Method for treating depression and pharmaceutical composition

Abstract

Provided is a method for treating depression in a patient by inhibiting Kir4.1 from binding to α-dystrobrevin. Further provided is a pharmaceutical composition for treating depression, comprising an agent for inhibiting Kir4.1 from binding to α-dystrobrevin.

Description

Treatment method and pharmaceutical composition for depression

[0001] The present application claims priority to the Chinese patent application No. 202411281349.7, filed on September 12, 2024, with the title of “Treatment method and pharmaceutical composition for depression”, the whole content of which is incorporated herein by reference. TECHNICAL FIELD

[0002] The present application relates to the field of disease treatment and medicine. In particular, the present application relates to the treatment of depression and pharmaceutical compositions for treating depression and methods of preparing the same. BACKGROUND

[0003] Depression is a common mental disorder, mainly manifested as long-term low mood, lack of interest in things, and severe depression can lead to suicide, causing huge losses and burdens to families and society.

[0004] As a mental disease, depression involves abnormal neuronal activity in specific brain regions and neural pathways. Recent studies have shown that dysfunction of glial cells is also an important cause of depression. Studies on various animal models of depression and patients with depression have shown that the activity of the lateral habenula increases.

[0005] Inward rectifier-type potassium channels (Kir) are potassium channels that are superactivated. There are seven protein family members of inward rectifier-type potassium channels (Kir1-Kir7) that have been reported. Kir channels of the same type can be divided into various subtypes due to splicing variance, and are distributed in various tissues and organs such as the heart, kidney, and nervous system. Kir4.1 (also known as potassium voltage-gated channel subfamily J member 10, Kcnj10) is one of the family members of inward rectifier-type potassium channels.

[0006] The inwardly rectifying potassium ion channel Kir4.1 of the astrocyte of the lateral habenula is increased in expression in depressive state (Li et al. 2013, Science.), and is distributed on the endfoot terminal of the astrocyte surrounding the neuron of the lateral habenula (Cui et al. 2018, Nature). This distribution structure forms a narrow space outside the cell body of the neuron, and Kir4.1 can buffer the potassium ion concentration in the space when the neuron is in high frequency activity, thereby regulating the membrane potential and firing pattern of the neuron. By reducing the number of Kir4.1 protein expression in the lateral habenula, the number of neuron cluster discharges can be significantly reduced, thereby reducing the depressive behavior phenotype. According to this theory, the distribution pattern and number increase of Kir4.1 are indispensable in the pathogenesis of depression.

[0007] However, the molecular mechanism of the distribution of Kir4.1 on the endfoot terminal of the astrocyte is still unclear in the art, and therefore needs to be further understood in order to design and develop new and better methods and drugs for treating depression, for example, with faster onset of action and safer dosage. SUMMARY

[0008] The present inventors have found for the first time that the C-terminal PDZ binding sequence of Kir4.1 protein is a necessary structure for maintaining the correct intracellular localization of Kir4.1 protein in the astrocyte of the lateral habenula, and that this sequence binds to the PDZ domain of the scaffold protein α-syntrophin, which plays a crucial role in the regulation of depression of Kir4.1 in the lateral habenula. The present inventors have thus discovered and demonstrated that an agent for reducing the function of Kir4.1 in the lateral habenula by inhibiting the binding of the PDZ binding sequence at the C-terminus of Kir4.1 to α-syntrophin can have an effect of inhibiting depression, thereby providing a method and a drug for treating (inhibiting) depression by inhibiting the activity of Kir4.1.

[0009] In particular, the present application provides a method for treating depression in a patient by inhibiting the binding of Kir4.1 to α-syntrophin. The present application also provides the use of an agent for inhibiting the binding of Kir4.1 to α-syntrophin in the manufacture of a medicament for treating depression in a patient.

[0010] "Kir4.1", or "inward rectifier potassium channel Kir4.1", also known as potassium voltage-gated channel subfamily J member 10 (Kcnj10), is one of the family members of inward rectifier potassium channels. Kir4.1 in glial cells can allow potassium ions to pass through the cell membrane, transport excess potassium ions in the extracellular environment to buffer the extracellular environment, control the level of resting membrane potential, maintain the homeostasis of the nervous system, and maintain the normal physiological activity of the nervous system by regulating the potassium ion concentration of the extracellular fluid around the nerve cells. In mammals, the protein sequence and the coding nucleic acid sequence of Kir4.1 are very conservative. The coding gene of human Kir4.1 protein (NP_002232) is Kcnj10 (Ensembl: ENSG00000177807). The human Kir4.1 protein has an amino acid sequence as shown in SEQ ID NO: 1. The coding gene of mouse Kir4.1 protein (NP_001034573) is Kcnj10 (Ensembl: ENSMSUSG00000044708). The mouse Kir4.1 protein has an amino acid sequence as shown in SEQ ID NO: 2

[0011] The patient in need of the methods and drugs (pharmaceutical compositions) described herein is a mammal. The patient can also be a human or a non-human primate such as a monkey. The mammal can be another animal, for example, a rat, a mouse, a rabbit, a pig, a dog, etc. The mammal can be a domesticated animal, for example, a cat or a dog.

[0012] In this context, the agents that inhibit the binding of Kir4.1 to dystrophin-associated protein alpha refer to agents that can reduce the binding of Kir4.1 to dystrophin-associated protein alpha in vivo or in vitro, in particular agents that can reduce the binding of Kir4.1 to dystrophin-associated protein alpha in the astrocytes of the lateral habenula. These inhibitors include compounds, complexes or mixtures that can reduce or lose the activity of Kir4.1 binding to dystrophin-associated protein alpha, or are compounds, complexes or mixtures that can reduce the amount of Kir4.1 binding to dystrophin-associated protein alpha.

[0013] The agents that inhibit the binding of Kir4.1 to dystrophin-associated protein alpha include small molecule compounds or complexes, or large molecule active ingredients such as proteins, nucleic acids, etc. The proteins or nucleic acids can be delivered to the target tissues or cells to play a role by means of techniques known in the art, for example, by being combined with suitable expression vectors.

[0014] In one aspect of the present application, the agent that inhibits the binding of Kir4.1 to dystrophin-associated protein alpha is an agent that inhibits the binding of the C-terminus of Kir4.1 to the PDZ domain of dystrophin-associated protein alpha.

[0015] In yet another aspect of the present application, the agent that inhibits the binding of the C-terminus of Kir4.1 to the PDZ domain of dystrophin-associated protein alpha is a competitive inhibitor of the binding of the C-terminus of Kir4.1 to the PDZ domain of dystrophin-associated protein alpha. In one embodiment, the competitive inhibitor is a mutant or fragment of Kir4.1 that lacks potassium ion channel activity but retains the ability to bind to the PDZ domain of dystrophin-associated protein alpha, or a nucleic acid encoding the mutant or fragment of Kir4.1.

[0016] Potassium ion channel activity refers to the activity of allowing potassium ions to pass through the cell membrane. Kir4.1 transports excess extracellular potassium ions to buffer the extracellular environment by regulating the potassium ion concentration of the extracellular fluid around the nerve cells, thereby controlling the resting membrane potential level and affecting the physiological activity of the nervous system.

[0017] The main function of the PDZ domain of dystrophin-associated protein alpha is to mediate the aggregation of proteins on the membrane by binding to the C-terminus of the target protein in the groove formed by the first alpha helix and the second beta sheet of the PDZ domain. The PDZ domain has the function of connecting scaffolding and adaptor proteins and other signaling pathways. The connection function of the PDZ domain comes from its inherent plasticity, which can change the function and binding mode by conformational change.

[0018] In yet another aspect of the present application, the competitive inhibitor of the binding of the C-terminus of Kir4.1 to the PDZ domain of dystrophin-associated protein alpha is a C-terminal fragment of Kir4.1 or a fusion protein thereof, or a nucleic acid encoding and expressing the C-terminal fragment or the fusion protein thereof. In one embodiment, the C-terminal fragment of Kir4.1 is a polypeptide fragment comprising at least 3-5 amino acids of the C-terminus of Kir4.1, preferably a polypeptide fragment having 5-30 amino acids, such as a polypeptide fragment having 5-15 amino acids. In another embodiment of the present application, the C-terminal fragment of Kir4.1 refers to a fragment having or comprising the amino acids 377-379 (i.e., SNV) of Kir4.1 with the amino acid sequence as shown in SEQ ID NO: 1.

[0019] In yet another aspect of the application, the fusion protein of the C-terminal fragment of Kir4.1 is a fusion protein of the C-terminal fragment of Kir4.1 and a cell-penetrating peptide (CPP). Cell-penetrating peptides include human immunodeficiency virus-1 transcription activator (HIV-1 TAT), such as TAT(48-60) HIV-1 protein, i.e., GRKKRRQRRRPPQQ, or TAT(47-57) HIV-1 protein, i.e., YGRKKRRQRRR, and sl Igl-1 homology domain, i.e., RVIRVWFQNKRCKDKK, transportan, i.e., GWTLNSAGYLLGKINLKALAALAKKIL, or P-alpha gp41-SV40, i.e., GALFLAFLAAALSLMGLWSQPKKKRRV, and the like.

[0020] In yet another aspect of the application, the fusion protein of the C-terminal fragment of Kir4.1 further comprises a polypeptide stability modification, such as a PEG modification or a somatostatin side chain modification.

[0021] In one embodiment of the application, the competitive inhibitor of the C-terminus of Kir4.1 binding to the PDZ domain of dystrophin-associated protein alpha is a C-terminal fragment of Kir4.1 having the following amino acid sequence:

[0022] YGRKKRRQRRRRISNV (SEQ ID NO: 3);

[0023] YGRKKRRQRRRALSVRISNV (SEQ ID NO: 4);

[0024] YGRKKRRQRRREGSALSVRISNV (SEQ ID NO: 5);

[0025] YGRKKRRQRRRAEKEGSALSVRISNV (SEQ ID NO: 6); or

[0026] YGRKKRRQRRRAEK(AEEAAEEAE-C18 diacid)EGSALSVRISNV.

[0027] In one aspect of the present application, the agent that inhibits the binding of Kir4.1 to dystrophin-associated protein a is an interfering RNA or a precursor thereof against the nucleotide sequence encoding the C-terminal end of Kir4.1. RNA interference (RNAi) is a process by which double-stranded RNA (dsRNA) induces the degradation of homologous mRNA, thereby reducing or eliminating the expression of the target gene. In the present application, the interfering RNA can include small interfering RNA (siRNA), short hairpin RNA (shRNA), and / or micro RNA (miRNA). One way of administering interfering RNA in vivo is by administering a precursor of siRNA, such as a short hairpin RNA that includes two short inverted repeat sequences. The siRNA sequence is cloned into a plasmid vector as a "short hairpin". When introduced into an animal, the hairpin sequence is expressed as a "double-stranded RNA", which is processed by Dicer in the cell to generate the corresponding siRNA, which exerts the RNAi effect.

[0028] In yet another aspect of the present application, the interfering RNA or a precursor thereof that can be used in the present application has a sequence that is identical or complementary, or 90% or more identical or complementary to a fragment of the mRNA encoding the C-terminal end of Kir4.1.

[0029] In yet another aspect of the present application, the agent that inhibits the binding of Kir4.1 to dystrophin-associated protein a is a specific antibody that recognizes and binds to the C-terminal fragment of Kir4.1, including a polyclonal antibody or a monoclonal antibody.

[0030] In the present application, the depression can particularly refer to "lateral habenula-mediated depression". Abnormal firing of neurons of the lateral habenula, particularly abnormal firing of burst discharges, plays an important role in the development of depression. Kir4.1 in the astrocytes of the lateral habenula is a crucial regulator of depression. The methods and drugs provided in the present application are particularly suitable for use in patients with depression that do not respond to other antidepressant methods and drugs.

[0031] In one aspect of the application, the methods and medicaments provided herein are methods and medicaments that are locally effective in the lateral habenula. For drugs used in neural tissue, particularly brain neural tissue, such as the lateral habenula, it is beneficial to limit the effect of the drug to the target tissue. Methods or medicaments for the LHb require consideration of whether the method or medicament is capable of exerting the effectiveness of the drug in the LHb, including whether the drug is able to reach the LHb, and whether an effective concentration is reached in the LHb, etc. In the present application, the medicament can be in a dosage form that is locally administered to the lateral habenula. The local administration of the drug can be used to limit the effect of the drug to the target tissue, such as by formulating the drug in a dosage form that is implanted through a cannula for local administration to the lateral habenula. For example, the drug can be formulated in a dosage form that is released slowly after implantation in the tissue, etc. The drug can also be formulated in a tissue-specific targeted drug delivery system. For example, a complex molecule can be formed by linking a small molecule compound or a biologically active molecule (a nucleic acid such as a protein-encoding DNA or mRNA molecule, a protein such as an antibody, etc.) that has the function of inhibiting clustered discharges to an antibody that is capable of specifically binding to a protein that is specifically expressed in the lateral habenula.

[0032] The present application also provides a pharmaceutical composition for treating depression. The present application provides a new pharmaceutical composition for treating depression, which comprises a therapeutically effective amount of a preparation that inhibits the binding of Kir4.1 to dystrophin-associated protein alpha, as defined above.

[0033] The present application also provides an isolated polypeptide comprising at least 3-5 amino acids of the C-terminal end of Kir4.1, preferably 5-30 amino acids of the C-terminal end of Kir4.1, such as 5-15 amino acids of the C-terminal end of Kir4.1, of the amino acid sequence shown in SEQ ID NO: 1.

[0034] In yet another aspect of the present application, the isolated polypeptide further comprises a cell-penetrating peptide; and the cell-penetrating peptide forms a fusion protein with the aforementioned polypeptide comprising the C-terminal amino acids of Kir4.1. Preferably, the cell-penetrating peptide is a type 1 human immunodeficiency virus transcriptional activator TAT, such as TAT(48-60)HIV-1 protein or TAT(47-57)HIV-1 protein, sl Igl-1 homology domain, transporter glycoptide, or P-alpha gp41-SV40, etc.

[0035] In yet another aspect of the present application, the isolated polypeptide further has a polypeptide stability modification, such as a PEG modification or a somatostatin side chain modification.

[0036] In yet another aspect of the present application, the isolated polypeptide has the following amino acid sequence:

[0037] YGRKKRRQRRRAEKEGSALSVRISNV (SEQ ID NO: 6); or

[0038] YGRKKRRQRRRAEKEGSALSVRISNV (SEQ ID NO: 6); or

[0039] YGRKKRRQRRRAEKEGSALSVRISNV (SEQ ID NO: 6); or

[0040] YGRKKRRQRRRAEKEGSALSVRISNV (SEQ ID NO: 6); or

[0041] YGRKKRRQRRRAEKEGSALSVRISNV (SEQ ID NO: 6); or

[0042] The present application also provides an isolated nucleic acid comprising a nucleotide sequence encoding an isolated polypeptide as previously described. The isolated polypeptide comprises at least 3-5 amino acids of the C-terminus of Kir4.1 having an amino acid sequence as set forth in SEQ ID NO: 1, and preferably further comprises a cell-penetrating peptide, and optionally further has a polypeptide stability modification.

[0043] As used herein, "nucleic acid" includes "polynucleotide," "oligonucleotide," and "nucleic acid molecule," and generally means a polymer of DNA or RNA, which can be single-stranded or double-stranded, synthesized or obtained (e.g., isolated and / or purified) from a natural source, which can contain natural, non-natural or altered nucleotides, and which can contain natural, non-natural or altered internucleotide linkages, such as phosphoramidate or phosphorothioate linkages in place of the phosphodiester linkages that exist between the nucleotides of an unmodified oligonucleotide. In some embodiments, the nucleic acid does not comprise any insertions, deletions, inversions, and / or substitutions. However, in some cases, a nucleic acid comprising one or more insertions, deletions, inversions, and / or substitutions can be suitable, as discussed herein. In some embodiments, the nucleic acid can encode an additional amino acid sequence that does not affect the function of the CAR and can or can not be translated following expression of the nucleic acid by a host cell.

[0044] Embodiments of the present application also provide an isolated or purified nucleic acid comprising a nucleotide sequence complementary to or hybridizing under stringent conditions to a nucleotide sequence of any of the nucleic acids described herein.

[0045] In embodiments of the application, the nucleic acids of the application can be incorporated into a recombinant expression vector. In this regard, embodiments of the application provide a recombinant expression vector comprising any of the nucleic acids of the application. For purposes herein, the term "recombinant expression vector" means a genetically modified oligonucleotide or polynucleotide construct that permits a host cell to express an mRNA, protein, polypeptide, or peptide when the construct comprises a nucleotide sequence encoding the mRNA, protein, polypeptide, or peptide and the vector is contacted with the cell under conditions sufficient for the mRNA, protein, polypeptide, or peptide to be expressed within the cell. The vectors of the application are not entirely naturally occurring. However, a portion of the vector can be naturally occurring. The recombinant expression vectors of the application can comprise any type of nucleotide, including but not limited to DNA and RNA, which can be single-stranded or double-stranded, partially synthesized or obtained from a natural source, and which can contain natural, non-natural or altered nucleotides.

[0046] In embodiments, the recombinant expression vectors of the application can be any suitable recombinant expression vector and can be used to transform or transfect any suitable host cell. Suitable vectors of the application include those designed for propagation and amplification or for expression or both, such as plasmids and viruses. The recombinant expression vectors can be viral vectors, such as retroviral vectors or lentiviral vectors.

[0047] The recombinant expression vectors can comprise a natural or non-natural promoter operably linked to a nucleotide sequence encoding a CAR, including functional portions and functional variants thereof, or to a nucleotide sequence that is complementary to or hybridizes to a nucleotide sequence encoding a polypeptide or protein of interest.

[0048] The formulations for inhibiting the binding of Kir4.1 to dystroglycan protein provided by the present application include small molecule compounds or complexes, or macromolecular active ingredients such as proteins, nucleic acids, etc., which can be formulated using methods known to those skilled in the art for preparing pharmaceutical compositions. When the compounds and complexes are used as active ingredients, they can be used in the form of solids, solutions, emulsions, dispersions, micelles, liposomes, etc., wherein the resulting formulations contain one or more compounds and complexes of the present application as active ingredients, and are mixed with organic or inorganic carriers or excipients suitable for enteral or parenteral application. In addition, adjuvants, stabilizers, thickeners, colorants, and flavorings can be used. The above pharmaceutical compositions can be prepared into dosage forms suitable for oral administration, such as tablets, pills, lozenges, aqueous or oily suspensions, dispersed powders or granules, emulsions, hard or soft capsules, or syrups. The oral formulations can be encapsulated to slow down decomposition and absorption in the gastrointestinal tract according to techniques known in the art, thereby providing a sustained effect for a longer period of time. The formulations can also be in the form of sterile injectable solutions or suspensions. The suspensions can be prepared using dispersing or wetting agents and suspending agents according to known methods.

[0049] The formulations provided herein for inhibiting the binding of Kir4.1 to dystrophin-associated protein alpha are mixtures of the macromolecular active ingredient, e.g., a protein, nucleic acid, etc., and a pharmaceutically acceptable carrier, which can be administered by dialysis of an aqueous solution of the pharmaceutical formulation containing the active ingredient. A typical pharmaceutical formulation comprises an effective amount of the subject matter, e.g., about 0.1 to 100 mg / ml, and a suitable amount of carrier. The pharmaceutical formulation can be administered parenterally. Preferably, it is administered intraperitoneally, subcutaneously, intravenously, or nasally.

[0050] Typically, a solution of the polypeptide as the active substance is dialyzed against the buffer to be used in the pharmaceutical composition, and the final concentration of the desired protein is adjusted by concentration or dilution.

[0051] Such pharmaceutical compositions can be used for injection or infusion, preferably intraperitoneal, subcutaneous, intravenous injection or nasal administration, and contain an effective amount of the polypeptide and pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and / or carriers. Such compositions include diluents of various buffer constituents (e.g., arginine, acetate, phosphate), pH and ionic strength, additives (e.g., detergents and solubilizers (e.g., Tween(TM) 80), antioxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (Timersol(TM), benzyl alcohol) and bulking substances (e.g., sucrose, mannitol), which are added to particulate formulations of polymeric compounds, e.g., polylactic acid, polyglycolic acid, etc., or to liposomes. Such compositions can affect the physical state, stability and clearance rate of the released polypeptide.

[0052] An "effective amount" of the formulations provided herein is an amount that, when administered to a patient in need, produces the desired therapeutic and / or prophylactic effect without causing any undesirable side effects. The effective dose will depend on many factors including, but not limited to, the gender, body weight, and age of the subject, the severity of the inability to regulate blood glucose, the route and bioavailability of administration, the pharmacokinetic profile of the fusion protein, potency.

[0053] The formulations of the present application can be administered to a patient in a single or multiple doses. For example, a patient can be treated with a dose of the polypeptide or nucleic acid of the present application in the range of 0.01 to 100 μmol, preferably 0.1 to 10 μmol per kilogram per week. The treatment can be administered over a period of time, e.g., for a week to about 3 months or more. The administration can be by subcutaneous, intravenous, intraperitoneal bolus or infusion on a weekly basis.

[0054] In another aspect of the present application, based on the finding and demonstration that the C-terminal PDZ binding sequence of Kir4.1 protein is the essential structure for its proper intracellular localization in the astrocytes of the lateral habenula and that this sequence binds to the PDZ domain of the scaffold protein dystrobrevin alpha, which is essential for the regulation of Kir4.1 in the lateral habenula for depression, there are also provided methods and kits for diagnosing depression by detecting Kir4.1 protein and / or dystrobrevin alpha, particularly methods and kits for diagnosing depression by detecting the C-terminal PDZ binding sequence of Kir4.1 protein and / or the PDZ domain of dystrobrevin alpha.

[0055] In one embodiment, there are provided kits or devices (including gene chips) for diagnosing depression, which comprise reagents for detecting the PDZ domain of dystrobrevin alpha and / or the C-terminal PDZ binding sequence of Kir4.1 protein.

[0056] In another aspect of the present application, the kits or devices comprise reagents for detecting the coding sequence of the PDZ domain of dystrobrevin alpha and / or the C-terminal PDZ binding sequence of Kir4.1 protein. In the present application, there are also provided uses of reagents for detecting the coding sequence of the PDZ domain of dystrobrevin alpha and / or the C-terminal PDZ binding sequence of Kir4.1 protein in the manufacture of the kits or devices for diagnosing depression according to the present aspect.

[0057] The term

[0058] In the present invention, "treatment" includes: ameliorating, alleviating, reducing or preventing the ongoing course or result of a symptom associated with depression; improving the ongoing course or result of a symptom associated with depression; normalizing the ongoing course or result of an organism's function in a disease or condition that leads to impairment of a particular organism function; or initiating the ongoing course or result of one or more clinically measurable parameters of a disease. In one embodiment, the goal of treatment is to prevent or slow (alleviate) an unwanted physiological condition, disorder or disease, or to obtain a beneficial or desired result. The result can be, for example, medical, physiological, clinical, physical therapy, occupational therapy, health professional or patient oriented; or a parameter understood in the art as "quality of life" or activities of daily living. In the present invention, a beneficial or desired clinical result includes, but is not limited to, alleviating symptoms; diminishing / reducing the extent of the condition, disorder or disease; stabilizing (i.e., not worsening) the state of the condition, disorder or disease; delaying the onset or slowing the progression of the condition, disorder or disease; ameliorating or palliating the condition, disorder or disease; and lessening (whether partial or total), whether detectable or undetectable; or enhancing or improving the condition, disorder or disease. In one embodiment, treatment includes eliciting a clinically significant response without excessive levels of side effects. In one embodiment, treatment also includes increasing survival as compared to the expected survival if treatment were not received. In one embodiment, treatment refers to administration of a drug or performance of a medical procedure on a patient. In the present invention, treatment can be prophylactic (preventative), curative of illness or disease, or ameliorative of a patient's clinical condition, including reducing the course or severity of a disease, or subjectively improving the quality of life of a patient or prolonging the survival of a patient.

[0059] The term "burst firing", or "burst firing", refers to a firing pattern in which a neuron produces two or more spikes simultaneously during a firing event.

[0060] Inhibiting burst firing refers to inhibiting the extent of burst firing, including reducing the frequency of burst firing or the number of spikes within a burst during a firing event, reducing the intensity of burst firing, or even eliminating the occurrence of burst firing. BRIEF DESCRIPTION OF DRAWINGS

[0061] Figure 1 shows micrographs and schematics of intracellular distribution of Kir4.1 expression after fusion of green fluorescent protein (GFP) at different positions of Kir4.1. (A) GFP fusion at the N-terminus of Kir4.1, Kir4.1 (green) can be distributed on the processes of glial cells that surround the neuronal cell body (red, immunofluorescence with NeuN antibody). (B) GFP fusion at the C-terminus of Kir4.1, Kir4.1 (green) is mainly distributed in the cell body of glial cells, with less Kir4.1 near the neuronal cell body. Scale bar, 10 μm. Blue is DAPI-labeled chromatin position of the nucleus.

[0062] Figure 2 shows the necessity of the C-terminal PDZ binding site of Kir4.1 for its normal intracellular distribution. (A) The three C-terminal amino acids of Kir4.1 native sequence, which are the PDZ binding sequence (motif), are marked in red. (B) Schematic diagram of the analysis pattern for the astrocytic structure, showing the different structures of astrocytic cell body and terminal foot. Scale bar, 5 μm. (C) Micrographs and schematic drawings of the intracellular distribution of Kir4.1 in the lateral habenula after missense or deletion mutation of the PDZ binding sequence of Kir4.1. Green is Kir4.1, and blue is DAPI-labeled chromatin position of the nucleus. Scale bar, 100 μm, and small window scale bar, 10 μm. (D) Statistics for the three Kir4.1 distribution patterns in Fig. C, including the proportion of signals of Kir4.1 accumulated in the cell body region to the total Kir4.1 signals, and the area covered by the terminal foot tips of the glial cells containing Kir4.1. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared with the control group. N.S. indicates no significant difference.

[0063] Figure 3 shows that Kir4.1 interacts with the PDZ domain-containing scaffold protein a-syntrophin in the habenula brain region. (A) Mass spectrometry analysis results of immunoprecipitation of habenula protein components with Kir4.1 antibody. According to the search and alignment of the native sequence, only a-syntrophin contains a PDZ structure. (B) Mass spectrometry analysis results of immunoprecipitation of habenula protein components with a-syntrophin antibody. The second most abundant component is Kir4.1. (C) Western blotting verification of immunoprecipitation of habenula protein components with a-syntrophin antibody. The presence of Kir4.1 and a-syntrophin in the precipitated elution components was indicated by the antibodies, respectively, proving the direct binding interaction between a-syntrophin and Kir4.1.

[0064] Figure 4 shows TAT-Kir4.1-C-terminal interfering peptides designed against a-syntrophin and Kir4.1 have direct effects on Kir4.1 function. (A) Amino acid sequences of four different TAT-Kir4.1-C-terminal peptides, named 5C, 9C, 12C and 15C, according to the homologous length with the C-terminal of native Kir4.1. (B) Design of biochemical experiments for functional verification of TAT-Kir4.1-C-terminal peptides. The peptides were dissolved in normal saline as 3 pmol / kg body weight injection, intraperitoneally injected into CRS mouse depression model animals, and the habenula was dissected for observation 2 hours later. (C) Immunoprecipitation of habenula protein components with a-syntrophin antibody showed that the binding of Kir4.1 to a-syntrophin was significantly reduced by TAT-Kir4.1-C-terminal peptides. (D) Immunoelectron microscopy showed that the number of Kir4.1 distributed along the glial cell membrane around the neuron cell body was significantly reduced after intraperitoneal injection of TAT-Kir4.1-C peptides. Each group contained 3 test animals, *P<0.05.

[0065] Figure 5A-D shows the effects of TAT-Kir4.1-C peptides on habenula neuron firing and depression-like behavior in mouse model animals. Figure 5A is a schematic diagram of the experimental mode of in vivo nerve recording electrodes, electrode surgical site and recording time course. Figure 5B shows schematic diagrams of firing unit signals and cluster discharge parameters recorded from habenula neurons after intraperitoneal injection of non-functional control peptides (upper row, gray) and TAT-Kir4.1-C-terminal peptides (lower row, red). Figure 5C and Figure 5D show that intraperitoneal injection of TAT-Kir4.1-C-terminal peptides has an antidepressant effect on depression-like behavior indicators in CRS mouse depression model (Figure 5C) and LPS mouse depression model (Figure 5D), including reduced forced swimming immobility time and increased sugar water preference. In the open field test, there was no significant difference in the total movement distance of mice, and the time spent in the central area was significantly increased, indicating that the peptide does not affect the movement ability of mice and has an anxiolytic effect. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 compared with the control group. N.S. indicates no significant difference.

[0066] Figure 6 shows long-term antidepressant effect of TAT-Kir4.1-15C-Somatostatin side chain. (A) TAT-Kir4.1-15C-Somatostatin side chain molecular structure, Somatostatin side chain is connected on lysine residue. (B) and (C) Intraperitoneal injection of TAT-Kir4.1-15C-Somatostatin side chain polypeptide on the depression-like behavior index test of CRS depression mouse model (C) and LPS mouse depression model (B), showing antidepressant effect, including 2-hour forced swimming immobility time reduction, 24-hour forced swimming or tail suspension test immobility time reduction. The antidepressant effect time is longer than that of the somatostatin side chain polypeptide without somatostatin side chain used in Figure 5. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 compared with the control group. N.S. indicates no significant difference. DETAILED DESCRIPTION

[0067] The essence and beneficial effects of the present application will be further illustrated below in conjunction with examples, which are only used to illustrate the present application and not to limit the present application. The experimental methods in the following examples without specific conditions are generally carried out according to conventional conditions, for example, the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer.

[0068] Materials and methods of Example 1

[0069] Animal materials

[0070] The mice used in the experiment are adult male (8-12 weeks old) C57BL / 6 mice, 4-5 per cage, 12-hour rhythm cycle (light from 7 am to 7 pm), and free access to stable water and food. All animal experiments are approved by the Zhejiang University Animal Protection and Use Committee.

[0071] Virus construction

[0072] The virus AAV2 / 5-gfaABC1D-EGFP-Kir4.1 used for studying the localization of Kir4.1 was coated by Shanghai Tailv Biological Technology Co., Ltd. The viruses AAV2 / 5-gfaABC1D-EGFP-Kir4.1-SNV (as a wild-type control, same as the AAV2 / 5-gfaABC1D-EGFP-Kir4.1 described above), AAV2 / 5-gfaABC1D-EGFP-Kir4.1-SAA (the last three amino acid residues of the Kir4.1 sequence were mutated from SNV to SAA), and AAV2 / 5-gfaABC1D-EGFP-Kir4.1-delta3 (mutant deletion of the last three amino acid residues of the Kir4.1 sequence), a total of three, were coated by the Wang Xuhua research group of Zhejiang University.

[0073] LPS (Lipopolysaccharide) induced depression model

[0074] C57BL / 6 male mice received a single LPS intraperitoneal injection (0.83 mg / kg body weight, solvent was normal saline) 24 hours after behavioral testing.

[0075] CRS (chronic restraint stress) chronic restraint depression model

[0076] C57BL / 6 male mice were restrained in a cylindrical transparent and breathable plastic tube with a volume of about 50 ml and a length of about 10 cm for 2-4 hours a day, and the start and end points were random but were all within the light feeding period. During this period, the mice could not move freely or eat, and could not observe other individuals (the line of sight was blocked by a partition). After 14 days, various biochemical or behavioral tests were performed.

[0077] Stereotactic injection

[0078] After the mice were anesthetized by intraperitoneal injection of 4% sodium pentobarbital, they were fixed on a stereotaxic instrument (RWD Life Science Co., Ltd.). About 100-200 nl of AAV virus (about 1011 infectious units per milliliter, which is lower than the general virus concentration, for sparse labeling to facilitate observation of individual astrocytes) was injected into each mouse on each side of the LHb. The stereotactic coordinates of the LHb (distance from Bregma: -1.60 mm (AP), left and right side open ±0.46 mm (ML), cortex surface down -2.60 mm (DV)). A glass microelectrode was used for slow injection (about 100-150 nl / min), the needle was left for 10 minutes after the injection was completed, and then the injection electrode was slowly removed. At least 21 days after the operation, dissection and brain tissue sample preparation were performed.

[0079] Immunohistochemistry

[0080] Mice were deeply anesthetized with intraperitoneal injection of 4% pentobarbital. Brains were fixed by cardiac perfusion with PBS and 4% PFA. Brains were removed and post-fixed overnight in 4% PFA and dehydrated in 30% sucrose-PBS solution for 1-3 days until the brains sink to the bottom of the tube. Brains were sectioned on a cryostat at 40 μιη thickness. Sections were processed in blocking solution (1% BSA, 10% goat serum, 0.03% Triton X-100) as follows: blocking (1-2 hours at room temperature), primary antibody incubation (4°C overnight, followed by two washes with PBS), secondary antibody incubation (room temperature for 1-2 hours, followed by two washes with PBS). Slides were finally mounted with DAPI-containing mounting medium (SouthernBiotech) and images were acquired using a Nikon Al laser confocal microscope. The primary antibodies used were as follows: anti-S100b (1 :500, Abeam), anti-GFP (1 : 1000, Abeam), anti-NeuN (1 :500, Millipore), anti-Kir4.1 (1 :500, Alomone APC-035). The secondary antibodies used were as follows: Alexa Fluor 488 goat anti-chicken IgG, Alexa Fluor 488 goat anti-rabbit IgG, Alexa Fluor 546 goat anti-mouse IgG Alexa Fluor Cy5 goat anti-mouse IgG (all secondary antibodies at 1 : 1000, Invitrogen). Images acquired were analyzed using imageJ software.

[0081] Harness protein extraction and co-immunoprecipitation

[0082] After isoflurane anesthesia, mice were not perfused and brain tissues were directly taken out, cortex and hippocampus were bluntly separated on ice and thalamus was exposed. From the top of the thalamus, the habenula was observed and taken out completely and grinded in RIPA solution (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 1 mM PMSF, 10 μg / ml aprotinin, 1 μg / ml pepstatin A and 1 μg / ml leupeptin) for 30 minutes on ice for lysis. Then, the supernatant was centrifuged at 4°C at 12000 g for 15 min to obtain the habenula total protein component. The α-syntrophin antibody (Santa-Cruz D-7, 10 μl per centrifugal column) or Kir4.1 antibody (Alomone APC-035, 10 μl per centrifugal column) was used in advance to covalently bind the centrifugal column resin (the steps followed the immunoprecipitation kit Thermo 63149), the total protein component solution was injected, and the resin-bound protein was dissociated using a reducing eluent, which was the immunoprecipitated component of the habenula corresponding to the antibody.

[0083] Protein mass spectrometry and immunoblotting

[0084] The habenula immunoprecipitated component was separated by 4-12% gradient SDS-PAGE gel using a fixed 20 μl volume.

[0085] For the component for protein mass spectrometry, the gel was subjected to Coomassie blue staining, and after the lane cutting showing the protein distribution was decolorized and digested with trypsin for dissociation. The dissociated sample was separated by a nanoflow HPLC liquid system Easy nLC. Buffer A was 0.1% formic acid aqueous solution, and buffer B was 0.1% formic acid acetonitrile aqueous solution (acetonitrile was 84%). The chromatographic column was equilibrated with 95% A liquid, and the sample was injected into the column (Thermo Scientific Acclaim PepMap100, 100 μm*2 cm, nanoViper C18) by an automatic sampler, separated by an analytical column (Thermo scientific EASY column, 10 cm, ID 75 μm, 3 μm, C18-A2), and subjected to mass spectrometry analysis by Q-Exactive mass spectrometer. The mass spectrometry experiment was performed by Shanghai ZK New Life Biotechnology Co., Ltd.

[0086] For the part of western blot, protein gel was transferred to PVDF membrane (400 mA, 25 min), blocked with skim milk and then subjected to western blot in 5% BSA solution. The primary antibodies used were anti-Kir4.1 (1 : 1000, Alomone APC-035), anti-a-syntrophin (1 : 1000, Santa-Cruz D-7), and the secondary antibodies were HRP-conjugated goat anti-mouse IgG (Biorad, 1 : 10000) and HRP-conjugated goat anti-rabbit IgG (Bio-rad, 1 : 10000). After chemiluminescence imaging, statistical analysis was performed using image J software.

[0087] Immunoelectron microscopy

[0088] Mice were deeply anesthetized by intraperitoneal injection of 4% pentobarbital. The mice were perfused with physiological saline and 4% PFA + 0.5% glutaraldehyde in PBS solution, and the brain tissue was removed and cut into square tissue blocks containing LHb with an edge length of not more than 5 mm, and fixed in 4% PFA + 0.5% glutaraldehyde in PBS solution for 24 hours. The tissue blocks were vibratome sectioned into 50 μm thin sections, treated with glycine and Triton X-100, and then incubated in BSA blocking solution overnight with primary and secondary antibodies (anti-Kir4.1, 1 : 200, Alomone APC-035; Nanogold-labeled Fab’ goat anti-rabbit IgG, 1 : 500, Nanoprobes, Inc) at 4°C. Subsequently, the samples were subjected to silver enhancement (HQ Silver Enhancer, Nanoprobes, Inc), treated with 1% osmium tetroxide and 2% uranyl acetate, dehydrated and embedded with Epon. The embedded samples were cut into 50 nm sections at the LHb position, observed using a Tecnai G2 spirit 120 kV transmission electron microscope, and statistical analysis was performed using image J software.

[0089] Forced swim test

[0090] The experiment was conducted during the light period of the mouse feeding cycle. The experimental environment was a normal human room lighting environment. The diameter of the mouse forced swim cylinder was 12 cm, and the height was 25 cm. The water depth of the test was 14 cm, and the water temperature was 22-24°C. A camera recorded the swimming of the mouse within 6 min from the side. The immobile time (periods when the animal was motionless or had only slight limb movements that did not cause displacement of the body's center of gravity) within the last 4 min of the 6 min recording was counted manually by double-blind observation.

[0091] tail suspension test

[0092] The experiment was performed during the light period of the mouse's housing cycle. The experimental environment was a normal human room lighting environment. The mouse was head down and the distal 1 cm of the tail was loosely fixed with medical tape to a plastic horizontal bar 35 cm above the floor. A video camera recorded the mouse's struggle from the side for 6 min. The immobility time (period of time when the animal was motionless) was manually counted by two blinded observers for the last 4 min of the 6 min video recording.

[0093] sucrose preference test

[0094] The experimental mice were housed individually and allowed to acclimate for four consecutive days. During the first two days of acclimation, the mice were given two bottles of regular drinking water. During the last two days of acclimation, the mice were given two bottles of 1% sucrose solution. During the acclimation period, the mice's water consumption was recorded by weighing the bottles to determine if the mice had a preference for the bottle spout and to correct for this by changing the apparatus. After the acclimation period, the mice were deprived of water for 24 h and then given a test of one bottle of regular water and one bottle of 1% sucrose solution for 2 h during the dark period of the mouse's housing cycle. The position of the water bottles was changed after the first hour and the water and sucrose solution consumption was recorded by weighing the bottles every hour.

[0095] open field test

[0096] The open field apparatus was a square field with a side length of 45 cm, surrounded by a 50 cm high fence on all four sides. The light intensity in the center of the open field was about 10-20 lux. A 22.5 cm square in the center was defined as the central zone. The mouse was lifted and placed in the central zone and a video camera was placed directly above the center of the open field to record the mouse's movement for 10 min. The distance traveled and the time spent in the central zone were analyzed using Anymaze software.

[0097] in vivo electrophysiological recording

[0098] The in vivo recording electrode was self-made and implanted in the LHb with the following stereotaxic coordinates: -1.60 mm (AP) from the bregma, ±0.46 mm (ML) from the midline, and -2.60 mm (DV) from the cortical surface. A stainless steel wire was connected to the skull screw for grounding. After 2 weeks of recovery, the in vivo recording system (Plexon) was used to collect and analyze the data. After the experiment, the brain tissue slices of the tested animals were collected to verify the accuracy of the electrode position. The data only included the data of animals with accurate implanted positions.

[0099] statistical analysis

[0100] All data are presented as mean ± SEM. All data significance was calculated using two-tailed Student's t-tests between two groups and one-way ANOVA among multiple groups.

[0101] Example 2 Location and mode of action of Kir4.1 expression in the habenula

[0102] Using stereotaxic injection, AAV2 / 5-gfaABC1D-EGFP-Kir4.1 (GFP directly linked and covers the N-terminal of Kir4.1) and AAV2 / 5-gfaABC1D-Kir4.1-EGFP (GFP directly linked and covers the C-terminal of Kir4.1) were expressed in mouse brain (Figure 1A). It was observed that the former GFP-Kir4.1 fusion protein can distribute on the endfoot terminal of astrocyte, while the latter Kir4.1-GFP fusion protein concentrated in the astrocyte cell body (Figure IB), and failed to distribute correctly on the endfoot terminal of astrocyte, indicating that the C-terminal amino acid residues of Kir4.1 directly participate in its intracellular localization.

[0103] The inventors found that the last three amino acid residues of the C-terminal of Kir4.1 is -SNV, which is consistent with the general rule of PDZ binding sequence under natural conditions (Figure 2A). To verify that the C-terminal of Kir4.1 is a PDZ binding sequence and plays a role in binding PDZ, the C-terminal -SNV amino acid of Kir4.1 in AAV2 / 5-gfaABC1D-EGFP-Kir4.1 was deleted (AAV2 / 5-gfaABC1D-EGFP-Kir4.1-delta3) or missense mutated (AAV2 / 5-gfaABC1D-EGFP-Kir4.1-SAA), and expressed in LHb (Figure 2C), respectively. The distribution of Kir4.1 in astrocyte in different locations was counted (Figure 2B), and it was observed that the deletion and missense mutation of Kir4.1 PDZ binding site significantly reduced the distribution ratio of Kir4.1 on the endfoot of astrocyte and increased the ratio of cell body accumulation (Figure 2D).

[0104] Using Kir4.1 antibody to immunoprecipitate the proteins combined with Kir4.1 in the habenula, mass spectrometry analysis found that α-syntrophin, a scaffold protein containing a PDZ domain, was the only scaffold protein (Figure 3A). Using α-syntrophin antibody to immunoprecipitate the protein components of the habenula, mass spectrometry analysis found that the main protein combined with α-syntrophin was Kir4.1 (Figure 3B). Immunoblotting experiments also verified that α-syntrophin and Kir4.1 have direct binding in the habenula (Figure 3C).

[0105] Based on the above experimental conclusions, it was found and proved that the combination of α-syntrophin and Kir4.1 is necessary for Kir4.1 to normally distribute on the endfoot terminal of lateral habenula astrocyte and to regulate the increase of neuron cluster discharge, thereby leading to depression.

[0106] Antidepressant therapeutic effect of polypeptide molecules targeting the binding of α-syntrophin and Kir4.1 in Example 3

[0107] The TAT polypeptide sequence naturally having a cell-penetrating effect was connected to the N-terminus of the polypeptide containing the PDZ binding sequence at the C-terminus of Kir4.1. According to the selection of different lengths at the C-terminus of Kir4.1, four TAT-Kir4.1-C polypeptides (Figure 4A) were constructed and synthesized, and the TAT sequence YGRKKRRQRRR (SEQ ID NO: 7) was added, which were named TAT-Kir4.1-5C, TAT-Kir4.1-9C, TAT-Kir4.1-12C and TAT-Kir4.1-15C, or simply 5C, 9C, 12C and 15C, respectively, each representing a polypeptide having 5, 9, 12 and 15 amino acids at the C-terminus of Kir4.1:

[0108] TAT-Kir4.1-5C: YGRKKRRQRRR-RISNV (SEQ ID NO: 3)

[0109] TAT-Kir4.1-9C: YGRKKRRQRRR-ALSVRISNV (SEQ ID NO: 4)

[0110] TAT-Kir4.1-12C: YGRKKRRQRRR-EGSALSVRISNV (SEQ ID NO: 5)

[0111] TAT-Kir4.1-15C: YGRKKRRQRRR-AEKEGSALSVRISNV (SEQ ID NO: 6).

[0112] In the biochemical experiment (Figure 4B), a polypeptide similar in structure to the TAT-Kir4.1-C polypeptide but not containing the PDZ binding sequence was used as a control group. The results showed that in the CRS depression mouse model, intraperitoneal injection of this polypeptide could reduce α-syntrophin and Kir4.1 in the habenula tissue (Figure 4C). At the same time, immunoelectron microscopy also showed that Kir4.1 on the cell membrane of glial cells around the neurons of the lateral habenula was significantly reduced (Figure 4D).

[0113] In addition, in vivo electrophysiology and behavioral tests were used to verify the effect of TAT-Kir4.1-C polypeptide on physiological indicators of depression mouse models. Through in vivo electrophysiological recording (Figure 5A), it was found that after intraperitoneal injection of TAT-Kir4.1-C polypeptide, the number and frequency of clustered discharges in the lateral habenula of CRS depression model mice were significantly reduced within 2 hours, and subsided after 4 hours (Figure 5B).

[0114] Correspondingly, in CRS mouse model, TAT-Kir4.1-C polypeptides can significantly reduce the time of immobility in forced swimming test (indicating behavioral despair) and significantly increase the proportion of sugar water selection in sugar water preference test (indicating lack of pleasure) at 2 hours time point after intraperitoneal injection, having an antidepressant effect (Figure 5C).

[0115] In LPS mouse model, the same effect of reducing behavioral despair is also achieved (Figure 5D).

[0116] Meanwhile, in the open field test, TAT-Kir4.1-C polypeptides do not affect the motor ability of mice, and significantly reduce the anxiety level of mice (Figure 5C).

[0117] Example 4 Somatostatin side chain modification enhances the antidepressant therapeutic effect of polypeptide molecules interfering with the binding of α-syntrophin and Kir4.1

[0118] Somatostatin side chains are added to the lysine residues of TAT-Kir4.1-15C (Figure 6A) to increase the in vivo stability of TAT-Kir4.1-C polypeptides and prolong the maintenance time of their antidepressant effect.

[0119] The following polypeptides with TAT sequence and 15 amino acids of Kir4.1, and lysine connecting fatty acid side chain (AEEA-AEEA-γE-C18 diacid) on the C-terminal sequence of Kir4.1 protein are synthesized:

[0120] TAT-Kir4.1-5C-somatostatin side chain: YGRKKRRQRRR-AEK(AEEA-AEEA-γE-C18 diacid)EGSALSVRISNV.

[0121] The results show that intraperitoneal injection of TAT-Kir4.1-15C-somatostatin side chain can not only reduce the behavioral despair phenotype of LPS depressed mouse model in forced swimming at 2 hours time point (Figure 6B), but also can be sustained to 24 hours, which is reflected in the tail suspension test of LPS model and the forced swimming test of CRS model (Figure 6B / 6C).

[0122] The above is a description of the present application, which cannot be regarded as a limitation of the present application. Unless otherwise indicated, the practice of the present application will employ conventional techniques of organic chemistry, polymer chemistry, biotechnology, etc. It is clear that the present application can be implemented in other ways in addition to those specifically described in the above description and examples. Other aspects and improvements within the scope of the present application will be apparent to those skilled in the art. Many changes and modifications are possible in light of the teachings of the present application, and therefore within the scope of the present application.

[0123] As used herein, the term "degrees," in reference to temperature, means degrees Celsius, i.e., °C, unless otherwise indicated.

Claims

1. A method of treating depression, comprising administering to a patient an agent that inhibits the binding of Kir4.1 to dystrophin-associated protein alpha.

2. The method of claim 1, wherein the agent that inhibits the binding of Kir4.1 to dystrophin-associated protein alpha is an agent that inhibits the binding of the C-terminus of Kir4.1 to the PDZ domain of dystrophin-associated protein alpha.

3. The method of claim 2, wherein the agent that inhibits the binding of the C-terminus of Kir4.1 to the PDZ domain of dystrophin-associated protein alpha is a competitive inhibitor of the binding of the C-terminus of Kir4.1 to the PDZ domain of dystrophin-associated protein alpha, such as a Kir4.1 mutant or fragment that lacks potassium ion channel activity but retains binding activity to the PDZ domain of dystrophin-associated protein alpha or a nucleic acid encoding the same.

4. The method of claim 3, wherein the competitive inhibitor of the binding of the C-terminus of Kir4.1 to the PDZ domain of dystrophin-associated protein alpha is a C-terminal fragment of Kir4.1 or a fusion protein thereof, or a nucleic acid encoding and expressing the C-terminal fragment or fusion protein thereof, wherein the C-terminal fragment of Kir4.1 is a polypeptide fragment comprising at least 3-5 amino acids of the C-terminus of Kir4.1, preferably a polypeptide fragment having 5-30 amino acids, such as a polypeptide fragment having 5-15 amino acids.

5. The method of claim 4, wherein the fusion protein of the C-terminal fragment of Kir4.1 is a fusion protein of the C-terminal fragment of Kir4.1 and a cell-penetrating peptide, preferably the cell-penetrating peptide is a human immunodeficiency virus type 1 transcriptional activator TAT such as TAT(48-60) HIV-1 protein, i.e., GRKKRRQRRRPPQQ or TAT(47-57) HIV-1 protein, i.e., YGRKKRRQRRR, sl Igl-1 homology domain, i.e., RVIRVWFQNKRCKDKK, transportan, i.e., GWTLNSAGYLLGKINLKALAALAKKIL, or P-alpha gp41-SV40, i.e., GALFLAFLAAALSLMGLWSQPKKKRRV, etc.

6. The method of claim 5, wherein the fusion protein of the C-terminal fragment of Kir4.1 further comprises a polypeptide stability modification, such as a PEG modification or a solabegum side chain modification.

7. The method of claim 4, wherein the competitive inhibitor of the binding of the C-terminus of Kir4.1 to the PDZ domain of dystrophin-associated protein alpha is a C-terminal fragment of Kir4.1 or a fusion protein thereof having the following amino acid sequence, or a nucleic acid encoding and expressing the C-terminal fragment or fusion protein thereof: YGRKKRRQRRRRISNV (SEQ ID NO: 3); YGRKKRRQRRRALSVRISNV (SEQ ID NO: 4); YGRKKRRQRRREGSALSVRISNV (SEQ ID NO: 5); YGRKKRRQRRRAEKEGSALSVRISNV (SEQ ID NO: 6); or YGRKKRRQRRRAEK(AEEAAEEAE-C18 diacid)EGSALSVRISNV.

8. The method according to claim 1 or 2, wherein the agent that inhibits the binding of Kir4.1 to dystrophin-associated protein a is an interfering RNA or a precursor thereof against the nucleotide sequence encoding the C-terminal end of Kir4.

1.

9. The method according to claim 1 or 2, wherein the agent that inhibits the binding of the C-terminal end of Kir4.1 to the PDZ domain of dystrophin-associated protein a is a specific antibody, including a polyclonal antibody or a monoclonal antibody, that recognizes and binds to the C-terminal end fragment of Kir4.

1.

10. The method according to claim 1 or 2, wherein the local administration is in the lateral habenula.

11. A pharmaceutical composition for the treatment of depression, comprising an agent that inhibits the binding of Kir4.1 to dystrophin-associated protein a, preferably wherein the agent that inhibits the binding of Kir4.1 to dystrophin-associated protein a is an agent that inhibits the binding of the C-terminal end of Kir4.1 to the PDZ domain of dystrophin-associated protein a.

12. The pharmaceutical composition according to claim 11, wherein the agent that inhibits the binding of the C-terminal end of Kir4.1 to the PDZ domain of dystrophin-associated protein a is a competitive inhibitor of the binding of the C-terminal end of Kir4.1 to the PDZ domain of dystrophin-associated protein a, preferably wherein the competitive inhibitor of the binding of the C-terminal end of Kir4.1 to the PDZ domain of dystrophin-associated protein a is a C-terminal end fragment of Kir4.1 or a fusion protein thereof, or a nucleic acid encoding and expressing said C-terminal end fragment or fusion protein thereof, wherein the C-terminal end fragment of Kir4.1 is a polypeptide fragment comprising at least 3-5 amino acids of the C-terminal end of Kir4.1, preferably a polypeptide fragment of 5-30 amino acids, such as a polypeptide fragment of 5-15 amino acids.

13. The pharmaceutical composition according to claim 12, wherein the fusion protein of the C-terminal end fragment of Kir4.1 is a fusion protein of the C-terminal end fragment of Kir4.1 and a cell-penetrating peptide, preferably wherein the cell-penetrating peptide is a human immunodeficiency virus type 1 transcriptional activator TAT such as TAT(48-60) HIV-1 protein GRKKRRQRRRPPQQ or TAT(47-57) HIV-1 protein YGRKKRRQRRR, sl Igl-1 homology domain RVIRVWFQNKRCKDKK, transportan GALFLAFLAAALSLMGLWSQPKKKRRV or P-alpha gp41-SV40 GALFLAFLAAALSLMGLWSQPKKKRRV.

14. The pharmaceutical composition according to claim 13, wherein the fusion protein of the C-terminal end fragment of Kir4.1 further comprises a polypeptide stability modification, such as a PEG modification or a somatostatin side chain modification.

15. The pharmaceutical composition of claim 14, wherein the competitive inhibitor of the binding of the C-terminal end of Kir4.1 to the PDZ domain of dystrophin-associated protein alpha is a C-terminal end fragment of Kir4.1 having the following amino acid sequence or a fusion protein thereof, or a nucleic acid encoding and expressing the C-terminal end fragment or fusion protein thereof: YGRKKRRQRRRRISNV (SEQ ID NO: 3); YGRKKRRQRRRALSVRISNV (SEQ ID NO: 4); YGRKKRRQRRREGSALSVRISNV (SEQ ID NO: 5); YGRKKRRQRRRAEKEGSALSVRISNV (SEQ ID NO: 6); or YGRKKRRQRRRAEK(AEEAAEEAE-C18 diacid)EGSALSVRISNV.

16. Use of an agent that inhibits the binding of Kir4.1 to dystrophin-associated protein alpha for the manufacture of a medicament for the treatment of depression, wherein the agent that inhibits the binding of Kir4.1 to dystrophin-associated protein alpha is as defined in claims 1-10.

17. An isolated polypeptide comprising at least 3-5 amino acids of the C-terminal end of Kir4.1 having the amino acid sequence of SEQ ID NO: 1, preferably 5-30 amino acids of the C-terminal end of Kir4.1, for example 5-15 amino acids of the C-terminal end of Kir4.1, more preferably the polypeptide further has a polypeptide stabilizing modification, for example a PEG modification or a somatostatin side chain modification.

18. The isolated polypeptide of claim 17, which is a fusion protein comprising a cell-penetrating peptide, preferably the cell-penetrating peptide is a human immunodeficiency virus type 1 transcriptional activator TAT such as TAT(48-60) HIV-1 protein GRKKRRQRRRPPQQ or TAT(47-57) HIV-1 protein YGRKKRRQRRR, sl Igl-1 homology domain RVIRVWFQNKRCKDKK, transportan glycylproline peptide GWTLNSAGYLLGKINLKALAALAKKIL or P-alpha gp41-SV40 GALFLAFLAAALSLMGLWSQPKKKRRV, etc.

19. The isolated polypeptide of claim 17, having the following amino acid sequence: YGRKKRRQRRRRISNV (SEQ ID NO: 3); YGRKKRRQRRRALSVRISNV (SEQ ID NO: 4); YGRKKRRQRRREGSALSVRISNV (SEQ ID NO: 5); YGRKKRRQRRRAEKEGSALSVRISNV (SEQ ID NO: 6); or YGRKKRRQRRRAEK(AEEAAEEAE-C18 diacid)EGSALSVRISNV. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 20. An isolated nucleic acid comprising a nucleotide sequence encoding a polypeptide according to any one of claims 17-19, wherein the polypeptide comprises at least 3-5 amino acids of the C-terminus of Kir4.1 having an amino acid sequence as set forth in SEQ ID NO: 1, preferably 5-30 amino acids of the C-terminus of said Kir4.1, for example 5-15 amino acids of the C-terminus of said Kir4.1.

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