Application of TRPM7 kinase domain M7CK in preparation of Alzheimer disease treatment medicine

Abstract

The invention relates to the technical field of biological medicines, in particular to application of a TRPM7 kinase structural domain M7CK in preparation of a medicine for treating Alzheimer's disease. The overexpressed M7CK can improve cognitive function impairment of AD model mice by increasing the phosphorylation level of MMP14, can improve the learning and memory ability of the mice, increase the synaptic density, increase the density of pre-synaptic vesicle protein synaptophysin and post-synaptic dense protein PSD-95, promote degradation of A beta in soluble and insoluble components, reduce accumulation of A beta amyloid plaques, and improve the cognitive function impairment of AD model mice. Therefore, the TRPM7 kinase structural domain provided by the invention has a great application prospect in gene therapy of patients with Alzheimer's disease.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the application of the kinase domain M7CK of TRPM7 in the preparation of a drug for treating Alzheimer's disease. Background Art

[0002] Alzheimer's disease (AD) is a neurodegenerative disease with a wide range of impacts. Its pathogenesis is extremely complex, and the main pathological manifestations are the loss of synapses in the brain, amyloid deposition, neurofibrillary tangles, etc. Clinical manifestations include progressive memory and cognitive decline, language function, personality changes, and difficulty in daily activities. It brings great mental and economic burdens to the patients themselves, their families, and even the entire society. So far, there is no effective cure. At present, one of the important targets for the development of Alzheimer's disease drugs is the deposition of amyloid proteins. Therefore, it has great application prospects to improve the pathological characteristics of Alzheimer's disease such as synaptic loss and cognitive impairment by targeting the clearance of amyloid proteins.

[0003] TRPM7 is a member of the M-type transient receptor potential family and a divalent ion channel protein widely expressed in various cell types. It contains a 6-transmembrane channel domain and a C-terminal kinase domain (M7CK), with both its N-terminus and C-terminus pointing to the cytoplasm. TRPM7 can selectively pass divalent cations, such as calcium ions, magnesium ions, and zinc ions, and plays an important role in maintaining intracellular and extracellular ion homeostasis and regulating downstream signaling pathways. It is expressed in a variety of neural cells, such as the hippocampus, cerebral cortex, cerebellum, and brainstem. The kinase end of TRPM7 (TRPM7 cleaved kinase fragments, M7CK) can be cleaved and exist independently, and participate in regulating important cellular processes such as chromatin remodeling and synaptic plasticity. Its unique dual domain structure means that it may be involved in a variety of cellular functions. Studies have shown that TRPM7 is involved in neurodegenerative diseases including Alzheimer's disease in various ways. However, some studies believe that inhibiting TRPM7 can be used as a therapeutic strategy, while other studies believe that activating TRPM7 may have a protective effect. In 2005, a study found that a missense mutation of TRPM7 can change the sensitivity of cells to magnesium ions, thereby affecting magnesium ion homeostasis, and thus causing the occurrence of neurodegenerative diseases. In 2010, a study found that the presenilin protein mutation associated with familial Alzheimer's disease inhibits the channel function of TRPM7. In 2017, a study found that pharmacological activation of the channel function of TRPM7 can promote the clearance of Aβ by activating autophagy. So far, there is no research on the protective effect of the kinase domain M7CK of TRPM7 on Alzheimer's disease. Summary of the invention

[0004] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide an application of the kinase domain M7CK of TRPM7 in the preparation of a drug for treating Alzheimer's disease, so as to solve the problems in the prior art.

[0005] To achieve the above objectives and other related objectives, the present invention provides the use of the kinase domain M7CK of TRPM7 in the preparation of a drug for treating Alzheimer's disease.

[0006] Preferably, the amino acid sequence of M7CK is selected from the following:

[0007] 1) A polypeptide sequence encoded by the nucleotide sequence SEQ ID No. 1;

[0008] 2) A polypeptide fragment having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% sequence identity with the polypeptide of 1) on the basis of having the same biological function as the polypeptide of 1).

[0009] Preferably, the polypeptide sequence is shown as SEQ ID No.3.

[0010] The present invention also provides a pAAV2 / 9-M7CK plasmid constructed based on the M7CK sequence of the kinase domain of TRPM7 and capable of overexpressing the M7CK sequence at positions 1299-1863 of TRPM7.

[0011] The present invention also provides a virus constructed based on the M7CK sequence of the kinase domain of TRPM7 and capable of overexpressing the M7CK sequence at positions 1299-1863 of TRPM7. The virus is obtained by viral packaging of the aforementioned pAAV2 / 9-M7CK plasmid.

[0012] The present invention also provides a method for preventing or treating Alzheimer's disease, comprising administering an effective dose of the aforementioned TRPM7 kinase domain M7CK sequence, the aforementioned pAAV2 / 9-M7CK plasmid or the aforementioned virus to a subject.

[0013] As described above, the use of the kinase domain M7CK of TRPM7 of the present invention in the preparation of a drug for treating Alzheimer's disease has the following beneficial effects:

[0014] In the present invention, overexpression of M7CK can improve cognitive impairment of AD model mice by increasing the phosphorylation level of MMP14, improve their learning and memory abilities, increase synaptic density, increase the density of presynaptic vesicle protein synaptophysin and postsynaptic density protein PSD-95, promote the degradation of Aβ in soluble and insoluble components, and reduce the accumulation of Aβ amyloid plaques. Therefore, the kinase domain of TRPM7 provided by the present invention has great application prospects in gene therapy for patients with Alzheimer's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Shown is a schematic diagram of the results of analyzing the TRPM7 expression levels in the hippocampus of Alzheimer's disease patients, 12-month-old 5XFAD mice, and 15-month-old APP / PS1 mice in the present invention.

[0016] Figure 2 Shown is a schematic diagram of synaptophysin / PSD95 immunofluorescence signals of primary hippocampal neurons overexpressing M7CK and treated with Aβ in the present invention and a schematic diagram of statistical results.

[0017] Figure 3 It is shown that in the present invention, AAV2 / 9-M7CK or AAV2 / 9-EGFP was injected into the hippocampus of 5-month-old WT and 5XFAD mice, and then their learning and memory functions were tested using the novel object recognition test, cognitive wall test and Y-maze test at 6 / 10 / 15 months of age.

[0018] Figure 4 Shown are the behavioral experimental test results of the present invention on 6 / 10 / 15 month old mice.

[0019] Figure 5 It is shown that before the injection of M7CK in the present invention, 14-month-old 5XFAD mice did not have the ability to recognize new objects. After overexpressing M7CK, the new object recognition index of 5XFAD mice increased significantly.

[0020] Figure 6 Shown is a schematic diagram and statistical results of immunofluorescence staining of synaptophysin and PSD95 in the hippocampus of 15-month-old 5XFAD mice overexpressing M7CK in the present invention.

[0021] Figure 7 Shown are schematic diagrams and statistical graphs of immunohistochemical staining of Aβ plaques in the hippocampus of 5XFAD mice overexpressing M7CK in the present invention.

[0022] Figure 8 The results show that the Aβ expression in the soluble protein fraction and insoluble protein fraction in the hippocampus of 5XFAD mice overexpressing M7CK in the present invention 1-40and Aβ 1-42 ELISA quantitative results.

[0023] Fig. 9 It is shown that overexpression of M7CK in the hippocampus of 5XFAD mice in the present invention increases the phosphorylation level of MMP14.

[0024] Fig.10 The present invention shows that M7CK can bind to and phosphorylate MMP14.

[0025] Fig.11 It was shown that knocking out TRPM7 in the present invention reduced the phosphorylation level of MMP14.

[0026] Fig.12 This shows that M7CK in the present invention promotes the degradation of Aβ by MMP14.

[0027] Fig.13 This shows that M7CK cannot restore the downregulation of synaptic density caused by Aβ after knocking down MMP14 in the present invention.

[0028] Fig.14 This shows that M7CK cannot promote Aβ degradation after knocking down MMP14 in the present invention.

[0029] Fig.15 Shown is a schematic diagram of the TRPM7 protein structure in the present invention.

[0030] Fig.16 Shown is a schematic diagram of the pAAV2 / 9-EGFP plasmid in the present invention.

[0031] Fig.17 Shown are the results of studies on the activity and specificity of the substance of the present invention that promotes the kinase cleavage of TRPM7 to produce M7CK.

[0032] Fig.18 The results show that chronic treatment with ATT, a substance of the present invention that promotes kinase cleavage of TRPM7 to produce M7CK, can rescue cognitive and memory functions of 5×FAD mice.

[0033] Fig.19 The results show that chronic treatment with ATT, a substance that promotes kinase cleavage of TRPM7 to produce M7CK, can protect the synaptic density and reduce Aβ plaques in 5×FAD mice.

[0034] Fig. 20 The results are shown as the results of changing the M7CK / TRPM7 ratio using a substance that promotes the kinase cleavage of TRPM7 to produce M7CK in the present invention. DETAILED DESCRIPTION

[0035] The present invention provides the use of the kinase domain M7CK of TRPM7 in preparing a drug for treating Alzheimer's disease, wherein the kinase domain M7CK is a polypeptide.

[0036] In some specific embodiments, the amino acid sequence of M7CK is a polypeptide sequence encoded by the nucleotide sequence SEQ ID No.1.

[0037] In some specific embodiments, the M7CK may be: a polypeptide fragment having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with the polypeptide encoded by the nucleotide sequence SEQ ID No. 1, on the basis of having the same biological function as the polypeptide encoded by the nucleotide sequence SEQ ID No. 1.

[0038] In some specific embodiments, the amino acid sequence of the kinase domain M7CK of TRPM7 includes the amino acid sequence of positions 1299-1863 of TRPM7. Preferably, the amino acid sequence of M7CK is shown in SEQ ID No.3.

[0039] In some embodiments, the kinase domain M7CK of the TRPM7 may include the amino acid sequence of a secretion tag. The secretion tag directs the kinase domain of TRPM7 to the periplasmic space of the host cell. In a specific embodiment, the signal peptide is derived from DsbA, PelB, OmpA, TolB, MalE, lpp, TorA or HylA. On the one hand, the secretion tag is attached to the kinase domain of TRPM7. On the other hand, the secretion tag is cut from the kinase domain of TRPM7.

[0040] In some embodiments, the kinase domain M7CK of the TRPM7 comprises a histidine tag. A histidine tag or a polyhistidine tag is a sequence of 2 to 20 histidine residues attached to collagen. The histidine tag comprises 2 to 20 histidine residues, 5 to 15 histidine residues, 5 to 18 histidine residues, 5 to 16 histidine residues, 5 to 15 histidine residues, 5 to 14 histidine residues, 5 to 13 histidine residues, 5 to 12 histidine residues, 5 to 11 histidine residues, 5 to 10 histidine residues, 6 to 12 histidine residues, 6 to 11 histidine residues or 7 to 10 histidine residues. The histidine tag can be used for purifying proteins by using a chromatographic method based on a nickel-based chromatographic medium.

[0041] In some embodiments, the Alzheimer's disease therapeutic drug includes an M7CK promoter.

[0042] In some specific embodiments, the M7CK promoter is selected from one or more of the following: a plasmid or virus that effectively overexpresses M7CK; and / or a substance that promotes the kinase cleavage of TRPM7 to produce M7CK.

[0043] In some specific embodiments, the plasmid or viral vector overexpressing M7CK comprises a nucleic acid fragment encoding M7CK.

[0044] In some specific embodiments, the substance that promotes the kinase cleavage of TRPM7 to produce M7CK is selected from one or more of the following compounds: resveratrol, zafirukast, TPCA-1, ATT, and glutamate or glutamate.

[0045] Further, the encoding nucleic acid fragment is as shown in SEQ ID No. 1. Preferably, the encoding nucleic acid fragment is a nucleic acid fragment optimized by any one or more of the following methods of the nucleic acid molecule as shown in SEQ ID No. 1: 1) the G / C content is increased compared with the nucleic acid molecule as shown in SEQ ID No. 1; 2) the nucleic acid molecule as shown in SEQ ID No. 1 is codon-optimized to adapt to the species it acts on, wherein the nucleic acid fragment does not change the encoded amino acid sequence after codon optimization or G / C content increase.

[0046] In some specific embodiments, the plasmid is selected from adenovirus packaging plasmid, adeno-associated virus packaging plasmid or lentivirus packaging plasmid; the virus is selected from adenovirus, adeno-associated virus or lentivirus.

[0047] In some specific embodiments, the plasmid is a pAAV2 / 9-M7CK plasmid. Preferably, the pAAV2 / 9-M7CK plasmid sequence is as shown in SEQ ID NO.2.

[0048] In some specific embodiments, the pAAV2 / 9-M7CK plasmid is used for AAV virus packaging to obtain packaged AAV virus.

[0049] In some specific embodiments, the method for using the Alzheimer's disease therapeutic drug is: the packaged AAV virus is injected into the brain by stereotaxic injection, specifically into the hippocampus region of the brain.

[0050] In some embodiments, the Alzheimer's disease therapeutic drug has one or more of the following functions:

[0051] 1) Increase the density of neuronal synapses;

[0052] 2) Improve learning and memory abilities;

[0053] 3) Reduction of Aβ plaques or Aβ protein;

[0054] 4) Activating MMP14 activity. Among them, increasing neuronal synaptic density means increasing neuronal synaptic density relative to Alzheimer's patients; improving learning and memory ability means improving learning and memory ability relative to Alzheimer's patients; reducing Aβ plaques means reducing Aβ plaques relative to Alzheimer's patients.

[0055] Furthermore, the Aβ plaque is composed of Aβ 1-40 Aβ 1-42 Protein aggregates form clumps of protein.

[0056] Furthermore, the activation of MMP14 is to phosphorylate the MMP14 protein.

[0057] In some specific embodiments, the Alzheimer's disease therapeutic drug also contains pharmaceutically acceptable excipients. Specifically, the excipients include various excipients and diluents, which are not necessary active ingredients and have no excessive toxicity after administration. The excipients include sterile water or saline, stabilizers, excipients, antioxidants (ascorbic acid, etc.), buffers (phosphoric acid, citric acid, other organic acids, etc.), preservatives, surfactants (PEG, Tween, etc.), chelating agents (EDTA, etc.) or adhesives. The excipients also include other low molecular weight polypeptides, serum albumin, glycine, glutamine, asparagine, arginine, polysaccharides, monosaccharides, mannitol or sorbitol. The excipients are selected from saline, glucose isotonic solution, D-sorbitol isotonic solution, D-mannose isotonic solution, D-manno or sugar alcohol isotonic solution when used for injection of aqueous solution. The injection aqueous solution contains a solubilizing agent. The solubilizing agent is selected from alcohol (ethanol), polyol (propylene glycol or PEG) and / or nonionic surfactant (Tween 80 or HCO-50). In the Alzheimer's disease therapeutic drug, the kinase domain M7CK sequence of TRPM7 is a single active ingredient, and can also be combined with one or more other active components useful for the treatment of Alzheimer's disease to form a combined preparation. The active component is other various drugs for the treatment of Alzheimer's disease. The content of the active ingredient in the pharmaceutical composition is a safe and effective amount, which should be adjustable by a person skilled in the art. For example, the dosage of the active ingredient of the aforementioned TRPM7 kinase domain M7CK sequence and the Alzheimer's disease treatment product depends on the patient's weight, the type of application, the condition and severity of the disease. For example, the dosage of the pharmaceutical composition as the active ingredient is 1-1000 mg / kg / day, 1-3 mg / kg / day, 3-5 mg / kg / day, 5-10 mg / kg / day, 10-20 mg / kg / day, 20-30 mg / kg / day, 30-40 mg / kg / day, 40-60 mg / kg / day, 60-80 mg / kg / day, 80-100 mg / kg / day, 100-200 mg / kg / day, 200-500 mg / kg / day, or greater than 500 mg / kg / day.

[0058] The present invention also provides a pAAV2 / 9-M7CK plasmid constructed based on the M7CK sequence of the kinase domain of TRPM7, which can effectively overexpress the M7CK sequence at positions 1299-1863 of TRPM7.

[0059] In some specific embodiments, the amino acid sequence of M7CK is a polypeptide sequence encoded by the nucleotide sequence SEQ ID No.1.

[0060] In some specific embodiments, the M7CK may be: a polypeptide fragment having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with the polypeptide encoded by the nucleotide sequence SEQ ID No. 1, on the basis of having the same biological function as the polypeptide encoded by the nucleotide sequence SEQ ID No. 1.

[0061] In some specific embodiments, the amino acid sequence of M7CK includes the amino acid sequence of positions 1299-1863 of TRPM7. Preferably, the amino acid sequence of M7CK is shown in SEQ ID No.3.

[0062] In some specific embodiments, the plasmid or viral vector overexpressing M7CK comprises a nucleic acid fragment encoding M7CK.

[0063] Further, the encoding nucleic acid fragment is as shown in SEQ ID No. 1. Preferably, the encoding nucleic acid fragment is a nucleic acid fragment optimized by any one or more of the following methods of the nucleic acid molecule as shown in SEQ ID No. 1: 1) the G / C content is increased compared with the nucleic acid molecule as shown in SEQ ID No. 1; 2) the nucleic acid molecule as shown in SEQ ID No. 1 is codon-optimized to adapt to the species it acts on, wherein the nucleic acid fragment does not change the encoded amino acid sequence after codon optimization or G / C content increase.

[0064] In some specific embodiments, the nucleotide sequence of the pAAV2 / 9-M7CK plasmid is shown as SEQ ID No.2.

[0065] The present invention also provides a virus constructed based on the M7CK sequence of the kinase domain of TRPM7 and capable of overexpressing the M7CK sequence at positions 1299-1863 of TRPM7, which is obtained by viral packaging of the aforementioned pAAV2 / 9-M7CK plasmid.

[0066] In some embodiments, the virus can be selected from one or more of adenovirus, adeno-associated virus or lentivirus.

[0067] Furthermore, the plasmid backbone of the virus can be selected from pLKO.1-CMV-tGFP, pLKO.1-puro-CMV-tGFP, pLKO.1-CMV-Neo, pLKO.1-Neo, pLKO.1-Neo-CMV-tGFP, pLKO.1-puro-CMV-TagCFP, pLKO.1-puro-CMV-TagYFP, pLKO.1-puro-CMV-TagRFP, pLKO.1-puro-CMV-TagFP635, pLKO.1-puro-Ub Any one or more of C-TurboGFP, pLKO.1-puro-UbC-TagFP635, pLKO-puro-IPTG-1xLacO, pLKO-puro-IPTG-3xLacO, pLP1, pLP2, pLP / VSV-G, pENTR / U6, pLenti6 / BLOCK-iT-DEST, pcDNA1.2 / V5-GW / lacZ, pLenti6.2 / N-Lumio / V5-DEST, pGCSIL-GFP, and pHR-SFFV-Puro.

[0068] In some specific embodiments, the aforementioned pAAV2 / 9-M7CK plasmid or virus is obtained by introducing the aforementioned M7CK sequence into an engineering cell and then replicating or assembling the cell. Specifically, the engineering cell can be selected from one or more of Escherichia coli, Lactobacillus, Bacillus licheniformis, Bacillus subtilis, Streptomyces, Saccharomyces cerevisiae, Candida, Torulopsis, Rhodotorula, Hansenula, Pichia pastoris, Kluyveromyces, 293, 293T, HUCCT1, HCCC-9810 or RBE.

[0069] The present invention also provides a method for preventing or treating Alzheimer's disease, specifically a method for preventing or treating a subject suffering from Alzheimer's disease, comprising administering an effective dose of the aforementioned M7CK promoter, the aforementioned pAAV2 / 9-M7CK plasmid or the aforementioned virus to the subject.

[0070] In some embodiments, the amount of administration is 1-1000 mg / kg / day. Specifically, the amount of administration is 1-3 mg / kg / day, 3-5 mg / kg / day, 5-10 mg / kg / day, 10-20 mg / kg / day, 20-30 mg / kg / day, 30-40 mg / kg / day, 40-60 mg / kg / day, 60-80 mg / kg / day, 80-100 mg / kg / day, 100-200 mg / kg / day, 200-500 mg / kg / day, or 500-1000 mg / kg / day.

[0071] In some embodiments, the subject of the method can be a mammal; preferably, the subject of the method is a human.

[0072] In the present invention, the term "identity" is generally the relationship between the sequences of two or more polypeptides or polynucleotides, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between them, as determined by the number of matches between the string segments with two or more amino acid residues or nucleotide residues. Identity measures the percentage of identical matches between two or more sequences relative to a smaller sequence, wherein a gap comparison processed by a specific mathematical model or computer program (e.g., "algorithm") is used. The identity of related peptides can be easily calculated by known methods. "Identity%" is defined as the percentage of residues (amino acid residues or nucleic acid residues) in a candidate amino acid or nucleic acid sequence that are identical to the residues in the amino acid sequence or nucleic acid sequence of the second sequence after aligning the sequences and, if necessary, introducing gaps to achieve the maximum identity percentage when it is applied to polypeptide or polynucleotide sequences. Methods and computer programs for comparison are well known in the art. It should be understood that identity depends on the calculation of the identity percentage, but may differ in numerical value due to the gaps and penalties introduced in the calculation. Typically, variants of a particular polynucleotide or polypeptide will have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity to that particular reference polynucleotide or polypeptide as determined by sequence alignment programs and parameters described herein and known to those of skill in the art.

[0073] In the present invention, the term "phosphorylation" generally refers to the process in which a phosphate group is added to a specific amino acid residue of a protein, such as serine, threonine, tyrosine or histidine, in an enzymatic reaction. This modification can lead to an increase or decrease in protein activity or change its interaction with other molecules.

[0074] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0075] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing the specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0076] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0077] The specific nucleotide sequences or amino acid sequences used in this application are as follows:

[0078] SEQ ID NO.1 (M7CK sequence):

[0079]

[0080]

[0081] MSLPQGDRESNNPFLCNIFMKDEKDPQYNLFGQDLPVIPQRKEFNIPEAGSSCGALFPSAVSPPELRQRRHGVEMLKIFNKNQKLGSSPNSSPHMSSPPTKFSVSTPSQPSCKSHLESTTKDQEPIFYKAAEGDNIEFGAF VGHRDSMDLQRFKETSNKIRELLSNDTPENTLKHVGAAGYSECCKTSTSSLHSVQESCSRRASTEDSPEVDSKAALLPDWLRDRPSNREMPSEGGTLNGLASPFKPVLDTNYYYSAVERNNLMRLSQSIPFVPVPPRGEPVT VYRLEESSPSILNNSMSSWSQLGLCAKIEFLSKEEMGGGLRRAVKVLCTWSEHDILKSGHLYIIKSFLPEVINTWSSIYKEDTVLHLCLREIQQQRAAQKLTFAFNQMKPKSIPYSPRFLEVFLLYCHSAGQWFAVEECMT GEFRKYNNNNGDEIIPTNTLEEIMLAFSHWTYEYTRGELLVLDLQGVGENLTDPSVIKAEEKRSCDMVFGPANLGEDAIKNFRAKHHCNSCCRKLKLPDLKRNDYTPDKIIFPQDESSDLNLQSGNSTKESEATNSVRLML

[0082] Example 1

[0083] The expression level of TRPM7 in the hippocampus of Alzheimer's disease patients and Alzheimer's disease transgenic model (5XFAD and APP / PS1) mice was significantly reduced.

[0084] The inventors of the present application used the western blotting method to observe that the expression level of TRPM7 protein in the hippocampus of patients with Alzheimer's disease was significantly lower than that in the control group; the same method was further used to observe that the expression level of TRPM7 in the hippocampus of 12-month-old 5XFAD and 15-month-old APP / PS1 mice was significantly lower than that in wild-type mice of the same age. Figure 1 shown.

[0085] The method used in this embodiment is as follows:

[0086] The hippocampal samples of Alzheimer's patients used in this example are from the National Health and Disease Human Brain Tissue Resource Bank (China Brain Bank, School of Medicine, Zhejiang University). The 5XFAD and APP / PS1 (purchased from Nanjing University & Nanjing Institute of Biomedical Research Center) used are both widely used Alzheimer's disease model mice. In this example, the method of western blotting analysis is as follows:

[0087] Take the frozen hippocampal tissue of Alzheimer's patients, fresh 5XFAD and APP / PS1 mouse hippocampal tissue and control group hippocampal tissue, place them in EP tubes, extract proteins with RIPA lysis buffer (100μL lysis buffer / 10mg tissue) containing phosphatase inhibitors and protease inhibitors, and quantify the protein concentration with BCA kit. Add loading buffer to the protein, and fully denature the protein at 100℃ for 5min; load the sample in 10% SDS-PAGE gel, with a loading amount of 20μg. Start electrophoresis, the electrophoresis time is upper gel: 80V 30min, lower gel: 120V 50min. Cut the PVDF membrane into the same size as the SDS-PAGE gel, activate it in methanol for a few seconds, and then sandwich the gel block and PVDF membrane between the multilayer filter paper of the transfer clip in the transfer solution precooled at 4°C (gel facing the black negative electrode, membrane facing the red positive electrode), and transfer the membrane at a constant voltage of 100V for 70min; after the transfer, the membrane was placed in a blocking solution made of 5% skim milk powder for 2 hours, and then transferred to the primary antibody diluted in the blocking solution (TRPM7 antibody, Alomone, ACC-047, 1:750; GAPDH antibody, Proteintech, 1:10000) for overnight incubation, washed three times with PBST, incubated with the secondary antibody for 1.5 hours, and washed three times with PBST. Drop the freshly prepared ECL developer, develop it in the Tianneng developer, select the appropriate exposure time and save the picture; use Image Pro Plus software to perform optical density quantitative analysis of the bands. The relative expression of TRPM7 was calculated using GAPDH as the internal reference.

[0088] Example 2

[0089] Construct an AAV2 / 9-M7CK adeno-associated virus that can effectively overexpress the M7CK sequence of positions 1299-1863 of TRPM7.

[0090] Vector construction:

[0091] 1) Using mouse hippocampal cDNA as template and Forward-ggatccatgtctcttcctcaaggtgat; Reverse-accggt cgtaacatcagacgaacagaa as primers (two restriction sites BamHI AgeI and corresponding protective bases at both ends of the primers) to clone TRPM7 positions 1299-1863, the PCR reaction system is as follows:

[0092] PRIME STAR MAX 12.5μL

[0093] Primer F / R 1μL / 1μL (0.2μM)

[0094] cDNA 100ng

[0095] Add ddH2O to 25 μL

[0096] PCR reaction conditions:

[0097]

[0098] 2) After the PCR reaction, use 1% agarose gel for electrophoresis, cut the band of about 1.7kb, and collect the M7CK fragment according to the instructions of the gel recovery kit: cut the gel at the location of the target band in the nucleic acid developer, collect it in an EP tube, weigh it, add PN solution (1μL / mg gel block), dissolve it in a 50℃ water bath, and shake it continuously until the gel block is completely dissolved; pre-add 500μL BL solution to the adsorption column, 12000rpm, 1min, and discard the waste liquid in the collection tube; restore the completely dissolved gel block solution to room temperature and add it to the adsorption column, let it stand at room temperature for 2min, 12000rpm, 1min, and discard the waste liquid; add PW600μL, let it stand for 4min, 12000rpm, 1min, discard the waste liquid, and repeat twice; empty centrifuge at 12000rpm, 2min, open the lid and dry for 9min; put the adsorption column into a new EP tube, add 30μL ddH2O, let stand for 2 min, 12000 rpm, 2 min, the collected liquid was loaded onto the column again, 12000 rpm, 2 min; the concentration and purity of the obtained DNA fragments were measured using a photometer.

[0099] 3) Enzyme digestion of the M7CK fragment and the vector pAAV2 / 9-EGFP (AAV2 / 92 / 9), the system is as follows:

[0100] Plasmid 3μg or M7CK fragment 1μg

[0101] BamHI 1 μL

[0102] AgeI 1μL

[0103] 10X CutSmart Buffer 5μL

[0104] Add dd H2O to 50 μL

[0105] 37℃ water bath for 1-2 hours

[0106] 4) Inactivate in a 65°C water bath for 20 min, add loading buffer, and perform agarose gel electrophoresis for 30 min at 120 V to recover fragments and vectors of corresponding sizes;

[0107] 5) Connection:

[0108] 10X ligase buffer 2μL

[0109] Carrier: 50ng

[0110] T4 Ligase 1 μL

[0111] Target gene fragment (~50ng / μL): make up to 20μL

[0112] 6) Connect overnight at room temperature (about 25°C);

[0113] 7) Transformation: Take 5 μL of the ligation product and add it to 50 μL of DH5 competent cells just taken out of the negative 80 refrigerator, and let it stand on ice for half an hour; after heat shock in a 42°C water bath for 45 seconds, immediately transfer it to ice for 2 minutes. This process needs to be operated gently and the centrifuge tube should not be shaken; then add 900 μL of LB medium without antibiotics and shake it at 37°C and 150 rpm for one hour to revive the bacteria; centrifuge at room temperature for 5 minutes at 4500 rpm, discard 900 μL of supernatant, resuspend the remaining 100 μL supernatant and the bottom bacterial sediment by suction, pour it on a plate containing ampicillin antibiotics, and evenly spread the bacterial solution with a glass rod that has been sterilized with an alcohol lamp and restored to room temperature, place the plate in a 37°C incubator until the liquid is absorbed, then invert the plate and culture overnight;

[0114] 8) The next day, use a 10 μL pipette tip to pick up a single clone colony, add it to a liquid culture medium containing ampicillin, and shake it at 37°C and 220 rpm for 8-12 hours. Then take 500 μL of the bacterial solution and send it to sequencing (Biological Engineering). If the sequencing is correct, the nucleotide sequence of the plasmid is shown in SEQ ID No. 2, and its structure is as follows Fig.16As shown, take the corresponding bacterial solution and add it to a 300mL culture bottle for expansion culture for 12 hours, then extract the plasmid according to the steps of plasmid extraction (Vazyme, #DC202-01): transfer the overnight cultured bacterial solution to a 50mL centrifuge tube, 11000rpm, 2min, discard the supernatant; add 7.5mL of P1 solution to the bacterial precipitate, vortex and oscillate to mix; continue to add 7.5mL of P2 solution, gently invert upside down to mix, and let stand at room temperature for 5min; add 7.5mL of P4 solution, immediately invert about 8 times, at this time a white flocculent precipitate appears, 11000rpm, 10min, aspirate the supernatant (avoid aspirating white precipitate) to a new 50mL round-bottom tube; add 0.1 times the volume of endotoxin remover, invert to mix, let stand on ice for 5 minutes, let stand at room temperature for 10 minutes, and invert to mix; centrifuge at 11000rpm at room temperature for 10 minutes, take the upper aqueous phase (containing DNA) to a new tube, add 0.5 times the volume of isopropanol, transfer to an adsorption column several times, 11000rpm, 1min, discard the waste liquid in the collection tube; use 10mL rinsing solution PW, 11000rpm, 1min, discard the waste liquid, repeat twice; centrifuge at 11000rpm for 3min; put the adsorption column into a new centrifuge tube, add 1mL ddH2O in the middle of the membrane, let stand at room temperature for 3min, and centrifuge at 11000rpm for 3min; measure the concentration and purity of the obtained plasmid with a photometer.

[0115] AAV2 / 9 virus packaging: 293 cells (6 15 cm culture dishes) transfected with helper plasmids and target plasmids 60 hours after transfection (VigoFect transfection reagent) were taken out of the incubator, the culture medium was removed, and PBS was gently added for washing once. Then, new PBS (25 mL / culture dish) was added and the cells were collected in three 50 mL tubes using a cell scraper; centrifuged at 400 g for 10 min at room temperature; the supernatant was removed, and 10 mL of saline solution (150 mM NaCl, 20 m M Tris-HCl), shake and resuspend the cell pellet and collect it in a 50mL tube; prepare 1.5mL of 10% sodium deoxycholate (prepared and used immediately, working concentration 0.5%) and add it all to 30mL of cell suspension, add Benzonase nuclease (working concentration 50U / mL), and lyse it in a 37℃ water bath for 2 hours; centrifuge at 3000g4℃ for 15min, take the supernatant and filter it with a 0.45μm filter membrane to remove cell debris; then connect the heparin tube (taken out of the refrigerator 2 hours in advance and placed under room temperature UV) to a 50mL syringe, pre-equilibrate the heparin tube with 10mL saline solution (150mM NaCl, 20mM Tris-HCl), and be careful to avoid bubbles in the tube; use a syringe to draw the liquid containing the virus (a total of 30mL), adjust the injection pump to 0.7mL / min, and start column passing; after the virus passes through the column, use a syringe to draw 20mL of saline solution (100mM NaCl, 20mM Use a 5mL syringe to draw 1mL of saline solution (200mM NaCl, 20mM Tris-HCI) to rinse the column; draw 1mL of saline solution (300mM NaCl, 20mM Tris-HCI) to rinse the column; draw 1.5mL (400mM NaCl, 20mM Tris-HCI), 3mL (450mM NaCl, 20mM Tris-HCI), 1.5mL (500mM NaCl, 20mM Tris-HCI) to elute the virus, collect the liquid in a 15mL tube; filter the virus with a 0.22μm filter membrane and add it to an Amicon Ultra 4mL filter, centrifuge at 2000g4℃ for 2min until the liquid in the tube is less than 200μL, rinse twice with 3.5mL PBS, dispense 5μL into each tube, and store at -80℃; use real-time quantitative The virus titer was quantified by PCR and then diluted to approximately 3-5×10 12 genomes / ml; and the virus was added to primary neurons or injected into the mouse brain to verify its infection efficiency.

[0116] Example 3

[0117] Overexpression of M7CK with the 1299-1863 sequence of TRPM7 in primary hippocampal neurons has a protective effect against Aβ-induced synaptic toxicity.

[0118] The inventors of the present application added AAV2 / 9-M7CK or AAV2 / 9-EGFP to primary hippocampal neurons cultured in vitro on the 7th day, and added Aβ on the 12th day. After 48 hours, the synaptic density marked by synaptophysin and PSD95 was observed to be significantly downregulated in the Aβ group by immunofluorescence staining, while the synaptic density of the Aβ group overexpressing M7CK was significantly restored ( Figure 2 ). Therefore, overexpression of M7CK has a protective effect against Aβ-induced synaptic toxicity.

[0119] The method used in this embodiment is as follows:

[0120] Culture of primary hippocampal neurons: Take mouse pups within 3 days of birth, quickly cut off the head, peel off the brain tissue, place in dissection buffer on ice, carefully separate the hippocampal tissue on both sides under a stereoscope, and try to peel off the blood cells on its surface; after the dissection, cut the hippocampal tissue into pieces to avoid tearing; wash the cut hippocampal tissue three times with dissection buffer; add trypsin lysis buffer, 37℃5min, gently shake 1-2 times during the period; wash once with dissection buffer; add dissociation solution containing DNase, and repeatedly blow with a pipette tip until there are no obvious tissue blocks; 4℃, 1500rpm, 15min; discard the supernatant, resuspend the precipitate with plating culture medium (400μL culture medium / pair of hippocampus), and add the cell suspension to the glass slide at the bottom of the four-well plate (100μL cell suspension / 1.8mm diameter hole, the glass slide needs to be pre-coated with matrix gel (matrix gel diluted 1:100 with buffer) at 37℃ for 1 hour). After culturing in a constant temperature incubator for 1.5-2 hours, the cells have basically completed attachment. At this time, 1 mL of plating medium / well is added. Two days later, the medium is replaced with serum-free medium, and the interval between half-changes is 3-4 days.

[0121] Cell immunofluorescence staining and quantification of neuronal synaptic density: After the neurons cultured in four-well plates (16 mm wells, with cell slides on the bottom) were taken out of the incubator, the culture medium was discarded, and they were quickly washed with PBS with gentle movements, and then 4% PFA 1 mL / well was added and fixed for 45 min; washed three times with PBS, 5 min / time; blocked with 5% goat serum diluted in 0.2% PBS-Triton on a shaker at room temperature for 2 hours, 0.2 mL / well; used blocking solution to dilute the primary antibody to the required concentration (synaptophysin, SYSY, 101011, 1:500; PSD95, SYSY, 124003, 1:200), and incubated in a shaker at 4°C overnight; washed three times with PBS, 5 min / time; used 0.2% Incubate the cells in a shaker at room temperature with fluorescent secondary antibodies diluted with PBS-Triton for 2 hours; wash three times with PBS, 5 minutes each time; carefully remove the cell slide from the bottom of the well with tweezers, cover it on a glass slide with a drop of sealing agent (containing dapi dye), wait for the sealing agent to dry for 10 minutes, and carefully apply nail polish around the slide. Let it dry. After completing the immunofluorescence staining of neurons cultured in vitro, place the slide under a confocal microscope and use a 60X oil lens (digital magnification 3 times at the same time) to take pictures. When shooting, the cell body of the neuron should be placed at the edge of the field of view, and then the synaptophysin or PSD-95 signal on the dendrite should be photographed, with a shooting thickness of 7μm and an interval of 1μm / layer. When counting, select the number of synaptopphysin / PSD-95puncta on the dendrites 10μm away from the cell body, and the number of puncta on the dendrites every 10μm is used as the dendrite density.

[0122] Example 4

[0123] Identification of substances that induce kinase cleavage of TRPM7 to produce M7CK and study its activity and specificity

[0124] Using a high-throughput screening platform, this example screened 80,000 compounds and identified four compounds / modulators (resveratrol, zafirukast, TPCA-1, and ATT) that can increase the activity of TRPM7 channels. To confirm the effects of these modulators, this example used whole-cell patch clamp technology in HEK cells transfected with TRPM7 to study their effects on TRPM7 channel currents. Once a stable TRPM7 current was obtained, this example applied these four compounds by perfusion. The results are shown in Figure 2. Fig.17As shown, these four compounds (resveratrol, zafirkast, TPCA-1 and ATT: 30 μM) significantly increased the TRPM7 current amplitude (resveratrol: 1.75±0.24-fold, p=0.015; zafirkast: 1.44±0.12-fold, p=0.007; TPCA-1: 1.34±0.07-fold, p=0.002; ATT: 1.41±0.11-fold, p<0.001). Figure 1 A). In addition, this example determined the median effective concentration (EC50) of these compounds (resveratrol: 28.1 μM; zafirukast: 1.4 μM; TPCA-1: 9.0 μM; ATT: 15.7 μM, Fig.17 C). Therefore, this example identified four substances that promote the kinase cleavage of TRPM7 to produce M7CK and confirmed their ability to activate TRPM7 channels. Next, this example examined the identified substances that promote the kinase cleavage of TRPM7 to produce M7CK. This example evaluated their effects on other TRPM ion channels, such as TRPM2, TRPM6, and TRPM8. For TRPM2, only TPCA-1 showed an excitatory effect on ion channel activity ( Fig.17 D). Regarding TRPM6, resveratrol and zafirukast activated the ion channel; meanwhile, ATT inhibited the channel activity ( Fig.17 E). For TRPM8, resveratrol, zafirukast, and ATT inhibited ion channel activity ( Fig.17 F). Thus, among the identified modulators, only ATT seems to specifically activate TRPM7; at the same time, the other identified modulators activate other TRPM ion channels. In contrast, ATT may inhibit both TRPM ion channels, TRPM6 and TRPM8.

[0125] The specific steps of the electrophysiological experiment in this embodiment are as follows: whole-cell patch clamp recordings were performed at room temperature using an EPC-10 amplifier and PatchMaster software (HEKA, Germany). The current was digitized at a sampling rate of 10 kHz and low-pass filtered at 2.0 kHz. Glass capillaries (World Precision Instruments, USA) with electrode resistances between 2 and 4 MΩ were prepared into needle tubes. The needle tube solution without Mg2+ contained (in mM): 145 CsCl, 8 NaCl, 10 HEPES and 10 EGTA (adjusted to pH 7.2 with CsOH). The bath solution contained (in mM): 140 NaCl, 5 KCl, 2 CaCl2, 20 HEPES and 10 glucose (adjusted to pH 7.4 with NaOH). While maintaining a potential of 0 mV, a 300 ms voltage ramp injection from -100 to +100 mV was repeated every 5 seconds to record TRPM7 currents. When the current reached a steady state (at least 30 ramps without change), the TRPM7 channel currents at +100 and -100 mV were obtained.

[0126] In addition, this example found that promoting neuronal activity by increasing extracellular glutamate concentration (for 1 hour) promoted the cleavage of TRPM7 in a dose-dependent manner, as indicated by the gradual increase in the M7CK / TRPM7 ratio ( Fig. 20 A). The minimum effective glutamate concentration (40 μM) was used to determine the time course of the cleavage process. This example found that glutamate-induced cleavage began after 30 minutes of glutamate incubation, reached a peak after 1 hour, and returned to baseline after 3 hours ( Fig. 20 B and Fig. 20 C).

[0127] in, Fig.17 The specific descriptions of each item are as follows:

[0128] (A). Left: Quantitative analysis of the increase in TRPM7 channel current amplitude by substances that promote the kinase cleavage of TRPM7 to produce M7CK (resveratrol: n=5, zafirlukast: n=5, TPCA-1: n=6 and ATT: n=8). They were analyzed using unpaired t-test (resveratrol and zafirlukast) and Mann-Whitney test (TPCA-1 and ATT). Right: Representative current-voltage relationship of substances that promote the kinase cleavage of TRPM7 to produce M7CK.

[0129] (B). Representative time course of the effects of substances that induce kinase cleavage of TRPM7 to generate M7CK (resveratrol, zafirlukast, TPCA-1, and ATT) on TRPM7 channels.

[0130] (C). Dose-response curves of substances (resveratrol, zolenkast, TPCA-1, and ATT) that induce kinase cleavage of TRPM7 to produce M7CK.

[0131] (D). Left: Quantitative analysis of the effects of substances that promote the kinase cleavage of TRPM7 to produce M7CK (resveratrol: n=4, zolenlast: n=7, TPCA-1: n=5 and ATT: n=5) on TRPM2 channel activity. They were analyzed by unpaired t-test. Right: Representative current-voltage relationship of the effects of substances that promote the kinase cleavage of TRPM7 to produce M7CK.

[0132] (E). Left: Quantitative analysis of the effects of substances that promote the kinase cleavage of TRPM7 to produce M7CK (resveratrol: n=6, zafirlukast: n=6, TPCA-1: n=6 and ATT: n=6) on TRPM6 channel activity. The analysis methods are Mann-Whitney test (resveratrol) and unpaired t-test (zafirlukast, TPCA-1 and ATT). Right: Representative current-voltage relationship of TRPM7 agonist effect.

[0133] (F). Left: Quantitative analysis of the effects of substances that promote the kinase cleavage of TRPM7 to produce M7CK (resveratrol: n=7, zafirlukast: n=6, TPCA-1: n=6 and ATT: n=7) on TRPM8 channel activity. Analyzed by unpaired t-test. Right: Representative current-voltage relationship of the effects of substances that promote the kinase cleavage of TRPM7 to produce M7CK. All recording experiments were performed in transfected HEK cells.

[0134] *P<0.05, **P<0.01, ***P<0.001.

[0135] Example 5

[0136] The AAV2 / 9-M7CK adeno-associated virus constructed by the present invention can effectively restore the impaired learning and memory functions of 5XFAD mice, restore synaptic density, and reduce Aβ levels.

[0137] The inventors of the present application injected the constructed AAV2 / 9-M7CK virus into the bilateral hippocampus of 5-month-old 5XFAD mice using mouse brain stereotaxic technology, and performed behavioral analysis (novel object recognition, Y-maze, cognitive wall test) at 6, 10, and 15 months of age. The results are as follows: Figure 3-5) found that overexpression of M7CK could restore the impaired learning and memory function of 5XFAD mice; compared with wild-type mice of the same age, the synaptic density marked by synaptophysin and PSD95 in the CA1 region of the hippocampus of 5XFAD mice was significantly downregulated, while overexpression of M7CK could significantly restore the synaptic density. Figure 6 As shown; Aβ plaques in the hippocampus of 5XFAD mice overexpressing M7CK were significantly downregulated. Figure 7 As shown; in the insoluble protein fraction of the hippocampus of 5XFAD mice, overexpression of M7CK significantly downregulated Aβ 1-40 and Aβ 1-42 In the soluble protein fraction, overexpression of M7CK significantly downregulated the concentration of Aβ1-42. Figure 8 shown.

[0138] The specific steps of this embodiment are as follows:

[0139] AAV2 / 9-M7CK and AAV2 / 9-EGFP viruses (as controls) were prepared and injected into the bilateral hippocampus of 5-month-old WT and 5XFAD mice. The experiment was divided into 4 groups: WT mice were given AAV2 / 9-EGFP; WT mice were given AAV2 / 9-M7CK; 5XFAD mice were given AAV2 / 9-EGFP; 5XFAD mice were given AAV2 / 9-M7CK. One month after the injection, behavioral experiments were started. After the behavioral experiments at 6 / 10 / 15 months of age, the mice were perfused and the brains were removed for frozen sections.

[0140] Stereotaxic injection of the hippocampus: anesthetize the mouse with 2% isoflurane; after the mouse is successfully anesthetized, fix the mouse head firmly with ear bars and nose bars; adjust the left and right and front and back so that the dorsal side of the mouse brain is on the same horizontal plane; wipe the surface skin of the mouse brain with alcohol and disinfect it, then cut the skin with scissors to fully expose the skull, and wipe off the blood on the surface with a cotton swab dipped in iodine; according to the position of the hippocampus in the mouse brain atlas, drill according to the following coordinates, and drill 2 holes on each side

[0141] CA1: AP=-2.06mm, ML=±1.4mm, DV=1.4mm

[0142] DG: AP=-2.06mm, ML=±1.4mm, DV=2.0mm

[0143] CA3: AP=-2.06mm, ML=±2.25mm, DV=2.1mm

[0144] After drilling, the injection needle was slowly inserted into the brain area of ​​the corresponding coordinates, and the injection was started at a speed of 1nl / s. The volume of the injected virus was 300nl per point. After the injection, the needle was retained for 10 minutes and then slowly removed. The scalp was sutured and the mouse was placed in a warm environment to wait for it to wake up from anesthesia.

[0145] New object recognition experiment: One week before the experiment, the mice were separated from multiple cages to single cages until the end of the experiment. The experimental device consists of a colorless, odorless, opaque box with a length, width and height of 50 cm and a camera hanging on the top, a computer connected to the camera, and objects ABC; at 9:00 am on the first day of the experiment, the mice were placed in the box (without any objects) and allowed to explore freely for 10 minutes to adapt to the environment. Before putting in the next mouse, the box needs to be cleaned with 30% odorless diluted hand soap to prevent the smell left by the previous mouse from affecting the behavior of the next mouse; at 9:00 am on the second day of the experiment, the mice were placed in the same box again (without objects) and allowed to explore freely for 5 minutes; at 9:00 am on the third day of the experiment, the same four objects A were placed at equal distances at the bottom of the box, and then the mice were placed in and allowed to explore freely for 5 minutes. At this time, the experimenter observed and recorded the number and time of each mouse's exploration of the four objects through the top camera. At 9:00 am on the fourth day, object A was replaced with a new object B, and the mice were placed in the environment and allowed to explore for 5 minutes. This period is called the 24-hour memory test. In the memory test phase, the novel object recognition index = the number of explorations of the novel object / the number of explorations of all objects × 100%.

[0146] Cognitive wall experiment: This experiment uses the PhenoTyper model 3000 device and fully automatic recording and analysis software of Noldus. At 21:00 the night before the experiment, the mice were placed in the PhenoTyper cage for 11 hours of adaptation, and they were fasted but not watered. At 8:00 a.m., the feeder was connected to the cage and the learning box was placed in it. About 10 sugar beans were manually given to the mice, and then the video tracking software was turned on. During this process, the software will track the number of times the mice shuttled in the learning box in real time and give sugar beans as rewards according to the following rules. If the mouse enters the learning box from the left hole for 5 consecutive times, a sugar bean reward will be given. If the mouse enters the hole from the left hole 24 times out of 30 times, it can be considered that the mouse has reached the standard of learning, that is, 80% correct rate. The learning curve of each group of mice can be drawn according to the number of holes required for the mouse to reach 80% correct rate. Before and after the experiment, the number of sugar beans remaining in the feeder needs to be counted and compared with the number of food automatically calculated by the software, and whether there is any software or hardware failure in time.

[0147] Y-maze experiment: Mice were placed in the center of a Y-shaped maze and allowed to explore freely for 8 minutes. The whole process was recorded with a camera. The order in which the mice entered the three arms of the Y-maze during this process (one time was counted when the mouse's hind legs completely entered the arm). Each time the mouse completed continuous and non-repeated entry into three different arms was counted as a spontaneous alternation (e.g., ABC / BCA / CAB, etc.). Spontaneous turning percentage = the number of combinations of continuous entry into three different arms / the total number of entry into the arm - 2 × 100%. The larger the percentage, the better the spatial memory ability of the mouse.

[0148] Quantification of synaptic density in mouse brain slices: Anesthetize mice with 2% isoflurane; after mice enter a fully anesthetized state, fix their limbs on a foam board, cut the chest skin to fully expose the heart, cut the right auricle, insert the syringe needle tip into the left ventricle, perfuse once with pre-cooled 20mL saline, and then perfuse once with 20mL 4% PFA. After the perfusion, carefully peel off the mouse brain tissue, soak it in 4% PFA overnight, and then transfer it to 20% and 30% sucrose solutions successively until the brain tissue is dehydrated and sinks to the bottom of the tube; embed the dehydrated brain tissue in OCT embedding agent, place it in a freezing microtome until it is completely solidified on the frozen head, and start freezing sectioning. The slice thickness is 16μm. The cut brain slices were stored in PBS, and then carefully attached to the glass slides using a brush, and dried at room temperature for 2-4 hours until the slices were firmly attached to the glass slides; a closed circle was painted around the brain slices with an immunohistochemistry pen and dried for 30 minutes; washed 3 times in PBS, 5 minutes each time; blocked in a wet box at room temperature for 2 hours with 5% goat serum (in 0.2% Triton-PBS); diluted the primary antibody (synaptophysin, SYSY, 101011, 1:500; PSD95, CST, 3450, 1:200) in the blocking solution, and incubated at 4°C overnight; washed 3 times in PBS, 5 minutes each time; diluted the secondary antibody (1:500) in 0.2% Triton-PBS, and incubated at room temperature for 2 hours; washed 3 times in PBS, 5 minutes each time; used anti-fading sealing agent to seal the slices, covered with a coverslip, and applied nail polish on the edges to fix them; and photographed using a laser confocal microscope. After completing the immunofluorescence staining of the brain slices, place the slides under a confocal microscope and use a 60X lens (digital magnification 3 times at the same time) to take pictures. The shooting thickness is 9μm, and the interval is 1μm / layer. The three layers with the brightest signals in the taken pictures are combined into one layer to export a tiff format picture with a resolution of 1024*1024. Use Image Pro Plus to automatically count puncta in the exported pictures: first apply the HiGauss enhancement filter twice to the picture; measure the area of ​​most typical puncta in the picture and set the appropriate threshold range (points greater than the maximum threshold will be segmented; points less than the minimum threshold will not be counted); finally, click the measurement button to get the number of puncta in the picture. Finally, calculate the number of puncta per 1000μm according to the ruler. 2 The number of puncta contained was used as an indicator of synaptic density.

[0149] Amyloid plaque immunohistochemical staining and quantification: Carefully stick the mouse brain slices soaked in PBS on the glass slide with a brush and let them dry for 2-3 hours; draw circles around the brain slices with a brush and let them dry for half an hour; wash three times with PBS; block with 5% goat serum diluted in 0.3% PBS-Triton on a shaker at room temperature for 2 hours, about 40μL / brain slice; incubate with Aβ antibody (CST, 9888S, 1:1600) diluted in blocking solution at 4℃ overnight; wash three times with PBS, 5 minutes each time; dilute the biotinylated secondary antibody (Vector Laboratories, PK-4000) to 1:200, incubate at room temperature for 30 minutes; wash three times with PBS, 5 minutes each time; dilute ABC reagent with PBS (A reagent 1:100; B reagent 1:100, prepared half an hour in advance), incubate at room temperature for 30 minutes; wash three times with PBS, 5 minutes each time; add DAB reagent (prepare immediately before use), observe the color development (about 5-10 minutes), and terminate the reaction by washing with running water; seal the slides with mounting medium and fix the edges with nail polish, and take bright-field photos with an Olympus vs120 microscope with a 10x lens; import the pictures into Image Pro Plus software, manually circle the hippocampus area as the target area (ROI), and set a unified color standard (to ensure that the color standard can cover all Aβ plaque signals), and the software will automatically calculate the area occupied by the Aβ signal and the optical density.

[0150] AβELISA: The extraction method of Aβ protein in the soluble part is the same as the extraction method used for protein immunoblotting experiments mentioned above. Add 5M guanidine hydrochloride (dissolved in 50mM Tris-HCl, pH8.0) to the precipitate after RIPA lysis and centrifugation, dissolve on a shaker at room temperature for 4 hours, and centrifuge at 12000rpm and 4℃ for 30min. At this time, almost no precipitate can be seen, and the supernatant can be used as the insoluble part; the protein in the soluble part is diluted 100 times and 50 times with the dilution buffer in the kit for Aβ 1-42 and Aβ 1-40 The insoluble protein was diluted 20000-fold and 2000-fold for Aβ 1-42 and Aβ 1-40Detection; Take the ELISA kit (Wako, 292-62301, 298-62401) and related liquids out of the 4℃ refrigerator and restore to room temperature for a while; Add a series of concentrations of standard substances and samples to be tested to the ELISA plate (the total volume of liquid in each well is 100μL), and make 3 duplicate wells for each unknown sample; Seal the ELISA plate with a sealing film and incubate at 4℃ overnight; Discard all liquids in the ELISA plate, and then wash it 5 times with rinse solution (200μL / well). Each time you wash, pour out the liquid in the well as much as possible (complete the plate on absorbent paper); Add 100μL / well of HRP-linked secondary antibody. Incubate in a 4°C refrigerator for 1 hour; discard the secondary antibody in the ELISA plate, then wash it with rinse solution (200 μL / well) 5 times; add 100 μL / well TMB solution (try to ensure that all wells are loaded in a short time), incubate at room temperature in the dark for 30 minutes; add stop solution to stop the reaction, read the absorbance at 450nm (reading needs to be completed within 30 minutes after adding the stop solution); draw a standard curve according to the standard concentration and the corresponding absorbance value, so as to calculate the Aβ in the unknown sample 1-42 and Aβ 1-40 concentration.

[0151] Example 6

[0152] ATT treatment protects memory, preserves synaptic density, and reduces Aβ plaques in the 5×FAD AD mouse model

[0153] This example investigates whether treatment with substances that promote the kinase cleavage of TRPM7 to produce M7CK can prevent AD pathology in the 5xFAD mouse model. Pharmacological and HPLC studies have shown that TPCA-1 cannot cross the blood-brain barrier, as no TPCA-1 or its metabolites were detected in brain tissue and cerebrospinal fluid samples from treated mice. Therefore, ATT was selected for in vivo studies in this example. Two groups of treated mice were prepared in this example and injected (ip) with two doses (50 or 75 mg / kg) of ATT daily. Untreated mice received a similar treatment regimen with an equal amount of vehicle. All treatments began when the mice were 5 months old and continued for 1 month before behavioral testing began. When the mice were 6 months old, open field tests showed that drug treatment did not affect the mice's movement or exploratory activities ( Fig.18 A). The nesting behavior of vehicle-treated 5×FAD mice was impaired. Low-dose (50 mg / kg) ATT also impaired the nesting behavior of mice. In contrast, high-dose (75 mg / kg) ATT successfully rescued the nesting behavior of mice ( Fig.18B and C). Memory function was assessed by the novel object recognition test (NORT). This example found that short-term memory was normal in all groups, including untreated 5×FAD mice. However, untreated 5×FAD mice showed long-term memory impairment ( Fig.18 D). One month of treatment with two doses of ATT (50 mg / kg and 75 mg / kg) improved memory impairment in 5×FAD mice ( Fig.18 D). These findings indicate that the TRPM7 modulator ATT can rescue memory and other cognitive deficits in 5×FAD mice. The data suggest that activation of TRPM7 prevents learning and memory impairments in this mouse model.

[0154] This example also investigated whether ATT, a substance that promotes the kinase cleavage of TRPM7 to produce M7CK, can protect the synaptic density and reduce Aβ plaques in the brains of 5×FAD mice. In this example, mice were perfused and the brains were collected for histological analysis. This example found that the punctate density of synaptophysin in the CA1 stratum radiatum of the hippocampus of 5×FAD mice that were not treated with ATT was significantly reduced. ATT treatment reversed the reduction in synaptic density in a dose-dependent manner. Only at a dose of 75 mg / kg did the synaptic density remain unchanged, while at a dose of 50 mg / kg it did not ( Fig.19 A and B). In addition, both doses of ATT treatment significantly reduced the Aβ plaque density in the hippocampus and cortex of 5×FAD mouse brain slices ( Fig.19 C). Therefore, ATT, a substance that promotes the kinase cleavage of TRPM7 to produce M7CK, can preserve the synaptic density and reduce Aβ plaque deposition in 5×FAD mice. The data in this example show that activation of TRPM7 can counteract AD pathology by protecting synapses and disease regulation mechanisms.

[0155] The behavioral experiment steps or histological staining steps in this embodiment can refer to other embodiments of the present invention that include behavioral experiments or histological staining.

[0156] Fig.18 The specific descriptions of each item are as follows:

[0157] A). Total distance moved freely in an open field for 5 min by wild-type (WT) mice and 5xFAD mice treated with vehicle and ATT (50 and 75 mg / kg) was measured. N = at least 10 mice per group, one-way ANOVA with Kruskal-Wallis test.

[0158] (B). Representative images of WT and 5×FAD mice during nesting social behavior at 6 months of age after treatment with vehicle and ATT (50 and 75 mg / kg), respectively.

[0159] (C). Nesting scores of WT and 5×FAD mice in nesting social behavior at 6 months of age, treated with vehicle and ATT (50 and 75 mg / kg), respectively. N = at least 10 mice per group, analyzed by one-way ANOVA Kruskal-Wallis test.

[0160] (D). The upper figure shows the experimental design of the novel object recognition test. The lower figure shows the recognition index of familiar objects and novel objects in WT mice and 5×FAD mice after vehicle and ATT (50 and 75 mg / kg) treatment during NORT training (left), short-term memory test two hours later (middle), and long-term memory test twenty-four hours later (right). N = at least 8 mice per group, two-way ANOVA followed by Bonferroni post-test.

[0161] *P<0.05 and ***P<0.001.

[0162] Fig.19 The specific descriptions of each item are as follows:

[0163] (A). Representative images of synaptophysin punctate density in the stratum radiatum of the CA1 region in wild-type (WT) mice and 5×FAD mice treated with drugs and ATT (50 and 75 mg / kg) at 6 months of age.

[0164] (B). Quantitative analysis of synaptophysin puncta density in the stratum radiatum of CA1 region of 6-month-old WT and 5×FAD mice treated with drugs and ATT (50 and 75 mg / kg). N = at least 22 brain sections from 6 mice per group, one-way ANOVA followed by Bonferroni post-test.

[0165] (C). The upper panel shows representative images of amyloid plaques in the hippocampus and cortex of 5×FAD mice treated with vehicle and ATT (50 and 75 mg / kg). The lower panel shows quantitative analysis of amyloid plaques in the hippocampus and cortex of 5×FAD mice treated with vehicle and ATT (50 and 75 mg / kg). N = at least 18 brain sections from 6 mice per group, analyzed by one-way ANOVA followed by Bonferroni post-test. At least three brain sections per mouse were used for quantitative immunohistochemical analysis.

[0166] *P<0.05 and ***P<0.001.

[0167] Example 7

[0168] In this example, the content of MMP14 was detected in WT and 5XFAD mice in Example 4, and it was determined that the expression level of Aβ-degrading enzyme MMP14 was upregulated in 5XFAD mice compared with WT mice, but its expression was not affected by the overexpression of M7CK. Compared with the 5XFAD-EGFP group, the phosphorylation level of MMP14 was significantly upregulated in the brain of 5XFAD-M7CK mice. The results are as follows: Fig. 9 As shown; Through the results of protein immunoprecipitation in this example, it is determined that MMP14 and M7CK have an interaction, and when MMP14 and M7CK coexist, the kinase activity color index is significantly higher than when the two exist alone. M7CK can bind to and phosphorylate MMP14, and the results are shown in Figure 10; knocking out TRPM7 in primary hippocampal neurons and HT22 cell lines does not affect the expression level of MMP14 itself, but reduces the level of p-MMP14 / MMP14. Therefore, M7CK plays an important role in regulating the phosphorylation level of MMP14, as shown in Figure 10. Fig.11 As shown; the in vitro Aβ degradation experiment in this example shows that M7CK phosphorylates MMP14 to improve the efficiency of MMP14 in degrading Aβ. When MMP14 is added to Aβ alone, Aβ degradation can occur (about 40% degradation). However, when MMP14+M7CK are added to Aβ at the same time, the degradation level of Aβ is significantly increased (about 70% degradation). That is, M7CK promotes the level of Aβ degradation by MMP14. The results are shown in Fig.12 As shown in the experiment of knocking down MMP14 in this example, it is shown that when MMP14 is knocked out, M7CK no longer has a protective effect on the decrease in synaptic density induced by Aβ. Regardless of whether exogenous Aβ is used, knocking out MMP14 alone will lead to a decrease in synaptic density. MMP14 itself also has an important regulatory effect on synaptic function. Therefore, the disappearance of the synaptic protective effect of M7CK after knocking out MMP14 may also be due to the effect of knocking out MMP14 itself on synapses. The results are shown in Fig.13 In addition, the experiment of knocking down MMP14 in this example also showed that when MMP14 was knocked out alone, the Aβ concentration increased by about 21%; when TRPM7 was knocked out alone, the Aβ concentration increased by about 43%; when M7CK was overexpressed and MMP14 was knocked out, M7CK could not reduce the Aβ concentration level. This shows that knocking out TRPM7 has a greater impact on the metabolic level of Aβ, as shown in Figure 14.

[0169] The specific steps of this embodiment are as follows:

[0170] Construction of recombinant MMP14-EGFP vector

[0171] 1) Using mouse hippocampal cDNA as a template, Forward-CCGCTCGAGATGTCT CCC GCC CCT CGA CC; Reverse-TGCTCTAGAGACCTTGTCCAGCAGCGAACG as primers (two restriction sites XhoIXbaI and corresponding protective bases at both ends of the primer) were used to clone the coding (CDS) region of MMP14. The PCR reaction system was as follows:

[0172] PRIME STAR MAX 12.5μL

[0173] Primer F / R 1μL / 1μL (0.2μM)

[0174] cDNA 100ng

[0175] Add ddH2O to 25 μL

[0176] PCR reaction conditions:

[0177] 98℃5min

[0178] 98℃10s

[0179] 55℃15s 35Cycles

[0180] 72℃30s

[0181] 72℃10min

[0182] 2) After the PCR reaction is completed, use 1% agarose gel for electrophoresis, cut the band of about 1.8 kb, and collect the MMP14 fragment according to the instructions of the gel recovery kit:

[0183] a) Cut the gel at the target band into pieces in a nucleic acid developer and collect them in an EP tube.

[0184] Weigh the gel, add PN solution (1 μL / mg gel), dissolve in a 50°C water bath, and continue

[0185] Shake until all the glue is dissolved;

[0186] b) Add 500 μL BL solution to the adsorption column in advance, spin at 12000 rpm for 1 min, and discard the collection tube

[0187] Wastewater in

[0188] c) After the completely dissolved gel solution is restored to room temperature, add it to the adsorption column and let it stand at room temperature for 2 minutes.

[0189] 12000rpm, 1min, discard waste liquid;

[0190] d) Add 600 μL of PW, let stand for 4 min, spin at 12000 rpm for 1 min, discard the waste liquid, and repeat twice;

[0191] e) Centrifuge at 12000 rpm for 2 min, and dry with the lid open for 9 min;

[0192] f) Place the adsorption column in a new EP tube, add 30 μL ddH2O, let stand for 2 min, and heat at 12000

[0193] rpm, 2 min, the collected liquid was loaded onto the column again, 12000 rpm, 2 min;

[0194] g) Measure the concentration and purity of the obtained DNA fragments using a photometer.

[0195] 3) Enzyme digestion of MMP14 fragment and vector Pegfp-N1, the system is as follows:

[0196] Plasmid 3 μg or MMP14 fragment 1 μg

[0197] XhoI 1 μL

[0198] XbaI 1 μL

[0199] 10X CutSmart Buffer 5μL

[0200] Add dd H2O to 50 μL

[0201] 37℃ water bath for 1-2 hours

[0202] 4) Inactivate in a 65°C water bath for 20 min, add loading buffer, and perform agarose gel electrophoresis for 30 min at 120 V to recover fragments and vectors of corresponding sizes;

[0203] 5) Connection:

[0204] 10X ligase buffer 2μL

[0205] Carrier: 50ng

[0206] T4 Ligase 1 μL

[0207] Target gene fragment (~50ng / μL): up to 20μL

[0208] 6) Connect overnight at room temperature (about 25°C);

[0209] 7) Transformation: Take 5 μL of the ligation product and add it to 50 μL of DH5 competent cells just taken out of the negative 80 refrigerator, and let it stand on ice for half an hour; after heat shock in a 42°C water bath for 45 seconds, immediately transfer it to ice for 2 minutes. This process needs to be operated gently and the centrifuge tube should not be shaken; then add 900 μL of LB medium without antibiotics and shake it at 37°C and 150 rpm for one hour to revive the bacteria; centrifuge at room temperature for 5 minutes at 4500 rpm, discard 900 μL of supernatant, resuspend the remaining 100 μL supernatant and the bottom bacterial sediment by suction, pour it on a plate containing ampicillin antibiotics, and evenly spread the bacterial solution with a glass rod that has been sterilized with an alcohol lamp and restored to room temperature, place the plate in a 37°C incubator until the liquid is absorbed, then invert the plate and culture overnight;

[0210] 8) The next day, use a 10μL pipette tip to pick up a single clone colony, add it to a liquid culture medium containing ampicillin, and shake it at 37°C and 220rpm for 8-12 hours. Then take 500μL of the bacterial solution and send it to sequencing (Biological Engineering). If the sequencing is correct, take the corresponding bacterial solution and add it to a 300mL culture bottle for expansion culture for 12 hours, and then extract the plasmid according to the steps of plasmid extraction (Vazyme, #DC202-01):

[0211] a) Transfer the overnight culture solution to a 50 mL centrifuge tube, centrifuge at 11000 rpm for 2 min, and discard the supernatant;

[0212] b) Add 7.5 mL of P1 solution to the bacterial pellet and vortex to mix;

[0213] c) Add another 7.5 mL of P2 solution, gently mix by inverting, and let stand at room temperature for 5 min;

[0214] d) Add 7.5 mL of P4 solution and immediately invert for about 8 times. A white flocculent precipitate will appear. Incubate at 11000 rpm for 10 min and aspirate the supernatant (avoid aspirating the white precipitate) into a new 50 mL round-bottom tube;

[0215] e) Add 0.1 volume of endotoxin remover, mix by inversion, let stand on ice for 5 min, let stand at room temperature for 10 min, and mix by inversion;

[0216] f) Centrifuge at 11000 rpm for 10 min at room temperature, take the upper aqueous phase (containing DNA) into a new tube, add 0.5 times the volume of isopropanol, transfer to an adsorption column several times, centrifuge at 11000 rpm for 1 min, and discard the waste liquid in the collection tube;

[0217] g) Use 10 mL of rinsing solution PW, 11000 rpm, 1 min, discard the waste liquid, and repeat twice;

[0218] h) Centrifuge at 11000 rpm for 3 min;

[0219] i) Place the adsorption column in a new centrifuge tube, add 1 mL of ddH2O in the middle of the membrane, let stand at room temperature for 3 min, and centrifuge at 11000 rpm for 3 min;

[0220] j) Measure the concentration and purity of the resulting plasmid using a photometer.

[0221] Recombinant protein purification:

[0222] 1) Add 1 mL of glycerol bacteria expressing MMP-14-EGFP or M7CK to 1 L of LB medium and shake at 37°C, 220 rpm overnight. Centrifuge the amplified bacterial solution at 4200 rpm, 4°C for several times until the bacterial precipitate is concentrated in the same 50 mL tube;

[0223] 2) Add bacterial lysis buffer (50 mM Tris HCI pH 8.0, 100 mM NaCl, 10 mM MgCl2, 1 mM EDTA, 10 mM 1-mercaptoethanol, 20% glycerol, 1 mg / μL lysozyme, 8 M urea, one Roche protease inhibitor tablet) to the precipitate and place on ice for 30 min;

[0224] 3) Centrifuge at 28,000 rpm, 4°C for 30 min;

[0225] 4) Pipette the supernatant and add it to the amicon ultra 15ML-30K protein ultrafiltration centrifuge tube. Centrifuge at 4200rpm, 4℃ for 60min. Repeat several times until the remaining liquid is less than 1mL. The liquid contains the concentrated protein and is used for subsequent in vitro enzyme activity test experiments.

[0226] Protein Co-immunoprecipitation (Co-IP)

[0227] 1) Collect 293 cells transfected with M7CK and MMP14-EGFP for 48 hours, wash the surface culture medium with PBS, then add PBS to the culture dish, and repeatedly blow and suck with the tip of the pipette to suspend the attached cells in PBS to obtain a cell suspension. Centrifuge at 4°C, 12000rpm for 5min, remove the supernatant, add IP-specific RIPA containing protease inhibitors and phosphatase inhibitors to the cell pellet, vortex and shake vigorously to fully lyse the cells, and let stand on ice for 30min. Centrifuge at 4°C, 12000rpm for 30min, and aspirate the supernatant into a new EP tube, which is the total protein extract;

[0228] 2) Pipette 50 μL of mixed magnetic beads into a 2 mL EP tube, place it on a magnetic stand, and remove the supernatant;

[0229] 3) Add 200 μL of antibody binding solution to the magnetic beads, then add antibody M7CK (dilution ratio 1:100) or MMP14 (dilution ratio 1:200) or control group IgG, and rotate at room temperature for 30-60 minutes;

[0230] 4) Place the EP tube on the magnetic stand and discard the supernatant. At this time, the antibody has been firmly bound to the magnetic beads;

[0231] 5) Add antibody binding solution, invert and mix, then place on a magnetic rack and discard the supernatant to wash away unbound antibodies

[0232] 6) Add total cell protein extract to the magnetic beads and incubate with rotation at 4°C for 42-48 hours;

[0233] 7) Place on a magnetic rack, remove the supernatant, add cleaning solution, rotate at room temperature for 1 min, discard the supernatant on the magnetic rack, and repeat three times;

[0234] 8) Add 20 μL of elution buffer to the magnetic beads, mix well and transfer to a 600 μL EP tube, then add 5 μL of loading buffer and boil at 100°C for 5 min;

[0235] 9) Western blotting was used to detect the relevant proteins in the liquid.

[0236] In vitro Aβ degradation assay:

[0237] MMP14 and M7CK recombinant proteins were purified from E. coli according to the above method. 80nM MMP14 / M7CK was mixed with 2μM Aβ1-42, zymogen buffer (50mM tris HCI, 200mM NaCl, 5mM CaCI 2, pH7.5), 10mM ATP, the system was 50μL, and incubated at 37℃ for 10 hours. The content of residual Aβ was detected by protein immunoblotting. The lower the content of residual Aβ, the higher the degradation efficiency.

[0238] The specific experimental steps for knocking out TRPM7 with AAV-TRPM7 shRNA are as follows:

[0239] 1. Primary Hippocampal Neuron Culture

[0240] 1) Take a mouse pup that is just 3 days old, quickly cut off the head, peel off the brain tissue, place it in a dissection buffer on ice, carefully separate the hippocampal tissue on both sides under a stereoscope, and try to peel off the blood cells on its surface;

[0241] 2) After the dissection is completed, cut the hippocampal tissue into small pieces to avoid tearing;

[0242] 3) Wash the chopped hippocampal tissue three times with dissection buffer;

[0243] 4) Add trypsin lysis buffer and incubate at 37°C for 5 min, shaking gently 1-2 times;

[0244] 5) Wash once with dissection buffer;

[0245] 6) Add dissociation solution containing DNase and repeatedly blow with a pipette tip until there are no obvious tissue blocks;

[0246] 7) 4°C, 1500 rpm, 15 min;

[0247] 8) Discard the supernatant, resuspend the pellet with plating medium (400 μL medium / pair of hippocampi), and add the cell suspension to the glass slide at the bottom of the four-well plate (100 μL cell suspension / hole with a diameter of 1.8 mm, the glass slide needs to be pre-coated with matrix gel (DM dilutes matrix gel 1:100) at 37°C for 1 hour). After culturing in a constant temperature incubator for 1.5-2 hours, the cells have basically completed the attachment to the wall. At this time, add 1 mL plating medium / well, and replace it with serum-free medium two days later. The interval between half-change of medium is 3-4 days.

[0248] 2. Insert the synthesized TRPM7-shRNA sequence (coding sequence 5153-5172 5′-AAT GCA TGA CTG GGGAAT TTC AAG AGA ATT CCC CAG TCA TGC at-3′) into the AAV-CAG-mCherry plasmid and package it into the AAV virus;

[0249] 3. Add AAV virus (1ul / ml culture medium) to the cultured mouse primary hippocampal neurons on the seventh day;

[0250] 4. When protein was extracted from mouse primary hippocampal neurons on the fourteenth day of culture, it was detected that the expression level of TRPM7 was significantly downregulated compared with the control group.

[0251] The specific experimental steps for knocking down MMP14 are as follows:

[0252] 1. Primary Hippocampal Neuron Culture

[0253] 1) Take a mouse pup that is just 3 days old, quickly cut off the head, peel off the brain tissue, place it in a dissection buffer on ice, carefully separate the hippocampal tissue on both sides under a stereoscope, and try to peel off the blood cells on its surface;

[0254] 2) After the dissection is completed, cut the hippocampal tissue into small pieces to avoid tearing;

[0255] 3) Wash the chopped hippocampal tissue three times with dissection buffer;

[0256] 4) Add trypsin lysis buffer and incubate at 37°C for 5 min, shaking gently 1-2 times;

[0257] 5) Wash once with dissection buffer;

[0258] 6) Add dissociation solution containing DNase and repeatedly blow with a pipette tip until there are no obvious tissue blocks;

[0259] 7) 4°C, 1500 rpm, 15 min;

[0260] 8) Discard the supernatant, resuspend the pellet with plating medium (400 μL medium / pair of hippocampi), and add the cell suspension to the glass slide at the bottom of the four-well plate (100 μL cell suspension / hole with a diameter of 1.8 mm, the glass slide needs to be pre-coated with matrix gel (DM dilutes matrix gel 1:100) at 37°C for 1 hour). After culturing in a constant temperature incubator for 1.5-2 hours, the cells have basically completed the attachment to the wall. At this time, add 1 mL plating medium / well, and replace it with serum-free medium two days later. The interval between half-change of medium is 3-4 days.

[0261] 2. The synthesized MMP14-shRNA sequence (#1 coding sequence 1026-1046, 5′-ACC GGG GGG GTG AGGAAT AAC CAA GTC TCG AGA CTT GGT TAT TCC TCA CCC GCT TTT T-3′. #2 coding sequence 788-808, 5′-ACC GGG GAT GGA CAC AGA GAA CTT CGC TCG AGC GAA GTT CTC TGT GTC CATCCT TTT T-3′) was inserted into the AAV-CAG-mCherry plasmid and packaged into the AAV virus;

[0262] 3. Add AAV virus (1ul / ml culture medium) to the cultured mouse primary hippocampal neurons on the seventh day;

[0263] 4. When protein was extracted from mouse primary hippocampal neurons on the fourteenth day of culture, it was detected that the expression level of MMP14 was significantly downregulated compared with the control group.

[0264] As can be seen from the above examples 1-5, the expression of TRPM7 is downregulated in the hippocampus of AD patients and two AD mouse models; overexpression of the kinase domain of TRPM7 in primary hippocampal neurons cultured in vitro can restore the decrease in synaptic density induced by Aβ, while the channel domain of TRPM7 has no such protective effect; in addition, overexpression of M7CK in the hippocampus of 5XFAD mice can improve its cognitive function damage, increase synaptic density, and reduce the accumulation of Aβ amyloid plaques; at the same time, M7CK also promotes the degradation of Aβ in soluble and insoluble components, and M7CK does not affect the production process of Aβ; further studies have shown that M7CK can promote the degradation process of Aβ by binding to and activating MMP14, which explains the mechanism of M7CK for AD protection to a certain extent. The experimental results of the present invention suggest the neuroprotective effect of TRPM7. In the study of the above examples, it was found that the kinase segment of overexpressing TRPM7 can promote the degradation of Aβ to offset the toxicity of Aβ to synapses and improve the cognitive function of AD mice. The loss of TRPM7 may promote the occurrence of AD pathology, while increasing the expression of TRPM7 kinase can improve AD pathology. The above examples show that the protein level and transcription level of TRPM7 in the hippocampus of AD patients are downregulated, and the expression of TRPM7 in the hippocampus of 5XFAD and APP / PS1 mice is also reduced. The kinase domain M7CK of TRPM7 can restore the Aβ-induced downregulation of synaptic density of primary hippocampal neurons; at the same time, overexpression of M7CK in the hippocampus of 5XFAD mice can improve their learning and memory abilities, and increase the density of presynaptic vesicle protein synaptophysin and postsynaptic density protein PSD-95. The channel domain of TRPM7 does not have this function. The above examples show that in in vivo and in vitro AD models, synaptic proteins synaptophysin and PSD-95 are reduced.

[0265] MMP14, as a member of the MMP family, is a special MMP located on the membrane and is active in itself. The experimental results of the above examples show that MMP14 interacts with M7CK, while no interaction is found between MMP2 and MMP9 and M7CK. Moreover, the activity of MMP14 is further enhanced when MMP14 and M7CK coexist. The protective effect of M7CK against Aβ toxicity also depends on MMP14 rather than MMP2 and MMP9. The above examples show that MMP14 can degrade exogenous Aβ 40 and Aβ 42: Under appropriate in vitro conditions, after the purified recombinant MMP14 protein was mixed and incubated with Aβ peptides for 10 hours, the Aβ level decreased by 50%, which is consistent with literature reports. The above examples show that in the hippocampus of 6-month-old 5XFAD mice, the protein level of MMP14 is higher than that of wild-type mice of the same age, which indicates that simply increasing the expression level of MMP14 is not enough to alleviate AD-related pathological characteristics, while overexpression of M7CK can promote Aβ degradation by increasing the phosphorylation level of MMP14, increase synaptic density and improve cognitive function of 5XFAD mice, which indicates that compared with the expression level of MMP14, the regulation of its phosphorylation level has a more obvious influence on improving AD pathology. However, no changes were found in the production process of APP (APP, βCTF). In addition, knocking out MMP14 in primary hippocampal neurons reduces synaptic density, indicating that MMP14 itself has an important regulatory role in synaptic function.

[0266] The above examples show that overexpression of M7CK in the hippocampus of wild-type mice does not enhance their learning and memory abilities and synaptic density, and only has an effect in 5XFAD mice. Therefore, the increase in synaptic density by M7CK occurs only under specific pathological conditions, rather than an indiscriminate overall enhancement. Combining previous studies with previously published studies, it was found that M7CK may undergo different shearing processes under physiological and pathological conditions, thereby affecting a series of downstream molecular mechanisms. The results of the above examples show that Aβ accumulation in vitro does not affect the shearing process; in addition, the above examples show that in Aβ-treated neurons, M7CK anchored on the cell membrane and free in the cytoplasm can restore Aβ-induced synaptic density reduction, indicating that under AD pathological conditions, the shearing state of M7CK does not affect its function of counteracting Aβ toxicity.

[0267] From the perspective of basic science, this invention will fill the major gaps in the prior art's understanding of the mechanism of action of the TRPM7 kinase domain in the central nervous system. This project will reveal new signaling pathways and mechanisms regulated by the TRPM7 kinase domain. This invention will demonstrate that TRPM7 uses physical and structural changes (cleavage and transport of peptides) rather than chemical modifications (phosphorylation) as triggers for the release and activation of kinases. Recently, structural changes in CaMKII rather than phosphorylation have been shown to trigger important functions for synaptic and cognitive functions. Therefore, this invention is introducing new concepts and discoveries that will expand the prior art's understanding of cellular, molecular and cognitive neuroscience. From the perspective of translational science, this invention combines computational, molecular, systems and biomedical research to achieve the transformation from basic science to translational science. In the future, these new mechanisms are expected to be transformed into potential drug targets for the development of new drugs for the treatment of neurodegenerative diseases (particularly Alzheimer's disease). In addition, this invention will provide evidence for the feasibility of TRPM7 as a therapeutic target for Alzheimer's disease. Provide a new gene therapy method for AD: viral-mediated overexpression of the enzyme domain of TRPM7.

[0268] In summary, the present invention explores in detail the possible role of TRPM7 in the course of AD and its specific mechanism of action, providing a potential target with important value for AD drug development.

[0269] The above examples are for the purpose of illustrating the embodiments disclosed by the present invention and are not to be construed as limiting the present invention. In addition, the various modifications listed herein and the variations of the methods in the invention are obvious to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of the present invention.

Claims

1. Application of the kinase domain M7CK of TRPM7 in the preparation of therapeutic drugs for Alzheimer's disease.

2. The use according to claim 1, characterized in that The amino acid sequence of the M7CK is selected from the following: 1) A polypeptide sequence encoded by the nucleotide sequence SEQ ID No. 1; 2) A polypeptide fragment having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% sequence identity with the polypeptide of 1) on the basis of having the same biological function as the polypeptide of 1).

3. The use according to claim 2, characterized in that 1) The polypeptide sequence comprises the amino acid sequence of positions 1299-1863 of TRPM7. Preferably, the polypeptide sequence is as shown in SEQ ID No.

3.

4. The use according to claim 1, characterized in that The Alzheimer's disease therapeutic drug includes an M7CK promoter.

5. The use according to claim 4, characterized in that The M7CK promoter is selected from one or more of the following: Plasmid or virus that effectively overexpresses M7CK; And / or, a substance that promotes the kinase cleavage of TRPM7 to produce M7CK.

6. The use according to claim 5, characterized in that The plasmid or viral vector overexpressing M7CK includes a nucleic acid fragment encoding M7CK; or, the substance that promotes the kinase cleavage of TRPM7 to produce M7CK is selected from one or more of the following compounds: resveratrol, zafirukast, TPCA-1, ATT, and glutamate or glutamate.

7. The use according to claim 6, characterized in that The sequence of the M7CK coding gene is shown in SEQ ID No.

1.

8. The use according to claim 6, characterized in that: The coding gene of M7CK is a nucleic acid molecule as shown in SEQ ID No.1, and the nucleic acid fragment is optimized by any one or more of the following methods: 1) the G / C content is increased compared with the nucleic acid molecule as shown in SEQ ID No.1; 2) the nucleic acid molecule as shown in SEQ ID No.1 is codon optimized; The nucleic acid fragment does not change the encoded amino acid sequence after codon optimization or G / C content improvement.

9. The use according to claim 5, characterized in that The plasmid is selected from adenovirus packaging plasmid, adeno-associated virus packaging plasmid or lentivirus packaging plasmid; the virus is selected from adenovirus, adeno-associated virus or lentivirus.

10. The use according to claim 5, characterized in that The plasmid is pAAV2 / 9-M7CK plasmid. Preferably, the pAAV2 / 9-M7CK plasmid sequence is shown in SEQ ID NO.

2.

11. The use according to claim 10, characterized in that: The pAAV2 / 9-M7CK plasmid is used for AAV virus packaging to obtain the packaged AAV virus.

12. The use according to claim 11, characterized in that: The method for using the Alzheimer's disease therapeutic drug is: the packaged AAV virus is injected into the brain by stereotaxic injection, specifically into the hippocampus region of the brain.

13. The use according to any one of claims 1 to 12, characterized in that: The Alzheimer's disease therapeutic drug has one or more of the following functions: 1) Increase the density of neuronal synapses; 2) Improve learning and memory abilities; 3) Reduction of Aβ plaques or Aβ protein; 4) Activating MMP14 activity, preferably, phosphorylating MMP14 protein.

14. A pAAV2 / 9-M7CK plasmid constructed based on the M7CK sequence of the kinase domain of TRPM7, which can overexpress the M7CK sequence at positions 1299-1863 of TRPM7.

15. The pAAV2 / 9-M7CK plasmid of claim 14, wherein the amino acid sequence of M7CK is selected from the following: 1) A polypeptide sequence encoded by the nucleotide sequence SEQ ID No. 1; 2) A polypeptide fragment having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% sequence identity with the polypeptide of 1) on the basis of having the same biological function as the polypeptide of 1).

16. The pAAV2 / 9-M7CK plasmid according to claim 14, characterized in that 1) The polypeptide sequence comprises the amino acid sequence of positions 1299-1863 of TRPM7. Preferably, the polypeptide sequence is as shown in SEQ ID No.

3.

17. The pAAV2 / 9-M7CK plasmid according to claim 14, characterized in that The pAAV2 / 9-M7CK plasmid includes a nucleic acid fragment encoding M7CK.

18. The pAAV2 / 9-M7CK plasmid according to claim 17, characterized in that The encoding nucleic acid fragment of the M7CK is: 1) Sequence shown in SEQ ID No.1; 2) A nucleic acid fragment optimized by any one or more of the following methods for the nucleic acid molecule shown in 1): 1) having an increased G / C content compared to the nucleic acid molecule shown in SEQ ID No. 1; 2) the nucleic acid molecule shown in SEQ ID No. 1 is codon optimized; wherein the amino acid sequence encoded by the nucleic acid fragment is not changed after codon optimization or increased G / C content.

19. A virus constructed based on the M7CK sequence of the kinase domain of TRPM7 and capable of overexpressing the M7CK sequence at positions 1299-1863 of TRPM7, which is obtained by viral packaging of the pAAV2 / 9-M7CK plasmid described in any one of claims 14 to 18.

20. The virus according to claim 19, characterized in that The virus is selected from one or more of adenovirus, adeno-associated virus or lentivirus.

21. A method for preventing or treating Alzheimer's disease, characterized in that: The method comprises administering an effective dose of the M7CK promoter according to any one of claims 4 to 13, the pAAV2 / 9-M7CK plasmid according to any one of claims 14 to 18, or the virus according to claim 19 or 20 to the subject.

22. The method according to claim 21, characterized in that The administered amount is 1-1000 mg / kg / day.

23. The method according to claim 21, characterized in that The subject of the method is a mammal; preferably, the subject of the method is a human.

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