Use of potassium channel inhibitor for treating depression

Abstract

In light of a discovery that astroglial Kir4.1 in lateral habenula drives neuronal bursts in depression, the present disclosure provides a pharmaceutical agent and a method of use thereof for treating depression. The pharmaceutical agent can inhibit an activity of an astroglial potassium channel, and especially suppress expression or functionality of Kir4.1, in astrocytes in the lateral habenula of a subject so that bursting activity of neurons in the lateral habenula of the subject can be suppressed. The pharmaceutical agent can include a vector expressing a target nucleotide sequence in the astrocytes in the lateral habenula, whose expression is configured to suppress Kir4.1 expression by RNA interference, or to block Kir4.1 functionality by a dominant negative effect of a mutant Kir4.1 protein. The pharmaceutical agent can alternatively comprise a small molecule compound, or an active macromolecule such as an anti-Kir4.1 antibody, configured to directly inhibit the astroglial potassium channel activity.

Claims

1. A pharmaceutical composition for treating depression in a subject, wherein the depression is characterized by abnormal burst firings of neurons in a lateral habenula (LHb) of the subject, the pharmaceutical composition comprising a therapeutically effective amount of a pharmaceutical agent, wherein the pharmaceutical agent comprises a recombinant vector configured to express in astrocytes in the LHb of the subject a short hairpin RNA (shRNA) molecule, wherein the shRNA molecule comprises two complementary sequences, and one of the two complementary sequences has a nucleotide sequence that is identical to the sequence as set forth in SEQ ID NO. 5.

2. The pharmaceutical composition of claim 1, wherein the recombinant vector is a recombinant viral vector capable of preferentially or specifically targeting the astrocytes of the subject.

3. The pharmaceutical composition of claim 2, wherein the recombinant vector is based on an adeno-associated virus (AAV) of 2/5 serotype (AAV2/5).

4. A method for treating depression in a subject, comprising: administering to the subject the pharmaceutical composition according to claim 1.

5. The method of claim 4, wherein the recombinant vector is a recombinant viral vector, wherein the administering to the subject the pharmaceutical composition comprises: obtaining virus particles carrying the recombinant viral vector; and administering the virus particles to the subject.

6. The method of claim 5, wherein the administering the virus particles to the subject is through an injection or an inhalation.

7. The method of claim 4, wherein the pharmaceutical agent in the pharmaceutical composition comprises a small molecule agent capable of inhibiting the activity of the astroglial potassium channel in the astrocytes in the lateral habenula of the subject, wherein the administering to the subject a pharmaceutical composition comprises: administering the pharmaceutical composition in a systemic manner; or administering the pharmaceutical composition locally to the lateral habenula of the subject.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIGS. 1A-1F show that Kir4.1 is upregulated in the LHb of the animal models of depression. FIGS. 1A and 1B: Western blot analysis showing upregulation of Kir4.1 protein in membrane fraction of habenula of cLH (FIG. 1A) and LPS-induced depression rat models (FIG. 1i). Tubulin is used as loading control. FIGS. 1C and 1D: I-V plot and bar graph showing Ba.sup.2+-sensitive current in cLH rats and their wild type controls at the age of P60-90 (FIG. 1C) and P30 (FIG. 1D). FIGS. 1E and 1F: Age-dependent learned helpless (FIG. 1E) and forced swim (FIG. 1F) phenotypes of cLH rats. Low number of lever press and high immobility time indicate depressive-like phenotype in P90 cLH rats. Data are means±SEM. Numbers in the bars indicate the number of animals used. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; n.s., not significant.

(2) FIGS. 2A-2D show that Kir4.1 is expressed on astrocytic processes tightly wrapping around neuronal soma. FIG. 2A: Immunohistochemistry signals of Kir4.1 envelope neuronal soma as indicated by white arrows. FIG. 2B: The pan-soma Kir4.1 signals remain intact in LHb of Kir4.1-floxed mice injected with AAV2/1-CaMKII-EGFP-Cre, but are eliminated when injected with AAV2/5-GFAP-EGFP-Cre, (GFAP: human astrocyte-specific GFAP promoter, gfaABC1D). FIG. 2C: Immunogold electron microscopy of Kir4.1. Red arrows indicate gold signals surrounding a neuronal soma. FIG. 2D: I-V plots of the Ba2+ sensitive Kir4.1 current recorded in LHb astrocytes and neurons, with representative traces shown in up-left and statistic bar graph of current recorded when cells are held at −120 mV shown in up-right. Data are means±SEM. ****P<0.0001; n.s., not significant.

(3) FIGS. 3A-3B show that Kir4.1 is expressed in astrocytic processes of LHb and astrocytic soma of hippocampus. FIGS. 3A and 3B: Kir4.1 co-immunostaining with neuronal marker (NeuN) or astrocytic marker (S100b and GFAP) in LHb (FIG. 3A) or hippocampus (FIG. 3B). Bottom two panels are staining with the same kir4.1 antibody pre-incubated with the antigen peptide, demonstrating Kir4.1 antibody.

(4) FIGS. 4A-4K show that extracellular potassium is highly correlated with the activity pattern and tightly control by astrocytic kir4.1. FIGS. 4A-4C: Changes of neuronal RMPs caused by BaCl.sub.2 (100 μM) in different neuronal types. FIGS. 4D-4E: Correlation between BaCl.sub.2-sensitive membrane potential and tonic firing frequency (FIG. 4D), inner burst firing frequency (FIG. 4E) and burst total spike frequency. FIGS. 4G-4H: Representative trace (FIG. 4G) and bar graph (FIG. 4H) shows BaCl.sub.2 effect onto bursting neurons. FIG. 4I: Example of a whole-cell patch recording showing LHb neuron spontaneous activity transformed from tonic- to burst-firing mode when Kout is switched from normal (2.75 mM) to half (1.4 mM). FIGS. 4J and 4K: Lowering Kout to half decreases neuronal RMPs (FIG. 4J) and increases the bursting population in neurons (FIG. 4K). *P<0.05, **P<0.01, ***P<0.001, compared with the control group; n.s., not significant.

(5) FIGS. 5A-5I show that overexpression of astrocytic kir4.1 increases neuronal bursts in LHb and causes depressive-like phenotypes. FIG. 5A: Schematics of AAV vectors engineered to overexpress a control construct and Kir4.1 under GFAP. FIG. 5B: Illustration of bilateral viral injection of AAV-GFAP-Kir4.1 in mouse LHb (stained with antibody against GFP and Hoechst). FIG. 5C: Experimental paradigm for electrophysiology and behavioral testing. FIGS. 5D-5F: Astrocytic overexpression of Kir4.1 decreases RMPs of both astrocytes (FIG. 5D) and neurons (FIG. 5E) and increases the bursting population in neurons (FIG. 5F). FIG. 5G: Histogram of inter-spike intervals (ISI, ms) distribution. The ISIs of LHb neurons in astrocytic Kir4.1-overexpressed mice exhibited a clear bimodal distribution with an extra sharp and condensed cluster of high frequency events centred around 40 ms, indicating a significant weight increase of burst firings. FIGS. 5H and 5I: Behavioral effects of expressing various viral constructs in LHb in forced swim test (FST) (FIG. 5H) and sucrose preference test (SPT) (FIG. 5I). Data are means±SEM. **P<0.01, ***P<0.001, ****P<0.0001; n.s., not significant.

(6) FIGS. 6A-6L show that loss of function of Kir4.1 in LHb decreases neuronal bursting and rescues depressive-like phenotypes. FIG. 6A: Schematics of the AAV vector engineered to overexpress shRNA or dominant negative form of Kir4.1. H1, human H1 promoter. CAG: The CMV early enhancer/chicken beta actin promoter. FIG. 6B: Western blot and quantification showing efficient knock-down of Kir4.1 by shRNA in HEK293T cells. FIGS. 6C-6H: Electrophysiological characterization in LHb of AAV-Kir4.1-shRNA and AAV-ctrl-shRNA injected rats. Floating bars for membrane slope conductance calculated from the I/V plots (between −120 and +40 mV). FIG. 6D: Experimental paradigm for behavioral testing of cLH rats infected by virus. FIG. 6E: I-V plots showing a shift of reverse potential from −72 mV to −40 mV in astrocytes infected by AAV-Kir4.1-shRNA. FIGS. 6F and 6G: AAV-Kir4.1-shRNA caused more depolarized RMP in astrocytes (FIG. 6F) and neurons in the viral infected region (FIG. 6G). FIG. 6H: AAV-Kir4.1-shRNA abolished neuronal bursting. FIGS. 6I-6L: Behavioral effects of expressing various viral constructs in the LHb of cLH rats in forced swim (FIG. 6I), learned helpless (FIGS. 6J and 6K) and sucrose preference (FIG. 6L) tests. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, n.s., not significant.

(7) FIG. 7 shows characterization of Kir4.1 loss-of-function constructs. Flag-tagged-Kir4.1 plasmid (pAAV-CMV-betaGlobin-Kir4.1-eGFP-3Flag) was co-transfected with pAAV-vector expressing 6 different shRNAs (see Methods) of Kir4.1 or the negative control (shRNA of luciferase) into HEK293TN cell. Based on the knock-down efficiency as shown in the western blot, Kir4.1-shRNA-5 was chosen for viral package.

(8) FIGS. 8A-8B show that overexpression of Kir4.1 or Kir4.1-shRNA in LHb does not affect locomotion in open field test. FIG. 8A: Overexpression of Kir4.in LHb does not affect locomotion activities. FIG. 8B: Overexpression of Kir41-shRNA in LHb does not affect locomotion activities. Data are means±SEM. n.s., not significant.

(9) FIGS. 9A-9C show the nucleotide sequence comparison between human, mouse and rat Kir4.1 mRNAs.

(10) FIG. 9D shows the amino acid sequence comparison between human, mouse and rat Kir4.1 proteins.

(11) FIG. 9E shows the sequence alignment results between the target sequence correspond to each of the shRNA No. 1-6 and the human Kir4.1 mRNA sequence.

DETAILED DESCRIPTION

(12) The nature and benefits of the present disclosure are further described with reference to the following examples, which are intended to illustrate the invention provided herein and not to limit the scope of the present disclosure.

Example 1. Materials and Methods

(13) Animals. Male Wistar rats (12 weeks) and Sprague Dawley rats (3-4 weeks or 12 weeks) were purchased from Shanghai SLAC Laboratory Animal Co. The cLH rats were introduced from Malinow's lab in Cold Spring Harbor of USA, and screened by learned helpless test for breeding as previously described (Schulz et al., 2010). Male cLH rats (3-4 weeks or 12 weeks) were used. Male adult (7-8 weeks of age) C57BL/6 mice (SLAC) were used for virus injection experiments. Animals were group-housed two/cage for rats and four/cage for mice under a 12-h light-dark cycle (light on from 7 a.m. to 7 p.m.). Animals were housed in stable conditions with food and water ad libitum. All animal studies and experimental procedures were approved by the Animal Care and Use Committee of the animal facility at Zhejiang University.

(14) Virus and Plasmid Construct.

(15) For knock-out tests, AAV5-gfaABC1D-GFP-CreMut (titer: 4.74×10.sup.12 v.g./ml) was ordered and prepared by Taitool Bioscience of China. AAV2/1-CamKII-HI-eGFP-Cre were purchase from University of Pennsylvania Vector core, Upenn, USA (Cat #: AV-1-PV2521). All viral vectors were aliquoted and stored at −80° C. until use.

(16) For overexpression of Kir4.1 tests, AAV2/5-gfaABC1D-EGFP-Kir4.1 was prepared, for which pZac2.1 gfaABC1D-eGFP-Kir4.1 plasmid (AddGene, Plasmid #52874) and the AAV virus (Taitool Bioscience of China, titer: 9.19×10.sup.12 v.g./ml) was used. The package and preparation of the Kir4.1 overexpression viral deliver system was ordered and prepared from Taitool Bioscience of China. A blank control, AAV-GFAP::GFP which expressed GFP but not Kir4.1 was also ordered and prepared from Taitool Bioscience of China.

(17) For loss-of-function of Kir4.1 tests, a Kir4.1 mutation construct and viral deliver system (namely AAV5-gfaABC1D-dnKir4.1-2A-eGFP, titer: 4.15×10.sup.13 v.g./ml) wherein GYG at position 130-132 are mutated to be AAA was ordered and prepared from Taitool Bioscience of China.

(18) For another loss-of-function of Kir4.1 test, shRNA constructs and viral deliver systems targeting Kir4.1 were ordered from and prepared by Taitool Bioscience of China.

(19) A total of 6 shRNA sequences were designed by using RNAi designer online software (Invitrogen), which are purported to target the following target sequence in the rat mRNA sequence of rat (as set forth in SEQ ID NO.:7):

(20) TABLE-US-00003 1) (SEQ ID No. 1) 5′-GGACGACCTTCATTGACAT-3′; 2) (SEQ ID No. 2) 5′-GCTACAAGCTTCTGCTCTTCT-3′; 3) (SEQ ID No. 3) 5′-GCTCTTCTCGCCAACCTTTAC-3′; 4) (SEQ ID No. 4) 5′-CCGGAACCTTCCTTGCAAA-3′; 5) (SEQ ID No. 5) 5′-GCGTAAGAGTCTCCTCATTGG-3′; or 6) (SEQ ID No. 6) 5′-GCCCTTAGTGTGCGCATTA-3′.

(21) Six shRNA plasmids were prepared accordingly. In particular, the six shRNA were cloned in a vector named WX231-L (Taitool Bioscience of China, Cat #: WX231) based on the sequences as shown in the following Table 1.

(22) TABLE-US-00004 TABLE 1 Sequences for shRNA cloning and construction (each sequence has a direction from 5′ end to 3′ end). shRNA Sticky No. end Target seq. Loop Rev seq. PolyT shRNA- TCC GGACGACCTTC TTCA ATGTCAATGA TTTTT 1 CC ATTGACAT AGAG AGGTCGTCC  (SEQ ID No. A (SEQ ID 1) No. 9) TCT GGACGACCTTC TCTC ATGTCAATGA G AAA ATTGACAT TTGA AGGTCGTCC  AAA (SEQ ID No. A (SEQ ID 1) No. 9) shRNA- TCC GCTACAAGCTT TTCA AGAAGAGCAG TTTTT 2 CC CTGCTCTTCT AGAG AAGCTTGTAG (SEQ ID No. A C (SEQ ID 2) No. 10) TCT GCTACAAGCTT TCTC AGAAGAGCAG G AAA CTGCTCTTCT TTGA AAGCTTGTAG AAA (SEQ ID No. A C (SEQ ID 2) No. 10) shRNA- TCC GCTCTTCTCGC TTCA GTAAAGGTTG TTTTT 3 CC CAACCTTTAC AGAG GCGAGAAGAG (SEQ ID No. A C (SEQ ID 3) No. 11) TCT GCTCTTCTCGC TCTC GTAAAGGTTG G AAA CAACCTTTAC TTGA GCGAGAAGAG AAA (SEQ ID No. A C (SEQ ID 3) No. 11) shRNA- TCC GCCGGAACCTT TTCA TTTGCAAGGA TTTTT 4 CC CCTTGCAAA AGAG AGGTTCCGGC (SEQ ID No. A (SEQ ID 19).sup.1 No. 20).sup.2 TCT GCCGGAACCTT TCTC TTTGCAAGGA G AAA CCTTGCAAA TTGA AGGTTCCGGC AAA (SEQ ID No. A (SEQ ID 19).sup.1 No. 20).sup.2 shRNA- TCC GCGTAAGAGTC TTCA CCAATGAGGA TTTTT 5 CC TCCTCATTGG AGAG GACTCTTACG (SEQ ID No. A C (SEQ ID 5) No. 13) TCT GCGTAAGAGTC TCTC CCAATGAGGA G AAA TCCTCATTGG TTGA GACTCTTACG AAA (SEQ ID No. A C (SEQ ID 5) No. 13) shRNA- TCC GCCCTTAGTGT TTCA TAATGCGCAC TTTTT 6 CC GCGCATTA AGAG ACTAAGGGC  (SEQ ID No. A (SEQ ID 6) No. 14) TCT GCCCTTAGTGT TCTC TAATGCGCAC G AAA GCGCATTA TTGA ACTAAGGGC  AAA  (SEQ ID No. A (SEQ ID 6) No. 14) Note: .sup.1The sequence as set forth in SEQ ID No. 19 is substantially the sequence as set forth in SEQ ID No. 4 plus an additional “G” at its 5′ end, in order to increase the cloning and/or transcription efficiency; .sup.2The sequence as set forth in SEQ ID No. 20 is the reverse complimentary of the sequence as set forth in SEQ ID No. 19, and the sequence as set forth in SEQ ID No. 20 consists substantially of a sequence (TTTGCAAGGAAGGTTCCGG, as set forth in SEQ ID No. 12), which is substantially the reverse complimentary of the sequence as set forth in SEQ ID No. 4, plus a “C” at its 3′ end. Such a design is purported to increase the cloning and/or transcription efficiency.

(23) The knocking down efficiency was tested by Western blot of Kir4.1 from HEK293TN cells which were co-transfected with Flag-tagged-Kir4.1 plasmid (pAAV-CMV-betaGlobin-Kir4.1-eGFP-3Flag) and each of the six shRNA plasmids. The fifth sequence, 5′-GCGTAAGAGTCTCCTCATTGG-3′, was chosen for use in Kir4.1-shRNA virus package.

(24) AAV5-H1-Kir4.1-shRNA-CAG-eGFP (titer: 3.04×10.sup.13 v.g./ml) and AAV5-H1-Luciferase-shRNA-CAG-eGFP (titer: 1.46×10.sup.13 v.g./ml) were ordered and prepared by Taitool Bioscience of China.

(25) LPS-induced depression model. The LPS-induced depression model was conducted as previously described (Adzic et al., 2015). Adult (3 months) Wistar male rats were used for the experiments. LPS (Sigma, L-2880) dissolved in sterile 0.9% saline was intraperitoneally injected into Wistar rats, at a dosage of 0.5 mg/kg. This dosage was used to stimulate a subclinical infection without inducing obvious inflammation and other apparent impairment in the animals. Saline or LPS was injected between 09:30 and 10:30 a.m. daily for 7 days. The forced swim test was performed 24 hours after the last injection. The habenular tissue was dissected 24 hours after the behavioral test.

(26) Stereotaxic surgery and virus injection. cLH rats or mice (postnatal 50-60 days) were deeply anesthetized by using 4% pentobarbital and placed in a stereotactic frame (RWD Instruments, China). All measurements were made relative to bregma for virus/implant surgeries (For rats, LHb: AP, −3.7 mm from bregma; ML, ±0.7 mm; DV, −4.1 mm from the brain surface; for mice: AP, −1.72 mm from bregma; ML, ±0.46 mm; DV, −2.62 mm from the brain surface). Virus injection was performed using a mircoinjection needle with a pressure microinjector (Picospritzer III, Parker, USA) delivering virus at a slow rate of 0.1 ul/min. After the injection was completed, two minutes were allowed to pass and leaving it for an additional 10 minutes before the needle was then slowly withdrawn completely. After surgery, mice recovered from anesthesia under a heat pad.

(27) At least 14 days after injection, mice or rats were used in behavioral or electrophysiological studies. All the injection sites will be checked by immunostaining after the behavioral experiments. Only mice with correct injection site will be counted into the behavioral statistics.

(28) Immunohistochemistry. Animals were anesthetized using 10% chloral hydrate, and then perfused transcardially with ice-cold PBS (pH 7.4) followed by 4% paraformaldehyde. After overnight post fix in 4% paraformaldehyde solution, brains were cryoprotected in 30% sucrose for 1 day (for mice) or 3 days (for rats). Coronal sections (40 μm) were cut on a microtome (Leica) and collected in PBS and stored at 4° C. for further using. The antibodies used were rabbit anti-Kir4.1 extracellular peptide (1:200, Alomone labs), mouse anti-GFAP (1:500, Sigma), mouse anti-NeuN (1:500, Millipore), mouse anti-S100b (1:500, Sigma), chicken anti-GFP (1:1000, Abcam), Alexa Fluor488 goat anti-rabbit IgG, Alexa Fluor488 goat anti-chicken IgG, Alexa Fluor594 goat anti-mouse IgG (all 1:1000, Invitrogen). Specifically, for Kir4.1 staining, the rabbit anti-Kir4.1 extracellular peptide antibody was incubated for 48-72 h and the other primary antibodies were incubated for 36-48 h. For the antibody absorption experiments, the rabbit anti-Kir4.1 extracellular peptide antibody was pre-adsorbed with the Kir4.1 antigen by mixing at the weight ratio of 1:2 for 24 h. Slices were counterstained with Hoechst in the final incubation step to check the injection site. Fluorescent image acquisition was performed with an Olympus Fluoview FV1000 confocal microscope and a Nikon A1 confocal microscope.

(29) Western Blot. The habenular membrane fraction and whole protein was extracted as previously described.sup.11. Animals were anesthetized using 10% chloral hydrate, and habenular tissue was quickly dissected from the brain and homogenized in lysis buffer (320 mM sucrose, 4 mM HEPES pH7.4, 1 mM MgCl.sub.2 and 0.5 mM CaCl.sub.2, 5 mM NaF, 1 mM Na.sub.3VO.sub.4, EDTA-free, Protease Inhibitor cocktail tablets (Roche)) on ice. The lysis buffer used for extracting the total protein of HEK293TN cell contained 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS and Protease Inhibitor cocktail tablets (Roche). After protein concentration measurement by BCA assay, 10-20 g proteins for each lane was separated on a 10% SDS-PAGE gel and transferred for western blot analysis. Anti-Kir4.1 (1:1000, Alomone labs), anti-GFAP (1:1000, Sigma) and anti-tubulin (1:5000, Bio-Rad) antibodies were used. High sensitive ECL reagent was used (GE Healthcare). All the bands were analyzed with Quantity one or Image J.

(30) LHb brain slice preparation. Rats (P25-30 or P60-90) and mice (P90) were anesthetized with isoflurane and 10% chloral hydrate, and then perfused with 20 ml ice-cold ACSF (oxygenated with 95% O.sub.2+5% CO.sub.2) containing (mM): 125 NaCl, 2.5 KCl, 25 NaHCO.sub.3, 1.25 NaH.sub.2PO.sub.4, 1 MgCl.sub.2, 2.5 CaCl.sub.2) and 25 Glucose, with 1 mM pyruvate added. The brain was removed as quickly as possible after decapitation and put into chilled and oxygenated ACSF. Coronal slices containing habenular (350 μm- and 300 μm-thickness for rats and mice, respectively) were sectioned in cold ACSF by a Leica2000 vibratome and then transferred to ASCF at 32° C. for incubation and recovery. ACSF was continuously gassed with 95% O.sub.2 and 5% CO.sub.2. Slices were allowed to recover for at least 1 hour before recording.

(31) In vitro electrophysiological recording. For LHb neuron recordings, currents were measured under whole-cell patch clamp using pipettes with a typical resistance of 4-6 MΩ filled with internal solution containing (mM) 105 K-Gluconate, 30 KCl, 4 Mg-ATP, 0.3 Na-GTP, 0.3 EGTA, 10 HEPES and 10 Na-phosphocreatine, with pH set to 7.35. The external ACSF solution contained (in mM) 125 NaCl, 2.5 KCl, 25 NaHCO.sub.3, 1.25 NaH.sub.2PO.sub.4, 1 MgCl.sub.2, 2.5 CaCl.sub.2 and 25 Glucose. Cells were visualized with infrared optics on an upright microscope (BX51WI, Olympus). AMultiClamp 700B amplifier and pCLAMP10 software were used for electrophysiology (Axon Instruments). The series resistance and capacitance was compensated automatically after stable Giga seal were formed. The spontaneous neuronal activity was recorded under current-clamp (I=0 pA) for consecutive 60 s. RMP was determined during the silent period of the neuronal spontaneous activity.

(32) To test TTX (1 μM, Sigma) and BaCl.sub.2 (100 μM, Sigma) effect onto neuronal RMP, baselines of RMP were recorded for at least for 3 min. Drug were then perfused, the arriving of the drug was precisely indicated with a bubble that pre-added before the transition from normal ACSF to drug added ACSF. TTX acts on LHb neuron as quickly as several minutes while BaCl.sub.2 takes more 10 min to affect neuronal RMP. The drug effect of TTX and BaCl.sub.2 onto neuronal RMP at the time point of 5 min and 15 min were then tested respectively.

(33) Astrocytic patch and Kir4.1 current isolation. Astrocytes were distinguished from neuron by their small (5-10 μm) oval shaped somata and by electrophysiological features: a hyperpolarized RMP and a low input resistance, a linear I-V relationship and an absence of action potentials in response to increased injection currents. BaCl.sub.2 (100 μM, Sigma) were applied to isolate Kir4.1 current which is subtracted from the IV curve recorded from −120 mV to 0 mV.

(34) Learned helpless test (LHT). Male juvenile (P30) or adult (P90) cLH rats were tested in a lever-pressing task to evaluate the learned helpless (LH) phenotype.sup.11. A cue-light-illuminated lever in the shock chamber was presented, which can terminate the shock when rats pressed the lever. 15 escapable shocks (0.8 mA) were delivered with a 24 s inter-shock interval were given. Each shock lasted up to 60 s unless the rat pressed the lever to terminate the shock. Out of the 15 trials, rats which failed to press the lever for more than 10 trials were defined as “learned helplessness” (LH), and rats with less than 5 failures were defined as “non-learned helplessness” (NLH).

(35) Forced swim test (FST). Animals were individually placed in a cylinder (12 cm diameter, 25 cm height for mice; 20 cm diameter, 50 cm height for rats) of water (23-25° C.) and swam for 6 min under normal light. Water depth was set to prevent animals from touching the bottom by tails and hind limbs. Animal behaviors were videotaped from the side. The immobility time during the last 4 min test was counted offline by an observer blind of the animal treatments. Immobility was defined as time when animals remained floating or motionless with only movements necessary for keeping balance in the water. For rats, an additional pre-test was conducted 24 h before the test, during which rats were individually placed in a cylinder of water with conditions described above for 15 min.

(36) Sucrose preference test (SPT). Animals were single housed and habituated with two bottles of water for 2 days, followed by two bottles of 2% sucrose for 2 days. Animals were then water deprived for 24 h and then exposed to one bottle of 2% sucrose and one bottle of water for 2 h in the dark phase. Bottle positions were switched after 1 h. Total consumption of each fluid was measured and sucrose preference was defined as the ratio of sucrose consumption divided by total consumptions of water and sucrose.

(37) Statistical analyses. Required sample sizes were estimated based on our past experience performing similar experiments. Animals were randomly assigned to treatment groups. Analyses were performed in a manner blinded to treatment assignments in all behavioral experiments. Statistical analyses were performed using GraphPad Prism software v6. By pre-established criteria, values were excluded from the analyses if the viral injection or drug delivering sites were out of LHb. All statistical tests were two-tailed, and significance was assigned at P<0.05. Normality and equal variances between group samples were assessed using the D'Agostino & Pearson omnibus normality test and Brown-Forsythe tests respectively. When normality and equal variance between sample groups was achieved, one-way ANOVAs (followed by Bonferroni's multiple comparisons test), or t test were used. Where normality or equal variance of samples failed, Mann-Whitney U test were performed. Linear regression test, Chi-square test was used in appropriate situations.

Example 2. Kir4.1 is Upregulated in LHb of Animal Models of Depression

(38) Western blot analysis confirmed that Kir4.1 had a significant increase (1.75-fold) in the membrane protein extraction of cLH habenulae (FIG. 1A). To test whether Kir4.1 upregulation is universal in depression, an additional depression animal model, the LPS (lipopolysaccharide)-induced depression was used. One week of LPS injection (0.5 mg/kg, i.p.) in 3-month-old Wistar rats was sufficient to cause strong depressive-like phenotype in the forced swim test (FST). The Kir4.1 level was also significantly increased in the LPS-depression rats (FIG. 1B, 1.87-fold).

(39) Kir4.1 is a principal component of the glial Kir channel and is largely responsible for mediating the K+ conductance and setting the RMP of astrocytes. To confirm that Kir4.1 function is indeed upregulated, whole-cell patch clamp was performed onto the astrocytes in brain slices made from the LHb of cLH or SD rats. Astrocytes were distinguished from neurons by their small (5-10 μM) oval shaped somata and electrophysiological features including a relatively hyperpolarized RMP (−74±1 mV), a low input resistance Rin (47±6 MΩ), a linear I-V relationship and an absence of action potentials in response to depolarizing current injections (FIG. 1C). Ba.sup.2+ (BaCl.sub.2, 100 μM) was then bath applied, which selectively blocks Kir channels at sub-mM concentrations, to isolate Kir4.1 current. The Ba.sup.2+-sensitive currents displayed a reversal potential close to Ek (−90 mV) (FIGS. 1E and 1F), indicating it represents the K+ conductance. It was found that Ba.sup.2+-sensitive currents in LHb astrocytes were almost doubled in cLH rats, compared with SD controls, at the age of P60-90 (FIG. 1C). Interestingly, the increase of Kir4.1 current was not obvious at P30 (FIG. 1D). At this age, cLH rats did not yet show depressive-like phenotypes in both learned helpless test (LHT, FIG. 1E) and the forced swim test (FST, FIG. 1F), suggesting that level of Kir4.1 overexpression correlated with the developmental onset of the depressive-like symptoms.

Example 3. Kir4.1 are Expressed on Astrocytic Processes Tightly Wrapping Around Neuronal Soma

(40) As an inwardly rectifying K.sup.+ channel, Kir4.1 has been strongly implicated in buffering excess extracellular K.sup.+ in tripartite synapses. Conventional model of K.sup.+ buffering suggests Kir4.1 to be highly expressed in astrocytic endfeet surrounding synapses. Surprisingly, with immunohistochemistry co-labeling, Kir4.1 staining in LHb appeared to overlap with the neuronal marker NeuN at low magnification (20×, FIG. 3A), although in the same brain slice Kir4.1 staining patterns in hippocampus were typical astrocytic-looking (FIG. 3B). However, higher magnification imaging with single layer scanning (0.76 μm per layer) revealed that Kir4.1 staining enveloped NeuN signals (FIG. 2A). To confirm that Kir4.1 indeed locates within astrocytes but not neurons in LHb, kir4.1 was separately knocked out in either neurons or astrocytes by injecting AAV virus expressing the cre recombinase under either the neuronal promoter CaMKII or glial promoter GFAP (gfaABC1D) into Kir4.1-floxed mice by using AAV5-gfaABC1D-GFP-CreMut (titer: 4.74×10.sup.12 v.g./ml), which was provided as mentioned in Example 1. The neural-surrounding staining of Kir4.1 remained intact with neuronal knock-out, but was completely eliminated with astrocytic knock-out (FIG. 2B). Electron microscopy imaging revealed that Kir4.1-positive gold particles were distributed encircling the membrane of neuronal cell bodies (FIG. 2C), as well as in the synapses. Consistently, whole-cell-patch recordings showed that Ba.sup.2+-sensitive currents are absent in neurons but abundant in astrocytes in LHb (FIG. 2D). Together these results suggest that Kir4.1 is mainly expressed in astrocytic processes tightly wrapping around neuronal soma and synapses in LHb.

Example 4. Kir4.1-Mediated K Buffering Regulates Neuronal RMP and Bursting Activity

(41) How does an astrocytic potassium channel regulate RMP and burst firing of the LHb neuron? The inventors hypothesize that within the highly confined extracellular space between neuronal soma and Kir4.1-positive astrocytic processes (FIGS. 2A-2D), the majority of constantly-released K+ from intrinsically active LHb neurons is quickly cleared by astrocytes through a Kir4.1-dependent mechanism. Accordingly, the Inventors predicted that blockade of Kir4.1 should compromise K+ spatial buffering, resulting in increased extracellular K+ (Kout), and according to Nernst Equation, depolarized neuronal RMPs. Consistent with this prediction, blocking Kir4.1 with BaCl.sub.2 depolarized LHb RMPs of tonic and burst-firing, but not silent neurons, after about 10 min bath perfusion of BaCl.sub.2 (FIGS. 4A-4C). The amount of changes in RMP positively correlated with the original firing rates of neurons (FIGS. 4D-4F), indicating the more active the neuron is, the larger contribution the K+ buffering to its RMP. Consequent to the RMP change, perfusion of BaCl.sub.2 caused a dramatic increase of firing frequency until the neuron reached a sustained plateau of a tetanus response and stopped firing (FIGS. 4G-4H).

Example 5. Enhanced Capacity of Extracellular K.SUP.+ Clearance Due to Kir4.1 Overexpression May Underlie the Neuronal Hyperpolarization Required for Burst Initiation

(42) To assess a causal relationship between Kout and firing mode, current-clamp recordings of LHb neurons were made while lowering Kout from 2.75 mM to 1.4 mM (FIG. 4I). This led to lowered neuronal RMP by 13.7+/−0.5 mV (FIG. 4J) and a direct shift of originally tonic-firing neurons into bursting mode (FIG. 4I). Consequently, percentage of bursting neurons was increased from 8% to 23% (FIG. 4K). In summary, by increasing astrocytic Kir4.1 expression or decreasing the extracellular K+ concentration, it was able to phenocopy in WT animals several key neuronal properties observed in the LHb of animal models of depression, namely hyperpolarized RMPs and enhanced bursts. These results indicate that enhanced capacity of extracellular K.sup.+ clearance due to Kir4.1 overexpression may underlie the neuronal hyperpolarization required for burst initiation.

Example 6. Overexpression of Kir4.1 in LHb Astrocytes Increases Neuronal Bursts and Causes Strong Depressive-Like Behaviors

(43) To test the effects of Kir4.1 upregulation in LHb, a Kir4.1 overexpression system AAV2/5-gfaABC1D-EGFP-Kir4.1 (namely “AAV-Kir4.1” or “AAV-GFAP::Kir4.1”) was prepared and assayed, which uses adeno-associated viruses of the 2/5 serotype (AAV2/5) that preferentially target astrocytes as the vector, together with the human GFAP (gfaABC1D) promoter for the expression. The Kir4.1 overexpression system GFP-tagged Kir4.1 channels (AAV-GFAP::Kir4.1) was prepared as described in Example 1, while AAV-GFAP::GFP which expressed GFP but not Kir4.1 was used as a control (FIG. 5A).

(44) 14 days after bilateral injection in the LHb at P50, AAV2/5-mediated viral transduction led to Kir4.1 and GFP expression in astrocytes throughout the LHb (FIG. 5B). Whole-cell recordings were made from either astrocytes or neurons surrounding the viral-transfected astrocytes in coronal LHb slices. The RMPs of both astrocytes and neurons were more hyperpolarized (FIGS. 5D and 5E) and the percentage of bursting neurons were significantly higher (FIG. 5F) in mice infected with AAV-GFAP::Kir4.1 than with AAV-GFAP::GFP.

(45) Depressive-like phenotypes were then assayed on and it was found that mice with AAV-GFAP::Kir4.1 infection in the LHb displayed severe depressive-like behaviors (FIGS. 5C-5I), including increased immobile duration and decreased latency to immobility in FST (FIG. 5H), and decreased sucrose preference in the sucrose preference test (SPT, FIG. 5I).

Example 7. Loss-of-Function of Kir4.1 in LHb Decreases Neuronal Bursting and Rescues Depressive-Like Phenotypes

(46) Next, to determine whether loss-of-function of Kir4.1 in LHb may reverse depressive phenotypes, two strategies were tried by using AAV2/5 viral vectors to express either a short hairpin RNA (shRNA) to knock down the level of Kir4.1, or a dominant negative construct to block its function in the LHb of cLH rats (FIG. 6A).

(47) Six shRNAs specifically targeting the Kir4.1 transcript in cell culture were tested. The results showed that the knock-off efficient of these 6 shRNAs were: Kir4.1-shRNA-1>Kir4.1-shRNA-4/Kir4.1-shRNA-5>Kir4.1-shRNA-2>Kir4.1-shRNA-6>Kir4.1-shRNA-3. The one with most efficient knock-down efficiency was chosen for viral package (FIG. 6B and FIG. 7). After viral expression of this shRNA under the H1 promoter in the LHb of cLH rats (FIG. 6C), its effect was examined on glial and neural electrophysiological properties. In astrocytes infected with the AAV-Kir4.1-shRNA vector, it was found that a dramatic change of I-V relation (FIG. 6E) and a 32 mV depolarization compared with neighboring non-infected astrocytes and 41 mV depolarization compared with ctrl-shRNA-GFP+ astrocytes (FIG. 6F). In neurons infected with the AAV-Kir4.1-shRNA vector, the RMPs did not differ from neighboring non-infected neurons (because neurons do not express Kir4.1 endogenously, FIG. 6G). However, RMPs of LHb neurons from AAV-Kir4.1-shRNA-infected brain slices were overall more depolarized than RMPs of those from AAV-luciferase-shRNA-infected rats (−43±2 mV vs. −53±2.7 Mv, FIG. 6G), suggesting that knock-down of Kir4.1 in astrocytes had a global impact on RMPs of neighboring neurons. Most importantly, bursting activity in LHb of cLH rats were completely eliminated by AAV-Kir4.1-shRNA viral infection (from 29% to 0%, FIG. 6H).

(48) Behaviorally, infection of AAV-Kir4.1-shRNA had a pronounced effect on rescuing the depressive-like phenotypes of cLH rats in three depression paradigms: reducing the immobility time and increasing latency to immobility in FST (FIG. 6I), increasing bar pressing number in the LHT (FIGS. 6J and 6K), and increasing the sucrose preference score in SPT (FIG. 6L).

(49) To avoid an off-target effect of shRNA, a dominant-negative form of Kir4.1, dnKir4.1, containing a GYG to AAA point mutation at the channel pore which blocks K.sup.+ channels was also tested. The preparation of the Kir4.1 mutation construct and viral deliver system (namely AAV-dnKir4.1, or AAV5-gfaABC1D-dnKir4.1-2A-eGFP) was prepared as described in Example 1. Infection of AAV-dnKir4.1 caused similarly strong anti-depression effects in cLH rats (FIGS. 6I-6L): reducing the immobility time and increasing latency to immobility in FST (FIG. 6I), increasing bar pressing number in the LHT (FIGS. 6J and 6K), and increasing the sucrose preference score in SPT (FIG. 6L).

Example 8. Kir4.1 is Highly Conserved Across the Species from Rat to Human

(50) The experiments as described above, which have largely been performed using rodent animal models (i.e. rat and mouse), have established a crucial role of the inward rectifier potassium (Kir) channel Kir4.1 (or KCNJ10) that is specifically expressed in the astroglial tissues or astrocytes in the lateral habenula (LHb) in causing the depression-like phenotype, at least in these rodent animal models. LHb Kir4.1 has been demonstrated to be able to regulate neuronal resting membrane potential (RMP) and bursting, and importantly, has been shown to be significantly upregulated in depression. The gain-of-function and the loss-of-function manipulations of Kir4.1 in the rodent animals have been shown to respectively cause and rescue depression, indicating that astroglial Kir4.1 is both necessary and sufficient for causing depression. Thus these above results point to Kir4.1 in the LHb as a potential target for treating clinical depression.

(51) In order to estimate whether, and to what extent, the above study results can be translated into other higher species like humans, a series of sequence conservation studies is performed.

(52) Firstly, a comparative study among the Kir4.1 mRNA sequences of rat (as set forth by SEQ ID NO. 7, see NCBI Reference Sequence: NM_031602.2), mouse (as set forth by SEQ ID NO. 15, see NCBI Reference Sequence: NM_001039484.1), and human (as set forth by SEQ ID NO. 17, see NCBI Reference Sequence: NM_002241.5) has shown that 86.7% of the nucleotide residues are identical, and 98.9% of the nucleotide residues show at least a consensus across the three species (FIGS. 9A-9C).

(53) Secondly, a comparative study among the Kir4.1 amino acid sequences of rat (as set forth by SEQ ID NO. 8, see NCBI Reference Sequence: NP_113790.2, which corresponds to NM_031602.2), mouse (as set forth by SEQ ID NO. 16, see NCBI Reference Sequence: NP_001034573.1, which corresponds to NM_001039484.1), and human (as set forth by SEQ ID NO. 18, see NCBI Reference Sequence: NP_002232.2, which corresponds to NM_002241.5) has shown that 97.9% of the amino acid residues are identical, and 99.7% of the amino acid residues show at least a consensus across the three species (FIG. 9D).

(54) Given the extremely high conservation between human Kir4.1 and rodent Kir4.1 in terms of the mRNA sequences (having 98.9% consensus positions) and the protein sequences (having 99.7% consensus positions), Kir4.1 shall play a similarly crucial role in the lateral habenula (LHb) in the pathogenesis and maintenance of depression.

(55) A survey over the above mentioned Kir4.1-targeting shRNAs (shRNA NOS. 1-6) has shown that each of their corresponding target sequences on the rat Kir4.1 mRNA sequence (i.e. as set forth in SEQ ID NO. 7), which are respectively set forth in SEQ ID NOS. 1-6, also represents a relatively high level of homology in the alignment (see FIG. 9E) with the human mRNA sequence of Kir4.1 (i.e. as set forth in SEQ ID NO. 17), with the 21-base target sequence for shRNA-2 (as set forth in SEQ ID NO. 2) being 100% identical, only one-base difference for both the 19-base target sequences of shRNA-1 and shRNA-6 (as set forth in SEQ ID NOS. 1 and 6 respectively), three-base difference for the 21-base target sequence of shRNA-5 (as set forth in SEQ ID NO. 5), and four-base difference for the 21-base target sequences of shRNA-3 and for the 19-base target sequences of shRNA-4 (as set forth in SEQ ID NOS. 3 and 4, respectively). As such, among the 6 shRNAs that have been utilized for suppressing expression of rat Kir4.1, at least the shRNA-2, likely the shRNA-1, 6 and 5, and potentially shRNA-4 as well, can be utilized to suppress the expression of human Kir4.1, and thus can serve as a component of a pharmaceutical agent capable of inhibiting the activity of the astroglial potassium channel Kir4.1 in a human subject with depression to suppress the bursting activity of neurons in a lateral habenula of the subject for treatment of the disease.

(56) Regarding the dominant negative mutant form of Kir4.1 protein (i.e. dnKir4.1), containing a GYG-to-AAA point mutation corresponding to position 130-132 of the amino acid sequence of wild-type Kir4.1 protein of rat Kir4.1 (as set forth in SEQ ID NO. 8), since the three-amino-acid sequence is strictly conserved within the 88-amino acid context region at positions 101-188 of all of the human, mouse and rat Kir4.1 proteins (as respectively set forth in SEQ ID NOS. 18, 16 and 8, see FIG. 9D), tissue-specific overexpression of such a mutant protein in the LHb of a human subject is expected to also interfere with the normal functionality of Kir4.1 in a dominant negative manner, and consequently, the bursting activity in the LHb of the human subject can be eliminated, thereby realizing an effective treatment of depression in the human subject. As such, the dnKir4.1 can similarly be used as a pharmaceutical agent capable of inhibiting the activity of the astroglial potassium channel Kir4.1 in a human subject with depression to suppress the bursting activity of neurons in a lateral habenula of the subject for treatment of the disease.

(57) Notably also, the vectors that have been utilized for the astrocyte-specific expression of shRNAs or dnKir4.1 in the rat studies as described above, such as AAV-GFAP, and esp. the AAV2/5 shall also be able to realize a similar astrocyte-specific expression of shRNAs (e.g. shRNA-2) and dnKir4.1 to be able to exert the therapeutic effects.

(58) The present disclosure for the first time and unexpectedly find that burst in neurons of the lateral habenula has an important role in the cause of depression, and identify key factors affecting the burst in the lateral habenula, including that the activation of NMDA receptors is the sufficient and necessary condition for inducing burst in the lateral habenula, and that burst in the lateral habenula need the participation of neuron membrane hyperpolarization and T-type low voltage activate calcium channel. Particularly, the present disclosure for the first time and unexpectedly find that it is the burst instead of whole neuron firing or neuron discharges that contribute to the cause of depression. The inventors provide a method and medicament for diagnosing and treating (inhibiting) depression by inhibiting burst in lateral habenula, especially method and medicament for rapidly treating (inhibiting) depression.

(59) Unless otherwise indicated, the practice of the present disclosure will employ common technologies of organic chemistry, polymer chemistry, biotechnology, and the like. It is apparently that in addition to the above description and examples than as specifically described, the present disclosure can also be achieved in other ways. Other aspects within the scope of the disclosure and improvement of the present disclosure will be apparent to the ordinary skilled in the art. According to the teachings of the present disclosure, many modifications and variations are possible, and therefore it is within the scope of the present disclosure.

(60) Unless otherwise indicated herein, the temperature unit “degrees” refers to Celsius degrees, namely ° C.

(61) All references that have been referred to in the present application are incorporated by reference in their entirety for all purposes.

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