CN104338135B - The regulatory factor of depression and its application - Google Patents

The regulatory factor of depression and its application Download PDF

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CN104338135B
CN104338135B CN201310347938.6A CN201310347938A CN104338135B CN 104338135 B CN104338135 B CN 104338135B CN 201310347938 A CN201310347938 A CN 201310347938A CN 104338135 B CN104338135 B CN 104338135B
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camkii
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depression
group
expression
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CN104338135A (en
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胡海岚
李坤
周涛
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Center for Excellence in Brain Science and Intelligence Technology Chinese Academy of Sciences
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Shanghai Institutes for Biological Sciences SIBS of CAS
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Abstract

The present invention provides the regulatory factor of depression and its applications.Specifically, the present invention provides β CaMKII inhibitor applies in preparing the drug for preventing and/or treating depression.The present invention's studies have shown that habenular nucleus overexpression β CaMKII (and non-alpha CaMKII) can induce out the core depressive symptom of anhedonia and behavioral despair on the outside.Habenular nucleus lowers the expression of β CaMKII on the outside, and the depressed phenotype of animal can be reversed by reducing its kinase activity or blocking its downstream molecules GluR1 then.

Description

The regulatory factor of depression and its application
Technical field
This field is related to biotechnology and field of medicaments, particularly, is related to regulatory factor and its application of depression.
Background technology
Major depressive disorder (MDD) is one of mental disease that is most common and most disabling, has the characteristics that, mood is low It falls, adynamia, it is desperate, it can not experience happy, also referred to as anhedonia (1).The reason of for leading to MDD, now viewpoint Think, when responding environmental stimuli such as pressure, the nervous activity of specific brain loop is changed.And it is this change be by Caused by the variation of specific molecular and cellular level.Recently, outside habenular nucleus (LHb), as from limbic forebrain to numerous lists The core brain area of information is transmitted at amine center, has become the crucial brain region of depression Pathological Physiology and Therapy study.LHb The mood that neuron is in detestation activates, including pressure, dejected, frightened or expected negative return.It is consistent therewith, neuroimaging Research has determined that the raising of the habenular nucleus liveness under depressive state.In addition, one recently is the study found that in animal depression In model, synaptic activity and the output of LHb neuron peak values are all enhanced.However in LHb these aberrant cellular processes molecule How the stimulation of mechanism and inducing depression such as pressure leads to the generation that these change, and there is still a need for determine.
The correlation factor adjusted there is an urgent need in the art to depression to be prevented and treated to depression and its effect Mechanism, and develop the drug that can be used for preventing and/or treat depression.
Invention content
It is an object of the invention to provide the active constituents and drug that can be used for preventing and/or treat depression
In the first aspect of the present invention, a kind of purposes of β CaMKII inhibitor is provided, be used to prepare prevention and/or is controlled Treat the drug of depression.
In another preferred example, the inhibitor is selected from the group：
(i) RNA interfering or its precursor of interference β CaMKII expression；
(ii) the saltant type β CaMKII albumen or its coded sequence that kinase activity declines or loses；
(iii) inhibit the active inhibitor of β CaMKII；
(iv) competitive inhibitor of β CaMKII；
(v) antibody of anti-β CaMKII；
(vi) antagonist of β CaMKII regulation and control AMPAR GluR1 accesses is checked.
In another preferred example, the RNA interfering is β RNAi (SEQ ID NO.:1,5'- GAGTATGCAGCTAAGATCA-3')。
In another preferred example, the saltant type β CaMKII albumen is the β CaMKII albumen dominant factors, preferably Ground is β K43R (Wayman GA et al.2011).
In another preferred example, the inhibitor of the β CaMKII includes KN-93, KN-62, AC3-I (Autocamtide-3Derived Inhibitory Peptide)、Ant-AIP-II(Autocamtide-2Related Inhibitory Peptide II,Cell-permeable)。
In another preferred example, the component (vi) is the dominant factor of AMPAR GluR1, preferably GluR1Ct。
In another preferred example, the inhibitor or antagonist include following form：Polynucleotides, polypeptide, expression carry Body, micromolecular compound, or combinations thereof.
In another preferred example, the expression vector is selected from the group：Viral vectors.
In another preferred example, the β CaMKII inhibitor is additionally operable to prepare the neuron peak electricity for reducing outside habenular nucleus The drug of the drug of position output and/or the synaptic efficacy of reduction outside habenular nucleus.
In another preferred example, the drug be targeting in the drug of outside habenular nucleus or the drug be to be applied to The drug of outside habenular nucleus region or near zone.
In the second aspect of the present invention, a kind of pharmaceutical composition that can be used for preventing and/or treat depression, institute are provided The pharmaceutical composition stated includes：
Pharmaceutically acceptable carrier；With
Effective quantity active constituent, wherein the active constituent is β CaMKII inhibitor or antagonist.
In another preferred example, the active constituent is to α CaMKII substantially unrestraint/antagonisms.
In another preferred example, the active constituent is to δ CaMKII substantially unrestraint/antagonisms.
In another preferred example, the active constituent is to γ CaMKII substantially unrestraint/antagonisms.Another excellent It selects in example, the active constituent has inhibition/antagonism to γ CaMKII.
In another preferred example, the active constituent selective depression β CaMKII, and to other CaMKII (including α CaMKII, γ CaMKII and δ CaMKII) free or substantially free of inhibition/antagonism.
In another preferred example, the β CaMKII inhibitor or antagonist are selected from the group：βCaMKII-RNAi、βK43R、 KN-93、KN-62、AC3-I(Autocamtide-3Derived Inhibitory Peptide)、Ant-AIP-II (Autocamtide-2Related Inhibitory Peptide II,Cell-permeable)。
In another preferred example, the pharmaceutical composition is oral preparation, injection, targeting preparation etc..
In the third aspect of the present invention, provides a kind of β CaMKII albumen or its coded sequence and/or β CaMKII promote The purposes of agent, it be used to prepare a reagent or composition, and the reagent or composition are for building animal models of depression.
In another preferred example, in the animal models of depression, β CaMKII are high expression.
In another preferred example, in the animal models of depression, β CaMKII are high expression in habenular nucleus on the outside.
In another preferred example, the high expression refers to >=150% (by mRNA level in-site or protein level), preferably >= 200%, more preferably >=200%.
In another preferred example, the upper limit of the high expression (by mRNA level in-site or protein level) is≤2000%, preferably ≤ 1000%, more preferably≤500%.
In the fourth aspect of the present invention, a kind of side screened for preventing and/or treating the potential substance of depression is provided Method, including step：
(1) tester to be screened is applied to animal models of depression, wherein in the outside of the animal models of depression β CaMKII are high expression in habenular nucleus；With
(2) related symptoms and/or index of the depression in the animal models of depression are observed, and are carried out with control group Compare；
Wherein, the related symptoms of depression are significantly improved in the animal models of depression, then it represents that the tester is It can be used for preventing and/or treat the potential substance of depression.
In another preferred example, the animal model is non-human mammal, such as rodent, rabbit, Primate.
In another preferred example, in step (2), pass through Animal Behavior Science Germicidal efficacy depressive symptom.
In another preferred example, Animal Behavior Science experiment is selected from the group：Xi Ze-zong experiment forces swimming real It tests, the experiment of sucrose preference, tail-suspention test, novel inhibit feeding experiment.
In another preferred example, the symptom is selected from the group：Xi Ze-zong (being reduced by lever number), abandoning swimming carries Morning and quiescent time increase, syrup consumption is reduced, struggle is reduced, food rcstriction increases.
In another preferred example, the index includes：The expression of β CaMKII, the activity level of β CaMKII.
In another preferred example, the application (or administration) includes being administered orally.
In another preferred example, the control group includes：Negative control group and/or positive controls.
In another preferred example, the negative control group includes：The control group of tester is not applied；Or apply tester Same test group before.
In the fifth aspect of the present invention, a kind of side screened for preventing and/or treating the potential substance of depression is provided Method, including step：
(1) in test group, tester to be detected is added into vitro detection system；With
(2) detect the expression and/or activity of β CaMKII in the vitro detection system of the test group, and with feminine gender Control group is compared；
Wherein, if compared with adding negative control group, the expression of β CaMKII is remarkably decreased in test group, and/ Or the activity of β CaMKII is remarkably decreased, then it represents that the tester is the potential substance for preventing and/or treating depression.
In another preferred example, the vitro detection system includes cell system；Preferably neuronal cell system； Preferably outside Habenular Neurons system.
In another preferred example, described to be remarkably decreased finger, compared with negative control, the expression of β CaMKII in test group Level or activity is the 5-90% of negative control group, preferably 10-60%, more preferably 20-50%.
In another preferred example, the step (2) further includes being compared with positive controls.
In another preferred example, the method further includes following one or more steps：
To the potential substance that previous step filters out, its influence to the membrane part of GluR1 is further tested；
To the potential substance that previous step filters out, it is applied to animal model, observes its influence to symptoms of depression.
In another preferred example, when testing its influence to the membrane part of GluR1, if (or empty with negative control group White control group) it compares, it is added or is reduced using GluR1 contents in the test group of the tester, then it represents that the tester is pre- Anti- and/or treatment depression potential substance.
In another preferred example, the GluR1 contents are the GluR1 contents on neuronal cell film.
In the sixth aspect of the present invention, a kind of purposes of GluR1 inhibitor is provided, prevention and/or treatment are used to prepare The drug of depression.
In another preferred example, the drug is additionally operable to reduce the neuron spike potential output of outside habenular nucleus and cynapse effect Energy.
In another preferred example, the GluR1 inhibitor is β CaMKII inhibitor.
In another preferred example, the GluR1 inhibitor is the competitive inhibitor of GluR1, including dominant because Son, preferably GluR1Ct.
In another preferred example, the GluR1 inhibitor is selected from the group：Polynucleotides, polypeptide, expression vector, small point Sub- compound, or combinations thereof.
In the seventh aspect of the present invention, a kind of method prevented and/or treat depression, including step are provided：To need The object wanted applies the β CaMKII inhibitor of safe and effective amount.
In another preferred example, the application is to be applied to outside habenular nucleus region.
In the eighth aspect of the present invention, a kind of method of reduction neuron spike potential output and synaptic efficacy, packet are provided Include step：The β CaMKII inhibitor of safe and effective amount is applied to the object of needs.
In another preferred example, the object is mammal, including rodent (such as rat, mouse), spirit are long dynamic Object (such as people).
In another preferred example, the application is to be applied to outside habenular nucleus region.
In the ninth aspect of the present invention, a kind of method prevented and/or treat depression, including step are provided：To need The object wanted applies the GluR1 inhibitor of safe and effective amount.
In another preferred example, the application is to be applied to outside habenular nucleus region.
It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and have in below (eg embodiment) It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist This no longer tires out one by one states.
Description of the drawings
Fig. 1 shows the up-regulation of β CaMKII in depression rat LHb.(A) the depression phenotype of cLH rats.Number represents institute Use size of animal.In LH tests, maximum pressure number is set as 15.(B) the high-throughput quantification albumen based on stable isotope labeling Matter group tests sketch map.Briefly, unlabelled habenular nucleus (14N) WT or cLH rats are dissected, and homogenize, and with 1:1 ratio With15The total brain tissue homogenate's mixing of rat of N labels.Membrane part is concentrated, 500g protein examples are used for standard mass spectral analysis.Meter Calculation has each identified peptide fragment14N/15N ratios.Compare cLH and each protein peptide fragment ratio details of control group look at method portion Point.(C) proteome analysis that β CaMKII are determined in 3 independent proteomics operations, based on total peptide fragment, or Unique peptide fragment (being not the peptide fragment common to other members in family).(D, E) Western blot are analysis shows that in cLH rats Reins (D) or hippocampus (E) membrane part β CaMKII variation.A large amount of tubulins of tissue are used as Loading Control.Protein expression Carry out quantitative criterion.(F) qPCR of β CaMKII mRNA is analyzed in reins.(G) CaMKII is horizontal in outside habenular nucleus part (LHb) Increase, as shown in left side IHC images and the right quantitatively figure.Engineer's scale, 200 μm.(H) in acute Xi Ze-zong and chronic stress (CMS) β CaMKII levels increase in depression model.ALH and ANLH be by LH pressure rat group, but ALH) performance LH diseases Shape, ANLH do not show LH symptoms.(I) Western blot are analysis shows that the third miaow of cLH rats physiological saline or antidepressants β CaMKII are horizontal in reins membrane part after piperazine processing.Data are average value ± SEM.*P<0.05, * * P<0.01, * * * P<0.001 with Control group is compared.N.S. indicate that difference is not notable.Other icons are identical.
Fig. 2 shows that the overexpression of β CaMKI (and non-alpha CaMKII) in mouse and rat causes Depressive behavior.(A) AAV is carried The engineered schematic diagram for being used for being overexpressed control structure, β CaMKII, α CaMKII or kinase dead mutant β CaMKII of body. ITR, inverted terminal repeat；Cytomegalovirus (CMV), cytomegalovirus promoter；Ubi：Ubiquitin promoter；2A：Allow The viral 2A connections peptide of a variety of non pregnant women translations.(B) WT mouse or rat are used for the experimental paradigm of performance testing.(C) small The bilateral AAV- β CaMKII virus injections diagrams of mouse LHb (use anti-GFP and core label Hoechst counterstain).Engineer's scale, 50 μM of (D-I) animal depression model, mouse (D-G) and rat (H, I), shadow of the expression different virus construction to behavior in animal LHb It rings.(F, G) compares being tested in forced swimming Plays dead time (F) or sucrose preference for LHb infection cell scores plotteds (G).Data are standardized using injection of AAV control mice average value.* P ＜ 0.05, * * P<0.01, * * * P<0.001 compares It is compareed with AAV in not injecting WT#P ＜ 0.05.
Fig. 3 shows the spike potential output of the overexpression enhancing LHb neuronal synapse activity sums of β CaMKII.(A) outside reins Core pair record schematic diagram (left figure).Right figure：β CaMKII infect under transmission (top) and fluorescence (bottom) light microscope LHb neurons pairing repairing (green arrow) and adjacent uninfection neuron (red arrow is pointed out).Engineer's scale, 10 μM of (B) the control group LHb neurons or AAV- β CaMKII, AAV- α CaMKII detected under full cell pattern infects neuron MEPSC trace examples.The cumulative distribution (left side) of phase and average frequency between (C, D) mEPSC, by AAV- β CaMKII (C) or AAV- α CaMKII (D) infects the mEPSC amplitudes (right side) of neuron.A pair of of control neuron and adjacent virus infection god are represented per a line Value through member.(E) the control group LHb neurons or AAV- β CaMKII, AAV- α CaMKII detected under full cell pattern infects The spontaneous discharge spike potential trace example of neuron.(F, G) infects nerve by AAV- β CaMKII (F) or AAV- α CaMKII (G) The average frequency of spontaneous discharges of member.
Fig. 4 shows that β CaMKII knock out the depression phenotype that can give treatment to cLH rats and reduce LHb neuronal synapses in LHb Activity.(A) engineering modification is used for being overexpressed the AAV carrier schematic diagrames of the RNAi of β CaMKII.H1：People's H1 promoters.(B) β is used CaMKII RNAi constructions specific knockdown β CaMKII rather than α CaMKII.The pSUPER- β CaMKII- in 293tn cells RNAi constructions be with AAV- β CaMKII or AAV- α CaMKII plasmid co-transfections, and first express before western analyses 48 small When.It is left：Represent western blot.It is right：Knock out effect quantitatively.(C) experimental paradigm of virus infection cLH rat behaviors test. (D-E) after cLH rat LHs b expression AAV- β RNAi and AAV- β K43R in forced swimming test (D) and Xi Ze-zong test (E, F) Behavioral implications.(F) per class animal percentage.LH：≤ 5 bars oppress Xi Ze-zong rat.NLH：>=10 bars oppress non-acquistion without Help rat.(G) subviral infection sub-region characterization.Upper figure：AAV- β RNAi viruses infect outside habenular nucleus in cLH rat brain slices (LHb-L) and medial habenular nucleus (LHb-m) subregion legend.Engineer's scale, 10010 μM.Figure below：Compare cLH rat LHs b-l and LHb-m The behavior response in Xi Ze-zong experiment that infection rate is drawn.(H) cLH rats AAV- β RNAi infect LHb neurons mEPSCs hairs It is raw to change.Upper figure：MEPSC trace examples.By phase and average frequency iterated integral between the mEPSC of AAV- β RNAi infection LHb neurons Cloth (lower-left), mEPSC amplitudes (bottom right).*P<0.05, * * P<0.01, * * * P<0.001 compares with cLH/AAV-
Fig. 5 shows that the cynapse for blocking GluR1-type ampa receptors can subtract in conjunction with (downstream molecules of a β CaMKII) Light depression.(A, B) Western blot analysis shows that the GluR1 of cLH rat habenular nucleus protein extract membrane parts expression Increase (A), is decreased obviously (B) after the processing of antidepressants imipramine.(C) the common table of AAV- β CaMKII-2A-GluR1Ct viruses is used The immunohistochemistry signal of CaMKII and GluR1 in LHb is enhanced up to β CaMKII and GluR1Ct.It is right：IHC signal quantizations. Engineer's scale, β CaMKII and GluR1Ct coexpressions are strong caused by having blocked β CaMKII to be overexpressed in 100 μM of (D, E) bilaterals LHb Compel swimming test (D) and anhedonia (E) phenotype.(F) model illustrates：In normal state, LHb has VTA and DRN opposite Weaker inhibition.The expression of the β CaMKII caused by depressive state, pressure raises, this causes the transport of GluR1 films to increase, and increases LHb neuronal synapses efficiency and spike potential is added to export.Therefore, LHb is enhanced for the inhibition of VTA and DRN, and then leads to pleasant sensation Shortage and behavioral despair.
Fig. 6 shows that proteomics data is analyzed.(A) data statistics flow chart (B) standardized data distribution (C) is This experimental technique is verified, a sample is divided into two same sections and is run twice on mass spectrograph.Experiment weight twice Complex data is compared to each other, and is shown highly relevant.
Fig. 7 shows that the candidate albumen expression by proteomics screening and identification eight raises (A), under four It adjusts (B).It is worth noting that, most up-regulated expression albumen are molecule abundant in neuron, overwhelming majority expression is lowered Albumen is the catalytic molecular for participating in basic metabolism function.
Fig. 8 shows the qRT-PCR verification results of expression up-regulation candidate albumen.*p<0.05, * * p<0.01 with compare Group is compared
Fig. 9 shows the measurement result of CamKII families the other three member.(A, B) western engram analysis shows cLH The variation of rat habenular nucleus (A) or α-, δ-and the γ-CaMKII in hippocampus (B) membrane part.
Figure 10 shows the result of the overexpression level estimation of Unilateral injection virus formulation object.(A) via these three viruses The coronal noisy slice schematic diagram of mouse of Unilateral injection.Top panel：With anti-GFP (green) dyeing or direct visualizztion tdTomato The brain section of (red), the brain section of Hoechst (blue) counterstain.It is left：Non-injection side.It is right：Injection side.D3V：Carry on the back third Room.Bottom panel：The adjacent brain section of same mouse is dyed with anti-CamKII antibody, for the quantification in B figures.(B) it notes It is horizontal to penetrate overexpression representated by the signal intensity ratio of the CaMKII of side and non-injection side.n=5for AAV-βCaMKII-2A-GFP, N=4forAAV- α CaMKII-2A-tdTomato and AAV- β CaMKII-K43R-2A-GFP.
Figure 11 shows the front and back region where the most of expressing virals of mouse LHb virus target area schematic diagram (A). Left figure is to come from the brain section schematic diagram of injection of AAV-β CaMKII virus mouse.Right figure is to adapt from Paxinos＆ The correspondence principle figure of Franklin2004 atlas.
Figure 12 shows the open field experimental result of injection different virus mouse.(A) total displacement distance.(B) open field is real Test middle center dwell time.
Figure 13 shows heterogeneity mEPSC response diagrams in LHb.(A) mEPSC is recorded in LHb differences subregion.Each point generation The record of one neuron of table.MEPSC response frequencies are marked by color.(B) each point represents a pair of nerve of the regional record Member.The value of each pair of neuron mEPSC frequency has all marked.(C) a neuron comparison is another in the pairs of neuron that figure B is recorded The mEPSC frequencies of one neuron.It is worth noting that, although position is different, the neuron from same a pair shows height Identical mEPSC frequencies.
Figure 14 shows the expression of habenular nucleus protein lysates whole or membrane part GluR1.(A) cLH rats are shown The western engram analysis schematic diagrames changed with the total component of habenular nucleus or membrane part GluR1 levels of control rats.(B)GluR1 The quantification of level variation.
Specific implementation mode
The present inventor after extensive and in-depth study, is surprised to find that β CaMKII are that one of depression is extremely closed for the first time Important regulatory factor combines the means such as molecule, behavior and electro physiology, it is determined that β CaMKII are to lead to habenular nucleus overacfivity With the key molecule of behavior depression, the present invention is completed on this basis.
Specifically, habenular nucleus is overexpressed β CaMKII (and non-alpha CaMKII) and prominent transmission effect can not only be remarkably reinforced on the outside The action potential of rate and outside Habenular Neurons exports, and can induce anhedonia and the core depression of behavioral despair Shape.Habenular nucleus lowers the expression of β CaMKII on the outside, reduces its kinase activity or blocks its downstream molecules GluR1 The depressed phenotype of animal can then be reversed.The above result shows that β CaMKII are to adjust LHb neurons in the pathology of depression to occur In the key molecule that functions.
Definition
As used herein, term CaMKII refers to cam kinase II (CaM kinase II).
As used herein, term " β CaMKII inhibitor " and " β CaMKII antagonists " are used interchangeably, and all refer to press down The expression or protein active and prevention of system and/or antagonism β CaMKII or the substance for checking β CaMKII functions.
As used herein, term mEPSCs refers to electric current after miniature excitability protrudes.
As used herein, AMPAR, alpha-amido -3- hydroxy-5-methyl base -4- isoxazoles propionic acid (α-amino-3-hydroxy- 5-methyl-4-isoxazolepropionic acid (AMPA)) receptor
Method
A kind of method the present invention provides screening for preventing and/or treating the potential substance of depression, including step：
(1) tester to be screened is applied to animal models of depression, wherein the β in the animal models of depression CaMKII is high expression；With
(2) related symptoms and/or index of the depression in the animal models of depression are observed, and are carried out with control group Compare；
Wherein, the related symptoms of depression are significantly improved in the animal models of depression, then it represents that the tester is It can be used for preventing and/or treat the potential substance of depression.
In addition, the animal model is non-human mammal, such as rodent, rabbit, Primate.
In addition, in step (2), pass through Animal Behavior Science Germicidal efficacy depressive symptom.The Animal Behavior Science experiment packet It includes：Forced swim test, the experiment of sucrose preference, open field experiment.The index includes：Expression, the β of β CaMKII The activity level of CaMKII.
Method the present invention also provides another screening for preventing and/or treating the potential substance of depression, including step Suddenly：
(1) in test group, tester to be detected is added into vitro detection system；With
(2) detect the expression and/or activity of β CaMKII in the vitro detection system of the test group, and with feminine gender Control group is compared；
Wherein, if compared with adding negative control group, the expression of β CaMKII is remarkably decreased in test group, and/ Or the activity of β CaMKII is remarkably decreased, then it represents that the tester is the potential substance for preventing and/or treating depression.It is described Vitro detection system include cell system；Preferably neuronal cell system.
In addition, described is remarkably decreased finger, and compared with negative control, the expression or activity of β CaMKII in test group It is the 5-90% of negative control group, preferably 10-60%, more preferably 20-50%.The step (2) further includes and positive controls It is compared.
In addition, the method further includes following one or more steps：
To the potential substance that previous step filters out, its influence to the membrane part of GluR1 is further tested；
Animal model is applied to the potential substance that previous step filters out, observes its influence to symptoms of depression.
When testing its influence to the membrane part of GluR1, if compared with negative control group (or blank control group), add Enter or reduced using GluR1 contents in the test group of the tester, then it represents that the tester is to prevent and/or treat depression Potential substance.The GluR1 contents are the GluR1 contents on neuronal cell film.
Using
Prevent present invention could apply to depression and/or treat, the screening and preparation of drug can also be applied to.
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip Part, such as Sambrook et al., molecular cloning：Laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.Unless otherwise stated, no Then percentage and number are weight percent and parts by weight.
Versatile material and method
Animal material
Male CHL diseases rat (4-7 week old) and Sprague Dawley rats of the same age.CHL rat feedings are as previously described (1).Two/cage of rat, 12 hours light and shade periods (7am-7pm has light).Grow up (10-14 week old) C57BL/6 mouse by with In performance testing：4/cage, 12 hours light and shade periods (5am-5pm has light).Rat and mouse can freely absorb stable Water and food, all zooperies are by Chinese Academy of Sciences's Neuroscience Research institute's animal protection and use the approval of the committee.
Protein science
Proteome analysis are carried out to three groups of brain samples, every group include wild type SD rat controls and congenital acquistion without (cHL) rat is helped, wherein congenital Xi Ze-zong rat all shows depressed row in Xi Ze-zong experiment and forced swim test For.Brain is removed after isoflurane anesthesia, stiff band tissue, liquid nitrogen frozen are removed in rapid dissection.The reins tissue and warp of different samples15N Mark (rat15In two generation of N feedings, makes atom be enriched with15N is separately added into precooling homogenate buffer up to rat whole brain tissue 95%) (320mM sucrose,4mM HEPES pH7.4,1mM MgCl2With 0.5mM CaCl2,5mM NaF,1mM Na3VO4,EDTA- Free, Protease Inhibitor cocktail tablets (Roche)), homogenate is made with glass homogenizer.With Bradford measure protein content (Bio-Rad), by each sample homogenate respectively and15The full brain homogenates of N press protein ratio (w/w) 1:1 Mixing.Mixture 800g, 4 DEG C of centrifugation 15min take supernatant, supernatant 1000g, 15min centrifuging and taking precipitation.Precipitation is cell membrane Part, 100ug precipitations are dissolved in homogenate buffer, and (MudPIT) (3) are analyzed with multidimensional protein identification techniques.For each Albumen is estimated in peptide level14N/15Then the ratio of N seeks the average value of protein level.The ratio of thousands of albumen is asked Log is converted into normal distribution, and normalization is so that intermediate value is 0.After normal state, the value of CHL divided by the value of control are exactly this Situation of change of a albumen under depression.It is exactly the notable of each albumen that mean ratio after normal state, which subtracts 2 times of standard deviations, Value.Variation >=50% and saliency value>0 albumen is as candidate, progress next step verification.The change after log is taken to each albumen Change multiple and carry out Student's t-test, p value is shown in Table 1.
Virus formulation
AAV- β CaMKII-2A-EGFP, AAV- α CaMKII-2A-tdTomato are designed and build by standard method, AAV- β CaMKII-K43R-2A-EGFP, AAV- β CaMKII-2A-Glu-R1Ct, AAV-GFP-CRE and AAV-GFP plasmids.β CaMKII and α CaMKII full-length cDNAs are obtained from SD rat cdnas library.2A signal peptide sequences are from plenti-2A-EGFP matter Infer in grain and obtain, AAV frame sequences are obtained by AAV-GFP-CRE plasmids (Lisa Monteggia give) clone.For AAV- β CaMKII-2A-EGFP plasmids,
1) β CaMKII sequences are first added on plenti-Ubiquitin-2A-EGFP plasmids, obtain Ubiquitin- β CaMKII-2A-EGFP plasmids, then AAV skeletons are introduced by BglII and XhoI restriction enzyme sites.
2) α CaMKII are cloned by BamH1 and Xba1 restriction enzyme sites on pLenti-2A-tdTomato carriers, and PCR expands Increase CaMKII-2A-tdTomato sections of α on lenti carriers, AAV carriers are then cloned by Xbal1 and BsrG1 restriction endonucleases On.
3) AAV- β CaMKII-K43R-2A-EGFP plasmids, β CaMKII-K43R segments pass through XbaL1 and EcoR1 digestions position Point is cloned on AAV-2A-EGFP.
4) AAV- β CaMKII-2A-EGFP-GluR1Ct plasmids are expanded from pSindbis-EGFP-GluR1Ct plasmids To EGFP-GluR1Ct segments, it is connect with AAV- β CaMKII-2A.
5) the bob folder oligonucleotide sequences (shRNAi) of β CaMKII are 5'-GAGTAT GCAGCTAA GATCA-3', AAV- β CaMKII-RNAi are synthesized (shanghai, china) by Neuron Biotech companies.
These constructions are generated with AAV2 methods noted earlier.
In short, by an AAV carrier and two assistant carriers (pXX680 and pXX2), it is thin that it is transfected into HEK293TN together Born of the same parents.Two days after transfection, with heparin-agarose post from cell pyrolysis liquid purified virus, and hyperconcetration obtains final volume.AAV2's Helper plasmid pXX680 and pXX2 is bought from University of North Carolina.With the virus sense of 10 times of gradient dilutions Contaminate HEK293TN raji cell assay Raji virus titers.
Anti depressant therapy
CLH rats pass through continuous 14 days injection imipramine (10mg/kg, sigma) progress anti depressant therapies.Divide after injection Analyse performance testing.
Stereotactic injection and histology
When to mouse injecting virus, after intraperitoneal injection of ketamine (100mg/kg weight) and Xylazine (8mg/kg) anesthesia, It is placed on stereotaxic frame (Stoelting instruments).All mouse of operation consent are 2 weeks or more gregarious.By 0.8-1ul Viral (~1012 infectious units/ml) bilaterals of AAV for purifying concentration are injected into the positions LHb (the anterior fontanelle coordinate bit of every mouse It sets：Outside (ML) in side (AP) before and after -1.32mm, ± 1.04mm, -2.48mm carry on the back veutro (DV), 14 ° of angles of coronal-plane center line), make It is slowly injected into (~100-150nl/min) with glass microelectrode, 5min injection electrodes are slowly recalled after the completion of infusion.
To rat injecting virus, 10% chloraldurate of intraperitoneal injection (4ml/kg weight) is anaesthetized, the positions LHb (SD rats 4 weeks Age, the AP-3.0mm from anterior fontanelle door, ML ± 0.7mm, the DV-4.4mm from anterior fontanelle door；4 week old of CHL rats, the AP- from anterior fontanelle door 3.0mm, ML ± 0.7mm, the DV-4.45mm from anterior fontanelle door；6 week old of CHL rats, the AP-3.3mm from anterior fontanelle door, ML ± 0.7mm, the DV-4.7mm from anterior fontanelle door) injection 1-1.5ul AAV viruses, be found that faint label effect on microelectrode beam. Postoperative at least 7 days, carry out behavioral experiment or electro physiology experiment.Injection position is checked after behavioral experiment, is only used correct Those of injection animal data.The rat of AAV- α CaMKII-tdTomato or the brain section of mouse have been injected in fluorescence microscopy It is checked under mirror, other viruses that GFP is marked check GFP albumen before micrography with antibody.The reins area of each brain is cut Continuously be sliced at 6 groups (slice of mouse 30um, every group 6；Rat 40um slices, every group of 8-9 piece).All slices are being pacified Hoechst counterstain is used before being attached to fixinig plate.Calculate the infection rate in reins area.Infection rate counting is by using ImageJ one It is carried out in the vertical brain section of group, total neural number in an area is that the NeuN in one group of slice with two control groups believes It number is averaged.It is exactly infection rate with infection cell number divided by total neuron number.
Immunohistochemistry and immunoblotting
Perfusion：10% chloraldurate deep anaesthesia of rat or mouse with phosphate buffer (PBS) and contains 4% poly first The phosphate of aldehyde (w/v) carries out cardiac perfusion.Brain fixing process after same 4% paraformaldehyde solution is stayed overnight, is then immersed in In 30% sucrose (PBS) solution 1 day (mouse) or 3 days (rat).The brain of frost uses sliding microtome on coronal-plane (CM1950, Leica) is cut into 30um (mouse) or 40um (rat) slices.Slice is immersed in PBS and refrigerates to be used after for 4 DEG C. Antibody is rabbit-anti CaKMII (1:300, Epitomics) rabbit-anti GluR1 (1:300, Chemicon), the anti-NeuN antibody of mouse (1:1000, Chemicon), rabbit-anti GFP antibody (1:1000, Invitrogen), the anti-GFP antibody of chicken (1: 1000, ABCAM), Alexa Fluor488 goat anti-rabbit iggs, Alexa Fluor488 sheep anti-chicken IgGs, Alexa Fluor594 sheep Anti- mouse IgG (all1:1000, Invitrogen), the goat anti-rabbit igg of biotin labeling, the sheep anti-mouse igg of biotin labeling (all1:250, Vector laboratories), Cy3- is coupled Streptavidin, and Cy2- is coupled Streptavidin (all1: 1000,Jackson ImmunoResearch).Specifically for the coloring of CaKMII and GluR1, we use three steps and put Big operation：Slice uses CaKMII antibody or GluR1 antibody, the secondary antibody of biotin labeling, Cy3 or Cy2 coupling strepto- parents successively It is incubated with element.All slices are finally incubated with Hoechst.Fluorescence imaging is using Olympus Fluoview FV1000 copolymerization Focusing microscope (10X, 20X object lens) and Olympus MVX10microscope (MV PLAPO2XC object lens).Image analysis software For Image-Pro Plus and ImageJ.The fluorescence intensity of CaKMII and GluR1 is quantitative：At 20X LHb is captured under identical setting It infects side and is uninfected by side Confocal Images, and further analyzed by Image-Pro Plus.The regional value of Hoechst signals Divided by the integrated optical density (IOD) of IHC signals obtains fluorescence intensity.Compare the fluorescence of virus the injection side and unimplanted side of LHb Intensity obtains overexpression level.
Immunoblotting：The processing for handling spectrum analysis therewith of reins or hippocampal tissue is identical.10%PAGE glue SDS electrophoresis, often swims Road applied sample amount 5-8ug albumen, revolving die do western traces, and antibody is anti-β CaMKII (1:4000,invitrogen), anti-αCaMKII(1:2000,millipore),anti-γCaMKII(1:1000,PTG),anti-δCaMKII(1:500, Santa Cruz) and anti-tubulin (1:10000,Bio-Rad).Use highly sensitive ECL reagents (GE Helthcare)。
Slice prepares
After SD rats (after birth 40-50 days) are anaesthetized with isoflurane, perfusion 20ml precooling dissection buffer solutions (25.0mM NaHCO3,1.25mM NaH2PO4,2.5mM KCl,0.5mM CaCl2,7.0mM MgCl2,25.0mM glucose,110.0mM Choline chloride, 11.6mM ascorbic acid and 3.1mM pyruvic acid, gassed with95%O2With 5%CO2), fast anatomical brain after broken end, coronal reins uses vibratome (DSK II) in the freezing dissection buffer solution of oxidation It is cut into 350 μm of slices.Reins is sliced in oxygen containing ACFS solution (118mM NaCl, 2.5mM KCl, 26mM NaHCO3,1mM NaH2PO4,10mM glucose,1.3mM MgCl2With 2.5mM CaCl2,gassed with95%O2And 5%CO2) in restore two Hour, it is transferred in the slot of the oxygen containing ACFS solution of sustainable supply.
Electro physiology
27 ± 1 DEG C of outside reins neuron patch-clamp experimental temperature, uses Multiclamp700B amplifiers (Molecular Devices) and it is equipped with the Olympus Optical microscope of infrared differential interference comparison.Electrode resistance is 3-4M Ω.For mEPSC It records, in the ACSF solution existing for TTX (1 μM) and picrotoxin (100 μM), clamps neuron at -60mv.Intracellular Solution includes CsMeSO3115mM,CsCl20mM,HEPES10mM,MgCl22.5mM,Na2-ATP4mM,Na-GTP0.4mM,Na- phosphocreatine10mM,EGTA0.6mM.Data are by Digidata1322A (Molecular Devices) in 2kHz It filters and in 10kHz random samplings.MEPSC analyses use Mini Analysis Program (Synaptosoft), threshold value width Degree is 5PA.In order to measure spontaneous discharge rate, neuron is connected into cell attachment device, there are picrotoxin (picrotoxin) in ACSF solution.Electrode resistance 6-7M Ω, solution is NaCl118mM, HEPES10mM in electrode.Each 50 scanning of cell record, every time scanning continue 7s, and data are analyzed by pClamp10 softwares.
Behavioral experiment
All behavioral experiments are all to carry out (mouse 18 in the circadian rhythm phase at night:00–23:00)；Rat 19:00-24: 00).
Xi Ze-zong experimental paradigm carries out (5) according to the specification of optimization noted earlier.Experiment is to brood cLH or wild Type SD rats carry out.Normal form includes two parts." training part " is included in stimulation room (coulbourn instruments), and 120 times are not Evitable, the uncontrolled volas 0.8mA electro photoluminescence is more than 40 minutes, and random stimulus duration and range are from 5 seconds to 15 second It is spaced stimulation time, total stimulus duration is 20 minutes." part of detecting " training carries out after 24 hours, and Xi Ze-zong phenotype is Task is oppressed by a bar to be assessed, one illumination indicating arm of period is placed into stimulation room.This part can including 15 times The 0.8mA intensity electro photoluminescence of escape and 24s trial intervals.Stimulation continue for 60 seconds every time, but can be oppressed and terminate by bar.It is more than Unsuccessfully it is defined as " Xi Ze-zong " (LH) for 10 times；It is unsuccessfully less than 5 times " non-Xi Ze-zong " (NLH).
In forced swim test, rat or mouse are placed on (23-25 DEG C of the water temperatures of 6min in water vat；For mouse, water vat Diameter 12cm, height 25cm；For rat, water vat a diameter of 20cm, height 40cm.The depth of water is that animal uses it in order to prevent Hind leg bottom out).Rat swims there are one additional 15min in the previous day and guides.Animal behavior is by from side video record. Each animal is in the dead time for testing last 4min by not knowing that the observer of animal processing mode calculates.Motionless definition is floating It is dynamic or stationary, it means that there is no any other movements for the action exposed the surface in addition to holding head.
In the test of sucrose preference, first mouse is individually disposed 1 week, then gets used to two bottle of 1% sucrose feeding 3 days, then two Bottle water feeding one day.Then mouse stops water inlet 24 hours, is then exposed to 2h before two bottled full 1% sucrose or water.Two bottles after 1h Location swap.The total flow of each fluid is measured, it is molten that the sucrose solution consumption that sucrose preference is defined in 2h tests accounts for sucrose The ratio of liquid and water total flow.
Open-field test is carried out after every other performance testing.In a bright room, rat or small Mouse is placed on the center of a plastic casing (for mouse：40cm×40cm×40.5cm；For rat 80cm × 80cm × 40cm) 6min, during test, animal behavior is videoed, and then uses EthoVision Pro video frequency following systems and software (Noldus) it analyzes.The total distance of mouse movement is calculated using software and the box center the time spent in.
Statistical analysis
All data are all with average value ± SEM.For all behavioral datas, using two-tailed Student's t-tests.The western blot data that AAV- β CaMKII-2A-EGFP and AAV- α CaMKII-2A-tdTomato are overexpressed And electric physiological data, using double tail paired t-tests.The electro physiology detection data of AAV- β CaMKIIRNAi, using paired Wilcoxon-tests。
Embodiment 1
As a result as shown in Figure 1A the cultivation phenotype Xi Zezong cLH rats of selectivity show as from can escape vola Under electric shock number is escaped to significantly reduce, this performance can by chronic antidepressant medicine treat reverse (imipramine, ip, 10mg/kg, 14 It) (Figure 1A).CLH rats also show as immobility in forced swimming test and increase (Figure 1A).
Embodiment 2
The bilateral habenular nucleus tissue of cLH and wild type control group rat are detached, quantitative proteomics analysis is carried out.
Extraction protein is used for15The quantitative proteomics analysis (Figure 1B) of N stable isotope labelings.In order to reduce sample Complexity, we be extracted memebrane protein component and carried out three times independent sample analysis (Fig. 6, Fig. 7, Fig. 8, table 1).Study table β CaMKII expression significantly up-regulation (being 1.9 times of wild type control, P=0.01, Fig. 1 C) in bright cLH rats habenular nucleus.To CaMKII Other hypotypes of family are also detected：Expressions of the α CaMKII in different samples differs greatly, but still observes Up-regulation trend；δ CaMKII are remained unchanged；γ CaMKII show 1.3 times of increase (P=0.0013) (Fig. 9).Due to brain Middle β CaMKII ratio γ CaMKII are more rich, we study for the hypotype of this CaMKII.Pass through western blot point Analyse secondary verification, it is determined that in cLH habenular nucleus sample film protein extracts β CaMKII compared with control group have 0.86 times increase (P= 0.003) (Fig. 1 D), but then without increasing (P=0.33, Fig. 1 E) in cLH hippocampus samples.Real-time quantitative PCR detection shows β The mRNA level in-site of CaMKII shows 0.37 times of increase (P=0.04) (Fig. 1 F), this shows that transcriptional control at least partly results in The variation of protein level.The reins staining brain sections of immunohistochemistry are shown：The increase of CaMKII protein expression levels is happened at outside Reins (Fig. 1 G).
Table 1
Embodiment 3
β CaMKII expressions are further had detected in other two depression model, one is acute Xi Ze-zong, It is induced and is generated by multiple inevitable and uncontrollable foot shock；Another kind is chronic mild stress, when passing through long Between be exposed to unpredictable slight stress-induced and generate.The expression of β CaMKII is also apparent under both Stress models Increase (Fig. 1 H), shows that the variation of the expression of β CaMKII is not limited to a kind of induction normal form of depression.In addition, imipramine Anti depressant therapy (can reverse cLH rat depression phenotypes (Figure 1A)), lead to the aobvious of β CaMKII protein expressions in cLH rat reins It writes and lowers (Fig. 1 I).The result shows that in the animal of depressive state and anti depressant therapy, β CaMKII show two-way level Variation.
Embodiment 4
Viral vectors (adeno-associated virus (AAV)) is built, scale is crossed in wild-type mice and Lateral Habenular Nucleus (LHb) Up to β-or α-CamKII, and test the effect (Fig. 2A-C) in various depression models.Because LHb neurons are almost paddy ammonia Sour energy, so we drive extensive and efficient gene expression using ubiquitin promoter.In order to estimate overexpression level, I Will express CaMKII virus injection habenular nucleus side.After injection 14 days, compared to non-injection side, injection side CaMKII water It is flat as follows：AAV- β CaMKII are 4.4 ± 0.6 times, and AAV- α CaMKII are 4.6 ± 0.9 times (Figure 10).We are again by virus injection Depressive behavior test (Fig. 2 B, 2C, Figure 11) is carried out to the both sides LHb and after expressing 10 days.
Embodiment 5
Viral vectors (adeno-associated virus (AAV)) is built, scale is crossed in wild-type mice and Lateral Habenular Nucleus (LHb) Up to β-or α-CamKII.Compared with injecting control group, the overexpression of β CaMKII in the mouse LHb for not living through stress stimulation Mouse dead time (P=0.003) in high-amplitude wave swimming test can be caused to dramatically increase, occur being ignorant of the shorter latencies of action (P=0.048).Open-field test shows that the spontaneous activity of these mouse without significant difference (Figure 12), shows that immobility less may be used Can be caused by movement defect.In addition, β CaMKII overexpressions can also cause anhedonia, source of evidence molten to sucrose in mouse The preference of liquid significantly reduces (P=0.01, Fig. 2 E).
Embodiment 6
In order to estimate to generate the minimum infection rate needed for depressive type, this research increases by one group of Bilateral injections 1:10 is diluted The mouse of AAV- β CaMKII viruses.Different from general injection group (infection rate is 38 ± 3%), this dilution injection group (sense Dye rate is 5 ± 0.6%) depressed phenotype (Fig. 2 F, 2G) is not shown.Therefore, depression severity and outside habenular nucleus β CaMKII infection rates are relevant.By contrast with β CaMKII, the overexpression of α CaMKII or GFP-Cre as a contrast, Under similar infection rate (β CaMKII36 ± 2.5%, GFP-CRE36 ± 3%), without result in similar depressed effect (Fig. 2 D-E).
Embodiment 7
CaMKII molecules are used as kinases and structure stand albumen simultaneously in cynapse, and research kinase activity is to β CaMKII work( Can importance, carrier construction AAV- β CaMKII-K43R-2A-EGFP, in rat by watchband β K43R in habenular nucleus.β CaMKII's One without kinase activity mutant, β K43R (being contained in a simple point mutation on lys43arg) though be overexpressed to β CaMKII Wild type similar level when (4.6 ± 0.5 times, infection rate be 40 ± 3%, Figure 10), do not cause depression phenotype (Fig. 2 D, 2E), this shows that the kinase function of β CaMKII is necessary for generating depression.In addition, the β CaMKII in Lateral Habenular Nucleus Overexpression equally dramatically increase forced swimming test in immobility (P=0.02) and reduction flight behavior, be embodied in acquistion Property helpless experiment in terminate the number of electric shock by pressing lever and reduce (Fig. 2 H, 2I).These results indicate that pressing down in multiple animal In strongly fragrant model (including rat and mouse), the expression of β CaMKII (rather than α CaMKII) rises in outside habenular nucleus (LHb), It is enough to lead to very strong depressive symptom.
Embodiment 8
Change the activity of LHb neurons and the cell mechanism of function to inquire into β CaMKII and be overexpressed, we are to wild type The LHb neurons of virus infection and adjacent uninfection carry out pairs of Whole-cell recording (figure in rat brain slice 3A).First, for the LHb neuronal synapse characteristics of detection, we measure miniature excitatory postsynaptic currents (mEPSCs) ( The picrotoxin of tetraodotoxin for preventing action potential and the synaptic currents for blocking GABAA mediations (picrotoxin) in the presence of), this electric current is the alpha-amido -3- hydroxy-5-methyl base -4- isoxazole propionic acid by glutamate receptor (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)) hypotype is mediated and body The single synaptic response being recorded on neuron is showed.
In the brain section being uninfected by, adjacent outside Habenular Neurons to showing the frequency of the similar mEPSC of height, Although subregions different LHb exists heterogeneous (Figure 13).In the neuron infected by AAV- β CaMKII, mEPSC frequencies are big It is big to increase (being 351 ± 51%, P=0.0004 of adjacent control group), mEPSC amplitudes it is same (be the 130 ± 10% of control group, P=0.003, Fig. 3 B, 3C).By contrast, AAV- α CaMKII infection can cause the faint decline of mEPSC frequencies (to be control group 81 ± 9%, P=0.047), mEPSC amplitudes are then without variation (being 87 ± 5%, P=0.12 of control group, Fig. 3 B, 3D).
Embodiment 9
In order to detect the output of these LHb neurons, we use the spontaneous of the method neuron of cell attachment formula record Granting rate.The neuron granting rate of AAV- β CaMKII infection is 3 times of (P=0.0005, figures of the neuron of periphery uninfection 3E, 3F), and this variation (P=0.1, Fig. 3 E, 3G) is not detected then in the neuron of AAV- α CaMKII infection.These knots Fruit shows increase significantly the cynapse efficiency of up-regulation LHb neurons and spike potential output of β CaMKII expressions, this is very big The neuron variation observed in the animal of depression is simulated in degree.
Embodiment 10
In order to determine that β CaMKII expressions are lowered in LHb or whether the function blocking of β CaMKII can reverse depressed table Type, we use RNA interference (RNAi) to reduce β CaMKII protein levels (Fig. 4 A) first.Previously have been reported that display specifically for β The short hairpin RNA of CaMKII transcriptions can effectively reduce β CaMKII protein levels, without influence α CaMKII (35, Fig. 4 B). Then this RNAi form of our the expression β CaMKII in AAV viral (AAV- β RNAi), and infected by Bilateral injections CLH rat LHs b carries out targeted expression (Fig. 4 C).It is shown in the forced swimming test dead time by the cLH rats of AAV- β RNAi infection It writes and reduces (P=0.0005), escape behavior (P=0.02, Fig. 4 D, 4E) is significantly increased in Xi Ze-zong test.Xi Ze-zong Animal percentage (being defined as be below 5 times by bar) drop to 25% (Fig. 4) from 83.3%.
Embodiment 11
In order to exclude potential RNAi undershooting-effects, we further test one of β CaMKII without kinase activity type, β K43R, it can block the function (33, Fig. 2A) of endogenous β CaMKII as a dominant invalid factor when being overexpressed.When After AAV- β K43R viruses are injected into cLH rat LHs b, forced swimming test and Xi Ze-zong test in all detect with Antidepressant effect (Fig. 4 D-4F) similar AAV- β RNAi.It is interesting that down to the AAV- β RNAi of 17 ± 3% infection rates or 30 ± The AAV- β K43R of 3% infection rate are enough to realize these strong antidepressant effects, prompt LHb neural network redundancies small, and change The activity of relatively small ratio LHb neurons is just enough to improve depression.
Embodiment 12
The further subregion of analysis infection LHb, the insides discovery LHb infection rate, rather than exterior portion, with improving depression Effect is closely related, this result prompts may be more important (Fig. 4 G) to the improvement of Xi Zezong behavior on the inside of LHb.It is big in cLH In mouse LHb brain pieces, significantly reduce (control group of adjacent uninfection by the neuron mEPSC frequencies of AAV- β RNAi infection 53.5 ± 15.3%, P=0.008, Fig. 4 H).In short, β CaMKII or blocking its effect these results indicate that knocking out, can obviously subtract The symptom of light depression.
Embodiment 13
It has detected the level of the GluR1 in cLH rat LHs b, finds to be adjusted to (the 222 of control group on horizontal on GluR1 films ± 41.7%, P=0.006, Fig. 5 A), be significantly higher than total GluR1 the increased multiple of level (147 ± 19%, P of control group= 0.02, Figure 14).On the contrary, the expression in the cLH rat membrane components GluR1 of antidepressants imipramine processing declines (control 72 ± 8%, the P=0.003, Fig. 5 B of group).
Embodiment 14
In order to detect effects of the GluR1 in the adjusting of β CaMKII depression, we use AAV- β CaMKII-2A- GluR1Ct viral vectors has been overexpressed a dominant factor of β CaMKII and GluR1, GluR1Ct, to hinder simultaneously The cynapse of GluR1 is transported.Expression testing result based on Unilateral injection show β CaMKII show 3.8 times and GluR1Ct is the overexpression (Fig. 5 C) of 3.7 times of Endogenous levels.When this viral bilateral is injected into wild-type mice LHb (infection rate is 37 ± 2%), two groups of mouse are all acted normally (Fig. 5 D, 5E) in forced swimming and the test of sucrose preference.Cause This, the coexpression of GluR1Ct prevents β CaMKII to be overexpressed caused depressed effect.These results indicate that AMPAR GluR1 The adjusting that cynapse is transmitted is the important downstream targets that β CaMKII show depressive symptom
All references mentioned in the present invention is incorporated herein by reference, independent just as each document It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can To be made various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims It encloses.

Claims (29)

1. a kind of purposes of β CaMKII inhibitor, which is characterized in that be used to prepare prevention and/or treat the drug of depression, institute The inhibitor stated is selected from the group：
(i) RNA interfering or its precursor of interference β CaMKII expression；
(ii) the saltant type β CaMKII albumen or its coded sequence that kinase activity declines or loses；
(iii) inhibit the active inhibitor of β CaMKII；
(iv) competitive inhibitor of β CaMKII；
(v) antibody of anti-β CaMKII；
(vi) antagonist of β CaMKII regulation and control AMPAR GluR1 accesses is checked；
Also, the drug be targeting in the drug of outside habenular nucleus or the drug be applied to outside habenular nucleus region or Near zone；The β CaMKII inhibitor selective depression β CaMKII, and to other CaMKII unrestraints/antagonisms.
2. purposes as described in claim 1, which is characterized in that the inhibitor is selected from the group：
(i) RNA interfering or its precursor of interference β CaMKII expression；
(iv) competitive inhibitor of β CaMKII；
(vi) antagonist of β CaMKII regulation and control AMPAR GluR1 accesses is checked.
3. purposes as described in claim 1, which is characterized in that the RNA interfering is β RNAi, such as SEQ ID NO.:1 institute Show, 5'-GAGTATGCAGCTAAGATCA-3'.
4. purposes as described in claim 1, which is characterized in that the saltant type β CaMKII albumen is that β CaMKII albumen is aobvious Property inactivation factor.
5. purposes as described in claim 1, which is characterized in that the component (vi) be AMPAR GluR1 dominant because Son.
6. purposes as claimed in claim 5, which is characterized in that the component (vi) is GluR1Ct.
7. purposes as described in claim 1, which is characterized in that the inhibitor includes following form：It is polynucleotides, more Peptide, expression vector, micromolecular compound, or combinations thereof.
8. purposes as claimed in claim 7, which is characterized in that the expression vector is selected from the group：Viral vectors.
9. purposes as described in claim 1, which is characterized in that the β CaMKII inhibitor, which is additionally operable to prepare, reduces outside The drug of the drug of the neuron spike potential output of habenular nucleus and/or the synaptic efficacy of reduction outside habenular nucleus.
10. purposes as described in claim 1, which is characterized in that the drug includes：
Pharmaceutically acceptable carrier；With
Effective quantity active constituent, wherein the active constituent is the β CaMKII inhibitor.
11. purposes as claimed in claim 10, which is characterized in that the active constituent makees α CaMKII unrestraints/antagonism With.
12. purposes as claimed in claim 10, which is characterized in that the active constituent makees δ CaMKII unrestraints/antagonism With.
13. purposes as claimed in claim 10, which is characterized in that the active constituent is to γ CaMKII unrestraints/antagonism Effect.
14. purposes as described in claim 1, it is characterised in that other described CaMKII include α CaMKII, γ CaMKII and δ CaMKII.
15. purposes as claimed in claim 10, which is characterized in that the drug is oral preparation, injection or targeting system Agent.
16. the purposes of a kind of β CaMKII albumen or its coded sequence and/or β CaMKII accelerating agents, which is characterized in that for making Standby a reagent or composition, the reagent or composition are moved for building animal models of depression, and in the depression In object model, β CaMKII are high expression in habenular nucleus on the outside, wherein the animal model is non-human mammal.
17. purposes as claimed in claim 16, which is characterized in that the high expression refers to >=150%, by mRNA level in-site or egg White level meter.
18. purposes as claimed in claim 17, which is characterized in that the high expression refers to >=200%.
19. purposes as claimed in claim 17, which is characterized in that the upper limit of the high expression is 2000%.
20. purposes as claimed in claim 16, which is characterized in that the animal models of depression is for screening for preventing And/or the animal model of the potential substance for the treatment of depression.
21. purposes as claimed in claim 16, which is characterized in that the non-human mammal is selected from the group：Rodent, spirit Long animal, or combinations thereof.
22. purposes as claimed in claim 19, which is characterized in that the upper limit of the high expression is 1000%.
23. purposes as claimed in claim 22, which is characterized in that the upper limit of the high expression is 500%.
24. a kind of method of screening for preventing and/or treating the potential substance of depression, which is characterized in that including step：
(1) in test group, tester to be detected is added into vitro detection system；With
(2) expression and/or activity of β CaMKII in the vitro detection system of the test group, and and negative control are detected Group is compared；
Wherein, if compared with adding negative control group, the expression of β CaMKII is remarkably decreased and/or β in test group The activity of CaMKII is remarkably decreased, then it represents that the tester is prevention and/or treats the potential substance of depression, also, institute The vitro detection system stated is outside Habenular Neurons system.
25. method as claimed in claim 24, which is characterized in that described is remarkably decreased finger, compared with negative control, test The expression of β CaMKII or activity are the 5-90% of negative control group in group.
26. method as claimed in claim 25, which is characterized in that described is remarkably decreased finger, compared with negative control, test The expression of β CaMKII or activity are the 10-60% of negative control group in group.
27. method as claimed in claim 26, which is characterized in that described is remarkably decreased finger, compared with negative control, test The expression of β CaMKII or activity are the 20-50% of negative control group in group.
28. method as claimed in claim 24, which is characterized in that the step (2) further includes being carried out with positive controls Compare.
29. method as claimed in claim 24, which is characterized in that the method further includes following one or more steps：
To the potential substance that previous step filters out, its influence to the membrane part of GluR1 is further tested；
When testing its influence to the membrane part of GluR1, if compared with negative control group, being added or applying the tester Test group in GluR1 contents reduce, then it represents that the tester be prevent and/or treatment depression potential substance；
The GluR1 contents are the GluR1 contents on neuronal cell film.
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