WO2023125744A1 - 质子泵调控剂在制备试剂中的用途 - Google Patents

质子泵调控剂在制备试剂中的用途 Download PDF

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WO2023125744A1
WO2023125744A1 PCT/CN2022/143119 CN2022143119W WO2023125744A1 WO 2023125744 A1 WO2023125744 A1 WO 2023125744A1 CN 2022143119 W CN2022143119 W CN 2022143119W WO 2023125744 A1 WO2023125744 A1 WO 2023125744A1
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atp6v1b2
use according
expression level
disease
subject
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PCT/CN2022/143119
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English (en)
French (fr)
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李开诚
李震
攸璞
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上海魁特迪生物科技有限公司
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Publication of WO2023125744A1 publication Critical patent/WO2023125744A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity

Definitions

  • the present application relates to the field of biomedicine, in particular to the use of a proton pump regulator in the preparation of reagents.
  • AD Alzheimer's disease
  • the ATP6V1B2 protein encoded by the ATP6V1B2 gene is an important structural protein of the proton pump driven by ATP hydrolysis. It is widely distributed in human tissues, but it is more abundant in brain tissues, kidneys, osteoclasts and other parts. It plays an important role in processes such as synaptic transmission and lysosome acidification. Mutations in its gene can cause DOORS syndrome (autosomal dominant congenital deafness of susceptibility with onychodysplasia syndrome). The decreased expression of ATP6V1B2 protein may be related to the pathogenesis of AD.
  • the present application provides a method for improving the subject's learning ability.
  • the present application discovers that ATP6V1B2 is a potential target for improving the subject's learning ability, and then discovers a reagent and/or that can increase the expression level and/or activity of ATP6V1B2.
  • Proton pump modulators can be used as potential drugs to improve the learning ability of subjects.
  • the present application provides a use of a proton pump modulator in the preparation of a reagent for improving learning ability.
  • the present application provides a use of a proton pump regulator in the preparation of a reagent for treating cognitive impairment.
  • the present application provides a use of a proton pump regulator in the preparation of a reagent for treating neurodegenerative diseases.
  • the agent increases the expression level and/or activity of a proton pump-related protein in the subject.
  • the proton pump modulator increases the expression level and/or activity of a proton pump-related protein in a subject.
  • the expression level of the proton pump-related protein comprises the expression level of the gene encoding the proton pump-related protein, the transcription level of the gene encoding the proton pump-related protein and/or the expression level of the proton pump-related protein.
  • the increase comprises an increase in the expression level and/or activity of the proton pump-related protein by at least about 10 compared to the expression level and/or activity of the original proton pump-related protein in the subject. %.
  • the expression level of said proton pump-associated protein is measured by performing an assay selected from the group consisting of qPCR, qRT-PCR, hybridization analysis, Northern blot, dot blot, in situ hybridization, gel electrophoresis , capillary electrophoresis, column chromatography, western blotting, immunohistochemistry, immunostaining, and mass spectrometry.
  • an assay selected from the group consisting of qPCR, qRT-PCR, hybridization analysis, Northern blot, dot blot, in situ hybridization, gel electrophoresis , capillary electrophoresis, column chromatography, western blotting, immunohistochemistry, immunostaining, and mass spectrometry.
  • the expression level of the proton pump-related protein is measured by using a substance selected from the group consisting of: a primer capable of specifically amplifying a gene encoding a proton pump-related protein, a primer that specifically binds to a gene encoding a proton pump-related protein Nucleic acid molecules specifically binding to proton pump-related proteins, nucleic acid molecules specifically binding to proton pump-related proteins, small molecules specifically binding to proton pump-related proteins, probes specifically binding to proton pump-related proteins, and polypeptides specifically binding to proton pump-related proteins .
  • the agent increases the expression level and/or activity of ATP6V1B2 in the subject.
  • the proton pump modulator increases the expression level and/or activity of ATP6V1B2 in the subject.
  • the expression level of ATP6V1B2 includes the expression level of ATP6V1B2 gene, the transcription level of ATP6V1B2 gene and/or the expression level of ATP6V1B2 protein.
  • said increasing comprises an increase in the expression level and/or activity of said ATP6V1B2 of at least about 10% compared to the expression level and/or activity of native ATP6V1B2 in said subject.
  • the expression level of ATP6V1B2 is measured by performing an assay selected from the group consisting of qPCR, qRT-PCR, hybridization analysis, Northern blot, dot blot, in situ hybridization, gel electrophoresis, capillary electrophoresis , column chromatography, western blotting, immunohistochemistry, immunostaining, and mass spectrometry.
  • an assay selected from the group consisting of qPCR, qRT-PCR, hybridization analysis, Northern blot, dot blot, in situ hybridization, gel electrophoresis, capillary electrophoresis , column chromatography, western blotting, immunohistochemistry, immunostaining, and mass spectrometry.
  • the expression level of ATP6V1B2 is measured by using a substance selected from the group consisting of primers capable of specifically amplifying the ATP6V1B2 gene, nucleic acid molecules specifically binding to the ATP6V1B2 gene, and ATP6V1B2 protein specifically binding Nucleic acid molecule, small molecule specifically binding to ATP6V1B2 protein, probe specifically binding to ATP6V1B2 protein and polypeptide specifically binding to ATP6V1B2 protein.
  • the proton pump modulator comprises a proton pump agonist.
  • the proton pump modulator comprises a vesicular proton pump agonist.
  • the proton pump modulator comprises an agonist of ATP6V1B2 protein.
  • the proton pump modulator is capable of binding an ATP6V1B2 protein.
  • the proton pump modulator can bind to at least part of the amino acid sequence from position 288 to position 512 of mouse ATP6V1B2 protein.
  • the proton pump modulator can bind to at least part of the amino acid sequence from position 288 to position 512 of human ATP6V1B2 protein.
  • the proton pump modulator comprises a nucleic acid or variant thereof, a polypeptide or variant thereof, and/or a small molecule.
  • the proton pump modulator is capable of increasing neuronal synaptic transmitter release.
  • the increase comprises an increase of at least about 10% compared to the level of primitive neuronal synaptic transmitter release in the subject.
  • the proton pump modulator is capable of increasing the firing frequency of excitatory postsynaptic currents.
  • said increase comprises an increase of at least about 10% compared to the level of firing frequency of primordial excitatory postsynaptic currents in said subject.
  • the learning ability comprises cognitive ability, motor ability, memory ability and/or spatial exploration ability.
  • the improvement in learning ability comprises an improvement in the subject's assessment of learning ability by at least about 50% compared to the subject's original assessment of learning ability.
  • said assessment score of learning ability is measured by performing a test selected from the group consisting of a novel object recognition test and a water maze test.
  • said novel object recognition test assesses said cognitive ability, motor ability and/or spatial exploration ability.
  • said water maze test assesses said memory ability, motor ability and/or spatial exploration ability.
  • the subject includes a mammal.
  • the subject comprises a human.
  • the subject includes a non-cognitively impaired patient and/or a non-neurodegenerative disease patient.
  • the subject comprises a neurodegenerative disease patient and/or a cognitively impaired patient.
  • the subject includes a patient with Alzheimer's disease.
  • the subject is aged.
  • the cognitive impairment comprises early cognitive impairment (MCI), intermediate cognitive impairment and late cognitive impairment.
  • the cognitive impairment comprises cognitive impairment due to normal aging, lews body dementia (LBD), frontotemporal dementia, and/or vascular dementia.
  • LBD lews body dementia
  • frontotemporal dementia frontotemporal dementia
  • vascular dementia vascular dementia
  • the inducing disease of cognitive impairment includes Alzheimer's disease, multiple infarct type, Parkinson's disease, AIDS and/or Creutzfeldt-Jakob disease (CJD).
  • the neurodegenerative disease includes acute neurodegenerative disease and chronic neurodegenerative disease.
  • the neurodegenerative disease includes neurodegenerative disease caused by neuronal death and glial cell homeostasis, neurodegenerative disease caused by aging, neurodegenerative disease caused by affected CNS cell function diseases, neurodegenerative diseases caused by abnormal communication between cells and/or neurodegenerative diseases caused by impaired cell motility.
  • the neurodegenerative disease comprises Alzheimer's disease, Parkinson's disease, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS) and/or Huntington's disease (HD ).
  • the agent is formulated for oral administration and/or injectable administration.
  • the agent is formulated for intravenous injection.
  • the present application provides a use of ATP6V1B2, its functionally active fragments or nucleic acid molecules encoding them in improving learning ability.
  • the present application provides a use of ATP6V1B2, its functionally active fragments or nucleic acid molecules encoding them in the treatment of cognitive disorders.
  • the present application provides a use of ATP6V1B2, its functionally active fragments or nucleic acid molecules encoding them in the treatment of neurodegenerative diseases.
  • the ATP6V1B2 is of human origin.
  • the ATP6V1B2 comprises the amino acid sequence shown in SEQ ID NO.8 or 16.
  • the functionally active fragment of ATP6V1B2 has the ability to specifically bind to the amino acid sequence shown in SEQ ID NO.5.
  • the functionally active fragment of ATP6V1B2 comprises a truncation of ATP6V1B2.
  • the functionally active fragment of ATP6V1B2 comprises at least part of the amino acid sequence from position 288 to position 512 of human ATP6V1B2 protein.
  • the functionally active fragment of ATP6V1B2 comprises at least part of the amino acid sequence from position 288 to position 512 of mouse ATP6V1B2 protein.
  • the functionally active fragment of ATP6V1B2 comprises the amino acid sequence shown in any one of SEQ ID NO.10-11.
  • the nucleic acid molecule comprises the nucleotide sequence shown in any one of SEQ ID NO.9,17.
  • the learning ability comprises cognitive ability, motor ability, memory ability and/or spatial exploration ability.
  • the improvement of learning ability comprises an increase of at least about 50% in the subject's assessment of learning ability after improvement compared to the subject's original assessment of learning ability.
  • said assessment score of learning ability is measured by performing a test selected from the group consisting of a novel object recognition test and a water maze test.
  • said novel object recognition test assesses said cognitive ability, motor ability and/or spatial exploration ability.
  • said water maze test assesses said memory ability, motor ability and/or spatial exploration ability.
  • the subject includes a mammal.
  • the subject comprises a human.
  • the subject includes a non-cognitively impaired patient and/or a non-neurodegenerative disease patient.
  • the subject comprises a neurodegenerative disease patient and/or a cognitively impaired patient.
  • the subject includes a patient with Alzheimer's disease.
  • the subject is aged.
  • the cognitive impairment comprises early cognitive impairment (MCI), intermediate cognitive impairment and late cognitive impairment.
  • the cognitive impairment comprises cognitive impairment due to normal aging, lews body dementia (LBD), frontotemporal dementia, and/or vascular dementia.
  • LBD lews body dementia
  • frontotemporal dementia frontotemporal dementia
  • vascular dementia vascular dementia
  • the inducing disease of cognitive impairment includes Alzheimer's disease, multiple infarct type, Parkinson's disease, AIDS and/or Creutzfeldt-Jakob disease (CJD).
  • the neurodegenerative disease includes acute neurodegenerative disease and chronic neurodegenerative disease.
  • the neurodegenerative disease includes neurodegenerative disease caused by neuronal death and glial cell homeostasis, neurodegenerative disease caused by aging, neurodegenerative disease caused by affected CNS cell function diseases, neurodegenerative diseases caused by abnormal communication between cells and/or neurodegenerative diseases caused by impaired cell motility.
  • the neurodegenerative disease comprises Alzheimer's disease, Parkinson's disease, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS) and/or Huntington's disease (HD ).
  • the present application provides a use of an ATP6V1B2 modulator in the preparation of a reagent for improving learning ability.
  • the present application provides a use of an ATP6V1B2 regulator in the preparation of a reagent for treating cognitive impairment.
  • the present application provides a use of an ATP6V1B2 regulator in the preparation of a reagent for treating neurodegenerative diseases.
  • the agent increases the expression level and/or activity of ATP6V1B2 in the subject.
  • the ATP6V1B2 modulator increases the expression level and/or activity of ATP6V1B2 in the subject.
  • the expression level includes the expression level of ATP6V1B2 gene, the transcription level of ATP6V1B2 gene and/or the expression level of ATP6V1B2 protein.
  • the activity increase includes increasing the expression level and/or activity of a proton pump-related protein.
  • said increasing comprises an increase in the expression level and/or activity of said ATP6V1B2 of at least about 10% compared to the expression level and/or activity of native ATP6V1B2 in said subject.
  • the expression level of ATP6V1B2 is measured by performing an assay selected from the group consisting of qPCR, qRT-PCR, hybridization analysis, Northern blot, dot blot, in situ hybridization, gel electrophoresis, capillary electrophoresis , column chromatography, western blotting, immunohistochemistry, immunostaining, and mass spectrometry.
  • an assay selected from the group consisting of qPCR, qRT-PCR, hybridization analysis, Northern blot, dot blot, in situ hybridization, gel electrophoresis, capillary electrophoresis , column chromatography, western blotting, immunohistochemistry, immunostaining, and mass spectrometry.
  • the expression level of ATP6V1B2 is measured by using a substance selected from the group consisting of primers capable of specifically amplifying the ATP6V1B2 gene, nucleic acid molecules specifically binding to the ATP6V1B2 gene, and ATP6V1B2 protein specifically binding Nucleic acid molecule, small molecule specifically binding to ATP6V1B2 protein, probe specifically binding to ATP6V1B2 protein and polypeptide specifically binding to ATP6V1B2 protein.
  • the modulator of ATP6V1B2 comprises an agonist of ATP6V1B2 protein.
  • the ATP6V1B2 modulator is capable of binding an ATP6V1B2 protein.
  • the ATP6V1B2 modulator can bind to at least part of the amino acid sequence from position 288 to position 512 of mouse ATP6V1B2 protein.
  • the ATP6V1B2 modulator can bind to at least part of the amino acid sequence from position 288 to position 512 of human ATP6V1B2 protein.
  • the modulator of ATP6V1B2 comprises a proton pump agonist.
  • the modulator of ATP6V1B2 comprises a vesicular proton pump agonist.
  • the ATP6V1B2 modulator includes a nucleic acid or variant thereof, a polypeptide or variant thereof, and/or a small molecule.
  • the modulator of ATP6V1B2 comprises a polypeptide or variant thereof.
  • the polypeptide or variant thereof is specifically capable of binding to an ATP6V1B2 protein.
  • the polypeptide or variant thereof is capable of increasing neuronal synaptic transmitter release.
  • the increase comprises an increase of at least about 10% compared to the level of primitive neuronal synaptic transmitter release in the subject.
  • the polypeptide or variant thereof is capable of increasing the firing frequency of excitatory postsynaptic currents.
  • said increase comprises an increase of at least about 10% compared to the level of firing frequency of primordial excitatory postsynaptic currents in said subject.
  • polypeptide or variant thereof comprises the amino acid sequence shown in SEQ ID NO.5.
  • polypeptide or variant thereof comprises the amino acid sequence shown in any one of SEQ ID NO.1,3.
  • the learning ability comprises cognitive ability, motor ability, memory ability and/or spatial exploration ability.
  • the improvement in learning ability comprises an improvement in the subject's assessment of learning ability by at least about 50% compared to the subject's original assessment of learning ability.
  • said assessment score of learning ability is measured by performing a test selected from the group consisting of a novel object recognition test and a water maze test.
  • said novel object recognition test assesses said cognitive ability, motor ability and/or spatial exploration ability.
  • said water maze test assesses said memory ability, motor ability and/or spatial exploration ability.
  • the subject includes a mammal.
  • the subject comprises a human.
  • the subject includes a non-cognitively impaired patient and/or a non-neurodegenerative disease patient.
  • the subject comprises a neurodegenerative disease patient and/or a cognitively impaired patient.
  • the subject includes a patient with Alzheimer's disease.
  • the subject is aged.
  • the cognitive impairment comprises early cognitive impairment (MCI), intermediate cognitive impairment and late cognitive impairment.
  • the cognitive impairment comprises cognitive impairment due to normal aging, lews body dementia (LBD), frontotemporal dementia, and/or vascular dementia.
  • LBD lews body dementia
  • frontotemporal dementia frontotemporal dementia
  • vascular dementia vascular dementia
  • the inducing disease of cognitive impairment includes Alzheimer's disease, multiple infarct type, Parkinson's disease, AIDS and/or Creutzfeldt-Jakob disease (CJD).
  • the neurodegenerative disease includes acute neurodegenerative disease and chronic neurodegenerative disease.
  • the neurodegenerative disease includes neurodegenerative disease caused by neuronal death and glial cell homeostasis, neurodegenerative disease caused by aging, neurodegenerative disease caused by affected CNS cell function diseases, neurodegenerative diseases caused by abnormal communication between cells and/or neurodegenerative diseases caused by impaired cell motility.
  • the neurodegenerative disease comprises Alzheimer's disease, Parkinson's disease, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS) and/or Huntington's disease (HD ).
  • the agent is formulated for oral administration and/or injectable administration.
  • the agent is formulated for intravenous injection.
  • the present application provides a method for screening drugs that can improve the learning ability of the subject, treat cognitive impairment, and/or treat neurodegenerative diseases, which includes the following steps: detecting the effect of the candidate drug on the subject Influence on the expression level and/or activity of proton pump-related protein, wherein after administration of the candidate drug, the expression level and/or activity of the proton pump-related protein is increased, then the candidate drug can improve the subject's study ability, treatment of cognitive impairment, and/or treatment of neurodegenerative diseases.
  • the present application provides a method for screening drugs that can improve the learning ability of the subject, treat cognitive impairment, and/or treat neurodegenerative diseases, which includes the following steps: detecting the effect of the candidate drug on the subject Influence on the expression level and/or activity of ATP6V1B2, wherein after administration of the candidate drug, the expression level and/or activity of the ATP6V1B2 is increased, then the candidate drug can improve the subject's learning ability and treat cognitive impairment , and/or treat neurodegenerative diseases.
  • the expression level of the proton pump-related protein includes the expression level of the gene encoding the proton pump-related protein, the transcription level of the gene encoding the proton pump-related protein, and/or the expression level of the proton pump-related protein.
  • the increase comprises an increase in the expression level and/or activity of the proton pump-related protein by at least about 10 compared to the expression level and/or activity of the original proton pump-related protein in the subject. %.
  • the expression level of said proton pump-associated protein is measured by performing an assay selected from the group consisting of qPCR, qRT-PCR, hybridization analysis, Northern blot, dot blot, in situ hybridization, gel electrophoresis , capillary electrophoresis, column chromatography, western blotting, immunohistochemistry, immunostaining, and mass spectrometry.
  • an assay selected from the group consisting of qPCR, qRT-PCR, hybridization analysis, Northern blot, dot blot, in situ hybridization, gel electrophoresis , capillary electrophoresis, column chromatography, western blotting, immunohistochemistry, immunostaining, and mass spectrometry.
  • the expression level of the proton pump-related protein is measured by using a substance selected from the group consisting of: a primer capable of specifically amplifying a gene encoding a proton pump-related protein, a primer that specifically binds to a gene encoding a proton pump-related protein Nucleic acid molecules specifically binding to proton pump-related proteins, nucleic acid molecules specifically binding to proton pump-related proteins, small molecules specifically binding to proton pump-related proteins, probes specifically binding to proton pump-related proteins, and polypeptides specifically binding to proton pump-related proteins .
  • the expression level of ATP6V1B2 includes the expression level of ATP6V1B2 gene, the transcription level of ATP6V1B2 gene and/or the expression level of ATP6V1B2 protein.
  • said increasing comprises an increase in the expression level and/or activity of said ATP6V1B2 by at least about 10% compared to the original ATP6V1B2 expression level and/or activity in said subject.
  • the expression level of ATP6V1B2 is measured by performing an assay selected from the group consisting of qPCR, qRT-PCR, hybridization analysis, Northern blot, dot blot, in situ hybridization, gel electrophoresis, capillary electrophoresis , column chromatography, western blotting, immunohistochemistry, immunostaining, and mass spectrometry.
  • an assay selected from the group consisting of qPCR, qRT-PCR, hybridization analysis, Northern blot, dot blot, in situ hybridization, gel electrophoresis, capillary electrophoresis , column chromatography, western blotting, immunohistochemistry, immunostaining, and mass spectrometry.
  • the expression level of ATP6V1B2 is measured by using a substance selected from the group consisting of primers capable of specifically amplifying the ATP6V1B2 gene, nucleic acid molecules specifically binding to the ATP6V1B2 gene, and ATP6V1B2 protein specifically binding Nucleic acid molecule, small molecule specifically binding to ATP6V1B2 protein, probe specifically binding to ATP6V1B2 protein and polypeptide specifically binding to ATP6V1B2 protein.
  • the learning ability comprises cognitive ability, motor ability, memory ability and/or spatial exploration ability.
  • the administering comprises oral and/or injection.
  • the administering comprises intravenous injection.
  • the present application provides a polypeptide or a variant thereof capable of regulating the expression level and/or activity of ATP6V1B2.
  • the polypeptide or variant thereof is capable of increasing neuronal synaptic transmitter release.
  • the increase comprises an increase of at least about 10% compared to the level of primitive neuronal synaptic transmitter release in the subject.
  • the polypeptide or variant thereof is capable of increasing the firing frequency of excitatory postsynaptic currents.
  • said increase comprises an increase of at least about 10% compared to the level of firing frequency of primordial excitatory postsynaptic currents in said subject.
  • the polypeptide or variant thereof is capable of binding an ATP6V1B2 protein.
  • the polypeptide or variant thereof is capable of increasing the expression level and/or activity of ATP6V1B2 in a subject.
  • the expression level of ATP6V1B2 includes the expression level of ATP6V1B2 gene, the transcription level of ATP6V1B2 gene and/or the expression level of ATP6V1B2 protein.
  • said increasing comprises an increase in the expression level and/or activity of said ATP6V1B2 of at least about 10% compared to the expression level and/or activity of native ATP6V1B2 in said subject.
  • the polypeptide or its variant can bind to at least part of the amino acid sequence from position 288 to position 512 of mouse ATP6V1B2 protein.
  • the polypeptide or variant thereof can bind to at least part of the amino acid sequence from position 288 to position 512 of human ATP6V1B2 protein.
  • polypeptide or variant thereof comprises the amino acid sequence shown in SEQ ID NO.5.
  • polypeptide or variant thereof comprises the amino acid sequence shown in any one of SEQ ID NO.1,3.
  • the polypeptide or variant thereof comprises a multimer.
  • the multimer comprises a homodimer.
  • cysteines in the amino acid sequence of the polypeptide or variant thereof do not have a sulfhydryl blocking modification.
  • the serine in the amino acid sequence of the polypeptide or variant thereof does not have a phosphorylated modification.
  • the present application provides a pharmaceutical combination comprising the reagent described in the present application and/or comprising the polypeptide described in the present application or a variant thereof.
  • the present application provides a pharmaceutical composition, which comprises the reagent described in the present application and/or, which comprises the polypeptide described in the present application or its variant, and a pharmaceutically acceptable carrier.
  • the present application provides a polypeptide described in the present application or its variant, the reagent described in the present application, the pharmaceutical combination described in the present application and/or the pharmaceutical composition described in the present application in the preparation of the reagent Use, the agent is used for improving learning ability, treating cognitive impairment and/or treating neurodegenerative diseases.
  • the learning ability comprises cognitive ability, motor ability, memory ability and/or spatial exploration ability.
  • the cognitive impairment comprises early cognitive impairment (MCI), intermediate cognitive impairment and late cognitive impairment.
  • the cognitive impairment comprises cognitive impairment due to normal aging, lews body dementia (LBD), frontotemporal dementia, and/or vascular dementia.
  • LBD lews body dementia
  • frontotemporal dementia frontotemporal dementia
  • vascular dementia vascular dementia
  • the inducing disease of cognitive impairment includes Alzheimer's disease, multiple infarct type, Parkinson's disease, AIDS and/or Creutzfeldt-Jakob disease (CJD).
  • the neurodegenerative disease includes acute neurodegenerative disease and chronic neurodegenerative disease.
  • the neurodegenerative disease includes neurodegenerative disease caused by neuronal death and glial cell homeostasis, neurodegenerative disease caused by aging, neurodegenerative disease caused by affected CNS cell function diseases, neurodegenerative diseases caused by abnormal communication between cells and/or neurodegenerative diseases caused by impaired cell motility.
  • the neurodegenerative disease comprises Alzheimer's disease, Parkinson's disease, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS) and/or Huntington's disease (HD ).
  • said polypeptide, said pharmaceutical combination and/or said pharmaceutical composition are formulated for oral administration and/or injection administration.
  • said polypeptide, said pharmaceutical combination and/or said pharmaceutical composition are formulated for intravenous injection.
  • the present application provides a non-human animal or part thereof comprising and/or expressing ATP6V1B2, functionally active fragments thereof and/or nucleic acid molecules encoding them.
  • the ATP6V1B2 is of human origin.
  • the ATP6V1B2 comprises the amino acid sequence shown in SEQ ID NO.8 or 16.
  • the functionally active fragment of ATP6V1B2 has the ability to specifically bind to the amino acid sequence shown in SEQ ID NO.5.
  • the functionally active fragment of ATP6V1B2 comprises a truncation of ATP6V1B2.
  • the functionally active fragment of ATP6V1B2 comprises at least part of the amino acid sequence from position 288 to position 512 of human ATP6V1B2 protein.
  • the functionally active fragment of ATP6V1B2 comprises at least part of the amino acid sequence from position 288 to position 512 of mouse ATP6V1B2 protein.
  • the functionally active fragment of ATP6V1B2 comprises the amino acid sequence shown in any one of SEQ ID NO.10-11.
  • the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO.9 or 17.
  • the non-human animal is, or is derived from, a gene-edited non-human animal.
  • the non-human animal is, or is derived from, a knock-in non-human animal.
  • the expression of the ATP6V1B2 and/or its functionally active fragments is regulated by the corresponding endogenous regulatory elements in the genome of the non-human animal.
  • the non-human animal is a non-human mammal.
  • the non-human animal is selected from the group consisting of mice, rats, rabbits, apes, monkeys, pigs, cows, sheep, horses, chickens, dogs, alpacas, and cats.
  • the non-human animal is a rodent or a primate.
  • said portion comprises an organ, tissue and/or cell of said non-human animal.
  • the portion includes bodily fluids.
  • the bodily fluid is selected from the group consisting of blood, plasma, serum, urine, sweat, tears, saliva, semen, and cerebrospinal fluid.
  • the plasma includes exosomes.
  • the portion comprises the brain or a portion thereof.
  • the portion of the brain comprises a brain region selected from the group consisting of olfactory bulb, amygdala, basal ganglia, hippocampus, thalamus, hypothalamus, cerebral cortex, medulla oblongata, and cerebellum.
  • the portion includes the olfactory mucosa.
  • the portion includes a portion of the central nervous system, a portion of the peripheral nervous system, skin tissue, muscle tissue, and/or an internal organ.
  • the central nervous system includes the spinal cord.
  • the portion comprises cells, and the cells comprise cells selected from the group consisting of primary neurons, microglia, astrocytes, oligodendrocytes, macrophages , perivascular epithelioid cells, B cells, T cells, adult stem cells, NK cells, totipotent stem cells, unipotent stem cells, embryonic stem cells, induced pluripotent stem cells, and gametes.
  • the gametes include sperm and/or oocytes.
  • the portion cannot develop into a complete non-human animal.
  • the offspring are obtained by crossing said non-human animal with another non-human animal, wherein said other non-human animal may or may not express said ATP6V1B2 and/or a functionally active fragment thereof.
  • said progeny or part thereof comprises and/or expresses said ATP6V1B2 and/or a functionally active fragment thereof.
  • the present application provides an isolated cell comprising and/or expressing ATP6V1B2, functionally active fragments thereof and/or nucleic acid molecules encoding them.
  • the ATP6V1B2 is of human origin.
  • the ATP6V1B2 and/or its functionally active fragments comprise the amino acid sequence shown in any one of SEQ ID NO:8, 10-11, and 16.
  • the gene encoding said ATP6V1B2 and/or a functionally active fragment thereof is homozygous or heterozygous.
  • the nucleic acid molecule encoding the ATP6V1B2 and/or its functionally active fragment comprises the nucleic acid sequence shown in SEQ ID NO:9 or 17.
  • the expression of the ATP6V1B2 and/or its functionally active fragments is regulated by the corresponding endogenous regulatory elements in the cell genome.
  • the cells are non-human animal cells.
  • the non-human animal is a non-human mammal.
  • the non-human animal is selected from the group consisting of mice, rats, rabbits, apes, monkeys, pigs, cows, sheep, horses, chickens, dogs, alpacas, and cats.
  • the non-human animal is a rodent or a primate.
  • the cells include cells selected from the group consisting of neuroblastoma cells, glioma cells, primary neurons, microglia, astrocytes, oligodendrocytes Plastid cells, macrophages, perivascular epithelioid cells, B cells, T cells, adult stem cells, NK cells, totipotent stem cells, unipotent stem cells, embryonic stem cells, induced pluripotent stem cells, and gametes.
  • the gametes include sperm and/or oocytes.
  • the cells are incapable of developing into a complete non-human animal.
  • the present application provides a tissue comprising and/or expressing ATP6V1B2, functionally active fragments thereof and/or nucleic acid molecules encoding them.
  • the ATP6V1B2 is of human origin.
  • the ATP6V1B2 and/or its functionally active fragments comprise the amino acid sequence shown in any one of SEQ ID NO:8, 10-11, and 16.
  • the gene encoding said ATP6V1B2 and/or a functionally active fragment thereof is homozygous or heterozygous.
  • the nucleic acid molecule encoding the ATP6V1B2 and/or its functionally active fragment comprises the nucleic acid sequence shown in SEQ ID NO:9 or 17.
  • the tissue is, or is derived from, a gene-edited tissue.
  • the tissue is, or is derived from, a knock-in tissue.
  • the expression of the ATP6V1B2 and/or its functionally active fragments is regulated by corresponding endogenous regulatory elements in the tissue genome.
  • the tissue is tissue of a non-human animal.
  • the non-human animal is a non-human mammal.
  • the non-human animal is selected from the group consisting of mice, rats, rabbits, apes, monkeys, pigs, cows, sheep, horses, chickens, dogs, alpacas, and cats.
  • the non-human animal is a rodent or a primate.
  • the tissue includes bodily fluids.
  • the bodily fluid is selected from the group consisting of blood, plasma, serum, urine, sweat, tears, saliva, semen, and cerebrospinal fluid.
  • the plasma includes exosomes.
  • the tissue comprises the brain or a portion thereof.
  • the portion of the brain comprises a brain region selected from the group consisting of olfactory bulb, amygdala, basal ganglia, hippocampus, thalamus, hypothalamus, cerebral cortex, medulla oblongata, and cerebellum.
  • the tissue includes the olfactory mucosa.
  • the tissue includes a portion of the central nervous system, a portion of the peripheral nervous system, skin tissue, muscle tissue, and/or internal organs.
  • the central nervous system includes the spinal cord.
  • the tissue cannot develop into a complete non-human animal.
  • the present application provides a cell line or cell culture derived from the non-human animal described herein or a part thereof, derived from the progeny described herein, derived from the cells described herein, and/or Derived from the organization described in this application.
  • the cell culture is a primary cell culture.
  • the cell culture comprises organoids.
  • the present application provides a composition comprising the non-human animal described in the present application or its part, the progeny described in the present application, the cell described in the present application, the tissue described in the present application, and/or the non-human animal described in the present application. Cell lines or primary cell cultures.
  • the present application provides a kit comprising the non-human animal described in the present application or its part, the progeny described in the present application, the cell described in the present application, the tissue described in the present application, and/or the non-human animal described in the present application.
  • a cell line or primary cell culture comprising one or more additional components selected from the group consisting of assay buffers, controls, substrates, standards, assay materials, laboratory supplies, equipment, instruments, cells , organs, tissues and user manuals or instructions.
  • the present application provides a method of screening drugs capable of improving the subject's learning ability, treating cognitive impairment, and/or treating neurodegenerative diseases, the method comprising administering the candidate drug to the Non-human animals or parts thereof, progeny described herein, cells described herein, tissues described herein, and/or cell lines or primary cell cultures described herein, and the effect of the candidate drug on the tested The expression level and/or activity of ATP6V1B2 in patients.
  • the present application provides a method for screening drugs capable of improving the learning ability of the subject, treating cognitive impairment, and/or treating neurodegenerative diseases, the method comprising: (i) administering the candidate drug to The non-human animal described in the application or part thereof, the progeny described in the application, the cell described in the application, the tissue described in the application, and/or the cell line or primary cell culture described in the application; The impact of the candidate drug on the expression level and/or activity of ATP6V1B2 in the subject; (iii) after administering the candidate drug, the expression level and/or activity of the ATP6V1B2 increase, then the candidate drug can Improving a subject's learning ability, treating cognitive impairment, and/or treating a neurodegenerative disease.
  • the expression level of ATP6V1B2 includes the expression level of ATP6V1B2 gene, the transcription level of ATP6V1B2 gene and/or the expression level of ATP6V1B2 protein.
  • said increasing comprises an increase in the expression level and/or activity of said ATP6V1B2 of at least about 10% compared to the expression level and/or activity of native ATP6V1B2 in said subject.
  • the learning ability comprises cognitive ability, motor ability, memory ability and/or spatial exploration ability.
  • the cognitive impairment comprises early cognitive impairment (MCI), intermediate cognitive impairment and late cognitive impairment.
  • the cognitive impairment comprises cognitive impairment due to normal aging, lews body dementia (LBD), frontotemporal dementia, and/or vascular dementia.
  • LBD lews body dementia
  • frontotemporal dementia frontotemporal dementia
  • vascular dementia vascular dementia
  • the inducing disease of cognitive impairment includes Alzheimer's disease, multiple infarct type, Parkinson's disease, AIDS and/or Creutzfeldt-Jakob disease (CJD).
  • the neurodegenerative disease includes acute neurodegenerative disease and chronic neurodegenerative disease.
  • the neurodegenerative disease includes neurodegenerative disease caused by neuronal death and glial cell homeostasis, neurodegenerative disease caused by aging, neurodegenerative disease caused by affected CNS cell function diseases, neurodegenerative diseases caused by abnormal communication between cells and/or neurodegenerative diseases caused by impaired cell motility.
  • the neurodegenerative disease comprises Alzheimer's disease, Parkinson's disease, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS) and/or Huntington's disease (HD ).
  • the present application provides a non-human animal or part thereof described in the present application, the progeny described in the present application, the cell described in the present application, the tissue described in the present application, and/or the cell line or primary generation described in the present application
  • the present application provides a non-human animal or part thereof described in the present application, the progeny described in the present application, the cell described in the present application, the tissue described in the present application, and/or the cell line or primary generation described in the present application Cell culture, which can be used to screen drugs and/or biomarkers that can improve the learning ability of subjects, treat cognitive impairment, and/or treat neurodegenerative diseases.
  • the present application provides a method for preparing the non-human animal or part thereof, the cell or the tissue described in the present application, the method comprising making the non-human animal or part thereof, cell or tissue comprise and /or express said ATP6V1B2 and/or its functionally active fragments.
  • the present application provides a method for preparing a disease model, the method comprising making a non-human animal or its part, cell or tissue contain and/or express ATP6V1B2 and/or its functionally active fragment.
  • the ATP6V1B2 and/or its functionally active fragments comprise the amino acid sequence shown in any one of SEQ ID NO:8, 10-11, and 16.
  • the method comprises transfecting a nucleic acid sequence encoding said ATP6V1B2 and/or a functionally active fragment thereof.
  • the nucleic acid sequence encoding the ATP6V1B2 and/or its functionally active fragment comprises the nucleic acid sequence shown in SEQ ID NO:9 or 17.
  • the transfection includes: transfecting an adeno-associated virus comprising the nucleic acid sequence.
  • FIGS 1a-1i show the effects of the polypeptides described herein on excitatory synaptic transmission.
  • Figure 2 shows that the polypeptide described in the present application can improve the cognitive behavior in the new object recognition test of AD mice through intragastric administration.
  • Fig. 3 shows that the polypeptide described in the present application can improve the learning ability in the water maze test of AD mice by intragastric administration.
  • Fig. 4 shows that the polypeptide described in the present application can improve the cognitive behavior in the new object recognition test of AD mice through intravenous administration.
  • Fig. 5 shows that the polypeptide described in the present application can improve the learning ability in the water maze test of AD mice through intravenous administration.
  • Figures 6a-6b show the co-immunoprecipitation results of the polypeptide described in this application and ATP6V1B2 protein.
  • Figures 7a-7e show that overexpression of ATP6V1B2 protein in the hippocampus of aged animals can improve learning and memory functions.
  • Figure 8 shows the effect of dimers of polypeptides described herein on excitatory synaptic transmission.
  • Figure 9 shows the effect of cysteine-modified polypeptides of the present application on excitatory synaptic transmission.
  • Figure 10 shows the effect of serine modified polypeptides of the present application on excitatory synaptic transmission.
  • Figure 11 shows the co-immunoprecipitation results of the polypeptide described in this application and ATP6V1B2 protein or its truncated fragments.
  • ATP6V1B2 generally refers to the ATPase H + transporting V1 subunit B2 (ATPase H+transporting V1subunit B2) protein (or ATP6B2, DOOD, HO57, VATB, VPP3, Vma2 or ZLS2), and the gene encoding the protein .
  • the ATP6V1B2 may be a multi-subunit enzyme that mediates the acidification of intracellular organelles in eukaryotic cells.
  • the ATP6V1B2 can participate in processes such as protein sorting, zymogen activation, receptor-mediated endocytosis, and synaptic vesicle proton gradient generation.
  • the ATP6V1B2 protein may comprise a cytoplasmic V1 domain and a transmembrane V0 domain.
  • the accession number of human ATP6V1B2 in GenBank is 526.
  • the accession number of human ATP6V1B2 in UniProt may be P21281.
  • expression level generally refers to the protein, RNA or mRNA level of a particular gene of interest. Any method known in the art can be used to determine the expression level of a particular related gene (eg, human ATP6V1B2 gene). In this application, the "expression” generally refers to the process of converting the information encoded by a gene into a structure that exists and operates in a cell.
  • Examples may include reverse transcription and amplification assays (e.g., PCR, ligated RT-PCR, or quantitative RT-PCT), hybridization assays, Northern blotting, dot blots, in situ hybridization, gel electrophoresis, capillary electrophoresis, Column chromatography, western blotting, immunohistochemistry, immunostaining, or mass spectrometry. Analysis can be performed directly on biological samples or on proteins/nucleic acids isolated from samples.
  • reverse transcription and amplification assays e.g., PCR, ligated RT-PCR, or quantitative RT-PCT
  • hybridization assays e.g., Northern blotting, dot blots, in situ hybridization, gel electrophoresis, capillary electrophoresis, Column chromatography, western blotting, immunohistochemistry, immunostaining, or mass spectrometry. Analysis can be performed directly on biological samples or on proteins/nucleic acids isolated from samples.
  • the term "activity" generally refers to any activity associated with a particular protein.
  • the activity may include, for example, any activity related to ATP6V1B2 protein.
  • the activity may include protease-associated enzymatic activity.
  • the activity may include biological activity.
  • the activity may involve binding of the protein to a receptor, for example, which binding may have measurable downstream effects.
  • the activity may include any activity that would be attributed to the protein by a person skilled in the art.
  • proton pump modulator generally refers to an agent capable of modulating proton pump activity and/or function.
  • proton pump agonist generally refers to an agent that activates a proton pump.
  • the proton pump agonist can increase the activity of the hydrogen ion pump.
  • vesicular proton pump agonist generally refers to an agent capable of increasing the activity of a vesicular proton pump.
  • Vesicular or vacuolar adenosine triphosphatase (V-ATPase) is an ATP-driven proton pump complexed by a cytosolic V1 complex for ATP hydrolysis and a membrane-embedded Vo for proton transfer composition.
  • V-ATPase vacuolar adenosine triphosphatase
  • the vesicular proton pump can play an important role in the acidification of eukaryotic intracellular vesicles, organelles and the extracellular environment.
  • proton pump-related protein generally refers to a protein that encodes and/or expresses a proton pump.
  • the proton pump can be a protein that actively transports hydrogen ions on the biological membrane against the electrochemical potential difference of hydrogen ions on both sides of the membrane.
  • the proton pumps may include Na-K pumps, Ca 2+ pumps, H + -ATP pumps and H + pyrophosphate pumps.
  • the term "learning ability” generally refers to all abilities related to or required for learning/cognitive processes.
  • the learning ability may include the ability to acquire new information, knowledge and/or skills through a process including experience, learning, or training.
  • the learning ability may include imagination, attention, perception and observation ability, reading ability, analysis ability, operation ability, adaptability, generalization ability, problem-solving ability or a combination thereof.
  • the term "cognitive ability” generally refers to the ability to process information through perception.
  • the cognitive ability may include the ability to grasp the composition of things, the relationship between performance and other things, the driving force of development, the direction of development and the basic laws.
  • athletic ability generally refers to the ability to play and train.
  • the athletic performance may include aerobic performance, muscular strength, body flexibility, balance and reflexes.
  • the athletic ability may be a comprehensive expression of various factors such as body shape, quality, skill, skill and mental ability.
  • the term "memory ability” usually refers to the ability to memorize, retain, re-recognize and reproduce the content and experience reflected in objective things.
  • the memory ability may include sensory memory ability, short-term memory ability and long-term memory ability.
  • spatial exploration capability generally refers to the ability to explore the shape and/or position of objects.
  • the spatial exploration ability includes seeing, thinking, imagining, recognizing and/or exploring the shape and/or location of objects.
  • the term "assessment score for learning ability” generally refers to a quantitative measurement score for a subject's learning ability.
  • the evaluation scores of the learning ability can be assessed by including attention/executive function assessment (such as Wechsler memory test), language ability assessment (such as language screening test (the language screening test, LAST)), visuospatial and structural ability assessment ( (eg, visuomotor integration test, Hooper visual organization test, object assembly test, figure arrangement test, clock drawing test), motor skills assessment, daily functioning assessment (eg, disability assessment for dementia (DAD)) and/or neuropsychological Scores obtained from the scale are obtained.
  • attention/executive function assessment such as Wechsler memory test
  • language ability assessment such as language screening test (the language screening test, LAST)
  • visuospatial and structural ability assessment eg, visuomotor integration test, Hooper visual organization test, object assembly test, figure arrangement test, clock drawing test
  • motor skills assessment eg, disability assessment for dementia (DAD)
  • the learning ability can be assessed by scores on the mini-mental state examination (MMSE), Montreal cognitive assessment (MoCA), Alzheimer's disease assessment Scale-cognitive part (Alzheimer disease assessment scale-cog, ADAS-cog) and clinical dementia rating scale (clinical dementia rating scale, CDR) test scores obtained.
  • MMSE mini-mental state examination
  • MoCA Montreal cognitive assessment
  • ADLS-cog Alzheimer's disease assessment Scale-cognitive part
  • CDR clinical dementia rating scale
  • the term "novel object recognition test” generally refers to a test for measuring the time required for an animal to learn the recognition of a new object by making the animal (such as a mouse) recognize the object in a specific space.
  • the new object cognition test can refer to the test method described in Ennaceur et al., Behav Brain Res 80 9-25,1996.
  • the novel object recognition test may include the following steps: putting two identical objects into a container with a fixed volume, putting mice into the container to recognize the two objects, and placing the mouse in the same container the next day. One of the two objects was replaced with a new object with a different shape, and the mice's search time for the new object was measured.
  • the term "water maze test” generally refers to a test in which an animal, such as a mouse, is forced to swim in order to learn to find a hidden platform in the water.
  • the water maze test (for example, Morris water maze) can test the learning ability and/or memory ability of mice for spatial position sense and direction sense.
  • the water maze test may also include acquisition training, exploration training, counterpoint training or counterpoint exploration training. If the time required for the animal (eg mouse) to find the platform from entering the water is shorter, and the distance traveled during this period is shorter, the assessment score of the learning ability of the animal (eg mouse) is correspondingly higher.
  • the water maze evaluation test can be an important test to evaluate learning ability.
  • neurodegenerative disease generally refers to cognitive disorders such as dementia that result from the gradual loss of neuronal structure and function, including neuronal death and glial homeostasis.
  • age eg, Alzheimer's disease (AD), Parkinson's disease (PD)
  • genetic mutations that affect CNS cell function eg, Huntington's disease, early-onset AD or PD, muscular dystrophy Lateral sclerosis (ALS)
  • AD Alzheimer's disease
  • PD Parkinson's disease
  • ALS muscular dystrophy Lateral sclerosis
  • the neurodegenerative disease may have changes and/or conditions selected from the group consisting of protein misfolding and aggregation; neuroinflammation (e.g., under signal stimuli such as toxic stimuli (such as protein aggregation), infection, traumatic injury, or autoimmunity) CNS inflammation); changes in cell signaling; acquired senescence/cell death (eg, disrupted apoptotic signaling, mitochondrial dysfunction, impaired autophagy, and activation of necrosomes by stress/inflammation); exercise Cell damage and epigenetic changes.
  • signal stimuli such as toxic stimuli (such as protein aggregation), infection, traumatic injury, or autoimmunity) CNS inflammation
  • changes in cell signaling e.g., acquired senescence/cell death (eg, disrupted apoptotic signaling, mitochondrial dysfunction, impaired autophagy, and activation of necrosomes by stress/inflammation); exercise Cell damage and epigenetic changes.
  • Alzheimer's disease generally refers to Alzheimer's disease, senile dementia, a neurodegenerative disease that develops slowly and worsens over time.
  • the most common early symptom is loss of short-term memory (difficulty remembering recent events), and as the disease progresses, at least one of the following symptoms may develop gradually: language disturbance, disorientation (for example, getting lost easily), emotional disturbance stability, loss of motivation, incapacity, and behavioral problems.
  • the true cause of Alzheimer's disease is still unknown, and its process may be related to the deposition of fibrous amyloid plaques and Tau protein in the brain. There are currently no treatments that can stop or reverse the course of the disease, and only a few methods may temporarily relieve or improve symptoms.
  • MCI middle cognitive impairment
  • the MCI may include cognitive impairment meeting criteria for dementia but exceeding normal aging.
  • MCI is diverse in clinical presentation, etiology, prognosis, and prevalence.
  • MCI can be a pathological stage of Alzheimer's disease.
  • Certain forms of cognitive impairment can be seen as early manifestations of neurodegenerative diseases that will eventually lead to dementia.
  • cognitive impairment due to normal aging generally refers to cognitive impairment due to normal aging.
  • the cognitive impairments caused by normal aging can manifest as memory loss, confusion about where familiar places are, taking longer than usual to complete everyday tasks, or changes in mood and personality.
  • lews body dementia usually refers to Lewy Body Detmentia, dementia with Lewy bodies.
  • Dementia with Lewy bodies is characterized by abnormal accumulations of protein that form masses called Lewy bodies. Dementia with Lewy bodies causes a gradual decline in mental ability. People with dementia with Lewy bodies may experience visual hallucinations and changes in alertness and concentration. Other effects include muscle stiffness, slowness of movement, difficulty walking, and tremors.
  • Patients with Lewy bodies in the brain can also have the plaques and tangles associated with Alzheimer's disease.
  • frontotemporal dementia generally refers to Pick's disease, a progressive rare disease in which the tau protein affects only the frontal and temporal lobes of the brain.
  • People with frontotemporal dementia have difficulties with higher-level reasoning, expressive language, language perception, and memory formation.
  • the frontal and temporal lobes of the brain in people with frontotemporal dementia can shrink over time.
  • vascular dementia generally refers to problems with reasoning, judgment, and memory due to impaired blood flow to the brain.
  • the vascular dementia may include dementia due to heart disease and stroke risk factors, such as high blood pressure and high cholesterol.
  • multiple infarct generally refers to small noncortical infarcts resulting from occlusion of a single perforating branch of a large cerebral artery.
  • the multiple infarction type may be a special type of cerebral infarction, also known as ischemic stroke.
  • the multi-infarct type may manifest as hemisensory impairment, aphasia, dysarthria, slowness of movement, and clumsiness (especially difficulty in fine movements such as writing).
  • Parkinson's disease generally refers to a progressive neurodegenerative disorder.
  • the clinical features of Parkinson's disease (PD) can include motor symptoms such as tremor, bradykinesia, muscle rigidity, and postural instability, as well as neuropsychiatric and other non-motor manifestations.
  • the non-motor manifestations may include cognitive dysfunction and dementia, mood disorders (eg, depression, anxiety, apathy), and sleep disorders.
  • AIDS generally refers to Acquired Immunodeficiency Syndrome (AIDS).
  • Clinical manifestations of AIDS include changes in memory, concentration, attention, and motor skills.
  • cognitive impairment can occur in AIDS patients, for example, about 50% of infected patients may further develop into HIV-associated neurocognitive disorders (HIV-associated neurocognitive disorders, HAND).
  • HIV-associated neurocognitive disorders HAND
  • CJD Creutzfeldt-Jakob disease
  • CJD is a transmissible spongiform encephalopathy that occurs in humans.
  • CJD is a disease caused by prion infection.
  • CJD patients can manifest paranoid behavior, confusion, loss of appetite and weight, depression, and a few patients have visual or auditory abnormalities; in the advanced stage, it manifests as progressive neurological deterioration (such as paresthesias, language disorders, and aphasia).
  • MS multiple sclerosis
  • the term “multiple sclerosis (MS)” generally refers to a demyelinating neuropathy.
  • the insulating substance (myelin sheath) on the surface of nerve cells in the brain or spinal cord of the MS patient is damaged, and the signal transduction of the nervous system is impaired, which can lead to a series of possible symptoms, affecting the patient's activities, mind, and even spirit. state. These symptoms can include double vision, impaired vision on one side, muscle weakness, dull sensations, or trouble with coordination.
  • ALS myotrophic lateral sclerosis
  • motor neuron disease a progressive and fatal neurodegenerative disease.
  • Frontotemporal dementia can occur in a minority of ALS patients. Some ALS patients experience degeneration of senses of sensation, vision, touch, smell and taste, and a very small number of ALS patients also develop dementia.
  • Huntington's disease usually refers to a genetic disorder that causes brain cell death.
  • HD patients as the disease progresses, incoordination of body movements becomes more pronounced, and abilities gradually deteriorate until movement becomes difficult and speech becomes impossible.
  • Mental capacity usually declines to dementia.
  • the term "aging stage” generally refers to the aging stage of a subject.
  • the aging stage can be above 60 years old, above 70 years old or above 75 years old; for mice, the old stage can be above 10 months old, for example, it can be above 13 months old or 18 months or older.
  • the elderly subject may have one or more symptoms of learning deficits, memory impairment, memory deficits, and/or brain dysfunction.
  • variant generally refers to a polypeptide comprising an amino acid sequence that differs by at least one amino acid residue from the amino acid sequence of a parent or reference polypeptide (eg, a wild-type polypeptide).
  • the variant may have a high (eg at least 80%) homology with the parent or reference polypeptide.
  • the homology may include sequence similarity or identity.
  • said homology can be determined using standard techniques known in the art (see, for example, Smith and Waterman, Adv. Appl. Math. "Advances in Applied Mathematics”); identity shared by polynucleotide or polypeptide sequences Percentages are determined by direct comparison of sequence information between molecules by aligning the sequences and determining identity using methods known in the art.
  • NCBI National Center for Biotechnology Information
  • the term "transmitter release” generally refers to the release of neurotransmitters, that is, the process in which a neuron transmits information by releasing neurotransmitters wrapped in vesicles to the synaptic cleft to act on another neuron.
  • the basic structure of the neural circuit, the synapse may be involved in the release of the transmitter.
  • the transmitter release may be referred to as synaptic transmission.
  • the release method of the transmitter may include synchronous release (Synchronous release), asynchronous release (Asynchronous release) and spontaneous release (Spontaneous release).
  • firing frequency generally refers to the firing frequency of an action potential.
  • the action potential can refer to a process of rapid and reversible reversal and recovery of the potential on both sides of the membrane generated on the basis of the resting potential when the excitable cell is stimulated.
  • Action potentials can be composed of spike potentials and afterpotentials, which correspond to depolarization and hyperpolarization processes, respectively.
  • the firing of action potentials can appear pulse-like. In some cases, the frequency of pulse firing is the ratio of the number of pulses fired to the time.
  • neuron generally refers to nerve cells, which are the main functional unit of the nervous system.
  • a neuron can consist of a cell body with its protruding axon and one or more dendrites. Neurons can transmit messages to other neurons or cells by releasing neurotransmitters at synapses.
  • multimer generally refers to a molecule having two or more polypeptide chains associated covalently, non-covalently, or by both covalent and non-covalent interactions.
  • the multimers may include dimers.
  • homodimer generally refers to a molecule formed from two identical monomers. These two identical monomers can aggregate, complex or associate with each other through covalent and/or non-covalent interactions.
  • thiol-blocking generally refers to blocking free thiol groups to make it difficult to form intramolecular and/or intermolecular disulfide bonds.
  • said sulfhydryl blocking may take place on cysteine residues.
  • the sulfhydryl blocking can prevent the formation of disulfide bonds between the cysteine residues of the protein and prevent the protein from being cross-linked or modified.
  • the blocking of the sulfhydryl group can be achieved by using a blocking agent, and the blocking agent can be a reducing agent.
  • the blocking agent may include dithiothreitol (DTT), ⁇ -mercaptoethanol (BME), and tris(2-carboxyethyl)phosphine hydrochloride (TCEP ⁇ HCl).
  • serine phosphorylation generally refers to a phosphorylation modification that occurs on a serine residue.
  • the serine phosphorylation may be a process of transferring a phosphate group of a donor (such as ATP or GTP) to a serine residue.
  • Said serine phosphorylation can be by means of protein kinases. Said serine phosphorylation can lead to changes in protein activity.
  • the term "about” generally refers to a numerical range of 20% more or less than the specified value.
  • “about X” includes a numerical range of ⁇ 20%, ⁇ 10%, ⁇ 5%, ⁇ 2%, ⁇ 1%, ⁇ 0.5%, ⁇ 0.2%, or ⁇ 0.1% of X, where X is a value.
  • the present application provides a use of a proton pump modulator in the preparation of a reagent for improving learning ability.
  • the present application provides a use of a proton pump regulator in the preparation of a reagent for treating cognitive impairment.
  • the present application provides a use of a proton pump regulator in the preparation of a reagent for treating neurodegenerative diseases.
  • the present application provides a use of an ATP6V1B2 modulator in the preparation of a reagent for improving learning ability.
  • the present application provides a use of an ATP6V1B2 regulator in the preparation of a reagent for treating cognitive impairment.
  • the present application provides a use of an ATP6V1B2 regulator in the preparation of a reagent for treating neurodegenerative diseases.
  • ATP6V1B2 when the expression level and/or activity of ATP6V1B2 in the subject increases, the learning ability of the subject can be significantly improved (for example, the cognitive ability, motor ability, memory ability And/or the ability to explore space can be significantly improved compared to when the expression level and/or activity of ATP6V1B2 in the subject is increased). Therefore, ATP6V1B2 can serve as a potential target to improve learning ability. For example, ATP6V1B2 may serve as a potential target for the treatment of neurodegenerative diseases (such as Alzheimer's disease) and/or cognitive impairment.
  • neurodegenerative diseases such as Alzheimer's disease
  • the agent can increase the expression level and/or activity of ATP6V1B2 in the subject.
  • the proton pump regulator can increase the expression level and/or activity of ATP6V1B2 in the subject.
  • the ATP6V1B2 regulator can increase the expression level and/or activity of ATP6V1B2 in the subject.
  • the expression level of ATP6V1B2 and/or its functionally active fragment may include the expression level of ATP6V1B2 gene, the transcription level of ATP6V1B2 gene and/or the expression level of ATP6V1B2 protein and/or its functionally active fragment.
  • the expression level may include the amount of polynucleotide, mRNA or amino acid product or protein of a particular gene (eg, human ATP6V1B2 gene).
  • the expression level may include the amount of transcribed polynucleotides, translated proteins, or fragments of post-translationally modified proteins of a particular gene (eg, human ATP6V1B2 gene).
  • the increase may comprise an increase of the expression level of ATP6V1B2 by at least about 10% compared to the original expression level of ATP6V1B2 in the subject. For example, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 200%, at least About 500% or more.
  • the expression level of ATP6V1B2 can be measured by using a substance selected from the following group: primers for specifically amplifying the ATP6V1B2 gene, nucleic acid molecules specifically binding to the ATP6V1B2 gene, and nucleic acid molecules specifically binding to the ATP6V1B2 protein , a small molecule specifically binding to the ATP6V1B2 protein, a probe specifically binding to the ATP6V1B2 protein, and a polypeptide specifically binding to the ATP6V1B2 protein.
  • the expression level of said ATP6V1B2 can be measured by performing a test selected from the group consisting of: reverse transcription and amplification analysis (such as PCR, ligated RT-PCR or quantitative RT-PCT), hybridization analysis, Northern blot ( Northern blotting), dot blot, in situ hybridization, gel electrophoresis, capillary electrophoresis, column chromatography, Western blotting, immunohistochemistry, immunostaining, or mass spectrometry.
  • the expression level of ATP6V1B2 described in the present application can be measured by qPCR, qRT-PCR, northern hybridization, western hybridization and/or ELISA detection.
  • the expression level of ATP6V1B2 can also be measured by performing an analysis directly on a biological sample or on a protein/nucleic acid isolated from a sample.
  • the ATP6V1B2 can be derived from human or mouse.
  • the ATP6V1B2 may comprise the amino acid sequence shown in SEQ ID NO.8 or 16.
  • the functionally active fragment of ATP6V1B2 may have the ability to specifically bind to the amino acid sequence shown in SEQ ID NO.5.
  • the functionally active fragment of ATP6V1B2 may include a truncation of ATP6V1B2.
  • the functionally active fragment of ATP6V1B2 may comprise at least part of the amino acid sequence from position 288 to position 512 of human ATP6V1B2 protein.
  • the functionally active fragment of ATP6V1B2 may comprise at least part of the amino acid sequence from position 288 to position 512 of mouse ATP6V1B2 protein.
  • the ATP6V1B2 and/or its functionally active fragment may comprise the amino acid sequence shown in any one of SEQ ID NO:8, 10-11, and 16.
  • nucleic acid sequence encoding the ATP6V1B2 and/or its functionally active fragment may comprise the nucleic acid sequence shown in SEQ ID NO:9 or 17.
  • the activity of ATP6V1B2 may include the biological activity of the ATP6V1B2 protein and/or its functionally active fragments (for example, may include measurable downstream effects caused by it).
  • the activity of ATP6V1B2 may include increasing the expression level and/or activity of the proton pump-related protein.
  • the increase may comprise an increase in the activity of the ATP6V1B2 of at least about 10% compared to the activity of the original ATP6V1B2 in the subject. For example, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 200%, at least About 500% or more.
  • the agent can increase the expression level and/or activity of the proton pump-related protein in the subject.
  • the proton pump regulator can increase the expression level and/or activity of proton pump-related proteins in the subject.
  • the ATP6V1B2 regulator can increase the expression level and/or activity of proton pump-related proteins in the subject.
  • the expression level of the proton pump related protein may include the expression level of the gene encoding the proton pump related protein, the transcription level of the gene encoding the proton pump related protein and/or the expression level of the proton pump related protein.
  • the expression level can include the amount of a polynucleotide, mRNA or amino acid product or protein of a particular gene (e.g., a gene encoding a proton pump-related protein).
  • the expression level can include the amount of a polynucleotide transcribed, a translated protein, or a fragment of a post-translationally modified protein of a particular gene (eg, a gene encoding a proton pump-related protein).
  • the increase may comprise an increase in the expression level of the proton pump-related protein by at least about 10% compared to the original expression level of the proton pump-related protein in the subject. For example, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 200%, at least About 500% or more.
  • the expression level of the proton pump-related protein can be measured by using a substance selected from the group consisting of primers capable of specifically amplifying the gene encoding the proton pump-related protein, primers that specifically bind to the gene encoding the proton pump-related protein Nucleic acid molecules, nucleic acid molecules specifically binding to proton pump-related proteins, small molecules specifically binding to proton pump-related proteins, probes specifically binding to proton pump-related proteins, and polypeptides specifically binding to proton pump-related proteins.
  • the expression level of the proton pump-related protein can be measured by performing an assay selected from the group consisting of: reverse transcription and amplification analysis (such as PCR, ligation RT-PCR or quantitative RT-PCT), hybridization analysis, RNA Northern blotting, dot blot, in situ hybridization, gel electrophoresis, capillary electrophoresis, column chromatography, western blotting, immunohistochemistry, immunostaining, or mass spectrometry.
  • the expression level of the proton pump-related protein described in this application can be measured by qPCR, qRT-PCR, northern hybridization, western hybridization and/or ELISA detection.
  • the expression level of the proton pump-related protein can also be measured by performing an analysis directly on a biological sample or on a protein/nucleic acid isolated from a sample.
  • the proton pump-related protein may include NADH dehydrogenase, coenzyme Q, succinate-coenzyme Q reductase, cytochrome c and/or coenzyme Q-cytochrome c reductase.
  • the activity of the proton pump-related protein may be the biological activity of the proton pump-related protein.
  • the activity of the proton pump-related protein can be carried out by the activity level of the hydrogen/potassium adenosine triphosphate enzyme system (also known as hydrogen/potassium ion ATPase, ie H + /K + ATPase); and/or the activity level of the H2 receptor measure.
  • the increase in the activity of the proton pump-related protein may comprise an increase in the activity of the proton pump-related protein by at least about 10% compared to the activity of the original proton pump-related protein in the subject.
  • the learning ability may include all abilities related or required to the learning/cognitive process.
  • the learning abilities may include cognitive abilities, motor abilities, memory abilities, and/or spatial exploration abilities.
  • the improvement of learning ability may comprise an improvement of at least about 50% in the assessment score of learning ability of the subject compared with the original assessment score of learning ability of the subject. For example, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least About 100%, at least about 200%, at least about 500%, or more.
  • the assessment scores of the learning ability can be assessed by including attention/executive function assessment (such as Wechsler memory test), language ability assessment (such as language screening test (the language screening test, LAST)), visuospatial And structural ability assessment (such as visual motor integration test, Hooper visual organization test, object assembly test, figure arrangement test, clock drawing test), motor ability assessment, daily function assessment (such as disability assessment for dementia (DAD)) and/or scores obtained on neuropsychological scales.
  • attention/executive function assessment such as Wechsler memory test
  • language ability assessment such as language screening test (the language screening test, LAST)
  • visuospatial And structural ability assessment such as visual motor integration test, Hooper visual organization test, object assembly test, figure arrangement test, clock drawing test
  • daily function assessment such as disability assessment for dementia (DAD)
  • said assessment score of learning ability can be measured by performing a test selected from the group consisting of: novel object recognition test and water maze test.
  • the novel object recognition test may evaluate the cognitive ability, motor ability and/or spatial exploration ability.
  • the new object recognition test can make the subjects (such as mice) explore and learn objects of specific shapes in a fixed container, and distinguish the objects in the fixed container according to the memory acquired by learning after a few days. Emerging new objects that have a different shape than previous objects. The shorter the time it took for the mice to distinguish the new object with a different shape after a few days, the higher the learning ability of the mice.
  • the water maze evaluation test can be an important test to evaluate the learning ability of the subjects.
  • the water maze test can evaluate the memory ability, motor ability and/or spatial exploration ability.
  • the water maze test (such as the Morris water maze) can force the subject (such as a mouse) to swim, so as to learn to find a platform hidden in the water, and then test the space of the mouse to the sense of spatial position and direction. Exploration skills and/or memory skills.
  • the water maze test may also include acquisition training, probing training, counterpoint training, or counterpoint probing training. If the time it takes for the mice to find the platform from entering the water is shorter, and the distance traveled during this time is shorter, the learning ability of the mice is correspondingly higher.
  • the water maze evaluation test can be an important test to evaluate the learning ability of the subjects.
  • the subject may include a mammal.
  • the subject can include a rodent and/or a primate, eg, the subject can include a human.
  • the subjects may include non-cognitive impairment patients and/or non-neurodegenerative disease patients.
  • the subject can be a normal and/or healthy person.
  • the subject may have a need and/or desire to further improve their learning abilities.
  • the neurodegenerative diseases may include acute neurodegenerative diseases and chronic neurodegenerative diseases.
  • the neurodegenerative disease may include neurodegenerative disease caused by neuronal death and glial cell homeostasis, neurodegenerative disease caused by aging, neurodegenerative disease caused by affected CNS cell function, neurodegenerative disease caused by Neurodegenerative diseases caused by abnormal communication between cells and/or caused by impaired cell motility.
  • the subjects may include patients with neurodegenerative diseases.
  • the subject can include an Alzheimer's disease patient.
  • the Alzheimer's disease patient may be in the early stage, early stage, middle stage or late stage of Alzheimer's disease.
  • the cognitive impairment may include early cognitive impairment (MCI), middle cognitive impairment and late cognitive impairment.
  • MCI early cognitive impairment
  • the cognitive impairment may include cognitive impairment due to normal aging, lews body dementia (LBD), frontotemporal dementia, and/or vascular dementia.
  • LBD lews body dementia
  • the inducing disease of cognitive impairment may include Alzheimer's disease, multi-infarct type, Parkinson's disease, AIDS and/or Creutzfeldt-Jakob disease (CJD).
  • the subjects may include patients with cognitive impairment.
  • the subject may suffer from early cognitive impairment (MCI) (e.g., loss of short-term memory, difficulty expressing or understanding abstract impairment and/or need for reminders for basic self-care), intermediate cognitive impairment (e.g., confusion between long-term memory and real-world situation memory, poor word expression, behavioral personality changes or emotional lability and/or need for assisted self-care) or late cognitive impairment
  • MCI early cognitive impairment
  • intermediate cognitive impairment e.g., confusion between long-term memory and real-world situation memory, poor word expression, behavioral personality changes or emotional lability and/or need for assisted self-care
  • Cognitive impairment eg, memory impairment, decline in physical activity and mental status, inability to express or communicate effectively, inability to care for oneself and/or disturbance of circadian clock.
  • the subject may suffer from a disease capable of inducing the cognitive impairment.
  • the subject may have Alzheimer's disease, multi-infarct type, Parkinson's disease, AIDS and/or Creutzfeldt-Jak
  • the subject may be at an aging stage.
  • the subject has exhibited cognitive impairment caused by normal aging.
  • the subject has exhibited symptoms of early cognitive impairment (MCI).
  • MCI early cognitive impairment
  • the subject has exhibited symptoms of a neurodegenerative disease such as Alzheimer's disease.
  • the proton pump modulator may include a proton pump agonist.
  • the proton pump modulator may include a vesicular proton pump agonist.
  • the proton pump regulator may include an agonist of ATP6V1B2 protein.
  • the proton pump modulator can bind to ATP6V1B2 protein.
  • the proton pump modulator can include a ligand for the ATP6V1B2 protein.
  • the proton pump modulator can include an agonist of the ATP6V1B2 protein.
  • the proton pump modulator can specifically bind to a specific position (eg, a specific epitope) in the ATP6V1B2 protein.
  • the proton pump modulator may include a nucleic acid or a variant thereof, a polypeptide or a variant thereof and/or a small molecule.
  • the proton pump modulator can increase neuron synaptic transmitter release.
  • said increase includes an increase of at least about 10% compared to the level of primitive neuronal synaptic transmitter release in said subject. For example, at least about 20%, at least about 50%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least About 95%, at least about 100%, at least about 200%, at least about 500%, or more.
  • the proton pump regulator can increase the firing frequency of excitatory postsynaptic current.
  • said increase includes an increase of at least about 10% compared to the level of firing frequency of primordial excitatory postsynaptic currents in said subject. For example, at least about 20%, at least about 50%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least About 95%, at least about 100%, at least about 200%, at least about 500%, or more.
  • the ATP6V1B2 regulator may include an ATP6V1B2 agonist.
  • the ATP6V1B2 can bind to the ATP6V1B2 protein.
  • the ATP6V1B2 modulator can include a ligand for the ATP6V1B2 protein.
  • the ATP6V1B2 modulator can include an agonist of the ATP6V1B2 protein.
  • the ATP6V1B2 modulator can specifically bind to a specific position (eg, a specific epitope) in the ATP6V1B2 protein.
  • the ATP6V1B2 regulator may include a proton pump agonist, for example, may include a vesicle-type proton pump agonist.
  • the ATP6V1B2 regulator may include nucleic acid or its variant, polypeptide or its variant and/or small molecule.
  • the ATP6V1B2 regulator can increase the release of neuron synaptic transmitters.
  • said increase includes an increase of at least about 10% compared to the level of primitive neuronal synaptic transmitter release in said subject. For example, at least about 20%, at least about 50%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least About 95%, at least about 100%, at least about 200%, at least about 500%, or more.
  • the ATP6V1B2 regulator can increase the firing frequency of excitatory postsynaptic current.
  • said increase includes an increase of at least about 10% compared to the level of firing frequency of primordial excitatory postsynaptic currents in said subject. For example, at least about 20%, at least about 50%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least About 95%, at least about 100%, at least about 200%, at least about 500%, or more.
  • the present application provides a polypeptide capable of regulating the expression level and/or activity of ATP6V1B2.
  • the polypeptide can increase the release of neuron synaptic transmitters.
  • said increase includes an increase of at least about 10% compared to the level of primitive neuronal synaptic transmitter release in said subject. For example, at least about 20%, at least about 50%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least About 95%, at least about 100%, at least about 200%, at least about 500%, or more.
  • the polypeptide can increase the firing frequency of excitatory postsynaptic current.
  • said increase includes an increase of at least about 10% compared to the level of firing frequency of primordial excitatory postsynaptic currents in said subject. For example, at least about 20%, at least about 50%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least About 95%, at least about 100%, at least about 200%, at least about 500%, or more.
  • the polypeptide or its variants can specifically bind to ATP6V1B2 protein.
  • the polypeptide or variant thereof may have a "ligand-receptor" relationship with the ATP6V1B2 protein.
  • the polypeptide or its variant can bind to at least part of the amino acid sequence from position 288 to position 512 of mouse ATP6V1B2 protein. In the present application, the polypeptide or its variant can bind to at least part of the amino acid sequence from position 288 to position 512 of human ATP6V1B2 protein.
  • the polypeptide or its variant may comprise a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds).
  • the polypeptide may include any chain of two or more amino acids and is not intended to be a specific length of product.
  • the variant may include a polypeptide comprising one or more amino acid substitutions, additions or deletions based on the polypeptide.
  • Said variants may include products with modifications on said polypeptide, for example said modifications may include glycosylation, acetylation, phosphorylation, acylation, derivatization by known protective/blocking groups, Proteolytic cleavage, and/or modification by non-naturally occurring amino acids.
  • the polypeptide or its variant can increase the expression level and/or activity of ATP6V1B2 in the subject.
  • the expression level of ATP6V1B2 may include the expression level of ATP6V1B2 gene, the transcription level of ATP6V1B2 gene and/or the expression level of ATP6V1B2 protein.
  • the increase may comprise an increase of the expression level and/or activity of the ATP6V1B2 by at least about 10% compared to the expression level and/or activity of the original ATP6V1B2 in the subject.
  • polypeptide may comprise the amino acid sequence shown in SEQ ID NO.5.
  • polypeptide may comprise the amino acid sequence shown in any one of SEQ ID NO.1,3.
  • the polypeptide or its variants may form multimers.
  • homodimers can be formed.
  • the homodimer may still have the ability to bind to the ATP6V1B2 protein.
  • the homodimer may be a tandem structure of two identical polypeptides.
  • the homodimer may comprise the amino acid sequence shown in SEQ ID NO.18.
  • the cysteine in the amino acid sequence of the polypeptide or its variant may not have the modification of sulfhydryl blocking.
  • the cysteine residue in the amino acid sequence of the polypeptide or its variant is blocked by a sulfhydryl group, it may lose the ability to bind to the ATP6V1B2 protein.
  • the serine in the amino acid sequence of the polypeptide or its variant may not have phosphorylation modification.
  • the serine residue in the amino acid sequence of the polypeptide or its variant is modified by phosphorylation, it may lose the ability to bind to the ATP6V1B2 protein.
  • the preparation (such as the polypeptide or its variant) can improve the learning ability of the subject (for example, compared with the administration of the polypeptide or its variant, the subject's learning ability is significantly improved). assessment score for the learning ability).
  • the preparation (such as the polypeptide or its variant) can regulate the expression level and/or activity of the gene related to learning ability in the subject.
  • the genes related to learning ability may include APP gene.
  • the APP gene is an amyloid precursor protein gene (amyloid precursor protein), and the gene may be related to Alzheimer's disease, such as familial Alzheimer's disease (FAD).
  • the learning ability-related genes may include presenilin 1 gene (presenilin1, PSEN1) and presenilin 2 gene (presenilin2, PSEN2).
  • the learning ability-related genes may include genes encoding glutamate receptors.
  • the glutamate receptors may include N-methyl-D-aspartate receptors (NMDAR), kainate receptors (KAR), and ⁇ -amino-3-hydroxy-5-methyl-4 Isoxazole receptors (AMPARs).
  • the learning ability-related genes may include genes encoding action receptors of memantine (eg, 1-amino-3,5-dimethylamantadine hydrochloride).
  • the receptors on which memantine acts can include NMDA receptors.
  • the polypeptide can reduce the expression level and/or activity of the above-mentioned genes in this paragraph.
  • the preparation described in this application, the ATP6V1B2 modulator, the proton pump modulator, and/or the polypeptide described in this application can be used as a potential drug for improving learning ability.
  • the preparation described in this application, the ATP6V1B2 regulator, the proton pump regulator, and/or the polypeptide described in this application can be used as a potential drug for treating cognitive disorders and/or treating neurodegenerative diseases.
  • the present application provides a pharmaceutical combination comprising the agents described herein.
  • the pharmaceutical combination may comprise pharmaceutical products suitable for pharmaceutical use such as improving learning ability, treating cognitive impairment and/or treating neurodegenerative diseases such as Alzheimer's disease.
  • the present application provides a pharmaceutical composition, which comprises the agent described in the present application and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition can be a composition comprising one or more active ingredients (such as the formulations described in the application) and one or more inert ingredients; or aggregation, or any product resulting directly or indirectly from the dissociation of one or more components, or from other types of reactions or interactions of one or more components.
  • the pharmaceutically acceptable carrier may include sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions and sterile powders for reconstitution into sterile injectable solutions or dispersions immediately before use.
  • suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles may include water, ethanol, polyols (such as propylene glycol, propylene glycol, polyethylene glycol, etc.), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic vinegars such as ethyl oleate.
  • the present application provides an application of the reagent described in the present application, the pharmaceutical combination described in the present application and/or the pharmaceutical composition described in the present application in the preparation of a drug for increasing the release of neuron synaptic transmitters.
  • the present application provides a use of the reagent described in the present application, the pharmaceutical combination described in the present application and/or the pharmaceutical composition described in the present application in the preparation of a drug for improving learning ability.
  • the application provides a reagent described in the application, a pharmaceutical combination described in the application and/or a pharmaceutical composition described in the application in the preparation of a drug for treating cognitive impairment and/or treating neurodegenerative diseases in the application.
  • the polypeptide described in the present application, the reagent described in the present application, the pharmaceutical combination described in the present application and/or the pharmaceutical composition described in the present application can be formulated to be suitable for oral and/or injection administration.
  • the polypeptides described herein, the agents described herein, the pharmaceutical combinations described herein and/or the pharmaceutical compositions described herein may be formulated to be suitable for intravenous administration.
  • the present application provides a method for screening drugs that can improve the learning ability of the subject, treat cognitive impairment, and/or treat neurodegenerative diseases, which includes the following steps: detecting the effect of the candidate drug on the subject Effects on the expression level and/or activity of a proton pump-related protein, wherein after administration of the candidate drug, the expression level and/or activity of the proton pump-related protein is increased, and the candidate drug can improve the subject's learning ability , treating cognitive impairment, and/or treating neurodegenerative diseases.
  • the present application provides a method for screening drugs that can improve the learning ability of the subject, treat cognitive impairment, and/or treat neurodegenerative diseases, which includes the following steps: detecting the effect of the candidate drug on the subject Influence on the expression level and/or activity of ATP6V1B2, wherein after administration of the candidate drug, the expression level and/or activity of the ATP6V1B2 is increased, then the candidate drug can improve the subject's learning ability and treat cognitive impairment , and/or treat neurodegenerative diseases.
  • the candidate drug may include an agent capable of regulating the expression level and/or activity of ATP6V1B2; reagents.
  • the drug candidate may include an agent capable of binding the ATP6V1B2 protein.
  • the administration may include oral administration and/or injection.
  • the administering may include intravenous injection.
  • the present application also provides a detection kit capable of detecting the expression level and/or activity of ATP6V1B2.
  • the detection kit may include instructions, which describe the specific steps of how to use the detection kit to detect the expression level and/or activity of proton pump-related proteins and/or ATP6V1B2, and/or how to use the detection results to judge the Specific steps in whether the drug candidate can improve the subject's ability to learn.
  • the detection kit may also include reagents capable of detecting other targets for judging whether the candidate drug can improve the subject's learning ability.
  • Non-human animals, their offspring or parts thereof are non-human animals, their offspring or parts thereof.
  • the non-human animal may be, or be derived from, a gene-edited non-human animal.
  • the non-human animal can be a knock-in non-human animal, or be derived from a knock-in non-human animal.
  • the non-human animal can be a non-human mammal.
  • the non-human animal is a rodent or a primate.
  • the non-human animal is selected from the group consisting of mice, rats, rabbits, apes, monkeys, pigs, cows, sheep, horses, chickens, dogs, alpacas, and cats.
  • the part of the non-human animal or progeny thereof may include organs, tissues and/or cells thereof.
  • the portion may include bodily fluids.
  • the bodily fluid may be selected from the group consisting of blood, plasma, serum, urine, sweat, tears, saliva, semen and cerebrospinal fluid.
  • the plasma includes exosomes.
  • the portion includes the brain (eg, brain tissue) or a portion thereof.
  • the portion of the brain may include a portion selected from the group consisting of olfactory bulb, amygdala, basal ganglia, hippocampus, thalamus, hypothalamus, cerebral cortex, medulla oblongata, and cerebellum.
  • the portion comprises the olfactory mucosa.
  • the portion includes a portion of the central nervous system, a portion of the peripheral nervous system, skin tissue, muscle tissue, and/or an internal organ.
  • the central nervous system may include the spinal cord.
  • the portion may comprise cells.
  • the cells may comprise cells selected from the group consisting of primary neurons, microglia, astrocytes, oligodendrocytes, macrophages, perivascular epithelioid cells, B cells, T cells, Adult stem cells, NK cells, totipotent stem cells, unipotent stem cells, embryonic stem cells, induced pluripotent stem cells, and gametes.
  • the gametes may include spermatozoa and/or oocytes.
  • the part eg, the organ, tissue or cell
  • the part cannot develop into a complete non-human animal.
  • said non-human animal, progeny thereof, or portion thereof comprises said ATP6V1B2 and/or a functionally active fragment thereof.
  • the ATP6V1B2 and/or functionally active fragments thereof may be expressed by the animal, the offspring or the cell, or from other sources, and then introduced (for example, injected) into the animal, the offspring or the cell, so that It comprises the ATP6V1B2 and/or its functionally active fragments.
  • said non-human animal, progeny thereof, or parts thereof express said ATP6V1B2 and/or functionally active fragments thereof.
  • the expression of ATP6V1B2 may include the expression of ATP6V1B2 gene, the expression of ATP6V1B2 gene transcript and/or the expression of ATP6V1B2 protein.
  • the expression may include the expression of a specific gene (eg, human ATP6V1B2 gene) polynucleotide, mRNA or amino acid product or protein.
  • Said expression may include the expression of fragments of transcribed polynucleotides, translated proteins, or post-translationally modified proteins of a particular gene (eg, human ATP6V1B2 gene).
  • the expression of the functionally active fragment of ATP6V1B2 may include the expression of the gene encoding the functionally active fragment of ATP6V1B2, the transcript of the gene encoding the functionally active fragment of ATP6V1B2 and/or the expression of the protein of the functionally active fragment of ATP6V1B2.
  • the expression may include the expression of a polynucleotide, mRNA or amino acid product or protein of a specific gene (such as a functionally active fragment encoding human ATP6V1B2 (such as the 288th to 512th amino acid sequence thereof).
  • the expression may include Expression of fragments of transcribed polynucleotides, translated proteins, or post-translationally modified proteins of a particular gene.
  • the present application also provides a method for preparing the non-human animal or part thereof, the cell described in the present application or the tissue described in the present application, the method comprising making the non-human animal or part thereof, cell or tissue comprise And/or express said ATP6V1B2 and/or its functionally active fragments.
  • the present application also provides a method for preparing a disease model, the method comprising making the non-human animal or its parts, cells or tissues contain and/or express ATP6V1B2 and/or its functionally active fragments.
  • the method may comprise introducing into said non-human animal or part thereof, said cell, tissue, cell line and/or cell culture said ATP6V1B2, a functionally active fragment thereof, a nucleic acid molecule encoding said ATP6V1B2 and /or a nucleic acid molecule encoding a functionally active fragment of ATP6V1B2.
  • Said introducing can comprise, for example, injecting said non-human animal, part thereof, said tissue or cells (eg, injecting a protein or nucleic acid molecule).
  • the introduction may also include nucleic acid molecule transfection, virus transduction and other means for introducing exogenous proteins, exogenous nucleic acid molecules, etc. into cells, tissues or animals.
  • the transfection can include transfection using an adeno-associated virus.
  • the method may comprise knocking in the non-human animal or part thereof, the cell, tissue, cell line and/or cell culture encoding the ATP6V1B2 and/or a functionally active fragment thereof nucleic acid sequence.
  • the method may further comprise identifying a modified cell or non-human animal comprising a knock-in heterologous nucleic acid sequence.
  • a donor nucleic acid molecule comprising a heterologous nucleic acid sequence may also comprise a 5' homology arm and a 3' homology arm.
  • the 5' and 3' homology arms may flank the heterologous nucleic acid sequence encoding said ATP6V1B2 and/or functionally active fragments thereof.
  • a homology arm eg, a 5' homology arm or a 3' homology arm
  • a homology arm in a donor nucleic acid molecule may be of sufficient size to facilitate interaction with an endogenous target site.
  • the homology arm and the target gene locus match or correspond to each other, and at this time the mutual consistency of the two local sequences is sufficient as The basis of the homologous recombination reaction.
  • "Homology” means that a DNA sequence is identical or has a certain sequence identity with a corresponding or matching sequence.
  • the sequence identity between a particular target site and the corresponding homology arms found in the donor nucleic acid molecule can be any degree of sequence identity that allows homologous recombination to occur.
  • the degree of sequence identity shared by the homology arms of the donor nucleic acid molecule (or fragment thereof) and the target site (or fragment thereof) may be at least about 50%, at least about 55%, at least about 60%, at least about 65% %, at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87% %, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100%, so that the sequences undergo homologous recombination.
  • the devices may include medical devices.
  • medical devices that are claimed to be effective in improving a subject's learning ability, treating cognitive impairment, and/or treating neurodegenerative diseases.
  • the identification, screening or design method of the present application may be an in vitro method, an ex vivo method or an in vivo method.
  • a candidate substance may be a synthetic compound, a peptide, a protein, a DNA library or a nucleic acid molecule in the library, an animal (for example, a mammal such as a mouse, rat, pig, cow, sheep, monkey or human) Tissue extracts or cell culture supernatants, extracts or culture products of plants or microorganisms, or any mixture of the above.
  • an animal for example, a mammal such as a mouse, rat, pig, cow, sheep, monkey or human
  • Tissue extracts or cell culture supernatants extracts or culture products of plants or microorganisms, or any mixture of the above.
  • Cognitive ability, motor ability, memory ability and/or spatial exploration ability can also be compared between the treatment group administered with the candidate drug and the control group, for example, by performing animal behavioral analysis.
  • a drug candidate may be selected for further study if a significant improvement in cognition, motor performance, memory, and/or spatial exploration is observed following treatment with the drug candidate compared to a control group (e.g. , as a potential therapeutic drug for improving the learning ability of subjects, treating cognitive impairment, and/or treating neurodegenerative diseases).
  • the method of screening can also be carried out using non-human animal tissues and/or cells according to the present application, such as neuronal cells, brain regions, or other tissues containing/corresponding to AD lesions, or parts thereof (eg olfactory bulb, amygdala, basal ganglia, hippocampus, thalamus, hypothalamus, cerebral cortex, medulla oblongata, central nervous system, peripheral nervous system, spinal cord, cerebellum, skin tissue, muscle tissue, and/or internal organs).
  • tissues and/or cells can be cultured in vitro or ex vivo, and then after an appropriate period of incubation, the candidate drug can be administered to the cultured tissues and/or cells (e.g. hours, days, weeks, or months), and the processed brain tissue/cells were examined using the method described above.
  • non-human animals derived from the present application can be collected in the presence of signs associated with neurological diseases or disorders (for example, A ⁇ deposition, neurological Samples (e.g., cells, tissues, or other DNA- or RNA-containing samples, protein-containing samples, and/or metabolite-containing samples), followed by comprehensive analysis of gene transcripts (transcriptome), gene translation products (proteome), lipids (lipidome) or metabolites (metabolome) from the samples , and identify substances that have changed before and after the associated indication.
  • neurological diseases or disorders for example, A ⁇ deposition, neurological Samples (e.g., cells, tissues, or other DNA- or RNA-containing samples, protein-containing samples, and/or metabolite-containing samples), followed by comprehensive analysis of gene transcripts (transcriptome), gene translation products (proteome), lipids (lipidome) or metabolites (metabolome) from the samples , and identify substances that have changed before and after the associated indication.
  • neurological Samples e.g., cells, tissues, or other DNA-
  • the sample may comprise a bodily fluid of the non-human animal, e.g., rat blood, plasma (e.g., plasma comprising exosomes), serum, urine, sweat, tears, saliva, semen , and/or cerebrospinal fluid.
  • a bodily fluid of the non-human animal e.g., rat blood, plasma (e.g., plasma comprising exosomes), serum, urine, sweat, tears, saliva, semen , and/or cerebrospinal fluid.
  • Nucleic acid microarrays such as DNA microarrays, can be used to analyze gene transcription products (such as transcriptome); gel electrophoresis (such as two-dimensional gel electrophoresis) or mass spectrometry (such as time-of-flight mass spectrometry, electron spray Ionization mass spectrometry, capillary HPLC/MS and LC/MS/MS analyze gene translation products (eg proteome)); NMR, capillary electrophoresis, LC/MS and LC/MS/MS can be used to analyze metabolites (metabolome).
  • gene transcription products such as transcriptome
  • gel electrophoresis such as two-dimensional gel electrophoresis
  • mass spectrometry such as time-of-flight mass spectrometry, electron spray Ionization mass spectrometry, capillary HPLC/MS and LC/MS/MS analyze gene translation products (eg proteome)
  • NMR capillary electrophoresis
  • LC/MS and LC/MS/MS can be used
  • the substance can be regarded as a biological agent for improving the learning ability of the subject, treating cognitive impairment, and/or treating neurodegenerative diseases. landmark.
  • mice 15-month-old mice were randomly divided into control group and experimental group, and the experimental group and the control group were injected with adeno-associated virus (pAAV2/9-hSyn -ATP6V1B2-Myc-2A-mCherry, wherein the amino acid sequence of the ATP6V1B2-mCherry fusion protein is shown in SEQ ID NO.6) or the control adeno-associated virus carrying mCherry to make the experimental group and the control group mouse brain bilateral dorsal hippocampus Neurons overexpress ATP6V1B2-mCherry protein or mCherry protein, respectively.
  • adeno-associated virus pAAV2/9-hSyn -ATP6V1B2-Myc-2A-mCherry, wherein the amino acid sequence of the ATP6V1B2-mCherry fusion protein is shown in SEQ ID NO.6
  • the control adeno-associated virus carrying mCherry to make the experimental group and the control
  • Example 2 The target is the polypeptide of ATP6V1B2 protein
  • Biotin-labeled QD202 and QD201 were intravenously administered to two 8-month-old C57BL/6 mice at a dose of 6.25 mg/kg. After 4 hours, the mice were anesthetized, and the hippocampus tissue was collected, and the lysis buffer was added, and the tissue was broken by mechanical grinding, and the supernatant was collected after centrifugation. Add biotin antibody and Protein G agarose beads to the supernatant for immunoprecipitation. For the test steps, refer to the book "Guidelines for Antibody Technology Experiments" published by Science Press in 2002. Proteins were eluted using protein loading buffer.
  • Polypeptide QD202 was synthesized according to the amino acid sequence shown in SEQ ID NO.1.
  • the peptide QD202N'-1 was synthesized according to the amino acid sequence shown in SEQ ID NO.2.
  • the peptide QD202C'-1 was synthesized according to the amino acid sequence shown in SEQ ID NO.3.
  • the peptide QD202C'-2 was synthesized according to the amino acid sequence shown in SEQ ID NO.4.
  • C57 mice aged 2-3 months were anesthetized by intraperitoneal injection of 0.1-0.15ml of 20% ureose, and the brain tissue was quickly decapitated and placed in an artificial atmosphere with mixed gas (95% O 2 and 5% CO 2 ).
  • Cerebrospinal fluid (ACSF) was placed in ice-water mixed liquid for 2 minutes.
  • the formula of ACSF was as follows: NaCl 11.7mM, KCl 0.36mM, NaH 2 PO 4 0.12mM, CaCl 2 0.25mM, MgCl 2 0.12mM, NaHCO 3 25mM, and glucose 11mM. Coronal slices of hippocampus of 350 ⁇ m were cut using a vibrating microtome.
  • the hippocampal slices were placed in ACSF at room temperature for more than 30 minutes for recovery, and then the brain slices were placed in the recording tank and continuously perfused with ACSF with mixed gas at a rate of 2-2.5ml/min.
  • the Hippocampal CA1 pyramidal neurons were blind-sealed, and Axon700B amplifiers and 1550B digital-to-analog converters were used for whole-cell patch-clamp recordings.
  • QD202 can increase the spontaneous excitatory synaptic transmitter release of hippocampal CA1 neurons, mainly manifested as increasing the firing frequency of excitatory postsynaptic currents, but has no effect on the amplitude of excitatory postsynaptic currents (see Figure 1a, b,c);
  • mice For new object recognition, refer to the behavior test program described in Leger, M., et al. Object recognition test in mice. Nat Protoc. made of opaque plastic.) for a novel object recognition test.
  • the mice On the first day, the mice were acclimatized in an open field box for 10 min.
  • each mouse On the second day, each mouse was gently placed in the center of the box, two similar objects (No. 1 battery) were placed in the central area, allowed to explore freely for 10 min, and then the mice were returned to their home cages. After 3 h, the mice were put back into the box again (one battery was replaced by a 10 cm high puppet toy) for 10 min to carry out the memory retention test. Videos were analyzed with Etho Vision XT 14 software, and the time spent by mice exploring new/old objects was recorded. The discrimination index was calculated as (Tnovel-Tfamliar)/(Tnovel+Tfamliar).
  • the Morris water maze refers to the experimental protocol described by Qing-Feng Wu et al. in Fibroblast growth factor 13 is a microtubule-stabilizing protein regulating neuronal polarization and migration. Cell. 149, 1549-1564 (2012).
  • the tests were carried out in a fixed environment room in a circular pool (diameter 120 cm, depth 30 cm, opaque by addition of titanium dioxide, kept at 21 ⁇ 1° C.) filled with water.
  • the experiment is divided into adaptation period (1 day), training period (5-6 days) and test period (1 day).
  • Adaptation period place the platform 0.5 cm above the water surface, and guide the mice to get on the platform.
  • Training day place the platform at 0.5 cm underwater, and train the mice to find the platform. Stop timing when the mouse reaches the platform.
  • mice do not reach the platform within 1 minute, guide it onto the platform and make it stay on the platform for 30 seconds.
  • QD202 group 3
  • memantine group 4
  • memantine group 4
  • memantine group 4
  • the dosage of memantine is 1.25mg/kg, wherein the concentration of memantine is 1 mM
  • 10-month-old C57BL/6 mice that were not given any reagent by intragastric administration were used as controls (group 1).
  • mice were subjected to the behavioral test described above.
  • mice were subjected to the behavioral test described above.
  • QD202 can significantly improve learning ability, especially memory ability and cognitive ability.
  • QD202 dimer Take QD202, dissolve it in water at a concentration of 10mg/ml, add dilute ammonia water to adjust the pH value between 8-9, stir and oxidize for 24 hours, during which the reaction end point is monitored by HPLC, after oxidation is complete, QD202 is obtained by HPLC purification dimer.
  • the QD202 dimer comprises the amino acid sequence shown in SEQ ID NO.18.
  • QD202 dimers were used to detect spontaneous excitatory postsynaptic currents of pyramidal neurons in mice.
  • the control refers to the result of spontaneous excitatory postsynaptic current recorded during normal artificial cerebrospinal fluid perfusion.
  • cysteine-modified QD202 was used to detect spontaneous excitatory postsynaptic currents of pyramidal neurons in mice.
  • the control refers to the result of spontaneous excitatory postsynaptic current recorded during normal artificial cerebrospinal fluid perfusion.
  • cysteine-modified QD202 increased the activity of spontaneous excitatory synaptic transmitter release in hippocampal CA1 neurons, mainly manifested as reducing the firing frequency of excitatory postsynaptic currents, and/or reducing excitatory synaptic After current amplitude.
  • Preparation of serine-modified QD202 replace Fmoc-Ser(tbu)-OH with Fmoc-Ser(HPO3Bzl)-OH, and directly synthesize according to the amino acid sequence of QD202, so that the 12th serine (S) from the N-terminus is phosphated B-modified serine-modified QD202.
  • the spontaneous excitatory post-synaptic current of pyramidal neurons was detected in mice using serine-modified QD202.
  • the control refers to the result of spontaneous excitatory postsynaptic current recorded during normal artificial cerebrospinal fluid perfusion.
  • the results are shown in Figure 10.
  • the results show that serine-modified QD202 increases the activity of spontaneous excitatory synaptic transmitter release in hippocampal CA1 neurons, mainly manifested as reducing the firing frequency of excitatory postsynaptic currents, and/or reducing the amplitude of excitatory postsynaptic currents .
  • the expression plasmids containing the full-length, 1-286aa truncated sequence or 1-172aa truncated sequence of the mouse ATP6V1B2 with Myc tag were respectively constructed, wherein the amino acid sequence of the full-length mouse ATP6V1B2 is shown in SEQ ID NO.8, and the mouse-derived The amino acid sequence of ATP6V1B2 1-286aa is shown in SEQ ID NO.10, and the amino acid sequence of mouse ATP6V1B2 1-172aa is shown in SEQ ID NO.11. These plasmids are called ATP6V1B2 full-length expression plasmids and ATP6V1B2 1-286aa expression plasmids respectively. Plasmid and ATP6V1B2 1-172aa expression plasmid. At the same time, the QD202 expression plasmid with EGFP tag was constructed.
  • Protein G agarose beads combined with Myc antibody to co-precipitate the full-length ATP6V1B2 protein, 1-286aa truncated sequence or 1-172aa truncated sequence and the proteins combined with each other, and finally detect the co-immunoprecipitated protein by Western blotting .
  • Atp6v1b2-Myc, b2-286 ⁇ -Myc and b2-172 ⁇ -Myc correspond to ATP6V1B2 full-length protein, 1-286aa truncated sequence or 1-172aa truncated sequence, respectively.
  • the results show that only the full-length ATP6V1B2 protein can bind to the QD202 polypeptide, so it can be seen that the binding site of ATP6V1B2 and QD202 is located in the region from the 288th to the 512th amino acid of the N-terminal of ATP6V1B2.

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Abstract

一种质子泵调控剂，以及所述质子泵调控剂在制备试剂中的用途，所述试剂用于改善学习能力、治疗认知障碍、和/或治疗神经退行性疾病。还涉及了一种ATP6V1B2调控剂及其在制备用于改善学习能力、治疗认知障碍、和/或治疗神经退行性疾病的试剂中的用途。

Description

质子泵调控剂在制备试剂中的用途 技术领域
本申请涉及生物医药领域，具体的涉及一种质子泵调控剂在制备试剂中的用途。
背景技术
阿尔茨海默病(AD)，一般由突触和神经元损伤、神经环路结构和功能异常所致。由于目前对突触和神经元损伤的机制仍然不够明确，因此AD的有效防治方法进展非常缓慢。
ATP6V1B2基因所编码的ATP6V1B2蛋白是ATP水解驱动的质子泵的重要组成结构蛋白。其在人体组织内广泛分布，但在脑组织中、肾脏、破骨细胞等部位分布更为丰富。其在突触传递及溶酶体酸化等过程中发挥重要功能。其基因突变可导致DOORS综合征(易感常染色体显性遗传先天性耳聋伴甲发育不良综合征)。ATP6V1B2蛋白表达降低可能与AD发病有相关性。
发明内容
本申请提供了一种改善受试者学习能力的方法，本申请通过发现ATP6V1B2为改善受试者学习能力的潜在靶点，进而发现能够提高ATP6V1B2的表达水平和/或活性提高的试剂和/或质子泵调控剂可以作为改善受试者学习能力的潜在药物。
一方面，本申请提供一种质子泵调控剂在制备试剂中的用途，所述试剂用于改善学习能力。
另一方面，本申请提供一种质子泵调控剂在制备试剂中的用途，所述试剂用于治疗认知障碍。
另一方面，本申请提供一种质子泵调控剂在制备试剂中的用途，所述试剂用于治疗神经退行性疾病。
在某些实施方式中，所述试剂使受试者中质子泵相关蛋白的表达水平和/或活性提高。
在某些实施方式中，所述质子泵调控剂使受试者中质子泵相关蛋白的表达水平和/或活性提高。
在某些实施方式中，所述质子泵相关蛋白的表达水平包括编码质子泵相关蛋白基因的 表达水平、编码质子泵相关蛋白基因的转录水平和/或质子泵相关蛋白的表达水平。
在某些实施方式中，所述提高包括与所述受试者中原始质子泵相关蛋白的表达水平和/或活性相比，所述质子泵相关蛋白的表达水平和/或活性提高至少约10％。
在某些实施方式中，所述质子泵相关蛋白的表达水平通过实施选自下组的试验衡量：qPCR、qRT-PCR、杂交分析、RNA印迹法、斑点印迹法、原位杂交、胶凝电泳、毛细管电泳、柱色谱、蛋白质印迹法、免疫组织化学、免疫染色和质谱。
在某些实施方式中，所述质子泵相关蛋白的表达水平通过利用选自下组的物质衡量：能够特异性扩增编码质子泵相关蛋白基因的引物、与编码质子泵相关蛋白基因特异性结合的核酸分子、与质子泵相关蛋白特异性结合的核酸分子、与质子泵相关蛋白特异性结合的小分子、与质子泵相关蛋白特异性结合的探针和与质子泵相关蛋白特异性结合的多肽。
在某些实施方式中，所述试剂使受试者中ATP6V1B2的表达水平和/或活性提高。
在某些实施方式中，所述质子泵调控剂使受试者中ATP6V1B2的表达水平和/或活性提高。
在某些实施方式中，所述ATP6V1B2的表达水平包括ATP6V1B2基因的表达水平、ATP6V1B2基因的转录水平和/或ATP6V1B2蛋白的表达水平。
在某些实施方式中，所述提高包括与所述受试者中原始ATP6V1B2的表达水平和/或活性相比，所述ATP6V1B2的表达水平和/或活性提高至少约10％。
在某些实施方式中，所述ATP6V1B2的表达水平通过实施选自下组的试验衡量：qPCR、qRT-PCR、杂交分析、RNA印迹法、斑点印迹法、原位杂交、胶凝电泳、毛细管电泳、柱色谱、蛋白质印迹法、免疫组织化学、免疫染色和质谱。
在某些实施方式中，所述ATP6V1B2的表达水平通过利用选自下组的物质衡量：能够特异性扩增ATP6V1B2基因的引物、与ATP6V1B2基因特异性结合的核酸分子、与ATP6V1B2蛋白特异性结合的核酸分子、与ATP6V1B2蛋白特异性结合的小分子、与ATP6V1B2蛋白特异性结合的探针和与ATP6V1B2蛋白特异性结合的多肽。
在某些实施方式中，所述质子泵调控剂包括质子泵激动剂。
在某些实施方式中，所述质子泵调控剂包括囊泡型质子泵激动剂。
在某些实施方式中，所述质子泵调控剂包括ATP6V1B2蛋白的激动剂。
在某些实施方式中，所述质子泵调控剂能够结合ATP6V1B2蛋白。
在某些实施方式中，所述质子泵调控剂能够与小鼠ATP6V1B2蛋白的第288位至第512位氨基酸序列中的至少部分序列结合。
在某些实施方式中，所述质子泵调控剂能够与人ATP6V1B2蛋白的第288位至第512位氨基酸序列中的至少部分序列结合。
在某些实施方式中，所述质子泵调控剂包括核酸或其变体、多肽或其变体和/或小分子。
在某些实施方式中，所述质子泵调控剂能够增加神经元突触递质释放。
在某些实施方式中，所述增加包括与所述受试者中原始神经元突触递质释放的水平相比，增加至少约10％。
在某些实施方式中，所述质子泵调控剂能够增加兴奋性突触后电流的发放频率。
在某些实施方式中，所述增加包括与所述受试者中原始兴奋性突触后电流的发放频率的水平相比，增加至少约10％。
在某些实施方式中，所述学习能力包括认知能力、运动能力、记忆能力和/或空间探索能力。
在某些实施方式中，所述学习能力的改善包括与受试者的原始的学习能力的评估分数相比，改善后的所述受试者的学习能力的评估分数提高至少约50％。
在某些实施方式中，所述学习能力的评估分数通过实施选自下组的试验衡量：新物体认知试验和水迷宫试验。
在某些实施方式中，所述新物体认知试验评价所述认知能力、运动能力和/或空间探索能力。
在某些实施方式中，所述水迷宫试验评价所述记忆能力、运动能力和/或空间探索能力。
在某些实施方式中，所述受试者包括哺乳动物。
在某些实施方式中，所述受试者包括人。
在某些实施方式中，所述受试者包括非认知障碍患者和/或非神经退行性疾病患者。
在某些实施方式中，所述受试者包括神经退行性疾病患者和/或认知障碍患者。
在某些实施方式中，所述受试者包括阿尔兹海默症患者。
在某些实施方式中，所述受试者处于老龄阶段。
在某些实施方式中，所述认知障碍包括早期认知障碍(MCI)、中期认知障碍和晚期认知障碍。
在某些实施方式中，所述认知障碍包括正常衰老引起的认知障碍、lews身体痴呆(LBD)、额颞叶痴呆和/或血管性痴呆。
在某些实施方式中，所述认知障碍的诱导疾病包括阿尔兹海默症、多发性梗死型、帕金森氏症、艾滋病和/或克雅氏病(CJD)。
在某些实施方式中，所述神经退行性疾病包括急性神经退行性疾病和慢性神经退行性疾病。
在某些实施方式中，所述神经退行性疾病包括由神经元死亡和胶质细胞稳态引起的神经退行性疾病、由衰老引起的神经退行性疾病、由CNS细胞功能被影响引起的神经退行性疾病、由细胞间异常通信引起的神经退行性疾病和/或由细胞运动受损引起的神经退行性疾病。
在某些实施方式中，所述神经退行性疾病包括阿尔兹海默症、帕金森氏症、多发性硬化(MS)、肌萎缩性脊髓侧索硬化症(ALS)和/或亨廷顿病(HD)。
在某些实施方式中，所述试剂被配制为适于口服施用和/或注射施用。
在某些实施方式中，所述试剂被配制为适于静脉注射。
另一方面，本申请提供一种ATP6V1B2、其功能活性片段或编码它们的核酸分子在改善学习能力中的用途。
另一方面，本申请提供一种ATP6V1B2、其功能活性片段或编码它们的核酸分子在治疗认知障碍中的用途。
另一方面，本申请提供一种ATP6V1B2、其功能活性片段或编码它们的核酸分子在治疗神经退行性疾病中的用途。
在某些实施方式中，所述ATP6V1B2来源于人。
在某些实施方式中，所述ATP6V1B2包含SEQ ID NO.8或16所示的氨基酸序列。
在某些实施方式中，所述ATP6V1B2的功能活性片段具有与SEQ ID NO.5所示的氨基酸序列特异性结合的能力。
在某些实施方式中，所述ATP6V1B2的功能活性片段包括ATP6V1B2的截短体。
在某些实施方式中，所述ATP6V1B2的功能活性片段包含人ATP6V1B2蛋白的第288位至第512位氨基酸序列中的至少部分序列。
在某些实施方式中，所述ATP6V1B2的功能活性片段包含小鼠ATP6V1B2蛋白的第288位至第512位氨基酸序列中的至少部分序列。
在某些实施方式中，所述ATP6V1B2的功能活性片段包含SEQ ID NO.10-11中任一项所示的氨基酸序列。
在某些实施方式中，所述核酸分子包含SEQ ID NO.9、17中任一项所示的核苷酸序列。
在某些实施方式中，所述学习能力包括认知能力、运动能力、记忆能力和/或空间探索能力。
在某些实施方式中，所述学习能力的改善包括与受试者的原始的学习能力的评估分数相 比，改善后的所述受试者的学习能力的评估分数提高至少约50％。
在某些实施方式中，所述学习能力的评估分数通过实施选自下组的试验衡量：新物体认知试验和水迷宫试验。
在某些实施方式中，所述新物体认知试验评价所述认知能力、运动能力和/或空间探索能力。
在某些实施方式中，所述水迷宫试验评价所述记忆能力、运动能力和/或空间探索能力。
在某些实施方式中，所述受试者包括哺乳动物。
在某些实施方式中，所述受试者包括人。
在某些实施方式中，所述受试者包括非认知障碍患者和/或非神经退行性疾病患者。
在某些实施方式中，所述受试者包括神经退行性疾病患者和/或认知障碍患者。
在某些实施方式中，所述受试者包括阿尔兹海默症患者。
在某些实施方式中，所述受试者处于老龄阶段。
在某些实施方式中，所述认知障碍包括早期认知障碍(MCI)、中期认知障碍和晚期认知障碍。
在某些实施方式中，所述认知障碍包括正常衰老引起的认知障碍、lews身体痴呆(LBD)、额颞叶痴呆和/或血管性痴呆。
在某些实施方式中，所述认知障碍的诱导疾病包括阿尔兹海默症、多发性梗死型、帕金森氏症、艾滋病和/或克雅氏病(CJD)。
在某些实施方式中，所述神经退行性疾病包括急性神经退行性疾病和慢性神经退行性疾病。
在某些实施方式中，所述神经退行性疾病包括由神经元死亡和胶质细胞稳态引起的神经退行性疾病、由衰老引起的神经退行性疾病、由CNS细胞功能被影响引起的神经退行性疾病、由细胞间异常通信引起的神经退行性疾病和/或由细胞运动受损引起的神经退行性疾病。
在某些实施方式中，所述神经退行性疾病包括阿尔兹海默症、帕金森氏症、多发性硬化(MS)、肌萎缩性脊髓侧索硬化症(ALS)和/或亨廷顿病(HD)。
另一方面，本申请提供一种ATP6V1B2调控剂在制备试剂中的用途，所述试剂用于改善学习能力。
另一方面，本申请提供一种ATP6V1B2调控剂在制备试剂中的用途，所述试剂用于治疗认知障碍。
另一方面，本申请提供一种ATP6V1B2调控剂在制备试剂中的用途，所述试剂用于治疗 神经退行性疾病。
在某些实施方式中，所述试剂使受试者中ATP6V1B2的表达水平和/或活性提高。
在某些实施方式中，所述ATP6V1B2调控剂使受试者中ATP6V1B2的表达水平和/或活性提高。
在某些实施方式中，所述表达水平包括ATP6V1B2基因的表达水平、ATP6V1B2基因的转录水平和/或ATP6V1B2蛋白的表达水平。
在某些实施方式中，所述活性提高包括使质子泵相关蛋白的表达水平和/或活性提高。
在某些实施方式中，所述提高包括与所述受试者中原始ATP6V1B2的表达水平和/或活性相比，所述ATP6V1B2的表达水平和/或活性提高至少约10％。
在某些实施方式中，所述ATP6V1B2的表达水平通过实施选自下组的试验衡量：qPCR、qRT-PCR、杂交分析、RNA印迹法、斑点印迹法、原位杂交、胶凝电泳、毛细管电泳、柱色谱、蛋白质印迹法、免疫组织化学、免疫染色和质谱。
在某些实施方式中，所述ATP6V1B2的表达水平通过利用选自下组的物质衡量：能够特异性扩增ATP6V1B2基因的引物、与ATP6V1B2基因特异性结合的核酸分子、与ATP6V1B2蛋白特异性结合的核酸分子、与ATP6V1B2蛋白特异性结合的小分子、与ATP6V1B2蛋白特异性结合的探针和与ATP6V1B2蛋白特异性结合的多肽。
在某些实施方式中，所述ATP6V1B2调控剂包括ATP6V1B2蛋白的激动剂。
在某些实施方式中，所述ATP6V1B2调控剂能够结合ATP6V1B2蛋白。
在某些实施方式中，所述ATP6V1B2调控剂能够与小鼠ATP6V1B2蛋白的第288位至第512位氨基酸序列中的至少部分序列结合。
在某些实施方式中，所述ATP6V1B2调控剂能够与人ATP6V1B2蛋白的第288位至第512位氨基酸序列中的至少部分序列结合。
在某些实施方式中，所述ATP6V1B2调控剂包括质子泵激动剂。
在某些实施方式中，所述ATP6V1B2调控剂包括囊泡型质子泵激动剂。
在某些实施方式中，所述ATP6V1B2调控剂包括核酸或其变体、多肽或其变体和/或小分子。
在某些实施方式中，所述ATP6V1B2调控剂包括多肽或其变体。
在某些实施方式中，所述多肽或其变体特异性能够结合ATP6V1B2蛋白。
在某些实施方式中，所述多肽或其变体能够增加神经元突触递质释放。
在某些实施方式中，所述增加包括与所述受试者中原始神经元突触递质释放的水平相比， 增加至少约10％。
在某些实施方式中，所述多肽或其变体能够增加兴奋性突触后电流的发放频率。
在某些实施方式中，所述增加包括与所述受试者中原始兴奋性突触后电流的发放频率的水平相比，增加至少约10％。
在某些实施方式中，所述多肽或其变体包含SEQ ID NO.5所示的氨基酸序列。
在某些实施方式中，所述多肽或其变体包含SEQ ID NO.1,3中任一项所示的氨基酸序列。
在某些实施方式中，所述学习能力包括认知能力、运动能力、记忆能力和/或空间探索能力。
在某些实施方式中，所述学习能力的改善包括与受试者的原始的学习能力的评估分数相比，改善后的所述受试者的学习能力的评估分数提高至少约50％。
在某些实施方式中，所述学习能力的评估分数通过实施选自下组的试验衡量：新物体认知试验和水迷宫试验。
在某些实施方式中，所述新物体认知试验评价所述认知能力、运动能力和/或空间探索能力。
在某些实施方式中，所述水迷宫试验评价所述记忆能力、运动能力和/或空间探索能力。
在某些实施方式中，所述受试者包括哺乳动物。
在某些实施方式中，所述受试者包括人。
在某些实施方式中，所述受试者包括非认知障碍患者和/或非神经退行性疾病患者。
在某些实施方式中，所述受试者包括神经退行性疾病患者和/或认知障碍患者。
在某些实施方式中，所述受试者包括阿尔兹海默症患者。
在某些实施方式中，所述受试者处于老龄阶段。
在某些实施方式中，所述认知障碍包括早期认知障碍(MCI)、中期认知障碍和晚期认知障碍。
在某些实施方式中，所述认知障碍包括正常衰老引起的认知障碍、lews身体痴呆(LBD)、额颞叶痴呆和/或血管性痴呆。
在某些实施方式中，所述认知障碍的诱导疾病包括阿尔兹海默症、多发性梗死型、帕金森氏症、艾滋病和/或克雅氏病(CJD)。
在某些实施方式中，所述神经退行性疾病包括急性神经退行性疾病和慢性神经退行性疾病。
在某些实施方式中，所述神经退行性疾病包括由神经元死亡和胶质细胞稳态引起的神经退行性疾病、由衰老引起的神经退行性疾病、由CNS细胞功能被影响引起的神经退行性疾病、由细胞间异常通信引起的神经退行性疾病和/或由细胞运动受损引起的神经退行性疾病。
在某些实施方式中，所述神经退行性疾病包括阿尔兹海默症、帕金森氏症、多发性硬化(MS)、肌萎缩性脊髓侧索硬化症(ALS)和/或亨廷顿病(HD)。
在某些实施方式中，所述试剂被配制为适于口服施用和/或注射施用。
在某些实施方式中，所述试剂被配制为适于静脉注射。
在另一方面，本申请提供一种筛选能够改善受试者学习能力、治疗认知障碍、和/或治疗神经退行性疾病的药物的方法，其包括以下的步骤：检测候选药物对受试者中质子泵相关蛋白的表达水平和/或活性的影响，其中在施用所述候选药物后，所述质子泵相关蛋白的表达水平和/或活性提高，则所述候选药物能够改善受试者学习能力、治疗认知障碍、和/或治疗神经退行性疾病。
在另一方面，本申请提供一种筛选能够改善受试者学习能力、治疗认知障碍、和/或治疗神经退行性疾病的药物的方法，其包括以下的步骤：检测候选药物对受试者中ATP6V1B2的表达水平和/或活性的影响，其中在施用所述候选药物后，所述ATP6V1B2的表达水平和/或活性提高，则所述候选药物能够改善受试者学习能力、治疗认知障碍、和/或治疗神经退行性疾病。
在某些实施方式中，所述质子泵相关蛋白的表达水平包括编码质子泵相关蛋白基因的表达水平、编码质子泵相关蛋白基因的转录水平和/或质子泵相关蛋白的表达水平。
在某些实施方式中，所述提高包括与所述受试者中原始质子泵相关蛋白的表达水平和/或活性相比，所述质子泵相关蛋白的表达水平和/或活性提高至少约10％。
在某些实施方式中，所述质子泵相关蛋白的表达水平通过实施选自下组的试验衡量：qPCR、qRT-PCR、杂交分析、RNA印迹法、斑点印迹法、原位杂交、胶凝电泳、毛细管电泳、柱色谱、蛋白质印迹法、免疫组织化学、免疫染色和质谱。
在某些实施方式中，所述质子泵相关蛋白的表达水平通过利用选自下组的物质衡量：能够特异性扩增编码质子泵相关蛋白基因的引物、与编码质子泵相关蛋白基因特异性结合的核酸分子、与质子泵相关蛋白特异性结合的核酸分子、与质子泵相关蛋白特异性结合的小分子、与质子泵相关蛋白特异性结合的探针和与质子泵相关蛋白特异性结合的多肽。
在某些实施方式中，所述ATP6V1B2的表达水平包括ATP6V1B2基因的表达水平、ATP6V1B2基因的转录水平和/或ATP6V1B2蛋白的表达水平。
在某些实施方式中，其中所述提高包括与所述受试者中原始ATP6V1B2的表达水平和/或活性相比，所述ATP6V1B2的表达水平和/或活性提高至少约10％。
在某些实施方式中，所述ATP6V1B2的表达水平通过实施选自下组的试验衡量：qPCR、qRT-PCR、杂交分析、RNA印迹法、斑点印迹法、原位杂交、胶凝电泳、毛细管电泳、柱色谱、蛋白质印迹法、免疫组织化学、免疫染色和质谱。
在某些实施方式中，所述ATP6V1B2的表达水平通过利用选自下组的物质衡量：能够特异性扩增ATP6V1B2基因的引物、与ATP6V1B2基因特异性结合的核酸分子、与ATP6V1B2蛋白特异性结合的核酸分子、与ATP6V1B2蛋白特异性结合的小分子、与ATP6V1B2蛋白特异性结合的探针和与ATP6V1B2蛋白特异性结合的多肽。
在某些实施方式中，所述学习能力包括认知能力、运动能力、记忆能力和/或空间探索能力。
在某些实施方式中，所述施用包括口服和/或注射。
在某些实施方式中，所述施用包括静脉注射。
另一方面，本申请提供一种能够调控ATP6V1B2的表达水平和/或活性的多肽或其变体。
在某些实施方式中，所述多肽或其变体能够增加神经元突触递质释放。
在某些实施方式中，所述增加包括与所述受试者中原始神经元突触递质释放的水平相比，增加至少约10％。
在某些实施方式中，所述多肽或其变体能够增加兴奋性突触后电流的发放频率。
在某些实施方式中，所述增加包括与所述受试者中原始兴奋性突触后电流的发放频率的水平相比，增加至少约10％。
在某些实施方式中，所述多肽或其变体能够结合ATP6V1B2蛋白。
在某些实施方式中，所述多肽或其变体能够使受试者中ATP6V1B2的表达水平和/或活性提高。
在某些实施方式中，所述ATP6V1B2的表达水平包括ATP6V1B2基因的表达水平、ATP6V1B2基因的转录水平和/或ATP6V1B2蛋白的表达水平。
在某些实施方式中，所述提高包括与所述受试者中原始ATP6V1B2的表达水平和/或活性相比，所述ATP6V1B2的表达水平和/或活性提高至少约10％。
在某些实施方式中，所述多肽或其变体能够与小鼠ATP6V1B2蛋白的第288位至第512位氨基酸序列中的至少部分序列结合。
在某些实施方式中，所述多肽或其变体能够与人ATP6V1B2蛋白的第288位至第512位 氨基酸序列中的至少部分序列结合。
在某些实施方式中，所述多肽或其变体包含SEQ ID NO.5所示的氨基酸序列。
在某些实施方式中，所述多肽或其变体包含SEQ ID NO.1,3中任一项所示的氨基酸序列。
在某些实施方式中，所述多肽或其变体包括多聚体。
在某些实施方式中，所述多聚体包括同二聚体。
在某些实施方式中，所述多肽或其变体的氨基酸序列中的半胱氨酸不具有巯基封闭的修饰。
在某些实施方式中，所述多肽或其变体的氨基酸序列中的丝氨酸不具有磷酸化的修饰。
另一方面，本申请提供一种药物组合，其包含本申请所述的试剂和/或，其包含本申请所述的多肽或其变体。
另一方面，本申请提供一种药物组合物，其包含本申请所述的试剂和/或，其包含本申请所述的多肽或其变体，以及药学上可接受的载体。
另一方面，本申请提供一种本申请所述的多肽或其变体、本申请所述的试剂、本申请所述的药物组合和/或本申请所述的药物组合物在制备试剂中的用途，所述试剂用于改善学习能力、治疗认知障碍和/或治疗神经退行性疾病。
在某些实施方式中，所述学习能力包括认知能力、运动能力、记忆能力和/或空间探索能力。
在某些实施方式中，所述认知障碍包括早期认知障碍(MCI)、中期认知障碍和晚期认知障碍。
在某些实施方式中，所述认知障碍包括正常衰老引起的认知障碍、lews身体痴呆(LBD)、额颞叶痴呆和/或血管性痴呆。
在某些实施方式中，所述认知障碍的诱导疾病包括阿尔兹海默症、多发性梗死型、帕金森氏症、艾滋病和/或克雅氏病(CJD)。
在某些实施方式中所述神经退行性疾病包括急性神经退行性疾病和慢性神经退行性疾病。
在某些实施方式中，所述神经退行性疾病包括由神经元死亡和胶质细胞稳态引起的神经退行性疾病、由衰老引起的神经退行性疾病、由CNS细胞功能被影响引起的神经退行性疾病、由细胞间异常通信引起的神经退行性疾病和/或由细胞运动受损引起的神经退行性疾病。
在某些实施方式中，所述神经退行性疾病包括阿尔兹海默症、帕金森氏症、多发性硬化(MS)、肌萎缩性脊髓侧索硬化症(ALS)和/或亨廷顿病(HD)。
在某些实施方式中，所述多肽、所述的药物组合和/或所述的药物组合物被配制为适于口服施用和/或注射施用。
在某些实施方式中，所述多肽、所述的药物组合和/或所述的药物组合物被配制为适于静脉注射。
另一方面，本申请提供一种非人动物或其部分，其包含和/或表达ATP6V1B2、其功能活性片段和/或编码它们的核酸分子。
在某些实施方式中，所述ATP6V1B2源自人。
在某些实施方式中，所述ATP6V1B2包含SEQ ID NO.8或16所示的氨基酸序列。
在某些实施方式中，所述ATP6V1B2的功能活性片段具有与SEQ ID NO.5所示的氨基酸序列特异性结合的能力。
在某些实施方式中，所述ATP6V1B2的功能活性片段包括ATP6V1B2的截短体。
在某些实施方式中，所述ATP6V1B2的功能活性片段包含人ATP6V1B2蛋白的第288位至第512位氨基酸序列中的至少部分序列。
在某些实施方式中，所述ATP6V1B2的功能活性片段包含小鼠ATP6V1B2蛋白的第288位至第512位氨基酸序列中的至少部分序列。
在某些实施方式中，所述ATP6V1B2的功能活性片段包含SEQ ID NO.10-11中任一项所示的氨基酸序列。
在某些实施方式中，所述核酸分子包含SEQ ID NO.9或17所示的核苷酸序列。
在某些实施方式中，所述非人动物为经基因编辑的非人动物，或源自经基因编辑的非人动物。
在某些实施方式中，所述非人动物是基因敲入的非人动物，或源自基因敲入的非人动物。
在某些实施方式中，所述ATP6V1B2和/或其功能活性片段的表达由所述非人动物基因组中相应的内源性调控元件调控。
在某些实施方式中，所述非人动物为非人哺乳动物。
在某些实施方式中，所述非人动物选自：小鼠，大鼠，兔，猿，猴，猪，牛，羊，马，鸡，狗，羊驼和猫。
在某些实施方式中，所述非人动物为啮齿类动物或灵长类动物。
在某些实施方式中，所述部分包括所述非人动物的器官、组织和/或细胞。
在某些实施方式中，所述部分包括体液。
在某些实施方式中，所述体液选自下组：血液、血浆、血清、尿液、汗液、眼泪、唾液、 精液和脑脊液。
在某些实施方式中，所述血浆包括外泌体。
在某些实施方式中，所述部分包括脑或其一部分。
在某些实施方式中，所述脑的一部分包括的脑部位选自下组：嗅球、杏仁核、基底神经节、海马、丘脑、下丘脑、大脑皮层、延髓和小脑。
在某些实施方式中，所述部分包括嗅粘膜。
在某些实施方式中，所述部分包括中枢神经系统的一部分、外周神经系统的一部分、皮肤组织、肌肉组织和/或内脏器官。
在某些实施方式中，所述中枢神经系统包括脊髓。
在某些实施方式中，所述部分包括细胞，且所述细胞包括选自下组的细胞：原代神经元、小胶质细胞、星形胶质细胞、少突胶质细胞、巨噬细胞、血管周上皮样细胞、B细胞、T细胞、成体干细胞、NK细胞、全能干细胞、单能干细胞、胚胎干细胞、诱导性多能干细胞和配子。
在某些实施方式中，所述配子包括精子和/或卵母细胞。
在某些实施方式中，所述部分不能发育成完整的非人动物。
在某些实施方式中，后代通过将所述非人动物与另一非人动物杂交而获得，其中所述另一非人动物可以表达或可以不表达所述ATP6V1B2和/或其功能活性片段。
在某些实施方式中，所述的后代或其部分包含和/或表达所述ATP6V1B2和/或其功能活性片段。
另一方面，本申请提供一种一种分离的细胞，其包含和/或表达ATP6V1B2、其功能活性片段和/或编码它们的核酸分子。
在某些实施方式中，所述ATP6V1B2源自人。
在某些实施方式中，所述ATP6V1B2和/或其功能活性片段包含SEQ ID NO:8、10-11、16中任一项所示的氨基酸序列。
在某些实施方式中，编码所述ATP6V1B2和/或其功能活性片段的基因是纯合的或杂合的。
在某些实施方式中，编码所述ATP6V1B2和/或其功能活性片段的核酸分子包含SEQ ID NO:9或17所示的核酸序列。
在某些实施方式中，所述ATP6V1B2和/或其功能活性片段的表达由所述细胞基因组中相应的内源性调控元件调控。
在某些实施方式中，所述的细胞为非人动物细胞。
在某些实施方式中，所述非人动物为非人哺乳动物。
在某些实施方式中，所述非人动物选自：小鼠，大鼠，兔，猿，猴，猪，牛，羊，马，鸡，狗，羊驼和猫。
在某些实施方式中，所述非人动物为啮齿类动物或灵长类动物。
在某些实施方式中，所述的细胞包括选自下组的细胞：神经母细胞瘤细胞，脑胶质瘤细胞，原代神经元、小胶质细胞、星形胶质细胞、少突胶质细胞、巨噬细胞、血管周上皮样细胞、B细胞、T细胞、成体干细胞、NK细胞、全能干细胞、单能干细胞、胚胎干细胞、诱导性多能干细胞和配子。
在某些实施方式中，所述配子包括精子和/或卵母细胞。
在某些实施方式中，所述的细胞不能发育成完整的非人动物。
另一方面，本申请提供一种组织，其包含和/或表达ATP6V1B2、其功能活性片段和/或编码它们的核酸分子。
在某些实施方式中，所述ATP6V1B2源自人。
在某些实施方式中，所述ATP6V1B2和/或其功能活性片段包含SEQ ID NO:8、10-11、16中任一项所示的氨基酸序列。
在某些实施方式中，编码所述ATP6V1B2和/或其功能活性片段的基因是纯合的或杂合的。
在某些实施方式中，编码所述ATP6V1B2和/或其功能活性片段的核酸分子包含SEQ ID NO:9或17所示的核酸序列。
在某些实施方式中，所述组织是经基因编辑的组织，或源自经基因编辑的组织。
在某些实施方式中，所述组织是基因敲入的组织，或源自基因敲入的组织。
在某些实施方式中，所述ATP6V1B2和/或其功能活性片段的表达由所述组织基因组中相应的内源性调控元件调控。
在某些实施方式中，所述组织为非人动物的组织。
在某些实施方式中，所述非人动物为非人哺乳动物。
在某些实施方式中，所述非人动物选自：小鼠，大鼠，兔，猿，猴，猪，牛，羊，马，鸡，狗，羊驼和猫。
在某些实施方式中，所述非人动物为啮齿类动物或灵长类动物。
在某些实施方式中，所述组织包括体液。
在某些实施方式中，所述体液选自下组：血液、血浆、血清、尿液、汗液、眼泪、唾液、精液和脑脊液。
在某些实施方式中，所述血浆包括外泌体。
在某些实施方式中，所述组织包括脑或其一部分。
在某些实施方式中，所述脑的一部分包括的脑部位选自下组：嗅球、杏仁核、基底神经节、海马、丘脑、下丘脑、大脑皮层、延髓和小脑。
在某些实施方式中，所述组织包括嗅粘膜。
在某些实施方式中，所述组织包括中枢神经系统的一部分、外周神经系统的一部分、皮肤组织、肌肉组织和/或内脏器官。
在某些实施方式中，所述中枢神经系统包括脊髓。
在某些实施方式中，所述组织不能发育成完整的非人动物。
另一方面，本申请提供一种细胞系或细胞培养物，其源自本申请所述的非人动物或其部分，源自本申请所述后代，源自本申请所述细胞，和/或源自本申请所述组织。
在某些实施方式中，所述细胞培养物为原代细胞培养物。
在某些实施方式中，所述细胞培养物包括类器官。
另一方面，本申请提供一种组合物，其包含本申请所述非人动物或其部分、本申请所述后代、本申请所述细胞、本申请所述组织，和/或本申请所述细胞系或原代细胞培养物。
另一方面，本申请提供一种试剂盒，其包含本申请所述非人动物或其部分、本申请所述后代、本申请所述细胞、本申请所述组织，和/或本申请所述细胞系或原代细胞培养物；和一种或多种选自下组的另外的组分：分析缓冲液、对照物、底物、标准品、检测材料、实验室用品、设备、仪器、细胞、器官、组织和用户手册或说明书。
另一方面，本申请提供一种筛选能够改善受试者学习能力、治疗认知障碍、和/或治疗神经退行性疾病的药物的方法，所述方法包括将候选的药物施用于本申请所述非人动物或其部分、本申请所述后代、本申请所述细胞、本申请所述组织，和/或本申请所述细胞系或原代细胞培养物，并测定所述候选药物对受试者中ATP6V1B2的表达水平和/或活性的影响。
另一方面，本申请提供一种筛选能够改善受试者学习能力、治疗认知障碍、和/或治疗神经退行性疾病的药物的方法，所述方法包括：(i)将候选的药物施用于本申请所述非人动物或其部分、本申请所述后代、本申请所述细胞、本申请所述组织，和/或本申请所述细胞系或原代细胞培养物；(ii)测定所述候选的药物对对受试者中ATP6V1B2的表达水平和/或活性的影响；(iii)在施用所述候选药物后，所述ATP6V1B2的表达水平和/或活性提高，则所述 候选药物能够改善受试者学习能力、治疗认知障碍、和/或治疗神经退行性疾病。
在某些实施方式中，所述ATP6V1B2的表达水平包括ATP6V1B2基因的表达水平、ATP6V1B2基因的转录水平和/或ATP6V1B2蛋白的表达水平。
在某些实施方式中，所述提高包括与所述受试者中原始ATP6V1B2的表达水平和/或活性相比，所述ATP6V1B2的表达水平和/或活性提高至少约10％。
在某些实施方式中，所述学习能力包括认知能力、运动能力、记忆能力和/或空间探索能力。
在某些实施方式中，所述认知障碍包括早期认知障碍(MCI)、中期认知障碍和晚期认知障碍。
在某些实施方式中，所述认知障碍包括正常衰老引起的认知障碍、lews身体痴呆(LBD)、额颞叶痴呆和/或血管性痴呆。
在某些实施方式中，所述认知障碍的诱导疾病包括阿尔兹海默症、多发性梗死型、帕金森氏症、艾滋病和/或克雅氏病(CJD)。
在某些实施方式中，所述神经退行性疾病包括急性神经退行性疾病和慢性神经退行性疾病。
在某些实施方式中，所述神经退行性疾病包括由神经元死亡和胶质细胞稳态引起的神经退行性疾病、由衰老引起的神经退行性疾病、由CNS细胞功能被影响引起的神经退行性疾病、由细胞间异常通信引起的神经退行性疾病和/或由细胞运动受损引起的神经退行性疾病。
在某些实施方式中，所述神经退行性疾病包括阿尔兹海默症、帕金森氏症、多发性硬化(MS)、肌萎缩性脊髓侧索硬化症(ALS)和/或亨廷顿病(HD)。
另一方面，本申请提供一种本申请所述非人动物或其部分、本申请所述后代、本申请所述细胞、本申请所述组织，和/或本申请所述细胞系或原代细胞培养物在制备鉴别和/或筛选系统中的用途，所述系统用于筛选能够改善受试者学习能力、治疗认知障碍、和/或治疗神经退行性疾病的药物和/或生物标志物。
另一方面，本申请提供一种本申请所述非人动物或其部分、本申请所述后代、本申请所述细胞、本申请所述组织，和/或本申请所述细胞系或原代细胞培养物，其可用于筛选能够改善受试者学习能力、治疗认知障碍、和/或治疗神经退行性疾病的药物和/或生物标志物。
另一方面，本申请提供制备本申请所述非人动物或其部分、本申请所述细胞或本申请所述组织的方法，所述方法包括使非人动物或其部分、细胞或组织包含和/或表达所述ATP6V1B2和/或其功能活性片段。
另一方面，本申请提供制备疾病模型的方法，所述方法包括使非人动物或其部分、细胞或组织包含和/或表达ATP6V1B2和/或其功能活性片段。
在某些实施方式中，所述ATP6V1B2和/或其功能活性片段包含SEQ ID NO:8、10-11、16中任一项所示的氨基酸序列。
在某些实施方式中，所述方法包括转染包含编码所述ATP6V1B2和/或其功能活性片段的核酸序列。
在某些实施方式中，编码所述ATP6V1B2和/或其功能活性片段的核酸序列包含SEQ ID NO:9或17所示的核酸序列。
在某些实施方式中，所述转染包括：转染包含所述核酸序列的腺相关病毒。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明书如下：
图1a-1i显示的是本申请所述多肽对兴奋性突触传递的影响。
图2显示的是本申请所述多肽通过灌胃施用能够改善AD小鼠新物体识别试验中的认知行为。
图3显示的是本申请所述多肽通过灌胃施用能够改善AD小鼠水迷宫试验中的学习能力。
图4显示的是本申请所述多肽通过静脉施用能够改善AD小鼠新物体识别试验中的认知行为。
图5显示的是本申请所述多肽通过静脉施用能够改善AD小鼠水迷宫试验中的学习能力。
图6a-6b显示的是本申请所述多肽与ATP6V1B2蛋白的免疫共沉淀结果。
图7a-7e显示的是在老龄动物海马中过表达ATP6V1B2蛋白能够改善学习记忆功能。
图8显示的是本申请所述多肽的二聚体对兴奋性突触传递的影响。
图9显示的是半胱氨酸经修饰的本申请所述多肽对兴奋性突触传递的影响。
图10显示的是丝氨酸经修饰的本申请所述多肽对兴奋性突触传递的影响。
图11显示的是本申请所述多肽与ATP6V1B2蛋白或其截短片段的免疫共沉淀结果。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
术语“ATP6V1B2”通常是指ATP酶H +运输V1亚基B2(ATPase H+transporting V1subunit B2)蛋白(或称为ATP6B2，DOOD，HO57，VATB，VPP3，Vma2或ZLS2)，以及编码该蛋白的基因。所述ATP6V1B2可以为一种介导真核细胞内细胞器酸化的多亚基酶。所述ATP6V1B2可以参与蛋白质分选、酶原激活、受体介导的内吞作用和突触囊泡质子梯度生成等过程。所述ATP6V1B2蛋白可以包括胞质V1结构域和跨膜V0结构域。人ATP6V1B2在GenBank中的登录号为526。人ATP6V1B2在UniProt中的登录号可以为P21281。
术语“表达水平”通常是指特定相关基因的蛋白质、RNA或mRNA水平。可以采用本领域中已知的任何方法来测定特定相关基因(例如人ATP6V1B2基因)的表达水平。在本申请中，所述“表达”通常是指将基因编码的信息转变成存在于细胞中并在细胞中操作的结构的过程。例如可以包括逆转录和扩增分析(例如PCR、连接RT-PCR或定量RT-PCT)、杂交分析、RNA印迹法(Northern blotting)、斑点印迹法、原位杂交、胶凝电泳、毛细管电泳、柱色谱、蛋白质印迹法、免疫组织化学、免疫染色或质谱。可以直接对生物样品或对从样品中分离出来的蛋白质/核酸执行分析。
术语“活性”通常是指与特定蛋白质相关的任何活性。在本申请中，所述活性可以包括例如ATP6V1B2蛋白相关的任何活性。所述活性可以包括蛋白酶相关的酶促活性。在某些情况下，所述活性可以包括生物活性。在某些情况下，所述活性可以包括蛋白质与受体的结合，例如，该结合可能产生可测量的下游效应。在本申请中，所述活性可以包括将被本领域技术人员归因于该蛋白的任何活性。
术语“质子泵调控剂”通常是指能够调理质子泵活性和/或功能的试剂。
术语“质子泵激动剂”通常是指激活质子泵的试剂。例如，所述质子泵激动剂可以提高氢离子泵的活性。
术语“囊泡型质子泵激动剂”通常是指能够对囊泡型质子泵起到提高其活性作用的试剂。囊泡型或空泡型腺苷三磷酸酶(V-ATPase)是一种ATP驱动的质子泵，由一个用于ATP水 解的胞浆V1复合物和一个用于质子转移的膜包埋Vo复合物组成。所述囊泡型质子泵可以在真核生物细胞内囊泡、细胞器和胞外环境的酸化中发挥重要作用。
术语“质子泵相关蛋白”通常是指编码和/或表达质子泵的相关蛋白。所述质子泵可以为在生物膜上逆膜两侧氢离子电化学势差主动运输氢离子的蛋白质。所述质子泵可以包括Na-K泵、Ca 2+泵、H +-ATP泵和H +焦磷酸泵。
术语“学习能力”通常是指一切与学习/认知过程相关或所需的能力。所述学习能力可以包括通过包括体验、学习或接受训练等的过程来获得新的信息、知识和/或技能的能力。所述学习能力可以包括想象力、注意力、感知观察能力、阅读能力、分析能力、操作能力、适应能力、归纳总结能力、解决问题能力或其组合。
术语“认知能力”通常是指通过感知处理信息的能力。所述认知能力可以包括对事物的构成、性能与他物的关系、发展的动力、发展方向以及基本规律的把握能力。
术语“运动能力”通常是指参加运动和训练所具备的能力。所述运动能力可以包括有氧运动能力、肌肉力量、身体柔韧性、平衡能力和反应能力。所述运动能力可以是身体形态、素质、技能、技能和心理能力等多种因素的综合表现。
术语“记忆能力”通常是指识记、保持、再认识和重现客观事物所反映的内容和经验的能力。所述记忆能力可以包括感觉记忆能力、短时记忆能力和长期记忆能力。
术语“空间探索能力”通常是指探索物体形状和/或位置的能力。所述空间探索能力包括观察、思考、想象、认识和/或摸索物体形状和/或位置。
术语“学习能力的评估分数”通常是指对受试者学习能力的定量测评分数。所述学习能力的评估分数可以通过进行包括注意/执行功能评估(例如韦氏记忆测验)、语言能力评估(例如语言筛查测验(the language screening test,LAST))、视空间和结构能力评估(例如视觉运动整合测验、Hooper视觉组织测验、物品拼凑测验、图形排列测验、画钟测验)、运用能力评估、日常功能评估(例如痴呆残疾评估(disability assessment for dementia，DAD))和/或神经心理量表所得到的分数来获得。在某些情况下，所述学习能力的评估分数可以通过进行简易精神状态检查(mini-mental state examination,MMSE)、蒙特利尔认知评估量表(Montreal cognitive assessment，MoCA)、阿尔茨海默病评估量表-认知部分(Alzheimer disease assessment scale-cog，ADAS-cog)和临床痴呆评定量表(clinical dementia rating scale,CDR)检测所获得的分数来获得。
术语“新物体认知试验”通常是指通过使动物(例如小鼠)在特定空间内识别物体，从而检测动物学习对新物体的识别所需时间的试验。在某些情况下，所述新物体认知试验可以 参考Ennaceur等，Behav Brain Res 80 9-25，1996所记载的试验方法。例如，所述新物体认知试验可以包括以下步骤：将两个相同的物体放入固定体积的容器中，将小鼠放入该容器中识别这两个物体，第二天，将同一容器中的两个物体之一替换为具有不同形状的新物体，然后测量小鼠对新物体的搜寻时间。
术语“水迷宫试验”通常是指强迫动物(例如小鼠)游泳，从而学习寻找隐藏在水中的平台的试验。所述水迷宫试验(例如Morris水迷宫)可以测试小鼠对空间位置感和方向感的学习能力和/或记忆能力。所述水迷宫试验也可包括获得性训练、探查训练、对位训练或对位探查训练。如果动物(例如小鼠)从进入水中到找到平台所需的时间越短，在此期间运动的距离约短，则动物(例如小鼠)的学习能力的评估分数相应约高。水迷宫评价试验可以是评价学习能力的重要实验。
术语“神经退行性疾病”通常是指由神经元结构和功能逐渐丧失，包括神经元死亡和胶质细胞平衡，所导致的痴呆等认知障碍。在某些情况下，年龄(例如阿尔茨海默病(AD)、帕金森病(PD))或影响CNS细胞功能的基因突变(例如亨廷顿氏舞蹈病、早发性AD或PD、肌萎缩性脊髓侧索硬化症(ALS))可以引起所述神经退行性疾病。所述神经退行性疾病可以具有选自以下的变化和/或病症：蛋白错误折叠和聚集；神经炎症(例如在毒刺激(如蛋白聚集)、感染、创伤性损伤或自身免疫等信号刺激下出现的CNS炎症)；细胞信号转导的改变；获得性衰老/细胞死亡(例如被中断的凋亡信号转导、线粒体功能障碍、自噬受损以及坏死小体被应激/炎症激活)；运动细胞受损以及表观遗传学变化。
术语“阿尔兹海默症”通常是指早老性痴呆、老年痴呆，是一种发病进程缓慢、随着时间不断恶化的神经退化性疾病。最常见的早期症状为丧失短期记忆(难以记住最近发生的事)，当疾病逐渐进展，可能逐渐出现下述症状中的至少一种：语言障碍、定向障碍(例如，容易迷路)、情绪不稳、丧失动机、无法自理和行为问题。阿尔茨海默病的真正成因至今仍然不明，其进程可能与大脑中的纤维状类淀粉蛋白质斑块沉积和Tau蛋白有关。目前并没有可以阻止或逆转病程的治疗，只有少数方法或许可以暂时缓解或改善症状。
术语“早期认知障碍(MCI)”通常是指一种介于正常认知与认知障碍的中间临床状态。在某些情况下，所述MCI可以包括满足痴呆标准但程度超过正常衰老的认知损害。MCI在临床表现、病因、预后和患病率方面存在多样化。在某些情况，MCI可以为阿尔茨海默症的一个病理阶段。某些形式的认知损害可视为神经变性疾病的早期表现，最终将导致痴呆。
术语“正常衰老引起的认知障碍”通常是指由于正常衰老引起的认知损害。例如，所述正常衰老引起的认知障碍可以表现为：记忆丧失、对熟悉地方的位置感到困惑、完成日常工 作需要比平时更长时间、或者情绪和性格的变化。
术语“lews身体痴呆(LBD)”通常是指Lewy Body Detmentia，路易体痴呆。路易体痴呆的特征是蛋白质异常积聚成为称为路易体的肿块。路易体痴呆症导致心智能力逐渐下降。路易体痴呆症患者可能会出现幻视以及警觉性和注意力的变化。其他影响包括肌肉僵硬、运动缓慢、行走困难和颤抖。大脑中有路易体的患者也可以有与阿尔茨海默病相关的斑块和缠结。
术语“额颞叶痴呆”通常是指皮克病，其是一种tau蛋白只影响大脑的额叶和颞叶的渐进性的罕见病。额颞叶痴呆患者在更高层次的推理、表达语言、语言感知和记忆形成方面都有困难。额颞叶痴呆患者的大脑的额叶和颞叶可以随着时间推移而萎缩。
术语“血管性痴呆”通常是指由于大脑血液流动受损而导致的推理、判断和记忆方面的问题。例如，所述血管性痴呆可以包括由于心脏病和中风风险的因素，如高血压和高胆固醇而导致的痴呆。
术语“多发性梗死型”通常是指由大型脑动脉的单个穿通支闭塞引起的非皮质性小梗死。所述多发性梗死型可以是脑梗塞的一种特殊类型，又称缺血性中风。所述多发性梗死型可以表现为偏身感觉障碍、失语、构音不全、动作缓慢、笨拙(尤以精细动作如书写更为困难)。
术语“帕金森氏症”通常是指一种进行性神经变性疾病。所述帕金森氏症(PD)的临床特征可以包括运动症状(例如震颤、运动徐缓、肌强直和姿势不稳)，以及神经精神性及其他非运动表现。例如，所述非运动表现可以包括认知功能障碍和痴呆、心境障碍(例如抑郁、焦虑、情感淡漠)和睡眠障碍。
术语“艾滋病”通常是指获得性免疫缺陷综合征(AIDS)。AIDS的临床表现包括记忆力、专注力、注意力和运动技能的改变。在某些情况下，AIDS患者可以出现认知障碍，例如，约50％的感染者可能进一步发展为HIV相关的神经认知功能障碍综合征(HIV-associated neurocognitive disorders，HAND)。
术语“克雅氏病(CJD)”通常是指一种发生在人类身上的传染性海绵状脑病。CJD是由朊病毒感染引起的疾病。CJD患者可以表现为偏执行为，意识模糊、食欲及体重下降、抑郁，少数患者有视觉或听觉异常；在进展期，表现为进行性的神经系统病情恶化(例如感觉异常、语言障碍和失语)。
术语“多发性硬化(MS)”通常是指一种脱髓鞘性神经病变。所述MS患者脑或脊髓中的神经细胞表面的绝缘物质(即髓鞘)受到破坏，神经系统的信号转导受损，可以导致一系列可能发生的症状，影响患者的活动、心智、甚至精神状态。这些症状可以包括复视、单侧 视力受损、肌肉无力、感觉迟钝，或协调障碍。
术语“肌萎缩性脊髓侧索硬化症(ALS)”通常是指渐冻人症、运动神经元病，是一种渐进且致命的神经退行性疾病。其中少数ALS患者可以出现额颞叶痴呆。部分ALS患者的觉、视觉、触觉、嗅觉和味觉会出现退化，极少数肌萎缩侧索硬化症患者会同时出现痴呆症。
术语“亨廷顿病(HD)”，即亨廷顿舞蹈症，通常是指一种会导致脑细胞死亡的遗传性疾病。HD患者随着疾病的进展，身体运动的不协调变得更加明显，能力逐渐恶化直到运动变得困难，无法说话。心智能力则通常会衰退为痴呆症。
术语“老龄阶段”通常是指受试者的老龄化阶段。例如，对人而言，所述老龄阶段可以为60岁以上，70岁以上或75岁以上；对小鼠而言，所述老龄阶段可以为10月龄以上，例如可以为13月龄以上或者18月龄以上。在某些情况下，所述老龄阶段的受试者可以具有学习缺陷、记忆力障碍、记忆力缺陷和/或脑功能障碍中的一种或多种症状。
术语“变体”通常是指包含与亲本或参考多肽(例如野生型多肽)的氨基酸序列相差至少一个氨基酸残基的氨基酸序列的多肽。在本申请中，所述变体可以与所述亲本或参考多肽具有较高的(例如至少80％)的同源性。所述同源性可以包括序列的相似性或同一性。在本申请中，所述同源性可以用本领域已知的标准技术确定(参见例如Smith和Waterman，Adv.Appl.Math.《应用数学进展》)；多核苷酸或多肽序列共有的同一性百分数通过分子之间序列信息的直接比较测定，所述比较通过序列比对并使用本领域已知的方法确定同一性来进行。适用于确定序列相似性的算法的例子是BLAST算法(参见Altschul等人，J.Mol.Biol.《分子生物学杂志》，215:403-410[1990])。用于进行BLAST分析的软件可通过国家生物技术信息中心(NCBI)公开获得。
术语“递质释放”通常是指神经递质释放，即一个神经元通过释放囊泡中包裹的神经递质(Neurotransmitter)到突触间隙，作用到另一个神经元上，从而传递信息的过程。在所述递质释放过程中，可以涉及神经环路的基本结构突触(Synapse)。在某些情况，所述递质释放可以被称为突触传递(Synaptic transmission)。所述递质释放的方法可以包括同步释放(Synchronous release)、非同步释放(Asynchronous release)和自发释放(Spontaneous release)。
术语“发放频率”通常是指动作电位的发放频率。动作电位可以是指可兴奋细胞受到刺激时在静息电位的基础上产生的一次膜两侧电位的快速而可逆转的倒转和复原的过程。动作电位可以由峰电位和后电位组成，分别对应去极化(Depolarisation)和超极化(Hyperpolarization)过程。动作电位的发放可以出现脉冲的特点。在某些情况下，脉冲的 发放频率为脉冲发放的数量与时间之比。
在本申请中，术语“神经元”通常是指神经细胞，其是神经系统主要的功能单元。神经元可以由细胞体及其突起轴突以及一个或多个树突组成。神经元可以通过在突触处释放神经递质向其他神经元或细胞传递信息。
术语“多聚体”通常是指具有共价、非共价或同时通过共价和非共价相互作用缔合的两个或多个多肽链的分子。所述多聚体可以包括二聚体。
术语“同二聚体”通常是指由两个相同的单体形成的分子。这两个相同的单体可以通过共价和/或非共价相互作用彼此聚集、络合或缔合。
术语“巯基封闭”通常是指使自由巯基被封闭而难以形成分子内和/或分子间的二硫键。在本申请中，所述巯基封闭可以发生在半胱氨酸残基上。所述巯基封闭可以使蛋白质的半胱氨酸残基之间无法形成二硫键而防止蛋白质进行交联或修饰。所述巯基封闭可以通过使用封闭剂实现，所述封闭剂可以为还原剂。所述封闭剂可以包括二硫苏糖醇(DTT)、β-巯基乙醇(BME)和三(2-羧乙基)膦盐酸盐(TCEP·HCl)。
术语“丝氨酸磷酸化”通常是指发生在丝氨酸残基上的磷酸化修饰。所述丝氨酸磷酸化可以为将供体(例如ATP或GTP)的磷酸基转移到丝氨酸残基上的过程。所述丝氨酸磷酸化可以借助蛋白质激酶。所述丝氨酸磷酸化可以导致蛋白质活性的变化。
术语“约”通常是指在比特定值多或少20％的数值范围。例如，“约X”包括为X的±20％、±10％、±5％、±2％、±1％、±0.5％、±0.2％或±0.1％的数值范围，其中X为数值。
发明详述
一方面，本申请提供一种质子泵调控剂在制备试剂中的用途，所述试剂用于改善学习能力。
另一方面，本申请提供一种质子泵调控剂在制备试剂中的用途，所述试剂用于治疗认知障碍。
另一方面，本申请提供一种质子泵调控剂在制备试剂中的用途，所述试剂用于治疗神经退行性疾病。
另一方面，本申请提供一种ATP6V1B2调控剂在制备试剂中的用途，所述试剂用于改善学习能力。
另一方面，本申请提供一种ATP6V1B2调控剂在制备试剂中的用途，所述试剂用于治疗认知障碍。
另一方面，本申请提供一种ATP6V1B2调控剂在制备试剂中的用途，所述试剂用于治疗神经退行性疾病。
在本申请中，当所述受试者中ATP6V1B2的表达水平和/或活性提高时，所述受试者的所述学习能力可以显著提高(例如，所述认知能力、运动能力、记忆能力和/或空间探索能力可以相较于当所述受试者中ATP6V1B2的表达水平和/或活性提高之前有显著提高)。因此，ATP6V1B2可以作为改善学习能力的潜在靶点。例如，ATP6V1B2可以作为治疗神经退行性疾病(例如阿尔兹海默症)和/或认知障碍的潜在靶点。
质子泵相关蛋白、ATP6V1B2的表达水平和/或活性提高
在本申请中，所述试剂可以使受试者中ATP6V1B2的表达水平和/或活性提高。
在本申请中，所述质子泵调控剂可以使受试者中ATP6V1B2的表达水平和/或活性提高。
在本申请中，所述ATP6V1B2调控剂可以使受试者中ATP6V1B2的表达水平和/或活性提高。
在本申请中，所述ATP6V1B2和/或其功能活性片段的表达水平可以包括ATP6V1B2基因的表达水平、ATP6V1B2基因的转录水平和/或ATP6V1B2蛋白和/或其功能活性片段的表达水平。例如，所述表达水平可以包括特定基因(例如人ATP6V1B2基因)多核苷酸、mRNA或氨基酸产物或蛋白质的量。所述表达水平可以包括特定基因(例如人ATP6V1B2基因)转录的多核苷酸、翻译的蛋白质或翻译后修饰的蛋白质的片段的量。
在本申请中，所述提高可以包括与所述受试者中原始ATP6V1B2的表达水平相比，所述ATP6V1B2的表达水平提高了至少约10％。例如，可以提高至少约15％、至少约20％、至少约25％、至少约30％、至少约35％、至少约40％、至少约45％、至少约50％、至少约55％、至少约60％、至少约65％、至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约100％、至少约200％、至少约500％或更多。
在本申请中，所述ATP6V1B2的表达水平可以通过利用选自下组的物质衡量：特异性扩增ATP6V1B2基因的引物、与ATP6V1B2基因特异性结合的核酸分子、与ATP6V1B2蛋白特异性结合的核酸分子、与ATP6V1B2蛋白特异性结合的小分子、与ATP6V1B2蛋白特异性结合的探针和与ATP6V1B2蛋白特异性结合的多肽。
在本申请中，所述ATP6V1B2的表达水平可以通过实施选自下组的试验衡量：逆转录和扩增分析(例如PCR、连接RT-PCR或定量RT-PCT)、杂交分析、RNA印迹法(Northern blotting)、斑点印迹法、原位杂交、胶凝电泳、毛细管电泳、柱色谱、蛋白质印迹法、免疫组织化学、免疫染色或质谱。例如，可以通过qPCR、qRT-PCR、northern杂交、western杂 交和/或ELISA检测对本申请所述的ATP6V1B2的表达水平进行测量。所述ATP6V1B2的表达水平也可以通过直接对生物样品或对从样品中分离出来的蛋白质/核酸执行分析来进行测量。
在本申请中，所述ATP6V1B2可以源自人或小鼠。
在本申请中，所述ATP6V1B2可以包含SEQ ID NO.8或16所示的氨基酸序列。
在本申请中，所述ATP6V1B2的功能活性片段可以具有与SEQ ID NO.5所示的氨基酸序列特异性结合的能力。例如，所述ATP6V1B2的功能活性片段可以包括ATP6V1B2的截短体。例如，所述ATP6V1B2的功能活性片段可以包含人ATP6V1B2蛋白的第288位至第512位氨基酸序列中的至少部分序列。例如，所述ATP6V1B2的功能活性片段可以包含小鼠ATP6V1B2蛋白的第288位至第512位氨基酸序列中的至少部分序列。
在本申请中，所述ATP6V1B2和/或其功能活性片段可以包含SEQ ID NO:8、10-11、16中任一项所示的氨基酸序列。
在本申请中，编码所述ATP6V1B2和/或其功能活性片段的核酸序列可以包含SEQ ID NO:9或17所示的核酸序列。
在本申请中，所述ATP6V1B2的活性可以包括ATP6V1B2蛋白和/或其功能活性片段的生物活性(例如可以包括其导致的可测量的下游效应)。例如，所述ATP6V1B2的活性可以包括使所述质子泵相关蛋白的表达水平和/或活性提高。
在本申请中，所述提高可以包括与所述受试者中原始ATP6V1B2的活性相比，所述ATP6V1B2的活性提高了至少约10％。例如，可以提高至少约15％、至少约20％、至少约25％、至少约30％、至少约35％、至少约40％、至少约45％、至少约50％、至少约55％、至少约60％、至少约65％、至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约100％、至少约200％、至少约500％或更多。
在本申请中，所述试剂可以使受试者中质子泵相关蛋白的表达水平和/或活性提高。
在本申请中，所述质子泵调控剂可以使受试者中质子泵相关蛋白的表达水平和/或活性提高。
在本申请中，所述ATP6V1B2调控剂可以使受试者中质子泵相关蛋白的表达水平和/或活性提高。
在本申请中，所述质子泵相关蛋白的表达水平可以质子泵相关蛋白的表达水平包括编码质子泵相关蛋白基因的表达水平、编码质子泵相关蛋白基因的转录水平和/或质子泵相关蛋白的表达水平。例如，所述表达水平可以包括特定基因(例如编码质子泵相关蛋白基因)多 核苷酸、mRNA或氨基酸产物或蛋白质的量。所述表达水平可以包括特定基因(例如编码质子泵相关蛋白基因)转录的多核苷酸、翻译的蛋白质或翻译后修饰的蛋白质的片段的量。
在本申请中，所述提高可以包括与所述受试者中原始的质子泵相关蛋白的表达水平相比，所述质子泵相关蛋白的表达水平提高了至少约10％。例如，可以提高至少约15％、至少约20％、至少约25％、至少约30％、至少约35％、至少约40％、至少约45％、至少约50％、至少约55％、至少约60％、至少约65％、至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约100％、至少约200％、至少约500％或更多。
在本申请中，所述质子泵相关蛋白的表达水平可以通过利用选自下组的物质衡量：能够特异性扩增编码质子泵相关蛋白基因的引物、与编码质子泵相关蛋白基因特异性结合的核酸分子、与质子泵相关蛋白特异性结合的核酸分子、与质子泵相关蛋白特异性结合的小分子、与质子泵相关蛋白特异性结合的探针和与质子泵相关蛋白特异性结合的多肽。
在本申请中，所述质子泵相关蛋白的表达水平可以通过实施选自下组的试验衡量：逆转录和扩增分析(例如PCR、连接RT-PCR或定量RT-PCT)、杂交分析、RNA印迹法(Northern blotting)、斑点印迹法、原位杂交、胶凝电泳、毛细管电泳、柱色谱、蛋白质印迹法、免疫组织化学、免疫染色或质谱。例如，可以通过qPCR、qRT-PCR、northern杂交、western杂交和/或ELISA检测对本申请所述的质子泵相关蛋白的表达水平进行测量。所述质子泵相关蛋白的表达水平也可以通过直接对生物样品或对从样品中分离出来的蛋白质/核酸执行分析来进行测量。
在本申请中，所述质子泵相关蛋白可以包括NADH脱氢酶、辅酶Q、琥珀酸-辅酶Q还原酶、细胞色素c和/或辅酶Q-细胞色素c还原酶。
在本申请中，所述质子泵相关蛋白的活性可以所述质子泵相关蛋白的生物活性。
所述质子泵相关蛋白的活性可以通过氢/钾三磷酸腺苷酵素系统(又称氢/钾离子ATP酶，即H +/K +ATPase)的活性水平；和/或H 2受体的活性水平来进行衡量。在本申请中，所述质子泵相关蛋白的活性的提高可以包括与所述受试者中原始质子泵相关蛋白的活性相比，所述质子泵相关蛋白的活性提高了至少约10％。例如，可以提高至少约15％、至少约20％、至少约25％、至少约30％、至少约35％、至少约40％、至少约45％、至少约50％、至少约55％、至少约60％、至少约65％、至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约100％、至少约200％、至少约500％或更多。
学习能力
在本申请中，所述学习能力可以包括一切与学习/认知过程相关或所需的能力。例如， 所述学习能力可以包括认知能力、运动能力、记忆能力和/或空间探索能力。
在本申请中，所述学习能力的改善可以包括与受试者的原始的学习能力的评估分数相比，改善后的所述受试者的学习能力的评估分数提高至少约50％。例如，可以提高至少约55％、至少约60％、至少约65％、至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约100％、至少约200％、至少约500％或更多。
在本申请中，所述学习能力的评估分数可以通过进行包括注意/执行功能评估(例如韦氏记忆测验)、语言能力评估(例如语言筛查测验(the language screening test,LAST))、视空间和结构能力评估(例如视觉运动整合测验、Hooper视觉组织测验、物品拼凑测验、图形排列测验、画钟测验)、运用能力评估、日常功能评估(例如痴呆残疾评估(disability assessment for dementia，DAD))和/或神经心理量表所得到的分数来获得。例如，所述学习能力的评估分数可以通过实施选自下组的试验衡量：新物体认知试验和水迷宫试验。
在本申请中，所述新物体认知试验可以评价所述认知能力、运动能力和/或空间探索能力。在本申请中，所述新物体认知试验可以使受试者(例如小鼠)在固定容器内探索并学习特定形状的物体，并根据学习获得的记忆在几天后区分出该固定容器内新出现的与之前物体形状不同的新物体。如果小鼠在几天后区分出该形状不同的新物体的时间越短，，则小鼠的学习能力相应越高。水迷宫评价试验可以是评价受试者习能力的重要实验。
在本申请中，所述水迷宫试验可以评价所述记忆能力、运动能力和/或空间探索能力。在本申请中，所述水迷宫试验(例如Morris水迷宫)可以强迫受试者(例如小鼠)游泳，从而学习寻找隐藏在水中的平台，进而测试小鼠对空间位置感和方向感的空间探索能力和/或记忆能力。水迷宫试验也可包括获得性训练、探查训练、对位训练或对位探查训练。如果小鼠从进入水中到找到平台所需的时间越短，在此期间运动的距离越短，则小鼠的学习能力相应越高。水迷宫评价试验可以是评价受试者学习能力的重要实验。
受试者和适应症
在本申请中，所述受试者可以包括哺乳动物。例如，所述受试者可以包括啮齿类动物和/或灵长类动物，例如所述受试者可以包括人。
在本申请中，所述受试者可以包括非认知障碍患者和/或非神经退行性疾病患者。例如，所述受试者可以为正常人和/或健康的人。例如，所述受试者可以具有进一步提高其学习能力的需求和/或愿望。
在本申请中，所述神经退行性疾病可以包括急性神经退行性疾病和慢性神经退行性疾病。例如，所述神经退行性疾病可以包括由神经元死亡和胶质细胞稳态引起的神经退行性疾病、 由衰老引起的神经退行性疾病、由CNS细胞功能被影响引起的神经退行性疾病、由细胞间异常通信引起的神经退行性疾病和/或由细胞运动受损引起的神经退行性疾病。
在本申请中，所述受试者可以包括神经退行性疾病患者。例如，所述受试者可以包括阿尔兹海默症患者。例如所述阿尔兹海默症患者可以处于阿尔兹海默症的前期、早期、中期或晚期。
在本申请中，所述认知障碍可以包括早期认知障碍(MCI)、中期认知障碍和晚期认知障碍。例如，所述认知障碍可以包括正常衰老引起的认知障碍、lews身体痴呆(LBD)、额颞叶痴呆和/或血管性痴呆。例如，所述认知障碍的诱导疾病可以包括阿尔兹海默症、多发性梗死型、帕金森氏症、艾滋病和/或克雅氏病(CJD)。
在本申请中，所述受试者可以包括认知障碍患者。例如，所述受试者可以患有早期认知障碍(MCI)(例如，失去短期记忆、表达或理解抽象事情时感困难、情绪或行为变幻无常、学习新事物及跟随复杂指令感到困难、判断力减退和/或基本自理需要旁人提醒)、中期认知障碍(例如，混淆远期记忆和现实情况记忆、词不达意、行为性格转变或情绪不稳和/或需要旁人协助自理)或晚期认知障碍(例如，记忆缺损、身体活动及精神状况出现衰退、不能有效表达或沟通、不能自理和/或生物钟混乱)。在本申请中，所述受试者可以患有能够引起诱导所述认知障碍的疾病。例如，所述受试者可以患有阿尔兹海默症、多发性梗死型、帕金森氏症、艾滋病和/或克雅氏病(CJD)。
在本申请中，所述受试者可以处于老龄阶段。例如，所述受试者已表现出了正常衰老引起的认知障碍。例如，所述受试者已表现出了早期认知障碍(MCI)的症状。例如，所述受试者已表现出了神经退行性疾病(例如阿尔兹海默症)的症状。
多肽或其变体、试剂和药物组合物及其应用
在本申请中，所述质子泵调控剂可以包括质子泵激动剂。
在本申请中，所述质子泵调控剂可以包括囊泡型质子泵激动剂。
在本申请中，所述质子泵调控剂可以包括ATP6V1B2蛋白的激动剂。
在本申请中，所述质子泵调控剂能够结合ATP6V1B2蛋白。例如，所述质子泵调控剂可以包括ATP6V1B2蛋白的配体。例如，所述质子泵调控剂可以包括ATP6V1B2蛋白的激动剂。例如，所述质子泵调控剂可以与所述ATP6V1B2蛋白中的特定位置(例如特定表位)特异性结合。
在本申请中，所述质子泵调控剂可以包括核酸或其变体、多肽或其变体和/或小分子。
在本申请中，所述质子泵调控剂能够增加神经元突触递质释放。
在本申请中，所述增加包括与所述受试者中原始神经元突触递质释放的水平相比，可以增加至少约10％。例如，可以提高至少约20％、至少约50％、至少约60％、至少约65％、至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约100％、至少约200％、至少约500％或更多。
在本申请中，所述质子泵调控剂能够增加兴奋性突触后电流的发放频率。
在本申请中，所述增加包括与所述受试者中原始兴奋性突触后电流的发放频率的水平相比，可以增加至少约10％。例如，可以增加至少约20％、至少约50％、至少约60％、至少约65％、至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约100％、至少约200％、至少约500％或更多。
在本申请中，所述ATP6V1B2调控剂可以包括ATP6V1B2激动剂。
在本申请中，所述ATP6V1B2能够结合ATP6V1B2蛋白。例如，所述ATP6V1B2调控剂可以包括ATP6V1B2蛋白的配体。例如，所述ATP6V1B2调控剂可以包括ATP6V1B2蛋白的激动剂。例如，所述ATP6V1B2调控剂可以与所述ATP6V1B2蛋白中的特定位置(例如特定表位)特异性结合。
在本申请中，所述ATP6V1B2调控剂可以包括质子泵激动剂，例如可以包括囊泡型质子泵激动剂。
在本申请中，所述ATP6V1B2调控剂可以包括核酸或其变体、多肽或其变体和/或小分子。
在本申请中，所述ATP6V1B2调控剂能够增加神经元突触递质释放。
在本申请中，所述增加包括与所述受试者中原始神经元突触递质释放的水平相比，可以增加至少约10％。例如，可以提高至少约20％、至少约50％、至少约60％、至少约65％、至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约100％、至少约200％、至少约500％或更多。
在本申请中，所述ATP6V1B2调控剂能够增加兴奋性突触后电流的发放频率。
在本申请中，所述增加包括与所述受试者中原始兴奋性突触后电流的发放频率的水平相比，可以增加至少约10％。例如，可以增加至少约20％、至少约50％、至少约60％、至少约65％、至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约100％、至少约200％、至少约500％或更多。
另一方面，本申请提供一种能够调控ATP6V1B2的表达水平和/或活性的多肽。
在本申请中，所述多肽能够增加神经元突触递质释放。在本申请中，所述增加包括与所 述受试者中原始神经元突触递质释放的水平相比，可以增加至少约10％。例如，可以提高至少约20％、至少约50％、至少约60％、至少约65％、至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约100％、至少约200％、至少约500％或更多。
在本申请中，所述多肽能够增加兴奋性突触后电流的发放频率。在本申请中，所述增加包括与所述受试者中原始兴奋性突触后电流的发放频率的水平相比，可以增加至少约10％。例如，可以增加至少约20％、至少约50％、至少约60％、至少约65％、至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约100％、至少约200％、至少约500％或更多。
在本申请中，所述多肽或其变体能够特异性结合ATP6V1B2蛋白。例如，所述多肽或其变体可以与所述ATP6V1B2蛋白具有“配体-受体”的关系。
在本申请中，所述多肽或其变体能够与小鼠ATP6V1B2蛋白的第288位至第512位氨基酸序列中的至少部分序列结合。在本申请中，所述多肽或其变体能够与人ATP6V1B2蛋白的第288位至第512位氨基酸序列中的至少部分序列结合。
在本申请中，所述多肽或其变体可以包括通过酰胺键(也称为肽键)线性连接的单体(氨基酸)构成的分子。所述多肽可以包括具有两个或更多个氨基酸的任意链，并且不指特定长度的产物。所述变体可以包括在所述多肽基础上包含一个或一个以上氨基酸取代、添加或缺失的多肽。所述变体可以包括在所述多肽基础上具有修饰的产物，例如所述修饰可以包括糖基化、乙酰化、磷酸化、酰化、通过已知的保护性/封闭性基团衍生化、蛋白水解分裂、和/或通过非天然存在的氨基酸修饰。
在本申请中，所述多肽或其变体能够使受试者中ATP6V1B2的表达水平和/或活性提高。其中，所述ATP6V1B2的表达水平可以包括ATP6V1B2基因的表达水平、ATP6V1B2基因的转录水平和/或ATP6V1B2蛋白的表达水平。在本申请中，所述提高可以包括与所述受试者中原始ATP6V1B2的表达水平和/或活性相比，所述ATP6V1B2的表达水平和/或活性提高至少约10％。例如，可以提高至少约20％、至少约50％、至少约60％、至少约65％、至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约100％、至少约200％、至少约500％或更多。
在本申请中，所述多肽可以包含SEQ ID NO.5所示的氨基酸序列。
在本申请中，所述多肽可以包含SEQ ID NO.1,3中任一项所示的氨基酸序列。
在本申请中，所述多肽或其变体可以形成多聚体。例如，可以形成同二聚体。在本申请 中，所述同二聚体可以仍具有与ATP6V1B2蛋白结合的能力。
所述同二聚体可以为2个完全相同的所述多肽的串联结构。例如，所述同二聚体可以包含SEQ ID NO.18所示的氨基酸序列。
在本申请中，所述多肽或其变体氨基酸序列中的半胱氨酸可以不具有巯基封闭的修饰。例如，如果所述多肽或其变体氨基酸序列中的半胱氨酸残基被巯基封闭，其可以丧失与ATP6V1B2蛋白结合的能力。
在本申请中，所述多肽或其变体氨基酸序列中的丝氨酸可以不具有磷酸化的修饰。例如，如果所述多肽或其变体氨基酸序列中的丝氨酸残基被磷酸化修饰，其可以丧失与ATP6V1B2蛋白结合的能力。
在本申请中，所述制剂(例如所述多肽或其变体)可以提高所述受试者的学习能力(例如相较于施用所述多肽或其变体前显著提高所述受试者的所述学习能力的评估分数)。
在本申请中，所述制剂(例如所述多肽或其变体)可以调节所述受试者中所述与学习能力相关的基因的表达量和/或活性。在本申请中，所述与学习能力相关的基因可以包括APP基因。APP基因为淀粉样前体蛋白基因(amyloid precursor protein)，该基因可以与阿尔茨海默病，例如家族性阿尔茨海默病(FAD)相关。此外，所述学习能力相关的基因可以包括早老素1基因(presenilin1，PSEN1)和早老素2基因(presenilin2，PSEN2)。例如，所述学习能力相关的基因可以包括编码谷氨酸受体的基因。例如，所述谷氨酸受体可以包括N-甲基-D-天冬氨酸受体(NMDAR)、海人藻酸受体(KAR)和α-氨基-3羟基-5甲基-4异恶唑受体(AMPAR)。例如，所述学习能力相关的基因可以包括编码美金刚(例如1-氨基-3，5-二甲基金刚烷胺盐酸盐)的作用受体的基因。例如，所述美金刚的作用受体可以包括NMDA受体。例如，所述多肽可以降低本段上述基因的表达量和/或活性。
在本申请中，本申请所述制剂、所述ATP6V1B2调控剂、所述质子泵调控剂、和/或本申请所述多肽可以作为改善学习能力的潜在药物。本申请所述制剂、所述ATP6V1B2调控剂、所述质子泵调控剂、和/或本申请所述多肽可以作为治疗认知障碍和/或治疗神经退行性疾病潜在药物。
另一方面，本申请提供一种药物组合，其本申请所述的试剂。
所述药物组合可以包括适用于药物用途(例如改善学习能力，治疗认知障碍和/或治疗神经退行性疾病(例如阿尔兹海默症))的药物产品。
另一方面，本申请提供一种药物组合物，其包含本申请所述的试剂以及药学上可接受的载体。
所述药物组合物可以为包含一种或多种活性成分(例如本申请所述的制剂)和一种或多种惰性成分的组合物；以及由任何两种或更多种成分的组合、复合或聚集，或由一种或多种成分的解离，或由一种或多种成分的其他类型的反应或相互作用直接或间接得到的任何产品。
所述药学上可接受的载体可以包括无菌的水或非水溶液、分散体、悬浮液或乳液及在使用前即刻用于重构成无菌可注射溶液或分散体的无菌粉末。合适的水和非水载体、稀释剂、溶剂或溶媒的实例可以包括水、乙醇、多元醇(例如丙二醇、丙二醇、聚乙二醇等等)、簇甲基纤维素及其合适的混合物、植物油(例如橄揽油)以及可注射的有机醋例如油酸乙醋。
另一方面，本申请提供一种本申请所述的试剂、本申请所述的药物组合和/或本申请所述的药物组合物在制备增加神经元突触递质释放的药物中的应用。
另一方面，本申请提供一种本申请所述的试剂、本申请所述的药物组合和/或本申请所述的药物组合物在制备改善学习能力的药物中的应用。
另一方面，本申请提供一种本申请所述的试剂、本申请所述的药物组合和/或本申请所述的药物组合物在制备治疗认知障碍和/或治疗神经退行性疾病的药物中的应用。
在本申请中，本申请所述多肽、本申请所述的试剂、本申请所述的药物组合和/或本申请所述的药物组合物可以被配制为适于口服和/或注射施用。在本申请中，本申请所述多肽、本申请所述的试剂、本申请所述的药物组合和/或本申请所述的药物组合物可以被配制为适于静脉注射施用。
药物筛选方法
另一方面，本申请提供一种筛选能够改善受试者学习能力、治疗认知障碍、和/或治疗神经退行性疾病的药物的方法，其包括以下的步骤：检测候选药物对受试者中质子泵相关蛋白的表达水平和/或活性的影响，其中在施用所述候选药物后，所述质子泵相关蛋白的表达水平和/或活性提高，则所述候选药物能够改善受试者学习能力、治疗认知障碍、和/或治疗神经退行性疾病。
在另一方面，本申请提供一种筛选能够改善受试者学习能力、治疗认知障碍、和/或治疗神经退行性疾病的药物的方法，其包括以下的步骤：检测候选药物对受试者中ATP6V1B2的表达水平和/或活性的影响，其中在施用所述候选药物后，所述ATP6V1B2的表达水平和/或活性提高，则所述候选药物能够改善受试者学习能力、治疗认知障碍、和/或治疗神经退行性疾病。
在某些情况下，所述候选药物可以包括能够调节ATP6V1B2的表达水平和/或活性的试剂；在某些情况下，所述候选药物可以包括能够调节质子泵相关蛋白的表达水平和/或活性 的试剂。在某些情况下，所述候选药物可以包括能够结合ATP6V1B2蛋白的试剂。
在本申请中，所述施用可以包括口服和/或注射。
在本申请中，所述施用可以包括静脉注射。
本申请还提供了一种可以检测ATP6V1B2表达水平和/或活性的检测试剂盒。所述检测试剂盒可以包含说明书，其中记载了如何采用所述检测试剂盒对质子泵相关蛋白和/或ATP6V1B2表达水平和/或活性进行检测的具体步骤，和/或如何利用检测结果判断所述候选药物能否改善受试者学习能力的具体步骤。
在本申请中，所述检测试剂盒还可以包括能够检测其他判断所述候选药物能否改善受试者学习能力的靶点的试剂。
非人动物、其后代或它们的部分
在本申请的任一方面中，所述非人动物可以是经基因编辑的非人动物，或源自经基因编辑的非人动物。例如，所述非人动物可以是基因敲入的非人动物，或源自基因敲入的非人动物。
例如，所述非人动物可以为非人哺乳动物。在某些实施方式中，所述非人动物为啮齿类动物或灵长类动物。在某些实施方式中，所述非人动物选自：小鼠，大鼠，兔，猿，猴，猪，牛，羊，马，鸡，狗，羊驼和猫。
在本申请的任一方面中，所述非人动物或其后代的部分可包括其器官、组织和/或细胞。
在某些实施方式中，所述部分可以包括体液。例如，所述体液可选自下组：血液、血浆、血清、尿液、汗液、眼泪、唾液、精液和脑脊液。在某些实施方式中，所述血浆中包括外泌体。在某些实施方式中，所述部分包括脑(例如，脑组织)或其一部分。所述脑的一部分可包括选自下组的部分：嗅球、杏仁核、基底神经节、海马、丘脑、下丘脑、大脑皮层、延髓和小脑。在某些实施方式中，所述部分包括嗅粘膜。在某些实施方式中，所述部分包括中枢神经系统的一部分、外周神经系统的一部分、皮肤组织、肌肉组织和/或内脏器官。例如，所述中枢神经系统可包括脊髓。
在某些实施方式中，所述部分可以包括细胞。所述细胞可包括选自下组的细胞：原代神经元、小胶质细胞、星形胶质细胞、少突胶质细胞、巨噬细胞、血管周上皮样细胞、B细胞、T细胞、成体干细胞、NK细胞、全能干细胞、单能干细胞、胚胎干细胞、诱导性多能干细胞和配子。例如，所述配子可包括精子和/或卵母细胞。
在某些实施方式中，所述部分(例如，所述器官、组织或细胞)不能发育成完整的非人动物。
在某些实施方式中，所述非人动物、其后代或它们的部分包含所述ATP6V1B2和/或其功能活性片段。例如，所述ATP6V1B2和/或其功能活性片段可以由该动物、该后代或该细胞表达，也可以来自其他来源，而后被引入(例如，注射到)所述动物、后代或细胞中，从而使其包含该ATP6V1B2和/或其功能活性片段。
在某些实施方式中，所述非人动物、其后代或它们的部分表达所述ATP6V1B2和/或其功能活性片段。
所述ATP6V1B2的表达可以包括ATP6V1B2基因的表达、ATP6V1B2基因的转录物的表达和/或ATP6V1B2蛋白的表达。例如，所述表达可以包括特定基因(例如人ATP6V1B2基因)多核苷酸、mRNA或氨基酸产物或蛋白质的表达。所述表达可以包括特定基因(例如人ATP6V1B2基因)转录的多核苷酸、翻译的蛋白质或翻译后修饰的蛋白质的片段的表达。
所述ATP6V1B2功能活性片段的表达可以包括编码ATP6V1B2的功能活性片段基因的表达、编码ATP6V1B2的功能活性片段基因的转录物和/或ATP6V1B2的功能活性片段蛋白的表达。例如，所述表达可以包括特定基因(例如编码人ATP6V1B2的功能活性片段(例如其中第288位至第512位氨基酸序列)的多核苷酸、mRNA或氨基酸产物或蛋白质的表达。所述表达可以包括特定基因转录的多核苷酸、翻译的蛋白质或翻译后修饰的蛋白质的片段的表达。
制备方法
本申请还提供了制备本申请所述的非人动物或其部分、本申请所述的细胞或本申请所述的组织的方法，所述方法包括使非人动物或其部分、细胞或组织包含和/或表达所述ATP6V1B2和/或其功能活性片段。
另一方面，本申请还提供了制备疾病模型的方法，所述方法包括使非人动物或其部分、细胞或组织包含和/或表达ATP6V1B2和/或其功能活性片段。
例如，所述方法可包括向所述非人动物或其部分、所述细胞、组织、细胞系和/或细胞培养物中引入所述ATP6V1B2、其功能活性片段、编码所述ATP6V1B2的核酸分子和/或编码ATP6V1B2的功能活性片段的核酸分子。所述引入可以包括例如，对所述非人动物、其部分、所述组织或细胞进行注射(例如，注射蛋白质或核酸分子)。例如，所述引入还可以包括核酸分子转染、病毒转导等其他将外源蛋白、外源核酸分子等引入细胞、组织或动物体内的手段。例如，所述转染可以包括使用腺相关病毒转染。
在某些实施方式中，所述方法可包括在所述非人动物或其部分、所述细胞、组织、细胞系和/或细胞培养物中敲入编码所述ATP6V1B2和/或其功能活性片段的核酸序列。
在某些实施方案中，该方法可以进一步包括鉴定含有敲入的异源核酸序列的经修饰的细胞或非人动物。
包含异源核酸序列的供体核酸分子也可以包含5'同源臂和3'同源臂。5′同源臂和3′同源臂可以位于编码所述ATP6V1B2和/或其功能活性片段的异源核酸序列的侧翼。供体核酸分子中的同源臂(例如5'同源臂或3'同源臂)可以具有足以促进与内源靶位点。
在本申请中，同源臂和靶基因位点(即内源ATP6V1B2基因位点内的同源基因区域或对应区域)彼此匹配或互相对应，这时两个局部序列的相互一致性就足够作为同源重组反应的基础。“同源性”是指DNA序列与对应或匹配序列相同或具有一定序列一致性。特定靶位点与在供体核酸分子中发现的相应同源臂之间的序列一致性可以是允许同源重组发生的任何程度的序列一致性。例如，供体核酸分子(或其片段)的同源臂与靶位点(或其片段)共有的序列同一性程度可以为至少约50％、至少约55％、至少约60％、至少约65％、至少约70％、至少约75％、至少约80％、至少约81％、至少约82％、至少约83％至少约84％、至少约85％、至少约86％％、至少约87％、至少约88％、至少约89％、至少约90％、至少约91％、至少约92％、至少约93％、至少约94％、至少约95％、至少约96％％、至少约97％、至少约98％、至少约99％、或至少约100％，以便对序列进行同源重组。
筛选/鉴别/设计方法
例如，所述装置可包括医疗装置。例如，被宣称在改善受试者学习能力、治疗认知障碍、和/或治疗神经退行性疾病方面有效的医疗设备。
本申请的所述鉴别、筛选或设计方法可以是体外方法、离体方法或体内方法。
在本申请中，候选物质可以是合成的化合物、肽，蛋白质，DNA文库或该文库中的核酸分子，动物(例如哺乳动物，如小鼠、大鼠、猪、牛、绵羊、猴子或人类)组织提取物或细胞培养上清液，植物或微生物的提取物或培养产物，或以上物质的任何混合物。
认知能力、运动能力、记忆能力和/或空间探索能力也可以在施用了候选的药物的治疗组和对照组之间进行比较，例如通过进行动物行为分析。例如，与对照组相比，在使用候选的药物处理后，如果观察到认知能力、运动能力、记忆能力和/或空间探索能力有显著改善，则可以选择该候选的药物进行进一步研究(例如，作为改善受试者学习能力、治疗认知障碍、和/或治疗神经退行性疾病的潜在治疗药物)。
在一些情况下，筛选的方法也可以使用根据本申请的非人动物组织和/或细胞进行，例如神经元细胞、脑部区域、或者包含/对应于AD病变的其他组织，或者其部分组织(例如嗅球、杏仁核、基底神经节、海马、丘脑、下丘脑、大脑皮层、延髓、中枢神经系统、周围 神经系统、脊髓、小脑、皮肤组织、肌肉组织、和/或内脏器官)。例如，可以在体外或离体(ex vivo)培养组织和/或细胞，然后在孵育适当的时间段后，将候选的药物施用于培养的组织和/或细胞(例如数小时、数天、数周、或数月)，并使用如上所述的方法对经处理的脑组织/细胞进行检查。
在某些情形中，例如在筛选生物标志物的方法中，可以采集源自本申请的非人动物、其部分、或其后代在出现神经系统疾病或病症相关指征(例如，Aβ沉积、神经原纤维缠结、形态或功能异常(萎缩)突触，神经元细胞死亡、或记忆力和学习功能受损)之前和之后的样品(例如细胞、组织或其他含DNA或RNA的样品、含蛋白质的样品、和/或含代谢物的样品)，然后对来自样品的基因转录产物(转录组)、基因翻译产物(蛋白质组)、脂质(脂质组)或代谢物(代谢组)进行全面分析，并鉴定出相关指征出现之前与之后发生了变化的物质。在某些实施方案中，所述样品可包含所述非人动物的体液，例如大鼠的血液、血浆(例如，包含外泌体的血浆)、血清、尿液、汗液、眼泪、唾液、精液、和/或脑脊髓液。
可以使用核酸微阵列芯片(microarray)，例如DNA微阵列芯片，来分析基因转录产物(例如转录组)；可以使用凝胶电泳(例如二维凝胶电泳)或质谱(例如飞行时间质谱，电子喷雾电离质谱，毛细管HPLC/MS和LC/MS/MS分析基因翻译产物(例如蛋白质组))；可以使用NMR，毛细管电泳，LC/MS和LC/MS/MS分析代谢物(代谢组)。
当某种物质的存在/含量在出现相关指征之前和之后表现出显著差异时，可以将该物质视为改善受试者学习能力、治疗认知障碍、和/或治疗神经退行性疾病的生物标志物。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的融合蛋白、制备方法和用途等，而不用于限制本申请发明的范围。
实施例
实施例1过表达ATP6V1B2蛋白能够改善学习记忆功能
试验步骤：
15月龄的老龄小鼠，随机分为对照组和实验组，分别向实验组和对照组小鼠脑双侧背侧海马注射携带有ATP6V1B2-mCherry融合蛋白的腺相关病毒(pAAV2/9-hSyn-ATP6V1B2-Myc-2A-mCherry，其中ATP6V1B2-mCherry融合蛋白的氨基酸序列为SEQ ID NO.6所示)或携带mCherry的对照腺相关病毒，使实验组和对照组小鼠脑双侧背侧海马的神经元分别过表达ATP6V1B2-mCherry蛋白或者mCherry蛋白。术后一个月，小鼠进行水迷宫实验。(试验步骤见实施例4)
结果发现，在15月龄的老龄小鼠海马内过表达ATP6V1B2蛋白(见图7a,b)，可明显改善老龄小鼠水迷宫行为实验中的学习记忆能力(见图7c,d,e)。
实施例2作用靶点为ATP6V1B2蛋白的多肽
试验步骤(参考E.哈洛D.莱恩,美E.哈洛D.莱恩,沈关心,等.抗体技术实验指南[J].科学出版社,2002)：
(1)根据SEQ ID NO.1所示的氨基酸序列合成多肽QD202，并在多肽氨基C’端加入生物素标记；将QD202的氨基酸序列随机打乱，并合成乱序对照QD201(氨基酸序列如SEQ ID NO.7所示)，并在多肽氨基C’端加入生物素标记。使用PBS溶解多肽QD202和QD201，配制储液浓度为500μM。
(2)将生物素标记的QD202和QD201分别通过静脉以6.25mg/kg的剂量给药2只8月龄C57BL/6小鼠。4小时后将小鼠麻醉，收集大脑海马组织，加入裂解缓冲液，通过机械研磨破碎组织，离心后收集上清。上清中加入生物素抗体和Protein G琼脂糖珠进行免疫沉淀，试验步骤参考2002年科学出版社《抗体技术实验指南》一书。使用蛋白上样缓冲液洗脱蛋白。
(3)配制SDS-PAGE蛋白电泳胶，下层分离胶浓度为10％，上层浓缩胶浓度为4％，将样品加入上样孔中，进行SDS-PAGE电泳。电泳结束后，将分离胶切下使用考马斯亮蓝染色，结果见图6a。
(4)将考马斯亮蓝染色显示的分子量大小约为55kDa的特异条带切下，进行质谱前处理，将蛋白酶解为肽段，并进行脱盐处理。样品经质谱的质量分析器将离子碎片按质量数分开，经检测器得到质谱图，通过搜索软件，在蛋白质数据库比对所鉴别的肽段序列，以鉴定蛋白种类。
(5)通过与乱序对照QD201的比较，检测到与QD202特异性结合的蛋白为ATP6V1B2。
(6)构建3’端带有myc标签的鼠源Atp6v1b2表达质粒，在HEK 293细胞中转染质粒，2天后收集细胞，并提取细胞蛋白。细胞裂解液中加入生物素抗体和Protein G琼脂糖珠进行免疫共沉淀 [1]，使用蛋白上样缓冲液洗脱蛋白。
(7)配制SDS-PAGE蛋白电泳胶，下层分离胶浓度为10％，上层浓缩胶浓度为4％，将样品加入上样孔中，进行免疫印迹法检测蛋白，结果见图6b。通过免疫共沉淀的方法可发现QD202与ATP6V1B2蛋白特异结合。
结果发现，通过蛋白质谱分析及免疫共沉淀的方法可发现QD202与ATP6V1B2蛋白结 合。结果如图6a,b所示。
实施例3多肽的生物学活性试验
试验步骤：
根据SEQ ID NO.1所示的氨基酸序列合成多肽QD202。
根据SEQ ID NO.2所示的氨基酸序列合成多肽QD202N’-1。
根据SEQ ID NO.3所示的氨基酸序列合成多肽QD202C’-1。
根据SEQ ID NO.4所示的氨基酸序列合成多肽QD202C’-2。
将2-3月龄C57小鼠腹腔注射20％乌来糖0.1-0.15ml麻醉后，快速断头剥离脑组织，并置于预先通混合气(95％O 2和5％CO 2)的人工脑脊液(ACSF)冰水混合态液体中，放置2min，ACSF配方如下：NaCl 11.7mM，KCl 0.36mM，NaH 2PO 4 0.12mM，CaCl 2 0.25mM，MgCl 20.12mM，NaHCO 3 25mM，葡萄糖11mM。利用振动切片机切取350μm的海马冠状脑片。海马脑片在室温ACSF中放置30min以上进行恢复，然后将脑片置于记录槽中，并用通混合气的ACSF以2-2.5ml/min的速度持续灌流，在Olympus BX51正置显微镜下，对海马CA1锥体神经元进行盲扎封接，利用Axon700B放大器和1550B数模转换器进行全细胞膜片钳记录，钳制电压为-70mV的状态下，电压钳记录锥体神经元自发兴奋性突触后电流(sEPSC)，记录5min sEPSC后给与5μM QD202或QD202N’-1，C’-1，C’-2等合成肽5min，然后以ACSF灌流冲洗10min。以Minianalysis软件对所记录的数据进行分析。
结果发现，QD202可增加海马CA1神经元自发的兴奋性突触递质释放，主要表现为增加兴奋性突触后电流的发放频率，但对兴奋性突触后电流幅度没有影响(参见图1a,b,c)；
与QD202影响兴奋性突触传递的作用相比较，在N端去掉一个氨基酸即观察不到与QD202类似的作用效果(参见图1d,e)；在C端去掉一个氨基酸仍可观察到与QD202类似的作用效果(参见图1f,g)；在C端去掉两个氨基酸则观察不到与QD202类似的作用效果(参见图1h,i)。
实施例4多肽对学习能力的改善作用
新物体识别测试
新物体识别参考Leger,M.,et al.Object recognition test in mice.Nat Protoc.8,2531-2537(2013)所记载的行为试验方案，使用旷场箱(40×40×35cm由蓝色的不透明塑料制成。)进行新物体识别测试。在第一天，将小鼠放入旷场箱中适应10min。在第二天，将每只小鼠轻轻放在箱的中央，在中央区域放置两个相似物体(一号电池)，让其自由探索10min，然后 将小鼠送回它们的饲养笼。3h后，将小鼠再次放回箱(将一个电池更换为一个10厘米高的人偶玩具)10min以进行记忆保持测试。用Etho Vision XT 14软件分析录像，记录小鼠探索新/旧物体所用时间。辨别指数计算为(Tnovel-Tfamliar)/(Tnovel+Tfamiliar)。
莫里斯水迷宫测试
莫里斯水迷宫参考Qing-Feng Wu等人于Fibroblast growth factor 13 is a microtubule-stabilizing protein regulating neuronal polarization and migration.Cell.149,1549-1564(2012)所记载的试验方案。测试是在固定环境的房间中，在装有水的圆形水池(直径120cm，深度30cm，通过添加二氧化钛使其不透明，保持在21±1℃)中进行的。实验分为适应期(1天)、训练期(5-6天)和测试期(1天)。适应期：将平台置于水面上方0.5厘米处，引导小鼠上平台。训练日：将平台置于水下0.5厘米处，训练小鼠找平台。当小鼠到达平台时停止计时，若在1分钟内小鼠没有到达平台，则引导上平台，并使其停留在平台上30s。每天训练4次，每天训练4次(8：00-16：00间进行)，每次从不同的入水点放下小鼠，每次训练间隔至少30min，共训练6天，每天的入水点顺序不同。实验日：训练日结束的24小时之后进行实验。移除平台，小鼠从未训练过的入水点入水，录像1分钟。用Etho Vision XT 14软件分析录像，记录参数包括进入目标象限的逃避潜伏期、穿越平台的次数、在目标象限的停留时间。
4.1口服多肽
选择10个月龄的APP/PS1转基因小鼠(购自南模生物)，将这些小鼠等分为3组，分别向这些小鼠灌胃服用PBS缓冲液(组2，
)、QD202(组3，QD202的给药剂量为6.25mg/kg(其中QD202的浓度为500μM))和美金刚(组4，美金刚的给药剂量为1.25mg/kg，其中美金刚的浓度为1mM)，没有灌胃服用任何试剂的10月龄C57BL/6小鼠作为对照(组1)。
将这四组小鼠进行上述的行为学试验。
结果发现，QD202连续灌胃1周，APP/PS1转基因AD小鼠在新物体识别(参见图2)及水迷宫行为(参见图3)检测中表现出明显的学习认知功能改善。
4.2静脉注射多肽
选择10个月龄的APP/PS1转基因小鼠(购自南模生物)，将这些小鼠等分为2组，分别向这些小鼠静脉注射PBS缓冲液(组2，PBS的浓度为500μM)、QD202(组3，QD202的给药剂量为1.25mg/kg，其中QD202的浓度为500μM)，没有静脉注射任何试剂的10月龄C57BL/6小鼠作为对照(组1)。
将这四组小鼠进行上述的行为学试验。
结果发现，QD202连续静脉注射1周，APP/PS1转基因AD小鼠在新物体识别(参见图4)及水迷宫行为(参见图5)检测中表现出明显的学习认知功能改善。
由此可见，QD202对学习能力，尤其是记忆能力和认知能力有显著的改善作用。
实施例5经修饰的多肽，及其对学习能力的改善作用
5.1二聚体形式的多肽
制备QD202二聚体：取QD202，10mg/ml的浓度溶于水，加稀氨水调PH值8-9之间，搅拌氧化24小时，期间HPLC监测反应终点，氧化完全后再经HPLC纯化得到QD202二聚体。QD202二聚体包含SEQ ID NO.18所示的氨基酸序列。
按照实施例4记载的方法，使用QD202二聚体对小鼠进行锥体神经元自发兴奋性突触后电流检测。其中，对照指在正常灌流人工脑脊液时所记录的自发兴奋性突触后电流结果。
结果如图8所示。结果说明，QD202二聚体可增加海马CA1神经元自发的兴奋性突触递质释放，主要表现为增加兴奋性突触后电流的发放频率，并且增加兴奋性突触后电流幅度。
5.2半胱氨酸修饰的多肽
制备半胱氨酸修饰的QD202：用Fmoc-Cys(CAm)-OH替代Fmoc-Cys(Trt)-OH，按照QD202的氨基酸序列，直接合成，得到包含半胱氨酸修饰的QD202。
按照实施例4记载的方法，使用半胱氨酸修饰的QD202对小鼠进行锥体神经元自发兴奋性突触后电流检测。其中，对照指在正常灌流人工脑脊液时所记录的自发兴奋性突触后电流结果。
结果如图9所示。结果说明，半胱氨酸修饰的QD202增加海马CA1神经元自发的兴奋性突触递质释放的活性消失，主要表现为减少兴奋性突触后电流的发放频率，和/或减少兴奋性突触后电流幅度。
5.3丝氨酸修饰的多肽
制备丝氨酸修饰的QD202：用Fmoc-Ser(HPO3Bzl)-OH替代Fmoc-Ser(tbu)-OH，按照QD202的氨基酸序列，直接合成，从而获得自N端起第12位丝氨酸(S)进行了磷酸化修饰的丝氨酸修饰的QD202。
按照实施例4记载的方法，使用丝氨酸修饰的QD202对小鼠进行锥体神经元自发兴奋性突触后电流检测。其中，对照指在正常灌流人工脑脊液时所记录的自发兴奋性突触后电流结果。
结果如图10所示。结果说明，丝氨酸修饰的QD202增加海马CA1神经元自发的兴奋 性突触递质释放的活性消失，主要表现为减少兴奋性突触后电流的发放频率，和/或减少兴奋性突触后电流幅度。
实施例6多肽与ATP6V1B2亚单位的结合位点
分别构建包含具有Myc标签的鼠源ATP6V1B2全长、1-286aa截短序列或1-172aa截短序列的表达质粒，其中鼠源ATP6V1B2全长的氨基酸序列如SEQ ID NO.8所示，鼠源ATP6V1B2 1-286aa的氨基酸序列如SEQ ID NO.10所示，鼠源ATP6V1B2 1-172aa的氨基酸序列如SEQ ID NO.11所示，分别称这些质粒为ATP6V1B2全长表达质粒、ATP6V1B2 1-286aa表达质粒和ATP6V1B2 1-172aa表达质粒。同时，构建带有EGFP标签的QD202表达质粒。
在293细胞(购自中国科学院细胞库/干细胞库)中同时转染QD202表达质粒，以及ATP6V1B2全长表达质粒、ATP6V1B2 1-286aa表达质粒或ATP6V1B2 1-172aa表达质粒，48小时后收取细胞裂解液，在裂解液中加入Myc抗体(购自Sigma-Aldrich)以富集ATP6V1B2全长蛋白、1-286aa截短序列或1-172aa截短序列。
而后加入Protein G琼脂糖珠结合Myc抗体，将ATP6V1B2全长蛋白、1-286aa截短序列或1-172aa截短序列及相互结合的蛋白共同沉淀下来，最后通过免疫印迹法检测免疫共沉淀的蛋白。
结果如图11所示，其中Atp6v1b2-Myc、b2-286Δ-Myc和b2-172Δ-Myc分别对应ATP6V1B2全长蛋白、1-286aa截短序列或1-172aa截短序列。结果显示：只有全长ATP6V1B2蛋白能与QD202多肽结合，由此可知ATP6V1B2与QD202的结合位点位于ATP6V1B2自N端的第288位至第512位氨基酸的区域范围中。
前述详细说明是以解释和举例的方式提供的，并非要限制所附权利要求的范围。目前本申请所列举的实施方式的多种变化对本领域普通技术人员来说是显而易见的，且保留在所附的权利要求和其等同方案的范围内。

Claims (230)

  1. 质子泵调控剂在制备试剂中的用途，所述试剂用于改善学习能力。
  2. 质子泵调控剂在制备试剂中的用途，所述试剂用于治疗认知障碍。
  3. 质子泵调控剂在制备试剂中的用途，所述试剂用于治疗神经退行性疾病。
  4. 根据权利要求1-3中任一项所述的用途，其中所述试剂使受试者中质子泵相关蛋白的表达水平和/或活性提高。
  5. 根据权利要求1-4中任一项所述的用途，其中所述质子泵调控剂使受试者中质子泵相关蛋白的表达水平和/或活性提高。
  6. 根据权利要求4-5中任一项所述的用途，其中，所述质子泵相关蛋白的表达水平包括编码质子泵相关蛋白基因的表达水平、编码质子泵相关蛋白基因的转录水平和/或质子泵相关蛋白的表达水平。
  7. 根据权利要求4-6中任一项所述的用途，其中所述提高包括与所述受试者中原始质子泵相关蛋白的表达水平和/或活性相比，所述质子泵相关蛋白的表达水平和/或活性提高至少约10％。
  8. 根据权利要求4-7中任一项所述的用途，其中所述质子泵相关蛋白的表达水平通过实施选自下组的试验衡量：qPCR、qRT-PCR、杂交分析、RNA印迹法、斑点印迹法、原位杂交、胶凝电泳、毛细管电泳、柱色谱、蛋白质印迹法、免疫组织化学、免疫染色和质谱。
  9. 根据权利要求4-8中任一项所述的用途，其中所述质子泵相关蛋白的表达水平通过利用选自下组的物质衡量：能够特异性扩增编码质子泵相关蛋白基因的引物、与编码质子泵相关蛋白基因特异性结合的核酸分子、与质子泵相关蛋白特异性结合的核酸分子、与质子泵相关蛋白特异性结合的小分子、与质子泵相关蛋白特异性结合的探针和与质子泵相关蛋白特异性结合的多肽。
  10. 根据权利要求1-9中任一项所述的用途，其中所述试剂使受试者中ATP6V1B2的表达水平和/或活性提高。
  11. 根据权利要求1-10中任一项所述的用途，其中所述质子泵调控剂使受试者中ATP6V1B2的表达水平和/或活性提高。
  12. 根据权利要求10-11中任一项所述的用途，其中，所述ATP6V1B2的表达水平包括ATP6V1B2基因的表达水平、ATP6V1B2基因的转录水平和/或ATP6V1B2蛋白的表达水平。
  13. 根据权利要求10-12中任一项所述的用途，其中所述提高包括与所述受试者中原始ATP6V1B2的表达水平和/或活性相比，所述ATP6V1B2的表达水平和/或活性提高至少 约10％。
  14. 根据权利要求10-13中任一项所述的用途，其中所述ATP6V1B2的表达水平通过实施选自下组的试验衡量：qPCR、qRT-PCR、杂交分析、RNA印迹法、斑点印迹法、原位杂交、胶凝电泳、毛细管电泳、柱色谱、蛋白质印迹法、免疫组织化学、免疫染色和质谱。
  15. 根据权利要求10-14中任一项所述的用途，其中所述ATP6V1B2的表达水平通过利用选自下组的物质衡量：能够特异性扩增ATP6V1B2基因的引物、与ATP6V1B2基因特异性结合的核酸分子、与ATP6V1B2蛋白特异性结合的核酸分子、与ATP6V1B2蛋白特异性结合的小分子、与ATP6V1B2蛋白特异性结合的探针和与ATP6V1B2蛋白特异性结合的多肽。
  16. 根据权利要求1-15中任一项所述的用途，其中所述质子泵调控剂包括质子泵激动剂。
  17. 根据权利要求1-16中任一项所述的用途，其中所述质子泵调控剂包括囊泡型质子泵激动剂。
  18. 根据权利要求1-17中任一项所述的用途，所述质子泵调控剂包括ATP6V1B2蛋白的激动剂。
  19. 根据权利要求1-18中任一项所述的用途，所述质子泵调控剂能够结合ATP6V1B2蛋白。
  20. 根据权利要求1-19中任一项所述的用途，所述质子泵调控剂能够与小鼠ATP6V1B2蛋白的第288位至第512位氨基酸序列中的至少部分序列结合。
  21. 根据权利要求1-20中任一项所述的用途，所述质子泵调控剂能够与人ATP6V1B2蛋白的第288位至第512位氨基酸序列中的至少部分序列结合。
  22. 根据权利要求1-21中任一项所述的用途，其中所述质子泵调控剂包括核酸或其变体、多肽或其变体和/或小分子。
  23. 根据权利要求1-22中任一项所述的用途，其中所述质子泵调控剂能够增加神经元突触递质释放。
  24. 根据权利要求23所述的用途，其中所述增加包括与所述受试者中原始神经元突触递质释放的水平相比，增加至少约10％。
  25. 根据权利要求1-24中任一项所述的用途，其中所述质子泵调控剂能够增加兴奋性突触后电流的发放频率。
  26. 根据权利要求25所述的用途，其中所述增加包括与所述受试者中原始兴奋性突触后电流的发放频率的水平相比，增加至少约10％。
  27. 根据权利要求1、4-26中任一项所述的用途，其中所述学习能力包括认知能力、运动能力、记忆能力和/或空间探索能力。
  28. 根据权利要求1、4-27中任一项所述的用途，其中所述学习能力的改善包括与受试者的原始的学习能力的评估分数相比，改善后的所述受试者的学习能力的评估分数提高至少约50％。
  29. 根据权利要求1、4-28中任一项所述的用途，其中所述学习能力的评估分数通过实施选自下组的试验衡量：新物体认知试验和水迷宫试验。
  30. 根据权利要求29所述的用途，其中所述新物体认知试验评价所述认知能力、运动能力和/或空间探索能力。
  31. 根据权利要求29-30中任一项所述的用途，其中所述水迷宫试验评价所述记忆能力、运动能力和/或空间探索能力。
  32. 根据权利要求4-31中任一项所述的用途，其中所述受试者包括哺乳动物。
  33. 根据权利要求4-32中任一项所述的用途，其中所述受试者包括人。
  34. 根据权利要求4-33中任一项所述的用途，其中所述受试者包括非认知障碍患者和/或非神经退行性疾病患者。
  35. 根据权利要求4-34中任一项所述的用途，其中所述受试者包括神经退行性疾病患者和/或认知障碍患者。
  36. 根据权利要求4-35中任一项所述的用途，其中所述受试者包括阿尔兹海默症患者。
  37. 根据权利要求4-36中任一项所述的用途，其中所述受试者处于老龄阶段。
  38. 根据权利要求2、4-37中任一项所述的用途，其中所述认知障碍包括早期认知障碍(MCI)、中期认知障碍和晚期认知障碍。
  39. 根据权利要求2、4-38中任一项所述的用途，其中所述认知障碍包括正常衰老引起的认知障碍、lews身体痴呆(LBD)、额颞叶痴呆和/或血管性痴呆。
  40. 根据权利要求2、4-39中任一项所述的用途，其中所述认知障碍的诱导疾病包括阿尔兹海默症、多发性梗死型、帕金森氏症、艾滋病和/或克雅氏病(CJD)。
  41. 根据权利要求3-40中任一项所述的用途，其中所述神经退行性疾病包括急性神经退行性疾病和慢性神经退行性疾病。
  42. 根据权利要求3-41中任一项所述的用途，其中所述神经退行性疾病包括由神经元死亡和胶质细胞稳态引起的神经退行性疾病、由衰老引起的神经退行性疾病、由CNS细胞功能被影响引起的神经退行性疾病、由细胞间异常通信引起的神经退行性疾病和/或由细胞运动受损引起的神经退行性疾病。
  43. 根据权利要求3-42中任一项所述的用途，其中所述神经退行性疾病包括阿尔兹海默症、帕金森氏症、多发性硬化(MS)、肌萎缩性脊髓侧索硬化症(ALS)和/或亨廷顿病 (HD)。
  44. 根据权利要求1-43中任一项所述的用途，其中所述试剂被配制为适于口服施用和/或注射施用。
  45. 根据权利要求1-44中任一项所述的用途，其中所述试剂被配制为适于静脉注射。
  46. ATP6V1B2、其功能活性片段或编码它们的核酸分子在改善学习能力中的用途。
  47. ATP6V1B2、其功能活性片段或编码它们的核酸分子在治疗认知障碍中的用途。
  48. ATP6V1B2、其功能活性片段或编码它们的核酸分子在治疗神经退行性疾病中的用途。
  49. 根据权利要求46-48中任一项所述的用途，其中所述ATP6V1B2来源于人。
  50. 根据权利要求46-49中任一项所述的用途，其中所述ATP6V1B2包含SEQ ID NO.8或16所示的氨基酸序列。
  51. 根据权利要求46-50中任一项所述的用途，其中所述ATP6V1B2的功能活性片段具有与SEQ ID NO.5所示的氨基酸序列特异性结合的能力。
  52. 根据权利要求46-51中任一项所述的用途，其中所述ATP6V1B2的功能活性片段包括ATP6V1B2的截短体。
  53. 根据权利要求46-52中任一项所述的用途，其中所述ATP6V1B2的功能活性片段包含人ATP6V1B2蛋白的第288位至第512位氨基酸序列中的至少部分序列。
  54. 根据权利要求46-53中任一项所述的用途，其中所述ATP6V1B2的功能活性片段包含小鼠ATP6V1B2蛋白的第288位至第512位氨基酸序列中的至少部分序列。
  55. 根据权利要求46-54中任一项所述的用途，其中所述ATP6V1B2的功能活性片段包含SEQ ID NO.10-11中任一项所示的氨基酸序列。
  56. 根据权利要求46-55中任一项所述的用途，其中所述核酸分子包含SEQ ID NO.9或17所示的核苷酸序列。
  57. 根据权利要求46、49-56中任一项所述的用途，其中所述学习能力包括认知能力、运动能力、记忆能力和/或空间探索能力。
  58. 根据权利要求46、49-57中任一项所述的用途，其中所述学习能力的改善包括与受试者的原始的学习能力的评估分数相比，改善后的所述受试者的学习能力的评估分数提高至少约50％。
  59. 根据权利要求46、49-58中任一项所述的用途，其中所述学习能力的评估分数通过实施选自下组的试验衡量：新物体认知试验和水迷宫试验。
  60. 根据权利要求59所述的用途，其中所述新物体认知试验评价所述认知能力、运动能力和/或空间探索能力。
  61. 根据权利要求59-60中任一项所述的用途，其中所述水迷宫试验评价所述记忆能力、运动能力和/或空间探索能力。
  62. 根据权利要求58-61中任一项所述的用途，其中所述受试者包括哺乳动物。
  63. 根据权利要求58-62中任一项所述的用途，其中所述受试者包括人。
  64. 根据权利要求58-63中任一项所述的用途，其中所述受试者包括非认知障碍患者和/或非神经退行性疾病患者。
  65. 根据权利要求58-64中任一项所述的用途，其中所述受试者包括神经退行性疾病患者和/或认知障碍患者。
  66. 根据权利要求58-65中任一项所述的用途，其中所述受试者包括阿尔兹海默症患者。
  67. 根据权利要求58-66中任一项所述的用途，其中所述受试者处于老龄阶段。
  68. 根据权利要求47、49-67中任一项所述的用途，其中所述认知障碍包括早期认知障碍(MCI)、中期认知障碍和晚期认知障碍。
  69. 根据权利要求47、49-68中任一项所述的用途，其中所述认知障碍包括正常衰老引起的认知障碍、lews身体痴呆(LBD)、额颞叶痴呆和/或血管性痴呆。
  70. 根据权利要求47、49-69中任一项所述的用途，其中所述认知障碍的诱导疾病包括阿尔兹海默症、多发性梗死型、帕金森氏症、艾滋病和/或克雅氏病(CJD)。
  71. 根据权利要求48-70中任一项所述的用途，其中所述神经退行性疾病包括急性神经退行性疾病和慢性神经退行性疾病。
  72. 根据权利要求48-71中任一项所述的用途，其中所述神经退行性疾病包括由神经元死亡和胶质细胞稳态引起的神经退行性疾病、由衰老引起的神经退行性疾病、由CNS细胞功能被影响引起的神经退行性疾病、由细胞间异常通信引起的神经退行性疾病和/或由细胞运动受损引起的神经退行性疾病。
  73. 根据权利要求48-72中任一项所述的用途，其中所述神经退行性疾病包括阿尔兹海默症、帕金森氏症、多发性硬化(MS)、肌萎缩性脊髓侧索硬化症(ALS)和/或亨廷顿病(HD)。
  74. ATP6V1B2调控剂在制备试剂中的用途，所述试剂用于改善学习能力。
  75. ATP6V1B2调控剂在制备试剂中的用途，所述试剂用于治疗认知障碍。
  76. ATP6V1B2调控剂在制备试剂中的用途，所述试剂用于治疗神经退行性疾病。
  77. 根据权利要求74-76中任一项所述的用途，其中所述试剂使受试者中ATP6V1B2的表达水平和/或活性提高。
  78. 根据权利要求74-77中任一项所述的用途，其中所述ATP6V1B2调控剂使受试者中 ATP6V1B2的表达水平和/或活性提高。
  79. 根据权利要求74-78中任一项所述的用途，其中，所述表达水平包括ATP6V1B2基因的表达水平、ATP6V1B2基因的转录水平和/或ATP6V1B2蛋白的表达水平。
  80. 根据权利要求77-79中任一项所述的用途，其中所述活性提高包括使质子泵相关蛋白的表达水平和/或活性提高。
  81. 根据权利要求77-80中任一项所述的用途，其中所述提高包括与所述受试者中原始ATP6V1B2的表达水平和/或活性相比，所述ATP6V1B2的表达水平和/或活性提高至少约10％。
  82. 根据权利要求77-81中任一项所述的用途，其中所述ATP6V1B2的表达水平通过实施选自下组的试验衡量：qPCR、qRT-PCR、杂交分析、RNA印迹法、斑点印迹法、原位杂交、胶凝电泳、毛细管电泳、柱色谱、蛋白质印迹法、免疫组织化学、免疫染色和质谱。
  83. 根据权利要求77-82中任一项所述的用途，其中所述ATP6V1B2的表达水平通过利用选自下组的物质衡量：能够特异性扩增ATP6V1B2基因的引物、与ATP6V1B2基因特异性结合的核酸分子、与ATP6V1B2蛋白特异性结合的核酸分子、与ATP6V1B2蛋白特异性结合的小分子、与ATP6V1B2蛋白特异性结合的探针和与ATP6V1B2蛋白特异性结合的多肽。
  84. 根据权利要求74-83中任一项所述的用途，所述ATP6V1B2调控剂包括ATP6V1B2蛋白的激动剂。
  85. 根据权利要求74-84中任一项所述的用途，所述ATP6V1B2调控剂能够结合ATP6V1B2蛋白。
  86. 根据权利要求74-85中任一项所述的用途，所述ATP6V1B2调控剂能够与小鼠ATP6V1B2蛋白的第288位至第512位氨基酸序列中的至少部分序列结合。
  87. 根据权利要求74-86中任一项所述的用途，所述ATP6V1B2调控剂能够与人ATP6V1B2蛋白的第288位至第512位氨基酸序列中的至少部分序列结合。
  88. 根据权利要求74-87中任一项所述的用途，其中所述ATP6V1B2调控剂包括质子泵激动剂。
  89. 根据权利要求74-88中任一项所述的用途，其中所述ATP6V1B2调控剂包括囊泡型质子泵激动剂。
  90. 根据权利要求74-89中任一项所述的用途，其中所述ATP6V1B2调控剂包括核酸或其变体、多肽或其变体和/或小分子。
  91. 根据权利要求74-90中任一项所述的用途，其中所述ATP6V1B2调控剂包括多肽或其变 体。
  92. 根据权利要求91所述的用途，其中所述多肽或其变体特异性能够结合ATP6V1B2蛋白。
  93. 根据权利要求91-92中任一项所述的用途，其中所述多肽或其变体能够增加神经元突触递质释放。
  94. 根据权利要求93所述的用途，其中所述增加包括与所述受试者中原始神经元突触递质释放的水平相比，增加至少约10％。
  95. 根据权利要求91-94中任一项所述的用途，其中所述多肽或其变体能够增加兴奋性突触后电流的发放频率。
  96. 根据权利要求95所述的用途，其中所述增加包括与所述受试者中原始兴奋性突触后电流的发放频率的水平相比，增加至少约10％。
  97. 根据权利要求91-96中任一项所述的用途，其中所述多肽或其变体包含SEQ ID NO.5所示的氨基酸序列。
  98. 根据权利要求91-97中任一项所述的用途，其中所述多肽或其变体包含SEQ ID NO.1,3中任一项所示的氨基酸序列。
  99. 根据权利要求74、77-98中任一项所述的用途，其中所述学习能力包括认知能力、运动能力、记忆能力和/或空间探索能力。
  100. 根据权利要求74、77-99中任一项所述的用途，其中所述学习能力的改善包括与受试者的原始的学习能力的评估分数相比，改善后的所述受试者的学习能力的评估分数提高至少约50％。
  101. 根据权利要求74、77-100中任一项所述的用途，其中所述学习能力的评估分数通过实施选自下组的试验衡量：新物体认知试验和水迷宫试验。
  102. 根据权利要求101所述的用途，其中所述新物体认知试验评价所述认知能力、运动能力和/或空间探索能力。
  103. 根据权利要求101-102中任一项所述的用途，其中所述水迷宫试验评价所述记忆能力、运动能力和/或空间探索能力。
  104. 根据权利要求77-103中任一项所述的用途，其中所述受试者包括哺乳动物。
  105. 根据权利要求77-104中任一项所述的用途，其中所述受试者包括人。
  106. 根据权利要求77-105中任一项所述的用途，其中所述受试者包括非认知障碍患者和/或非神经退行性疾病患者。
  107. 根据权利要求77-106中任一项所述的用途，其中所述受试者包括神经退行性疾病患者和/或认知障碍患者。
  108. 根据权利要求77-107中任一项所述的用途，其中所述受试者包括阿尔兹海默症患者。
  109. 根据权利要求77-108中任一项所述的用途，其中所述受试者处于老龄阶段。
  110. 根据权利要求75、77-109中任一项所述的用途，其中所述认知障碍包括早期认知障碍(MCI)、中期认知障碍和晚期认知障碍。
  111. 根据权利要求75、77-110中任一项所述的用途，其中所述认知障碍包括正常衰老引起的认知障碍、lews身体痴呆(LBD)、额颞叶痴呆和/或血管性痴呆。
  112. 根据权利要求75、77-111中任一项所述的用途，其中所述认知障碍的诱导疾病包括阿尔兹海默症、多发性梗死型、帕金森氏症、艾滋病和/或克雅氏病(CJD)。
  113. 根据权利要求76-112中任一项所述的用途，其中所述神经退行性疾病包括急性神经退行性疾病和慢性神经退行性疾病。
  114. 根据权利要求76-113中任一项所述的用途，其中所述神经退行性疾病包括由神经元死亡和胶质细胞稳态引起的神经退行性疾病、由衰老引起的神经退行性疾病、由CNS细胞功能被影响引起的神经退行性疾病、由细胞间异常通信引起的神经退行性疾病和/或由细胞运动受损引起的神经退行性疾病。
  115. 根据权利要求76-114中任一项所述的用途，其中所述神经退行性疾病包括阿尔兹海默症、帕金森氏症、多发性硬化(MS)、肌萎缩性脊髓侧索硬化症(ALS)和/或亨廷顿病(HD)。
  116. 根据权利要求74-115中任一项所述的用途，其中所述试剂被配制为适于口服施用和/或注射施用。
  117. 根据权利要求74-116中任一项所述的用途，其中所述试剂被配制为适于静脉注射。
  118. 筛选能够改善受试者学习能力、治疗认知障碍、和/或治疗神经退行性疾病的药物的方法，其包括以下的步骤：检测候选药物对受试者中质子泵相关蛋白的表达水平和/或活性的影响，其中在施用所述候选药物后，所述质子泵相关蛋白的表达水平和/或活性提高，则所述候选药物能够改善受试者学习能力、治疗认知障碍、和/或治疗神经退行性疾病。
  119. 筛选能够改善受试者学习能力、治疗认知障碍、和/或治疗神经退行性疾病的药物的方法，其包括以下的步骤：检测候选药物对受试者中ATP6V1B2的表达水平和/或活性的影响，其中在施用所述候选药物后，所述ATP6V1B2的表达水平和/或活性提高，则所述候选药物能够改善受试者学习能力、治疗认知障碍、和/或治疗神经退行性疾病。
  120. 根据权利要求118所述的方法，其中，所述质子泵相关蛋白的表达水平包括编码质子泵相关蛋白基因的表达水平、编码质子泵相关蛋白基因的转录水平和/或质子泵相关蛋白的表达水平。
  121. 根据权利要求118、120中任一项所述的方法，其中所述提高包括与所述受试者中原始质子泵相关蛋白的表达水平和/或活性相比，所述质子泵相关蛋白的表达水平和/或活性提高至少约10％。
  122. 根据权利要求118、120-121中任一项所述的方法，其中所述质子泵相关蛋白的表达水平通过实施选自下组的试验衡量：qPCR、qRT-PCR、杂交分析、RNA印迹法、斑点印迹法、原位杂交、胶凝电泳、毛细管电泳、柱色谱、蛋白质印迹法、免疫组织化学、免疫染色和质谱。
  123. 根据权利要求118、120-121中任一项所述的方法，其中所述质子泵相关蛋白的表达水平通过利用选自下组的物质衡量：能够特异性扩增编码质子泵相关蛋白基因的引物、与编码质子泵相关蛋白基因特异性结合的核酸分子、与质子泵相关蛋白特异性结合的核酸分子、与质子泵相关蛋白特异性结合的小分子、与质子泵相关蛋白特异性结合的探针和与质子泵相关蛋白特异性结合的多肽。
  124. 根据权利要求119-123中任一项所述的方法，其中，所述ATP6V1B2的表达水平包括ATP6V1B2基因的表达水平、ATP6V1B2基因的转录水平和/或ATP6V1B2蛋白的表达水平。
  125. 根据权利要求119-124中任一项所述的方法，其中所述提高包括与所述受试者中原始ATP6V1B2的表达水平和/或活性相比，所述ATP6V1B2的表达水平和/或活性提高至少约10％。
  126. 根据权利要求119-125中任一项所述的方法，其中所述ATP6V1B2的表达水平通过实施选自下组的试验衡量：qPCR、qRT-PCR、杂交分析、RNA印迹法、斑点印迹法、原位杂交、胶凝电泳、毛细管电泳、柱色谱、蛋白质印迹法、免疫组织化学、免疫染色和质谱。
  127. 根据权利要求119-126中任一项所述的方法，其中所述ATP6V1B2的表达水平通过利用选自下组的物质衡量：能够特异性扩增ATP6V1B2基因的引物、与ATP6V1B2基因特异性结合的核酸分子、与ATP6V1B2蛋白特异性结合的核酸分子、与ATP6V1B2蛋白特异性结合的小分子、与ATP6V1B2蛋白特异性结合的探针和与ATP6V1B2蛋白特异性结合的多肽。
  128. 根据权利要求118-127中任一项所述的方法，其中所述学习能力包括认知能力、运动能力、记忆能力和/或空间探索能力。
  129. 根据权利要求118-128中任一项所述的方法，其中所述施用包括口服和/或注射。
  130. 根据权利要求118-129中任一项所述的方法，其中所述施用包括静脉注射。
  131. 能够调控ATP6V1B2的表达水平和/或活性的多肽或其变体。
  132. 根据权利要求131所述的多肽或其变体，其能够增加神经元突触递质释放。
  133. 根据权利要求132所述的多肽或其变体，其中所述增加包括与所述受试者中原始神经元突触递质释放的水平相比，增加至少约10％。
  134. 根据权利要求131-133中任一项所述的多肽或其变体，其增加兴奋性突触后电流的发放频率。
  135. 根据权利要求134所述的多肽或其变体，其中所述增加包括与所述受试者中原始兴奋性突触后电流的发放频率的水平相比，增加至少约10％。
  136. 根据权利要求131-135中任一项所述的多肽或其变体，其能够结合ATP6V1B2蛋白。
  137. 根据权利要求131-136中任一项所述的多肽或其变体，其能够使受试者中ATP6V1B2的表达水平和/或活性提高。
  138. 根据权利要求137所述的多肽或其变体，其中所述ATP6V1B2的表达水平包括ATP6V1B2基因的表达水平、ATP6V1B2基因的转录水平和/或ATP6V1B2蛋白的表达水平。
  139. 根据权利要求137-138中任一项所述的多肽或其变体，其中所述提高包括与所述受试者中原始ATP6V1B2的表达水平和/或活性相比，所述ATP6V1B2的表达水平和/或活性提高至少约10％。
  140. 根据权利要求131-139中任一项所述的多肽或其变体，其能够与小鼠ATP6V1B2蛋白的第288位至第512位氨基酸序列中的至少部分序列结合。
  141. 根据权利要求131-140中任一项所述的多肽或其变体，其能够与人ATP6V1B2蛋白的第288位至第512位氨基酸序列中的至少部分序列结合。
  142. 根据权利要求131-141中任一项所述的多肽或其变体，其包含SEQ ID NO.5所示的氨基酸序列。
  143. 根据权利要求131-142中任一项所述的多肽或其变体，其包含SEQ ID NO.1,3中任一项所示的氨基酸序列。
  144. 根据权利要求131-143中任一项所述的多肽或其变体，其包括多聚体。
  145. 根据权利要求144所述的多肽或其变体，其中所述多聚体包括同二聚体。
  146. 根据权利要求131-145中任一项所述的多肽或其变体，其氨基酸序列中的半胱氨酸不具有巯基封闭的修饰。
  147. 根据权利要求131-146中任一项所述的多肽或其变体，其氨基酸序列中的丝氨酸不具有磷酸化的修饰。
  148. 药物组合，其包含权利要求131-147中任一项所述的多肽或其变体。
  149. 药物组合物，其包含权利要求131-147中任一项所述的多肽或其变体以及药学上可接受的载体。
  150. 权利要求131-147中任一项所述的多肽或其变体、权利要求148所述的药物组合和/或权利要求149所述的药物组合物在制备试剂中的用途，所述试剂用于改善学习能力、治疗认知障碍和/或治疗神经退行性疾病。
  151. 根据权利要求150所述的用途，其中所述学习能力包括认知能力、运动能力、记忆能力和/或空间探索能力。
  152. 根据权利要求150-151中任一项所述的用途，其中所述认知障碍包括早期认知障碍(MCI)、中期认知障碍和晚期认知障碍。
  153. 根据权利要求150-152中任一项所述的用途，其中所述认知障碍包括正常衰老引起的认知障碍、lews身体痴呆(LBD)、额颞叶痴呆和/或血管性痴呆。
  154. 根据权利要求150-153中任一项所述的用途，其中所述认知障碍的诱导疾病包括阿尔兹海默症、多发性梗死型、帕金森氏症、艾滋病和/或克雅氏病(CJD)。
  155. 根据权利要求150-154中任一项所述的用途，其中所述神经退行性疾病包括急性神经退行性疾病和慢性神经退行性疾病。
  156. 根据权利要求150-155中任一项所述的用途，其中所述神经退行性疾病包括由神经元死亡和胶质细胞稳态引起的神经退行性疾病、由衰老引起的神经退行性疾病、由CNS细胞功能被影响引起的神经退行性疾病、由细胞间异常通信引起的神经退行性疾病和/或由细胞运动受损引起的神经退行性疾病。
  157. 根据权利要求150-156中任一项所述的用途，其中所述神经退行性疾病包括阿尔兹海默症、帕金森氏症、多发性硬化(MS)、肌萎缩性脊髓侧索硬化症(ALS)和/或亨廷顿病(HD)。
  158. 根据权利要求150-157中任一项所述的用途，其中所述多肽、所述的药物组合和/或所述的药物组合物被配制为适于口服施用和/或注射施用。
  159. 根据权利要求150-158中任一项所述的用途，其中所述多肽、所述的药物组合和/或所述的药物组合物被配制为适于静脉注射。
  160. 一种非人动物或其部分，其包含和/或表达ATP6V1B2、其功能活性片段和/或编码它们的核酸分子。
  161. 根据权利要求160所述的非人动物或其部分，其中所述ATP6V1B2源自人。
  162. 根据权利要求160-161中任一项所述的非人动物或其部分，其中所述ATP6V1B2包含 SEQ ID NO.8或16所示的氨基酸序列。
  163. 根据权利要求160-162中任一项所述的非人动物或其部分，其中所述ATP6V1B2的功能活性片段具有与SEQ ID NO.5所示的氨基酸序列特异性结合的能力。
  164. 根据权利要求160-163中任一项所述的非人动物或其部分，其中所述ATP6V1B2的功能活性片段包括ATP6V1B2的截短体。
  165. 根据权利要求160-164中任一项所述的非人动物或其部分，其中所述ATP6V1B2的功能活性片段包含人ATP6V1B2蛋白的第288位至第512位氨基酸序列中的至少部分序列。
  166. 根据权利要求160-165中任一项所述的非人动物或其部分，其中所述ATP6V1B2的功能活性片段包含小鼠ATP6V1B2蛋白的第288位至第512位氨基酸序列中的至少部分序列。
  167. 根据权利要求160-166中任一项所述的非人动物或其部分，其中所述ATP6V1B2的功能活性片段包含SEQ ID NO.10-11中任一项所示的氨基酸序列。
  168. 根据权利要求160-167中任一项所述的非人动物或其部分，其中所述核酸分子包含SEQ ID NO.9或17所示的核苷酸序列。
  169. 根据权利要求160-168中任一项所述的非人动物或其部分，其中所述非人动物为经基因编辑的非人动物，或源自经基因编辑的非人动物。
  170. 根据权利要求160-169中任一项所述的非人动物或其部分，其中所述非人动物是基因敲入的非人动物，或源自基因敲入的非人动物。
  171. 根据权利要求160-170中任一项所述的非人动物或其部分，其中所述ATP6V1B2和/或其功能活性片段的表达由所述非人动物基因组中相应的内源性调控元件调控。
  172. 根据权利要求160-171中任一项所述的非人动物或其部分，其中所述非人动物为非人哺乳动物。
  173. 根据权利要求160-172中任一项所述的非人动物或其部分，其中所述非人动物选自：小鼠，大鼠，兔，猿，猴，猪，牛，羊，马，鸡，狗，羊驼和猫。
  174. 根据权利要求160-173中任一项所述的非人动物或其部分，其中所述非人动物为啮齿类动物或灵长类动物。
  175. 根据权利要求160-174中任一项所述的非人动物或其部分，其中所述部分包括所述非人动物的器官、组织和/或细胞。
  176. 根据权利要求160-175中任一项所述的非人动物或其部分，其中所述部分包括体液。
  177. 根据权利要求176所述的非人动物或其部分，其中所述体液选自下组：血液、血浆、血清、尿液、汗液、眼泪、唾液、精液和脑脊液。
  178. 根据权利要去160-177中任一项所述的非人动物或其部分，其中所述部分包括脑或其一部分。
  179. 根据权利要求160-178中任一项所述的非人动物或其部分，其中所述部分不能发育成完整的非人动物。
  180. 一种分离的细胞，其包含和/或表达ATP6V1B2、其功能活性片段和/或编码它们的核酸分子。
  181. 根据权利要求180所述的细胞，其中所述ATP6V1B2源自人。
  182. 根据权利要求180-181中任一项所述的细胞，其中所述ATP6V1B2和/或其功能活性片段包含SEQ ID NO:8、10-11、16中任一项所示的氨基酸序列。
  183. 根据权利要求180-182中任一项所述的细胞，其中编码所述ATP6V1B2和/或其功能活性片段的基因是纯合的或杂合的。
  184. 根据权利要求180-183中任一项所述的细胞，其中编码所述ATP6V1B2和/或其功能活性片段的核酸分子包含SEQ ID NO:9或17所示的核酸序列。
  185. 根据权利要求180-184中任一项所述的细胞，其中所述ATP6V1B2和/或其功能活性片段的表达由所述细胞基因组中相应的内源性调控元件调控。
  186. 根据权利要求180-185中任一项所述的细胞，其为非人动物细胞。
  187. 根据权利要求186所述的细胞，其中所述非人动物为非人哺乳动物。
  188. 根据权利要求180-187中任一项所述的细胞，其中所述非人动物选自：小鼠，大鼠，兔，猿，猴，猪，牛，羊，马，鸡，狗，羊驼和猫。
  189. 根据权利要求180-188中任一项所述的细胞，其中所述非人动物为啮齿类动物或灵长类动物。
  190. 根据权利要求180-189中任一项所述的细胞，其不能发育成完整的非人动物。
  191. 一种组织，其包含和/或表达ATP6V1B2、其功能活性片段和/或编码它们的核酸分子。
  192. 根据权利要求191所述的组织，其中所述ATP6V1B2源自人。
  193. 根据权利要求191-192中任一项所述的组织，其中所述ATP6V1B2和/或其功能活性片段包含SEQ ID NO:8、10-11、16中任一项所示的氨基酸序列。
  194. 根据权利要求191-193中任一项所述的组织，其中编码所述ATP6V1B2和/或其功能活性片段的基因是纯合的或杂合的。
  195. 根据权利要求191-194中任一项所述的组织，其中编码所述ATP6V1B2和/或其功能活性片段的核酸分子包含SEQ ID NO:9或17所示的核酸序列。
  196. 根据权利要求191-195中任一项所述的组织，其是经基因编辑的组织，或源自经基因编辑的组织。
  197. 根据权利要求191-196中任一项所述的组织，其是基因敲入的组织，或源自基因敲入的组织。
  198. 根据权利要求191-197中任一项所述的组织，其中所述ATP6V1B2和/或其功能活性片段的表达由所述组织基因组中相应的内源性调控元件调控。
  199. 根据权利要求191-198中任一项所述的组织，其为非人动物的组织。
  200. 根据权利要求199所述的组织，其中所述非人动物为非人哺乳动物。
  201. 根据权利要求191-200中任一项所述的组织，其中所述非人动物选自：小鼠，大鼠，兔，猿，猴，猪，牛，羊，马，鸡，狗，羊驼和猫。
  202. 根据权利要求191-201中任一项所述的组织，其中所述非人动物为啮齿类动物或灵长类动物。
  203. 根据权利要求191-202中任一项所述的组织，其包括体液。
  204. 根据权利要求203所述的组织，其中所述体液选自下组：血液、血浆、血清、尿液、汗液、眼泪、唾液、精液和脑脊液。
  205. 根据权利要求191-204中任一项所述的组织，其包括脑或其一部分。
  206. 根据权利要求191-205中任一项所述的组织，其不能发育成完整的非人动物。
  207. 一种细胞系或细胞培养物，其源自权利要求160-179中任一项所述的非人动物或其部分，源自权利要求180-190中任一项所述的细胞，和/或源自权利要求191-206中任一项所述的组织。
  208. 根据权利要求207所述的细胞系或细胞培养物，其中所述细胞培养物为原代细胞培养物。
  209. 根据权利要求207-208中任一项所述的细胞系或细胞培养物，其中所述细胞培养物包括类器官。
  210. 组合物，其包含权利要求160-179中任一项所述的非人动物或其部分、权利要求180-190中任一项所述的细胞、权利要求191-206中任一项所述的组织，和/或权利要求207-209中任一项所述的细胞系或原代细胞培养物。
  211. 试剂盒，其包含权利要求160-179中任一项所述的非人动物或其部分、权利要求180-190中任一项所述的细胞、权利要求191-206中任一项所述的组织，和/或权利要求207-209中任一项所述的细胞系或原代细胞培养物；和一种或多种选自下组的另外的组分： 分析缓冲液、对照物、底物、标准品、检测材料、实验室用品、设备、仪器、细胞、器官、组织和用户手册或说明书。
  212. 一种筛选能够改善受试者学习能力、治疗认知障碍、和/或治疗神经退行性疾病的药物的方法，所述方法包括将候选的药物施用于权利要求160-179中任一项所述的非人动物或其部分、权利要求180-190中任一项所述的细胞、权利要求191-206中任一项所述的组织，和/或权利要求207-209中任一项所述的细胞系或原代细胞培养物，并测定所述候选药物对受试者中ATP6V1B2的表达水平和/或活性的影响。
  213. 一种筛选能够改善受试者学习能力、治疗认知障碍、和/或治疗神经退行性疾病的药物的方法，所述方法包括：(i)将候选的药物施用于权利要求160-179中任一项所述的非人动物或其部分、权利要求180-190中任一项所述的细胞、权利要求191-206中任一项所述的组织，和/或权利要求207-209中任一项所述的细胞系或原代细胞培养物；(ii)测定所述候选的药物对对受试者中ATP6V1B2的表达水平和/或活性的影响；(iii)在施用所述候选药物后，所述ATP6V1B2的表达水平和/或活性提高，则所述候选药物能够改善受试者学习能力、治疗认知障碍、和/或治疗神经退行性疾病。
  214. 根据权利要求212-213中任一项所述的方法，其中，所述ATP6V1B2的表达水平包括ATP6V1B2基因的表达水平、ATP6V1B2基因的转录水平和/或ATP6V1B2蛋白的表达水平。
  215. 根据权利要求212-214中任一项所述的方法，其中所述提高包括与所述受试者中原始ATP6V1B2的表达水平和/或活性相比，所述ATP6V1B2的表达水平和/或活性提高至少约10％。
  216. 根据权利要求212-215中任一项所述的方法，其中所述学习能力包括认知能力、运动能力、记忆能力和/或空间探索能力。
  217. 根据权利要求212-216中任一项所述的方法，其中所述认知障碍包括早期认知障碍(MCI)、中期认知障碍和晚期认知障碍。
  218. 根据权利要求212-217中任一项所述的方法，其中所述认知障碍包括正常衰老引起的认知障碍、lews身体痴呆(LBD)、额颞叶痴呆和/或血管性痴呆。
  219. 根据权利要求212-218中任一项所述的方法，其中所述认知障碍的诱导疾病包括阿尔兹海默症、多发性梗死型、帕金森氏症、艾滋病和/或克雅氏病(CJD)。
  220. 根据权利要求212-219中任一项所述的方法，其中所述神经退行性疾病包括急性神经退行性疾病和慢性神经退行性疾病。
  221. 根据权利要求212-220中任一项所述的方法，其中所述神经退行性疾病包括由神经元死亡和胶质细胞稳态引起的神经退行性疾病、由衰老引起的神经退行性疾病、由CNS细胞功能被影响引起的神经退行性疾病、由细胞间异常通信引起的神经退行性疾病和/或由细胞运动受损引起的神经退行性疾病。
  222. 根据权利要求212-221中任一项所述的方法，其中所述神经退行性疾病包括阿尔兹海默症、帕金森氏症、多发性硬化(MS)、肌萎缩性脊髓侧索硬化症(ALS)和/或亨廷顿病(HD)。
  223. 权利要求160-179中任一项所述的非人动物或其部分、权利要求180-190中任一项所述的细胞、权利要求191-206中任一项所述的组织，和/或权利要求207-209中任一项所述的细胞系或原代细胞培养物在制备鉴别和/或筛选系统中的用途，所述系统用于筛选能够改善受试者学习能力、治疗认知障碍、和/或治疗神经退行性疾病的药物和/或生物标志物。
  224. 权利要求160-179中任一项所述的非人动物或其部分、权利要求180-190中任一项所述的细胞、权利要求191-206中任一项所述的组织，和/或权利要求207-209中任一项所述的细胞系或原代细胞培养物，其可用于筛选能够改善受试者学习能力、治疗认知障碍、和/或治疗神经退行性疾病的药物和/或生物标志物。
  225. 制备权利要求160-179中任一项所述的非人动物或其部分、权利要求180-190中任一项所述的细胞、权利要求191-206中任一项所述的组织的方法，所述方法包括使非人动物或其部分、细胞或组织包含和/或表达所述ATP6V1B2和/或其功能活性片段。
  226. 制备疾病模型的方法，所述方法包括使非人动物或其部分、细胞或组织包含和/或表达ATP6V1B2和/或其功能活性片段。
  227. 根据权利要求225-226中任一项所述的方法，其中所述ATP6V1B2和/或其功能活性片段包含SEQ ID NO:8、10-11、16中任一项所示的氨基酸序列。
  228. 根据权利要求225-227中任一项所述的方法，其包括转染包含编码所述ATP6V1B2和/或其功能活性片段的核酸序列。
  229. 根据权利要求228所述的方法，其中编码所述ATP6V1B2和/或其功能活性片段的核酸序列包含SEQ ID NO:9或17所示的核酸序列。
  230. 根据权利要求228-229中任一项所述的方法，其中所述转染包括：转染包含所述核酸序列的腺相关病毒。
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