CN116763922A

CN116763922A - Application of PACSIN2 inhibitor in preparation of antidepressant drugs

Info

Publication number: CN116763922A
Application number: CN202310449952.0A
Authority: CN
Inventors: 于书彦; 王昌民; 李晔; 常梦妮; 陈晓; 兰天; 王文静
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2023-04-20
Filing date: 2023-04-20
Publication date: 2023-09-19

Abstract

The invention relates to application of a PACSIN2 inhibitor in preparation of antidepressant drugs. The invention adopts a classical depression animal model of chronic unpredictability Wen Heying stress (CUMS), proves that external environment stimulation can induce a rat depression-like phenotype and is accompanied by synaptic plasticity injury, and experiments are carried out on the basis of proteomics and transcriptomics result analysis so as to find a new strategy for treating depression more effectively. The invention firstly proves that PACSIN2 induces disorder of synaptic structure and function plasticity of hippocampal neurons by mediating endocytosis and endocytosis of AMPA receptor, finally participates in depression-like behavior caused by chronic stress stimulation, further perfects the pathogenesis of MDD, proves the feasibility of PACSIN2 as a new target for treating major depressive disorder, and provides a new strategy and scientific basis for clinical treatment of major depressive disorder.

Description

Application of PACSIN2 inhibitor in preparation of antidepressant drugs

Technical Field

The invention belongs to the technical field of targeted therapy of major depression, and particularly relates to application of a PACSIN2 inhibitor in preparation of antidepressant drugs.

Background

The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.

Major depressive disorder (Major depressive disorder, MDD) is a common and serious multifactorial mood disorder-like psychotic disorder, associated with plastic destruction of neurons in specific brain regions, and characterized primarily by sustained mood swings and brain dysfunction. For patients with major depressive disorder, depression not only brings about profound mental pain, but also causes pathophysiological disorders, increases susceptibility to certain diseases, such as heart diseases and cerebrovascular diseases, and brings about heavy burden to patients and families.

The etiology and pathogenesis of major depressive disorder are not clear, and many factors such as biology, psychology and environment are possibly involved, and the main biological basic hypothesis of the pathogenesis of MDD studied at present comprises monoamines, stress, neurotrophins and neurogenesis, excitatory and inhibitory neurotransmission, mitochondrial dysfunction, genetics, inflammation, opiate system, myelination, intestinal brain axis and the like. There are documents showing that the risk factors for the onset of major depressive disorder include sex, age, race, socioeconomic status, personality traits, social environment, abuse and dependence of psychoactive substances, and pharmaceutical factors, etc. At present, the clinical medicine for treating major depressive disorder has poor treatment effect and a plurality of side effects, so that the pathogenesis of the major depressive disorder is further clarified, and the searching of a new therapeutic medicine target point is urgent.

There is increasing evidence that the structure and function of neurons in major depressive patients have been altered. In neural networks, neurons are the basic functional units that integrate and transmit signals in response to internal and external information. Synaptic plasticity is an important feature of the nervous system and also an important guarantee of the nervous system to perform its function.

Neuronal protein kinase C casein kinase substrate 2 (PACSIN 2) is also called Syndapin II, belongs to the super-protein family of BAR structural domain, and is involved in regulating various cell processes of cytoskeleton, receptor transport, intracellular substance transport, signal transduction, endocytosis and the like. PACSIN2 has been shown to have unique functions in mediating AMPA receptor trafficking and synaptic plasticity in cerebellar purkinje cells. Tetramers of AMPA receptors consisting of 4 subunits, gluA1, gluA2, gluA3 and GluA4, mediate rapid excitatory synaptic transmission of more than 90% of glutamatergic synapses, and dynamic regulation of postsynaptic neuronal AMPA receptor numbers is a central mechanism of synaptic plasticity.

Based on the research background, the inventor considers that the exploration and perfection of the potential pathogenesis of the major depressive disorder and the provision of a new drug target and a new therapeutic scheme for treating the major depressive disorder have important significance.

Disclosure of Invention

The invention is proved by a series of experiments: overexpression of PACSIN2 protein expression levels in the hippocampus is effective to induce a depression-like phenotype in rats; protein expression of PACSIN2 knockdown in the hippocampus of depressed animal models can improve their depression-like behavior. In view of the findings, the invention carries out intensive research on PACSIN2 protein, and proves that the expression elevation and depression expression are positively correlated, so that the expression content of the PACSIN2 protein in the organism is expected to be used as an index for diagnosis or prognosis judgment of depression. The invention also proves that the protein can be used as a new target for treating major depressive disorder by constructing a PACSIN2 protein over-expression and knock-down model.

The invention provides a new mechanism for regulating the plastic function of the nerve synapse in the onset of MDD, and further improves the pathogenesis of depression; the PACSIN2 has great research and development potential as a drug target for treating major depressive disorder, and the protein expression of the PACSIN2 can be used as an effective treatment mode for the major depressive disorder through targeted regulation for the first time.

The invention specifically provides the following technical scheme:

in a first aspect, the invention provides the use of a PACSIN2 inhibitor in the manufacture of an antidepressant.

The PACSIN2 inhibitor comprises a small molecular compound, a high molecular polymer, a polypeptide or a nucleic acid substance capable of regulating the over-expression condition of the PACSIN2 in a subject, wherein the nucleic acid substance also comprises related substances for silencing, knocking out or partially knocking out the PACSIN2 based on a genetic engineering mode, and specific examples include plasmids, phage, lentiviruses and the like.

In a preferred embodiment, the PACSIN2 inhibitor targets the brain of the subject, and further targets the hippocampal gyrus domain of the subject; such targeting means include, but are not limited to, localized injection, or by binding a targeting group, etc.

In one embodiment of the present invention, the PACSIN2 inhibitor is a lentivirus carrying PACSIN2 silencing sequences that localizes the hippocampal return structure in a subject by injection.

The antidepressant can be applied to various types of depression, such as endogenous depression, reactive depression, drug secondary depression and the like; aiming at the depression caused by different causes, the medicament can be applied to the treatment of mild depression or even major depression, and in a more preferable scheme, the inhibitor is applied to the preparation of medicaments for resisting major depression.

In a second aspect, the invention provides the use of PACSIN2 assay reagents for the preparation of a diagnostic or prognostic assay product for depression.

The PACSIN2 detection reagent comprises relevant reagents required for detecting the content of the PACSIN2 based on antigen-antibody binding technology or PCR principle. The depression diagnosis or prognosis products include products for depression screening, diagnosis, auxiliary diagnosis or prognosis evaluation, and may be in the form of reagents or devices, specific examples being kits.

The kit comprises a related reagent for detecting the content of PACSIN2 in a sample, and if the detection result shows that the content with statistical significance is increased compared with a control group, the condition of depression or poor prognosis is judged.

In a third aspect of the invention, a method of treatment of depression is provided, including but not limited to the use of a pharmaceutical formulation or surgery to improve PACSIN2 overexpression in a subject's body.

In the above treatment regimen, the treatment regimen should be determined by the physician, taking into account factors including, but not limited to, the individual condition of the patient, the severity of the illness, etc.

In addition, the pharmaceutical preparation should have an active dose of PACSIN2 inhibitor, wherein the dose may be determined according to the administration mode, the delivery site and other factors; in some embodiments, the pharmaceutical formulation may also be used to prevent the occurrence of depression, since the prophylactic dose is for the subject prior to the onset of the disease or early in the disease, the effective amount for prevention is often less than the effective amount for treatment in a product that prevents depression.

The beneficial effects of the above technical scheme are:

(1) PACSIN2 was demonstrated for the first time to mediate transport and long-term inhibition of AMPA receptors in hippocampal pyramidal neuronal cells, involved in synaptic plasticity regulation.

(2) PACSIN2 was first shown to be involved in regulating the occurrence of plastic lesions and depressive-like phenotypes in the hippocampal synapses of depressed rats by regulating the activity of AMPA receptors on the membrane.

(3) The first time of gene regulation of the target PACSIN2 can effectively improve the depression-like behavior of a CUMS animal experimental model, and prove the beneficial effect of the target PACSIN2 in treating major depressive disorder.

(4) The adenovirus related virus designed for PACSIN2 and the inhibitor used for regulating the endocytic process of the AMPA receptor have no adverse effects such as lethality and the like in animal experiments.

(5) The invention further perfects the pathogenesis of MDD and provides a new method and strategy for the research and development and application of clinical antidepressant drugs.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.

FIG. 1A schematic diagram of the mechanism by which PACSIN2 is involved in regulating the occurrence of plastic lesions and depressive-like phenotypes of the hippocampal synapses in depressed rats by regulating the activity of AMPA receptors on the membrane.

FIG. 2 is a schematic representation of synaptic plastic damage and depression-like behavior in the hippocampal region of a CUMS group of rats;

FIG. 2A is a schematic diagram of Chronic Unpredictability Mild Stimulation (CUMS) modeling;

FIG. 2B is a lens electron microscope result for neuronal synaptic locations;

FIG. 2C is a graph showing the results of the expression level of GluR1 protein on rat hippocampal membranes;

FIG. 2D is the results of the forced swim test, sucrose preference test, open field test and elevated plus maze test for rats;

FIG. 2E is a graph showing the results of a rat Sholl analysis;

FIG. 2F is a graph of frequency and amplitude results for rat sEPSCs;

FIG. 3 is a schematic representation of a significant increase in PACSIN2 expression in the CA1 region of the hippocampus of depressed rats in the CUMS group;

FIGS. 3A and 3B are results of proteomic analysis of the CA1 region of the hippocampus of normal rats and depressed rats from the CUMS group;

FIG. 3C shows the results of GO, KEGG enrichment analysis of candidate proteins;

FIGS. 3D and 3E are Western blotting results of PACSIN2 at the hippocampal site of normal rats and depressed rats in the CUMS group;

FIG. 3F shows immunofluorescence results of hippocampal sites in normal rats and in CUMS group depressed rats;

FIG. 4 is an up-regulated pre-normal rat hippocampal site PACSIN2 expression;

FIG. 4A is a flow chart of the overexpression of PACSIN2 at the hippocampal site in rats;

FIG. 4B is a schematic diagram of the construction of adeno-associated virus;

FIG. 4C is a schematic view of the injection site of adeno-associated virus and a fluorescence image of infection efficiency;

FIGS. 4D and 4E are, in order, a rat hippocampal region PACSIN2 western blot banding pattern and an expression level histogram;

FIG. 5 is a schematic representation of normal rat hippocampal CA1 region overexpression of PACSIN2 inducing depressive-like behavior in animals;

FIG. 5A is a graph showing the results of open field experiments;

FIG. 5B is an elevated plus maze result;

FIG. 5C is a tail-out test result;

FIG. 5D is a forced swimming outcome;

FIG. 5E is a sucrose preference experiment;

FIG. 6 is a schematic representation of knock-down of PACSIN2 in the CA1 region of the hippocampus of depressed rats to improve depressive-like behavior in animals;

FIG. 6A is an experimental flow for brain stereotactic injection of knock-down PACSIN2 adeno-associated virus;

FIG. 6B is a schematic diagram of the construction of AAV adeno-associated viral vectors;

FIG. 6C shows the results of a western blot of PACSIN2 at the hippocampal site in rats;

fig. 6D shows the results of the forced swimming experiment.

Detailed Description

It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present invention. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.

In order to enable those skilled in the art to more clearly understand the technical scheme of the present invention, the technical scheme of the present invention will be described in detail with reference to specific embodiments.

1. Construction of depressive animal models and behavioral tests

Male Wistar rats of appropriate ages were purchased from Shandong university animal centers and fed for one week in single cages and on normal diet and light. The second week was followed by a Chronic Unpredictable Mild Stimulation (CUMS) model, with a stimulation given randomly to CUMS rats daily, including 24h of water-deprivation fasting, 5min of plantar electric shock, 45 ° of cage inclination, 12h of wet cage, 12h of diurnal inversion, 3min of tail clamping, 5min of ice water swimming, and 2h of restraint. The normal control group did not require any treatment. After 5 weeks of CUMS stimulation, 4 behavioural tests of a syrup preference experiment, a forced swimming experiment, an overhead plus maze experiment and an open field experiment were performed. As shown in fig. 2, the CUMS rats were continuously given CUMS treatment for 5 weeks, and the control rats were subjected to conventional treatment. After the modeling was completed, a behavioral test was performed to evaluate the depressed phenotype (fig. 2A), and the transmission electron microscope results were shown (fig. 2B): compared to Control group, the number of synapses at the hippocampal sites was significantly reduced in the CUMS group. The behavior test results (FIG. 2C) indicate that. The chronic unpredictable mild stress employed in this example successfully induced rats to develop depressive-like symptoms such as behavioural despair, absence of pleasure, etc.

2. Proteomics

Proteomic analysis was performed on the CA1 region of hippocampus in normal rats and depressed rats (fig. 3A, 3B), and it was found that the PACSIN2 protein (protein kinase C and casein kinase substrate in neurons, protein kinase C and casein kinase substrate 2 in neurons) was differentially expressed between the two groups in normal Control and depressed models, and according to the results of GO, KEGG enrichment analysis, PACSIN2 protein was significantly associated with endocytic processes (fig. 3C), and the results of western blotting (fig. 3D-3E) showed that the PACSIN2 protein levels were significantly increased in the hippocampal sites of the CUMS rats in comparison with the Control group, consistent with the proteomic results; immunofluorescence results (FIG. 3F) showed that the level of PACSIN2 expression was increased at the CA1 site in the hippocampus of the rats in the CUMS group as compared to the Control group.

3. Brain stereotactic and adeno-associated virus injection

Adeno-associated viruses that overexpressed and knocked down PACSIN2 were constructed from Shanghai and metas (fig. 4A-4B). Prior to virus injection, rats were fixed on a brain stereotactic apparatus after deep anesthesia. Exposed bones were exposed with tissue scissors and injection points were drilled according to the coordinates of the hippocampal region in the rat brain map. The bilateral hippocampal CA1 region was slowly injected with adeno-associated virus by microinjector, left at least 5-10 minutes after injection, and then slowly withdrawn. Infection efficiency and injection site detection (fig. 4C) were performed 3 weeks after viral infection, and various behavioral test analyses were performed, followed by sacrifice of the material.

4. Western blot (Western blot) experiment

WB experiments were used to detect PACSIN2 expression levels, the collected samples were placed on ice boxes, added with RIPA buffer, protease inhibitor and proteasome inhibitor PMSF solution, placed in a refiner for full lysis, collected with a pipette into EP tubes prepared in advance, and then placed in a centrifuge at 4 ℃ for centrifugation at 12000rpm for 20min. After centrifugation, the protein supernatant was aspirated and transferred to two sets of prepared EP tubes, one set was stored in a refrigerator at-80℃for further use, and the protein concentration of the sample was measured according to the BCA protein concentration measurement kit for the other set, and the protein loading was adjusted. Then the protein supernatant and a1×loading buffer are loaded according to a ratio of 1:1, shaking, mixing, centrifuging, heating in a metal bath (100deg.C) for 5min, cooling, packaging at-20deg.C, and storing. The subsequent operations were performed according to Western blot (Western blot). According to FIGS. 4 and 6A-6C, the present example successfully constructs a rat hippocampal CA1 region over-expression and knock-down PACSIN2 model by adenovirus vectors.

The results of the above two groups of model behaviours are shown in fig. 5, and according to the open field experimental results (fig. 5A), the total movement distance of the PACSIN2 overexpressing group rats in open field is not significantly different from that of the control group rats, but the residence time in the central area is significantly reduced, which indicates that the up-regulation of the rat hippocampal region PACSIN2 expression induces anxiety-like behaviors in rats.

The elevated plus maze results (fig. 5B) showed a significant decrease in the number of times PACSIN2 overexpressing group rats entered the open arms and the residence time in the open arms compared to control rats, indicating that upregulation of PACSIN2 expression at the hippocampal sites of rats induces anxiety-like behavior in the rats.

The tail-suspension test results (fig. 5C) showed a significant increase in resting time in PACSIN2 overexpressing rats compared to control rats, indicating that upregulation of PACSIN2 expression at the hippocampal sites in rats induces behavior destimation in rats.

The forced swimming test results (fig. 5D) showed a significant increase in resting time in PACSIN2 overexpressing rats compared to control rats, indicating that upregulation of PACSIN2 expression at the hippocampal sites in rats induces behavior destimation in rats.

Sucrose preference experimental results (fig. 5E) show that compared with rats in the control group, the sugar preference degree of the rats in the PACSIN2 overexpression group is significantly reduced, which indicates that up-regulating the expression of PACSIN2 at the hippocampal site of the rats induces the rats to generate depression-like behaviors such as sense of pleasure and the like.

5. Golgi staining and Sholl analysis

The golgi staining technique allows for the observation of small morphological changes in neuronal dendrites and dendritic spines. Brain slices were stained by a rapid golgi staining kit (FD NeuroTechnologies). The specific operation flow is as follows: preparing an AB mixed solution one day in advance, and mixing the solution A and the solution B according to the following formula 1:1, and is gently mixed, not stirrable, and requires at least 5ml of solution per cubic centimeter of tissue. The experimental animals were anesthetized by intraperitoneal injection of 2% sodium pentobarbital (0.1 g/Kg), and after anesthesia, the cranium top skin of the animals was cut with surgical scissors, the mouse brain was peeled off rapidly and carefully, the blood stain on the surface of the mouse brain was rinsed off with single distilled water, and the mouse brain was immersed in the AB mixture. The next day, the new AB mix was changed and stored for two weeks in a dark ambient temperature environment. After two weeks, the tissues were transferred to fluid C for a minimum of 72 hours and a maximum of one week. After 24h, change the new C liquid. Tissue sections were sectioned using an oscillating microtome to a thickness of 100-200 μm. And (3) dripping the solution C onto a gelatin-coated glass slide in advance, transferring the cut tissue onto the glass slide by using a thin brush, sucking away excessive liquid by using filter paper, preserving at room temperature, and airing. Stored under dark examination for up to three days. After the sections were air-dried, the sections were blocked with neutral resin by passing through xylene solution. Storage in dark room temperature environment. A panoramic digital slice scanning microscope (VS 120) was used for imaging and analysis using Fiji software.

6. Brain patch clamp recording

Changes in synaptic function plasticity index are observed by electrophysiological techniques. Firstly, preparing brain slices, injecting 2% pentobarbital sodium into the abdominal cavity of an animal for anesthesia, rapidly breaking the head of the animal by using a broken head table, transferring the animal into a pre-prepared ice box for cooling, shearing the skin to expose the skull by using scissors, rapidly and carefully lifting the skull by using rongeurs to avoid damaging brain tissues, picking up the brain, taking out the animal brain, placing the animal brain on a clean ice box, trimming the tissues by using an inlet blade, fixing the coronal surface in a slicing machine, pouring ice-sand slicing liquid, and continuously introducing a mixed gas of 95% oxygen and 5% carbon dioxide. The parameters such as amplitude and speed of the Leica microtome are adjusted, and the hippocampal brain sections of the rats or mice with the thickness of 300mM are prepared. After slicing, rapidly transferring the brain slices in the slicing liquid to a recovery liquid for incubation, continuously introducing a mixed gas of 95% oxygen and 5% carbon dioxide, placing an incubation cylinder in a constant-temperature water bath at 32.5 ℃ for incubation for 30min, and then transferring to room temperature for incubation for 1h. And opening the devices such as a patch clamp amplifier MultiClamp 700B, a digital-to-analog converter Axon Digidata 1440A, a microscope and the like, sequentially opening the multi clamp 700B software and the pCLAMP 10 recording software on a computer, clamping cells and rupture membranes, and recording the change of excitatory postsynaptic current and action potential.

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

Use of pacsin2 inhibitor in the preparation of antidepressant.
2. The use of a PACSIN2 inhibitor in the manufacture of an antidepressant medicament according to claim 1, wherein the PACSIN2 inhibitor comprises a small molecule compound, a high molecular polymer, a polypeptide or a nucleic acid substance capable of modulating PACSIN2 overexpression in a subject, wherein the nucleic acid substance further comprises a related substance that silences, knocks out or partially knocks out PACSIN2 based on genetic engineering.
3. The use of PACSIN2 inhibitor in the preparation of antidepressant drug as in claim 2, wherein the nucleic acid substance is a plasmid, phage or lentivirus.
4. The use of a PACSIN2 inhibitor in the preparation of an antidepressant drug according to claim 1, wherein the PACSIN2 inhibitor targets the hippocampal gyrus domain of the brain of a subject.
5. The use of a PACSIN2 inhibitor in the preparation of an antidepressant according to claim 3, wherein the PACSIN2 inhibitor is a lentivirus carrying PACSIN2 silencing sequences to localize the hippocampus structure in a subject by injection.
6. The use of PACSIN2 inhibitor in the manufacture of an antidepressant drug according to claim 1, wherein the antidepressant drug is useful in endogenous depression, reactive depression or drug-secondary depression, including mild to severe depression.
7. The use of PACSIN2 inhibitor in the manufacture of an antidepressant according to claim 6 for the manufacture of a medicament for the treatment of major depressive disorder.
Application of PACSIN2 detection reagent in preparing products for diagnosis or prognosis of depression.
9. The use of PACSIN2 detection reagent according to claim 8 for preparing diagnostic or prognostic products for depression, wherein the PACSIN2 detection reagent comprises the relevant reagents required for detecting PACSIN2 content based on antigen-antibody binding technology or PCR principles.
10. The use of PACSIN2 detection agent in the manufacture of a diagnostic or prognostic product for depression of claim 8, wherein the diagnostic or prognostic product for depression comprises a product for screening, diagnosis, auxiliary diagnosis or prognostic assessment of depression, including in the form of an agent or instrument.