CN119161442A - A TrkB targeting polypeptide and its application - Google Patents

Abstract

The present invention discloses a TrkB targeting polypeptide and its application, which is composed of 21-55 consecutive amino acids of MDGA2 protein and contains KARLLSPVFSIAPKNPYGPTN (SEQ ID NO.08). The TrkB targeting polypeptide and fusion protein in the present invention can significantly inhibit the activity of TrkB, and thus can be used to treat diseases related to excessive TrkB activity (such as autism and other nervous system diseases and tumors), and has great clinical value.

Description

TrkB targeting polypeptide and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a TrkB targeting polypeptide and application thereof.

Background

After BDNF and TrkB are combined, trkB signal channels are activated, and different channels including PI3K/Akt/mTOR, MAPK/ERK, PLC gamma and the like are included in the downstream of the BDNF, so that the BDNF plays an important role in the development and multiple functions of a nervous system. However, abnormal activation of TrkB signaling pathways may also lead to dysfunction of the nervous system, leading to disease. For example, the BDNF/TrkB signaling pathway can promote the plasticity of the "reward mechanism" in the brain and is therefore associated with drug addiction. In an animal model of cocaine addiction, inhibition of BDNF/TrkB can improve cocaine exploration behavior and withdrawal response in animals. In neuropathic pain, the BDNF/TrkB signaling pathway of the dorsal horn of the spinal cord may be involved in pain by affecting KCC2 expression, whereas the use of TrkB inhibitors can block KCC2 expression down-regulation and alleviate neuropathic pain. Elevated levels of BDNF were found in the serum of most epileptic patients, while significant increases in BDNF and TrkB were found in some epileptic animal models in the temporal lobe and hippocampus. By deleting or blocking TrkB, epileptogenesis in these mice can be improved. Administration of BDNF to the hippocampus of rats induces epileptogenesis. In addition, elevated levels of BDNF were also found in autistic pediatric patients, suggesting that abnormalities in BDNF/TrkB signaling pathways may also be involved in autism.

Still other studies have found that BDNF/TrkB signaling can promote cancerous, invasive and metastatic events in cells and may be responsible for the development of chemotherapy resistance. For example, transplantation of neural crest-derived cells overexpressing TrkB into mice can lead to rapid tumor growth. Furthermore, tumor cells appear to be able to place PI3K kinase in a long-term activated state by activating TrkB, thereby increasing resistance to anoikis and success rate of metastasis.

Taken together, inhibition of BDNF/TrkB activity may be a new strategy for the treatment of a variety of neurological diseases and tumors.

Disclosure of Invention

The invention aims at providing a TrkB targeting polypeptide.

It is a further object of the present invention to provide the use of a TrkB targeting polypeptide as described above.

The technical scheme of the invention is as follows:

A TrkB targeting polypeptide consisting of 21-55 contiguous amino acids of MDGA protein and comprising KARLLSPVFSIAPKNPYGPTN (SEQ ID No. 08).

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.

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

Further preferred, the amino acid sequence is KARLLSPVFSIAPKNPYGPTNT (human, SEQ ID NO. 07) or KARLLSPVFSIAPKNPYGPTNS (murine, SEQ ID NO. 09).

A conjugate having a TrkB targeting polypeptide as described above and a modifying moiety selected from an additional polypeptide selected from CPP (enhancing its ability to penetrate a cell membrane), a targeting moiety (making it targeted) and/or a protein tag (to facilitate its expression, detection, tracking and/or purification), a detectable label (making it useful for detection), or any combination thereof.

A fusion protein having a TrkB targeting polypeptide as described above and an additional polypeptide selected from CPPs (cell membrane penetrating peptides, enhancing their ability to penetrate cell membranes), targeting moieties (to be targeted), protein tags (to facilitate their expression, detection, tracking and/or purification), or any combination thereof.

For the above conjugates and fusion proteins:

Preferably, the modification is linked to the N-terminus or the C-terminus of the TrkB targeting polypeptide, optionally via a linker.

Further preferred, the CPP is a Tat derivative peptide, e.g., having the sequence shown in SEQ ID NO. 11.

Further preferred, the targeting moiety is a ligand, receptor or antibody.

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

Further preferred, the protein tag is HA, myc, GFP or biotin.

An isolated nucleic acid molecule having the nucleotide sequence of the TrkB targeting polypeptide described above, or the fusion protein described above.

A vector comprising the isolated nucleic acid molecule. The vector is a cloning vector or an expression vector.

In a preferred embodiment of the invention, the vector comprises a plasmid, a cosmid, a phage, and a cosmid.

Further preferred, the vector is capable of expressing a TrkB targeting polypeptide of the invention or a fusion protein of the invention in a subject (e.g., a mammal, e.g., a human).

A host cell having the isolated nucleic acid molecule described above or the vector described above. Such host cells include prokaryotic cells such as E.coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (e.g., mammalian cells such as mouse cells, human cells, etc.). The cells of the invention may also be cell lines, such as 293T cells.

The preparation method of the TrkB target polypeptide or the fusion protein comprises culturing the host cell under proper conditions and recovering the TrkB target polypeptide or the fusion protein from the culture of the host cell.

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

Use of the TrkB targeting polypeptide, the conjugate, the fusion protein, the isolated nucleic acid molecule, the vector or the host cell described above in the preparation of a pharmaceutical composition.

The pharmaceutical composition is used for treating diseases related to the excessive activity of TrkB or inhibiting the activity of TrkB.

Diseases associated with the above-mentioned TrkB activity include neurological diseases and tumors.

The above-mentioned nervous system diseases are characterized by abnormal activation of BDNF/TrkB signaling pathways, and preferably autism.

The tumor is preferably a brain tumor.

Preferably, the pharmaceutical composition may further comprise an additional pharmaceutically active ingredient;

The additional pharmaceutically active component is a drug having therapeutic activity for neurological diseases (e.g., epilepsy and neuropathic pain).

Or the additional pharmaceutically active component is selected from omega-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.

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

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

The TrkB targeting polypeptides, fusion proteins or pharmaceutical compositions of the invention may be formulated in any dosage form known in the medical arts, e.g., in the form of tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injectable solutions, lyophilized powders), and the like. In some embodiments, the TrkB targeting polypeptides, fusion proteins, or pharmaceutical compositions of the invention may be formulated as an injection or lyophilized powder.

Furthermore, the TrkB targeting polypeptides or fusion proteins of the invention may be present in pharmaceutical compositions in unit dosage form for ease of administration.

The TrkB targeting polypeptides, fusion proteins or pharmaceutical compositions of the invention may be administered by any suitable method known in the art, including oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic, inguinal, intravesical, topical (e.g., powder, ointment or drops), or nasal route. For many therapeutic uses, however, the preferred route/mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). The skilled artisan 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 invention is administered by intravenous infusion or injection.

The TrkB targeting polypeptides, fusion proteins or pharmaceutical compositions provided herein may be used alone or in combination, or in combination with additional pharmaceutically active components (e.g., drugs having activity in the treatment of neurological disorders). In certain preferred embodiments, the TrkB targeting polypeptides or fusion proteins of the invention are used in combination with other agents having activity in the treatment of neurological disorders to prevent and/or treat disorders associated with excessive TrkB activity (e.g., neurological disorders). Such additional pharmaceutically active components may be administered prior to, simultaneously with, or after administration of the TrkB targeting polypeptides, fusion proteins, or pharmaceutical compositions of the invention.

The pharmaceutical compositions of the invention may comprise a "therapeutically effective amount" or a "prophylactically effective amount" of a TrkB targeting polypeptide or fusion protein of the invention. By "prophylactically effective amount" is meant an amount sufficient to prevent, arrest, or delay the onset of a disease (e.g., a disease associated with TrkB activity). By "therapeutically effective amount" is meant an amount sufficient to cure or at least partially arrest 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 invention may vary depending on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient, such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, and the like.

In the present invention, the dosing regimen may be adjusted to achieve the optimal target response (e.g., therapeutic or prophylactic response). For example, the dosage may be administered in a single dose, may be administered multiple times over a period of time, or may be proportionally reduced or increased as the degree of urgency of the treatment situation.

Typical non-limiting ranges for therapeutically or prophylactically effective amounts of the TrkB targeting polypeptides or fusion proteins of the invention are 0.001-100mg/kg body weight, e.g. 0.01-50mg/kg body weight, 0.1-25mg/kg body weight.

It should be noted that the dosage may vary with the type and severity of the condition being treated.

Furthermore, those skilled in the art will appreciate that for any particular patient, the particular dosage regimen should be adjusted over time according to the needs of the patient and the professional judgment of the physician, and that the dosage ranges given herein are for illustrative purposes only and are not limiting of the use or scope of the pharmaceutical compositions of the invention.

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

Definition of terms

In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Moreover, the cell culture, molecular genetics, nucleic acid chemistry, immunological laboratory procedures used in the present invention are all conventional procedures widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.

As used herein, the term "cell penetrating peptide (CELL PENETRATING PEPTIDE, CPP)" also known as "cell penetrating peptide," protein transduction domain (protein translocation domain, PTD), "Trojan horse peptides," or "transduction peptide (transduction peptide)" and the like, refers to polypeptides capable of promoting cellular uptake of various molecules (e.g., various macromolecules including proteins or nucleic acids; e.g., trkB targeting polypeptides of the invention or variants thereof). Such polypeptides are well known in the art and are described, for example, in Stewart, et al, 2008 and CN101490081a (which are incorporated herein by reference in their entirety), or may be obtained by methods known in the art, such as the methods described in detail in US 2008/023483 A1, which are incorporated herein by reference in their entirety.

In the present invention, examples of the CPP include (i) protein derived peptide (protein DERIVED CPPS) such as a sequence derived from a gene controlling antenna (antenna peptide), for example pAntp (43-58), a sequence derived from HIV-1 such as a Tat derived peptide such as amino acid residues 37-72, amino acid residues 37-60, amino acid residues 48-60 or amino acid residues 49-57 from TAT, (hCT (9-32), pVEC, (plSL), mouse PRP (1-28), E ^ms (194-220), or Restricocin L (60-73), etc., (ii) model peptide (model peptides) such as VT5, MAP, or arginine elongation sequence (ARGININE STRETCH), etc., (iii) design peptide (DESIGNED CPPS) such as MPG, transport 10, pep-1, peptide selected from KALA, or peptide selected from Bulforin2, etc.

Furthermore, the CPP used in the conjugates of the invention may also be selected from polypeptide sequences having about 60, 70, 80, 90, 95, 99% or 100% sequence identity to any of the polypeptide sequences described above, so long as the polypeptide sequence still retains its biological activity, i.e., promotes cellular uptake of the isolated polypeptide (or variant thereof) of the invention and/or promotes permeation of the isolated polypeptide (or variant thereof) of the invention through the blood-brain barrier.

As used herein, the term "targeting moiety" refers to a domain capable of directing a TrkB targeting polypeptide (or variant thereof) of the invention to a desired location, which may 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 the corresponding targeting domains by the nature of the desired location.

As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide may be inserted. When a vector enables expression of a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction or transfection such that the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include plasmids, phagemids, cosmids, artificial chromosomes, such as Yeast Artificial Chromosomes (YACs), bacterial Artificial Chromosomes (BACs) or P1-derived artificial chromosomes (PACs), phages, such as lambda or M13 phages, animal viruses and the like. Animal viruses that may be used as vectors include, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papilloma-vacuolated viruses (e.g., SV 40). A vector may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain a replication origin.

As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, and includes, for example, prokaryotic cells such as E.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, HEK 293 cells or human cells.

As used herein, the term "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared 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 "percent identity" between two sequences is a function of the number of matched positions shared by the two sequences divided by the number of positions to be compared x 100. For example, if 6 out of 10 positions of two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 positions in total are matched). Typically, the comparison is made when two sequences are aligned to produce maximum identity. Such alignment may be achieved using, for example, the method of Needleman et al (1970) J.mol.biol.48:443-453, which may be conveniently performed by a computer program such as the Align program (DNAstar, inc.). The algorithm of E.Meyers and W.Miller (Comput. Appl biosci.,4:11-17 (1988) that has been incorporated into the ALIGN program (version 2.0) can also be used to determine the percent identity between two amino acid sequences using the PAM120 weight residue table (weight residue table), the GAP length penalty of 12 and the GAP penalty of 4. Furthermore, needleman and Wunscch (J MoI biol.48) that has been incorporated into the GAP program of the GCG software package (available on www.gcg.com) can be used: 444-453 (1970) algorithm, blossum 62 matrix or PAM250 matrix and 16, 14, 12, 10, 8, 6 or 4 GAP weights (GAP WEIGHT) and 1, 2, 3, 4, 5 or 6 length weights were used to determine the percent identity between two amino acid sequences.

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

As used herein, the term "pharmaceutically acceptable carrier and/or excipient" refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and active ingredient, as 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 include: for example, pH adjusting agents 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 sugar, naCl and the like, agents for delaying absorption include monostearates and gelatin, diluents include water, aqueous buffers (e.g., buffered saline), alcohols and polyols (e.g., glycerol), and the like, adjuvants include aluminum adjuvants (e.g., aluminum hydroxide), stabilizers have the meaning commonly understood by those skilled in the art that they are capable of stabilizing the desired activity of the active ingredient in the drug (e.g., inhibitory activity against 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 degradation products thereof (e.g., lactalbumin hydrolysate), and the like.

As used herein, the term "treating" refers to 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).

As used herein, the term "preventing" refers to preventing, inhibiting or delaying the onset of a disease (e.g., a neurological disease).

As used herein, the term "effective amount" refers to an amount that is effective to achieve the intended purpose. For example, a therapeutically effective amount may be an amount effective or sufficient to treat or cure a disease (e.g., a neurological disease), delay the onset of symptoms of a disease (e.g., a neurological disease), and/or delay the progression of a disease (e.g., a neurological disease). A prophylactically effective amount may be an amount effective or sufficient to prevent, inhibit or delay the occurrence of a disease (e.g., a neurological disease). Such effective amounts can be readily determined by one of skill in the art or a physician, and can be related to the intended purpose (e.g., treatment or prevention), the general health of the subject, the age, sex, weight, severity of the disease to be treated, complications, mode of administration, and the like. Determination of such effective amounts is well within the ability of those skilled in the art.

As used herein, the term "subject" refers to a mammal, such as a primate mammal, e.g., a human. In certain embodiments, the subject (e.g., human) has, or is at risk of having, a disease associated with TrkB activity (e.g., a neurological disease).

As used in the present invention, the biological functions of the TrkB targeting polypeptides of the present invention include one or more selected from the group consisting of:

1) Ability to specifically bind TrkB;

2) Ability to inhibit TrkB activity;

3) Ability to reduce TrkB activity in a subject (optionally, after conjugation of the polypeptide to a CPP);

4) An ability to ameliorate synaptic and/or social dysfunction and repetitive patterning behavior caused by excessive levels of TrkB in a subject (optionally, after conjugation of the polypeptide to a CPP);

5) Ability to treat a disease associated with an excessive TrkB activity (e.g., a neurological disease) in a subject (optionally, after conjugation of the polypeptide to a CPP).

The beneficial effects of the invention are that the TrkB targeting polypeptide and the fusion protein can obviously inhibit the activity of TrkB, so 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 MDGA over-activity, and have great clinical value.

Drawings

FIG. 1 shows increased BDNF/TrkB activity in MDGA2 heterozygous deletion mice. The MDGA2 heterozygous deletion (HET) and wild-type control (WT) mouse brain tissues were isolated, synaptic components were extracted, protein levels were analyzed MDGA, mature and immature BDNF (mBDNF and ImBDNF), trkB/pTrkB, akt/pAkt, mTOR/pmTOR, S6/pS6, caMK2/pCaMK2 by WB analysis and compared. The statistical analysis graphs all use Image J software to perform gray scale analysis on protein levels in the graphs. Results are expressed as mean ± standard error (s.e.m). * P <0.05, < P <0.01, < P <0.001, "ns" means no significant difference (2-measured Student's t test).

FIGS. 2A-2G evaluation of MDGA2 interaction with TrkB. FIG. 2A is a schematic diagram of full length MDGA2, deleted MAM region (ΔMAM), deleted M1 region (ΔM1), and deleted M2 region (ΔM2) MDGA. FIG. 2c shows the correlation of the overexpression of TrkB and the different MDGA truncations in HEK293T cells by IP-WB analysis. FIG. 2C is a schematic diagram of M1, L1 and L2, and fragments of P1 and P2 of MDGA2. FIG. 2D GST-tagged L1 and L2 fragments were incubated with TrkB protein, respectively, and pulled down with Glutathione sepharose B. The pulled down protein was assayed for binding of TrkB to the different fragments using WB. FIG. 2E shows that biotin-labeled P1 and P2 fragments were injected into the hippocampus of mice, and that the protein bound to biotin was pulled down by STREPTAVIDIN AGAROSE after tissue lysis of the hippocampus after 24h, and that binding of TrkB to the different fragments was detected by WB. FIG. 2E FITC-labeled P2 fragment incubated with SH-SY5Y cells followed by immunofluorescent staining with TrkB antibody and microscopic observation of co-localization between the two. Scale 10 μm. FIG. 2G prediction of RPS23RG1 and MDGA2 interaction relationship and interaction region using PYMOL molecular docking software.

FIG. 3 influence of P2 polypeptide on BDNF-TrkB binding. P2 and control polypeptide were added at different concentrations (+0.1. Mu.M; +: 1. Mu.M) to MDGA heterozygous deleted neurons overexpressing TrkB-Flag, respectively, and after 16h, BDNF binding to TrkB-Flag was detected by the IP-WB method and the protein levels in the figures were analyzed in gray scale using Image J software. Results are expressed as mean ± standard error (s.e.m), < P <0.05, < P <0.01 (one-way ANOVA with Tukey's multiple comparisons test).

FIGS. 4A-4D evaluation of therapeutic efficacy of MDGA-based P2 polypeptide in MDGA-deleted ASD model mice. FIG. 4A is a free social experiment of mice, analyzing the time of sniffing in free space exploration by mice treated with P2 or control polypeptides in communication with strangers. Fig. 4B, three-box social experiments of mice, in which the time of mice sniffing strangers (S1) and empty cages (E) was analyzed in the social preference test phase, while the social preference index pi=t (S1-E)/T (s1+e) was analyzed. The mice were then analyzed for the time to sniff another strange mouse (S2) and (S1) mice during the social novelty test phase. Social novelty preference index pi=t (S2-S1)/T (s1+s2) is analyzed simultaneously. FIG. 4C shows the protein levels of TrkB/pTrkB, akt/pAkt, mTOR/pmTOR analyzed by WB after lysis of the brains of mice treated with P2 or control polypeptides and compared. FIG. 4D shows the protein levels of TrkB binding to BDNF in mouse brain lysates by IP-WB analysis and comparison. * P <0.05, < P <0.01, < P <0.001 (one-way ANOVA with Tukey's multiple comparisons test).

Detailed Description

The technical scheme of the invention is further illustrated and described below by the specific embodiments in combination with the accompanying drawings.

Sequence information

Information on the sequences to which the present invention relates is provided in table 1 below.

TABLE 1 description of the sequences

Unless otherwise indicated, the molecular biology experimental methods and immunoassays used in the present invention are essentially described in J.Sambrook et al, molecular cloning, in laboratory Manual, 2 nd edition, cold spring harbor laboratory Press, 1989, and F.M. Ausubel et al, fine-programming molecular biology experimental guidelines, 3 rd edition, john Wiley & Sons, inc.,1995, and the use of restriction enzymes is in accordance with conditions recommended by the manufacturer of the product. The reagents of non-noted origin in the examples are all conventional reagents in the art or commercially available reagents.

Experimental materials and methods

Real-time fluorescence quantitative PCR (qRT-PCR)

Total RNA was extracted from mouse tissues using TRIzol reagent and the concentration was determined by an enzyme-labeled instrument, and mRNA was reverse transcribed into cDNA using the kit. qRT-PCR experiments were performed using FastStartUniversalSYBRGreenMaste (Roche). After the qRT-PCR experiment is finished, a small amount of PCR products are taken and analyzed in 1.5% agarose gel electrophoresis to determine whether the sizes of the PCR product fragments are correct or not and whether the PCR product fragments are specifically amplified or not.

Protein extraction and immunoblotting (Westernblot)

1. Extraction of cell sample proteins:

(1) Cell collection, namely, discarding culture solution, washing cells for 2 times by using PBS, discarding the PBS, scraping the cells by using a cell scraper, collecting the cells into a centrifuge tube, centrifuging at 1000rpm for 5min, collecting the cells at the bottom of the tube, and discarding supernatant.

(2) Cell lysis by adding TNEN cell lysate containing cocktail protease inhibitor and phosphatase inhibitor (added before use) to the cell mass collected in (1), shaking on ice for 30min or slowly lysing overnight at 4 ℃ with a mute mixer, centrifuging at 12000rpm for 10min, collecting supernatant, and measuring protein concentration.

2. Extraction of tissue sample proteins:

Fresh tissue is taken out and immediately put into liquid nitrogen for freezing, and then stored in an ultralow temperature refrigerator at-80 ℃ for standby. The tissue was minced with surgical scissors before homogenization, then electrohomogenized with polytron in RIPA lysate, after sufficient lysis, centrifuged at 4℃and 12000rpm for 15min, the supernatant was collected and centrifuged once again to determine protein concentration.

3. Determination of protein concentration:

(1) Preparing BCA protein assay solution, and mixing solution A and solution B at a ratio of 50:1 for use;

(2) Taking BSA as a standard sample, and preparing a protein standard curve;

(3) Protein samples are respectively added into 96-well plates, TNEN/RIPA lysate is used as a blank control, and 3 holes are arranged in each group;

(4) 200 μl of the prepared protein assay solution is added into each hole, and the mixture is uniformly mixed and reacted for 30min in a 37 ℃ incubator;

(5) OD562 was measured using a microplate reader and the resulting values were substituted into a protein standard curve to calculate protein sample concentrations.

4.Westernblot:

(1) Protein electrophoresis, namely taking 30-40 mug protein sample, adding 1/4 volume of 5 XSDS loading buffer solution, boiling for 3-5min at 100 ℃, and electrophoresis in Tris-Glycine electrophoresis buffer solution by using SDS-PAGE protein gel.

(2) And (3) carrying out electrophoresis transfer on proteins, namely precooling an electrophoresis transfer buffer solution at 4 ℃, cutting off PVDF membranes with proper sizes, soaking the PVDF membranes with methanol, soaking the PVDF membranes and filter paper in the electrophoresis transfer buffer solution for 10min, and simultaneously cutting the gel after electrophoresis and soaking the gel in the electrophoresis transfer buffer solution for 5min. And then, pasting PVDF film on the adhesive, covering filter paper on both sides, expelling bubbles, and placing the film in an electrotransport groove in the order of the film towards the positive electrode, and carrying out constant current electrotransport at 4 ℃ (300 mA,90 min).

(3) Antigen-antibody reaction:

a. Sealing, namely sealing 5% skimmed milk powder for 1h at room temperature;

b. Primary antibody reaction, namely diluting the primary antibody in a sealing liquid in a proper proportion, and standing at 4 ℃ for night;

c. the secondary antibody reaction, PBST washing three times, 10min each time, the primary antibody is diluted in a sealing liquid according to a proper proportion, and the primary antibody is incubated for 1h at room temperature.

(4) ECL detection:

PBST is washed three times for 10-15min each time, the solution A and the solution B in the ECL kit are mixed at a ratio of 1:1 (V/V), ECL is dripped into a darkroom to the surface of the membrane, the membrane is incubated for 5min and then exposed, and the exposure time is adjusted according to the fluorescence intensity.

Immunoprecipitation (IP) or co-immunoprecipitation (CoIP)

(1) Protein samples were collected, 10. Mu.l Protein-ASepharose was added to the Protein samples and incubated at 4℃for 1-2h with rotation.

(2) Centrifuge at 5000rpm for 2min at 4 ℃. The supernatant was transferred to a new EP tube, made up to 700ul with 0.5% TNEN lysate, 20 ul Protein-GSephase and corresponding antibodies were added and incubated overnight at 4℃with spin.

(3) Centrifuge at 4 ℃,5000rpm for 2min, after aspiration of the supernatant, add pre-chilled 1% TNEN solution, incubate at 4 ℃ for 8min with spin.

(4) The step (3) was repeated three times, and the supernatant was carefully blotted dry with a microinjector for the last time, then 20. Mu.L of 2 Xloading buffer was added, and after mixing well, the sample was boiled at 100℃for 5min. The resulting samples were used for immunoblot analysis.

Immunofluorescence

(1) The treated cover glass is placed into a 24-well plate (one for each well), uniformly divided into cells with a certain density (the density is 5% -10%), and cultured for 24 hours.

(2) The corresponding plasmid was transferred according to lipo2000 transfection method.

(3) After 24h of transfection, the medium was aspirated and the cells were washed 3 times with pre-chilled PBS. The PBS was blotted off and fixed with 4% paraformaldehyde for 10min at room temperature.

(4) 0.1% Triton X00-PBS was penetrated for 5-10min at room temperature (time should be strictly controlled here).

(5) Blocking with 3% BSA-PBS was performed for 1h.

(6) Incubate overnight with different primary antibodies (1:100 diluted in 3% BSA-PBS) at 4 ℃.

(7) The plates were left to stand without shaking when washed 4 times with PBS for 5min each.

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

(9) If necessary, DAPI was incubated at room temperature for 3-5min.

(10) PBS was washed 4 times, each for 5min.

(11) The tablets were blocked overnight at room temperature, observed under confocal microscopy after the tablet had dried and photographed.

Separation of lysosomes

1. Tissue (Bei Bo organism, lysosome extraction kit, BB-3603)

(1) Taking out brain tissue of a mouse, firstly cutting the brain tissue into pieces as much as possible by using surgical scissors, washing the pieces twice by using cold PBS, adding 400 mu l of cold reagent A, placing the pieces on ice for standing for 10min, and homogenizing the pieces under 30-40 by using a tight Dounce homogenizer;

(2) The homogenate was centrifuged at 1000 Xg for 5min at 4 ℃. Discarding the precipitate, and collecting the supernatant;

(3) The supernatant was centrifuged at 4℃for 10min at 3000 Xg force. Discarding the precipitate, and collecting the supernatant;

(4) The supernatant was centrifuged at 4℃under 5000 Xg force for 10min. Discarding the precipitate, and collecting the supernatant;

(5) The supernatant was centrifuged at 20000-30000 Xg force for 20min at 4 ℃. Discarding the supernatant, collecting the precipitate, adding 400 μl of cold reagent B into the precipitate, and mixing;

(6) Centrifuging at 4deg.C under 20000-30000 Xg force for 20min. Discarding the supernatant, collecting the precipitate, and re-suspending the precipitate with lysosome preservation solution C to obtain a lysosome sample, and preserving the lysosome sample in a refrigerator for standby or directly using the lysosome sample in downstream experiments.

2. Cell (LysoIP)

(1) Using HeLa cells stably transformed with TMEM192-3xHA, the treated cells were washed twice with PBS and then collected with KPBS (136mM KCl,10mM KH2PO4,pH 7.25, conditioned with KOH), leaving a fraction of the cell lysis as whole protein fraction;

(2) KPBS the cell suspension was slowly homogenized with a tight Dounce homogenizer at 30℃and centrifuged at 1000g for 2min;

(3) Collecting supernatant and KPBS pre-washing anti-HA beads for incubation on a gentle rotary shaker for 30min;

(4) The immunoprecipitate was washed three times with KPBS and resuspended in TNEN buffer to obtain the lysosome sample, which was stored in a refrigerator for use or directly used in downstream experiments.

Cell surface protein biotin labeling

Dissolving Biotin (EZ-LinkSulfo-NHS-SS-Biotin) in freshly prepared PBS/CM buffer solution with the final concentration of 0.5mg/mL, placing on ice for standby, taking out cells from an incubator, placing on ice, sucking off the culture medium, adding a proper amount of precooled PBS/CM buffer solution to wash the cells for 3 times, sucking off the buffer solution, adding a Biotin solution into a cell culture dish, culturing for 20min under ice bath conditions, sucking off the Biotin solution once, adding PBS/CM buffer solution containing 50mM NH ₄ Cl for stopping the reaction (10 min), lysing the cells by TNEN lysate (containing protease inhibitor), centrifuging for 10min at 4 ℃, transferring supernatant into a new centrifuge tube at 12000RPM, measuring the protein concentration by a BCA method, adding an equal volume of 2X loading buffer solution, uniformly mixing, 100 ℃ for 5min, detecting protein expression by an immunoblotting method, adding streptavidin beads (STREPTAVIDIN BEADS) into the equal amount of the rest protein sample, carrying out 4 ℃ for 20min, sucking off the protease solution for 2 ℃ for 2min, and carrying out immunoblotting for 2% by a micro-channel assay method, and carrying out 2% centrifugation for 20min, and carrying out a 20% Western blotting method, respectively.

Plasma membrane separation

The cell membrane preparation procedure used a cell membrane protein extraction kit (Phygene, pH 0710). Taking out cells from an incubator, placing on ice, sucking off the culture medium, adding a proper amount of precooled PBS buffer solution to wash the cells for 2 times, re-suspending the collected cells in precooled extraction reagent A and incubating for 15min, slowly homogenizing for 200 min by using a pine type Dounce homogenizer, stopping grinding when about 70% of the cells are broken, centrifuging the cell suspension at 700g for 10min, collecting the supernatant, and centrifuging at 120,000g for 30min to collect membrane sediment. Finally, the membrane proteins in the pellet were resuspended and solubilized using lysis buffer containing protease inhibitors, and collected by centrifugation at 12,000g for 10min for further experiments.

Protein interaction structure prediction

The researchers of the present invention selected PDB files referencing MDGA2 (AF-Q7Z 553-F1) suitable for the studies of the present invention in SWISS-MODEL protein structure data and the protein structure of MDGA (C-score= -4.62) was predicted by Zhang Lab_I-TASSER (MUNIVERSITY OF MICHIGAN). Protein-to-protein interactions were predicted by GRAMM: docking web server (http:// grad. Combbio. Ku. Edu/gramm, vakser laboratories), and finally structural visualization and molecular docking analysis was performed on three-dimensional protein models and their interactions using PYMOL.

Tail vein injection

Dissolving the synthesized polypeptide in physiological saline, adding dimethyl sulfoxide with concentration not exceeding 2% to assist in dissolving, and placing the fully dissolved polypeptide on ice for standby. When injecting the mice, the mice were ensured to be comfortably placed in the mouse tube and positioned with the tail vein syringe barrel while the tail of the mice was placed in the extrusion plate groove. Regulating light to make blood vessel clearly visible, then starting injection from bottom end of tail portion, slowly pushing polypeptide solution in the course of injection, observing health condition of mouse, stopping needle after injection so as to ensure normal absorption of medicine, after needle-drawing, using cotton swab to make compression hemostasis for 1min, continuously injecting for 5 days at the same time.

Animal behavioural experiments

All mice behavioural experiments were data collection and analytical processing using SmartVideoTrackingSoftware (Panlab, harvard apparatus). Animal behavioural experiments were performed between 9:00a.m. -18:00p.m. per day, with a light intensity of 650lux in the laboratory.

(1) Touching the mice 3 days before the experiment starts, touching one mouse each time once a day, gently grabbing the mouse with the tail, allowing the mouse to stay on the hand for 30 seconds, marking the mouse with a marker pen, placing the mouse in the palm after marking, grabbing the tail, and gently placing the mouse back into the mouse cage;

(2) On the day of the experiment, the experimental mice were transferred to the preparation room before the experiment, and were allowed to acclimate for 60min. The box and maze used for the experiment were cleaned with 70% alcohol before the experiment was ready to begin. After each experiment is finished, the box body and the maze are cleaned by using 70% alcohol so as to remove the excreted excrement and urine of the mice in the experiment process and eliminate the interference of the residual smell of the mice on the experiment.

1. Open field experiment (Open FIELD TEST)

The method is used for researching the autonomous locomotor ability and anxiety behavior of the mice, and is mainly based on the evasiveness of the mice to light and open space. In the open field experiment, the mice were placed in the center of a box (40 cm (L). Times.40 cm (W). Times.40 cm (H)), allowed to freely explore in the maze for 10min, and the Total movement distance (Total distance) of the mice and the time of middle area movement (TIME IN CENTER) were recorded.

2. Self-combing test (Self-grooming test)

The mice were placed in the center of the box (40 cm (L). Times.40 cm (W). Times.40 cm (H)), allowed to freely explore in the maze for 15min, and the entire procedure was recorded using a Smart3.0 video tracking system. The first 5min is considered to be the adaptation phase of the mice into the new environment, so the total number of times and total time each experimental mouse combed its own hair with limbs is counted by observing the video data of the mice for the last 10 min.

3. Free social connection (Social affiliation)

The experimental mice were placed in the center of a box (40 cm (L) ×40cm (W) ×40cm (H)), one wild-type mouse (stimulated mouse) of the same sex and age as the experimental mice was placed in a metal cage before the experiment was started, then the experimental mice were placed in the box, allowed to freely explore for 10min, and the whole process was recorded using a smart3.0 video tracking system. The time spent by mice in the range of 2cm around and around the metal cage was recorded.

4. Three-box social experiment (Three chamber sociability test)

The box (60 x 40 x 22cm deep) has two transparent baffles separating the left, middle and right chambers (20 x 40cm each) and each baffle has a small opening (5 x 5 cm) for access to each chamber. A fixed wire cylinder (8.5 cm in diameter) was placed in each of the left and right chambers. The test includes three phases, adaptation, social ability test, and social novelty test. In the adaptation phase, both the left and right cylinders were empty, and the test mice were placed in the middle, leaving them free to explore for 10min. In the social ability test, a sex-matched strange mouse (S1) was enclosed in an empty cylinder, which was free to explore for 10min. 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 by subtracting the time to explore E from the time to explore S1 and dividing by the time to explore S1 and E. During the social novelty test, another sex-matched strange mouse (S2) was placed in the empty canister, leaving the mouse free to explore for 10min. The time the mice explored S1 or S2 was measured and social novelty preference index (T (S2-S1)/T (S2+S1)) was calculated by subtracting the time to explore S1 from the time to explore S2 and dividing by the time to explore S2 and S1.

Experimental materials and methods

Protein extraction and immunoblotting (Western blot, WB)

1. Extraction of cell sample proteins:

(1) Cell collection, namely, discarding culture solution, washing cells for 2 times by using PBS, discarding the PBS, scraping the cells by using a cell scraper, collecting the cells into a centrifuge tube, centrifuging at 1000rpm for 5min, collecting the cells at the bottom of the tube, and discarding supernatant.

(2) Cell lysis by adding TNEN cell lysate containing cocktail protease inhibitor and phosphatase inhibitor (added before use) to the cell mass collected in (1), shaking on ice for 30min or slowly lysing overnight at 4 ℃ with a mute mixer, centrifuging at 12000rpm for 10min, collecting supernatant, and measuring protein concentration.

2. Extraction of tissue sample proteins:

Fresh tissue is taken out and immediately put into liquid nitrogen for freezing, and then stored in an ultralow temperature refrigerator at-80 ℃ for standby. The tissue was minced with surgical scissors before homogenization, then electrohomogenized with polytron in RIPA lysate, after sufficient lysis, centrifuged at 4℃and 12000rpm for 15min, the supernatant was collected and centrifuged once again to determine protein concentration.

3. Determination of protein concentration:

(1) Preparing BCA protein assay solution, and mixing solution A and solution B at a ratio of 50:1 for use;

(2) Taking BSA as a standard sample, and preparing a protein standard curve;

(3) Protein samples are respectively added into 96-well plates, TNEN/RIPA lysate is used as a blank control, and 3 holes are arranged in each group;

(4) 200 μl of the prepared protein assay solution is added into each hole, and the mixture is uniformly mixed and reacted for 30min in a 37 ℃ incubator;

(5) OD562 was measured using a microplate reader and the resulting values were substituted into a protein standard curve to calculate protein sample concentrations.

4.WB:

(1) Protein electrophoresis, namely taking 30-40 mug protein sample, adding 1/4 volume of 5 XSDS loading buffer solution, boiling for 3-5min at 100 ℃, and electrophoresis in Tris-Glycine electrophoresis buffer solution by using SDS-PAGE protein gel.

(2) And (3) carrying out electrophoresis transfer on proteins, namely precooling an electrophoresis transfer buffer solution at 4 ℃, cutting off PVDF membranes with proper sizes, soaking the PVDF membranes with methanol, soaking the PVDF membranes and filter paper in the electrophoresis transfer buffer solution for 10min, and simultaneously cutting the gel after electrophoresis and soaking the gel in the electrophoresis transfer buffer solution for 5min. And then, pasting PVDF film on the adhesive, covering filter paper on both sides, expelling bubbles, and placing the film in an electrotransport groove in the order of the film towards the positive electrode, and carrying out constant current electrotransport at 4 ℃ (300 mA,90 min).

(3) Antigen-antibody reaction:

a. Sealing, namely sealing 5% skimmed milk powder for 1h at room temperature;

b. Primary antibody reaction, namely diluting the primary antibody in a sealing liquid in a proper proportion, and standing at 4 ℃ for night;

c. the secondary antibody reaction, PBST washing three times, 10min each time, the primary antibody is diluted in a sealing liquid according to a proper proportion, and the primary antibody is incubated for 1h at room temperature.

(4) ECL detection:

PBST is washed three times for 10-15min each time, the solution A and the solution B in the ECL kit are mixed at a ratio of 1:1 (V/V), ECL is dripped into a darkroom to the surface of the membrane, the membrane is incubated for 5min and then exposed, and the exposure time is adjusted according to the fluorescence intensity.

Immunoprecipitation (IP) or co-immunoprecipitation (CoIP)

(1) Protein samples were collected, 10. Mu.l Protein-ASepharose was added to the Protein samples and incubated at 4℃for 1-2h with rotation.

(2) Centrifuge at 5000rpm for 2min at 4 ℃. The supernatant was transferred to a new EP tube, made up to 700ul with 0.5% TNEN lysate, 20 ul Protein-GSephase and corresponding antibodies were added and incubated overnight at 4℃with spin.

(3) Centrifuge at 4 ℃,5000rpm for 2min, after aspiration of the supernatant, add pre-chilled 1% TNEN solution, incubate at 4 ℃ for 8min with spin.

(4) The step (3) was repeated three times, and the supernatant was carefully blotted dry with a microinjector for the last time, then 20. Mu.L of 2 Xloading buffer was added, and after mixing well, the sample was boiled at 100℃for 5min. The resulting samples were used for immunoblot analysis.

Immunofluorescence

(1) The treated cover glass is placed into a 24-well plate (one for each well), uniformly divided into cells with a certain density (the density is 5% -10%), and cultured for 24 hours.

(2) The corresponding plasmid was transferred according to lipo2000 transfection method.

(3) After 24h of transfection, the medium was aspirated and the cells were washed 3 times with pre-chilled PBS. The PBS was blotted off and fixed with 4% paraformaldehyde for 10min at room temperature.

(4) 0.1% Triton X00-PBS was penetrated for 5-10min at room temperature (time should be strictly controlled here).

(5) Blocking with 3% BSA-PBS was performed for 1h.

(6) Incubate overnight with different primary antibodies (1:100 diluted in 3% BSA-PBS) at 4 ℃.

(7) The plates were left to stand without shaking when washed 4 times with PBS for 5min each.

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

(9) If necessary, DAPI was incubated at room temperature for 3-5min.

(10) PBS was washed 4 times, each for 5min.

(11) The tablets were blocked overnight at room temperature, observed under confocal microscopy after the tablet had dried and photographed.

Protein interaction structure prediction

The researchers of the present invention selected PDB files in ALPHAFOLD protein structure data that reference protein structures of MDGA (AF-Q7Z 553-F1) and TrkB (AF-Q16620-F1) suitable for the studies of the present invention. Protein-to-protein interactions were predicted by GRAMM: docking web server (http:// grad. Combbio. Ku. Edu/gramm, vakser laboratories), and finally structural visualization and molecular docking analysis was performed on three-dimensional protein models and their interactions using PYMOL.

Brain stereotactic injection

After the mice were anesthetized, the sleeves were embedded and fixed in the hippocampal locations on both sides, respectively, with embedded coordinates of AP, -2.0mm, L, + -2.3 mm, DV, -2.0mm. After 3 days of recovery of the mice, the synthesized polypeptide was dissolved in physiological saline at a concentration of 1. Mu.g/. Mu.L, and 1. Mu.L of the polypeptide was injected into the hippocampus of the mice via a cannula over a period of 5min, once daily for 7 consecutive days.

Animal behavioural experiments

All mice behavioural experiments were data collection and analytical processing using SmartVideoTrackingSoftware (Panlab, harvard apparatus). Animal behavioural experiments were performed between 9:00a.m. -18:00p.m. per day, with a light intensity of 650lux in the laboratory.

(1) Touching the mice 3 days before the experiment starts, touching one mouse each time once a day, gently grabbing the mouse with the tail, allowing the mouse to stay on the hand for 30 seconds, marking the mouse with a marker pen, placing the mouse in the palm after marking, grabbing the tail, and gently placing the mouse back into the mouse cage;

(2) On the day of the experiment, the experimental mice were transferred to the preparation room before the experiment, and were allowed to acclimate for 60min. The box and maze used for the experiment were cleaned with 70% alcohol before the experiment was ready to begin. After each experiment is finished, the box body and the maze are cleaned by using 70% alcohol so as to remove the excreted excrement and urine of the mice in the experiment process and eliminate the interference of the residual smell of the mice on the experiment.

1. Free social connection (Social affiliation)

The experimental mice were placed in the center of a box (40 cm (L) ×40cm (W) ×40cm (H)), one wild-type mouse (stimulated mouse) of the same sex and age as the experimental mice was placed in a metal cage before the experiment was started, then the experimental mice were placed in the box, allowed to freely explore for 10min, and the whole process was recorded using a smart3.0 video tracking system. The time spent by mice in the range of 2cm around and around the metal cage was recorded.

2. Three-box social experiment (Three chamber sociability test)

The box (60 x 40 x 22cm deep) has two transparent baffles separating the left, middle and right chambers (20 x 40cm each) and each baffle has a small opening (5 x 5 cm) for access to each chamber. A fixed wire cylinder (8.5 cm in diameter) was placed in each of the left and right chambers. The test includes three phases, adaptation, social ability test, and social novelty test. In the adaptation phase, both the left and right cylinders were empty, and the test mice were placed in the middle, leaving them free to explore for 10min. In the social ability test, a sex-matched strange mouse (S1) was enclosed in an empty cylinder, which was free to explore for 10min. 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 by subtracting the time to explore E from the time to explore S1 and dividing by the time to explore S1 and E. During the social novelty test, another sex-matched strange mouse (S2) was placed in the empty canister, leaving the mouse free to explore for 10min. The time the mice explored S1 or S2 was measured and social novelty preference index (T (S2-S1)/T (S2+S1)) was calculated by subtracting the time to explore S1 from the time to explore S2 and dividing by the time to explore S2 and S1.

Materials and reagents

Example 1 MDGA2 heterozygous deletions promote BDNF/TrkB Activity

First, the present investigators detected an increase in BNDF protein levels in synaptosomes of MDGA2 heterozygous deletion mice (as shown in FIG. 1). BDNF is a receptor for TrkB, and BDNF/TrkB binding activates the downstream mTOR-Akt signaling pathway. Thus, the present inventors have further examined that the phosphorylation levels of both TrkB, mTOR, akt, S and CaMK2 proteins in MDGA-heterozygous mice are increased (as shown in FIG. 1), and that these increased levels of protein phosphorylation represent activation of TrkB and downstream mTOR-Akt signaling, indicating that the MDGA2 deletion promotes BDNF/TrkB and its downstream signaling pathway activity.

Example 2 analysis of MDGA2 interaction with TrkB and determination of MDGA core sequence

The researchers of the present invention predicted that there was an interaction relationship between the MAM region of MDGA and TrkB by protein-protein interaction analysis (fig. 2G). To further confirm, the present inventors constructed expression plasmids of GFP modified human full length MDGA2 (amino acid sequence shown as SEQ ID NO.01, cDNA sequence shown as SEQ ID NO. 03), human MDGA2 deleted of MAM region (aa 746-921), and human MDGA2 deleted of MAM amino terminus (M1, aa 746-833) and MAM carboxy terminus (M2, aa 834-921), respectively (see Table 2 and FIGS. 2A and B). These expression plasmids were co-transfected into HEK293T cells with Flag-modified human full-length TrkB expression plasmids, respectively, and their interactions were analyzed by co-immunoprecipitation-immunoblotting. As a result, full length MDGA2 and M2 deleted MDGA were able to interact with TrkB, whereas MAM deleted MDGA2 and M1 deleted were unable to interact with TrkB, as shown in fig. 2B. These results indicate that MDGA2 interacts with TrkB through the M1 region.

The present inventors further divided the M1 region of MDGA2 into two fragments, L1 (aa 746-766) and L2 (aa 767-833), constructed GST-modified L1 and L2 expression plasmids, respectively (see Table 2 and FIG. 2C, D), and after incubation of GST-fused L1 and L2 polypeptides expressed in cells with human TrkB protein, respectively, pulled down by Glutathione sepharose B, showing that only L2 is able to bind to the TrkB protein and pull it down (FIG. 2D).

Furthermore, the present inventors have divided the L2 fragment into two segments, P1 (aa 787-811, SEQ ID NO. 06) and P2 (aa 812-833, SEQ ID NO. 07), and synthesized polypeptides and labeled with biotins, respectively (see Table 2 and FIGS. 2C and E). The two polypeptides and the biotin-labeled disordered polypeptide were injected into the hippocampus of mice respectively, and after 24h, the tissue of the hippocampus was taken for cleavage, and then the biotin-bound protein was pulled down by STREPTAVIDIN AGAROSE, and only the biotin-labeled P2 polypeptide was found to pull down the TrkB protein in the brains of mice (FIG. 2E). In addition, co-localization was found between FITC-labeled P2 polypeptide (green) and SH-SY5Y cells after incubation with TrkB antibody for immunofluorescent staining (red). The above results indicate that the 812-833 sequence region of MDGA is the critical region for interaction with TrkB.

TABLE 2 truncations of human full length MDGA2

Example 3 inhibition of BDNF binding to TrkB in cells by MDGA-based Polypeptides

To further determine whether the core region polypeptide of MDGA2 that interacted with TrkB would affect the function of TrkB, the present inventors have artificially synthesized the P2 polypeptide of human MDGA (KARLLSPVFSIAPKNPYGPTNT; SEQ ID NO. 07) and the out-of-order polypeptide (PYPPNTTLKSLPVISFNGKRAA; SEQ ID NO. 05). The polypeptides are synthesized by biological companies. The above polypeptides were added to MDGA heterozygous deleted neurons overexpressing TrkB-Flag at different concentrations (+0.1. Mu.M; +: 1. Mu.M), and after 16h, BDNF binding to TrkB-Flag was detected by immunoprecipitation-immunoblotting, and it was found that the P2 polypeptide was able to inhibit BDNF binding to TrkB in a concentration-dependent manner (as shown in FIG. 3), indicating that the P2 polypeptide had an effect of inhibiting BDNF/TrkB activity.

EXAMPLE 4 evaluation of therapeutic efficacy of MDGA-based Polypeptides in Autism (ASD) model mice

MDGA2 is believed to be a susceptibility gene associated with ASD disease, and insufficient MDGA2 protein results in increased excitatory synaptic transmission, and mice exhibit anxiety, social disorders and notch behaviors, so MDGA heterozygous deletion mice are widely used as a model of typical autism.

The above experimental results have shown that BDNF/TrkB activity is abnormally increased in MDGA heterozygous mice, while the P2 polypeptide can inhibit the combination of BDNF and TrkB, and can be used as a medicament for treating diseases. Thus, this example further evaluates the therapeutic effect of MDGA-based polypeptides on autism model mice.

The researchers of the present invention synthesized human P2 polypeptide (KARLLSPVFSIAPKNPYGPTNT; SEQ ID NO. 07) and disordered polypeptide (PYPPNTTLKSLPVISFNGKRAA; SEQ ID NO. 05) of MDGA. The polypeptides are synthesized by biological companies. In Mdga/^+/- mice of 1 month old or littermate control WT mice, mice were treated continuously for one week at 1 μg/day by bilateral hippocampal stereotactic injection, and after one week of rest, were subjected to behavioral detection and biochemical analysis.

The mouse behavioral test results showed that Mdga 2. 2 ^+/- mice given the control polypeptide showed significant social impairment in common social experiments (fig. 4A) and also social interest and impairment of social preference in three-box social experiments (fig. 4B) relative to wild-type mice. Whereas social disorders in Mdga2 ^+/- mice were significantly improved upon the MDGA-based polypeptide administration (fig. 4A, B).

By immunoblotting experiments after brain lysis of experimental mice, it was found that the abnormally elevated TrkB-mTOR-Akt signaling pathway activity in Mdga2, 2 ^+/- mice was significantly restored to normal levels after treatment with the P2 polypeptide (fig. 4C). Furthermore, using co-immunoprecipitation-immunoblotting experiments, it was found that BDNF binding to TrkB was increased in Mdga2 ^+/- mice, whereas BDNF binding to TrkB was reduced following treatment with P2 polypeptide (fig. 4D). The above results demonstrate that MDGA 2-based polypeptides can significantly improve disease-related phenotypes in autistic mice by targeting TrkB.

In addition to ASD caused by MDGA loss of 2, aberrant activation of the BDNF/TrkB signaling pathway has also been found to be involved in other diseases, such as BDNF/TrkB signaling pathway associated with drug addiction, inhibition of BDNF/TrkB can improve cocaine exploration behavior and withdrawal response in animals, BDNF/TrkB signaling pathway in the dorsal horn of the spinal cord is involved in neuropathic pain, and use of TrkB inhibitors can alleviate neuropathic pain, and elevated levels of BDNF in serum in most epileptic patients, while significant increases in BDNF and TrkB are found in temporal lobes and hippocampal regions in some epileptic animal models. By deleting or blocking TrkB, epileptogenesis in these mice can be improved. In addition, there have been studies to find that BDNF/TrkB signaling can promote cancerous, invasive and metastatic events in cells and may be responsible for the development of chemotherapy resistance. Thus, the polypeptides of the invention that are capable of targeted modulation of MDGA's 2 metabolism, thereby increasing their expression levels, are also useful in the treatment of other MDGA-inactivating-induced neurological disorders (e.g., autism, epilepsy) and tumors (e.g., brain tumors).

The foregoing description is only illustrative of the preferred embodiments of the present invention and is not to be construed as limiting the scope of the invention, i.e., the invention is not to be limited to the details of the invention.

Claims (12)

1. A TrkB targeting polypeptide, which is characterized in that the TrkB targeting polypeptide consists of 21-55 continuous amino acids of MDGA protein and comprises KARLLSPVFSIAPKNPYGPTN (SEQ ID NO. 08).

2. A TrkB targeting polypeptide according to claim 1 in which the amino acid sequence comprises KARLLSPVFSIAPKNPYGPTNX (SEQ ID NO. 10) wherein X is T or S.

3. A TrkB targeting polypeptide according to claim 2 having an amino acid sequence of KARLLSPVFSIAPKNPYGPTNT (SEQ ID NO. 07) or KARLLSPVFSIAPKNPYGPTNS (SEQ ID NO. 09).

4. A conjugate comprising a TrkB targeting polypeptide according to any one of claims 1 to 3 and a modifying moiety selected from a further polypeptide selected from CPP, a targeting moiety and/or a protein tag, a detectable label or any combination thereof.

5. A fusion protein comprising the TrkB targeting polypeptide according to any one of claims 1 to 3 and a further polypeptide selected from the group consisting of CPP, targeting moiety, protein tag and any combination thereof.

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

7. A vector comprising the isolated nucleic acid molecule according to claim 6.

8. A host cell comprising the isolated nucleic acid molecule of claim 6 or the vector of claim 7.

9. A method of producing a TrkB targeting polypeptide according to any one of claims 1 to 3 or a fusion protein according to claim 5 comprising culturing a host cell according to claim 8 under suitable conditions and recovering said TrkB targeting polypeptide or said fusion protein from the culture of said host cell.

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

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

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