WO2018205927A1 - 钾离子通道抑制剂治疗抑郁症的用途和药物组合物 - Google Patents

钾离子通道抑制剂治疗抑郁症的用途和药物组合物 Download PDF

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WO2018205927A1
WO2018205927A1 PCT/CN2018/086021 CN2018086021W WO2018205927A1 WO 2018205927 A1 WO2018205927 A1 WO 2018205927A1 CN 2018086021 W CN2018086021 W CN 2018086021W WO 2018205927 A1 WO2018205927 A1 WO 2018205927A1
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seq
depression
protein
inhibitor
sequence
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French (fr)
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胡海岚
崔一卉
杨艳
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浙江大学
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Priority to EP18798117.0A priority Critical patent/EP3622958B1/en
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Priority to US16/679,197 priority patent/US11326168B2/en
Priority to US17/717,140 priority patent/US20230050684A1/en

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Definitions

  • the present invention relates to the field of disease treatment and medicine.
  • the present invention relates to the treatment of depression and pharmaceutical compositions for the treatment of depression and methods for their preparation.
  • Depression is a chronic mental disorder characterized by significant and persistent low mood, lack of motivation, behavioral despair and loss of pleasure as the main clinical features, and severe suicidal tendency.
  • Lateral nucleus has been considered as a key brain area for studying the pathophysiology of depression in recent years. Significant elevations in lateral nucleus activity have been found in many animal models of depression and depression.
  • the abnormal activity of neurons is mainly attributed to abnormal synaptic transmission, changes in physiological characteristics of the body, and changes in the environment inside the neurons.
  • Astrocytes are involved in the regulation of neural cell autoactivity, neurotransmitter release, and play an important role in a range of diseases including schizophrenia, epilepsy, senile dementia, depression, etc. (Hamilton et al., Frontiers in neuroenergetics 2 , 2010).
  • Antidepressants can directly act on astrocytes, significantly affecting their morphology and function. This phenomenon is thought to be one of the pathways by which antidepressants play an antidepressant effect in the central nervous system (Czeh et al., the journal of the European College of Neuropsychopharmacology 23, 171-185, 2013). These evidences suggest that in addition to neurons, glial cells play a role that cannot be ignored in mental illness.
  • the inward rectifier-type potassium channel refers to a super-activated potassium ion channel.
  • the inwardly-rectifying potassium ion channel has been reported to have seven protein family members (Kir1 to Kir7). Similar Kir channel can be divided into multiple subtypes due to the splicing variance, which is distributed in various tissues and organs such as 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 the inward rectifier type potassium ion channel. In the nervous system, Kir4.1 is specifically expressed in glial cells.
  • Kir4.1 in glial cells allows potassium ions to pass through the cell membrane, regulates the extracellular environment by regulating the potassium ion concentration of the outer fluid around the nerve cells, and transports extracellular potassium ions to control the resting membrane potential level. Maintains the homeostasis of the nervous system and maintains the normal physiological activity of the nervous system.
  • the abnormal function of Kir4.1 will have a great influence on the function of glial cells and neurons, and thus exhibit a variety of neurological diseases. In mammals, the protein sequence and coding nucleic acid sequence of Kir4.1 are very conservative.
  • Kir4.1 in astrocytes of the lateral habenular nucleus is a crucial regulator of depression, and uses molecular, behavioral, and electrophysiological methods to determine the lateral iliac crest.
  • Kir4.1 expressed by astrocytes exists in the lateral nucleus in a manner that closely surrounds the nucleus of the nucleus of the nucleus, regulating the extracellular potassium balance and changing the release characteristics of the lateral nucleus neurons. Lead to excessive hyperactivity of the lateral nucleus, which in turn regulates the phenotype of depression.
  • the present inventors have also discovered and demonstrated various agents that block the function of Kir4.1 in the lateral nucleus, thereby providing methods and medicaments for treating (suppressing) depression by inhibiting the activity of Kir4.1.
  • the present invention provides a method of treating depression in a patient by inhibiting the activity of Kir4.1.
  • the invention also provides the use of a Kir4.1 inhibitor for the manufacture of a medicament for treating depression in a patient.
  • mammals including humans or non-human primates such as monkeys.
  • the mammal can be other animals such as rats, mice, rabbits, pigs, dogs, and the like.
  • the mammal can be a domestic animal such as a cat or a dog.
  • a Kir4.1 inhibitor refers to an agent capable of reducing or losing the activity of the Kir4.1 channel.
  • the activity of the Kir4.1 channel refers to the activity that allows potassium ions to pass through the cell membrane.
  • Kir4.1 buffers the extracellular environment by regulating the potassium ion concentration of the external fluid around the nerve cells and transporting extracellular potassium ions to control the resting membrane potential level and affect the physiological activity of the nervous system.
  • Kir4.1 inhibitors include compounds, complexes or mixtures which are capable of reducing or losing the activity of the Kir4.1 channel, as well as preparations used in methods for inhibiting Kir4.1 activity (including surgical methods) and the like.
  • the Kir4.1 inhibitor is capable of affecting Kir4.1 protein An agent that affects the activity of the Kir4.1 channel.
  • the reagent includes a small molecule compound or complex, or a macromolecular active ingredient such as a protein or a nucleic acid, for example, an antagonist such as an antibody that binds to the Kir4.1 protein, or a nucleic acid that affects the expression level of the Kir4.1 protein.
  • proteins or nucleic acids can be delivered to a target tissue or cell by techniques well known in the art, for example, in conjunction with a suitable expression vector.
  • the Kir4.1 inhibitor is an inhibitor that specifically inhibits Kir4.1.
  • a specific Kir4.1 inhibitor generally means that the Kir4.1 inhibitor has no inhibitory activity against other Kir proteins, or has a significantly less inhibitory activity against other Kir proteins than for Kir4.1, for example against other Kir proteins.
  • the inhibitory activity is 50% or less, preferably 20% or less, more preferably 5% or less, of the inhibitory activity against Kir4.1.
  • the Kir4.1 inhibitor is an interfering RNA or a precursor thereof that interferes with the expression of Kir4.1.
  • RNA interference induces efficient and specific degradation of homologous mRNA by double-stranded RNA (dsRNA), thereby reducing or even eliminating the expression of the target gene.
  • the interfering RNA may include small interfering RNA (siRNA), short hairpin RNA (shRNA), and/or microRNA (miRNA).
  • siRNA small interfering RNA
  • shRNA short hairpin RNA
  • miRNA microRNA
  • One way to administer interfering RNA in vivo is by administration of a precursor shRNA to the siRNA, such as a short hairpin RNA comprising two short inverted repeats.
  • siRNA sequence was cloned into a plasmid vector as a "short hair clip". When introduced into an animal, the hairpin sequence is expressed to form a "double-stranded RNA", and the corresponding siRNA is generated by the intracellular Dicer enzyme to exert an RNAi effect.
  • an interfering RNA or a precursor thereof useful in the invention has a sequence that is identical or complementary to, or more than 90% identical or complementary to, a fragment of a target Kir4.1 mRNA.
  • the interfering RNA that interferes with the expression of Kir4.1 or a precursor thereof has the following sequence:
  • the above interfering RNA or a precursor thereof is directed against the sequence of the corresponding mRNA fragment of the rat Kir4.1 mRNA sequence having SEQ ID No. 7 (ie, the CDS region sequence in the sequence of Genebank No. NM_031602.2), ie, the target rat Kir4.
  • the sequence of the 1 mRNA fragment is identical or complementary to the sequence of the interfering RNA of SEQ ID No. 1-6, or more than 90% of the sequences are identical or complementary.
  • the Kir4.1 inhibitor is a mutant Kir4.1 protein having a decreased or lost potassium channel activity or a coding sequence thereof. Mutant proteins can compete with normal proteins, thereby reducing the activity of normal proteins.
  • the mutant protein can be expressed in a target tissue or cell by administering a vector expressible in a target tissue or cell (the vector carries an expressible mutant protein gene and/or an expression factor thereof).
  • the mutant Kir4.1 protein is in the nuclear pore region of the Kir4.1 protein, for example, at positions 130-132 in the amino acid sequence corresponding to the Kir4.1 sequence of SEQ ID No.
  • a mutant Kir4.1 protein with a mutation in GYG such as a mutant Kir4.1 protein with a GYG mutation to AAA.
  • Kir4.1 having the amino acid sequence of SEQ ID No. 8 is Kir4.1 (NP_113790.2) of rat.
  • Kir4.1 NP_113790.2
  • Those skilled in the art can obtain corresponding ones in other mammals based on existing reports (e.g., Hiroshi et al., 2010. Physiological Reviews 90, 291-366, 2010) and understanding of the conservation of the sequence of Kir4.1.
  • the Kir4.1 inhibitor is a specific antibody to Kir4.1, including a polyclonal antibody or a monoclonal antibody. .
  • the present invention provides a method for treating depression by inhibiting the activity of Kir4.1 and a Kir4.1 inhibitor in the preparation of a treatment for depression, in one aspect of the present invention.
  • the Kir4.1 inhibitor is not buspirone, mianserin, fluoxetine, sertraline, fluvoxamine ( Fluvoxamine) or nortriptyline.
  • the present invention provides a method for treating depression by inhibiting the activity of Kir4.1 and a Kir4.1 inhibitor in the preparation of a treatment for depression, in one aspect of the present invention.
  • the Kir4.1 inhibitors are not selective serotonin reuptake inhibitors (SSRIs) or tricyclic antidepressants (TCAs).
  • the depression may specifically refer to "lateral lateral nucleus mediated depression".
  • the inventors of the present application have found that abnormal distribution of neurons of the lateral nucleus, particularly abnormal distribution of cluster discharges, plays an important role in the generation of depression.
  • the inventors of the present application also found that Kir4.1 in astrocytes of the lateral habenular nucleus is a crucial regulator of depression and has been found and demonstrated to block Kir4 in the lateral habenular nucleus.
  • a functional reagent thereby providing a method and a medicament for treating (suppressing) depression by inhibiting the activity of Kir4.1 in astrocytes of the lateral nucleus.
  • the methods and medicaments provided by the invention are methods and medicaments for topical action in the lateral nucleus.
  • drugs for nerve tissue particularly brain nerve tissue, such as the lateral nucleus
  • the method or drug for LHb needs to consider whether the method or drug can exert the efficacy of the drug in LHb, including whether the drug can reach LHb, and whether the effective concentration can be achieved in LHb.
  • the drug may be in the form of a topical administration to the lateral nucleus.
  • the action of the drug can be limited to the target tissue by topical administration, for example by making the drug into a dosage form that can be administered topically to the lateral nucleus by cannulation.
  • the drug is prepared into a dosage form or the like which is sustained release after being implanted into a tissue.
  • the above drugs can also be formulated in the form of tissue-specific targeted drug delivery systems.
  • an antibody which binds to a small molecule compound or a biologically active molecule nucleic acid such as a protein-encoding DNA or mRNA molecule, a protein such as an antibody, etc.
  • the complex molecules that form cells capable of recognizing and binding to the lateral nucleus are joined.
  • the above-described method for treating depression by locally inhibiting the activity of Kir4.1 in the lateral nucleus and the Kir4.1 inhibitor are locally effective in the preparation of the lateral nucleus in the present invention.
  • the Kir4.1 inhibitor may also be a selective serotonin reuptake inhibitor (buspirone, mianserin, fluoxetine). ), sertraline or fluvoxamine, and tricyclic antidepressants (nortriptyline, etc.).
  • the invention also provides a pharmaceutical composition for treating depression.
  • the present invention provides a novel pharmaceutical composition for treating depression comprising a therapeutically effective amount of a Kir4.1 inhibitor.
  • the Kir4.1 inhibitor is as defined above.
  • the Kir4.1 inhibitor is an interfering RNA or a precursor thereof that interferes with the expression of Kir4.1, which has a target Kir4.1 mRNA Fragments are identical or complementary, or more than 90% identical or complementary sequences.
  • the interfering RNA or its precursor has the following sequence:
  • the Kir4.1 inhibitor in the pharmaceutical composition for treating depression, is a mutant Kir4.1 protein having a decreased or lost potassium channel activity or a coding sequence thereof, the mutation
  • the type Kir4.1 protein is preferably a mutant Kir4.1 protein which is mutated in the nuclear pore region of the Kir4.1 protein and corresponds to the GYG at positions 130-132 in the amino acid sequence of SEQ ID No. 8 of the Kir4.1 sequence, for example, Mutant Kir4.1 protein mutated to AAA.
  • the pharmaceutical composition for treating depression provided by the present invention is a specific antibody of Kir4.1, including a polyclonal antibody or a monoclonal antibody.
  • the Kir4.1 inhibitor is not buspirone, miansine Mianserin, fluoxetine, sertraline, fluvoxamine or nortriptyline.
  • the pharmaceutical composition for treating depression described above the Kir4.1 inhibitor is not a selective serotonin reuptake inhibitor (SSRIs) or tricyclic antidepressants (TCAs).
  • compositions provided by the present invention are particularly suitable for use in other depression patients and drug-ineffective depression patients.
  • the pharmaceutical composition provided by the present invention is a pharmaceutical composition provided by the present invention which is locally active in the lateral nucleus.
  • it is a dosage form for topical administration to the lateral nucleus.
  • the Kir4.1 inhibitor may also be a selective serotonin reuptake inhibitor (fluoxetine, sertraline, fluvoxamine, butyl) Buspirone, mianserin, etc. and tricyclic antidepressants (nortriptyline, etc.).
  • the invention also provides an animal model of depression, preferably a rat or a mouse.
  • the animal model of depression according to the present invention has a characteristic of depression, and Kir4.1 is highly expressed in the lateral habenular nucleus.
  • the present invention also provides a method for screening potential substances for treating depression using the above animal model, comprising the steps of:
  • test substance is a potential substance that can be used to treat depression.
  • a method of screening a potential substance for treating depression comprising the steps of:
  • the test substance is prevention and/or treatment of depression. Potential substance.
  • the method of screening for a potential substance for treating depression further comprises one or more of the following steps:
  • Test substances are potential substances for the treatment of depression.
  • Kir4.1 potassium voltage-gated channel subfamily J member 10
  • Kcnj10 potassium voltage-gated channel subfamily J member 10
  • Kir4.1 in glial cells allows potassium ions to pass through the cell membrane, regulates the extracellular environment by regulating the potassium ion concentration of the outer fluid around the nerve cells, and transports extracellular potassium ions to control the resting membrane potential level. Maintains the homeostasis of the nervous system and maintains the normal physiological activity of the nervous system. In mammals, the protein sequence and encoding nucleic acid sequence of Kir4.1 are both conserved.
  • the gene encoding the human Kir4.1 protein is KCNJ10 (Ensembl: ENSG00000177807).
  • the gene encoding the rat Kir4.1 protein is KCNJ10 (Ensembl: ENSMUSG00000044708).
  • treatment includes: an ongoing process or result of ameliorating, alleviating, reducing or preventing symptoms associated with depression; an ongoing process or result of ameliorating symptoms associated with depression; causing a particular organism to be caused An ongoing process or result of normalization of the body's function in a disease or condition that is impaired; or an ongoing process or result that results in the improvement of one or more clinically measurable parameters of the disease.
  • the therapeutic goal is to prevent or slow (reduce) undesired physiological conditions, conditions or diseases, or to obtain beneficial or desired results.
  • the result may be, for example, medical, physiological, clinical, physical therapy, occupational therapy, to a health care professional or patient; or as a parameter in the art as "quality of life" or activities of daily living.
  • beneficial or desired clinical outcomes include, but are not limited to, alleviating symptoms; reducing/reducing the extent of the condition, disorder or disease; stabilizing (ie, not worsening) the condition, condition or state of the disease; delaying the condition , the onset or slowing of the progression of the condition or disease; the improvement or alleviation of the condition, disorder or disease; and the reduction (whether partial or general), whether detectable or undetectable; or the enhancement or amelioration of the condition, condition Or disease.
  • the treatment comprises eliciting a clinically effective response without excessive levels of side effects.
  • the treatment also includes prolonging the survival period as compared to the expected survival if not receiving treatment.
  • treatment refers to administering a drug or performing a medical procedure on a patient.
  • the treatment may be prevention (prevention), cure of weakness or disease, or improvement of the clinical condition of the patient, including lowering the course of the disease or the severity of the disease, or subjectively improving the quality of life of the patient or prolonging the survival of the patient.
  • cluster discharge refers to a discharge pattern in which two or more spikes are simultaneously generated by a neuron during discharge.
  • Suppressing cluster discharge refers to suppressing the degree of distribution of cluster discharge, including reducing the frequency of cluster discharge or the number of peak potentials in the cluster during discharge, reducing the intensity of cluster discharge, and even eliminating the occurrence of cluster discharge.
  • single dispense or “single discharge” is a discharge pattern in which a neuron issues a spike each time during discharge.
  • a method of diagnosing depression comprising detecting the expression of Kir4.1 of the lateral nucleus of the patient.
  • the method of diagnosing depression is to detect an increase in the expression of Kir4.1 of the lateral nucleus of the patient.
  • the method of diagnosing depression comprises comparing Kir4.1 of the lateral nucleus of the patient at different times (eg, at different stages of depression, or before or after treatment) The expression, or the expression of Kir4.1 in the lateral habenular nucleus of a normal person in a general or a specific population, is significantly increased, and the subject is judged to be a depressed patient.
  • expression of Kir4.1 can be detected by any method known in the art for detecting protein (expression) in a sample.
  • the method can check for the presence or absence of Kir4.1.
  • the method can also quantitatively detect the amount of expression of Kir4.1.
  • a method for detecting a protein (expression) in a sample usable in the present invention includes an immunoassay.
  • ELISA or Western blotting is performed by an antibody that specifically recognizes Kir4.1.
  • Antibodies can be monoclonal or polyclonal.
  • the method for detecting a protein (expression) in a sample usable in the present invention further comprises detecting the presence or amount of mRNA of Kir4.1, for example, detecting the amount of mRNA of Kir4.1 or a fragment thereof in the sample by RT-PCR.
  • the test sample is from the lateral nucleus of the subject. In one aspect of the invention, the test sample is from an ex vivo sample. In one aspect of the invention, detection of the expression of Kir4.1 can also be performed by in vivo observation and detection of the amount of Kir4.1 of the lateral nucleus of the subject. For example by imaging the lateral nucleus of the patient, for example for PET imaging. The positron emission tomography (PET) scan was performed on the lateral nucleus by identifying and displaying a positive electron emission radionuclide tracer of Kir4.1 in an intravenous injection.
  • PET positron emission tomography
  • Figure 1 shows the up-regulation of Kir4.1 expression and function in the lateral habenular nucleus of depressed rats.
  • A, B Western Blot experiments showed that the expression of Kir4.1 in the nucleus of rats with cLH rats and LPS (lipopolysaccharide)-induced depression was significantly higher than that of the control group.
  • the tubulin Tubulin which is abundantly expressed in tissues, is used for loading control. Protein expression was quantified by quantification.
  • C qPCR analysis of Kir4.1 mRNA in the nucleus.
  • D, E adult cLH rats (60-90 days old) but not in juvenile rats (30 days old), the Ba+-sensitive current (mediated by the Kir4.1 channel) in the lateral habenula was significantly increased.
  • FIG. 2 shows that Kir4.1 is expressed on the neurite outgrowth of the axillary nucleus and tightly surrounds the neuronal cell body.
  • A Immunofluorescence double-labeled staining showed that the Kir4.1 immunopositive signal was wrapped around the cell body of the neuron.
  • FIG. 3 Cellular localization of Kir4.1 in the lateral nucleus and hippocampus.
  • A Colocalization of Kir4.1 with the neuronal marker NeuN, astrocyte markers GFAP and S100b in the lateral habenular nucleus;
  • B Kir4.1 and neuronal marker NeuN in the hippocampal CA1 region, star Colocalization of glial cell markers GFAP and S100b.
  • the bottom two panels show that the Kir4.1 antibody was not adsorbed by the Kir4.1 extracellular peptide antigen in the lateral nucleus and hippocampal CA1, and no positive signal was detected, suggesting that the antibody used is specific for Kir4.1.
  • Figure 4 shows that the extracellular potassium concentration of the lateral nucleus is highly correlated with neuronal activity and is regulated by astrocyte Kir4.1.
  • AC Extracellular perfusion of artificial cerebrospinal fluid containing BaCl2 blocks the Kir4.1 channel on the star glue, which can significantly increase the resting membrane potential of clustered and tonic neurons in the lateral nucleus. The meta is expressed as depolarization.
  • D-F The degree of depolarization of neurons is positively correlated with the frequency of self-issue.
  • G A typical map of the effects of BaCl2 on neuronal firing.
  • BaCl2 significantly reduces the frequency of clustered dispensing per minute of clustered cells.
  • FIG. 5 Overexpression of astrocyte Kir4.1 increases the proportion of neuron clustered cells and leads to a depressive phenotype.
  • A Schematic diagram of viral vector construction of astrocyte Kir4.1 overexpression.
  • B Immunofluorescence showed that the virus in the lateral habenular nucleus overexpresses Kir4.1 protein in astrocytes.
  • C, D Kir4.1 overexpression significantly decreased the resting membrane potential of both neurons and astrocytes, showing hypersensitivity.
  • E Overexpression of astrocyte Kir4.1 increases the proportion of neuron clustered cells
  • F Overexpression of astrocyte Kir4.1 increases the proportion of high frequency release cells
  • G astrocyte Cell Kir4.1 Overexpressed Mouse Depression Phenotype Detection Timeline
  • H Astrocyte Kir4.1 Overexpressing Mice showed a significant depressive phenotype in both forced swimming and sacrifice preference experiments. All data are expressed as mean ⁇ SEM. *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001, ****P ⁇ 0.0001 compared to the control group. N.S. indicates that the difference is not significant.
  • FIG. 6 shows that down-regulating the function of Kir4.1 in LHb can reduce the proportion of cluster-distributed neurons in neurons and effectively alleviate depressive symptoms.
  • A Schematic representation of AAV viral vectors for down-regulating RNAI expression levels of Kiri and Kir4.1 dominant mutations downregulating Kir4.1 function.
  • B Western blot analysis showed that Kir4.1-shRNA can effectively reduce Kir4.1 overexpressing cells in HEK293 cells cultured in vitro.
  • C The above figure shows that Kir4.1 function down-regulates rats for electrophysiological experiments and The timeline of behavioral testing. The immunofluorescence shown below shows that Kir4.1 dominant mutant virus is expressed in lateral axillary astrocytes.
  • D Kir4.1 down-regulated astrocyte reversal point elevation.
  • FIG. 7 In vitro validation of different Kir4.1 shRNA knockout efficiencies. Western blotting was used to detect the exogenously expressed Kir4.1 in HEK293 cells cultured in vitro, and the efficiency of knockdown of shRNA with different sequences of Kir4.1 by 6 pairs was detected.
  • FIG. 8 LHb virus overexpression of Kir4.1 or down-regulation of Kir4.1 did not affect the animal's ability to exercise.
  • A AAV2/5-gfaABC1D-EGFP-Kir4.1 virus expression, after LHb overexpressed Kir4.1, there was no significant difference in the total distance of movement and central zone residence time of mice in the mine compared with the GFP control group.
  • B AAV2/5-Kir4.1 shRNA virus expression, after the down-regulation of Kir4.1 in LHb, the total movement distance and central zone residence time of cLH rats in the open field were no different from the control virus.
  • cLH rats Male cLH rats (4-12 weeks old), Sprague Dawley rats (4-12 weeks old) and male Wistar rats (12 weeks old).
  • cLH rats are a selectively cultured animal model of depression with a congenital acquired helpless depression phenotype (D. Schulz, M. M. Mirrione, F. A. Henn, Neurobiol Learn Mem 93, 291, Feb, 2010).
  • the cLH rats of this experiment were introduced from the Malinow Laboratory in Cold Spring Harbor, USA. CLH rat feeding and propagation are described in the aforementioned D. Schulz, et al, Feb, 2010. Rats 4/cage, 12-hour light-dark cycle (light at 7am-7pm).
  • One cLLH rat/cage was used for cannulation experiments.
  • Kir4.1 overexpressed the virus AAV2/5-gfaABC1D-EGFP-Kir4.1.
  • Kir4.1 overexpression plasmid pZac2.1gfaABC1D-EGFP-Kir4.1, purchased from AddGene, Cat. No.: Plasmid #52874. It was coated by Shanghai Taiting Biotechnology Co., Ltd. into AAV2/5 virus, namely AAV2/5-gfaABC1D-EGFP-Kir4.1.
  • AAV2/1-CamKII-HI-eGFP-Cre purchased from the University of Pennsylvania Vector Core, Upenn, USA, Cat#: AV-1-PV2521.
  • Kir4.1 down-regulates virus AAV2/5-H1-Kir4.1-shRNA-gfaABC1D-EGFP (interfering RNA-shRNA of Kir4.1: 5'-GCGTAAGAGTCTCCTCATTGG-3'), control virus AAV2/5-H1-Luciferase-shRNA -gfaABC1D-EGFP; Kir4.1 dominant mutant virus AAV2/5-gfaABC1D-Kir4.1dn-2A-EGFP (the nuclear region of the Kir4.1 sequence has a GYG mutation of AAA to achieve a dominant mutation of Kir4.1). Plasmids and coatings were prepared by Shanghai Taiting Biotechnology Co., Ltd.
  • 12-week-old male Wistar rats were intraperitoneally injected with LPS (500 ⁇ g/kg body weight) once a day for 7 days, and 24 hours after the last injection, FST test was performed. The nucleus tissue was removed 3 days after the end of the test. Volume analysis.
  • mice were anesthetized with a mixture of ketamine (100 mg/kg body weight) and xylazine (8 mg/kg) and fixed on a stereotaxic instrument (Stoelting instruments). Each mouse was injected with 0.1-0.2 ul of purified AAV virus ( ⁇ 1013 infectious units/ml) per LHb, LHb stereotactic coordinates (front and back distance Bregma: -1.7 mm (AP), left and right side ⁇ 0.46 mm (ML ), the surface of the cortex is down -2.56 mm (DV)). Slowly inject ( ⁇ 100-150nl/min) using a self-drawn glass microelectrode, leave the needle at the end of the injection for 5 min, and then slowly remove the injection electrode within 5 min.
  • AAV virus ⁇ 1013 infectious units/ml
  • LHb stereotactic coordinates front and back distance Bregma: -1.7 mm (AP), left and right side ⁇ 0.46 mm (ML )
  • ML
  • AAV virus ⁇ 10 13 infectious units/ml
  • LHb stereotactic coordinates front and back distance Bregma: -3.7 mm (AP), ⁇ 0.7 mm left and right (ML), the surface of the cortex is down - 4.55
  • Behavioral experiments or electrophysiological experiments were performed at least 14 days after surgery. At the end of the behavioral experiment, the injection site was examined and only the animal data for the correct injection was used.
  • the GFP-labeled virus was injected with an antibody to examine the GFP protein prior to microscopic examination.
  • the sacral region of each brain was cut into 6 consecutive sections (30 ⁇ m sections of mice, 6 per group; 40 ⁇ m of rats)
  • the antibody concentrations were as follows: anti-Kir4.1 (1:200, Alomone labs), anti-NeuN (1:500, chemicon), anti-GFAP (1:500, Chemicon), anti-S100b (1:500, Invitrogen) , ABCAM), anti-GFP (1:1000, abCaM). Alexa Fluor 488 goat anti-rabbit IgG, Alexa Fluor 488 goat anti-chicken IgG, Alexa Fluor 594 goat anti-mouse IgG (all 1: 1000, Invitrogen), Hoechst (1:5000) fluorescence image taken with Olympus Fluoview FV1000 and NiconA1 laser confocal microscope image.
  • the brain was decapitated.
  • the nucleus tissue is rapidly separated on ice and frozen in liquid nitrogen.
  • the sample was subjected to tissue disruption in the homogenate.
  • the tissue homogenate was centrifuged at 800 g for 15 minutes at 4 degrees.
  • the supernatant was taken and centrifuged at 10,000 g for 15 min.
  • the precipitate is the membrane protein component which is then dissolved in the homogenate.
  • Samples of the 293TN cell line were subjected to 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 ⁇ g/ml aprotinin, 1 ⁇ g/ml pepstatin).
  • 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 ⁇ g/ml aprotinin, 1 ⁇ g/ml pepstatin.
  • the supernatant was taken by centrifugation at 10,000 g for 15 min.
  • the protein concentration was determined by the BCA method, 8-15 ⁇ g of protein was loaded per well, and 10% SDS-PAGE gel was used for separation,
  • the primary antibodies used were anti-Kir4.1-introcellular (1:1000, Alomone labs), anti-tubulin (1:10000, Bio-Rad), and the quantitative analysis of immunoblot results was performed using Quantity One or image J software for statistical analysis. .
  • Rats 40-50 days after birth or mice 8 weeks old were anesthetized with isoflurane and perfused with 20 ml of ice-cold oxygenated section.
  • the brain is quickly decapitated and placed in an oxygenated section.
  • a 350 ⁇ m coronal section was then sectioned in an oxygenated ice-cold slice using a Leica vibratome.
  • the nucleus slices were recovered in an oxygenated 34 ° C ACSF (118 mM NaCl, 2.5 mM KCl, 26 mM NaHCO 3 , 1 mM NaH 2 PO 4 , 10 mM glucose, 1.3 mM MgCl 2 and 2.5 mM CaCl 2 , gassed with 95% O 2 and 5% CO 2 ). After two hours, transfer to room temperature for recording.
  • the patch clamp recording of the lateral habenular nucleus was recorded using an Axon Multiclamp 700B amplifier under an Olympus microscope equipped with an infrared differential interference phase contrast optical lens at 32 ⁇ 1 °C.
  • the impedance of the astrocyte recording electrode is 7-10 M ⁇ , and the impedance of the neuron recording electrode is 4-6 M ⁇ . All cells were recorded in whole cell mode. Based on the morphology of the recorded cells, the electrophysiological properties were recorded to determine whether they were neurons (15-20 micrometer diameter) or astrocytes (5-10 micrometer diameter).
  • Kir4.1 current calculation method subtract the voltage and current curves of strontium chloride (100Mm, which specifically antagonizes Kir4.1 current) from the voltage and current curves recorded in normal artificial cerebrospinal fluid. The data was filtered at 2 kHz and recorded using a Digidata 1322A at 10 kHz. Data were analyzed using the pClamp 10 software.
  • LH learned helpless
  • NLH non-acquired helpless
  • the experiment was carried out under normal fluorescent lighting.
  • the mouse forced swimming cylindrical container has a diameter of 12 cm and a height of 25 cm.
  • the test water depth is 14 cm and the water temperature is 23-24 °C.
  • the camera recorded the swimming of the mice within 6 min from the side.
  • the double-blind method was used to count the immobility time (the floating position of the animal or the time when the limbs were completely inactive) after 4 minutes of swimming in the mouse.
  • mice were housed separately for 1 week, and then the mice were given two bottles of normal water for 2 consecutive days, and then the water was exchanged for two bottles of 2% sucrose water for training for two days. After the training, give the animal a bottle of normal water and a bottle of 2% sucrose water for testing, exchange the position of the water bottle every 12 hours, record the consumption of water and sugar water every 24 hours (weigh the water bottle), record a total 48 hours.
  • LPS lipopolysaccharide
  • a and B in Figure 1 are the results of Western blotting experiments, showing that the expression of Kir4.1 in the lateral habenula of rats with depression induced by cLH rats and LPS (lipopolysaccharide) was significantly higher than that of the control group.
  • a in Figure 1 shows that the expression level of Kir4.1 protein in the nucleus membrane component of cLH rats is 1.75 times higher than that of the control.
  • B in Figure 1 shows that the expression level of Kir4.1 in the lateral nucleus is also significantly increased (1.87 fold) in LPS-induced depression rats.
  • Kir4.1 was distributed in the lateral nucleus in a manner that was expressed around the cell body of the neuron.
  • the expression of Kir4.1 is mainly star-shaped. (As shown in Figure 3)
  • Figure 2 shows that Kir4.1 is expressed on the neurite outgrowth of the axillary nucleus and tightly surrounds the neuronal cell body.
  • FIG 2 A shows that immunofluorescence double-labeled staining shows that the Kir4.1 immunopositive signal is wrapped around the cell body of the neuron.
  • Kir4.1 was observed and judged by applying a conditional knockout method.
  • Conditional knockout was achieved by injection of adenovirus AAV2/1-CaMKII-EGFP-Cre into the lateral nucleus of Kir4.1 conditional knockout mice.
  • conditional knockout of Kir4.1 in the nucleus of the lateral nucleus the phenomenon of Kir4.1 surrounding the neuron cell body can still be seen; but the injection of AAV2/5-GFAP-EGFP-Cre virus After knocking out Kir4.1 in the axillary nucleus of the lateral nucleus, the wrapping phenomenon disappeared.
  • Kir4.1 immunogold particles were distributed around the neuronal cell membrane and less at the synaptic sites of the neurons (as shown in C of Figure 2).
  • Figure 3 shows the cellular localization of Kir4.1 in the lateral nucleus and hippocampus.
  • Figure 3 A shows colocalization of Kir4.1 in the lateral nucleus with the neuronal marker NeuN, astrocyte markers GFAP and S100b;
  • B in Figure 3 shows Kir4.1 and neuronal markers in the hippocampal CA1 region NeuN, colocalization of astrocyte markers GFAP and S100b.
  • the bottom two panels in Figure 3 show that the Kir4.1 antibody was not adsorbed by the Kir4.1 extracellular peptide antigen, and no positive signal was detected in the lateral nucleus and hippocampal CA1, suggesting that the antibody used is specific for Kir4.1. .
  • Example 5 Kir4.1 causes changes in electrophysiological function of neurons and astrocytes by modulating extracellular potassium concentration
  • Example 6 In vivo experiments in animals showed that overexpression of Kir4.1 in the lateral habenular nucleus caused symptoms of depression
  • the Kir4.1 dominant mutant virus pAAV-gfaABC1D-Kir4.1dn-2A-EGFP was constructed (the GYG mutation in the nuclear pore region of the Kir4.1 sequence was AAA to achieve a dominant mutation of Kir4.1).
  • gfaABC1D Fusion Fw a linear fragment of pZac2.1 gfaABC1D deleted from EGFP-Kir4.1 was amplified; then pAAV-Ubi-Kir4.1dn-2A-EGFP plasmid was used as a template, Kir4.1dn-2A-eEGFP Fusion Rev and Kir4 .1dn-2A-eEGFP Fusion Fw is a primer that amplifies the Kir4.1dn-2A-EGFP fragment.
  • the pAAV-gfaABC1D-Kir4.1dn-2A-EGFP plasmid was obtained after homologous recombination of the above two linearized fragments.
  • FIG. 5 shows a schematic representation of the construction of a viral vector for overexpression of Kir4.1 in astrocytes.
  • B of Figure 5 is an immunofluorescence showing that the virus in the lateral purine nucleus overexpresses the Kir4.1 protein in astrocytes.
  • LHb brain slices overexpressing Kir4.1 were recorded using whole-cell patch clamp, and the resting membrane potentials of astrocytes and neurons were significantly superimposed (as shown in D and E in Figure 5), and the nerves were The proportion of metacluster distribution was also significantly higher than that of the control GFP virus group (shown as F, G in Figure 5).
  • the overexpression of Kir4.1 in LHb also significantly increased the immobility of mice in forced swimming, which significantly reduced the preference of mice for syrup (as shown in H, I in Figure 5). There was no change in exercise capacity (shown in A of Figure 8).
  • Example 7 Down-regulation of Kir4.1 expression level in lateral nucleus or Kir4.1 function to reverse depression phenotype
  • the short hairpin RNA (shRNA) of Kir4.1 was expressed by AAV2/5 virus to down-regulate the expression level of LHb Kir4.1 protein in cLH rats.
  • the shRNAs of the six Kir4.1 interfering RNAs constructed according to the following table were cloned into the WX231-L vector (purchased from Shanghai Taiting Biotechnology Co., Ltd., Cat#: WX231):
  • the primer sequences used for cloning are as follows:
  • Figure 7 is the result of Western blotting.
  • a protein luciferase not expressed in animals was used as a negative control (NC).
  • AAV2/5-H1-Luciferase (luciferase)-shRNA-gfaABC1D-EGFP expresses NC-shRNA as a control virus.
  • the knockout efficiency of Kir4.1shRNA is: Kir4.1-shRNA-1>Kir4.1-shRNA-4, Kir4.1-shRNA-5>Kir4.1-shRNA-2>Kir4.1-shRNA-6>Kir4 .1-shRNA-3.
  • Kir4.1-shRNA-5 was packaged into a virus with AAV2/5 as a carrier for down-regulating rat experiments with Kir4.1 function.
  • Figure 6 A is a schematic representation of the AAV viral vector for down-regulating RNAI expression levels of RNAi and down-regulating Kir4.1 function of Kir4.1 dominant mutations.
  • Figure 6B shows the results of Western blot analysis, showing that Kir4.1-shRNA can effectively reduce Kir4.1 overexpressed on cells in HEK293 cells cultured in vitro.
  • the upper panel of Figure 6 shows that the Kir4.1 function down-regulates the time axis of electrophysiological and behavioral testing in rats; the immunofluorescence in the lower panel shows that Kir4.1 dominant mutant virus is expressed in lateral axillary astrocytes. .
  • FIG. 6A shows the structure of the Kir4.1 dominant mutant virus AAV2/5-gfaABC1D-Kir4.1dn-2A-EGFP (the GYG mutation of the nuclear pore region amino acid sequence 130-132 of the Kir4.1 sequence is AAA to achieve Kir4. a dominant mutation in 1).
  • AAV-dnKir4.1-EGFP virus significantly reversed the depression phenotype of cLH rats: the forced swimming inactivity of the rats was significantly reduced (as shown in I of Figure 6), learned helpless
  • the rod behavior is significantly increased (as shown by J, K in Fig. 6), and the syrup preference is also significantly increased (as shown by L in Fig. 6).
  • Kir4.1 in astrocytes of the lateral habenular nucleus is a crucial regulator of depression, and uses molecular, behavioral, and electrophysiological methods to discover Kir4.1.
  • a tightly wrapped nucleus of the nucleus of the nucleus it exists in the lateral nucleus, regulates the extracellular potassium balance, changes the distribution characteristics of the lateral nucleus, and leads to excessive activation of the lateral nucleus, which in turn regulates the phenotype of depression.
  • the present inventors have also discovered and demonstrated various agents which block the activity of Kir4.1 in the lateral nucleus, thereby providing methods and medicaments for treating (suppressing) depression by inhibiting the activity of Kir4.1.
  • the unit "degree” of temperature appearing in this document refers to degrees Celsius, or °C.

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Abstract

本发明提供了钾离子通道蛋白 Kir4.1 抑制剂在制备治疗抑郁症的药物中的用途。本发明还提供了治疗抑郁症的药物组合物，其中包含 Kir4.1 抑制剂。本发明还提供了筛选治疗抑郁症的潜在物质的方法，包括给抑郁症动物模型施用待筛选的测试物的步骤，其中所述抑郁症动物模型的外侧缰核中 Kir4.1 是高表达的。

Description

钾离子通道抑制剂治疗抑郁症的用途和药物组合物
本申请要求2017年5月9日提交的、申请号为201710322245.X、发明名称为“Kir4.1抑制剂治疗抑郁症的用途和药物组合物”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及疾病治疗和药物领域。具体的，本发明涉及抑郁症的治疗和用于治疗抑郁症的药物组合物和其制备方法。
背景技术
抑郁症是一种慢性精神障碍性疾病，以显著而持久的心境低落,动力缺失，行为绝望以及快感缺失为主要临床特征，严重的可表现出自杀倾向。
外侧缰核(LHb)近年来被认为是研究抑郁症病理生理学的关键脑区，在众多抑郁症的动物模型以及抑郁症病人中都发现了外侧缰核活性的显著升高。
神经元的异常活动主要归因于突触传递异常、自身生理特性变化以及神经元内环境的改变。星形胶质细胞参与调节神经细胞的自身活性，递质释放，而且在一系列包括精神分裂、癫痫、老年痴呆、抑郁症等在内的疾病中扮演重要角色(Hamilton et al.,Frontiers in neuroenergetics2,2010)。对抑郁症死者大脑的解剖学研究表明，其前脑边缘系统中，胶 质细胞的数目，形态及功能均发生显著变化(Cotter et al.,Archives of general psychiatry 58,545-553,2001；Coyle et al.,Archives of general psychiatry 57,90-93,2000；Rajkowska et al.,CNS&neurological disorders drug targets 6,219-233,2007)。利用化学手段诱导星形胶质细胞凋亡足以引起抑郁症状(Banasr et al.,Biological psychiatry 64,863-870,2008)。抗抑郁药物能直接作用于星形胶质细胞，显著影响其形态及功能。该现象被认为可能是抗抑郁药物在中枢神经系统中起到抗抑郁效应的途径之一(Czeh et al.,the journal of the European College of Neuropsychopharmacology 23,171-185,2013)。这些证据表明，除了神经元，胶质细胞在精神疾病中也扮演了不容忽视的角色。
内向整流型钾离子通道(inward rectifier-type potassium channel，Kir)是指超级化激活的钾离子通道。已报道的内向整流型钾离子通道有七个蛋白家族成员(Kir1～Kir7)。同类Kir通道因为存在RNA剪接的差异(splicing variance)又可分为多种亚型，在心脏，肾脏，神经系统等多种组织和器官中分布。Kir4.1(也称为potassium voltage-gated channel subfamily J member 10，Kcnj10)是内向整流型钾离子通道的家族成员之一。在神经系统中，Kir4.1在胶质细胞特异性表达。胶质细胞中的Kir4.1可允许钾离子在细胞膜上通过，通过调节神经细胞周围外液的钾离子浓度，转运胞外过多的钾离子来缓冲细胞外环境，达到控制静息膜电位水平，维持神经系统的内稳态，保持神经系统的正常生理活性的作用。Kir4.1的功能异常将对胶质细胞和神经元的功能产生很大的影响，进而表现出多种神经系统病变。在哺乳动物中，Kir4.1的蛋白序列和编码核酸序列 都很保守。
本领域已经具有一些常用的抗抑郁药物，但这些药物通常在比较长的一段时间后才能见效。而且导致抑郁症的病理机制还未完全被认识。本领域还需要新的，或是起效更快速、使用剂量更安全的治疗抑郁症的方法和药物。
发明内容
本发明人经过深入研究，首次发现外侧缰核的星形胶质细胞中的Kir4.1是抑郁症的一个至关重要的调节因子，运用分子、行为和电生理等手段，确定了在外侧缰核中，星形胶质细胞表达的Kir4.1，以一种紧密包绕缰核神经元胞体的方式存在于外侧缰核中，调节胞外钾平衡，改变外侧缰核神经元的发放特性，导致外侧缰核过度活跃，进而调节抑郁表型。本发明人还发现和证明了多种可以阻断外侧缰核中Kir4.1功能的试剂，由此提供了通过抑制Kir4.1的活性来治疗(抑制)抑郁症的方法和药物。
具体的，本发明提供了通过抑制Kir4.1的活性来治疗患者的抑郁症的方法。本发明还提供了Kir4.1抑制剂在制备治疗患者的抑郁症的药物中的用途。
需要本文所述的方法和药物(药物组合物)的患者为哺乳动物，包括人或者非人灵长类如猴。哺乳动物可以是其它动物，例如大鼠、小鼠、兔、猪、狗等。所述哺乳动物可以是家养动物，例如猫或者狗。
Kir4.1抑制剂是指能够使得Kir4.1通道的活性下降或者丧失的试剂。 Kir4.1通道的活性是指允许钾离子在细胞膜上通过的活性。Kir4.1通过调节神经细胞周围外液的钾离子浓度，转运胞外过多的钾离子来缓冲细胞外环境，达到控制静息膜电位水平，影响神经系统的生理活性。Kir4.1抑制剂包括能够使得Kir4.1通道的活性下降或者丧失的化合物、复合物或混合物，以及在抑制Kir4.1活性的方法(含外科手术方法)中使用的制剂等。
在本发明的通过抑制Kir4.1的活性来治疗抑郁症的方法和Kir4.1抑制剂在制备治疗抑郁症的药物中的用途中，所述Kir4.1抑制剂是指能够影响Kir4.1蛋白的活性从而影响Kir4.1通道的活性的试剂。所述试剂包括小分子化合物或复合物，或是蛋白、核酸等大分子活性成分，例如与Kir4.1蛋白结合的拮抗剂如抗体，或是影响Kir4.1蛋白的表达水平的核酸等。这些蛋白或核酸可通过本领域公知的技术，例如与合适的表达载体结合传送到目标组织或细胞发挥作用。
在本发明的其中又一个方面，Kir4.1抑制剂是特异性抑制Kir4.1的抑制剂。特异性Kir4.1抑制剂一般是指，所述Kir4.1抑制剂对其它Kir蛋白没有抑制活性，或是对其它Kir蛋白的抑制活性显著小于对Kir4.1的抑制活性，例如对其它Kir蛋白的抑制活性为对Kir4.1的抑制活性的50％以下，优选20％以下，更优选5％以下。
在本发明的其中又一个方面，所述Kir4.1抑制剂为干扰Kir4.1表达的干扰RNA或其前体。RNA干扰(RNAi)是通过双链RNA(double-stranded RNA，dsRNA)来诱发同源mRNA高效特异性降解，从而使得目标基因的表达降低甚至消除。在本发明中，干扰RNA可包括 小干扰RNA(Small interfering RNA，siRNA)、短发夹RNA(shRNA)和/或微小RNA(miRNA)。在体内给予干扰RNA的一种方式是通过给予siRNA的前体shRNA实现，例如包括两个短反向重复序列的短发夹RNA。将siRNA序列作为“短发夹”克隆进质粒载体中。当送入动物体内时，该发夹序列被表达出来，形成一个“双链RNA”，利用细胞内的Dicer酶，生成相应的siRNA，发挥RNAi作用。
在本发明的其中又一个方面，可用于本发明的干扰RNA或其前体具有与目标Kir4.1 mRNA的片段具有相同或互补，或90％以上相同或互补的序列。例如，在本发明的其中又一个方面，所述干扰Kir4.1表达的干扰RNA或其前体具有以下序列：
5’-GGACGACCTTCATTGACAT-3’(SEQ ID No.1)；
5’-GCTACAAGCTTCTGCTCTTCT-3’(SEQ ID No.2)；
5’-GCTCTTCTCGCCAACCTTTAC-3’(SEQ ID No.3)；
5’-CCGGAACCTTCCTTGCAAA-3’(SEQ ID No.4)；
5’-GCGTAAGAGTCTCCTCATTGG-3’(SEQ ID No.5)；或
5’-GCCCTTAGTGTGCGCATTA-3’(SEQ ID No.6)。
上述干扰RNA或其前体针对具有SEQ ID No.7(即Genebank编号NM_031602.2的序列中的CDS区序列)的大鼠Kir4.1 mRNA序列的对应mRNA片段的序列，即目标大鼠Kir4.1 mRNA片段的序列与所述SEQ ID No.1-6的干扰RNA的序列相同或互补，或90％以上序列相同或互补。
本领域的技术人员可以理解和得到在其它哺乳动物(如人、小鼠等)中对应的所述Kir4.1的干扰RNA序列片段。
在本发明的其中又一个方面，所述Kir4.1抑制剂为钾离子通道活性下降或丧失的突变型Kir4.1蛋白或其编码序列。突变型蛋白可与正常蛋白发生竞争性作用，从而降低正常蛋白发挥的活性。突变型蛋白可通过给予在目标组织或细胞可表达的载体(载体上携带可表达的突变型蛋白基因和/或其表达因子)等方式在目标组织或细胞表达。在本发明的其中又一个方面，所述突变型Kir4.1蛋白为在Kir4.1蛋白的核孔区，例如在对应Kir4.1序列为SEQ ID No.8的氨基酸序列中第130-132位的GYG发生突变的突变型Kir4.1蛋白，例如GYG突变为AAA的突变型Kir4.1蛋白。具有序列为SEQ ID No.8的氨基酸序列的Kir4.1是大鼠的Kir4.1(NP_113790.2)。本领域的技术人员可以根据已有的报道(例如Hiroshi et al.,2010.Physiological Reviews 90,291-366,2010)，以及对Kir4.1的序列的保守性的理解，得到在其它哺乳动物中对应的所述Kir4.1的核孔区突变位点的序列以及其它突变方式。
在本发明的其中又一个方面，所述Kir4.1抑制剂为Kir4.1的特异性抗体，包括多克隆抗体或单克隆抗体。。
本领域存在一些个别的报道某些化合物可用于抗抑郁。例如，丁螺环酮(buspirone)，米安色林(mianserin)，氟西汀(fluoxetine)，舍曲林(sertraline)，氟伏沙明(fluvoxamine)或去甲替林(nortriptyline)。但在这些报道中，其抗抑郁的机制与本发明发现的机制，即外侧缰核的星形胶质细胞表达的Kir4.1通过抑制外侧缰核神经元的异常发放，特别是簇状放电的异常发放来抑制抑郁症，完全不同。在不破坏本发明的创新性的情况下，在本发明的其中一个方面，本发明提供的上述通过抑制 Kir4.1的活性来治疗抑郁症的方法和Kir4.1抑制剂在制备治疗抑郁症的药物中的用途中，所述Kir4.1抑制剂不为丁螺环酮(buspirone)，米安色林(mianserin)，氟西汀(fluoxetine)，舍曲林(sertraline)，氟伏沙明(fluvoxamine)或去甲替林(nortriptyline)。在不破坏本发明的创新性的情况下，在本发明的其中一个方面，本发明提供的上述通过抑制Kir4.1的活性来治疗抑郁症的方法和Kir4.1抑制剂在制备治疗抑郁症的药物中的用途中，所述Kir4.1抑制剂不为选择性五羟色胺重摄取抑制剂(SSRIs)或三环类抗抑郁药(TCAs)。
在本发明中，所述抑郁症可以特别指“外侧缰核介导的抑郁症”。本申请的发明人发现了外侧缰核的神经元的异常发放，特别是簇状放电的异常发放在抑郁症的产生中具有重要作用。本申请的发明人还发现了外侧缰核的星形胶质细胞中的Kir4.1是抑郁症的一个至关重要的调节因子，并发现和证明了多种可以阻断外侧缰核中Kir4.1功能的试剂，由此提供了通过抑制外侧缰核的星形胶质细胞中的Kir4.1的活性来治疗(抑制)抑郁症的方法和药物。这是本领域已知的治疗抑郁症的机制和药物未能针对的抑郁症病理机制和治疗抑郁症的脑部靶组织或其分子水平上的靶目标。因此，本发明提供的方法和药物特别适合用于在其它抗抑郁方法和药物不起效的抑郁症患者中使用。
在本发明的其中一个方面，本发明提供的方法和药物为在外侧缰核中局部起效的方法和药物。对于用于神经组织的药物，特别是脑部神经组织，例如外侧缰核来说，将药物的作用限定在目标组织是有益的。用于LHb的方法或药物需要考虑该方法或药物是否能够在LHb发挥药物的有 效性，包括药物是否能到达LHb，以及在LHb中是否能达到起效的浓度等。在本发明中，所述药物可以为在外侧缰核局部给药的剂型。可以通过局部给药的方式来达到将药物作用限定在目标组织，例如通过将药物制成可通过套管植入外侧缰核局部给药的剂型。又例如，将药物制成植入组织后缓释的剂型等。另外还可将上述药物制成组织特异性的靶向药物递送系统的形式。例如可以通过将具有抑制簇状放电功能的小分子化合物或生物活性分子(核酸如蛋白编码DNA或mRNA分子、蛋白如抗体等)与能够特异性结合在外侧缰核特异性表达的蛋白结合的抗体连接形成能够识别和结合外侧缰核的细胞的复合分子。
在本发明的其中一个方面，在本发明提供的上述在通过在外侧缰核中局部抑制Kir4.1的活性来治疗抑郁症的方法和Kir4.1抑制剂在制备在外侧缰核中局部起效的治疗抑郁症的药物的用途中，所述Kir4.1抑制剂也可为选择性的五羟色胺重摄取抑制剂(丁螺环酮(buspirone)，米安色林(mianserin)，氟西汀(fluoxetine)，舍曲林(sertraline)或氟伏沙明(fluvoxamine)等)和三环类抗抑郁药(去甲替林(nortriptyline)等)。
本发明还提供了用于治疗抑郁症的药物组合物。本发明提供新的治疗抑郁症的药物组合物，其包含治疗有效量的Kir4.1抑制剂。所述Kir4.1抑制剂如前面所定义。
在其中一个方面，本发明提供的用于治疗抑郁症的药物组合物中，所述Kir4.1抑制剂为干扰Kir4.1表达的干扰RNA或其前体，其具有与目标Kir4.1 mRNA的片段相同或互补，或90％以上相同或互补的序列。优选的，所述干扰RNA或其前体具有以下序列：
5’-GGACGACCTTCATTGACAT-3’(SEQ ID No.1)；
5’-GCTACAAGCTTCTGCTCTTCT-3’(SEQ ID No.2)；
5’-GCTCTTCTCGCCAACCTTTAC-3’(SEQ ID No.3)；
5’-CCGGAACCTTCCTTGCAAA-3’(SEQ ID No.4)；
5’-GCGTAAGAGTCTCCTCATTGG-3’(SEQ ID No.5)；或
5’-GCCCTTAGTGTGCGCATTA-3’(SEQ ID No.6)。
在其中一个方面，本发明提供的用于治疗抑郁症的药物组合物中，所述Kir4.1抑制剂为钾离子通道活性下降或丧失的突变型Kir4.1蛋白或其编码序列，所述突变型Kir4.1蛋白优选为在Kir4.1蛋白的核孔区，对应Kir4.1序列为SEQ ID No.8的氨基酸序列中第130-132位的GYG发生突变的突变型Kir4.1蛋白，例如突变为AAA的突变型Kir4.1蛋白。
在其中一个方面，本发明提供的用于治疗抑郁症的药物组合物中，所述Kir4.1抑制剂为Kir4.1的特异性抗体，包括多克隆抗体或单克隆抗体。
在不破坏本发明的创新性的情况下，在本发明的其中一个方面，本发明提供的上述药物组合物中，所述Kir4.1抑制剂不为丁螺环酮(buspirone)，米安色林(mianserin)，氟西汀(fluoxetine)，舍曲林(sertraline)，氟伏沙明(fluvoxamine)或去甲替林(nortriptyline)。在不破坏本发明的创新性的情况下，在本发明的其中一个方面，本发明提供的上述治疗抑郁症的药物组合物中，所述Kir4.1抑制剂不为选择性五羟色胺重摄取抑制剂(SSRIs)或三环类抗抑郁药(TCAs)。
本发明提供的药物组合物特别适合用于在其它抗抑郁方法和药物不起效的抑郁症患者中使用。
在本发明的其中一个方面，本发明提供的药物组合物为在外侧缰核中局部起效的本发明提供的药物组合物。例如为在外侧缰核局部给药的剂型。在本发明的这一个方面，所述Kir4.1抑制剂也可为选择性地五羟色胺重摄取抑制剂(氟西汀(fluoxetine)，舍曲林(sertraline)，氟伏沙明(fluvoxamine)，丁螺环酮(buspirone)，米安色林(mianserin)等)和三环类抗抑郁药(去甲替林(nortriptyline)等)。
本发明还提供了一种抑郁症动物模型，优选为大鼠或小鼠。本发明所述抑郁症动物模型具有抑郁症特征，其外侧缰核中Kir4.1是高表达的。
本发明还提供了采用上述动物模型筛选用于治疗抑郁症的潜在物质的方法，包括步骤：
(1)给抑郁症动物模型施用待筛选的测试物；和
(2)观察所述抑郁症动物模型中的抑郁症的相关症状和/或指标，并与对照组进行比较。
其中，如果所述抑郁症动物模型中抑郁症的相关症状有显著改善，则表示该测试物是可用于治疗抑郁症潜在物质。
在本发明的其中又一个方面，提供了一种筛选用于治疗抑郁症的潜在物质的方法，其特征在于，包括步骤：
(1)在测试组中，向体外检测体系中加入待检测的测试物；和
(2)检测所述测试组的体外检测体系中Kir4.1的表达水平和/或活性，并与阴性对照组进行比较。
其中，如果与加入了阴性对照组相比，测试组中Kir4.1的表达水平显著下降，和/或Kir4.1的通道功能显著下降，则表示所述测试物是预防 和/或治疗抑郁症的潜在物质。
在本发明的其中又一个方面，所述筛选用于治疗抑郁症的潜在物质的方法还包括以下一个或多个步骤：
进一步测试所述潜在物质对神经元簇状发放的影响；和/或
将所述潜在物质施用于动物模型，观察其对抑郁症症状的影响；
在测试其对神经元簇状发放的影响时，如果与阴性对照组(或空白对照组)相比，加入或施用所述测试物的测试组中神经元簇状发放比例显著降低，则表示该测试物是治疗抑郁症的潜在物质。
术语
“Kir4.1”，或“内向整流型钾离子通道(inward rectifier potassium channel)Kir4.1”，也称为potassium voltage-gated channel subfamily J member 10(Kcnj10)，是内向整流型钾离子通道的家族成员之一。胶质细胞中的Kir4.1可允许钾离子在细胞膜上通过，通过调节神经细胞周围外液的钾离子浓度，转运胞外过多的钾离子来缓冲细胞外环境，达到控制静息膜电位水平，维持神经系统的内稳态，保持神经系统的正常生理活性的作用。在哺乳动物中，Kir4.1的蛋白序列和编码核酸序列都很保守。人类的Kir4.1蛋白(NP_002232)的编码基因是KCNJ10(Ensembl:ENSG00000177807)。大鼠Kir4.1蛋白(NP_113790)的编码基因是KCNJ10(Ensembl:ENSMUSG00000044708)。
本发明中，“治疗”包括：改良、减轻、减少或预防与抑郁症相关的症状的进行中的过程或结果；改善与抑郁症相关的症状的进行中的过程或结果；使处于导致特定机体功能损伤的疾病或病症中的机体功能正常 化的进行中的过程或结果；或者引发疾病的一种或多种临床可测定的参数改善的进行中的过程或结果。在一个实施方案中，治疗目的是预防或减慢(减轻)不希望的生理情况、病症或疾病，或获得有益的或期望的结果。该结果可以是，例如医学的、生理学的、临床的、物理治疗、职业治疗，面向保健人员或患者；或本领域理解为“生活品质”或日常生活活动的参数。本发明中，有益的或期望的临床结果包括但不限于，减轻症状；减小/缩小该情况、病症或疾病的程度；稳定(即非恶化)该情况、病症或疾病的状态；延迟该情况、病症或疾病的开始或减慢其进展；改善或缓和该情况、病症或疾病；和减轻(无论部分或总体)、无论可检测出的或不可检测出的；或增强或改善该情况、病症或疾病。在一个实施方案中，治疗包括引发临床有效响应而没有过度水平的副作用。在一个实施方案中，治疗也包括与如果不接受治疗的预期的存活期相比延长存活期。在一个实施方案中，治疗指给药药物或对患者执行医疗程序。本发明中，治疗可以是预防(防止)，治愈虚弱或病，或改良患者的临床情况，包括降低病程或疾病严重度，或主观改善患者的生活品质或延长患者的存活期。
术语“簇状发放”，或“簇状放电”，是指神经元在放电过程中同时产生两个或两个以上锋电位的放电模式。
抑制簇状放电是指抑制簇状放电的发放程度，包括减少簇状放电的频率或放电过程中簇内峰电位的个数，降低簇状放电的强度，甚至是消除簇状放电的发生。
术语“单个发放”，或“单个放电”，是神经元在放电过程中每次发放 一个锋电位的放电模式。
在本发明的一个方面，还提供了诊断抑郁症的方法，其包括检测患者的外侧缰核的Kir4.1的表达。在本发明的其中一个方面，所述诊断抑郁症的方法是检测患者的外侧缰核的Kir4.1的表达的增加。在本发明的其中又一个方面，所述诊断抑郁症的方法包括比较患者在不同时期(例如在不同抑郁症发病阶段，或是在发病前或治疗后)所述外侧缰核的Kir4.1的表达，或是与一般或特定群体中正常人的外侧缰核的Kir4.1的表达进行比较，出现显著增加的，就判断所述受试者为抑郁症患者。
在本发明的其中一个方面，Kir4.1的表达可以通过本领域已知的任何检测样品中蛋白质(表达)的方法进行检测。所述方法可以检查Kir4.1的存在或不存在。所述方法也可以定量检测Kir4.1的表达的量。
在本发明中可用的检测样品中蛋白质(表达)的方法包括免疫测定(immunoassay)。例如通过特异性识别Kir4.1的抗体进行ELISA或蛋白质印迹。抗体可以是单克隆或多克隆的。
在本发明中可用的检测样品中蛋白质(表达)的方法还包括检测Kir4.1的mRNA的存在或其数量，例如通过RT-PCR检测样品中Kir4.1的mRNA或其片段的量。
在本发明的其中一个方面，所述检测样品来自受试者的外侧缰核。在本发明的其中一个方面，所述检测样品来自离体样品。在本发明的其中一个方面，对Kir4.1的表达的检测还可以通过对受试者的外侧缰核的Kir4.1的量进行活体观察和检测进行。例如通过对患者的外侧缰核进行成像，例如为PET成像。其中通过在静脉注射识别和显示Kir4.1的正电 子发射放射性核素示踪剂，然后对外侧缰核进行正电子发射断层显像(PET)扫描。
附图说明
图1显示了抑郁大鼠外侧缰核中Kir4.1表达和功能的上调。(A，B)Western Blot实验显示cLH大鼠和LPS(脂多糖)诱导的抑郁大鼠缰核Kir4.1表达显著高于对照组。组织中大量表达的微管蛋白Tubulin被用于加载控制。蛋白表达进行定量标准化。(C)缰核中Kir4.1 mRNA的qPCR分析。(D，E)成年cLH大鼠(60-90天龄)而非未成年大鼠(30天龄)中，外侧缰核Ba+敏感的电流(多由Kir4.1通道介导)显著增高。(F,G)成年大鼠而非未成年cLH大鼠在习得性无助和强迫游泳行为测试中表现出了显著的抑郁表型。所有数据均表示为平均值±SEM。*P<0.05，**P<0.01，***P<0.001，****P<0.0001与对照组相比。N.S.表示差异不显著。其他的图标相同。
图2显示了外侧缰核中Kir4.1表达在星形胶质细胞的突起上并紧密包裹神经元胞体。(A)免疫荧光双标染色显示Kir4.1免疫阳性信号包绕在神经元的胞体周围。(B)注射腺相关病毒AAV2/1-CaMKII-EGFP-Cre到Kir4.1条件性敲除小鼠的外侧缰核，条件性敲除外侧缰核神经元中的Kir4.1，依然可以看到Kir4.1包绕神经元胞体的现象；但注射AAV2/5-GFAP-EGFP-Cre病毒敲除外侧缰核星形胶质细胞中的Kir4.1后包绕现象消失。(C)免疫电镜观测到标记了金颗粒的Kir4.1信号分布在神经元胞体的周围。(D)全细胞膜片钳电生理技术记录到星形胶质细胞 而非神经元上有Ba2+敏感的Kir4.1电流。所有数据均表示为平均值±SEM。****P<0.0001与对照组相比。N.S.表示差异不显著。
图3 Kir4.1在外侧缰核和海马中的细胞定位。(A)外侧缰核中Kir4.1与神经元标志物NeuN，星形胶质细胞标志物GFAP和S100b的共定位；(B)海马CA1区中Kir4.1与神经元标志物NeuN，星形胶质细胞标志物GFAP和S100b的共定位。最下方两幅图显示Kir4.1抗体被Kir4.1胞外肽段抗原吸附后在外侧缰核和海马CA1中，均检测不到阳性信号，提示所用抗体是特异性针对Kir4.1的。
图4外侧缰核细胞外钾浓度与神经元活性高度相关且被星形胶质细胞Kir4.1所调节。(A-C)胞外灌流含BaCl2的人工脑脊液阻断星胶上Kir4.1通道，可使外侧缰核中簇状发放(burst)和基础发放(tonic)神经元的静息膜电位显著增高，神经元表现为去极化。(D-F)神经元去极化的程度与自身发放频率成正相关。(G)BaCl2对神经元发放作用的典型图。(H)BaCl2显著降低簇状发放细胞的每分钟内簇状发放的频率。(I-K)降低胞外钾可以显著地降低神经元的静息膜电位，增加簇状发放细胞的比例。*P<0.05，**P<0.01，***P<0.001，与对照组相比。N.S.表示差异不显著。
图5星形胶质细胞Kir4.1过量表达增加神经元簇状发放细胞的比例且导致抑郁表型。(A)星形胶质细胞Kir4.1过量表达的病毒载体构建示意图。(B)免疫荧光显示外侧缰核中病毒在星形胶质细胞中过量表达Kir4.1蛋白。(C，D)Kir4.1过表达使神经元和星形胶质细胞的静息膜电位都显著下降，表现为超级化。(E)星形胶质细胞Kir4.1过量表达增加 神经元簇状发放细胞的比例(F)星形胶质细胞Kir4.1过量表达增加了高频发放细胞的比例(G)星形胶质细胞Kir4.1过量表达小鼠抑郁表型检测时间轴(H，I)星形胶质细胞Kir4.1过量表达小鼠在强迫游泳和糖水偏好实验中都表现出显著的抑郁表型。所有数据均表示为平均值±SEM。*P<0.05，**P<0.01，***P<0.001，****P<0.0001与对照组相比。N.S.表示差异不显著。
图6下调LHb中Kir4.1的功能能降低神经元中簇状发放神经元的比例且有效的缓解抑郁症状。(A)用于下调Kir4.1表达水平的RNAi和下调Kir4.1功能的Kir4.1显性突变的AAV病毒载体示意图。(B)Western blot实验显示体外培养的HEK293细胞中，Kir4.1-shRNA可有效降低过表达在细胞上的Kir4.1.(C)上图显示Kir4.1功能下调大鼠进行电生理学实验和行为学检测的时间轴。下图免疫荧光显示Kir4.1显性突变病毒在外侧缰核星形胶质细胞中表达。(D)Kir4.1下调的星形胶质细胞反转点位升高。(E)Kir4.1下调的星形胶质细胞静息膜电位显著增高，神经元的静息膜电位也显著增高，但周围星胶是否有Kir4.1的下调，神经元的膜电位变化没有差异。(G)下调Kir4.1的表达显著降低外侧缰核簇状发放细胞比例。(H)下调Kir4.1表达及功能均显著降低cLH大鼠在强迫游泳中的不动时间。(I，J)下调Kir4.1表达及功能反转cLH在习得性无阻行为检测中的抑郁表型。(K)下调Kir4.1表达及功能显著反转cLH大鼠的糖水偏好缺失。
图7不同Kir4.1 shRNA敲除效率的体外验证。运用蛋白免疫印迹的方法，检测体外培养的HEK293细胞中外源性表达的Kir4.1，被6条针 对Kir4.1不同序列的shRNA敲除的效率。
图8 LHb病毒注射过表达Kir4.1或下调Kir4.1均不影响动物的运动能力。(A)AAV2/5-gfaABC1D-EGFP-Kir4.1病毒表达，LHb过表达Kir4.1后，小鼠在矿场中总的运动距离和中央区停留时间与GFP对照组相比均无显著差异。(B)AAV2/5-Kir4.1 shRNA病毒表达，LHb中Kir4.1表达下调后，cLH大鼠在旷场中的总运动距离和中央区停留时间与对照病毒相比无差异。
具体实施方式
下面将结合实施例进一步说明本发明的实质内容和有益效果，该实施例仅用于说明本发明而非对本发明的限制。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。
实施例1材料和方法
动物材料
雄性cLH大鼠(4-12周龄)，Sprague Dawley大鼠(4-12周龄)和雄性Wistar大鼠(12周龄)。cLH大鼠是一个选择性培育的具有先天习得性无助抑郁表型的抑郁症动物模型(D.Schulz,M.M.Mirrione,F.A.Henn,Neurobiol Learn Mem 93,291，Feb,2010)。本实验的cLH大鼠从美国冷泉港Malinow实验室引进。cLH大鼠饲养和繁殖如前述D.Schulz,et al， Feb,2010中描述。大鼠4只/笼，12小时的明暗周期(7am-7pm有光)。用于套管实验的cLH大鼠1只/笼饲养。成年(8-12周龄)C57BL/6小鼠被用于行为测试：4只/笼，12小时明暗周期(5am-5pm有光)。大鼠和小鼠都能够自由摄取稳定的水和食物，所有的动物实验经过浙江大学动物保护和使用委员会的批准。
病毒构建
Kir4.1的过表达病毒AAV2/5-gfaABC1D-EGFP-Kir4.1。Kir4.1过表达质粒：pZac2.1gfaABC1D-EGFP-Kir4.1，购自于AddGene公司，货号：Plasmid#52874。并由上海泰廷生物科技有限公司包被成AAV2/5型病毒，即AAV2/5-gfaABC1D-EGFP-Kir4.1。
AAV2/1-CamKII-HI-eGFP-Cre，购自于美国宾夕法尼亚大学载体中心(University of Pennsylvania Vector core,Upenn,USA)，Cat#：AV-1-PV2521。
Kir4.1下调病毒AAV2/5-H1-Kir4.1-shRNA-gfaABC1D-EGFP(Kir4.1的干扰RNA-shRNA：5’-GCGTAAGAGTCTCCTCATTGG-3’)，对照病毒AAV2/5-H1-Luciferase-shRNA-gfaABC1D–EGFP；Kir4.1显性突变病毒AAV2/5-gfaABC1D-Kir4.1dn-2A-EGFP(Kir4.1序列的核孔区GYG突变为AAA而实现Kir4.1的显性突变)。均由由上海泰廷生物科技有限公司制备质粒和包被。
LPS(脂多糖)诱导抑郁模型
12周龄的雄性Wistar大鼠，每天腹腔注射1次LPS(500μg/kg体重)， 连续注射7天，最后一次注射结束后24h，进行FST测试，测试结束后3天取出缰核组织，进行表达量分析。
立体定位注射和组织学
小鼠注射病毒:小鼠腹腔注射氯胺酮(100mg/kg体重)和赛拉嗪(8mg/kg)混合液麻醉后，固定于立体定位仪上(Stoelting instruments)。每只小鼠每侧LHb注入0.1-0.2ul纯化浓缩的AAV病毒(～1013感染单位/ml)，LHb立体定位坐标(前后距离Bregma：-1.7mm(AP)，左右旁开±0.46mm(ML)，皮层表面往下-2.56mm(DV))。使用自行拉制的玻璃微电极缓慢注入(～100-150nl/min)，注射结束留针5min，然后再5min内缓慢移出注射电极。
大鼠注射病毒：大鼠腹腔注射4％戊巴比妥那(60mg/kg体重)麻醉后，固定于大鼠立体定位仪上。每只大鼠每测LHb注射入0.1-0.2ul纯化浓缩的AAV病毒(～10 13感染单位/ml)，LHb立体定位坐标(前后距离Bregma：-3.7mm(AP)，左右旁开±0.7mm(ML)，皮层表面往下-4.55mm(DV))。使用自行拉制的玻璃微电极缓慢注入(～100-150nl/min)，注射结束留针5min，然后再5min内缓慢移出注射电极。
术后至少14天，开展行为实验或者电生理实验。行为实验结束后检查注射位置，只使用正确注射的那些动物数据。注射了GFP标记的病毒在显微检查之前用抗体检查GFP蛋白。每个大脑的缰区切成6组连续的切片(小鼠30μm的切片，每组6片；大鼠40μm
切片，每组8-9片)。所有的切片在安装到固定片之前用Hoechst复染色。
免疫组化
首先腹腔注射4％的戊巴比妥那让动物进入深度麻醉。然后分别用PBS和冰预冷4％的PFA进行灌流固定，取出动物脑组织经4％的PFA后固定过夜。30％的蔗糖溶液脱水处理1-3天，待脑组织沉到管底，可进行冰冻切片，切片厚度为40μm，并-20度冻存于切片保护液中。抗体的浓度如下：anti-Kir4.1(1：200，Alomone labs)，anti-NeuN(1：500，chemicon)，anti-GFAP(1:500,Chemicon),anti-S100b(1:500,Invitrogen,ABCAM),anti-GFP(1:1000,abCaM)。Alexa Fluor488 goat anti-rabbit IgG,Alexa Fluor488 goat anti-chicken IgG,Alexa Fluor594 goat anti-mouse IgG(all1:1000,Invitrogen),Hoechst(1:5000)荧光图片利用Olympus Fluoview FV1000和NiconA1激光共聚焦显微镜采集图像。
蛋白免疫印迹
大鼠经异氟烷麻醉后，断头取脑。冰上快速分离缰核组织并冻于液氮中。样品在匀浆液内进行组织破碎。组织匀浆物以800g在4度离心15分钟。取上清并以10000g离心15min。沉淀物即为膜蛋白组分，然后将它们溶解在匀浆液中。293TN细胞系的样品经过RIPA溶液(20mM Tris-HCl[pH 7.5]、150mM NaCl、1mM EDTA、1mM EGTA、1％NP-40、1％sodium deoxycholate、1mM PMSF、10μg/ml aprotinin、1μg/ml pepstatin A和1μg/ml leupeptin)裂解后，10000g离心15min取上清。用BCA法测定蛋白浓度后，以8-15μg蛋白每孔上样，10％SDS-PAGE胶进行分离，PVDF膜转印做Western印迹。所用一抗为anti-Kir4.1-introcellular(1：1000，Alomone labs)，anti-tubulin(1:10000， Bio-Rad)，对于免疫印迹结果的定量，使用Quantity One或image J软件进行统计分析。
电生理
出生后40-50天的大鼠或出生8周的小鼠经异氟烷麻醉后，用20ml冰冷充氧的切片液进行灌流。快速断头取出大脑，放进充氧的切片液中。随后利用Leica振动切片机在充氧的冰冷的切片液中，进行350μm的冠状切面切片。缰核脑片在充氧的34℃的ACSF(118mM NaCl,2.5mM KCl,26mM NaHCO3,1mM NaH2PO4,10mM glucose,1.3mM MgCl2 and 2.5mM CaCl2,gassed with 95％O2 and 5％CO2)中，恢复两小时后转移到室温进行记录。
外侧缰核脑片的膜片钳记录采用Axon Multiclamp 700B放大器，在32±1℃环境下，在装配红外微分干涉相差光学镜头的Olympus显微镜下，进行记录。星形胶质细胞记录电极的阻抗为7-10MΩ，神经元记录电极阻抗为4-6MΩ。所有细胞均在全细胞模式下记录。根据所记录细胞的形态，记录电生理特性判断是神经元(15-20微米直径)还是星形胶质细胞(5-10微米直径)。神经元记录参数包括细胞膜电位，膜阻抗，动作电位自发放频率(I＝0记录)，神经元电压电流曲线以及神经元输入输出兴奋性。星形胶质细胞记录参数包括膜电位，膜阻抗，电压电流曲线(电压钳下从-130mV至40mV电压，每次记录增加10mV，记录各个电位下的电流反应)。Kir4.1电流计算方法：由正常人工脑脊液中记录得到的电压电流曲线减去加入氯化钡(100Mm,该浓度特异性拮抗Kir4.1电流)的电压电流曲线。数据经过2kHz过滤后使用Digidata 1322A在10kHz下 采样记录。数据使用pClamp 10软件进行分析。
行为学实验
习得性无助实验
实验是对同窝cLH或野生型SD大鼠进行。分两部分进行：“训练部分”包括在刺激室(coulbourn仪器)中，120次不可避免的、不受控的0.8mA足底电刺激超过40分钟，随机刺激持续时间和范围从5秒到15秒间隔刺激时间，总刺激持续时间为20分钟。“测试部分”训练24小时后进行，习得无助表型是由一个杆压迫任务进行评估，期间一个光照指示杆被放入刺激室中。这一部分包括15次可逃避的0.8mA强度电刺激和24s实验间隔。每次刺激持续了60秒，但是能被杆压迫终止。超过10次失败被定义为“习得无助”(LH)；少于5次失败为“非习得无助”(NLH)。
强迫游泳测试
实验在正常日光灯下进行。小鼠强迫游泳圆柱形容器的直径为12cm，高25cm。测试水深为14cm，水温23-24℃。摄像头从侧边记录小鼠在6min内的游泳情况。采用双盲方式统计小鼠游泳6min内后4min的不动时间(动物的漂浮姿势或者四肢完全没有活动的时间)。
糖水偏好测试
实验小鼠单独饲养1周，然后连续2天给予小鼠两瓶普通水，之后两天将水换为两瓶2％的蔗糖水进行训练。训练结束后，给予动物一瓶普通水和一瓶2％的蔗糖水进行测试，每12小时交换一次水瓶的位置，每24小时记录一次水和糖水的消耗量(对水瓶称重)，共记录48小时。
统计分析
所有的数据都以平均值±SEM。对于所有的行为数据，采用two-tailed Student's t-tests或Mann-Whitney test。
实施例2抑郁大鼠缰核中Kir4.1表达和活性上调
如图1所示。在动物体内观察抑郁大鼠外侧缰核中Kir4.1表达和活性。分别在先天抑郁(cLH)大鼠和另一种抑郁症的动物模型——脂多糖(LPS)诱导的大鼠抑郁模型。脂多糖(LPS)诱导的大鼠抑郁模型是对3月龄的雄性Wistar大鼠，连续7天每天腹腔注射500ug/kg的LPS，最后一次注射后24小时，检测到大鼠在强迫游泳中的不动性显著增加。
图1中的A和B是蛋白免疫印迹实验结果，显示cLH大鼠和LPS(脂多糖)诱导的抑郁大鼠的外侧缰核中的Kir4.1表达显著高于对照组。图1中的A显示cLH大鼠缰核膜组分中Kir4.1蛋白表达水平比对照增高了1.75倍。图1中的B显示外侧缰核中Kir4.1的表达水平在LPS诱导的抑郁大鼠中也显著增高(1.87倍)。
为了确定抑郁大鼠中Kir4.1的功能是否也增强，运用全细胞膜片钳记录技术检测了cLH和SD大鼠LHb脑片中星形胶质细胞和神经元的变化。根据星形胶质细胞胞体较小(5-10μm)的形态学特征，和一系列电生理学的特征来分离星形胶质细胞。这些电生理特征包括：相对超级化的静息膜电位(-74±1mV)；低的输入阻抗Ri(47±6MΩ)；线性的电流-电压曲线关系和去极化电流不能使它产生动作电位。运用Ba2+去选择性地阻断Kir通道的电流，进而分离出Kir4.1的电流。电生理记录发现， 在成年(60-90天)cLH大鼠LHb的星形胶质细胞中，Ba2+敏感的Kir4.1电流几乎是对照SD组电流的两倍(图1的C)。同时，在未成年的cLH大鼠中Kir4.1的电流没有增高(图1的D)，且未成年大鼠的强迫游泳的不动时间和习得性无助实验中为了逃脱电击而按指示杆的次数与对照组都没有差异(图1的E和F)。这显示，Kir4.1的过表达与抑郁症状的起始有关联。
实施例3 Kir4.1在外侧缰核的表达分布特性
运用免疫荧光双标的方法，发明人出乎意料地首次发现Kir4.1以包绕在神经元的胞体上表达的方式在外侧缰核中分布。而在海马中，Kir4.1的表达主要是星形状的。(如图3所示)
图2显示了外侧缰核中Kir4.1表达在星形胶质细胞的突起上并紧密包裹神经元胞体。
图2的A表示，免疫荧光双标染色显示Kir4.1免疫阳性信号包绕在神经元的胞体周围。
另外，通过运用条件性敲除的方法来来观察和判断Kir4.1的表达。通过注射腺病毒AAV2/1-CaMKII-EGFP-Cre到Kir4.1条件性敲除小鼠的外侧缰核来实现条件性敲除。如图2的B所示，条件性敲除外侧缰核神经元中的Kir4.1，依然可以看到Kir4.1包绕神经元胞体的现象；但注射AAV2/5-GFAP-EGFP-Cre病毒来敲除外侧缰核星形胶质细胞中的Kir4.1后，包绕现象消失。
运用免疫电镜的方法也检测到Kir4.1免疫金颗粒在神经元细胞膜周 围分布，而在神经元突触部位较少(如图2的C所示)。
电生理证据也同样地发现Ba 2+敏感的Kir4.1电流主要在星形胶质细胞被记录到而非神经元(如图2的D所示)。
图3给出的是Kir4.1在外侧缰核和海马中的细胞定位。图3的A显示外侧缰核中Kir4.1与神经元标志物NeuN，星形胶质细胞标志物GFAP和S100b的共定位；图3的B显示海马CA1区中Kir4.1与神经元标志物NeuN，星形胶质细胞标志物GFAP和S100b的共定位。图3最下方两幅图显示Kir4.1抗体被Kir4.1胞外肽段抗原吸附后，在外侧缰核和海马CA1中均检测不到阳性信号，提示所用抗体是特异性针对Kir4.1的。
以上结果均说明LHb中星形胶质细胞突起上的Kir4.1，主要以紧密包绕在神经元胞体周围的表达形式存在。
实施例4 Kir4.1对神经元活性的调节作用
运用膜片钳电生理记录的方法对Kir4.1对神经元活性的调节作用进行研究。首先通过胞外灌流含有BaCl 2的人工脑脊液，阻断Kir4.1缓冲钾离子的功能。发现BaCl 2处理能显著去极化除了无自发放的细胞外的所有LHb神经元。如图4的A-C所示，胞外灌流含BaCl 2的人工脑脊液阻断星胶上Kir4.1通道，可使外侧缰核中簇状发放(burst)和基础发放(tonic)神经元的静息膜电位显著增高，神经元表现为去极化。这与Nernst方程推算的增多胞外钾浓度降低，引起神经元的膜电位超级化相一致。且BaCl 2处理对LHb自发放细胞去极化的程度与细胞自身发放频率正相关(如图4的D-F所示)。BaCl 2处理长时间过度兴奋簇状发放神 经元，使得神经元处于强直状态，最终使神经元簇状发放数量显著降低(如图4的G-H所示)。
实施例5 Kir4.1通过调节胞外钾浓度而引起神经元和星形胶质细胞的电生理功能的变化
在正常大鼠的LHb脑片上孵育钾浓度下降(2.75mM-1.4mM)的人工脑脊液，这使得神经元的静息膜电位超级化了10.3mV(如图4的I,J所示)，且使簇状发放细胞比例从8％增高到了23％(如图4的K所示)。这些结果显示Kir4.1的过表达通过增加胞外钾离子的清除而超级化神经元，引发簇状发放。
实施例6动物体内实验显示在外侧缰核过表达Kir4.1引发抑郁症症状
构建Kir4.1显性突变病毒pAAV-gfaABC1D-Kir4.1dn-2A-EGFP(Kir4.1序列的核孔区GYG突变为AAA而实现Kir4.1的显性突变)。
pAAV-gfaABC1D-Kir4.1dn-2A-EGFP质粒构建：
1.首先构建pAAV-Ubi-Kir4.1-2A-EGFP质粒，用pAAV-Ubi-CaMKIIb-2A-EGFP质粒(Li et al.,2013)为模板，用表1中引物pAAV-ubi Fusion Fw和pAAV-ubi Fusion Rev扩增出一个线性化载体；再用pZac2.1-gfaABC1D-EGFP-Kir4.1(购自AddGene,Plasmid#52874)为模板，用pAAV-ubi-Kir4.1Fusion Fw和Rev扩增出kir4.1的片段。以上获得的两个线性化片段重组成pAAV-Ubi-Kir4.1-2A-EGFP。
表1
Figure PCTCN2018086021-appb-000001
2.构建pAAV-Ubi-Kir4.1dn-2A-EGFP质粒：以pAAV-Ubi-Kir4.1-2A-EGFP质粒为模板，用表2中的引物Kir4.1 GYG-AAA Fusion Fw和Kir4.1 GYG-AAA Fusion Rev扩增出一个线性化的突变片段，然后自身重组成完整的AAA突变的pAAV-Ubi-Kir4.1dn-2A-EGFP质粒。
表2
Figure PCTCN2018086021-appb-000002
3.构建pAAV-gfaABC1D-Kir4.1dn-2A-EGFP质粒,方法是：以pZac2.1-gfaABC1D-EGFP-Kir4.1质粒为模板，运用表3所示的引物pZac2.1gfaABC1D Fusion Rev和pZac2.1 gfaABC1D Fusion Fw，扩增出EGFP-Kir4.1缺失的pZac2.1 gfaABC1D线性片段；再以pAAV-Ubi-Kir4.1dn-2A-EGFP质粒为模板，Kir4.1dn-2A-eEGFP Fusion Rev和Kir4.1dn-2A-eEGFP Fusion Fw为引物，扩增出Kir4.1dn-2A-EGFP片段。以上两个线性化片段同源重组后可获得pAAV-gfaABC1D -Kir4.1dn-2A-EGFP质粒。
表3
Figure PCTCN2018086021-appb-000003
运用立体定位注射的方法，在外侧缰核注射pAAV-gfaABC1D-Kir4.1dn-2A-EGFP，表达14天，使Kir4.1在LHb星形胶质细胞里过表达。图5的A显示用于在星形胶质细胞过量表达Kir4.1的病毒载体构建示意图。图5的B是免疫荧光图，显示外侧缰核中病毒在星形胶质细胞中过量表达Kir4.1蛋白。采用全细胞膜片钳记录过表达Kir4.1的LHb脑片，发现其中的星形胶质细胞和神经元的静息膜电位都显著超级化(如图5的D和E所示)，且神经元簇状发放的比例也显著高于注射对照GFP病毒组(如图5的F,G所示)。同时Kir4.1在LHb的过表达也显著增高了小鼠在强迫游泳中的不动性，显著降低了小鼠对糖水的偏好性(如图5的H,I所示)，而小鼠的运动能力没有发生改变(图8的A所示)。这些实验证明LHb Kir4.1过表达小鼠表现出了典型的抑郁症状。
实施例7外侧缰核的Kir4.1表达水平的下调或Kir4.1功能逆转抑郁表型
采用AAV2/5病毒表达Kir4.1的短发夹RNA(shRNA)来下调cLH大鼠LHb Kir4.1蛋白表达水平。
将根据下表中构建的6个Kir4.1的干扰RNA的shRNA克隆到WX231-L载体(购自于上海泰廷生物科技有限公司，Cat#：WX231)上：
Figure PCTCN2018086021-appb-000004
克隆采用的引物序列如下表：
Figure PCTCN2018086021-appb-000005
在体外培养的HEK293细胞中，分别共表达Kir4.1和所述6个shRNA。
图7是蛋白免疫印迹结果。用动物体内不表达的蛋白荧光素酶，作为 阴性对照(negative control，NC)。AAV2/5-H1-Luciferase(荧光素酶)-shRNA-gfaABC1D–EGFP作为对照病毒表达NC-shRNA。Kir4.1shRNA的敲除效率为：Kir4.1-shRNA-1>Kir4.1-shRNA-4，Kir4.1-shRNA-5>Kir4.1-shRNA-2>Kir4.1-shRNA-6>Kir4.1-shRNA-3。
由上海泰廷生物科技有限公司包被，将Kir4.1-shRNA-5包装成载体为AAV2/5的病毒用于Kir4.1功能下调大鼠实验。
图6的A是用于下调Kir4.1表达水平的RNAi和下调Kir4.1功能的Kir4.1显性突变的AAV病毒载体示意图。图6的B是Western blot实验结果，显示体外培养的HEK293细胞中，Kir4.1-shRNA可有效降低过表达在细胞上的Kir4.1。
图6的C的上图显示Kir4.1功能下调大鼠进行电生理学实验和行为学检测的时间轴；下图免疫荧光显示Kir4.1显性突变病毒在外侧缰核星形胶质细胞中表达。
电生理记录AAV-Kir4.1-shRNA病毒表达的大鼠LHb脑片，表达shRNA的星形胶质细胞静息膜电位显著去极化，而表达shRNA的神经元静息膜电位与相邻的不表达shRNA的神经元一样，但比表达对照shRNA的神经元显著的超级化(如图6的D-G所示)。提示星形胶质细胞中Kir4.1的敲除对所有的神经元静息膜电位均有影响。Kir4.1 shRNA的表达，也使LHb中簇状发放的神经元比例从29％显著降低到0％(如图6的H所示)。同时，下调cLH大鼠LHb Kir4.1蛋白表达水平显著反转cLH大鼠的抑郁表型：大鼠的强迫游泳不动性显著降低(如图6的I所示)，习得性无助按杆行为显著增高(如图6的J,K所示)，糖水偏好也显著增高(如 图6的L所示)，而大鼠的运动能力没有发生改变(如图8的B所示)。
另外，通过病毒表达Kir4.1的显性突变体去抑制cLH大鼠LHb Kir4.1通道的功能。图6的A显示Kir4.1显性突变病毒AAV2/5-gfaABC1D-Kir4.1dn-2A-EGFP的结构(Kir4.1序列的核孔区氨基酸序列130-132的GYG突变为AAA而实现Kir4.1的显性突变)。在LHb注射AAV-dnKir4.1-EGFP病毒都显著地反转cLH大鼠的抑郁表型：大鼠的强迫游泳不动性显著降低(如图6的I所示)，习得性无助按杆行为显著增高(如图6的J,K所示)，糖水偏好也显著增高(如图6的L所示)。
结论
本发明人经过深入研究，首次发现外侧缰核的星形胶质细胞中的Kir4.1是抑郁症的一个至关重要的调节因子，并运用分子、行为和电生理等手段，发现Kir4.1以一种紧密包绕缰核神经元胞体的方式存在于外侧缰核中，调节胞外钾平衡，改变外侧缰核神经元的发放特性，导致外侧缰核过度活跃，进而调节抑郁表型。本发明人还发现和证明了多种可以阻断外侧缰核中Kir4.1活性的试剂，由此提供了通过抑制Kir4.1的活性来治疗(抑制)抑郁症的方法和药物。
上面是对本发明进行的说明，不能将其看成是对本发明进行的限制。除非另外指出，本发明的实践将使用有机化学、聚合物化学、生物技术等的常规技术，显然除在上述说明和实施例中所特别描述之外，还可以 别的方式实现本发明。其它在本发明范围内的方面与改进将对本发明所属领域的技术人员显而易见。根据本发明的教导，许多改变和变化是可行的，因此其在本发明的范围之内。
如无特别表示，本文中出现的温度的单位“度”是指摄氏度，即℃。

Claims (17)

  1. 治疗抑郁症的方法，其中包括对患者施用Kir4.1抑制剂。
  2. 权利要求1的方法，其中所述Kir4.1抑制剂为干扰Kir4.1表达的干扰RNA或其前体，优选为针对Kir4.1的对应序列为SEQ ID No.7的mRNA序列中以下序列片段的干扰RNA或其前体：
    5’-GGACGACCTTCATTGACAT-3’(SEQ ID No.1)；
    5’-GCTACAAGCTTCTGCTCTTCT-3’(SEQ ID No.2)；
    5’-GCTCTTCTCGCCAACCTTTAC-3’(SEQ ID No.3)；
    5’-CCGGAACCTTCCTTGCAAA-3’(SEQ ID No.4)；
    5’-GCGTAAGAGTCTCCTCATTGG-3’(SEQ ID No.5)；或
    5’-GCCCTTAGTGTGCGCATTA-3’(SEQ ID No.6)。
  3. 权利要求1的方法，其中所述Kir4.1抑制剂为钾离子通道活性下降或丧失的突变型Kir4.1蛋白或其编码序列，优选为在Kir4.1蛋白的核孔区，对应Kir4.1序列为SEQ ID No.8的氨基酸序列中第130-132位的GYG发生突变的突变型Kir4.1蛋白，例如突变为为AAA的突变型Kir4.1蛋白。
  4. 权利要求1的方法，其中所述Kir4.1抑制剂为Kir4.1的特异性抗体，包括多克隆抗体或单克隆抗体。
  5. 权利要求1的方法，其中在外侧缰核局部给药。
  6. Kir4.1抑制剂在制备治疗抑郁症的药物中的用途。
  7. 权利要求6的用途，其中所述Kir4.1抑制剂为干扰Kir4.1表达的 干扰RNA或其前体，优选为针对Kir4.1的对应序列为SEQ ID No.7的mRNA序列中以下序列片段的干扰RNA或其前体：
    5’-GGACGACCTTCATTGACAT-3’(SEQ ID No.1)；
    5’-GCTACAAGCTTCTGCTCTTCT-3’(SEQ ID No.2)；
    5’-GCTCTTCTCGCCAACCTTTAC-3’(SEQ ID No.3)；
    5’-CCGGAACCTTCCTTGCAAA-3’(SEQ ID No.4)；
    5’-GCGTAAGAGTCTCCTCATTGG-3’(SEQ ID No.5)；或
    5’-GCCCTTAGTGTGCGCATTA-3’(SEQ ID No.6)。
  8. 权利要求6的用途，其中所述Kir4.1抑制剂为钾离子通道活性下降或丧失的突变型Kir4.1蛋白或其编码序列，优选为在Kir4.1蛋白的核孔区，对应Kir4.1序列为SEQ ID No.8的氨基酸序列中第130-132位的GYG发生突变的突变型Kir4.1蛋白，例如突变为为AAA的突变型Kir4.1蛋白。
  9. 权利要求6的用途，其中所述Kir4.1抑制剂为Kir4.1的特异性抗体，包括多克隆抗体或单克隆抗体。
  10. 权利要求6的用途，其中所述药物为在外侧缰核局部给药的剂型。
  11. 治疗抑郁症的药物组合物，其中包含Kir4.1抑制剂。
  12. 权利要求11的药物组合，其中所述Kir4.1抑制剂为干扰Kir4.1表达的干扰RNA或其前体，优选为针对Kir4.1的对应序列为SEQ ID No.7的mRNA序列中以下序列片段的干扰RNA或其前体：
    5’-GGACGACCTTCATTGACAT-3’(SEQ ID No.1)；
    5’-GCTACAAGCTTCTGCTCTTCT-3’(SEQ ID No.2)；
    5’-GCTCTTCTCGCCAACCTTTAC-3’(SEQ ID No.3)；
    5’-CCGGAACCTTCCTTGCAAA-3’(SEQ ID No.4)；
    5’-GCGTAAGAGTCTCCTCATTGG-3’(SEQ ID No.5)；或
    5’-GCCCTTAGTGTGCGCATTA-3’(SEQ ID No.6)。
  13. 权利要求11的药物组合，其中所述Kir4.1抑制剂为钾离子通道活性下降或丧失的突变型Kir4.1蛋白或其编码序列，优选为在Kir4.1蛋白的核孔区，对应Kir4.1序列为SEQ ID No.8的氨基酸序列中第130-132位的GYG发生突变的突变型Kir4.1蛋白，例如突变为AAA的突变型Kir4.1蛋白。
  14. 权利要求11的药物组合，其中所述Kir4.1抑制剂为Kir4.1的特异性抗体，包括多克隆抗体或单克隆抗体。
  15. 权利要求11的药物组合物，其中所述药物为在外侧缰核局部给药的剂型。
  16. 一种筛选治疗抑郁症的潜在物质的方法，其特征在于，包括步骤：
    (1)给抑郁症动物模型(优选的，所述动物模型为大鼠或小鼠)施用待筛选的测试物，其中所述抑郁症动物模型的外侧缰核中Kir4.1是高表达的；和
    (2)观察所述抑郁症动物模型中的抑郁症的相关症状和/或指标，并与对照组进行比较；
    其中，所述抑郁症动物模型中抑郁症的相关症状有显著改善，则表 示该测试物是可用于治疗抑郁症的潜在物质。
  17. 筛选用于治疗抑郁症的潜在物质的方法，其特征在于，包括步骤：
    (1)在测试组中，向体外检测体系中加入待检测的测试物；和
    (2)检测所述测试组的体外检测体系中Kir4.1的表达水平和/或活性，并与阴性对照组进行比较，
    优选的，所述方法还包括以下一个或多个步骤：
    进一步测试所述潜在物质对神经元簇状发放的影响；和/或
    将所述潜在物质施用于动物模型，观察其对抑郁症症状的影响。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114788868A (zh) * 2021-01-15 2022-07-26 山东大学齐鲁医院 一种基因抑制剂在制备抑郁症治疗药物中的用途

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005054848A2 (en) * 2003-11-25 2005-06-16 Bayer Healthcare Ag Diagnostics and therapeutics for diseases associated with g-protein-coupled inwardly rectifying potassium channel (girk2)
ATE486606T1 (de) * 2004-09-18 2010-11-15 Univ Maryland Therapeutische mittel zum targeting des ncca-atp- kanals und verwendungsverfahren dafür
EP2530088A1 (en) * 2011-05-30 2012-12-05 Klinikum rechts der Isar der Technischen Universität München Means and methods for diagnosing and treating multiple sclerosis
EP2887806B1 (en) * 2012-07-20 2019-11-13 University Of Rochester Method of treating and preventing brain impairment using na+-k+ -2ci- cotransporter isoform 1 inhibitors
CN104338135B (zh) * 2013-08-09 2018-08-24 中国科学院上海生命科学研究院 抑郁症的调节因子及其应用
EP3636281B1 (en) * 2017-05-09 2023-11-01 Zhejiang University Method for treating depression, and pharmaceutical composition

Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
BANASR ET AL., BIOLOGICAL PSYCHIATRY, vol. 64, 2008, pages 863 - 870
COTTER ET AL., ARCHIVES OF GENERAL PSYCHIATRY, vol. 58, 2001, pages 545 - 553
COYLE ET AL., ARCHIVES OF GENERAL PSYCHIATRY, vol. 57, 2000, pages 90 - 93
CZEH ET AL., THE JOURNAL OF THE EUROPEAN COLLEGE OF NEUROPSYCHOPHARMACOLOGY, vol. 23, 2013, pages 171 - 185
FURUTANI, K. ET AL.: "2401-Pos Board B371. The Structural Basis for Antidepressants Blok being Confined to Kir4.1", BIOPHYSICAL JOURNAL, vol. 96, no. 3, 3 March 2009 (2009-03-03), pages 465a, XP055547634 *
HIGASHI, K ET AL.: "An Inwardly Rectifying K+ Channel, kir4.1, Expressed in Astrocytes Surrounds Synapses and Blood Vessels in Brain", AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY, vol. 281, no. 3, 30 September 2001 (2001-09-30), pages C922 - C931, XP055547655 *
HIROSHI ET AL., PHYSIOLOGICAL REVIEWS, vol. 90, 2010, pages 291 - 366
LI, FAN: "Role of Inwardly Rectifying Potassium Channel Kir4.1 in Oligodendrocyte Development and Myelin Formation", MASTER THESIS, 15 March 2017 (2017-03-15), pages 1 - 65, XP009517920 *
RAJKOWSKA ET AL., CNS & NEUROLOGICAL DISORDERS DRUG TARGETS, vol. 6, 2007, pages 219 - 233
SAMBROOK ET AL.: "Molecular Cloning: Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
SU , S. ET AL.: "Inhibition of Astroglial Inwardly Rectifying Kir4.1 Channels by a Tricyclic Antidepressant, Nortriptyline", JOURNAL OF PHARMACOLOGY & EXPERIMENTAL THERAPEUTICS, vol. 320, no. 2, 1 February 2007 (2007-02-01), pages 573 - 580, XP055547641 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114788868A (zh) * 2021-01-15 2022-07-26 山东大学齐鲁医院 一种基因抑制剂在制备抑郁症治疗药物中的用途
CN114788868B (zh) * 2021-01-15 2024-06-21 北京钟楣科技有限公司 一种基因抑制剂在制备抑郁症治疗药物中的用途

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