A TrkB-targeting peptide and its application

Abstract

This invention discloses a TrkB-targeting polypeptide and its applications, which consists of 21-55 consecutive amino acids of the MDGA2 protein and contains KARLLSPVFSIAPKNPYGPTN (SEQ ID NO. 08). The TrkB-targeting polypeptide and fusion protein of this invention can significantly inhibit TrkB activity, and therefore can be used to treat diseases associated with excessive TrkB activity (such as autism and other neurological disorders and tumors), possessing significant clinical value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a TrkB-targeting polypeptide and its applications. Background Technology

[0002] BDNF binding to TrkB activates the TrkB signaling pathway, whose downstream pathways include PI3K / Akt / mTOR, MAPK / ERK, and PLCγ, playing crucial roles in nervous system development and various functions. However, abnormal activation of the TrkB signaling pathway can also lead to nervous system dysfunction and disease. For example, the BDNF / TrkB signaling pathway promotes the plasticity of the brain's reward mechanism and is therefore associated with drug addiction. In animal models of cocaine addiction, inhibiting BDNF / TrkB can improve cocaine exploration behavior and withdrawal responses. In neuropathic pain, the BDNF / TrkB signaling pathway in the dorsal horn of the spinal cord may participate in pain by affecting KCC2 expression, and TrkB inhibitors can block the downregulation of KCC2 expression and alleviate neuropathic pain. BDNF levels are elevated in the serum of most epilepsy patients, and significant increases in BDNF and TrkB have been found in the temporal lobe and hippocampus in some animal models of epilepsy. The occurrence of epilepsy in these mice was improved by deleting or blocking TrkB. Administration of BDNF to the hippocampus of rats induced epilepsy. Furthermore, elevated BDNF levels were found in children with autism, suggesting that abnormalities in the BDNF / TrkB signaling pathway may also be involved in autism.

[0003] Other studies have found that BDNF / TrkB signaling can promote cell carcinogenesis, invasion, and metastasis, and may be a cause of chemotherapy resistance. For example, transplanting neural crest-derived cells with TrkB overexpression into mice can lead to rapid tumor growth. Moreover, tumor cells appear to be able to activate TrkB to keep PI3K kinase in a long-term activated state, thereby increasing resistance to anoikis and the success rate of metastasis.

[0004] In conclusion, inhibiting BDNF / TrkB activity may be a novel strategy for treating a variety of neurological diseases and tumors. Summary of the Invention

[0005] The purpose of this invention is to provide a TrkB-targeting polypeptide.

[0006] Another object of the present invention is to provide the use of the above-mentioned TrkB-targeting peptide.

[0007] The technical solution of the present invention is as follows:

[0008] A TrkB-targeting polypeptide consisting of 21-55 consecutive amino acids of the MDGA2 protein and containing KARLLSPVFSIAPKNPYGPTN (SEQ ID NO.08).

[0009] Preferably, the TrkB-targeting polypeptide is composed of 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, or 21 consecutive amino acid residues.

[0010] In a preferred embodiment of the present invention, the amino acid sequence comprises KARLLSPVFSIAPKNPYGPTNX (SEQ ID NO.10), wherein X is T or S.

[0011] Further preferred amino acid sequences are KARLLSPVFSIAPKNPYGPTNT (human, SEQ ID NO.07) or KARLLSPVFSIAPKNPYGPTNS (mouse, SEQ ID NO.09).

[0012] A conjugate having the above-described TrkB-targeting polypeptide and a modifying moiety selected from other polypeptides, detectable labels (for detection), or any combination thereof, wherein the other polypeptide is selected from CPP (to enhance its ability to penetrate cell membranes), a targeting moiety (to make it targeted), and / or a protein tag (to facilitate its expression, detection, tracing, and / or purification).

[0013] A fusion protein having the above-mentioned TrkB targeting peptide and another peptide selected from CPP (cell membrane permeation peptide, which enhances its ability to penetrate cell membranes), a targeting moiety (to make it targeted), a protein tag (to facilitate its expression, detection, tracing and / or purification), or any combination thereof.

[0014] For the above conjugates and fusion proteins:

[0015] Preferably, the modified portion is optionally linked to the N-terminus or C-terminus of the TrkB-targeting peptide via a linker.

[0016] More preferably, the CPP is a Tat-derived peptide; for example, the CPP has a sequence as shown in SEQ ID NO.11.

[0017] Further preferred options include targeting ligands, receptors, or antibodies.

[0018] Further preferred, the detectable label is a fluorescent dye, such as FITC.

[0019] More preferably, the protein tag is HA, Myc, GFP, or biotin.

[0020] An isolated nucleic acid molecule having the nucleotide sequence of the aforementioned TrkB targeting polypeptide or the aforementioned fusion protein.

[0021] A vector having the isolated nucleic acid molecules described above. This vector is a cloning vector or an expression vector.

[0022] In a preferred embodiment of the present invention, the vector includes plasmids, granules, bacteriophages, and Cos plasmids.

[0023] More preferably, the vector is capable of expressing the TrkB-targeting polypeptide of the present invention or the fusion protein of the present invention in a subject (e.g., a mammal, such as a human).

[0024] A host cell having the isolated nucleic acid molecules or the aforementioned carriers. The host cell includes: prokaryotic cells such as *Escherichia coli* cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.). The cell of the present invention can also be a cell line, such as 293T cells.

[0025] The method for preparing the above-mentioned TrkB-targeting polypeptide or the above-mentioned fusion protein includes: culturing the above-mentioned host cells under suitable conditions, and recovering the TrkB-targeting polypeptide or fusion protein from the culture of the host cells.

[0026] A pharmaceutical composition comprising the above-described TrkB-targeting polypeptide, the above-described conjugate, the above-described fusion protein, the above-described isolated nucleic acid molecule, the above-described carrier or the above-described host cell, and a pharmaceutically acceptable carrier and / or excipient.

[0027] The use of the above-mentioned TrkB-targeting polypeptide, the above-mentioned conjugate, the above-mentioned fusion protein, the above-mentioned isolated nucleic acid molecule, the above-mentioned carrier or the above-mentioned host cell in the preparation of pharmaceutical compositions.

[0028] This pharmaceutical composition is used to treat diseases associated with excessive TrkB activity or to inhibit TrkB activity.

[0029] The diseases associated with excessive TrkB activity include neurological disorders and tumors.

[0030] The aforementioned neurological disorders are characterized by abnormal activation of the BDNF / TrkB signaling pathway, with autism being a preferred example.

[0031] The preferred tumor mentioned above is a brain tumor.

[0032] Preferably, the pharmaceutical composition may also contain additional pharmaceutically active components;

[0033] The additional pharmaceutically active component is a drug with therapeutic activity for neurological disorders (e.g., epilepsy and neuropathic pain).

[0034] Alternatively, the other pharmaceutically active ingredient may be selected from ω-3 fatty acids, vitamin B12, intravenous immunoglobulins, hyperbaric oxygen therapy, glutamate receptor antagonists, catechol-O-methyltransferase (COMT) inhibitors, dopa decarboxylase inhibitors, or any combination thereof.

[0035] A method for in vitro inhibition of TrkB activity for non-diagnostic and therapeutic purposes, comprising contacting the aforementioned TrkB-targeting peptide, the aforementioned conjugate, or the aforementioned fusion protein with cells in need.

[0036] Preferably, the cells are neuronal cells or tumor cells (preferably brain tumor cells).

[0037] The TrkB-targeting peptides, fusion proteins, or pharmaceutical compositions of the present invention can be formulated into any dosage form known in the medical field, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, and injections (including injectable solutions and lyophilized powders). In some embodiments, the TrkB-targeting peptides, fusion proteins, or pharmaceutical compositions of the present invention can be formulated into injectable solutions or lyophilized powders.

[0038] Furthermore, the TrkB-targeting polypeptide or fusion protein of the present invention can be present in the pharmaceutical composition in unit dose form for ease of administration.

[0039] The TrkB-targeting peptides, fusion proteins, or pharmaceutical compositions of the present invention can be administered by any suitable method known in the art, including oral, oral, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum groove, groin, intravesical, topical (e.g., powder, ointment, or drops), or nasal routes. However, for many therapeutic uses, the preferred route / method of administration is parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). Those skilled in the art will understand that the route and / or method of administration will vary depending on the intended purpose. In a preferred embodiment, the TrkB-targeting peptides, fusion proteins, or pharmaceutical compositions of the present invention are administered by intravenous infusion or injection.

[0040] The TrkB-targeting peptides, fusion proteins, or pharmaceutical compositions provided by this invention can be used alone or in combination, or in combination with other pharmaceutically active components (e.g., drugs with activity for treating neurological disorders). In some preferred embodiments, the TrkB-targeting peptides or fusion proteins of this invention are used in combination with other drugs with activity for treating neurological disorders to prevent and / or treat diseases associated with excessive TrkB activity (e.g., neurological disorders). Such additional pharmaceutically active components can be administered before, simultaneously with, or after the administration of the TrkB-targeting peptides, fusion proteins, or pharmaceutical compositions of this invention.

[0041] The pharmaceutical compositions of the present invention may include a “therapeutic effective amount” or a “preventive effective amount” of the TrkB-targeting polypeptide or fusion protein of the present invention. A “preventive effective amount” refers to an amount sufficient to prevent, stop, or delay the onset of a disease (e.g., a disease associated with excessive TrkB activity). A “therapeutic effective amount” refers to an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. The therapeutic effective amount of the TrkB-targeting polypeptide or fusion protein of the present invention may vary depending on factors such as the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general characteristics such as age, weight, and sex, the method of drug administration, and other concurrent treatments, etc.

[0042] In this invention, the dosing regimen can be adjusted to obtain the optimal target response (e.g., therapeutic or preventative response). For example, it can be administered as a single dose, multiple times over a period of time, or the dose can be reduced or increased proportionally according to the urgency of the treatment situation.

[0043] The typical non-limiting range of the therapeutic or preventive effective amount of the TrkB-targeting peptide or fusion protein of the present invention is 0.001-100 mg / kg body weight, for example 0.01-50 mg / kg body weight, 0.1-25 mg / kg body weight.

[0044] It should be noted that the dosage may vary depending on the type and severity of the symptoms that need to be treated.

[0045] Furthermore, those skilled in the art will understand that for any given patient, a specific dosing regimen should be adjusted over time based on the patient's needs and the physician's professional evaluation; the dosage ranges given herein are for illustrative purposes only and do not limit the use or scope of the pharmaceutical compositions of the present invention.

[0046] In this invention, the subject can be a mammal, such as a human.

[0047] Terminology Definition

[0048] In this invention, unless otherwise stated, the scientific and technical terms used have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory procedures used in this invention are all conventional procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0049] As used herein, the term "cell penetrating peptide (CPP)," also known as "cell-penetrating peptide," "protein translocation domain (PTD)," "Trojan horsepeptides," or "transduction peptide," refers to a polypeptide capable of promoting cellular uptake of various molecules (e.g., various macromolecules including proteins or nucleic acids; for example, the TrkB targeting peptide of the present invention or a variant thereof). Such polypeptides are well known in the art and described, for example, in Stewart, et al., 2008 and CN101490081A (all of which are incorporated herein by reference); or can be obtained by methods known in the art, such as those described in detail in US2008 / 0234183A1, all of which are incorporated herein by reference.

[0050] In this invention, examples of the CPP include: (i) protein-derived CPPs: such as sequences derived from genes controlling antennae (Antennapedia), e.g., pAntp (43-58); sequences derived from HIV-1, e.g., Tat-derived peptides, such as amino acid residues 37-72, 37-60, 48-60, or 49-57 from TAT; hCT (9-32); pVEC; plSL; mouse PRP (1-28); E ms (194-220); or Restricocin L3 (60-73), etc.; (ii) model peptides: such as VT5; MAP; or arginine stretch, etc.; (iii) designed CPPs: such as MPG; Transportan; Transportan 10; Pep-1; peptides selected from KALA; or peptides selected from Bulforin2, etc.

[0051] Furthermore, the CPP used in the conjugates of the present invention may also be selected from polypeptide sequences having approximately 60, 70, 80, 90, 95, 99% or 100% sequence identity with any polypeptide sequence as described above, provided that the polypeptide sequence still retains its biological activity, i.e., promoting cellular uptake of the isolated polypeptide (or its variants) of the present invention and / or promoting the isolated polypeptide (or its variants) of the present invention across the blood-brain barrier.

[0052] As used herein, the term "targeting portion" refers to a domain capable of directing the TrkB targeting polypeptide (or a variant thereof) of the present invention to a desired location, which may be a specific tissue, a specific cell, or even a specific intracellular location (e.g., the nucleus, ribosome, endoplasmic reticulum, lysosome, or peroxisome). Those skilled in the art know how to design corresponding targeting domains based on the characteristics of the desired location.

[0053] As used herein, 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, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include: plasmids; phage particles; Cosmids; 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 retroviruses (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 promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0054] As used in this invention, the term "host cell" refers to a cell that can be used to introduce a vector, including prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, or human cells.

[0055] As used herein, the term "identity" refers to the sequence matching between two polypeptides or two nucleic acids. When a position in two compared sequences is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percentage identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared × 100. For example, if six out of ten positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT have 50% identity (three out of six positions match). Typically, two sequences are compared to produce the maximum identity. Such comparisons can be performed using, for example, a computer program such as the Align program (DNAstar, Inc.) Needleman et al. (1970) J. Mol. Biol. 48: 443-453. The percentage identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)) integrated into the ALIGN program (version 2.0), which uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percentage identity between two amino acid sequences can be determined using the Needleman and Wunsch (J MoIBiol. 48:444-453 (1970) algorithm in the GAP program integrated into the GCG software package (available at www.gcg.com), which uses a Blossum 62 matrix or a PAM250 matrix, along with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0056] As used herein, the term "isolated" means that the target analyte (e.g., a polypeptide) has been purified from contaminants present in a sample, such as a sample containing the target analyte obtained from a natural source. The term "isolated" does not necessarily exclude the presence of other components intended to act in conjunction with the isolated analyte. For example, the TrkB-targeting polypeptide of the present invention may be described as isolated, although it may be linked to cell-penetrating peptides.

[0057] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" means a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes: pH adjusters, surfactants, ionic strength enhancers, agents for maintaining osmotic pressure, agents for delaying absorption, diluents, adjuvants, preservatives, stabilizers, etc. For example, pH adjusters include phosphate buffers. Surfactants include cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include sodium chloride. Agents for maintaining osmotic pressure include sugars, NaCl, and the like. Agents for delaying absorption include monostearates and gelatin. Diluents include water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol), etc. Adjuvants include aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., complete Freund's adjuvant), etc. Preservatives include various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, p-hydroxybenzoate, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning commonly understood by those skilled in the art for stabilizing the desired activity of the active ingredient in a drug (e.g., inhibitory activity against TrkB), including monosodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein) or their degradation products (such as lactalbumin hydrolysate), etc.

[0058] As used in this invention, the term "treatment" means treating or curing a disease (e.g., a neurological disease), delaying the onset of symptoms of a disease (e.g., a neurological disease), and / or delaying the progression of a disease (e.g., a neurological disease).

[0059] As used in this invention, the term "prevention" means preventing, inhibiting, or delaying the onset of a disease (e.g., a neurological disease).

[0060] As used herein, the term "effective amount" refers to an amount that can effectively achieve the intended purpose. For example, a therapeutically effective amount can be an amount that effectively or sufficiently treats or cures a disease (e.g., a neurological disorder), delays the onset of symptoms of a disease (e.g., a neurological disorder), and / or delays the progression of a disease (e.g., a neurological disorder). A preventatively effective amount can be an amount that effectively or sufficiently prevents, inhibits, or delays the occurrence of a disease (e.g., a neurological disorder). Such effective amounts can be readily determined by those skilled in the art or by a physician and can be related to the intended purpose (e.g., treatment or prevention), the general health condition of the subject, age, sex, weight, severity of the disease to be treated, complications, method of administration, etc. The determination of such effective amounts is entirely within the capabilities of those skilled in the art.

[0061] As used in this invention, the term "subject" refers to a mammal, such as a primate mammal, like a human. In some embodiments, the subject (e.g., a human) suffers from a disease (e.g., a neurological disorder) associated with excessive TrkB activity, or is at risk of suffering from a disease (e.g., a neurological disorder) associated with excessive TrkB activity.

[0062] As used in this invention, the biological functions of the TrkB-targeting peptide of this invention include one or more selected from the following:

[0063] 1) The ability to specifically bind to TrkB;

[0064] 2) The ability to inhibit TrkB activity;

[0065] 3) The ability to reduce TrkB activity in subjects (optionally, after conjugating the peptide to CPP);

[0066] 4) The ability to improve synaptic dysfunction and / or social dysfunction and repetitive stereotyped behaviors caused by excessive TrkB levels in subjects (optionally, after conjugating the peptide to CPP);

[0067] 5) The ability to treat diseases associated with excessive TrkB activity (e.g., neurological disorders) in subjects (optionally, after conjugating the peptide to CPP).

[0068] The beneficial effects of this invention are: the TrkB-targeting peptide and fusion protein in this invention can significantly inhibit the activity of TrkB, and therefore can be used to treat diseases associated with excessive MDGA2 activity (such as autism and other neurological diseases and tumors), which has significant clinical value. Attached Figure Description

[0069] Figure 1Increased BDNF / TrkB activity was observed in MDGA2 heterozygous deletion mice. Brain tissues from MDGA2 heterozygous deletion (HET) and wild-type control (WT) mice were isolated, and synaptic components were extracted. Western blotting was used to analyze and compare the protein levels of MDGA2, mature and immature BDNF (mBDNF and ImBDNF), TrkB / pTrkB, Akt / pAkt, mTOR / pmTOR, S6 / pS6, and CaMK2 / pCaMK2. Statistical analysis of the graphs was performed using ImageJ software to analyze protein levels at grayscale. Results are expressed as mean ± standard error (SEM). *P<0.05, **P<0.01, ***P<0.001, “ns” indicates no significant difference (2-tailed Student's t-test).

[0070] Figure 2 A-2G: Evaluation of the interaction between MDGA2 and TrkB. Figure 2 A: A schematic diagram of the full-length MDGA2, the missing MAM region (ΔMAM), the missing M1 region (ΔM1), and the missing M2 region (ΔM2). Figure 2 c: TrkB and different truncated forms of MDGA2 were overexpressed in HEK293T cells, and their relationship was analyzed by IP-WB. Figure 2 C: A schematic diagram of the segments M1, L1, L2, P1, and P2 of MDGA2. Figure 2 D: GST-labeled L1 and L2 fragments were incubated with TrkB protein and then pulled down with Glutathione sepharose B. The protein pulled down was then analyzed by Western blotting to detect the binding of TrkB to different fragments. Figure 2 E: Biotin-labeled P1 and P2 fragments were injected into the hippocampus of mice. After 24 hours, the hippocampal tissue was lysed and the biotin-bound proteins were pulled down using Streptavidin Agarose. The binding of TrkB to different fragments was detected by Western blotting. Figure 2 E: FITC-labeled P2 fragments were incubated with SH-SY5Y cells, followed by immunofluorescence staining with TrkB antibody. Colocalization between the two was observed under a microscope. Scale bar: 10 μm. Figure 2 G: Predict the interaction relationship and interaction region between RPS23RG1 and MDGA2 using PYMOL molecular docking software.

[0071] Figure 3The effect of P2 peptide on BDNF-TrkB binding. P2 and control peptides were added at different concentrations (+: 0.1 μM; ++: 1 μM) to MDGA2 heterozygous neurons overexpressing TrkB-Flag. After 16 h, the binding of BDNF to TrkB-Flag was detected by IP-WB, and protein levels in the images were analyzed using ImageJ software. Results are expressed as mean ± standard error (SEM), *P<0.05, **P<0.01 (one-way ANOVA with Tukey's multiple comparisons test).

[0072] Figure 4 A-4D: Evaluation of the efficacy of MDGA2-based P2 peptides in MDGA2-deficient ASD model mice. Figure 4 A: Mouse free socialization experiment, analyzing the time mice treated with P2 or control peptides spent interacting and sniffing with unfamiliar mice in free space exploration. Figure 4 B: In the three-box social experiment for mice, the time spent by mice sniffing a strange mouse (S1) and an empty cage (E) during the social preference test phase was analyzed, along with the social preference index PI = T(S1-E) / T(S1+E). Then, the time spent by mice sniffing another strange mouse (S2) and mouse (S1) during the social novelty test phase was analyzed, along with the social novelty preference index PI = T(S2-S1) / T(S1+S2). Figure 4 C: The brains of mice treated with P2 or control peptides were lysed and analyzed by Western blotting to determine the protein levels of TrkB / pTrkB, Akt / pAkt, and mTOR / pmTOR, and the results were compared. Figure 4 D: The protein levels of TrkB bound to BDNF in mouse brain lysates were analyzed by IP-WB and compared. *P<0.05,**P<0.01,***P<0.001 (one-way ANOVA with Tukey's multiple comparisons test). Detailed Implementation

[0073] The technical solution of the present invention will be further explained and described below with reference to specific embodiments and accompanying drawings.

[0074] Sequence information

[0075] Information about the sequence involved in this invention is provided in Table 1 below.

[0076] Table 1: Sequence Description

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in this invention are substantially in accordance with the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F.M. Susubel et al., A Concise Guide to Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995; the use of restriction endonucleases is in accordance with the manufacturer's recommendations. Reagents not otherwise specified in the examples are conventional reagents in the art or commercially available reagents.

[0085] Experimental Materials and Methods

[0086] Real-time quantitative PCR (qRT-PCR)

[0087] Total RNA was extracted from mouse tissues using TRIzol reagent according to the manufacturer's instructions. After determining the concentration using a microplate reader, the mRNA was reverse transcribed into cDNA using a kit. qRT-PCR experiments were performed using a FastStart Universal SYBR Green Master (Roche). After the qRT-PCR experiments, a small amount of PCR product was analyzed by 1.5% agarose gel electrophoresis to determine if the PCR product fragment size was correct and if it was a specific amplification.

[0088] Protein extraction and Western blot

[0089] 1. Extraction of proteins from cell samples:

[0090] (1) Cell collection: Discard the culture medium and wash the cells twice with PBS; discard the PBS, scrape off the cells with a cell scraper and collect them into a centrifuge tube; centrifuge at 1000 rpm for 5 min, collect the cells at the bottom of the tube, and discard the supernatant.

[0091] (2) Cell lysis: Add TNEN cell lysis buffer containing cocktail protease inhibitor and phosphatase inhibitor (added immediately before use) to the cell clusters collected in (1), shake on ice for 30 min or place in a silent mixer at 4°C for slow lysis overnight, centrifuge at 12000 rpm at 4°C for 10 min, collect the supernatant and measure the protein concentration.

[0092] 2. Protein extraction from tissue samples:

[0093] Fresh tissue was immediately frozen in liquid nitrogen after extraction and then stored in an ultra-low temperature freezer at -80°C for later use. Before homogenization, the tissue was minced with surgical scissors and then homogenized in RIPA lysis buffer using a polytron electric homogenizer. After complete lysis, the tissue was centrifuged at 4°C and 12,000 rpm for 15 min, and the supernatant was collected. The centrifugation was repeated once, and the protein concentration was measured.

[0094] 3. Determination of protein concentration:

[0095] (1) Prepare the BCA protein assay solution by mixing solution A and solution B in a 50:1 ratio and set aside.

[0096] (2) Using BSA as the standard sample, a protein standard curve was prepared;

[0097] (3) Add protein samples to 96-well plates, with TNEN / RIPA lysis buffer as a blank control, and set up 3 wells for each group;

[0098] (4) Add 200 μl of the prepared protein assay solution to each well, mix well, and react in a constant temperature oven at 37°C for 30 min.

[0099] (5) Use an enzyme-linked immunosorbent assay (ELISA) reader to measure OD562, and substitute the obtained value into the protein standard curve to calculate the protein sample concentration.

[0100] 4. Western blot:

[0101] (1) Protein electrophoresis: Take 30-40 μg of protein sample, add 1 / 4 volume of 5×SDS loading buffer, boil at 100℃ for 3-5 min, use SDS-PAGE protein gel, and electrophoresis in Tris-Glycine electrophoresis buffer.

[0102] (2) Electrophoretic transfer of proteins: Pre-cool the electrophoresis transfer buffer at 4°C, cut out an appropriate size PVDF membrane, moisten it with methanol, and soak it together with filter paper in the electrophoresis transfer buffer for 10 min. At the same time, cut the electrophoretically processed gel and soak it in the electrophoresis transfer buffer for 5 min. Then, attach the PVDF membrane to the gel, cover both sides with filter paper, remove all air bubbles, and place the membranes in the electrophoresis tank with the positive electrode facing up. Electrophoresis is performed at a constant current (300 mA, 90 min) at 4°C.

[0103] (3) Antigen-antibody reaction:

[0104] a. Sealing: Seal 5% skim milk powder at room temperature for 1 hour;

[0105] b. Primary antibody reaction: Dilute the primary antibody in the blocking buffer at an appropriate ratio and incubate overnight at 4°C;

[0106] c. Secondary antibody reaction: Wash three times with PBST for 10 min each time, dilute the primary antibody in blocking buffer at an appropriate ratio, and incubate at room temperature for 1 h.

[0107] (4) ECL detection:

[0108] Wash three times with PBST, 10-15 min each time; mix solutions A and B from the ECL kit at a 1:1 (V / V) ratio, add ECL to the membrane surface in a dark room, incubate for 5 min, and then expose to light. Adjust the exposure time according to the fluorescence intensity.

[0109] Immunoprecipitation (IP) or co-immunoprecipitation (CoIP)

[0110] (1) Collect protein samples, add 10 μl Protein-ASepharose to the protein samples, and incubate at 4℃ for 1-2 h.

[0111] (2) Centrifuge at 5000 rpm for 2 min at 4℃. Transfer the supernatant to a new EP tube, add 0.5% TNEN lysis buffer to a final volume of 700 μl, add 20 μl Protein-GSepharose and the corresponding antibody, and incubate overnight at 4℃ by rotation.

[0112] (3) Centrifuge at 5000 rpm for 2 min at 4℃, remove the supernatant, add pre-cooled 1% TNEN solution, and incubate at 4℃ for 8 min by rotation.

[0113] (4) Repeat step (3) three times. On the last time, carefully aspirate the supernatant with a microsyringe, then add 20 μL of 2× loading buffer, mix well, and boil at 100°C for 5 min. The resulting sample is used for immunoblotting analysis.

[0114] Immunofluorescence

[0115] (1) Place the treated coverslip into a 24-well plate (one per well), and evenly distribute the cells at a certain density (5% to 10%), and culture for 24 hours.

[0116] (2) Transfect the corresponding plasmid according to the lipo2000 transfection method.

[0117] (3) 24 h after transfection, remove the culture medium and wash the cells 3 times with pre-cooled PBS. Remove the PBS and fix with 4% paraformaldehyde at room temperature for 10 min.

[0118] (4) 0.1% TritonX00-PBS, room temperature, 5-10 min (the time should be strictly controlled here).

[0119] (5) Block with 3% BSA-PBS for 1 hour.

[0120] (6) Incubate overnight at 4°C with different primary antibodies (1:100 diluted in 3% BSA-PBS).

[0121] (7) Wash with PBS 4 times, 5 minutes each time. Let the culture dish stand still during this time without shaking.

[0122] (8) Incubate with the corresponding fluorescent secondary antibody at room temperature for 60 min.

[0123] (9) If necessary, incubate DAPI at room temperature for 3-5 minutes.

[0124] (10) Wash with PBS 4 times, 5 min each time.

[0125] (11) Mount the slide at room temperature overnight. After the mounting medium dries, observe and photograph it under a confocal microscope.

[0126] Lysosome isolation

[0127] 1. Tissue (BioBio, Lysosome Extraction Kit, BB-3603)

[0128] (1) Remove mouse brain tissue, cut it into small pieces as much as possible with surgical scissors, wash it twice with cold PBS, add 400 μl of cold reagent A, place it on ice and let it stand for 10 min, and homogenize it 30-40 times with a tight Dounce homogenizer.

[0129] (2) The homogenate was centrifuged at 4℃ and 1000×g for 5 min. The precipitate was discarded and the supernatant was collected;

[0130] (3) Centrifuge the supernatant at 4℃ and 3000×g for 10 min. Discard the precipitate and collect the supernatant;

[0131] (4) Centrifuge the supernatant at 4℃ and 5000×g for 10 min. Discard the precipitate and collect the supernatant;

[0132] (5) Centrifuge the supernatant at 4℃ and 20000-30000×g for 20 min. Discard the supernatant, collect the precipitate, add 400 μl of cold reagent B to the precipitate, and mix well;

[0133] (6) Centrifuge at 4℃ and 20000-30000×g for 20 min. Discard the supernatant, collect the precipitate, and resuspend the precipitate in lysosome preservation solution C to obtain the lysosome sample. Store in a refrigerator for later use or use directly in downstream experiments.

[0134] 2. Cells (LysoIP)

[0135] (1) Using HeLa cells stably transfected with TMEM192-3xHA, the treated cells were washed twice with PBS, and then collected with KPBS (136mM KCl, 10mM KH2PO4, pH 7.25, adjusted with KOH), and a portion of the cell lysate was retained as the total protein component.

[0136] (2) The KPBS cell suspension was slowly homogenized for 30 cycles using a tight-fitting Dounce homogenizer, and then centrifuged at 1000g for 2 minutes at 4°C.

[0137] (3) Collect the supernatant and pre-wash the anti-HA beads with KPBS and incubate them on a gentle rotating shaker for 30 min;

[0138] (4) Wash the immunoprecipitate three times with KPBS and resuspend it in TNEN buffer to obtain the lysosome sample. Store it in a refrigerator for later use or use it directly in downstream experiments.

[0139] Cell surface protein biotin labeling

[0140] Biotin (EZ-LinkSulfo-NHS-SS-Biotin) was dissolved in freshly prepared PBS / CM buffer to a final concentration of 0.5 mg / mL and kept on ice. Cells were removed from the incubator, placed on ice, and the culture medium was aspirated. Cells were washed three times with an appropriate amount of pre-chilled PBS / CM buffer, and the buffer was aspirated. Biotin solution was added to the cell culture dish, and the cells were incubated on ice for 20 min. The biotin solution was aspirated, and the process was repeated once. The reaction was terminated by adding PBS / CM buffer containing 50 mM NH4Cl (10 min). Cells were lysed with TNEN lysis buffer (containing protease inhibitors), centrifuged at 12000 rpm for 10 min at 4 °C, and the supernatant was transferred to a new centrifuge tube. After determining the protein concentration by the BCA method, an equal volume of protein sample was added to an equal volume of 2× loading buffer, mixed well, and boiled at 100 °C for 5 min. Protein expression was detected by Western blotting. Streptavidin beads were added to an equal volume of the remaining protein sample. The streptavidin beads were incubated overnight at 4°C to precipitate biotinylated protein. The next day, the beads were centrifuged at 5000 RPM for 2 min at 4°C, and the supernatant was removed. The streptavidin beads were washed three times with 1% TNEN for 10 min each time. The residual liquid was removed with a microsyringe, and 20 μL of 2× loading buffer was added. The beads were boiled at 100°C for 5 min, and the content of the target protein was determined by Western blotting.

[0141] plasma membrane separation

[0142] Cell membrane preparation was performed using a cell membrane protein extraction kit (Phygene, PH0710). Cells were removed from the incubator, placed on ice, and the culture medium was aspirated. The cells were washed twice with pre-chilled PBS buffer. The collected cells were resuspended in pre-chilled extraction reagent A and incubated for 15 min. Then, the cells were slowly homogenized 200 times using a loose-fitting Dounce homogenizer until approximately 70% of the cells were ruptured. The cell suspension was then centrifuged at 700 g for 10 min, and the supernatant was collected. The membrane precipitate was then collected by centrifugation at 120,000 g for 30 min. Finally, the membrane proteins in the precipitate were resuspended and dissolved using lysis buffer containing protease inhibitors. The precipitate was then collected by centrifugation at 12,000 g for 10 min for further experiments.

[0143] Protein-protein interaction structure prediction

[0144] The researchers of this invention selected the MDGA2 (AF-Q7Z553-F1) PDB file, suitable for this study, from the SWISS-MODEL protein structure data. The protein structure of MDGA2 (C-score = -4.62) was predicted by Zhang Lab_I-TASSER (MUNIVERSITY OF MICHIGAN). Protein-protein interactions were predicted by the GRAMM:Docking web server (http: / / gramm.compbio.ku.edu / gramm, Vacker Lab). Finally, PYMOL was used to visualize the three-dimensional protein model and its interactions, and to analyze molecular docking.

[0145] Tail vein injection

[0146] The synthesized peptides were dissolved in physiological saline, with no more than 2% dimethyl sulfoxide added to aid dissolution. The fully dissolved peptides were then placed on ice for later use. When injecting mice, ensure the mice were comfortably positioned in the syringe and located using the tail vein syringe barrel, while simultaneously placing the mouse's tail in the groove of the compression plate. Adjust the lighting to make the veins clearly visible, then begin injection from the base of the tail, slowly advancing the peptide solution while constantly monitoring the mouse's health. After injection, leave the needle in place to ensure proper drug absorption. After needle removal, apply pressure with a cotton swab for 1 minute to stop bleeding. Injections were repeated at the same time for 5 consecutive days.

[0147] Animal behavioral experiments

[0148] All mouse behavioral experiments were conducted using SmartVideoTracking Software (Panlab, Harvard Apparatus) for data acquisition and analysis. Animal behavioral experiments were performed daily between 9:00 a.m. and 6:00 p.m., with a laboratory light intensity of 650 lux.

[0149] (1) Three days before the start of the experiment, touch the mice once a day, touch one mouse each time, gently grasp the mouse by the tail and pick it up, let the mouse stay in your hand for 30 seconds, then mark the mouse by drawing a line on the tail with a marker. After marking the mouse, keep it in your palm, grasp the tail, and then gently put it back into the cage.

[0150] (2) On the day of the experiment, the mice were transferred to the preparation room before the experiment and allowed to acclimatize for 60 minutes. Before the experiment began, the test chamber and maze were cleaned with 70% alcohol. After each experiment, the test chamber and maze were cleaned with 70% alcohol to remove the feces and urine excreted by the mice during the experiment and to eliminate the interference of residual mouse odor on the experiment.

[0151] 1. Open field test

[0152] This study investigates the voluntary movement ability and anxiety behavior of mice, primarily based on their tendency to avoid bright light and open spaces. In the open field experiment, mice are placed in the center of a maze (40cm (L) × 40cm (W) × 40cm (H)) and allowed to freely explore the maze for 10 minutes. The total distance traveled and the time spent in the center are recorded.

[0153] 2. Self-grooming test

[0154] Mice were placed in the center of a maze (40cm (L) × 40cm (W) × 40cm (H)) and allowed to explore freely for 15 minutes. The entire process was recorded using the Smart3.0 video tracking system. It is generally believed that the first 5 minutes are the adaptation phase for mice entering a new environment; therefore, by observing the video data of the mice in the last 10 minutes, the total number of times and the total time each mouse groomed itself with its limbs were recorded.

[0155] 3. Free social affiliation

[0156] The experimental mouse was placed in the center of a box (40cm (L) × 40cm (W) × 40cm (H)). Before the experiment, a wild-type mouse of the same sex and age as the experimental mouse (the stimulation mouse) was placed in a metal cage. The experimental mouse was then placed in the box and allowed to explore freely for 10 minutes. The entire process was recorded using the Smart3.0 video tracking system. The exploration time of the mouse within the metal cage and its surrounding 2cm area was recorded.

[0157] 4. Three-chamber sociability test

[0158] The test chamber (60cm wide × 40cm deep × 22cm high) contains two transparent partitions that separate the left, middle, and right chambers (each 20cm × 40cm). Each partition has a small opening (5cm × 5cm) for access to each chamber. A fixed wire cylinder (8.5cm in diameter) is placed in each of the left and right chambers. The test consists of three phases: adaptation, social skills test, and social novelty test. In the adaptation phase, both cylinders are empty, and the test mouse is placed in the middle and allowed to explore freely for 10 minutes. In the social skills test, a sex-matched unfamiliar mouse (S1) is confined in an empty cylinder and allowed to explore freely for 10 minutes. The time spent exploring S1 or the other empty cylinder (E) is measured, and the social preference index (T(S1-E) / T(S1+E)): the time spent exploring S1 minus the time spent exploring E, then divided by the time spent exploring both S1 and E). During the social novelty test, another sex-matched unfamiliar mouse (S2) was placed in an empty container and allowed to explore freely for 10 minutes. The time spent exploring S1 or S2 was measured, and the social novelty preference index (T(S2-S1) / T(S2+S1)) was calculated: the time spent exploring S2 minus the time spent exploring S1, then divided by the time spent exploring S2 and S1.

[0159] Experimental Materials and Methods

[0160] Protein extraction and Western blot (WB)

[0161] 1. Extraction of proteins from cell samples:

[0162] (1) Cell collection: Discard the culture medium and wash the cells twice with PBS; discard the PBS, scrape off the cells with a cell scraper and collect them into a centrifuge tube; centrifuge at 1000 rpm for 5 min, collect the cells at the bottom of the tube, and discard the supernatant.

[0163] (2) Cell lysis: Add TNEN cell lysis buffer containing cocktail protease inhibitor and phosphatase inhibitor (added immediately before use) to the cell clusters collected in (1), shake on ice for 30 min or place in a silent mixer at 4°C for slow lysis overnight, centrifuge at 12000 rpm at 4°C for 10 min, collect the supernatant and measure the protein concentration.

[0164] 2. Protein extraction from tissue samples:

[0165] Fresh tissue was immediately frozen in liquid nitrogen after extraction and then stored in an ultra-low temperature freezer at -80°C for later use. Before homogenization, the tissue was minced with surgical scissors and then homogenized in RIPA lysis buffer using a polytron electric homogenizer. After complete lysis, the tissue was centrifuged at 4°C and 12,000 rpm for 15 min, and the supernatant was collected. The centrifugation was repeated once, and the protein concentration was measured.

[0166] 3. Determination of protein concentration:

[0167] (1) Prepare the BCA protein assay solution by mixing solution A and solution B in a 50:1 ratio and set aside.

[0168] (2) Using BSA as the standard sample, a protein standard curve was prepared;

[0169] (3) Add protein samples to 96-well plates, with TNEN / RIPA lysis buffer as a blank control, and set up 3 wells for each group;

[0170] (4) Add 200 μl of the prepared protein assay solution to each well, mix well, and react in a constant temperature oven at 37°C for 30 min.

[0171] (5) Use an enzyme-linked immunosorbent assay (ELISA) reader to measure OD562, and substitute the obtained value into the protein standard curve to calculate the protein sample concentration.

[0172] 4. WB:

[0173] (1) Protein electrophoresis: Take 30-40 μg of protein sample, add 1 / 4 volume of 5×SDS loading buffer, boil at 100℃ for 3-5 min, use SDS-PAGE protein gel, and electrophoresis in Tris-Glycine electrophoresis buffer.

[0174] (2) Electrophoretic transfer of proteins: Pre-cool the electrophoresis transfer buffer at 4°C, cut out an appropriate size PVDF membrane, moisten it with methanol, and soak it together with filter paper in the electrophoresis transfer buffer for 10 min. At the same time, cut the electrophoretically processed gel and soak it in the electrophoresis transfer buffer for 5 min. Then, attach the PVDF membrane to the gel, cover both sides with filter paper, remove all air bubbles, and place the membranes in the electrophoresis tank with the positive electrode facing up. Electrophoresis is performed at a constant current (300 mA, 90 min) at 4°C.

[0175] (3) Antigen-antibody reaction:

[0176] a. Sealing: Seal 5% skim milk powder at room temperature for 1 hour;

[0177] b. Primary antibody reaction: Dilute the primary antibody in the blocking buffer at an appropriate ratio and incubate overnight at 4°C;

[0178] c. Secondary antibody reaction: Wash three times with PBST for 10 min each time, dilute the primary antibody in blocking buffer at an appropriate ratio, and incubate at room temperature for 1 h.

[0179] (4) ECL detection:

[0180] Wash three times with PBST, 10-15 min each time; mix solutions A and B from the ECL kit at a 1:1 (V / V) ratio, add ECL to the membrane surface in a dark room, incubate for 5 min, and then expose to light. Adjust the exposure time according to the fluorescence intensity.

[0181] Immunoprecipitation (IP) or co-immunoprecipitation (CoIP)

[0182] (1) Collect protein samples, add 10 μl Protein-ASepharose to the protein samples, and incubate at 4℃ for 1-2 h.

[0183] (2) Centrifuge at 5000 rpm for 2 min at 4℃. Transfer the supernatant to a new EP tube, add 0.5% TNEN lysis buffer to a final volume of 700 μl, add 20 μl Protein-GSepharose and the corresponding antibody, and incubate overnight at 4℃ by rotation.

[0184] (3) Centrifuge at 5000 rpm for 2 min at 4℃, remove the supernatant, add pre-cooled 1% TNEN solution, and incubate at 4℃ for 8 min by rotation.

[0185] (4) Repeat step (3) three times. On the last time, carefully aspirate the supernatant with a microsyringe, then add 20 μL of 2× loading buffer, mix well, and boil at 100°C for 5 min. The resulting sample is used for immunoblotting analysis.

[0186] Immunofluorescence

[0187] (1) Place the treated coverslip into a 24-well plate (one per well), and evenly distribute the cells at a certain density (5% to 10%), and culture for 24 hours.

[0188] (2) Transfect the corresponding plasmid according to the lipo2000 transfection method.

[0189] (3) 24 h after transfection, remove the culture medium and wash the cells 3 times with pre-cooled PBS. Remove the PBS and fix with 4% paraformaldehyde at room temperature for 10 min.

[0190] (4) 0.1% TritonX00-PBS, room temperature, 5-10 min (the time should be strictly controlled here).

[0191] (5) Block with 3% BSA-PBS for 1 hour.

[0192] (6) Incubate overnight at 4°C with different primary antibodies (1:100 diluted in 3% BSA-PBS).

[0193] (7) Wash with PBS 4 times, 5 minutes each time. Let the culture dish stand still during this time without shaking.

[0194] (8) Incubate with the corresponding fluorescent secondary antibody at room temperature for 60 min.

[0195] (9) If necessary, incubate DAPI at room temperature for 3-5 minutes.

[0196] (10) Wash with PBS 4 times, 5 min each time.

[0197] (11) Mount the slide at room temperature overnight. After the mounting medium dries, observe and photograph it under a confocal microscope.

[0198] Protein-protein interaction structure prediction

[0199] The researchers of this invention selected PDB files of the protein structures MDGA2 (AF-Q7Z553-F1) and TrkB (AF-Q16620-F1) from the ALPHAFOLD protein structure data, which were suitable for the research of this invention. Protein-protein interactions were predicted using the GRAMM:Docking web server (http: / / gramm.compbio.ku.edu / gramm, Vakser lab). Finally, PYMOL was used to visualize the three-dimensional protein models and their interactions, and to analyze molecular docking.

[0200] Stereotactic injection of the brain

[0201] After anesthetizing the mice, cannulas were implanted and fixed in the bilateral hippocampi at the following coordinates: AP, -2.0 mm; L, ±2.3 mm; DV, -2.0 mm. Three days after recovery, the synthesized peptide was dissolved in physiological saline at a concentration of 1 μg / μL. 1 μL of the peptide was then injected into the mouse hippocampus through the cannulas over a 5-minute period, once daily for seven consecutive days at the same time.

[0202] Animal behavioral experiments

[0203] All mouse behavioral experiments were conducted using SmartVideoTracking Software (Panlab, Harvard Apparatus) for data acquisition and analysis. Animal behavioral experiments were performed daily between 9:00 a.m. and 6:00 p.m., with a laboratory light intensity of 650 lux.

[0204] (1) Three days before the start of the experiment, touch the mice once a day, touch one mouse each time, gently grasp the mouse by the tail and pick it up, let the mouse stay in your hand for 30 seconds, then mark the mouse by drawing a line on the tail with a marker. After marking the mouse, keep it in your palm, grasp the tail, and then gently put it back into the cage.

[0205] (2) On the day of the experiment, the mice were transferred to the preparation room before the experiment and allowed to acclimatize for 60 minutes. Before the experiment began, the test chamber and maze were cleaned with 70% alcohol. After each experiment, the test chamber and maze were cleaned with 70% alcohol to remove the feces and urine excreted by the mice during the experiment and to eliminate the interference of residual mouse odor on the experiment.

[0206] 1. Social affiliation

[0207] The experimental mouse was placed in the center of a box (40cm (L) × 40cm (W) × 40cm (H)). Before the experiment, a wild-type mouse of the same sex and age as the experimental mouse (the stimulation mouse) was placed in a metal cage. The experimental mouse was then placed in the box and allowed to explore freely for 10 minutes. The entire process was recorded using the Smart3.0 video tracking system. The exploration time of the mouse within the metal cage and its surrounding 2cm area was recorded.

[0208] 2. Three-chamber sociability test

[0209] The test chamber (60cm wide × 40cm deep × 22cm high) contains two transparent partitions that separate the left, middle, and right chambers (each 20cm × 40cm). Each partition has a small opening (5cm × 5cm) for access to each chamber. A fixed wire cylinder (8.5cm in diameter) is placed in each of the left and right chambers. The test consists of three phases: adaptation, social skills test, and social novelty test. In the adaptation phase, both cylinders are empty, and the test mouse is placed in the middle and allowed to explore freely for 10 minutes. In the social skills test, a sex-matched unfamiliar mouse (S1) is confined in an empty cylinder and allowed to explore freely for 10 minutes. The time spent exploring S1 or the other empty cylinder (E) is measured, and the social preference index (T(S1-E) / T(S1+E)): the time spent exploring S1 minus the time spent exploring E, then divided by the time spent exploring both S1 and E). During the social novelty test, another sex-matched unfamiliar mouse (S2) was placed in an empty container and allowed to explore freely for 10 minutes. The time spent exploring S1 or S2 was measured, and the social novelty preference index (T(S2-S1) / T(S2+S1)) was calculated: the time spent exploring S2 minus the time spent exploring S1, then divided by the time spent exploring S2 and S1.

[0210] Materials and reagents

[0211]

[0212]

[0213]

[0214] Example 1: MDGA2 heterozygous deletion promotes BDNF / TrkB activity

[0215] First, the researchers of this invention detected increased levels of BNDF protein in the synaptosomes of MDGA2 heterozygous mice (e.g., Figure 1 (As shown). BDNF is the receptor for TrkB, and BDNF / TrkB binding activates the downstream mTOR-Akt signaling pathway. Therefore, the researchers of this invention further discovered that the phosphorylation levels of proteins such as TrkB, mTOR, Akt, S6, and CaMK2 were increased in MDGA2 heterozygous mice (e.g., Figure 1 As shown in the figure, the increased phosphorylation levels of these proteins indicate that TrkB and downstream mTOR-Akt signaling are activated, suggesting that MDGA2 deficiency promotes the activity of BDNF / TrkB and its downstream signaling pathways.

[0216] Example 2: Interaction analysis between MDGA2 and TrkB and determination of the MDGA2 core sequence

[0217] The researchers of this invention predicted an interaction between the MAM region of MDGA2 and TrkB through protein-protein interaction analysis. Figure 2 G). To further confirm, the researchers of this invention constructed GFP-modified full-length human MDGA2 (amino acid sequence as shown in SEQ ID NO. 01, cDNA sequence as shown in SEQ ID NO. 03), human MDGA2 with the MAM region (aa746-921) deleted, and expression plasmids of human MDGA2 with the MAM amino terminus (M1, aa746-833) and MAM carboxyl terminus (M2, aa834-921) deleted respectively (see Table 2 and...). Figure 2 (A and B). These expression plasmids were co-transfected into HEK293T cells with the Flag-modified human full-length TrkB expression plasmid, and their interactions were analyzed by immunoprecipitation-Western blotting. The results are as follows. Figure 2 As shown in Figure B, full-length MDGA2 and MDGA2 lacking M2 can interact with TrkB, while MDGA2 lacking MAM and M1 cannot interact with TrkB. These results indicate that MDGA2 interacts with TrkB through the M1 region.

[0218] The researchers of this invention further divided the M1 region of MDGA2 into two fragments, L1 (aa746-766) and L2 (aa767-833), and constructed GST-modified L1 and L2 expression plasmids respectively (see Table 2 and...). Figure 2 (C, D) The L1 and L2 peptides, expressed in cells and fused with GST, were incubated with human TrkB protein and then pulled down using Glutathione sepharose B. The results showed that only L2 could bind to and pull down the TrkB protein. Figure 2 D).

[0219] The researchers of this invention further divided the L2 fragment into two segments, P1 (aa787-811, SEQ ID NO.06) and P2 (aa812-833, SEQ ID NO.07), synthesized polypeptides from each segment, and labeled them with biotin (see Table 2 and...). Figure 2 C and E). These two peptides, along with a biotin-labeled disordered peptide, were injected into the hippocampus of mice. After 24 hours, hippocampal tissue was lysed and the biotin-bound protein was pulled down using streptavidin agarose. It was found that only the biotin-labeled P2 peptide could pull down the TrkB protein from the mouse brain. Figure 2E). Furthermore, incubation of FITC-labeled P2 peptide (green) with SH-SY5Y cells followed by immunofluorescence staining with TrkB antibody (red) revealed co-localization between the two. These results indicate that the 812-833 sequence region of MDGA2 is a key region for interaction with TrkB.

[0220] Table 2: Truncated human full-length MDGA2

[0221]

[0222]

[0223] Example 3: MDGA2-based peptides inhibit the binding of BDNF and TrkB in cells.

[0224] To further determine whether the core region peptide of MDGA2 that interacts with TrkB affects TrkB function, the researchers of this invention artificially synthesized the human MDGA2 P2 peptide (KARLLSPVFSIAPKNPYGPTNT; SEQ ID NO. 07) and the disordered peptide (PYPPNTTLKSLPVISFNGKRAA; SEQ ID NO. 05). Both peptides were synthesized by Sangon Biotech. The above peptides were added at different concentrations (+: 0.1 μM; ++: 1 μM) to MDGA2 heterozygous neurons overexpressing TrkB-Flag. After 16 hours, the binding of BDNF to TrkB-Flag was detected by immunoprecipitation-Western blotting. It was found that the P2 peptide could inhibit the binding of BDNF to TrkB in a concentration-dependent manner (e.g., ...). Figure 3 As shown in the figure, this indicates that the P2 peptide has the effect of inhibiting BDNF / TrkB activity.

[0225] Example 4: Evaluation of the therapeutic effect of MDGA2-based peptides in autism (ASD) model mice

[0226] MDGA2 is considered a susceptibility gene associated with ASD. MDGA2 protein deficiency leads to increased excitatory synaptic transmission, and mice exhibit anxiety, social impairment, and stereotyped behaviors. Therefore, MDGA2 heterozygous mice are widely used as a typical autism model.

[0227] The above experimental results have shown that BDNF / TrkB activity is abnormally elevated in MDGA2 heterozygous deletion mice, and the P2 peptide can inhibit the binding of BDNF and TrkB, potentially making it a drug for disease treatment. Therefore, this embodiment further evaluates the therapeutic effect of MDGA2-based peptides on autism model mice.

[0228] The researchers of this invention artificially synthesized the P2 peptide (KARLLSPVFSIAPKNPYGPTNT; SEQ ID NO.07) and the disordered peptide (PYPPNTTLKSLPVISFNGKRAA; SEQ ID NO.05) of human MDGA2. Both peptides were synthesized by Sangon Biotech. + / - In mice or littermate control WT mice, the mice were treated with bilateral hippocampal stereotactic injection at a dose of 1 μg / day for one week, followed by a one-week rest period. After this treatment, the mice underwent behavioral testing and biochemical analysis.

[0229] Behavioral testing results in mice showed that Mdga2 administered with the control peptide... + / - Compared to wild-type mice, these mice exhibited significant social impairment in standard socialization tests. Figure 4 A), also showed barriers to social interest and social preference in the three-box social experiment ( Figure 4 B). Administration of MDGA2-based peptides significantly improved Mdga2 levels. + / - Social impairment in mice Figure 4 A, B).

[0230] After lysing the brains of experimental mice, immunoblotting experiments revealed that Mdga2... + / - The abnormally elevated TrkB-mTOR-Akt signaling pathway activity in mice was significantly restored to normal levels after treatment with P2 peptide. Figure 4 C). Furthermore, using immunoprecipitation-immunoblotting assays, it was found that in Mdga2... + / - In mice, the binding of BDNF to TrkB was increased, while treatment with P2 peptide decreased the binding of BDNF to TrkB. Figure 4 D). The above results confirm that MDGA2-based peptides can significantly improve disease-related phenotypes in autistic mice by targeting TrkB.

[0231] Besides ASD caused by MDGA2 deficiency, abnormal activation of the BDNF / TrkB signaling pathway has also been found to be involved in other diseases. For example, the BDNF / TrkB signaling pathway is associated with drug addiction; inhibiting BDNF / TrkB can improve cocaine exploration behavior and withdrawal responses in animals. The BDNF / TrkB signaling pathway in the dorsal horn of the spinal cord is involved in neuropathic pain; using TrkB inhibitors can alleviate neuropathic pain. BDNF levels are elevated in the serum of most epilepsy patients, and BDNF and TrkB have been significantly increased in the temporal lobe and hippocampus in some animal models of epilepsy. Deleting or blocking TrkB can improve the occurrence of epilepsy in these mice. Furthermore, some studies have found that BDNF / TrkB signaling can promote cell carcinogenesis, invasion, and metastasis, and may be a cause of chemotherapy resistance. Therefore, the peptides of this invention that can target and regulate MDGA2 metabolism to increase its expression levels are also suitable for treating other neurological diseases (e.g., autism, epilepsy) and tumors (e.g., brain tumors) caused by MDGA2 inactivation.

[0232] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A TrkB-targeting polypeptide, characterized in that: Its amino acid sequence is KARLLSPVFSIAPKNPYGPTNT.

2. A conjugate, characterized in that: It consists of the TrkB-targeting peptide as described in claim 1 and a modified portion, wherein the modified portion is a detectable marker.

3. A fusion protein, characterized in that: It has the TrkB-targeting polypeptide of claim 1 and an additional portion, which is GST-labeled.

4. An isolated nucleic acid molecule, characterized in that: The nucleic acid molecule is a nucleotide encoding the TrkB targeting polypeptide of claim 1, or a nucleotide encoding the fusion protein of claim 3.

5. A carrier, characterized in that: It contains the isolated nucleic acid molecules as described in claim 4.

6. A host cell, characterized in that: It contains the isolated nucleic acid molecule as described in claim 4 or the vector as described in claim 5, wherein the host cell is not a plant cell.

7. A method for preparing the TrkB-targeting polypeptide of claim 1 or the fusion protein of claim 3, characterized in that: include: The host cell of claim 6 is cultured under suitable conditions, and the TrkB targeting polypeptide or the fusion protein is recovered from the culture of the host cell.

8. A pharmaceutical composition, characterized in that: It comprises the TrkB-targeting polypeptide of claim 1, the conjugate of claim 2, the fusion protein of claim 3, the isolated nucleic acid molecule of claim 4, the vector of claim 5 or the host cell of claim 6, and a pharmaceutically acceptable carrier and / or excipient.

9. Use of the TrkB-targeting polypeptide of claim 1 in the preparation of a pharmaceutical composition for treating autism.

Images