is administered to an individual in need by direct targeted administration to the affected site of a nerve damage disease.

25. The method according to any one of claims 1 to 24, characterized in that: The modulator is administered to a subject in need thereof by cell transfection, cell infection, cell endocytosis, injection, intracranial administration, spinal administration, inhalation, parenteral administration, intravenous administration, intramuscular administration, intradermal administration, topical administration, local administration, targeted administration, subretinal injection, intravitreal injection, or oral administration.

26. The method of claim 25, wherein: The nerve injury disease is a central nervous system injury disease, and the regulator is injected into the striatum, hippocampus or substantia nigra.

27. The method according to claims 1 to 26, characterized in that The modulator is capable of increasing the proportion of neuroprotective astrocytes.

28. The method according to claim 1 or 2, characterized in that The TMEM164 modulator is co-administered with the SOX18 modulator, or the TMEM164 modulator is co-administered with the CAPN15 modulator.

29. Use of a TMEM164 modulator that upregulates the expression level of TMEM164, a SOX18 modulator that upregulates the expression level of SOX18, or a CAPN15 modulator that downregulates the expression level of CAPN15 for preparing a drug for preventing, delaying, or treating diseases related to nerve damage or neuronal aging.

30. Use of an AAV vector carrying a TMEM164 modulator that upregulates the expression level of TMEM164, a SOX18 modulator that upregulates the expression level of SOX18, or a CAPN15 modulator that downregulates the expression level of CAPN15 for preparing a drug for preventing, delaying, or treating diseases related to neural injury or neuronal aging, or alleviating the symptoms of neural injury or neuronal aging.

31. The use according to claim 29 or 30, characterized in that The neurological injury disease is brain injury.

32. The use according to claim 31, characterized in that The brain damage includes Alzheimer's disease (AD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), multiple sclerosis (MS), and FTLD.

33. Use of a TMEM164 modulator that upregulates the expression level of TMEM164, a SOX18 modulator that upregulates the expression level of SOX18, or a CAPN15 modulator that downregulates the expression level of CAPN15 for preparing a neuroprotective agent.

34. A method for regulating the CAPN15-ELOVL1 pathway using a TMEM164 modulator that upregulates the expression level of TMEM164, a SOX18 modulator that upregulates the expression level of SOX18, or a CAPN15 modulator that downregulates the expression level of CAPN15; preferably, the regulation is to reduce the expression level of the neurotoxic saturated lipid synthase Elovl1.

35. Use of a TMEM164 modulator that upregulates the expression level of TMEM164, a SOX18 modulator that upregulates the expression level of SOX18, or a CAPN15 modulator that downregulates the expression level of CAPN15, for downregulating the expression of CAPN15, Kidinis220, Zfp1, or Zfp626.

36. The use according to claim 34, characterized in that A TMEM164 modulator that upregulates the expression level of TMEM164 can downregulate the expression of CAPN15.

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