TrkB targeting polypeptide and application thereof

Abstract

The invention discloses a TrkB targeting polypeptide and application thereof, the TrkB targeting polypeptide is composed of 21-55 continuous amino acids of MDGA2 protein, and the TrkB targeting polypeptide contains KARLLSPVFSIAPKNPYGPTN (SEQ ID NO.08). The TrkB targeting polypeptide and the fusion protein can significantly inhibit the activity of TrkB, so that the TrkB targeting polypeptide and the fusion protein can be used for treating diseases (such as nervous system diseases and tumors such as autism) related to too high TrkB activity, and have great clinical value.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a TrkB targeting polypeptide and an application thereof. Background Art

[0002] After BDNF binds to TrkB, it activates the TrkB signaling pathway, whose downstream pathways include PI3K / Akt / mTOR, MAPK / ERK, PLCγ and other different pathways, which play an important role in the development and various functions of the nervous system. However, when the TrkB signaling pathway is abnormally activated, it can also lead to dysfunction of the nervous system and cause the occurrence of diseases. For example, the BDNF / TrkB signaling pathway can promote the plasticity of the "reward mechanism" in the brain, and is therefore related to drug addiction. In animal models of cocaine addiction, inhibiting BDNF / TrkB can improve the animal's cocaine exploration behavior and withdrawal reaction. In neuropathic pain, the BDNF / TrkB signaling pathway in the dorsal horn of the spinal cord may be involved in pain by affecting the expression of KCC2, and the use of TrkB inhibitors can block the downregulation of KCC2 expression and relieve neuropathic pain. The level of BDNF is increased in the serum of most epilepsy patients, and some animal models of epilepsy have found that BDNF and TrkB are significantly increased in the temporal lobe and hippocampus. By deleting TrkB or blocking TrkB, epilepsy in these mice can be improved. Administration of BDNF to the hippocampus of rats can induce epilepsy. In addition, elevated levels of BDNF have also been found in children with autism, indicating 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 the cause of chemotherapy resistance. For example, transplanting neural crest-derived cells that overexpress TrkB into mice can lead to rapid tumor growth. Moreover, tumor cells seem to be able to activate TrkB to keep PI3K kinase in a long-term activated state, thereby improving resistance to anoikis and the success rate of metastasis.

[0004] In summary, inhibiting the activity of BDNF / TrkB may become a new strategy for treating a variety of neurological diseases and tumors. Summary of the invention

[0005] The object of the present invention is to provide a TrkB targeting polypeptide.

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

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

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

[0009] Preferably, the TrkB targeting polypeptide consists 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 preferably, the amino acid sequence is KARLLSPVFSIAPKNPYGPTNT (human origin, SEQ ID NO.07) or KARLLSPVFSIAPKNPYGPTNS (mouse origin, SEQ ID NO.09).

[0012] A conjugate, which has the above-mentioned TrkB targeting polypeptide and a modification part, wherein the modification part is selected from another polypeptide, a detectable label (to make it detectable) or any combination thereof, and the other polypeptide is selected from CPP (to improve its ability to penetrate the cell membrane), a targeting part (to make it targeted) and / or a protein tag (to facilitate its expression, detection, tracing and / or purification).

[0013] A fusion protein, which has the above-mentioned TrkB targeting polypeptide and another polypeptide, wherein the other polypeptide is selected from CPP (cell membrane penetrating peptide, which improves its ability to penetrate the cell membrane), a targeting part (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 modification portion is optionally linked to the N-terminus or C-terminus of the TrkB targeting polypeptide via a linker.

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

[0017] Further preferably, the targeting moiety is a ligand, a receptor or an antibody.

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

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

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

[0021] A vector having the above-mentioned isolated nucleic acid molecule is a cloning vector or an expression vector.

[0022] In a preferred embodiment of the present invention, the vector includes plasmid, cosmid, phage and cosmid.

[0023] Further 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 (eg, a mammal, such as a human).

[0024] A host cell having the above-mentioned isolated nucleic acid molecule or the above-mentioned vector. 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 TrkB targeting polypeptide or the fusion protein comprises: culturing the host cell under appropriate conditions, and recovering the TrkB targeting polypeptide or fusion protein from the culture of the host cell.

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

[0027] Use of the TrkB targeting polypeptide, the conjugate, the fusion protein, the isolated nucleic acid molecule, the vector or the host cell in preparing a pharmaceutical composition.

[0028] The pharmaceutical composition is used for treating diseases associated with excessive TrkB activity or inhibiting TrkB activity.

[0029] The above-mentioned diseases associated with excessive TrkB activity include nervous system diseases and tumors.

[0030] The above-mentioned nervous system disease is characterized by abnormal activation of the BDNF / TrkB signaling pathway, preferably autism.

[0031] The above-mentioned tumor is preferably a brain tumor.

[0032] Preferably, the pharmaceutical composition may further comprise additional pharmaceutically active ingredients;

[0033] The additional pharmaceutically active ingredient is a drug having therapeutic activity for nervous system disorders (eg, epilepsy and neuropathic pain).

[0034] Or the additional pharmaceutically active ingredient is selected from omega-3 fatty acids, vitamin B12, intravenous immunoglobulin, hyperbaric oxygen therapy, glutamate receptor antagonists, catechol-O-methyltransferase (COMT) inhibitors, dopa decarboxylase inhibitors or any combination thereof.

[0035] A method for inhibiting TrkB activity in vitro for non-diagnostic therapeutic purposes, comprising contacting the TrkB targeting polypeptide, the conjugate or the fusion protein with a cell in need thereof.

[0036] Preferably, the cell is a neuronal cell or a tumor cell (the tumor cell is preferably a brain tumor cell).

[0037] The TrkB targeting polypeptide, fusion protein or pharmaceutical composition of the present invention can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, lyophilized powders), etc. In some embodiments, the TrkB targeting polypeptide, fusion protein or pharmaceutical composition of the present invention can be formulated into injection solutions or lyophilized powders.

[0038] In addition, the TrkB targeting polypeptide or fusion protein of the present invention can be present in a pharmaceutical composition in the form of a unit dosage for easy administration.

[0039] The TrkB targeting polypeptide, fusion protein or pharmaceutical composition 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, inguinal, intravesical, topical (e.g., powder, ointment or drops), or nasal route. However, for many therapeutic uses, the preferred route / mode of administration is parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). The skilled person will appreciate that the route and / or mode of administration will vary depending on the intended purpose. In a preferred embodiment, the TrkB targeting polypeptide, fusion protein or pharmaceutical composition of the present invention is administered by intravenous infusion or injection.

[0040] The TrkB targeting polypeptide, fusion protein or pharmaceutical composition provided by the present invention can be used alone or in combination, or can be used in combination with another pharmaceutically active component (e.g., a drug having the activity of treating a nervous system disease). In certain preferred embodiments, the TrkB targeting polypeptide or fusion protein of the present invention is used in combination with other drugs having the activity of treating a nervous system disease to prevent and / or treat diseases associated with excessive TrkB activity (e.g., nervous system diseases). Such another pharmaceutically active component can be administered before, simultaneously with, or after the administration of the TrkB targeting polypeptide, fusion protein or pharmaceutical composition of the present invention.

[0041] The pharmaceutical composition of the present invention may include a "therapeutically effective amount" or a "prophylactically effective amount" of the TrkB targeting polypeptide or fusion protein of the present invention. A "prophylactically effective amount" refers to an amount sufficient to prevent, prevent, or delay the occurrence of a disease (e.g., a disease associated with excessive TrkB activity). A "therapeutically effective amount" refers to an amount sufficient to cure or at least partially prevent the disease and its complications in a patient already suffering from the disease. The therapeutically effective amount of the TrkB targeting polypeptide or fusion protein of the present invention may vary depending on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and gender, the mode of administration of the drug, and other treatments administered simultaneously, etc.

[0042] In the present invention, the dosage regimen can be adjusted to obtain the best desired response (e.g., therapeutic or preventive response). For example, the dosage can be administered once, multiple times over a period of time, or the dosage can be reduced or increased in proportion to the urgency of the treatment situation.

[0043] A typical non-limiting range for a therapeutically or prophylactically effective amount of a TrkB targeting polypeptide or fusion protein of the invention is 0.001-100 mg / kg body weight, such as 0.01-50 mg / kg body weight, 0.1-25 mg / kg body weight.

[0044] It should be noted that dosage may vary with the type and severity of the condition requiring treatment.

[0045] In addition, those skilled in the art will appreciate that for any particular patient, the 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 composition of the present invention.

[0046] In the present invention, the subject may be a mammal, such as a human.

[0047] Definition of terms

[0048] In the present invention, unless otherwise specified, the scientific and technical terms used in the present invention have the meanings commonly understood by those skilled in the art. In addition, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory operation steps used in the present invention are all conventional steps widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0049] As used in the present invention, the term "cell penetrating peptide (CPP)" is also called "cell penetrating peptide", "protein translocation domain (PTD), "Trojan horsepeptides" or "transduction peptide (transduction peptide)", etc., which refers to a polypeptide that can promote the cellular uptake of various molecules (e.g., various macromolecules including proteins or nucleic acids; for example, the TrkB targeting polypeptide of the present invention or its variant). Such polypeptides are well known in the art and are described in, for example, 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 the method described in detail in US2008 / 0234183A1, all of which are incorporated herein by reference.

[0050] In the present invention, examples of the CPP include: (i) protein derived CPPs: such as sequences derived from the gene controlling antennae (Antennapedia), such as pAntp (43-58); sequences derived from HIV-1, such as 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: for example VT5; MAP; or arginine stretch, etc.; (iii) designed peptides: for example MPG; Transportan; Transportan 10; Pep-1; a peptide selected from KALA; or a peptide selected from Bulforin2, etc.

[0051] In addition, the CPP used in the conjugate of the present invention can also be selected from a polypeptide sequence having about 60, 70, 80, 90, 95, 99% or 100% sequence identity with any polypeptide sequence as described above, as long as the polypeptide sequence still retains its biological activity, i.e., promoting the cellular uptake of the isolated polypeptide of the present invention (or its variant) and / or promoting the isolated polypeptide of the present invention (or its variant) to pass through the blood-brain barrier.

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

[0053] As used in the present invention, the term "vector" refers to a nucleic acid carrier into which a polynucleotide can be inserted. When a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art, including: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses. Animal viruses that can be used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40). A vector can contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site.

[0054] As used in the present 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 in the present invention, the term "identity" is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. When a position in both compared sequences is occupied by the same base or amino acid monomer subunit (for example, a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent 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 6 out of 10 positions of the two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of a total of 6 positions match). Usually, the comparison is made when the two sequences are aligned to produce maximum identity. Such an alignment can be achieved by using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). The percent 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), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol. Biol. 48: 444-453 (1970) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

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

[0057] As used in the present invention, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with a subject and an active ingredient, which is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes: pH regulators, surfactants, ionic strength enhancers, agents that maintain osmotic pressure, agents that delay absorption, diluents, adjuvants, preservatives, stabilizers, and the like. For example, pH regulators include phosphate buffers. Surfactants include cationic, anionic or nonionic surfactants, such as Tween-80. Ionic strength enhancers include sodium chloride. Agents that maintain osmotic pressure include sugars, NaCl, and the like. Agents that delay absorption include monostearate and gelatin. Diluents include water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), and the like. 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, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meanings generally understood by those skilled in the art, which can stabilize the desired activity of the active ingredient in the drug (e.g., inhibitory activity on TrkB), including sodium glutamate, gelatin, SPGA, sugars (e.g., sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (e.g., glutamic acid, glycine), proteins (e.g., dried whey, albumin or casein) or their degradation products (e.g., lactalbumin hydrolyzate), etc.

[0058] As used herein, the term "treat" refers to treating or curing a disease (eg, a nervous system disease), delaying the onset of symptoms of a disease (eg, a nervous system disease), and / or delaying the progression of a disease (eg, a nervous system disease).

[0059] As used in the present invention, the term "prevention" refers to preventing, inhibiting or delaying the occurrence of a disease (eg, a nervous system disease).

[0060] As used in the present invention, 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 is effective or sufficient to treat or cure a disease (e.g., a nervous system disease), delay the onset of symptoms of a disease (e.g., a nervous system disease) and / or delay the development of a disease (e.g., a nervous system disease). A preventive effective amount can be an amount that is effective or sufficient to prevent, inhibit or delay the occurrence of a disease (e.g., a nervous system disease). Such an effective amount can be easily determined by a person skilled in the art or a doctor, and can be related to the intended purpose (e.g., treatment or prevention), the general health of the subject, age, sex, weight, severity of the disease to be treated, complications, mode of administration, etc. The determination of such an effective amount is fully within the capabilities of those skilled in the art.

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

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

[0063] 1) Ability to specifically bind to TrkB;

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

[0065] 3) the ability to reduce TrkB activity in a subject (optionally after conjugating the polypeptide to a CPP);

[0066] 4) the ability to improve synaptic dysfunction and / or social dysfunction and repetitive stereotyped behaviors caused by excessive levels of TrkB in a subject (optionally after conjugating the polypeptide to a CPP);

[0067] 5) The ability to treat a disease associated with excessive TrkB activity (eg, a neurological disease) in a subject (optionally after conjugating the polypeptide to a CPP).

[0068] The beneficial effect of the present invention is that the TrkB targeting polypeptide and fusion protein of the present invention can significantly inhibit the activity of TrkB, and thus can be used to treat diseases associated with excessive MDGA2 activity (such as autism and other nervous system diseases and tumors), and has great clinical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1:BDNF / TrkB activity is increased in MDGA2 heterozygous deletion mice. Brain tissues of MDGA2 heterozygous deletion (HET) and wild-type control (WT) mice were isolated, synaptic components were extracted, and the protein levels of MDGA2, mature and immature BDNF (mBDNF and ImBDNF), TrkB / pTrkB, Akt / pAkt, mTOR / pmTOR, S6 / pS6, and CaMK2 / pCaMK2 were analyzed by WB and compared. Statistical analysis graphs were analyzed by grayscale analysis of protein levels in the figures using Image J software. The 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: Schematic diagram of full-length MDGA2, MDGA2 lacking the MAM region (ΔMAM), MDGA2 lacking the M1 region (ΔM1), and MDGA2 lacking the M2 region (ΔM2). Figure 2 c: TrkB and different truncated forms of MDGA2 were overexpressed in HEK293T cells, and their interaction was analyzed by IP-WB. Figure 2 C: Schematic representation of the M1, L1 and L2, and P1 and P2 fragments 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 pulled down proteins were used to detect the binding of TrkB with different fragments by WB. Figure 2 E: Biotin-labeled P1 and P2 fragments were injected into the mouse hippocampus. After 24 hours, the hippocampal tissue was lysed and the biotin-bound proteins were pulled down with Streptavidin Agarose. The binding of TrkB to different fragments was detected by WB. Figure 2 E: FITC-labeled P2 fragments were incubated with SH-SY5Y cells and then immunofluorescently stained with TrkB antibody, and the co-localization between the two was observed under a microscope. Scale bar: 10 μm. Figure 2 G: PYMOL molecular docking software was used to predict the interaction relationship and interaction region between RPS23RG1 and MDGA2.

[0071] Figure 3:The effect of P2 peptide on BDNF-TrkB binding. P2 and control peptide were added to MDGA2 heterozygous null neurons overexpressing TrkB-Flag at different concentrations (+: 0.1μM; ++: 1μM). After 16 hours, the binding of BDNF and TrkB-Flag was detected by IP-WB method, and the grayscale analysis of protein levels in the figure was performed using Image J software. The 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 therapeutic efficacy of MDGA2-based P2 peptide in MDGA2-deficient ASD model mice. Figure 4 A: Mouse free social interaction experiment, analyzing the time that mice treated with P2 or control peptide spend interacting and sniffing with stranger mice during free space exploration. Figure 4 B: In the three-box social experiment of mice, the time that mice sniffed the stranger mouse (S1) and the empty cage (E) was analyzed in the social preference test phase, and the social preference index PI = T (S1-E) / T (S1+E) was analyzed at the same time. Then, the time that mice sniffed another stranger mouse (S2) and (S1) mice was analyzed in the social novelty test phase. The social novelty preference index PI = T (S2-S1) / T (S1+S2) was also analyzed. Figure 4 C: The brains of mice treated with P2 or control peptide were lysed and analyzed by WB to compare the protein levels of TrkB / pTrkB, Akt / pAkt, and mTOR / pmTOR. Figure 4 D: The protein levels of TrkB bound to BDNF were analyzed by IP-WB in mouse brain lysate and compared. *P<0.05, **P<0.01, ***P<0.001 (one-way ANOVA with Tukey's multiple comparisons test). DETAILED DESCRIPTION

[0073] The technical solution of the present invention is further illustrated and described below through specific implementation modes in combination with the accompanying drawings.

[0074] Sequence information

[0075] The information of the sequences involved in the present invention is provided in Table 1 below.

[0076] Table 1: Description of sequences

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in the present invention are basically carried out with reference to the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989, and FM Ausubel et al., A Compendium of Molecular Biology Experiments, 3rd edition, John Wiley & Sons, Inc., 1995; the use of restriction endonucleases is in accordance with the conditions recommended by the product manufacturers. The reagents whose sources are not indicated in the examples are all 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 the concentration was determined by a microplate reader, the mRNA was reverse transcribed into cDNA using a kit. FastStart Universal SYBR Green Master (Roche) was used for qRT-PCR experiments. After the qRT-PCR experiment, a small amount of PCR product was analyzed by 1.5% agarose gel electrophoresis to determine whether the PCR product fragment size was correct and whether it was specifically amplified.

[0088] Protein extraction and Western blot

[0089] 1. Extraction of cell sample protein:

[0090] (1) Cell collection: discard the culture medium and wash the cells twice with PBS; discard the PBS, scrape the cells with a cell scraper and collect them in 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 inhibitors and phosphatase inhibitors (added just before use) to the cell mass collected in (1), shake on ice for 30 min or place in a silent mixer at 4°C for slow lysis overnight, centrifuge at 12,000 rpm at 4°C for 10 min, collect the supernatant, and measure the protein concentration.

[0092] 2. Extraction of tissue sample protein:

[0093] Fresh tissues were immediately frozen in liquid nitrogen after being taken out, and then stored in a -80°C ultra-low temperature refrigerator for later use. The tissues were minced with surgical scissors before homogenization, and then homogenized with a polytron in RIPA lysis buffer. After sufficient lysis, the tissues were centrifuged at 4°C, 12,000 rpm for 15 minutes, the supernatant was collected, and the centrifugation was repeated once to measure the protein concentration.

[0094] 3. Determination of protein concentration:

[0095] (1) Prepare BCA protein assay solution by mixing solution A and solution B at a ratio of 50:1 and set aside;

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

[0097] (3) Protein samples were added to a 96-well plate, and TNEN / RIPA lysis buffer was used as a blank control. Three wells were set up in each group.

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

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

[0100] 4.Westernblot:

[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, and run electrophoresis in Tris-Glycine electrophoresis buffer using SDS-PAGE protein gel.

[0102] (2) Electrophoretic transfer of proteins: Precool the electrophoretic transfer buffer at 4°C, cut a PVDF membrane of appropriate size, soak it with methanol, and soak it in the electrophoretic transfer buffer together with filter paper for 10 minutes. At the same time, cut the gel after electrophoresis and soak it in the electrophoretic transfer buffer for 5 minutes. Then, stick the PVDF membrane on the gel, cover both sides with filter paper, remove all bubbles, and install it in the electrotransfer tank with the membrane facing the positive electrode, and perform constant current electrotransfer (300mA, 90min) at 4°C.

[0103] (3) Antigen-antibody reaction:

[0104] a. Blocking: Block with 5% skim milk powder at room temperature for 1 hour;

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

[0106] c. Secondary antibody reaction: Wash three times with PBST, 10 min each time, dilute the primary antibody in blocking solution 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 solution A and solution B in the ECL kit at a ratio of 1:1 (V / V), add ECL to the membrane surface in a dark room, incubate for 5 min and then expose, adjusting the exposure time according to the fluorescence intensity.

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

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

[0111] (2) Centrifuge at 4°C, 5000 rpm for 2 min. Transfer the supernatant to a new EP tube, fill up to 700 ul with 0.5% TNEN lysis buffer, add 20 μl Protein-GSepharose and corresponding antibodies, and incubate at 4°C with rotation overnight.

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

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

[0114] Immunofluorescence

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

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

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

[0118] (4) 0.1% TritonX00-PBS penetration at room temperature for 5-10 minutes (the time should be strictly controlled here).

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

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

[0121] (7) Wash with PBS 4 times, 5 min each time. Let the culture plate stand still without shaking.

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

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

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

[0125] (11) Seal the slides at room temperature overnight. After the sealant is dry, observe and photograph under a confocal microscope.

[0126] Isolation of lysosomes

[0127] 1. Tissue (Bebo Biotechnology, lysosome extraction kit, BB-3603)

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

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

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

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

[0132] (5) Centrifuge the supernatant at 4°C, 20,000-30,000 × 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°C, 20,000-30,000 × g for 20 min. Discard the supernatant, collect the precipitate, and resuspend the precipitate in Lysosome Preservation Buffer C to obtain a 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 rinsed twice with PBS, then collected with KPBS (136 mM KCl, 10 mM KH2PO4, pH 7.25, adjusted with KOH), and a portion of the cells were lysed as the whole protein fraction;

[0136] (2) The KPBS cell suspension was homogenized slowly with a tight Dounce homogenizer for 30 seconds, and centrifuged at 1000 g for 2 min at 4°C;

[0137] (3) The supernatant was collected and incubated with KPBS pre-washed anti-HA beads 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 the refrigerator for later use or use it directly in downstream experiments.

[0139] Biotin labeling of cell surface proteins

[0140] Dissolve biotin (EZ-Link Sulfo-NHS-SS-Biotin) in freshly prepared PBS / CM buffer to a final concentration of 0.5 mg / mL and place on ice for later use; remove cells from the incubator, place on ice, remove the culture medium, add an appropriate amount of pre-cooled PBS / CM buffer to wash the cells three times, remove the buffer, add biotin solution to the cell culture plate, culture for 20 minutes under ice bath conditions, remove the biotin solution, and repeat once; add PBS / CM buffer containing 50 mM NH4Cl to terminate the reaction (10 minutes); lyse the cells with TNEN lysis buffer (containing protease inhibitors), centrifuge at 4°C, 12000 rpm for 10 minutes, transfer the supernatant to a new centrifuge tube, determine the protein concentration by BCA method, take an equal amount of protein sample, add an equal volume of 2× loading buffer, mix well, boil at 100°C for 5 minutes, and detect protein expression by immunoblotting analysis; add streptavidin beads (Streptavidin The beads were centrifuged at 4°C for 2 min at 5000 RPM on the next day, the supernatant was removed, the streptavidin beads were rinsed with 1% TNEN for 3 times, each time for 10 min, the residual liquid was removed with a microinjector, 20 μL of 2× loading buffer was added, the beads were boiled at 100°C for 5 min, and the target protein content was determined by protein immunoblotting analysis.

[0141] Plasma membrane separation

[0142] The cell membrane preparation process uses a cell membrane protein extraction kit (Phygene, PH0710). Remove the cells from the incubator, place them on ice, remove the culture medium, add an appropriate amount of pre-cooled PBS buffer to wash the cells twice, resuspend the collected cells in pre-cooled extraction reagent A and incubate for 15 minutes, then use a loose Dounce homogenizer to slowly homogenize 200 times, stop grinding when about 70% of the cells are broken, centrifuge the cell suspension at 700g for 10 minutes, collect the supernatant, and then centrifuge at 120,000g for 30 minutes to collect the membrane precipitate. Finally, resuspend the membrane protein in the precipitate and dissolve it with a lysis buffer containing protease inhibitors, centrifuge at 12,000g for 10 minutes, and collect it for the next experiment.

[0143] Protein interaction structure prediction

[0144] The researchers of the present invention selected the PDB file of MDGA2 (AF-Q7Z553-F1) suitable for the present invention in 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). The interaction between proteins was predicted by GRAMM: Docking network server (http: / / gramm.compbio.ku.edu / gramm, Vakser laboratory), and finally PYMOL was used to perform structural visualization and molecular docking analysis on the three-dimensional protein model and its interaction.

[0145] Tail vein injection

[0146] Dissolve the synthesized peptide in physiological saline, add no more than 2% dimethyl sulfoxide to help dissolve, and place the fully dissolved peptide on ice for later use. When injecting mice, make sure the mouse is comfortably placed in the mouse tube and positioned with the tail vein syringe barrel, while placing the mouse tail in the groove of the squeeze plate. Adjust the light so that the blood vessels are clearly visible, and then start the injection from the bottom of the tail. During the injection process, slowly push the peptide solution and always observe the health of the mouse. After the injection, the needle stays to ensure the normal absorption of the drug. After the needle is removed, use a cotton swab to press and stop bleeding for 1 minute. Inject at the same time for 5 consecutive days.

[0147] Animal behavior experiments

[0148] All mouse behavioral experiments were performed using Smart Video Tracking Software (Panlab, Harvard Apparatus) for data collection and analysis. Animal behavioral experiments were conducted between 9:00 a.m. and 18:00 p.m. every day, with a light intensity of 650 lux in the laboratory.

[0149] (1) Touch and stroke the mice once a day for 3 days before the start of the experiment. Gently grasp the mouse's tail and pick it up. Let the mouse stay in your hand for 30 seconds, then use a marker to draw a line on the mouse's tail. After marking the mouse, still hold it in your palm, grasp its 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 and allowed to adapt for 60 minutes. Before the experiment, the boxes and mazes were cleaned with 70% alcohol. After each experiment, the boxes and mazes were cleaned with 70% alcohol to remove the feces and urine excreted by the mice during the experiment and to eliminate the interference of the residual smell of the mice on the experiment.

[0151] 1. Open field test

[0152] Used to study the autonomous movement ability and anxiety behavior of mice, mainly based on the avoidance of mice to bright light and open space. In the open field test, the mice were placed in the center of the box (40cm (L) × 40cm (W) × 40cm (H)), and allowed to freely explore the maze for 10 minutes, and the total movement distance (Total distance) and the time spent in the middle area (Time in center) were recorded.

[0153] 2. Self-grooming test

[0154] The mice were placed in the center of the box (40cm (L) × 40cm (W) × 40cm (H)) and allowed to explore the maze freely for 15 minutes. The Smart3.0 video tracking system was used to record the entire process. It is generally believed that the first 5 minutes is the adaptation stage for mice to enter a new environment. Therefore, by observing the video data of the mice in the last 10 minutes, the total number of times and total time that each experimental mouse combed its own hair with its limbs were counted.

[0155] 3. Social affiliation

[0156] The experimental mouse was placed in the center of the box (40 cm (L) × 40 cm (W) × 40 cm (H)). Before the experiment, a wild-type mouse (stimulus mouse) of the same gender and age as the experimental mouse was placed in a metal cage. The experimental mouse was then placed in the box and allowed to explore freely for 10 min. The Smart3.0 video tracking system was used to record the entire process. The exploration time of the mouse in the metal cage and within 2 cm around it was recorded.

[0157] 4. Three chamber sociability test

[0158] There are two transparent partitions in the box (60 width × 40 depth × 22 height cm) that separate the left, middle and right chambers (20 × 40 cm each), and each partition has a small opening (5 × 5 cm) for access to each chamber. A fixed wire cylinder (8.5 cm in diameter) is placed in each of the left and right chambers. The test consists of three stages: adaptation, sociability test, and social novelty test. In the adaptation stage, both the left and right cylinders are empty, and the test mouse is placed in the middle and allowed to explore freely for 10 minutes. In the sociability test, a gender-matched stranger mouse (S1) is enclosed in an empty cylinder, and the mouse can explore freely for 10 minutes. The time the mouse explores S1 or another empty cylinder (E) is measured, and the social preference index (T(S1-E) / T(S1+E)) is calculated: the time exploring S1 minus the time exploring E, divided by the time exploring S1 and E). During the social novelty test, another unfamiliar mouse (S2) of the same sex was placed in the empty cylinder and allowed to explore freely for 10 min. The time the mouse explored 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, 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 cell sample protein:

[0162] (1) Cell collection: discard the culture medium and wash the cells twice with PBS; discard the PBS, scrape the cells with a cell scraper and collect them in 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 inhibitors and phosphatase inhibitors (added just before use) to the cell mass collected in (1), shake on ice for 30 min or place in a silent mixer at 4°C for slow lysis overnight, centrifuge at 12,000 rpm at 4°C for 10 min, collect the supernatant, and measure the protein concentration.

[0164] 2. Extraction of tissue sample protein:

[0165] Fresh tissues were immediately frozen in liquid nitrogen after being taken out, and then stored in a -80°C ultra-low temperature refrigerator for later use. The tissues were minced with surgical scissors before homogenization, and then homogenized with a polytron in RIPA lysis buffer. After sufficient lysis, the tissues were centrifuged at 4°C, 12,000 rpm for 15 minutes, the supernatant was collected, and the centrifugation was repeated once to measure the protein concentration.

[0166] 3. Determination of protein concentration:

[0167] (1) Prepare BCA protein assay solution by mixing solution A and solution B at a ratio of 50:1 and set aside;

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

[0169] (3) Protein samples were added to a 96-well plate, and TNEN / RIPA lysis buffer was used as a blank control. Three wells were set up in each group.

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

[0171] (5) Use an 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, and run electrophoresis in Tris-Glycine electrophoresis buffer using SDS-PAGE protein gel.

[0174] (2) Electrophoretic transfer of proteins: Precool the electrophoretic transfer buffer at 4°C, cut a PVDF membrane of appropriate size, soak it with methanol, and soak it in the electrophoretic transfer buffer together with filter paper for 10 minutes. At the same time, cut the gel after electrophoresis and soak it in the electrophoretic transfer buffer for 5 minutes. Then, stick the PVDF membrane on the gel, cover both sides with filter paper, remove all bubbles, and install it in the electrotransfer tank with the membrane facing the positive electrode, and perform constant current electrotransfer (300mA, 90min) at 4°C.

[0175] (3) Antigen-antibody reaction:

[0176] a. Blocking: Block with 5% skim milk powder at room temperature for 1 hour;

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

[0178] c. Secondary antibody reaction: Wash three times with PBST, 10 min each time, dilute the primary antibody in blocking solution 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 solution A and solution B in the ECL kit at a ratio of 1:1 (V / V), add ECL to the membrane surface in a dark room, incubate for 5 min and then expose, adjusting the exposure time according to the fluorescence intensity.

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

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

[0183] (2) Centrifuge at 4°C, 5000 rpm for 2 min. Transfer the supernatant to a new EP tube, fill up to 700 ul with 0.5% TNEN lysis buffer, add 20 μl Protein-GSepharose and corresponding antibodies, and incubate at 4°C with rotation overnight.

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

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

[0186] Immunofluorescence

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

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

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

[0190] (4) 0.1% TritonX00-PBS penetration at room temperature for 5-10 minutes (the time should be strictly controlled here).

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

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

[0193] (7) Wash with PBS 4 times, 5 min each time. Let the culture plate stand still without shaking.

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

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

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

[0197] (11) Seal the slides at room temperature overnight. After the sealant is dry, observe and photograph under a confocal microscope.

[0198] Protein interaction structure prediction

[0199] The researchers of the present invention selected the PDB files of the protein structures of MDGA2 (AF-Q7Z553-F1) and TrkB (AF-Q16620-F1) suitable for the present invention from the ALPHAFOLD protein structure data. The protein-protein interactions were predicted by the GRAMM:Docking web server (http: / / gramm.compbio.ku.edu / gramm, Vakser Laboratory), and finally PYMOL was used to perform structural visualization and molecular docking analysis on the three-dimensional protein model and its interactions.

[0200] Stereotaxic brain injection

[0201] After the mice were anesthetized, cannulas were implanted and fixed in the hippocampus on both sides, with the embedding coordinates as follows: AP, -2.0mm; L, ±2.3mm; DV, -2.0mm. After the mice recovered for 3 days, the synthesized peptide was dissolved in physiological saline at a concentration of 1μg / μL, and 1μL of the peptide was injected into the hippocampus of the mice through the cannula within 5 minutes, once a day, and at the same time for 7 consecutive days.

[0202] Animal behavior experiments

[0203] All mouse behavioral experiments were performed using Smart Video Tracking Software (Panlab, Harvard Apparatus) for data collection and analysis. Animal behavioral experiments were conducted between 9:00 a.m. and 18:00 p.m. every day, with a light intensity of 650 lux in the laboratory.

[0204] (1) Touch and stroke the mice once a day for 3 days before the start of the experiment. Gently grasp the mouse's tail and pick it up. Let the mouse stay in your hand for 30 seconds, then use a marker to draw a line on the mouse's tail. After marking the mouse, still hold it in your palm, grasp its 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 and allowed to adapt for 60 minutes. Before the experiment, the boxes and mazes were cleaned with 70% alcohol. After each experiment, the boxes and mazes were cleaned with 70% alcohol to remove the feces and urine excreted by the mice during the experiment and to eliminate the interference of the residual smell of the mice on the experiment.

[0206] 1. Social affiliation

[0207] The experimental mouse was placed in the center of the box (40 cm (L) × 40 cm (W) × 40 cm (H)). Before the experiment, a wild-type mouse (stimulus mouse) of the same gender and age as the experimental mouse was placed in a metal cage. The experimental mouse was then placed in the box and allowed to explore freely for 10 min. The Smart3.0 video tracking system was used to record the entire process. The exploration time of the mouse in the metal cage and within 2 cm around it was recorded.

[0208] 2. Three chamber sociability test

[0209] There are two transparent partitions in the box (60 width × 40 depth × 22 height cm) that separate the left, middle and right chambers (20 × 40 cm each), and each partition has a small opening (5 × 5 cm) for access to each chamber. A fixed wire cylinder (8.5 cm in diameter) is placed in each of the left and right chambers. The test consists of three stages: adaptation, sociability test, and social novelty test. In the adaptation stage, both the left and right cylinders are empty, and the test mouse is placed in the middle and allowed to explore freely for 10 minutes. In the sociability test, a gender-matched stranger mouse (S1) is enclosed in an empty cylinder, and the mouse can explore freely for 10 minutes. The time the mouse explores S1 or another empty cylinder (E) is measured, and the social preference index (T(S1-E) / T(S1+E)) is calculated: the time exploring S1 minus the time exploring E, divided by the time exploring S1 and E). During the social novelty test, another unfamiliar mouse (S2) of the same sex was placed in the empty cylinder and allowed to explore freely for 10 min. The time the mouse explored 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, divided by the time spent exploring S2 and S1.

[0210] Materials and reagents

[0211]

[0212]

[0213]

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

[0215] First, the researchers of the present invention detected an increase in the level of BNDF protein in the synaptosomes of MDGA2 heterozygous deletion mice (e.g. Figure 1 BDNF is a receptor for TrkB, and BDNF / TrkB binding activates the downstream mTOR-Akt signaling pathway. Therefore, the researchers of the present invention further detected that the phosphorylation levels of TrkB, mTOR, Akt, S6, and CaMK2 proteins in MDGA2 heterozygous mice were increased (as shown in Figure 2A). Figure 1 The increase in the phosphorylation levels of these proteins represents the activation of TrkB and downstream mTOR-Akt signaling, indicating that MDGA2 deficiency promotes the activity of BDNF / TrkB and its downstream signaling pathways.

[0216] Example 2: Analysis of the interaction between MDGA2 and TrkB and determination of the core sequence of MDGA2

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

[0218] The researchers of the present 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 (see Table 2 and Figure 2 C, D), L1 and L2 peptides fused with GST expressed in cells were incubated with human TrkB protein and then pulled down with Glutathione sepharose B. The results showed that only L2 could bind to TrkB protein and pull it down ( Figure 2 D).

[0219] The researchers of the present 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 peptides respectively and labeled them with biotin (see Table 2 and Figure 2 C and E). The two peptides and the biotin-labeled scrambled peptide were injected into the mouse hippocampus, and 24 hours later, the hippocampus tissue was lysed and the biotin-bound protein was pulled down with Streptavidin Agarose. It was found that only the biotin-labeled P2 peptide could pull down the TrkB protein in the mouse brain ( Figure 2E). In addition, after incubating SH-SY5Y cells with FITC-labeled P2 polypeptide (green) and then using TrkB antibody for immunofluorescence staining (red), co-localization was found between the two. The above results indicate that the 812-833 sequence region of MDGA2 is the key region for interaction with TrkB.

[0220] Table 2: Truncations of human full-length MDGA2

[0221]

[0222]

[0223] Example 3: MDGA2-based polypeptides inhibit the binding of BDNF to TrkB in cells

[0224] In order to further determine whether the core region polypeptide of MDGA2 that interacts with TrkB will affect the function of TrkB, the researchers of the present invention artificially synthesized the P2 polypeptide (KARLLSPVFSIAPKNPYGPTNT; SEQ ID NO.07) and the scrambled polypeptide (PYPPNTTLKSLPVISFNGKRAA; SEQ ID NO.05) of human MDGA2. The above polypeptides were synthesized by Sangon Biotech Co., Ltd. The above polypeptides were added to MDGA2 heterozygous null neurons overexpressing TrkB-Flag at different concentrations (+: 0.1 μM; ++: 1 μM), and the binding of BDNF and TrkB-Flag was detected by immunoprecipitation-immunoblotting method 16 hours later. It was found that the P2 polypeptide could inhibit the binding of BDNF and TrkB in a concentration-dependent manner (such as Figure 3 This indicates that the P2 polypeptide has the effect of inhibiting BDNF / TrkB activity.

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

[0226] MDGA2 is considered to be a susceptibility gene associated with ASD. MDGA2 protein deficiency can lead to increased excitatory synaptic transmission, and mice will show anxiety, social disorders and stereotyped behaviors. Therefore, MDGA2 heterozygous deletion mice are widely used as a typical autism model.

[0227] The above experimental results have shown that the activity of BDNF / TrkB is abnormally increased in MDGA2 heterozygous deletion mice, and P2 polypeptide can inhibit the binding of BDNF and TrkB, and may become a drug for disease treatment. Therefore, this example further evaluates the therapeutic effect of MDGA2-based polypeptides on autism model mice.

[0228] The researchers of the present invention artificially synthesized the P2 polypeptide (KARLLSPVFSIAPKNPYGPTNT; SEQ ID NO.07) and the disordered polypeptide (PYPPNTTLKSLPVISFNGKRAA; SEQ ID NO.05) of human MDGA2. The above polypeptides were synthesized by Sangon Biotech Co., Ltd. + / - Mice or littermate control WT mice were treated with 1 μg / day by stereotaxic injection into the bilateral hippocampus for one week. After one week of rest, the mice underwent behavioral testing and biochemical analysis.

[0229] The results of behavioral tests on mice showed that Mdga2 + / - Compared with wild-type mice, mice showed obvious social impairment in common social experiments ( Figure 4 A), and also showed impairment of social interest and preference in the three-box social experiment ( Figure 4 B) Administration of MDGA2-based peptides significantly improved the + / - Social impairment in mice Figure 4 A, B).

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

[0231] In addition to ASD caused by MDGA2 deficiency, for example, abnormal activation of the BDNF / TrkB signaling pathway has also been found to be involved in other diseases, such as the BDNF / TrkB signaling pathway is related to drug addiction, and inhibition of BDNF / TrkB can improve the cocaine exploration behavior and withdrawal reaction of animals; the BDNF / TrkB signaling pathway in the dorsal horn of the spinal cord is involved in neuropathic pain, and the use of TrkB inhibitors can relieve neuropathic pain; the level of BDNF in the serum of most epilepsy patients is elevated, and some epilepsy animal models have found that BDNF and TrkB are significantly increased in the temporal lobe and hippocampus. By deleting TrkB or blocking TrkB, the occurrence of epilepsy in these mice can be improved. In addition, some studies have found that BDNF / TrkB signals can promote cell carcinogenesis, infiltration and metastasis, and may be the cause of chemotherapy resistance. Therefore, the polypeptide of the present invention that can target and regulate MDGA2 metabolism to increase its expression level is also suitable for treating other nervous system diseases (such as autism, epilepsy) and tumors (such as brain tumors) caused by MDGA2 inactivation.

[0232] The above description is only a preferred embodiment of the present invention, and therefore cannot be used to limit the scope of the present invention. That is, equivalent changes and modifications made according to the patent scope of the present invention 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: It consists of 21-55 consecutive amino acids of MDGA2 protein and contains KARLLSPVFSIAPKNPYGPTN (SEQ ID NO. 08).

2. A TrkB targeting polypeptide according to claim 1, characterized in that: The amino acid sequence thereof comprises KARLLSPVFSIAPKNPYGPTNX (SEQ ID NO. 10), wherein X is T or S.

3. A TrkB targeting polypeptide as claimed in claim 2, wherein the amino acid sequence is KARLLSPVFSIAPKNPYGPTNT (SEQ ID NO.07) or KARLLSPVFSIAPKNPYGPTNS (SEQ ID NO.09).

4. A conjugate, characterized in that: It comprises the TrkB targeting polypeptide according to any one of claims 1 to 3 and a modification portion, wherein the modification portion is selected from another polypeptide, a detectable label or any combination thereof, and the other polypeptide is selected from CPP, a targeting portion and / or a protein tag.

5. A fusion protein, characterized in that: The invention comprises the TrkB targeting polypeptide according to any one of claims 1 to 3 and another polypeptide, wherein the other polypeptide is selected from CPP, a targeting moiety, a protein tag or any combination thereof.

6. An isolated nucleic acid molecule, characterized in that: The polypeptide has the nucleotide sequence of the TrkB targeting polypeptide according to any one of claims 1 to 3, or the fusion protein according to claim 5.

7. A carrier, characterized in that: It has the isolated nucleic acid molecule according to claim 6.

8. A host cell, characterized in that: It has the isolated nucleic acid molecule of claim 6 or the vector of claim 7.

9. A method for preparing the TrkB targeting polypeptide according to any one of claims 1 to 3 or the fusion protein according to claim 5, characterized in that: include: The host cell of claim 8 is cultured under suitable conditions, and the TrkB targeting polypeptide or the fusion protein is recovered from the culture of the host cell.

10. A pharmaceutical composition, characterized in that: It comprises the TrkB targeting polypeptide according to any one of claims 1 to 3, the conjugate according to claim 4, the fusion protein according to claim 5, the isolated nucleic acid molecule according to claim 6, the vector according to claim 7 or the host cell according to claim 8, and a pharmaceutically acceptable carrier and / or excipient.

11. Use of the TrkB targeting polypeptide according to any one of claims 1 to 3, the conjugate according to claim 4, the fusion protein according to claim 5, the isolated nucleic acid molecule according to claim 6, the vector according to claim 7 or the host cell according to claim 8 in the preparation of a pharmaceutical composition for treating a disease associated with excessive TrkB activity or inhibiting the activity of TrkB.

12. A method for inhibiting TrkB activity in vitro for non-diagnostic therapeutic purposes, characterized in that: The TrkB targeting polypeptide according to any one of claims 1 to 3, the conjugate according to claim 4 or the fusion protein according to claim 5 is contacted with a cell in need thereof.

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