Application of TRPM7 activator in preparation of Alzheimer disease treatment product

Abstract

The invention relates to the field of biological medicine, in particular to application of a TRPM7 activator in preparation of a product for treating Alzheimer's disease. The TRPM7 activator provided by the invention can be a regulator of extracellular divalent cations or a metal salt containing the divalent cations. The TRPM7 activator provided by the invention can retain the synaptic density of an Alzheimer's disease model mouse and reduce A beta plaque deposition, and the morphological and organ weight analysis of the heart, liver and pancreas shows that the application of the TRPM7 activator is safe. Besides, the TRPM7 gene or the protein coded by the TRPM7 gene can be used as a treatment target of the Alzheimer's disease, and a high-throughput method for screening potential drug targets of the Alzheimer's disease by screening a TRPM7 activator through the TRPM7 protein broadens thoughts for discovery and development of new drugs for the Alzheimer's disease and provides a new direction.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the application of TRPM7 activators in the preparation of Alzheimer's disease treatment products. Background Technology

[0002] TRPM7 is a ubiquitous divalent cation channel with a functional serine / threonine kinase at its C-terminus. TRPM7 can transport magnesium (Mg) 2+ ), Zinc (Zn) 2+ ) and calcium (Ca 2+ TRPM7 is a divalent cation, including α-kinase ...

[0003] In the mammalian brain, the kinase domain itself has been shown to be essential for synaptic density, synaptic plasticity, and learning and memory, all mediated through cofilin-dependent mechanisms. The free TRPM7 kinase domain is part of the Rac1-SSH2-cofilin complex, which regulates actin filament polymerization in neural dendrites. Unlike cell lines, the kinase domain does not regulate epigenetic modifications in neurons. Under pathological conditions, the kinase domain interacts with and activates the metalloproteinase MMP14, promoting Aβ degradation, protecting synaptic density, and maintaining memory function in an Alzheimer's disease mouse model. Therefore, cleavage of TRPM7 to release the kinase domain appears to occur in the central nervous system. However, the current technology remains inconclusive regarding where the free kinase domain is transported in neurons and what cellular and molecular mechanisms regulate this cleavage. Summary of the Invention

[0004] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide the application of TRPM7 activator in the preparation of Alzheimer's disease treatment products, in order to solve the problems in the prior art.

[0005] To achieve the above and other related objectives, the present invention provides the use of TRPM7 activator in the preparation of Alzheimer's disease treatment products.

[0006] Preferably, the TRPM7 activator is a divalent cation regulator or a metal salt containing a divalent cation.

[0007] Preferably, the TRPM7 activator is selected from one or more of the following compounds: resveratrol, zalfilocaster, TPCA-1, ATT, and glutamic acid or glutamate.

[0008] The present invention also provides the use of TRPM7 protein or its mutant protein in the preparation of screening MEF2C protein activator products and / or RPS6 protein activator products.

[0009] Preferably, the Alzheimer's disease treatment product is an activator of MEF2C protein and / or RPS6 protein.

[0010] The present invention also provides a method for screening MEF2C protein activators and / or RPS6 protein activators using TRPM7 protein or its mutant protein, characterized in that the method comprises the following steps:

[0011] 1) After mixing the activator to be tested with cells expressing TRPM7 protein, the reagents that phosphorylate S222 and / or S396 residues in MEF2C protein or S235 and / or S236 residues in RPS6 protein were screened to obtain the reagents.

[0012] 2) Mix the reagents selected in step 1) with cells expressing TRPM7 protein and detect the membrane potential of the cells. If the change in membrane potential increases, an effective MEF2C protein activator or / and RPS6 protein activator is selected.

[0013] As described above, the application of the TRPM7 activator of the present invention in the preparation of Alzheimer's disease treatment products has the following advantages:

[0014] Beneficial effects:

[0015] This invention provides a TRPM7 activator that can preserve synaptic density and reduce Aβ plaque deposition in Alzheimer's disease model mice. Morphological and organ weight analyses of the heart, liver, and pancreas demonstrate the safety of TRPM7 activator administration. Furthermore, the TRPM7 gene or its encoded protein can serve as a therapeutic target for Alzheimer's disease. This invention provides a high-throughput method for screening potential drug targets for Alzheimer's disease by screening TRPM7 activators, broadening the horizons and providing new directions for the discovery and development of new Alzheimer's drugs, and potentially reducing the development costs and timelines. Attached Figure Description

[0016] Figure 1The results are shown in the study of the activity and specificity of the TRPM7 activator in this invention.

[0017] Figure 2 The results show that different modulators activated the TRPM7 ion channel in this invention can prevent Aβ-induced loss of synapses in cultured neurons.

[0018] Figure 3 The results show that chronic treatment with the TRPM7 activator ATT in this invention can salvage the cognitive and memory functions of 5×FAD mice.

[0019] Figure 4 The results show that chronic treatment with the TRPM7 activator ATT in this invention can protect synaptic density and reduce Aβ plaques in 5×FAD mice.

[0020] Figure 5 The results of a toxicity study of ATT, the TRPM7 activator in this invention, are shown.

[0021] Figure 6 This is shown as the result that TRPM7 is mainly cleaved in excitatory neurons and transported to synapses, cytoplasm, and nuclei in this invention.

[0022] Figure 7 The results show the effects of Calpain 1, neuronal activity, and the regulation of M7CK release and activity by calcium and magnesium concentrations in this invention.

[0023] Figure 8 The results shown are from a spatial proteomics study of the M7CK regulatory signal in a cell compartment-specific manner, as presented in this invention.

[0024] Figure 9 The results show the effects of TRPM7 cleavage, free kinase, and M7CK translocation to the nucleus on synaptic density in this invention.

[0025] Figure 10 The image shows the results of TRPM7 lysis in different brain regions during this invention.

[0026] Figure 11 This is shown as a result in nerve cells, where Caspase3 does not cleave TRPM7 to release M7CK.

[0027] Figure 12 The results show that inhibiting TRPM7 and its kinase domain in this invention affects activity-dependent protein synthesis.

[0028] Figure 13 The image shows the results of M7CK being located in the cell nucleus in this invention. Detailed Implementation

[0029] This invention provides the application of TRPM7 activator in the preparation of Alzheimer's disease treatment products.

[0030] In some specific embodiments, the TRPM7 activator can activate or upregulate TRPM7 gene expression, and / or activate or enhance the activity of the protein product encoded by the TRPM7 gene. Specifically, enhancing the activity of the protein product encoded by the TRPM7 gene involves opening the ion channels of the protein product encoded by the TRPM7 gene.

[0031] In some specific embodiments, the TRPM7 activator can be a regulator of divalent cations or a metal salt containing divalent cations. Specifically, the divalent cation is selected from one or more of magnesium ions, calcium ions, zinc ions, copper ions, or ferrous ions. Preferably, the divalent cation is one or more of magnesium ions or calcium ions. Specifically, the regulator of divalent cations has the function of regulating extracellular divalent cations in cells.

[0032] In some specific embodiments, the TRPM7 activator is selected from low molecular weight chemical compounds or small molecule compounds. Specifically, the molecular weight of the low molecular weight chemical compound or small molecule compound is no greater than 3000 Da. More specifically, the molecular weight of the low molecular weight chemical compound or small molecule compound can be 10-100, 100-200, 200-400, 400-800, 800-1000, 1000-1200, 1200-1500, 1500-1750, 1750-2000, 2000-2500, or 2500-3000 Da. The low molecular weight chemical compound or small molecule compound can bind to and enhance the function of the TRPM7 gene or its expression product.

[0033] Furthermore, the TRPM7 activator may be selected from one or more of the following compounds: resveratrol, zarfilocaster, TPCA-1 (CAS No.:507475-17-4), ATT (Anethole trithione CAS No.:532-11-6), and glutamic acid or glutamate.

[0034] In some specific embodiments, the TRPM7 activator is selected from the protein product of the TRPM7 gene or its active fragment, or a nucleic acid molecule encoding the protein product or its active fragment, or a vector containing the nucleic acid molecule, or a trans-regulatory element of the TRPM7 gene, or a nucleic acid molecule encoding the trans-regulatory element or its active fragment, or a vector (e.g., a cloning vector or an expression vector) containing the nucleic acid molecule. Specifically, the trans-regulatory element may be selected from one or more of the following: Calpain1 protein or its mutant, a CRISPR / dCas9 system containing the VP64 domain, a CRISPR / dCas9 system containing the p65 activation domain, a CRISPR / dCas9 system containing the Rta47 domain, or a CRISPR / dCas9 system containing the VP16 domain. Specifically, the Calpain1 protein comprises a polypeptide whose amino acid sequence includes that shown in SEQ ID No. 1; a Calpain1 protein mutant, having the same biological function as the polypeptide whose amino acid sequence is shown in SEQ ID No. 1, has a polypeptide fragment with 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 shown in SEQ ID No. 1.

[0035] In some specific embodiments, the Alzheimer's disease treatment product is selected from drugs having one or more of the following functions:

[0036] 1) Increase neuronal synapse density;

[0037] 2) Improve learning and memory abilities;

[0038] 3) Reduce Aβ plaques. This includes increasing neuronal synaptic density relative to Alzheimer's patients; improving learning and memory abilities relative to Alzheimer's patients; and reducing Aβ plaques relative to Alzheimer's patients.

[0039] In some specific embodiments, the Alzheimer's disease treatment drug further comprises pharmaceutically acceptable excipients. Specifically, the excipients include various excipients and diluents that are not essential active ingredients and do not cause excessive toxicity after administration. The excipients include sterile water or physiological saline, stabilizers, excipients, antioxidants (ascorbic acid, etc.), buffers (phosphate, citric acid, other organic acids, etc.), preservatives, surfactants (PEG, Tween, etc.), chelating agents (EDTA, etc.), or binders. The excipients also include other low molecular weight peptides, serum albumin, glycine, glutamine, asparagine, arginine, polysaccharides, monosaccharides, mannitol, or sorbitol. When the excipients are used in an aqueous solution for injection, they are selected from physiological saline, glucose isotonic solution, D-sorbitol isotonic solution, D-mannose isotonic solution, D-mannitol or sugar alcohol isotonic solution. The aqueous solution for injection contains a solubilizer. The solubilizer is selected from alcohols (ethanol), polyols (propylene glycol or PEG), and / or nonionic surfactants (Tween 80 or HCO-50). In the Alzheimer's disease treatment drug, the aforementioned TRPM7 activator is a single active ingredient, or it can be combined with one or more other active ingredients useful for Alzheimer's disease treatment to form a combination formulation. The active ingredients are various other drugs used 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 those skilled in the art. For example, the dosage of the active ingredient in the aforementioned TRPM7 activator and Alzheimer's disease treatment products depends on the patient's weight, the type of application, the condition and severity of the disease. For example, the dosage of the bifunctional compound 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.

[0040] The present invention also provides a treatment method for Alzheimer's disease, wherein the treatment method comprises administering an effective dose of the aforementioned TRPM7 activator to the patient.

[0041] This invention also provides the use of TRPM7 protein or its mutant protein in the preparation of screening products for MEF2C protein activators and / or RPS6 protein activators. The screening can be performed in an in vitro cell-free culture environment or an in vitro cell culture environment.

[0042] In some specific embodiments, the MEF2C protein activator and / or RPS6 protein activator are Alzheimer's disease treatment products.

[0043] Furthermore, the MEF2C protein activator and / or RPS6 protein activator is a reagent that phosphorylates the MEF2C protein or the RPS6 protein.

[0044] Further, the MEF2C protein activator is a reagent that phosphorylates residues S222 and / or S396 of the MEF2C protein; or, the RPS6 protein activator is a reagent that phosphorylates residues S235 and / or S236 of the RPS6 protein. The amino acid sequence of the MEF2C protein is shown in SEQ ID No. 2, and the amino acid sequence of the RPS6 protein is shown in SEQ ID No. 3.

[0045] In some specific embodiments, the TRPM7 protein comprises a polypeptide whose amino acid sequence includes the one shown in SEQ ID No. 4.

[0046] In some specific embodiments, the TRPM7 protein mutant is a polypeptide fragment that, while having the same biological function as the polypeptide with the amino acid sequence shown in SEQ ID No. 4, has 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 shown in SEQ ID No. 4.

[0047] Further, the TRPM7 protein mutant may be selected from one or more of the following mutants: TRPM7(P1040R), whose amino acid sequence is shown in SEQ ID No. 5; or TRPM7(LQRFK+LHSVQ), whose amino acid sequence is shown in SEQ ID No. 6; or TRPM7(K1646R), whose amino acid sequence is shown in SEQ ID No. 7.

[0048] This invention also provides a method for screening MEF2C protein activators and / or RPS6 protein activators using the aforementioned TRPM7 protein or its mutant protein, the method comprising the following steps:

[0049] 1) After mixing the activator to be tested with cells expressing TRPM7 protein, the reagents that phosphorylate S222 and / or S396 residues in MEF2C protein or S235 and / or S236 residues in RPS6 protein were screened to obtain the reagents.

[0050] 2) Mix the reagents selected in step 1) with cells expressing TRPM7 protein, and detect the membrane potential of the cells. An increase in the magnitude of the membrane potential change indicates the selection of an effective MEF2C protein activator and / or RPS6 protein activator. This method can be used for in vitro screening of TRPM7 activators.

[0051] In some specific embodiments, the cells expressing the TRPM7 protein are obtained by transfecting or infecting eukaryotic cells with a nucleic acid molecule encoding the TRPM7 protein product or an active fragment thereof, or a vector containing the nucleic acid molecule.

[0052] In some specific embodiments, the eukaryotic cells may be selected from one or more of HEK293, HEK293T, HUCCT1, HT-22, HCCC-9810, or RBE.

[0053] In this invention, the terms "low molecular weight chemical compound" or "small molecule compound" refer to organic non-protein compounds.

[0054] In this invention, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell through transformation, transduction, or transfection, allowing the genetic material elements it carries to be expressed in the host cell. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, the vector may also contain a replication initiation site.

[0055] In this invention, the term "CRISPR / dCas9 system" generally refers to the complex formed by the binding of the dCas9 protein to a designed crRNA. In the CRISPR / dCas9 system, the dCas9 protein loses its cleavage function, but the binding of dCas9 to crRNA can precisely locate and bind to the target site.

[0056] In this invention, the term "metal salt" generally refers to a compound formed by the combination of metal ions (usually cations) and non-metal ions (usually anions) through ionic bonds. Metal salts can be generated by the reaction of metals with acids (such as hydrochloric acid, sulfuric acid, nitric acid, etc.) or certain organic acids (such as acetic acid, citric acid, etc.).

[0057] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0058] Before further describing 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 terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0059] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0060] The specific amino acid or nucleotide sequence information used in this application is as follows:

[0061] Calpain1 protein: SEQ ID No. 1

[0062] MTEELITPVYCTGVSAQVQKKRDKELGLGRHENAIKYLGQDYETLRARCLQSGVLFQDEAFPPVSHSLGFKELGPHSSKTYGIKWKRPTELMSNPQFIVDGATRTDICQGALGDCWLLAAIASLTLNETILHRVVPYGQSFQDGYAGIFHFQLWQFGEWVDVVIDDLLPTKDGKLVFVHSAQGNEFWSALLEKAYAKVNGSYEALSGGCTSEAFEDFTGGVTEWYDLQKAPSDLYQIILKALERGSLLGCSINISDIRDLEAITFKNLVRGHAYSVTGAKQVTYQGQRVNLIRMRNPWGEVEWKGPWSDSSYEWNKVDPYEREQLRVKMEDGEFWMSFRDFIREFTKLEICNLTPDALKSRTLRNWNTTFYEGTWRRGSTAGGCRNYPATFWVNPQFKIRLEEVDDADDYDNRESGCSFLLALMQKHRRRERRFGRDMETIGFAVYQVPRELAGQPVHLKRDFFLANASRAQSEHFINLREVSNRIRLPPGEYIVVPSTFEPNKEGDFLLRFFSEKKAGTQELDDQIQANLPDEKVLSEEEIDDNFKTLFSKLAGDDMEISVKELQTILNRIISKHKDLRTNGFSLESCRSMVNLMDRDGNGKLGLVEFNILWNRIRNYLTIFRKFDLDKSGSMSAYEMRMAIEAAGFKLNKKLHELIITRYSEPDLAVDFDNFVCCLVRLETMFRFFKLLDTDLDGVVTFDLFKWLQLTMFA

[0063] MEF2C protein: SEQ ID No.2

[0064] MGRKKIQITRIMDERNRQVTFTKRKFGLMKKAYELSVLCDCEIALIIFNSTNKLFQYASTDMDKVLLKYTEYNEPHESRTNSDIVETLRKKGLNGCDSPDPDADDSVGHSPESEDKYRKINEDIDLMISRQRLCAVPPPSFEMPVTIPVSSHNSLVYSNPVSTLGNPNLLPLAHPSLQRNSMSPGVTHRPPSAGNTGGLMGGDLTSGAGTSAGNGYGNPRNSPGLLVSPGNLNKNIQAKSPPPMNLGMNNRKPDLRVLIPPGSKNTMPSVSEDVDLLLNQRINNSQSAQSLATPVVSVATPTLPGQGMGGYPSAISTTYGTEYSLSSADLSSLSGFNTASALHLGSVTGWQQQHLHNMPPSALSQLGDRTTTPSRYPQHTTRHEAGRSPVDSLSSCSSSYDGSDREDHRNEFHSPIGLTRPSPDERESPSVKRMRLSEGWAT

[0065] RPS6 protein: SEQ ID No.3

[0066] MKLNISFPATGCQKLIEVDDERKLRTFYEKRMATEVAADALGEEWKGYVVRISGGNDKQGFPMKQGVLTHGRVRLLLSKGHSCYRPRRTGERKRKSVRGCIVDANLSVLNLVIVKKGEKDIPGLTDTTVPRRLGPKRASRIRKLFNLSKEDDVRQYVVRKPLNKEGKKPRTKAPKIQRLVTPRVLQHKRRRIALKKQRTKKNKEEAAEYAKLLAKRMKEAKEKRQEQIAKRRRLSSLRASTSKSESSQK

[0067] TRPM7 protein: SEQ ID No.4

[0068]

[0069] TRPM7 protein mutant

[0070] P1040R: SEQ ID No. 5

[0071]

[0072] LQRFK+LHSVQ: SEQ ID No.6

[0073]

[0074] K1646R:SEQ ID No.7

[0075]

[0076] The embodiments of this application can be accomplished through the following experimental methods:

[0077] Drug preparation

[0078] NS8593 and NDGA, from Sigma-Aldrich (USA), were dissolved in DMSO to prepare a 20 mM solution and stored at -20°C until use. The concentration of DMSO in the bath solution did not exceed 0.05%. Resveratrol, Zafirukast, TPCA-1, and anisole were from MCE (China), dissolved in DMSO to prepare a 30 mM solution and stored at -20°C until use. Anisole was prepared as a 20 mg / ml solution in animal studies using 10% DMSO and 90% corn oil (carrier). It was administered intraperitoneally at doses of 2.5 and 3.75 μl / g, resulting in final concentrations of 50 and 75 mg / kg. For toxicity studies, anisole was administered intraperitoneally at doses of 2.5, 3.75, 5, 10, 15, 20, and 25 μl / g, resulting in final concentrations of 50, 75, 100, 200, 300, 400, and 500 mg / kg.

[0079] laboratory animals

[0080] Five-fold FAD male mice (B6SJL-Tg(APP-SwFlLon, PS1*M146L*L286V)6799Vas / J, JAX Stock No.: 034840) were mated with wild-type female mice (C57BL / 6). Offspring remained hemizygous and were genotyped according to the Jackson Laboratory protocol. Wild-type mice used for breeding the five-fold FAD mice were purchased from J.C. Labs in China. Mice were housed in controlled environments at the Fudan University Laboratory Animal Center (three to four mice per cage) at a temperature of 23℃±1°C and humidity of 50±10%, using a 12-hour / 12-hour inverted light-dark cycle. All experiments involving the animals were approved by the Fudan University Animal Health and Use Committee (License No.: SYXK-2020-0032). Unless otherwise stated, all mice used for behavioral and biochemical analyses were male.

[0081] Identical TRPM7-flox mice (TRPM7 flox / flox Crossing 2-5 month old mice with calcium / calmodulin kinase II (CaMKII)-Cre mice (2-5 months old) can produce antibodies in CaMKIIα-positive glutamatergic neurons (CaMKII-TRPM7). - / -Brain-specific mice lacking TRPM7 were used. Newborn (0-3 days old) primary cultured hippocampal neurons or adult (2-5 months old) wild-type mice (C57BL / 6J) were purchased from J.C.J. Laboratory Animal Co., Ltd. (Shanghai, China). Mice were housed in groups (3-4 mice / cage) under controlled conditions of temperature (21℃±1), humidity (50±10%), and an inverted light cycle (12:12 hours, lights on at 9:00 AM), with free access to food and water. All animal experiments were approved by the Animal Husbandry and Use Committee (License No.: CH-A-001: SYXK-2020-0032). Male mice (2-5 months old) were used.

[0082] clones

[0083] To express these cells in mammalian cells, TRPM7 (NM_021450.2), TRPM2 (NM_138301), TRPM6 (NM_153417.2), TRPM8 (NM_134252.5), and potassium voltage-gated channel subfamily H member 2 (KCNH2, NM_000238.4) were cloned into the pcDNA3.1 vector (Invitrogen, USA) and validated by double-stranded DNA sequencing.

[0084] Caloplasmin 1 (GenBank ID: NM_001110504.1) and caloplasmin 2 (GenBank ID: NM_009794.4) siRNA sequences and scrambled miRNA sequences (negative control) were provided by Hanbi Biotech (China) and cloned into the pcDNA3.1 vector (Orbio Technologies, China). The oligonucleotide sequences are as follows: Caloplasmin 1 siRNA sequence: positive strand

[0085] The calpain 2 siRNA sequence is as follows: 5'-GGA ACU ACC CAG CUA CCU UTT-3'; antisense strand, 5'-AAG GUA GCU GGG UAG UUCCTT-3'; calpain 2 siRNA sequence: sense strand, 5'-CCG GGA AGU GGA AUG ACA ATT-3'; antisense strand, 5'-UUG UCA UUC CAC UUC CCG GTT-3'; interference siRNA sequence: sense strand, 5'-UUC UCC GAA CGU GUCACG UdT dT-3'; antisense strand, 5'-ACG UGA CAC GUU CGG AGA AdT dT-3'. The calpain 1 clone used for overexpression experiments was generated and cloned using the pcDNA3.1 vector (Orbio Technologies, China). The primer sequences for calpain 1 are as follows: positive primer, 5'-ATG ACA GAG GAG TTA ATC ACC C-3'; antisense primer, 5'-GGC AAA CATAGT CAG CTG GA-3'. The full-length TRPM7 (GenBank ID: NM_001164325.1) used to construct EGFP-TRPM7, TRPM7-EGFP, and EGFP-TRPM7-mCherry plasmids was generated and cloned in pEGFP-N1 or pEGFP-C1 vectors (OBiO Technology, China). The primer sequences for the full-length TRPM7 are as follows: positive primer, 5'-ATG TCC CAG AAA TCCTGG ATA G-3'; antisense primer, 5'-TAA CAT CAG ACG AAC AGA ATT TGT TGC-3'. For the mutant TRPM7 clones (TRPM7P1040R-EGFP and TRPM7K1646R-EGFP), the following methods were used: Max Ultra Fidelity DNA Polymerase Kit (Vazyme Biotechnology Co., Ltd., #P505-d1, China). Then, use In- The TRPM7 mutant plasmid was constructed using a rapid assembly master mixture (Takara Biomedical Technology, #638948, USA). Primer sequences are as follows: TRPM7P1040R: Sensitive strand, 5'-GCT AAA GAT ATA GTT TTT CAT CGA TAC TGG ATG ATT TTT GGTG-3'; Antisense strand, 5'-ATG AAA AAC TAT ATC TTT AGC AAG AGA CCA AGA TGG TTC TT-3'; TRPM7K1646R: Sensitive strand, 5'-GGG CAT CTC TAT ATC ATT CGG TCA TTT CTT CCT GAG GT-3'; Antisense strand, 5'-ATG ATA TAG AGA TGC CCT GAC TTC AGG ATA TCG TG-3'. A membrane-bound M7CK clone (GPI-M7CK-EGFP-GPI) was synthesized and cloned into the N-terminus and C-terminus of the original M7CK clone. The N-GPI sequence was: 5'-ATG GGA ATC CAA GGA GGA GGG TCT GTC CTG TTCGGG CTG CTG CTC GTC CTG GCT GTC TTC TGC CAT TCA GGT CAT AGC-3' and the C-GPI sequence was: 5'-CTT GAA AAT GGT GGG ACA TCC TTA TCA GAG AAA ACA GTT CTT CTG CTG GTG ACT CCATTT CTG GCA GCA GCC TGG AGC CTT CAT CCC TAA-3'. Similarly, M7CK clones of the nuclear localization signal (NLS) and nuclear output signal (NES) (3×NLS-M7CK-EGFP and 3×NES-M7CK-EGFP) were synthesized (Shanghai Sangon Biotech Co., Ltd., China) and cloned at the N-terminus of M7CK. The NLS sequence is as follows: 5'-CCT GCC GCC AAG AGG GTG AAG CTG GAT-3', and the NES sequence is as follows: 5'-CTG CCT CCACTT GAA AGA CTG ACA CTG-3'.

[0086] Quantitative reverse transcription polymerase chain reaction (qRT-PCR)

[0087] Total RNA was extracted from and homogenized from HEK 293T cell lysates or hippocampal tissues using TRIzol reagent (Ambion, #15596018, USA) from TRPM7flox / flox and CaMKII-TRPM7- / - mice, according to the manufacturer's instructions. One microgram of total RNA was converted to cDNA using SuperScript II reverse transcriptase (Invitrogen, #18064-014, USA). Primer sequences designed using Primer-BLAST (National Center for Biotechnology Information, USA) are as follows: TRPM7 primer sequences: positive primer, 5'-GAC CCC AAC GAG AAG CG-3'; antisense primer, 5'-CCA AAC GAC CAC AGA AAC A-3'; GAPDH primer sequences: positive strand, 5'-AGA GTG TTT CCT CGT CCC GTA-3'; antisense strand, 5'-TCG CTC CTGGAA GAT GGT GAT-3'. GAPDH was used as an internal reference in HEK 293T cell transfection validated by different cleavage mutant TRPM7 clones and RNA-seq data.

[0088] To validate the RNA-seq data, the following primers were used for the selected 12 genes: AQP4 primer sequences: positive primer, 5'-GCT AAG TCC GTC TTC TAC ATC-3'; antisense primer, 5'-CAG TGG TTT GCC CAG TTT-3'; ATP9A primer sequences: positive primer, 5'-GTA GAG GAG ATC CGA TGT TAT G-3'; ESD primer sequences: positive primer, 5'-TGT GAA TGC CAC TGA AGA-3'; antisense primer, 5'-GGA TGT CAA TCT GGG AAC-3'; ITGAM primer sequences: positive primer, 5'-CGA CAC CAT CGC ATC TAA-3'; antisense primer, 5'-ACC CAG GTA AGC ATCATT CA-3'; MAP2K1 primer sequences: positive primer, 5'-TTC AAG GTC TCC CAC AAG C-3'; antisense, 5'-TTC TCCCGA AGA TAG GTC A-3'; MRPS6 primer sequence: positive, 5'-GGA GGG TAT TTC CTG GTG-3'; antisense, 5'-GTG GGA CTG GGA CTA TGC-3'; NIBAN2 primer sequence: positive, 5'-TCC AGT ACC AGG AAG ACAATA A-3', antisense. 5'-agg atg cca cag gat gag a-3'; OLFML3 primer sequence: positive primer, 5'-CACTTG GCA AAC CGA ACA-3'; antisense primer, 5'-TGA ATA CGA GCC CTA CTG G-3'; PEBP1 primer sequence: positive primer, 5'-GCA GCA TTT CAT GGG ACG-3'; antisense primer, 5'-CAC CAG CCA GAC ATAGCG-3'; STAT3 primer sequence: positive primer, 5'-TTC AGA CCC GCC AAC AAA 3'; antisense primer, 5'-CCAGCA ACC TGA CTT TCG-3'; SYNGR1 primer sequence: positive primer, 5'-CAT CGT GGT CTT CGG CTC T3'; antisense primer, 5'-GGT TGT CCT TGG GCT TGG-3'; TPRG1L primer sequences: positive, 5'-TTC CTC CGAAGT CGC TCA A-3'; negative, 5'-GCT TTC CAA ATG GCA CAG A-3'.PCR was performed using Premix Taq (Takara, #RR901, USA) with equal volumes of cDNA and primers. After electrophoresis, the bands were visualized using a Tanon 1600 gel imaging system (Tanon, China) and analyzed using ImageJ software (v1.53, Bethesda, NIH, USA). The quantitative signals were normalized to the corresponding GAPDH band signals.

[0089] Cell culture and transfection

[0090] Human embryonic kidney (HEK) cells and hippocampal neural cell line (HT-22, Zhongqiao Xinzhou Biotechnology Co., Ltd., China) were cultured in Dulbecco modified eagletone medium (DMEM, Gibco, USA) containing 10% fetal bovine serum (FBS, Gibco, USA). Chinese hamster ovary (CHO) cells were cultured in DMEM / F12 medium supplemented with 10% FBS. Cells (approximately 60% density, 25 flasks) were transiently transfected using Lipofectamine 3000 reagent (Thermo Fisher Scientific, USA) with 3.6 μg cDNA carrying TRPM7, TRPM2, TRPM6, TRPM8 (HEK cells), or KCNH2. Control cells were transfected with 0.4 μg EGFP cDNA. All cells were maintained at 37°C in a humidified incubator containing 5% CO2. Electrophysiological recordings were performed 18–24 hours post-transfection.

[0091] In the calpain assay: HEK 293T or HT-22 cells were transfected with 4 μg of DNA plasmid using Lipofectamine 3000 (Invitrogen, #L3000015, USA) to cover 70% to 80% of the surface area of ​​a cell culture plate (35 mm in diameter, Thermo Fisher Scientific, #140675, USA). For pharmacological inhibition of TRPM7 cleavage, different concentrations of the calpain inhibitor Ac-LLnL-CHO (ALLN: 0, 2.5, 5, 10, 20, 40, and 100 μM) or different concentrations (1, 10, 25, and 100 μM) of the caspase inhibitor III (Calbiochem, #218745, Germany) were added to the culture medium, and imaging was performed 30 hours later. For siRNA of calpain 1 / 2, calpain 1 overexpression, and co-immunoprecipitation experiments, cells were collected 48 hours after transfection for Western blot analysis.

[0092] Hippocampal neuron culture, incubation, and transfection

[0093] Hippocampal bodies of neonatal C57BL / 6 wild-type mice (days 0-3) were carefully dissected in cold dissection medium (Sigma-Aldrich, #M0518, USA). The hippocampal bodies were then fragmented and digested with trypsin (Sigma-Aldrich, #T1005) and deoxyribonuclease (Sigma-Aldrich, #D5025, USA) at 37°C for 5 minutes, followed by chemical and mechanical cell dissociation. Cell suspensions were dropped onto coverslips (14 mm diameter, 0.17 mm thickness) and cultured in vitro for 14 days (DIV) before use. Cultures were incubated at 37°C in a humidified environment of 5% CO2. The same batch of homologous culture was used for each experiment, and treatments were performed on the same day (control and experimental groups). Results of hippocampal neuron culture experiments were obtained from at least three independent cell cultures in each group. In neuronal transfection experiments, DIV5 cultures were transfected with Lipofectamine 2000 (Invitrogen, #11668027, USA) as control (-EGFP), TRPM7-EGFP, EGFP-TRPM7, EGFP-TRPM7-mCherry, TRPM7P1040R-EGFP, and TRPM7K1646R-EGFP, and cultured for 48-72 hours, followed by imaging at DIV7-8. In TRPM7 inhibition experiments, DIV7 cultures were transduced with AAV-tdTomato-TRPM7shRNA (1 μl / ml medium) for neuronal viral infection to knock out TRPM7. The same cultures were cultured with AAV-M7CK-EGFP, AAV-M7CK-EGFP-GPI, AAV-3×NLS-M7CK-EGFP, or AAV-3×NES-M7CK-EGFP, and rescue experiments were performed at DIV7. Seven days later (DIV14), synaptophysin immunostaining or spike density assays were performed. Following neuronal activity, cultures of neurons differentiated for 14 days were treated for 1 hour with different concentrations of glutamate (0, 10, 20, 40, and 100 μM). These cultures were then collected for Western blot analysis to detect TRPM7 cleavage. During the time course of neuronal activity effects, 40 μM glutamate was added at 0, 5, 10, 15, 20, 30, 60, and 180 minutes, and cultures were then collected for Western blot analysis. To investigate the regulation of TRPM7 cleavage by calcium and magnesium, DIV12 cultures were cultured for 48 hours in media containing different concentrations of calcium (0.4, 0.8, 1.2, 1.8, 2.2, or 2.6 mM) or magnesium (0.2, 0.4, 0.8, 1.0, 1.4, and 2.0 mM), and cultures were then collected at DIV14 for Western blot analysis.In a pharmacological inhibition of TRPM7 cleavage assay, hippocampal neuronal cultures (DIV12) were cultured for 30 hours with the calpain 1 inhibitor Ac-LLnL-CHO (Calbiochem, product number #479975, Germany) and then imaged to monitor the distribution of mCherry signals.

[0094] Electrophysiological techniques

[0095] Whole-cell patch-clamp recordings were performed at room temperature using an EPC-10 amplifier and Patch Master software (HEKA, Germany). Currents were digitized at a sampling rate of 10 kHz and low-pass filtered at 2.0 kHz. Needles were fabricated using glass capillaries (World Precision Instruments, USA) with electrode resistances between 2 and 4 MΩ. The Mg2+-free syringe solution 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). TRPM7 currents were recorded by 300 ms voltage ramp injections from -100 to +100 mV, repeated every 5 seconds, while maintaining a potential of 0 mV. When the current reaches a steady state (no change for at least 30 ramps), the TRPM7 channel current at +100 and -100mV is obtained.

[0096] Primary hippocampal neuron culture and Aβ treatment

[0097] Hippocampus and cerebellum of WT mice on day 1 after birth were isolated in ice-cold separation medium (Sigma-Aldrich, #M0518). The tissues were then cut and chemically and mechanically separated by digestion with trypsin (Sigma-Aldrich, #T1005) and deoxyribonuclease (Sigma-Aldrich, #D5025) at 37°C for 5 minutes. The cell suspension was seeded onto glass slides with a diameter of 14 mm and a thickness of 0.17 mm and cultured in vitro for 14 days (DIV) before use. Cultures were performed at 37°C in a CO2 atmosphere with 5% humidity. Sister cultures from the same batch were always used and treated on the same day (divided into control and experimental groups). Aβ1–42 peptide (Tocris, #1428) was dissolved at a concentration of 1 μg / μl (200 μM) in 50 mM tris(hydroxymethyl)aminomethane (pH 7.4) buffer and stored as small aliquots at -20°C until use. At DIV12, a small amount of 2.5 μl of Aβ peptide was added to the neuron culture medium, resulting in a final concentration of 500 nM. The same volume of 50 mM tris(hydroxymethyl)aminomethane buffer was added to the control group culture medium. At DIV14, immunostaining of neuron cultures was performed.

[0098] Fluorescent immunostaining

[0099] For immunostaining of brain sections, anesthetized mice were perfused with 0.9% saline via the heart and then fixed with 4% PFA. The brains were removed, post-fixed overnight with 4% PFA, and dehydrated in a sucrose gradient. Next, the brains were embedded in a compound for optimal cutting temperature (Tissue Tech, #4583) and stored at -80°C. Frozen coronal sections (20 μm) were cut, mounted on slides, and allowed to dry for 5–6 hours. Hippocampal neuronal cultures were fixed in PBS containing 4% glutaraldehyde (PFA) at room temperature for 30 minutes. Then, the mounted brain sections or neuronal cultures were blocked for 2 hours at room temperature with a freshly prepared blocking solution (5% goat serum and 0.2% Triton X-100 in PBS), followed by overnight incubation at 4°C with a primary antibody diluted in blocking buffer. After washing with PBS, the slides were incubated at room temperature with a CF dye-labeled secondary antibody for 2 hours. The slides were incubated with 40,6-diamino-2-phenylindole (DAPI; 1:1000; Sigma-Aldrich, #D9542) at room temperature for 10 minutes. Finally, the sections and cultures were covered with an appropriate amount of anti-quenching encapsulation solution (Sangon Biotech, E675005-0010). The primary antibodies used include: anti-M7CK (1:200, a custom-designed polyclonal antibody targeting the TRPM7 kinase domain, Abclonal, China), anti-TRPM7 (1:50, Alomone, #ACC-047, a commercially available polyclonal antibody targeting the TRPM7 ion channel portion), anti-calpain 1 (1:100, Cell Signaling Technology, #2556, USA), anti-calpain 2 (1:100, Cell Signaling Technology, #2539, USA), anti-cleavage-caspase 3 (1:400, Cell Signaling Technology, #9661S, USA), synaptophysin (1:500; Synaptic Systems, #101011), Map-2 (1:500; Cell Signaling Technology, #4542S), anti-CaMKII (1:200, R&D Systems, #AF2086, USA), and anti-MEF2C (1:1000, Cell Signaling Technology, #4542S). Technology, #5030S, USA) and Anti-RPS6 (1:1000, Cell Signaling Technology, #2317S, USA).The secondary antibodies used were: CF-488 goat anti-mouse secondary antibody (1:500; Biotium, #20010), CF-555 goat anti-rabbit secondary antibody (1:500; Biotium, #20033), CF533 or CF647 (Biotium).

[0100] Microscopy, imaging and spot analysis

[0101] In hippocampal neuron cultures, images of synaptophysin spots were captured using an Olympus FluoView FV3000 confocal microscope with a 60×Numerical Aperture (NA) 1.42 oil immersion lens, digital zoom 3, and a resolution of 1024×1024. Synaptophysin spots located directly on individual dendrites were counted to measure density. Results are presented as the number of spots per 10 μm dendrite per neuron. In brain slices, images of synaptophysin were captured in the radial layer region of CA1 using an Olympus FluoView FV3000 confocal microscope with a 60×NA 1.42 oil immersion lens, digital zoom 3, and a resolution of 1024×1024. Continuous z-sections were captured in 1 μm steps. The three z-sections with the strongest signal were then superimposed into a single image. Spot counts were analyzed using Image-Pro Plus 6.0 (Media Cybernetics). The synaptophysin signal was enhanced using a HiGauss filter. Furthermore, the same finite watershed segmentation range was used to segment the spots in each image. The software calculated the total number of spots. The spot density, i.e., the total number of spots per 1000 μm², was calculated and presented. Three slices were used for quantitative analysis for each mouse.

[0102] Images were captured at 1024×1024 resolution with a 60×NA 1.42 oil immersion objective on an OLYMPUS FLUOVIEW FV1000 (Japan) at 3x magnification to observe TRPM7 cleavage and the distribution of fluorescence signals in hippocampal neuron cultures. The localization and distribution of EGFP or mCherry fluorescence signals indicated TRPM7 cleavage and fragment transport. For M7CK signaling in excitatory neurons (CaMKII), mouse cortical sections were imaged at 1024×1024 resolution with a 60×NA 1.42 oil immersion objective on an OLYMPUS confocal microscope at 1x magnification. The ratio of intranuclear to extranuclear M7CK fluorescence signals was used as a quantitative measure to compare kinase cleavage and transport in excitatory neurons. To co-localize TRPM7 with caspase 3, calpain 2, and calpain 1 in hippocampal neuron cultures, images were captured at 1024 × 1024 resolution using a 60 × NA 1.42 oil immersion objective lens at 3x magnification. For HT-22 cell or hippocampal neuron cultures pharmacologically inhibiting caspase 3 or calpain 1, images were captured at 1024 × 1024 resolution using a 60 × NA 1.42 oil immersion objective lens at 3x magnification. Nucleus fluorescence was used to indicate lysis. In all experiments, sequential Z-sections were captured in 1 μm steps. The central Z-section (including the central portion of the nucleus with the strongest signal) was then used for analysis and visualization. To co-localize M7CK and MEF2C (in the nucleus) or RPS6 (in the cell body) in excitatory neurons of the mouse cerebral cortex, images were captured at 1024×1024 resolution at 5x magnification using a 60×NA 1.42 oil immersion objective in the same microscope. The step size of consecutive z-slices was always 1 μm. The three middle z-sections with the strongest signal were then superimposed to generate a confocal image. To analyze synaptophysin punctate processes in hippocampal neuron cultures, images were captured at 1024×1024 resolution at 3x magnification using a 60×NA 1.42 oil immersion objective in a confocal microscope (OLYMPUS FLUOVIEW FV1000 or FV3000). The step size of consecutive z-slices was always 1 μm. The three z-sections with the strongest signal were then superimposed to form a single image. Synaptophysin points directly located on individual dendrites were counted to measure density. Results are expressed as the number of points per 10 μm dendrites per neuron.

[0103] Immunostaining and quantification of Aβ plaques

[0104] After blocking with 5% goat serum, frozen sections were mounted on slides and incubated overnight in moistened culture dishes at 4°C with primary antibody against Aβ (1:1000; Cell Signaling Technology, #9888). After washing with PBS, sections were incubated with biotinylated secondary antibody for 30 minutes. Slides were then incubated at room temperature with VECTASTAIN ABC reagent (Vector Laboratories, #PK-4000) for 30 minutes. Next, sections were incubated at room temperature for 4 minutes in peroxidase substrate staining agent 3,30-diaminobiphenyl (Sangon, #E670033). Sections were then gently rinsed with tap water to reduce background color. The stained sections were imaged using an Olympus VS120 microscope with a 10× objective. Aβ plaque density in the hippocampus, cortex, or entire section was analyzed using Image-Pro Plus 6.0. Aβ plaques were manually delineated until all plaques were included. The same parameters were then applied to all images from all 5×FAD groups (treated with saline, 50 or 75 mg / kg ATT). Aβ plaque density was measured from three slices from each mouse.

[0105] Activity-dependent protein synthesis assay

[0106] Hippocampal neuronal cultures from DIV7 were treated with AAV-tdTomato-ControlSCR and AAV-tdTomato-TRPM7 shRNA viruses. On DIV14, four cultures were prepared: naïve AAV-tdTomato-ControlSCR and naïve AAV-tdTomato-TRPM7 shRNA, activated AAV-tdTomato-ControlSCR and activated AAV-tdTomato-TRPM7 shRNA (n=4 per culture group). 50 mM KCl was added to the culture medium, and the cultures were incubated for 10 minutes to activate neurons. Click-iT was used... TM HPG Alexa Fluor TM The 488 Protein Synthesis Detection Kit (Invitrogen, #C10428, USA) labels nascent proteins. HPG (component A) was diluted 1:1000 in preheated medium containing 200 μM L-cysteine, 2 mM L-glutamine, and 10 mM HEPES, but without L-methionine (Dulbecco's Modified Eagle Medium, #21013024, USA, without L-glutamine, sodium pyruvate, L-methionine, and L-cysteine) to prepare a 50 μM final working solution. Neuronal cultures were incubated with 1 mL of component A final working solution for 90 min. The cultures were washed once with PBS and then fixed in PBS with 4% paraformaldehyde for 30 min. The medium was blocked with blocking solution and then washed twice at room temperature for 20 min each time. 1X HPG reaction buffer was diluted with DD water at a ratio of 1:10. The final volume of the mixed solution was 16 mL, prepared as follows: 1X HPG reaction buffer (13.76 mL), copper(II) sulfate (CuSO4) (component D, 0.64 mL), Alexa azide (Component B, 0.04 ml) and 1X HPG buffer additive (1.6 mL). After inhibition, the culture was washed twice in PBS with 3% BSA, and then incubated at room temperature for 30 minutes in a mixed solution (1 mL / coverslip). Wash once with reaction wash buffer (component F) for 5 minutes each time. For staining the cell nuclei, use HCS NuclearMask. TM The Blue Stain (G component) solution was diluted 1:2000 in PBS, and the culture was incubated at room temperature in the dark for 30 minutes. After washing (twice, 5 minutes each time), an appropriate amount of anti-quenching mounting medium (Sangon Biotech, E675005-0010) was added, and a coverslip containing the culture was fixed onto a glass slide.

[0107] Fluorescence imaging was performed at 1024×1024 resolution using a confocal microscope (OLYMPUS FLUOVIEW FV1000) with a 20×NA 0.75 objective lens and 1x magnification. Serial z-slices were captured in 1 μm increments. The three strongest z-slices were then stacked for subsequent quantitative analysis. Four cultures were used per group, and each culture was divided into 10–15 equal regions (size: 1024×1024) for imaging. Fluorescence intensity was quantified using ImageJ software (v1.53, NIH, Bethesda, USA) as an indicator of protein synthesis.

[0108] Protein cloning, purification, and enzymatic activity of kinases

[0109] The M7CK sequence (amino acids 1299 to 1863 of the TRPM7 sequence), cofilin sequence (Genebank ID: NM_007687.5), myocyte-specific enhancer 2C (MEF2C) sequence (Genebank ID: NM_001347571.2), ribosomal protein S6 (RPS6) (Genebank ID: NM_009096.3), myelin basic protein (MBP) sequence (GenBank ID: NM_001025245.1), and matrix metalloproteinase 2 (MMP2) sequence (GenBank ID: NM_008610.3) were amplified from WT mouse hippocampal cDNA and cloned into the pcDNA3.1+ vector (Aobio Technology, China). The constructed plasmid was transformed into DH5α fertile cells (Weidi Biotech, #DL1001, China), and then evenly dropped onto Luria-Bertani buffered agar plates containing ampicillin (100 μg / ml; Sangon Biotech, #A610029, China) (10 g tryptone, 5 g yeast extract, 10 g sodium chloride and 15 g agar powder were added to 1 liter double distilled water), and cultured overnight at 37°C.

[0110] Five or six single colonies were selected for sequencing (Sangon Biotech, China), and the correct colonies were used to amplify the protein overnight at 37°C for protein extraction. The amplified bacteria were centrifuged at 4200 rpm at 4°C and then resuspended in a lysis buffer containing protease and phosphatase inhibitors (50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 10 mM MgCl2, 1 mM EDTA, 10 mM β-mercaptoethanol, 20% glycerol, 1 mg / μl lysozyme). Dissolved urea (8 mol / L) was added to the lysis buffer, and the mixture was incubated on ice for 30 minutes, followed by ultracentrifugation at 28000 rpm at 4°C (Optima XPN-100 ultracentrifuge, Beckman Coulter, USA) for 30 minutes. After obtaining the supernatant, it was condensed at 4200 rpm at 4°C using an Amicon Ultra 15ML-10K and 30K protein separation centrifuge filter (Millipore, #UFC901024, #UFC903024, Germany) until less than 1 mL of liquid remained. The ultrafiltrate for each protein was collected and used to determine kinase activity. The ultrafiltrate from untransformed bacteria served as a negative control for the enzyme assay (no kinases or substrate).

[0111] According to the user manual, the kinase activity of M7CK was determined using a universal kinase activity kit (R&D Systems, #EA004, USA). These experiments used purified protein. In the metal ion concentration kinase activity assay, cofilin protein was used as the reaction substrate. The effects of different metal ions on kinase activity were investigated by varying the concentrations of magnesium, calcium, or zinc ions in the reaction system. The assay kit was reconfigured to accommodate different magnesium ion concentrations. M7CK and cofilin protein were diluted to 0.4 μg / μl. The protein was added to reaction systems containing 0, 0.8, 1.6, 3.2, and 4.8 mM magnesium and incubated for 20 minutes at room temperature. Then, malachite green reagents A and B were added to each reaction system and incubated for 20 minutes at room temperature to stop the enzyme reaction upon color formation. The optical density of each reaction was detected at 620 nm using a MultiScan Go (Thermo Fisher Scientific, USA). The concentrations of calcium and zinc in the assay were varied by using different concentrations of chelating agents (such as EGTA for calcium chelation and TPEN for zinc chelation). Then, kinase activity was measured as described above. All data were calculated as a percentage of the control (M7CK + substrate) reaction. For MEF2C and RPS6 proteins, kinase assays were performed using a blank control, an M7CK-only reaction (substrate blank control), an M7CK + MMP2 reaction (substrate negative control), an M7CK + MBP reaction (substrate positive control), and a reaction involving both M7CK and the target substrate (RPS6 or MEF2C). Except for data calculated as a percentage of the substrate negative control reaction (M7CK only), all conditions were performed according to the standard kinase reactions described above. Calibration curves were constructed for each experiment according to the kit user manual.

[0112] Fluorescence recovery after photobleaching

[0113] DIV5 hippocampal neuron cultures were transfected according to the transfection protocol. After 60 hours, the cultures were transferred to a live-cell imaging system. The photobleaching recovery procedure was performed using the FRAP module of a Leica TCS SP5 microscope (Leica, Germany). EGFP-positive cells were selected, and images were taken at 1400 Hz, 6x magnification, and a resolution of 256 × 256 using a 63× oil immersion objective. During FRAP analysis, images were taken every 0.115 seconds (×10) before photobleaching; during photobleaching under high laser stimulation, images were taken every 0.115 seconds (×200); and after bleaching, images were taken every 0.115 seconds (×100), 1 second (×10), and 5 seconds (×10). EGFP-positive cells-EGFP (n = 7 cells), EGFP-TRPM7, and TRPM7-EGFP (n = 5 cells / group) were observed. Approximately three cellular regions per cell were locked and decomposed (19, 18, and 16 regions, respectively). Photobleaching recovery time was statistically analyzed. Fluorescence recovery values ​​and areas were measured using ImageJ software (v1.53, Bethesda, NIH). The diffusion coefficient (D) is the ratio of the relevant region area to 4 × τ. τ is the time required for fluorescence recovery to half of its maximum value.

[0114] Western blot

[0115] Total protein was extracted from hippocampal neurons and brain tissue using radioimmunoprecipitation (RIPA) buffer (Beyotime, #P0013B, China) supplemented with protease and phosphatase inhibitors (Roche, 04906837001 and 04693159001, Germany, respectively). After freezing for 30 minutes, homogenates were centrifuged (4°C, 13000g, 10 minutes), and the supernatant was collected as total protein. Total protein concentration was determined using a bisquinolinic acid (BCA) kit (Thermo Fisher Scientific, #23225, USA). For standard Western blotting, equal volumes of total protein were separated using 10% or 12% SDS-PAGE gels. For cell region-specific detection of M7CK, equal volumes of proteins prepared from nuclei, cytoplasm, or synapses were loaded onto SDS gels and separated. The separated bands were transferred to a 0.45-μm polyvinylidene fluoride membrane (Millibor, #IPVH00010, USA). After blocking (5% skim milk and 0.1% Tween-20 added to PBS), the membrane was incubated overnight at 4°C with primary antibody. After rinsing, the membrane was incubated for 2 hours at room temperature with an anti-rabbit antibody (1:5000; SAB, #L3012, USA) or an anti-mouse antibody (1:5000; Cell Signaling Technology, #7076S, USA) conjugated with horseradish peroxidase (HRP). The membrane was then incubated with enhanced chemiluminescence solution (Tanon, #180-5001, China) and imaged using a Tanon 5200 multi-imaging system. Furthermore, the images were analyzed using Gel Pro Analyzer software (Media Cybernetics, USA). The integrated optical density (IOD) of the target protein bands was measured and normalized to the IOD of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or β-actin (β-ACTIN). Postsynaptic density protein 95 (PSD95) and histone deacetylase 2 (HDAC2) were co-detected in synaptosome, cytoplasmic, and nuclear preparations as internal quality controls. The primary antibodies used were: anti-TRPM7 (1:500, Alomone, #ACC-047), anti-M7CK (1:500, custom-made, Abclonal, China), anti-PSD95 (1:1000, Abclonal, China; Cell Signaling Technology, #3450S, USA), anti-HDAC2 (1:1000; 1:1000, Cell Signaling Technology, #2540S, USA), and anti-calpain 1 (1:1000, Cell Signaling Technology, #2556S).Anti-calpain 2 (1:1000, Cell Signaling Technology, #2539S, USA), anti-lysing aspase 3 (1:1000, Cell Signaling Technology, #9661S, USA), anti-RPS6 (1:1000, Cell Signaling Technology, #2317S, USA), anti-Phos-RPS6 (Ser235 / 236) (1:1000, Cell Signaling Technology, #4858S, USA), anti-Phos-RPS6 (Ser240 / 244) (1:1000, Cell Signaling Technology, #5364S, USA), anti-MEF2C (1:1000, Cell Signaling Technology, #5030S, USA), anti-Phos-MEF2C (S222) (1:1000, Novus) Biologicals, #NBP2-60774, USA), Anti-Phos-MEF2C (S396) (1:1000, Abcam, #ab78888, USA), Anti-Phos-MEF2C (S387) (1:1000, Bioss, #bs-5481R, China), Anti-MAP3K1 (1:1000, Proteintech, #19970-1-AP, China), Anti-Thr1400-MAP3K1 (1:1000, Abcam, #ab138662, China), Anti-GSK3β (1:1000, Santa, #sc-377213, China), Anti-Phos-GSK3β (1:1000, CellSignaling Technology, #9323S, USA), Anti-m-TOR (1:500, Novus) (Biologicals, #BP1-19855, USA), anti-Phos-m-TOR (1:500, Novus Biologicals, #BP1-1934, USA), anti-P70 S6 kinase (1:1000, Millipore, #04-391, USA), anti-Phos-p70 S6 kinase (Thr389) (1:1000, Millipore, #04-392, USA), anti-β-ACTIN (1:5000, Cell Signaling Technology, #4970L, USA), and anti-GAPDH (1:10000, Proteintech, #10494-1-AP, USA).

[0116] Combined immunoprecipitation

[0117] HEK 293T cells were cultured and transfected with 4 μg of DNA carrying plasmids M7CK, MEF2C, and RPS6. After 48 hours, cells were collected and homogenized in RIPA buffer (Beyondem, #P0013C, China) containing protease and phosphatase inhibitors, incubated on ice for 30 minutes, and then centrifuged at 12,000 rpm for 10 minutes at 4°C. The supernatant was collected for the following steps. Co-immunoprecipitation of MEF2C or RPS6 and M7CK was performed according to the manufacturer's instructions of the Dynabeads Protein G Immunoprecipitation Kit (Invitrogen, #10007D). In the co-immunoprecipitation assay, a custom rabbit polyclonal M7CK antibody (1:80 dilution) targeting the kinase domain was used to elicit M7CK and its interacting proteins. The antibody had previously undergone rigorous validation, following the procedure: First, 2.5 μl of M7CK antibody was mixed with 200 μl of antibody-binding buffer containing 1.5 mg Dynabeads, and the mixture was incubated at room temperature for 30 minutes at 60 rpm on a vortex mixer. After washing, 150 μl of total protein was added to the Dynabead antibody complex, and the mixture was incubated at 4°C for 36 hours. The Dynabead antibody-antigen complex was washed and transferred to a new test tube. Elution buffer and loading buffer were added, and the mixture was boiled at 99°C for 5 minutes. The eluted proteins were separated by 12% SDS-PAGE and transferred to a PVDF membrane for detection using the Western blot method described above.

[0118] Space proteomics

[0119] Expression of M7CK-EGFP in the hippocampus of transgenic mice. Three-month-old CaMKII-TRPM7- / - mice (n=12) were divided into two groups, and either AAV-EGFP or AAV-M7CK-EGFP (n=6 per group) were injected into the hippocampus. Mice were anesthetized with 2% isoflurane and fixed in a stereotactic frame (RWD Instruments, China). Injection coordinates were as follows: CA1: anteroposterior coordinate (AP) = -2.06 mm, medial-lateral coordinate (ML) = ±1.4 mm, dorsolateral coordinate (DV) = 1.4 mm, dentate gyrus coordinate = 2.0 mm. CA3: AP = -2.06 mm, ML = ±2.25 mm, DV = 2.1 mm. 300 nanoliters of virus (100 nanoliters per site) were injected into each side at a rate of 0.01 mL / min using a glass capillary driven by a Nanoject III pump (DRUMMOND, USA). The capillaries were removed after more than 10 minutes in situ. After suturing, the mice were placed in a warm room to recover from anesthesia before being returned home. One month later, the hippocampus was dissected and the tissue was collected for protein extraction.

[0120] Synaptosome protein preparation. Hippocampal tissue was homogenized in 5 volumes of extraction buffer (5 mM HEPES, 1 mM MgCl2, 0.5 mM CaCl2, 1 mM DTT, 0.32 M sucrose, protease inhibitor, pH 7.4). The homogenate was then centrifuged (4°C, 1400 g, 10 min), and the supernatant was collected and reserved. The particles were then resuspended in 20 mL of extraction buffer and centrifuged (4°C, 710 g, 10 min). The supernatant was collected again and mixed with the previously reserved supernatant sample. The collected supernatant was ultracentrifuged (4°C, 13800 g, 10 min). The supernatant was then collected as cytoplasmic proteins. The precipitate was resuspended in 3 mL of synaptosome extraction buffer (6 mM Tris, 0.32 M sucrose, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and protease inhibitor). The suspended particles were added to test tubes containing gradient sucrose concentrations (1.2, 1.0, and 0.85 M sucrose and 6 mM Tris buffer, pH 8.1). The tubes were centrifuged at 82,500 g for 2 hours at 4°C. Finally, the 0.85 M and 1.0 M sucrose were aspirated. The 1.0 / 1.2 M interfacial material was collected as the synaptosome preparation. All protein preparations were stored at -80°C before use.

[0121] Nucleoprotein preparation. Hippocampal tissue was homogenized in 5 volumes of extraction buffer (10 mM HEPES, 10 mM KCl, 0.1 mM MgCl2, 0.1 mM EDTA, 0.1 mM DTT, 5 mM PMSF, pH 7.9). The sample was incubated on ice for 10 min, centrifuged (500 g, 4 °C, 10 min), and the supernatant was collected and stored as cytoplasmic protein. The precipitate was resuspended in 500 μL of nuclear extraction buffer (10 mM HEPES, 100 mM NaCl, 1.5 mM MgCl2, 0.1 mM EDTA, 0.1 mM DTT, 5 mM PMSF, pH 7.9). The suspended particles were stored in the buffer at 4 °C for 20 min. The sample was centrifuged (10000 g, 4 °C) for 20 min. The supernatant was collected and stored as the nucleoprotein preparation. All protein preparations were stored at -80 °C before use.

[0122] Cytoplasmic protein preparation. The collected cytoplasmic protein preparations (from the nucleus or synaptosome) were stored at -80°C before use. Cytoplasmic samples were analyzed individually. The results of interacting proteins from both samples were then combined. To identify substances that specifically interact with M7CK in the cytoplasm, all proteins found in the nucleus or synaptosome interactome lists were excluded.

[0123] Co-immunoprecipitation and liquid chromatography-mass spectrometry (CO-IP / LC-MS) were used. Quality control of all samples was performed using Western blotting. Synaptic protein PSD95 was predominantly detected in the synaptosome preparation, while the ribozyme HDAC2 was predominantly detected in the nucleus, ensuring the preparation's suitability for subsequent spatial proteomics analysis. All protein samples were quantified using the BCA kit (Thermo Fisher Scientific, #23225, USA) according to the manufacturer's instructions. M7CK and its interacting substances were extracted from the three preparations using mouse monoclonal EGFP antibody (1:500, Proteintech, #66002-1-Ig, USA) and the Dynabeads Protein G Immunoprecipitation Kit (Invitrogen, #10007D, USA) (Figure S3A). Briefly, 4 μl of EGFP antibody was mixed with 400 μl of antibody-binding buffer containing 3.0 mg of Dynabeads, and the mixture was incubated at room temperature for 30 minutes at 60 rpm on a vortex mixer. After washing, protein samples were added to Dynabead antibody-antigen complexes and the mixture was incubated at 4°C for 36 hours. The Dynabead antibody-antigen complexes were washed and transferred to new tubes. Elution buffer and loading buffer were added, and the mixture was boiled at 99°C for 5 minutes. The eluted proteins were separated by 10% SDS-PAGE and stained with Coomassie dye to observe the proteins in each preparation. Finally, the SDS-gels containing proteins in each preparation were carefully cut. Gel fragments were collected in 1.5 mL centrifuge tubes. The samples were carefully labeled and delivered to the Fudan University Proteomics Core Laboratory. Each sample was run three times independently before further data analysis. Proteins with varying concentrations (>2-fold) in the M7CK samples were identified and listed. Final functional analysis (GO analysis) of cell-specific interacting proteins was performed using a public database (https: / / cn.string-db.org / ).

[0124] Transcriptomics and proteomics

[0125] Expression of M7CK-EGFP in the hippocampus of transgenic mice. Four groups of 3-month-old mice were prepared for transcriptomic and proteomic studies: TRPM7 flox / flox CaMKII-TRPM7 - / - Hippocampal injection of AAV-EGFP-containing CaMKII-TRPM7 - / - -EGFP group or hippocampal injection of AAV-M7CK-EGFP CaMKII-TRPM7 - / -(M7CK-EGFP group, n=4 per group). As described above, the virus was injected into the left and right hippocampi (CA1 / DG / CA3). One month later, hippocampal tissue was dissected and collected, and RNA (right hippocampus) and protein (left hippocampus) were extracted.

[0126] RNA-seq. RNA was isolated using Trizol as described above. RNA samples were sent to a sequencing facility (BGI, Shenzhen, China). All samples underwent quality control tests, including RIN / RQN and 28S / 18S standard values. Data were generated on the BGIseq500 platform, filtered using SOAPnuke (v1.5.2), and clean reads were obtained and saved in raw FASTQ format. The clean reads were aligned to a reference coding mouse genome using HISAT2 (v2.0.4) and Bowtie2 (v2.2.5). Gene expression levels were calculated using RSEM (v1.2.12). Differential gene expression for Q values ​​≤ 0.05 was then analyzed using DEseq2 (v.1.4.5).

[0127] To identify genes regulated by kinase domains (in a bidirectional manner), those in TRPM7 are listed. flox / flox vs. CaMKII-TRPM7 - / -EGFP vs. M7CK-EGFP: Differentially expressed genes that are identical but have opposite regulatory directions in two comparisons. These genes (68 in total) are listed, and their biological functions were determined using the NCBI and MGI databases.

[0128] Liquid chromatography-tandem mass spectrometry (LC-MS / MS). Left hippocampal tissue was homogenized in RIPA buffer (Beyotime, #P0013B, China) containing a mixture of phosphatase and protease inhibitors (Roche, #04906837001, #04693159001, Switzerland). Samples were centrifuged at 12,000 rpm for 15 minutes at 4°C. The supernatant was collected and delivered to the Core Laboratory of Proteomics, Fudan University. Each sample was repeated twice to obtain the final list of differentially expressed proteins. Experiments were performed on an Orbitrap Exploris 480 and an Easy-nLC 1200 (Thermo Fisher Scientific, USA). LC-MS / MS scans were performed at a resolution of 15,000 m / z 200, with an automatic gain control (AGC) target of 75% and a maximum injection time of 22 ms. Proteins were searched using Proteome Discoverer 2.4. This is a database of all audited SWISSPROT entries from musmusculus (http: / / uniprot.org / ). The maximum number of missed cleavage sites was set to 2. A percolator was used to reassess the confidence level of identification, with an acceptance criterion of a false discovery rate (FDR) of less than 1%. Protein abundance was compared using embedded minola characterization and precursor quantification methods. The data were analyzed using the R package Limma (v3.46.0) to identify differentially expressed proteins in the experimental groups. An adjusted p-value (≤0.05) was set, and proteins showing significant differences between the comparison groups were selected.

[0129] To identify proteins regulated by kinase domains (in a bidirectional manner), two differentially expressed proteins that are identical in appearance but have opposite regulatory directions are listed: TRPM7 flox / flox vs. CaMKII-TRPM7 - / - -EGFP vs. M7CK-EGFP. Protein genes (2022 proteins) were listed, and GO analysis was performed using the R package cluster-profiler (v3.18.1) to determine their biological functions. Additionally, KEGG signaling pathway analysis was performed using Cluego in Cytoscape. Adjusted p-values ​​(≤0.05) were calculated using the Bonferroni downgrading method. Finally, genes and proteins regulated by kinase domains were compared simultaneously to identify cellular molecules regulated by M7CK at both the gene and protein levels.

[0130] Nest building test

[0131] Each mouse was housed individually for one week prior to the test. The nesting test was conducted from 11:00 AM to 11:00 PM. Before the task began, each cage was covered with a 5x5 cm square of paper towel. Images were taken at the end of the test. Nesting behavior was scored according to the following rules: 5 = all paper towels were torn into small pieces and appropriately gathered in one corner; 4 = most paper towels were torn and gathered; 3 = some paper towels were gathered and moderately torn; 2 = some paper towels were torn but not gathered; 1 = no paper towels were torn or gathered.

[0132] Novel Object Recognition Test (NORT)

[0133] Mice were allowed free exploration of an open-field apparatus (50×50×50 cm PVC arena, white walls and floor) for 10 minutes on day one (“acclimatization period”). The sessions were videotaped and later analyzed using Limelight software (Coulbourn Instruments) to monitor the mice’s exploration and movement activities. On day two, mice were placed individually in the experimental apparatus, facing two identical objects (objects A, 1, and 2), positioned at two oblique angles, 10 cm from the walls. Animals were allowed free exploration of the objects for 5 minutes. Test sessions were conducted at 2 hours and 24 hours post-training. At 2 hours post-training, a Short-Term Memory (STM) test was performed, with mice exploring the objects (familiar [A] and novel [B]) for 5 minutes. At 24 hours post-training, a Long-Term Memory (LTM) test was performed, with the same mice allowed free exploration of the objects (familiar [A] and novel [C]) for 5 minutes. All presented objects had similar texture, color, and size, but unique shapes. Exploration was defined as sniffing an object with the nose at a distance of no more than 2 cm and touching the object with the nose or forepaws. Sitting on an object was not considered exploration. The recognition index is calculated as the percentage of times each object is explored out of all objects explored. After each animal is explored, the area and objects are wiped clean with 70% ethanol.

[0134] Toxicity studies

[0135] Toxicity studies were conducted on male SPF C57 / BL6 mice weighing 20–25 g. A two-week dose-range toxicity study was performed. C57 / BL6 mice were randomly assigned to eight groups, including one vehicle group and seven intraperitoneal injection groups (n=6 per group); ATT doses were 50, 75, 100, 200, 300, 400, and 500 mg / kg. All animals were observed at least once daily for at least 14 days to monitor for toxicity symptoms, including body weight, food and water intake. Survival rates were calculated over 14 days, and the 50% lethal dose was determined. Liver, heart, and pancreas were dissected from animals receiving standard treatment doses of ATT (50 and 75 mg / kg) to monitor their morphology, size, and weight.

[0136] Statistical analysis

[0137] All data were analyzed using GraphPad Prism (GraphPad). Normality and homogeneity of variance were first tested. The Shapiro-Wilk normality test was used to check if the data followed a normal distribution. Homogeneity of variance was assessed using the F-test for two-group experiments and the Brown-Forsythe test for multiple-group experiments. For two-group comparisons, a two-tailed independent samples t-test was used for normally distributed data; an independent samples t-test with Welch correction was used for data with significantly different variances; and a Whitney test was used for non-normally distributed data. For multiple-group comparisons, a Bonferroni post-hoc test was performed after one-way ANOVA for normally distributed data; and a Dunn post-hoc test was performed after one-way ANOVA with Kruskal-Wallis analysis for non-normally distributed data. Experiments involving two-factor analyses were tested using two-way ANOVA. Bonferroni post-hoc tests were used for comparisons in two-way ANOVA. Survival curve data were analyzed using log-rank statistical analysis (Mantel-Cox test). All data are expressed as mean ± standard error (SEM). Statistical significance was defined as P < 0.05.

[0138] Example 1

[0139] Identification of TRPM7 activators and study of their activity and specificity

[0140] Using a high-throughput screening platform, this embodiment screened 80,000 compounds and identified four compounds / regulators (resveratrol, zalfilocaster, TPCA-1, and ATT) that could increase TRPM7 channel activity. To confirm the effects of these regulators, this embodiment studied their influence on TRPM7 channel currents using whole-cell patch-clamp techniques in TRPM7-transfected HEK cells. Once stable TRPM7 currents were obtained, these four compounds were applied via perfusion. The results are as follows: Figure 1 As shown, these four compounds (resveratrol, zaffirukast, TPCA-1, and ATT: 30 μM) significantly increased the TRPM7 current amplitude (resveratrol: 1.75 ± 0.24 times, p = 0.015; zaffirukast: 1.44 ± 0.12 times, p = 0.007; TPCA-1: 1.34 ± 0.07 times, p = 0.002; ATT: 1.41 ± 0.11 times, p < 0.001). Figure 1A). Furthermore, this embodiment determined the median effective concentrations (EC50) of these compounds (resveratrol: 28.1 μM; zalfilocaster: 1.4 μM; TPCA-1: 9.0 μM; ATT: 15.7 μM). Figure 1 C). Therefore, this embodiment identified four TRPM7 agonists and confirmed their ability to activate TRPM7 channels. Next, this embodiment examined the specificity of the identified TRPM7 modulators. This embodiment 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 (C). Figure 1 D). Regarding TRPM6, resveratrol and zalfilocaster activate the ion channel; simultaneously, ATT inhibits channel activity ( Figure 1 E). For TRPM8, resveratrol, zarfilocastanol, and ATT inhibited ion channel activity ( Figure 1 (F). Therefore, among the identified regulators, only ATT appears to specifically activate TRPM7; meanwhile, other identified regulators activate other TRPM ion channels. Conversely, ATT may inhibit two TRPM ion channels, namely TRPM6 and TRPM8.

[0141] Figure 1 The specific details for each item are as follows:

[0142] (A). Left figure: Quantitative analysis of the increase in TRPM7 channel current amplitude by TRPM7 agonists (resveratrol: n=5, zafirlukast: n=5, TPCA-1: n=6, and ATT: n=8). Unpaired t-tests (resveratrol and zafirlukast) and Mann-Whitney tests (TPCA-1 and ATT) were used for analysis. Right figure: Representative current-voltage relationships for TRPM7 agonists.

[0143] (B). Representative time processes of TRPM7 agonists (resveratrol, zafirlukast, TPCA-1 and ATT) on the TRPM7 channel.

[0144] (C). Dose-response curves of TRPM7 agonists (resveratrol, zolpidem, TPCA-1 and ATT).

[0145] (D). Left figure: Quantitative analysis of the effects of TRPM7 agonists (resveratrol: n=4, zolpidem: n=7, TPCA-1: n=5, and ATT: n=5) on TRPM2 channel activity. They were analyzed using unpaired t-tests. Right figure: Representative current-voltage relationships of the TRPM7 agonist effect.

[0146] (E). Left panel: Quantitative analysis of the effects of TRPM7 agonists (resveratrol: n=6, zafirlukast: n=6, TPCA-1: n=6, and ATT: n=6) on TRPM6 channel activity. The analytical methods used were the Mann-Whitney test (resveratrol) and unpaired t-tests (zafirlukast, TPCA-1, and ATT). Right panel: Representative current-voltage relationships of the TRPM7 agonist effect.

[0147] (F). Left panel: Quantitative analysis of the effects of TRPM7 agonists (resveratrol: n=7, zafirlukast: n=6, TPCA-1: n=6, and ATT: n=7) on TRPM8 channel activity. Analysis was performed using an unpaired t-test. Right panel: Representative current-voltage relationships of the TRPM7 agonist effect. All recorded experiments were performed in transfected HEK cells.

[0148] *P<0.05, **P<0.01, ***P<0.001.

[0149] Example 2

[0150] Activation of TRPM7 can prevent Aβ-induced synaptic loss in neuronal culture.

[0151] This embodiment tested whether activating ion channels to promote kinase cleavage and release could effectively intervene in preventing Aβ-induced synaptic toxicity, reducing Aβ plaques, preserving synaptic density, and protecting memory function, using in vitro and in vivo models.

[0152] First, this example tests which of the four compounds in Example 1 can prevent Aβ-induced synaptic damage in hippocampal neuron cultures. (Adding A...) 1-42 Extracellular concentrations of exogenous Aβ decrease synaptic density in primary neuronal cultures. Exposure of hippocampal neuronal cultures (DIV14) to high concentrations of exogenous Aβ (500 nM, for 48 hours) reduced synaptic density by approximately 44%. Figure 2 This study found that resveratrol (30 and 100 μM) did not prevent Aβ-induced synaptic damage. Meanwhile, zalfilocaster, TPCA-1, and ATT prevented Aβ-induced synaptic damage in a dose-dependent manner. Figure 2 A, the culture was incubated with the compound for 24 hours.

[0153] Next, this embodiment explores the relationship between the protective effect of the modulator from Example 1 and the activity of the TRPM7 ion channel. In this section, this embodiment utilizes TPCA-1 and ATT as AI-based structure-function analyses, and literature analysis indicates that they are safer than zarfilocaster. The results show that TPCA-1 effectively prevents Aβ-induced synaptic damage in a dose-dependent manner. This protective effect is completely blocked by the TRPM7 inhibitor NS8593. Figure 2 B). Similarly, the addition of ATT to the culture medium prevented Aβ-induced synaptic damage in a dose-dependent manner. This protective effect was completely blocked by the TRPM7 inhibitor NS8593 and NDGA. Figure 2 C. Cultures were incubated with regulators and / or inhibitors for 24 hours. This example demonstrates that the TRPM7 regulators TPCA-1 and ATT alleviated Aβ-induced synaptic loss in vitro via a TRPM7 activity-dependent mechanism.

[0154] Figure 2 The specific details for each item are as follows:

[0155] (A) Fluorescence images (left) and quantitative analysis (right) of synaptophysin punctate processes in hippocampal neuron cultures cultured in Tris buffer or Aβ (500 nM, 48 h) and treated with different doses of resveratrol, zalfillustar, TPCA-1, and ATT. N = at least 4 neurons per group. One-way ANOVA was performed, followed by Bonferroni post-test.

[0156] (B). Fluorescence imaging and quantitative analysis of synaptophysin punctate processes in hippocampal neuronal cultures, which were cultured in Tris buffer or Aβ and treated with different TPCA-1 and TRPM7 inhibitors (NS8593). N = at least 11 neurons per group. One-way ANOVA was performed, followed by Bonferroni post-test.

[0157] (C). Fluorescence images and quantitative analysis of synaptophysin punctate processes in hippocampal neuron cultures cultured with Tris buffer or Aβ and treated with different doses of ATT and TRPM7 inhibitors (NS8593 and NDGA). N = 15 neurons per group. One-way ANOVA was performed, followed by Bonferroni post-test.

[0158] All neurons were derived from three separate neuronal cultures.

[0159] *P<0.05, **P<0.01, ***P<0.001.

[0160] Example 3

[0161] ATT treatment can protect memory, preserve synaptic density, and reduce Aβ plaques in a 5×FAD AD mouse model.

[0162] This study investigated whether treatment with a TRPM7 activator could prevent Alzheimer's disease (AD) lesions in a 5xFAD mouse model. Pharmacological and high-performance liquid chromatography (HPLC) studies showed 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 chosen for in vivo studies in this study. Two groups of mice were treated and injected (ip) daily with two doses (50 or 75 mg / kg) of ATT. Untreated mice received a similar treatment regimen using an equal volume of the carrier. All treatments were initiated at 5 months of age and continued for one month before behavioral testing began. At 6 months of age, open-field testing showed that the drug treatment did not affect the mice's movement or exploratory activities. Figure 3 A). Nesting behavior was impaired in 5×FAD mice treated with the vehicle. Low-dose (50 mg / kg) ATT also affected nesting behavior in the mice. Conversely, high-dose (75 mg / kg) ATT successfully rescued nesting behavior in the mice. Figure 3 B and C). Memory function was assessed using the Novel Object Recognition Test (NORT). This study found that short-term memory was normal in all groups, including untreated 5×FAD mice. However, untreated 5×FAD mice exhibited long-term memory impairment. Figure 3 D). After one month of treatment with two doses (50 mg / kg and 75 mg / kg) of ATT, memory impairment was improved in 5×FAD mice. Figure 3 (D). These findings indicate that the TRPM7 modulator ATT can rescue memory and other cognitive deficits in 5×FAD mice. Data suggest that activation of TRPM7 can prevent learning and memory impairment in this mouse model.

[0163] This embodiment also investigated whether the TRPM7 ion channel modulator ATT could protect synaptic density and reduce Aβ plaques in the brains of 5×FAD mice. Mice were perfused, and brain tissue was collected for histological analysis. This embodiment found that the punctate density of synaptophysin radiculars in the CA1 layer of the hippocampus was significantly reduced in untreated 5×FAD mice. ATT treatment reversed the reduction in synaptic density in a dose-dependent manner. Synaptic density remained unchanged only at a dose of 75 mg / kg, and not at a dose of 50 mg / kg. Figure 4 A and B). Furthermore, both doses of ATT treatment significantly reduced Aβ plaque density in the hippocampus and cortex of 5×FAD mouse brain slices. Figure 4(C) Therefore, the TRPM7 modulator ATT can preserve synaptic density and reduce Aβ plaque deposition in 5×FAD mice. Data from this example suggest that activating TRPM7 can combat AD lesions by protecting synapses and disease regulatory mechanisms.

[0164] Figure 3 The specific details for each item are as follows:

[0165] A) Measure the total distance traveled by wild-type (WT) mice and 5xFAD mice treated with vehicles and ATT (50 and 75 mg / kg) for 5 minutes in an open field. N = at least 10 mice per group, measured by one-way ANOVA and Kruskal-Wallis test.

[0166] (B). Representative images of nesting social behavior in WT and 5×FAD mice at 6 months of age after treatment with vehicles and ATT (50 and 75 mg / kg), respectively.

[0167] (C). Nesting scores in nesting social behavior of WT and 5×FAD mice at 6 months of age were obtained by vehicle and ATT (50 and 75 mg / kg) treatments, respectively. N = at least 10 mice per group. Analysis was performed by one-way ANOVA and Kruskal-Wallis test.

[0168] (D). The top figure is a schematic diagram of the experimental design for the novel object recognition test. The bottom figure shows the recognition indices of WT mice and 5×FAD mice after receiving vector and ATT (50 and 75 mg / kg) treatment during NORT training (left), short-term memory test after two hours (middle), and long-term memory test after twenty-four hours (right), for familiar and novel objects. N = at least 8 mice per group. Two-way ANOVA was used, followed by Bonferroni post-test.

[0169] *P<0.05 and ***P<0.001.

[0170] Figure 4 The specific details for each item are as follows:

[0171] (A). Representative images of punctate density of synaptophysin in the CA1 region after treatment with drugs and ATT (50 and 75 mg / kg) at 6 months of age in wild-type (WT) mice and 5×FAD mice.

[0172] (B). Quantitative analysis of punctate density of synaptophysin in the 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 slices from 6 mice per group. One-way ANOVA was performed, followed by Bonferroni post-test.

[0173] (C). The top image shows representative images of hippocampal and cortical amyloid plaques in 5×FAD mice after treatment with the vector and ATT (50 and 75 mg / kg). The bottom image shows the quantitative analysis of hippocampal and cortical amyloid plaques in 5×FAD mice after treatment with the vector and ATT (50 and 75 mg / kg). N = at least 18 brain slices from 6 mice per group, analyzed using one-way ANOVA followed by Bonferroni post-test. At least three brain slices from each mouse were used for immunohistochemical quantitative analysis.

[0174] *P<0.05 and ***P<0.001.

[0175] Example 4

[0176] To assess the safety of ATT, organ morphology and toxicity studies were conducted in this embodiment. The heart, liver, and pancreas of untreated and treated 5×FAD mice were dissected in this embodiment. Morphological and organ weight analyses indicated no significant effect of treatment. Figure 5 A, B, and C). In addition, this embodiment also included an in vivo toxicity study of ATT in wild-type mice. Seven new groups of mice were prepared and treated for 14 days with different doses of ATT (50, 75, 100, 200, 300, 400, and 500 mg / kg). All animals were administered an intraperitoneal injection once daily, and clinical observation was performed at least once daily. No significant toxicity was observed in the 50, 75, and 100 mg / kg groups. No significant toxicity was observed in the 200 mg / kg group, but one animal died on day 13. Higher doses of ATT (including 300, 400, and 500 mg / kg) caused significant mortality in mice. Figure 5 D). Further data analysis (curve fitting analysis) conducted to determine the median lethal dose (LD50) showed that ATT's LD50... 50 It is 246.1 mg / kg ( Figure 5 E). Therefore, the dose of ATT that protects mouse memory, maintains synaptic density, reduces Aβ plaque deposition, and reduces toxicity is safe.

[0177] The human ether-à-go-go-related gene (hERG) encodes the channel pore portion of the KCNH2 channel, a key regulator of the heartbeat that controls the rapidly delayed rectified current during the repolarization phase of the cardiac action potential. Inhibition or blockage of the KCNH2 channel prolongs cardiac repolarization, manifested on an electrocardiogram as a prolonged QT interval. This blockage can trigger life-threatening ventricular tachyarrhythmias, leading to sudden cardiac death. Therefore, the KCNH2 channel is a crucial parameter for assessing cardiotoxicity during drug development. In this embodiment, the safety of ATT in terms of potential cardiotoxicity was evaluated by investigating its effect on channel current and comparing it with mibepridine. After the KCNH2 current reached a steady state, the effects of different concentrations of ATT or mibepridine on the current were examined. Figure 5 F and G). The results showed that the TRPM7 regulator ATT ( Figure 5 F) and Mibefradil ( Figure 5 G) reduced the KCNH2 current in a dose-dependent manner. At the same concentration, ATT's inhibitory effect was weaker than Mibefradil because ATT's half-maximum inhibitory concentration (IC50) was lower. 50 Approximately 57.42 μM ( Figure 5 F); and Mibefradil's IC 50 0.93 μM ( Figure 5 G). Therefore, in terms of cardiotoxicity, ATT has a higher safety profile than mibefradil. It is noteworthy that the in vitro neuroprotective doses of ATT at 30 μM and 50 μM ( Figure 2 C) All are within the established safety range. Furthermore, ATT's EC50 is 15.7 μM ( Figure 1 C), less than two other well-known but less safe TRPM7 activators, namely naltraben (20 μM) and mibeladil (50 μM).

[0178] Figure 5 The specific details for each item are as follows:

[0179] Toxicity studies of (A, B, C). Representative images (top) and quantitative analysis (bottom) of the heart (A), liver (B), and pancreas (C) of mice treated with the drug and ATT (50 and 75 mg / kg). N = at least 5 mice per group. Analysis was performed using one-way ANOVA followed by Bonferroni post-test (heart and liver) and one-way ANOVA Kruskal-Wallis test (pancreas).

[0180] (D). Survival curves of wild-type mice after treatment with the vector and ATT (50, 75, 100, 200, 300, 400 and 500 mg / kg). Six mice per group.

[0181] (E). Dose-survival curves of mice after treatment with the vector and ATT (50, 75, 100, 200, 300, 400 and 500 mg / kg). Six mice per group.

[0182] (F). Dose-response curve of ATT inhibition of KCNH2 current in CHO cells. N = at least 3 cells per group.

[0183] (G). Dose-response curve of imibedil inhibiting KCNH2 current in CHO cells. N = at least 3 cells per group.

[0184] Example 5

[0185] The TRPM7 kinase domain is cleaved and transported to different neuronal compartments.

[0186] To investigate whether TRPM7 is cleaved to release the C-terminus-containing α-kinase (M7CK), two TRPM7 clones (EGFP-TRPM7 or TRPM7-EGFP) with the fluorescent protein EGFP linked to their N-terminus or C-terminus were constructed in this study. Hippocampal neuronal cultures were transfected with the TRPM7 clones, and the expression and distribution of fluorescence signals were analyzed by fluorescence imaging. The fluorescence signal of the EGFP-TRPM7 clone was localized to the cell membrane, similar to other typical ion channels; simultaneously, signals from the TRPM7-EGFP clone were observed in the cell membrane, cytoplasm, nucleus, and dendrites. Figure 6 A). To rule out the possibility of TRPM7 C-terminal EGFP being cleaved or interfering with membrane localization, this embodiment used fluorescence recovery after photobleaching (FRAP) technology to detect protein migration kinetics. Hippocampal neurons transfected with EGFP-TRPM7, TRPM7-EGFP, or EGFP were photobleached, and the fluorescence recovery of each group was quantified and used as an indicator of protein size. Diffusion coefficient analysis showed that the control EGFP had the fastest recovery rate, followed by TRPM7-EGFP, while EGFP-TRPM7 had the slowest recovery rate. Figure 6 B). Furthermore, in this embodiment, hippocampal neurons were transfected with full-length TRPM7 (EGFP-TRPM7-mCherry) double-labeled with N-terminal EGFP and C-terminal mCherry. It was found that the N-terminal EGFP fluorescence signal remained localized to the cell membrane; simultaneously, the C-terminal mCherry signal diffused throughout the cell body, including the nucleus. Figure 6 C and Figure 10A). Next, this embodiment used previously developed and validated M7CK-specific antibodies (Chang, Chen et al. 2023, Zhang, Cao et al. 2023) to confirm the presence of M7CK in different cellular regions. In this embodiment, cell region-specific proteins were extracted from mouse hippocampal tissue and subjected to Western blot analysis, revealing M7CK bands in the nucleus, cytoplasm, and synaptic protein preparations. Figure 6 D). Data indicate that TRPM7 is cleaved in nerve cells, releasing its C-terminal portion, including M7CK, and transported to different cellular compartments, including the nucleus, cytoplasm, and dendrites. TRPM7 cleavage occurs in different brain regions ( Figure 10 B).

[0187] Example 6

[0188] Calpain1, neuronal activity, and calcium and magnesium concentration regulate the release and activity of M7CK.

[0189] In cell lines, TRPM7 is cleaved by caspase 3 or 8, releasing its active kinase domain, thereby regulating cellular processes, including Fas-dependent apoptosis. Furthermore, TRPM7 can regulate cell adhesion and spheroidization via the calcium-dependent protease calpain2 (activated at extremely high intracellular calcium concentrations). Under physiological conditions, neurons cannot activate this cleavage mechanism. Therefore, the cleavage mechanism of neurons under physiological conditions remains unknown. First, this example identifies the protease that cleaves TRPM7. Co-immunoimmunoassay of TRPM7 and caspase-3 showed no confocalization in neurons. Figure 7 A). Furthermore, in neuroblastoma cells transfected with the EGFP-TRPM7-mCherry clone, drug inhibition of Caspase could not prevent the cleavage and spread of the M7CK-mCherry moiety. Figure 11 A). Therefore, in neuronal cells, caspase 3 does not cleave TRPM7 to release M7CK. Calpain 1 and calpain 2 are two major protease isoforms in the brain, which are activated by low and high concentrations (micromolar or millimolar) of intracellular calcium, respectively, during various physiological and pathological processes, catalyzing the cleavage of target proteins. Co-immunoimmunoassay experiments in neuronal cells showed that TRPM7 and calpain 2 did not co-localize; however, a strong co-localization with calpain 1 was also observed. Figure 7A). In neuroblastoma cells transfected with the EGFP-TRPM7-mCherry clone, this example found that drug inhibition of calpain1 (30 hours) prevented the cleavage and release of the M7CK-mCherry moiety in a dose-dependent manner. Figure 7 B). Calpain 1 inhibitor (40 μM, duration 30 h) also prevented the cleavage and release of the M7CK-mCherry fraction in hippocampal neuron cultures. Figure 7 C). Furthermore, this embodiment also constructed two small interfering RNAs (siRNAs) to knock out calpain1 or calpain2 proteins. Figure 11 B), the results showed that knocking out calpain 1 with siRNA reduced the free M7CK in neuroblastoma cells. Figure 7 D), while knocking out calpain2 does not. On the other hand, overexpression of calpain1 in neuroblastoma cells increases free M7CK (D). Figure 7 E). Therefore, the calcium-dependent protease calpain1 (regulated by intracellular physiological calcium concentration) cleaves TRPM7, releasing the kinase domain.

[0190] Secondly, this embodiment investigated the factors regulating the lysis rate of hippocampal neuron cultures. To ensure that different experimental cultures started culturing at the same M7CK / TRPM7 ratio baseline, the cultures were first cultured with a Caplain1 inhibitor (40 μM, incubated for 1 hour) before adding glutamate to the culture medium. This embodiment found that promoting neuronal activity by increasing the extracellular glutamate concentration (continuously for 1 hour) promoted TRPM7 lysis in a dose-dependent manner, as evidenced by the gradual increase in the M7CK / TRPM7 ratio. Figure 7 F). The minimum effective glutamate concentration (40 μM) was used to determine the time course of the cleavage process. In this example, it was found that glutamate-induced cleavage began after 30 minutes of glutamate incubation, peaked after 1 hour, and returned to baseline after 3 hours. Figure 7 G and Figure 11 C). To explore the effect of divalent cations on cleavage rate, this study varied the concentrations of extracellular calcium or magnesium (for 48 hours) and calculated the M7CK / TRPM7 ratio using Western blotting. This study found that increasing extracellular calcium concentration within physiological limits increased TRPM7 cleavage in a concentration-dependent manner. Figure 7 H). Conversely, increasing the extracellular magnesium concentration did not alter the M7CK / TRPM7 ratio, indicating that magnesium ions had no effect on the cleavage of TRPM7. Figure 7H). Therefore, neural activity and extracellular calcium concentration regulate the cleavage of TRPM7 by the calpain1 calpain.

[0191] Finally, this embodiment identified which divalent cation might be an essential cofactor for M7CK activity. This embodiment used a standard kinase activity assay kit and cofilin as a standard substrate to quantitatively detect M7CK enzyme activity in vitro. In this embodiment, different concentrations of EGTA or TPEN were used to gradually decrease the concentration of calcium or zinc in the enzymatic reaction. This embodiment found that the activity of the M7CK enzyme remained unchanged. Figure 11 (D and 11E). To understand the effect of magnesium ions, enzyme activity reactions were generated with different concentrations of magnesium ions (final concentrations in the reaction): 0.0, 0.8, 1.6, 3.2, and 4.8 mM). It was found that increasing the magnesium ion concentration gradually increased the enzyme activity. The activity peaked at 1.6 mmol, and the higher the concentration, the lower the activity. Figure 7 I). Therefore, the above results indicate that magnesium ions can regulate the catalytic activity of the cleaved TRPM7 kinase domain.

[0192] Example 7

[0193] Spatial proteomics study of M7CK regulatory signaling in a cell region-specific manner

[0194] To elucidate the M7CK-regulated signaling pathway in a cell lineage-specific manner, this embodiment performed spatial proteomics studies to identify the interacting partners of the kinase in the cytoplasm, nucleus, and synaptosome. Here, this embodiment utilized transgenic mice to investigate the calmodulin (CaM)-dependent protein kinase II-positive neurons (CaMKII-TRPM7). - / - Conditional gene knockout of TRPM7 was achieved in mice (see the aforementioned experimental methods). This embodiment uses adeno-associated viruses (AAV-EGFP, AAV-M7CK-EGFP) in CaMKII-TRPM7 mice. - / - EGFP-linked M7CK was expressed in the hippocampus of mice. As a control, AAV-EGFP was injected into the hippocampus of another group of transgenic mice. One month after injection, the hippocampal tissue was dissected, and protein preparations were isolated from different cellular regions (cytoplasm and nucleus, cytoplasm and synaptosome). Cytoplasmic samples collected from both preparation methods were pooled together for subsequent analysis. M7CK-EGFP or control EGFP was extracted from each sample using an anti-EGFP protein antibody, and the co-precipitated proteins were analyzed by liquid chromatography-mass spectrometry (LC-MS). Figure 8 A, Figure 12A and 12B). A total of 293 proteins were differentially enriched in the cytoplasm (defined as >2-fold enrichment in M7CK-EGFP), 48 in the nucleus, and 208 in the synaptosome. To identify the cellular processes regulated by this enzyme, this embodiment increased the rigor of the spatial proteomics analysis, including only proteins found in the M7CK-EGFP group, while none were found in the control group. Furthermore, because the collected cytoplasmic preparations may be contaminated with synaptosome or nuclear protein residues, this embodiment only included proteins identified in the cytoplasm, while none were found in the nuclear or synaptosome preparations. This embodiment identified 15 proteins regulating transcription, actin filaments, and microtubules in the nucleus. 70 interacting proteins were identified in the cytoplasm, regulating ion binding and transport, protein transport, and synaptic transmission. In the synaptosome preparation, this embodiment identified 92 proteins, most of which are related to energy / metabolism / microsomes, actin filament regulation, and protein synthesis processes. Figure 8 B).

[0195] This embodiment identified positive transcriptional regulators in the cell nucleus, including the synaptic density and plasticity-regulating transcription factor myocyte-specific enhancer factor 2C (MEF2C). Furthermore, ribosomal protein S6 (RPS6), a downstream member of the mTOR signaling pathway, was found in the synaptosome, mediating the synthesis of dendritic proteins required for synaptic plasticity and learning / memory. Western blot analysis revealed that this embodiment lacked TRPM7 compared to the control group. flox / flox Compared to mice, CaMKII-TRPM7 - / - The phosphorylation (activation) of MEF2C at residues S222 and S396 in mice is reduced. Figure 8 C); meanwhile, the phosphorylation of S387 residues remained unchanged. Figure 12 C). Similarly, this example found that in TRPM7 knockout mice, RPS6 was present at S235 and S236 ( Figure 8 Phosphorylation (activation) at D) is reduced, while phosphorylation (activation) at S240 / S244 is not reduced. Figure 12 D). The reduction in RPS6 activation is not due to mTOR signaling inactivation, because the phosphorylation of all upstream signaling molecules, including mitogen-activated protein kinase kinase 1 (MAP3K1), glycogen synthase kinase 3β (GSK3β), mammalian target of rapamycin (mTOR), and p70 ribosomal protein S6 kinase (p70s6k), remains unchanged. Figure 12 E). Through co-immunoprecipitation, anti-M7CK specific antibody, and transfection of the 293T cell line, this example revealed the interaction between M7CK and MEF2C and RPS6 (E). Figure 8E). Co-immunostained sections of mouse (wild-type) cortex showed that M7CK signaling (detected by kinase domain-specific antibodies) co-localized with MEF2C in the nucleus and RPS6 in the cytoplasm of excitatory neurons. Figure 8 F). Next, this example used a kinase activity assay kit to detect whether MEF2C and RPS6 are phosphorylation targets of M7CK. In this example, a blank control, an M7CK-only negative control, an M7CK+ metalloproteinase MMMP2 as a substrate negative control, and an M7CK+ myelin basic protein MBP as a substrate positive control were included. The results showed that MEF2C and RPS6 are phosphorylation substrates of the TRPM7 kinase domain, similar to myelin basic protein MBP, but opposite to metalloproteinase 2 MMP2. Figure 8 G). The results indicate that the TRPM7 cleavage kinase domain may regulate various cellular processes in a cell-specific manner, including gene expression, protein synthesis, cytoskeleton, and protein transport. For example, cleaved M7CK may be transported to the nucleus, interact with MEF2C, and phosphorylate it (activate) to regulate gene expression. Furthermore, when M7CK is cleaved and transported to the synaptic / dendritic compartment, it may regulate locally activity-dependent protein synthesis by interacting with and phosphorylating RPS6. To support this latter conclusion, this embodiment used a protein synthesis fluorescence sensor in neuronal cell cultures and found that inhibiting TRPM7 and its kinase domain impaired activity-dependent protein synthesis (…). Figure 12 F).

[0196] Example 8

[0197] TRPM7 cleavage, free kinase, and M7CK translocation to the nucleus are important for synaptic density.

[0198] M7CK (not the ion channel portion of TRPM7) is a crucial regulator of synaptic density, plasticity, and learning and memory. The kinase domain is cleaved and transported to different cellular regions to regulate gene expression (in the nucleus) and protein synthesis (in dendrites). This mechanism is a key regulator of synaptic density, plasticity, learning, and memory. Next, this example presents experiments linking kinase cleavage, transport, cellular compartments, and cellular function.

[0199] In this embodiment, TRPM7 shRNA was used to inhibit TRPM7 and its kinase domain in hippocampal neuron cultures, and a significant reduction in synaptic density was observed. Figure 9 and Figure 13 A). Inhibition of TRPM7 was sufficient to salvage synaptic density by overexpression of the kinase domain alone. Figure 9A). Importantly, overexpression of membrane-anchored M7CK via the addition of glycosylphosphatidylinositol residues (GPI) failed to salvage synaptic density ( Figure 13 A). Next, this example tested whether overexpression of full-length TRPM7 could salvage synaptic density, thereby allowing normal cellular processes to control the cleavage of the enzyme. Full-length TRPM7 salvaged synaptic density ( Figure 9 B). Similarly, overexpression of the ion channel mutant TRPM7 (P1040R) can also salvage synaptic density (B). Figure 9 C). However, overexpression of the kinase mutant TRPM7 (K1646R) failed to salvage synaptic density (C). Figure 9 D). Finally, this embodiment found that the non-cleavable mutant TRPM7 (LQRFK+LHSVQ: a mutation that prevents kinase cleavage) failed to salvage synaptic density ( Figure 9 E). Therefore, maintaining synapse density requires a cleaved, free, and active kinase domain. Next, this example tested whether maintaining synapse density requires the transport of the enzyme into or outside the nucleus. In this example, the M7CK clone was fused with a nuclear localization signal (NLS) or nuclear export signal (NES). This example found that M7CK localizes to the nucleus (E). Figure 13 B), rather than the extranuclear region ( Figure 13 C) can salvage synaptic density after inhibiting TRPM7. Figure 9 F). Therefore, to maintain synaptic density via M7CK, TRPM7 must be cleaved and its enzyme domain transported to the nucleus. It is noteworthy that in the control experiment of this example, overexpression of the kinase domain or any full-length TRPM7 clone did not affect the synaptic density readings of cultures transfected with control shRNA. Figure 13 D and 13E).

[0200] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications and variations of the methods listed herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the 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 this invention.

Claims

1. Application of TRPM7 activator in the preparation of Alzheimer's disease treatment products.

2. The application according to claim 1, characterized in that, The TRPM7 activator is a regulator of divalent cations or a metal salt containing divalent cations; preferably, the divalent cation is selected from one or more of magnesium ions, calcium ions, zinc ions, copper ions or ferrous ions; more preferably, the divalent cation is one or more of magnesium ions or calcium ions.

3. The application according to claim 1, characterized in that, The TRPM7 activator is selected from low molecular weight chemical compounds or small molecule compounds, wherein the molecular weight of the low molecular weight chemical compounds or small molecule compounds is not greater than 3000 Da.

4. The application according to claim 3, characterized in that, The TRPM7 activator is selected from one or more of the following compounds: resveratrol, zarfilocaster, TPCA-1, ATT, and glutamic acid or glutamate.

5. The application according to claim 1, characterized in that, The TRPM7 activator is selected from the protein product of the TRPM7 gene or its active fragment, or a nucleic acid molecule encoding the protein product or its active fragment, or a vector containing the nucleic acid molecule, or a trans-regulatory element of the TRPM7 gene, or a nucleic acid molecule encoding the trans-regulatory element or its active fragment, or a vector containing the nucleic acid molecule.

6. The application according to claim 5, characterized in that, The trans-regulatory element is selected from one or more of the following: Calpain1 protein or its mutants, a CRISPR / dCas9 system containing the VP64 domain, a CRISPR / dCas9 system containing the p65 activation domain, a CRISPR / dCas9 system containing the Rta47 domain, or a CRISPR / dCas9 system containing the VP16 domain.

7. The application according to claim 1, characterized in that, The Alzheimer's disease treatment product is selected from drugs that have one or more of the following functions: 1) Increase neuronal synapse density; 2) Improve learning and memory abilities; 3) Reduce Aβ plaques.

8. Application of TRPM7 protein or its mutant protein in the preparation of screening MEF2C protein activator products and / or RPS6 protein activator products.

9. The application according to claim 8, characterized in that, The MEF2C protein activator and / or RPS6 protein activator are Alzheimer's disease treatment products; preferably, the MEF2C protein activator and / or RPS6 protein activator are reagents that phosphorylate MEF2C protein or RPS6 protein; more preferably, the MEF2C protein activator is a reagent that phosphorylates S222 and / or S396 residues in MEF2C protein; or, the RPS6 protein activator is a reagent that phosphorylates S235 and / or S236 residues in RPS6 protein.

10. The screening method for MEF2C protein activator and / or RPS6 protein activator in applications as described in claim 8 or 9, characterized in that, The method includes the following steps: 1) After mixing the activator to be tested with cells expressing TRPM7 protein, the reagents that phosphorylate S222 and / or S396 residues in MEF2C protein or S235 and / or S236 residues in RPS6 protein were screened to obtain the reagents. 2) Mix the reagents selected in step 1) with cells expressing TRPM7 protein and detect the membrane potential of the cells. If the change in membrane potential increases, an effective MEF2C protein activator or / and RPS6 protein activator is selected.

11. The method according to claim 10, characterized in that, The method also includes one or more of the following features: 1) MEF2C protein activator and / or RPS6 protein activator can be TRPM7 activators; 2) The cells expressing TRPM7 protein are obtained by transfecting or infecting eukaryotic cells with a nucleic acid molecule encoding the TRPM7 protein product or its active fragment, or a vector containing the nucleic acid molecule; preferably, the eukaryotic cells are selected from one or more of HEK293, HEK293T, HUCCT1, HT-22, HCCC-9810 or RBE.

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