CN116397021A - Application of PPP4r3a and/or Kif2a in diagnosis and treatment of progressive supranuclear palsy - Google Patents
Application of PPP4r3a and/or Kif2a in diagnosis and treatment of progressive supranuclear palsy Download PDFInfo
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- CN116397021A CN116397021A CN202310251005.0A CN202310251005A CN116397021A CN 116397021 A CN116397021 A CN 116397021A CN 202310251005 A CN202310251005 A CN 202310251005A CN 116397021 A CN116397021 A CN 116397021A
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- ppp4r3a
- kif2a
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- progressive supranuclear
- supranuclear palsy
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Abstract
本发明属于生物医药和分子生物学技术领域，具体涉及PPP4r3a和/或Kif2a在进行性核上性麻痹诊治中的应用。本发明通过研究发现，PPP4r3a在老年大脑中表达水平明显降低，在进行性核上性麻痹患者的血清中，Ppp4r3a的转录水平较正常对照下降。Ppp4r3a缺失被证明具有导致小鼠出现运动功能障碍及认知功能障碍，病理水平可以观察到大脑中多个脑区有Tau蛋白的过度磷酸化。此外，Ppp4r3a可通过调节Kif2a/AKT/GSK3β介导Tau蛋白磷酸化水平。通过促进Ppp4r3a或抑制Kif2a的表达，可改善神经元内Tau蛋白的磷酸化水平，起到保护、治疗的作用，因此具有良好的实际应用之价值。
The invention belongs to the technical field of biomedicine and molecular biology, and specifically relates to the application of PPP4r3a and/or Kif2a in the diagnosis and treatment of progressive supranuclear palsy. The present invention finds through research that the expression level of PPP4r3a in aged brains is significantly reduced, and in the serum of patients with progressive supranuclear palsy, the transcription level of Ppp4r3a is lower than that of normal controls. Ppp4r3a deletion has been shown to cause motor dysfunction and cognitive dysfunction in mice, and hyperphosphorylation of Tau protein can be observed in multiple brain regions at the pathological level. In addition, Ppp4r3a can mediate the phosphorylation level of Tau protein by regulating Kif2a/AKT/GSK3β. By promoting the expression of Ppp4r3a or inhibiting the expression of Kif2a, the phosphorylation level of Tau protein in neurons can be improved, and it can protect and treat, so it has good practical application value.
Description
技术领域technical field
本发明属于生物医药和分子生物学技术领域，具体涉及PPP4r3a和/或Kif2a在进行性核上性麻痹诊治中的应用。The invention belongs to the technical field of biomedicine and molecular biology, and specifically relates to the application of PPP4r3a and/or Kif2a in the diagnosis and treatment of progressive supranuclear palsy.
背景技术Background technique
公开该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解，而不必然被视为承认或以任何形式暗示该信息构成已经成为本领域一般技术人员所公知的现有技术。The information disclosed in this background section is only intended to increase the understanding of the general background of the present invention, and is not necessarily taken as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.
Tau蛋白病是一组神经变性病,特征为神经元或胶质细胞内病理性tau蛋白异常聚集、沉积。根据tau异构体为3R或4R，对此可进一步分类为3R-tau蛋白病、4R-tau蛋白病或混合性3R/4R-tau蛋白病。其中4R-tau蛋白病包括非典型帕金森综合征进行性核上性麻痹(progressive supranuclear palsy,PSP)。进行性核上性麻痹是一种相对少见的神经系统变性病，常见于60岁及以上人群，典型临床表现包括垂直性核上性凝视麻痹、姿势不稳、轴性肌张力障碍、假性球麻痹以及认知功能障碍。尽管有多数患者有着经典的临床表现，仍有部分不典型临床患者，难以与帕金森病、皮质基底节变性、多系统萎缩等疾病相鉴别。疾病的临床异质性使得诊断更加困难多样化。Tauopathy is a group of neurodegenerative diseases characterized by abnormal aggregation and deposition of pathological tau protein in neurons or glial cells. Depending on whether the tau isoform is 3R or 4R, it can be further classified as 3R-tauopathy, 4R-tauopathy, or mixed 3R/4R-tauopathy. Among them, 4R-tau protein disease includes atypical parkinsonism progressive supranuclear palsy (progressive supranuclear palsy, PSP). Progressive supranuclear palsy is a relatively rare neurodegenerative disease that is common in people aged 60 and over. Typical clinical manifestations include vertical supranuclear gaze palsy, postural instability, axial dystonia, pseudobulbar Paralysis and cognitive impairment. Although most patients have classic clinical manifestations, there are still some atypical clinical patients, which are difficult to distinguish from Parkinson's disease, corticobasal degeneration, multiple system atrophy and other diseases. The clinical heterogeneity of the disease makes diagnosis more difficult and diverse.
Tau蛋白过度磷酸化是Tau蛋白病的主要病理表型，对于进行性核上性麻痹患者，可在神经元、星形胶质细胞及少突胶质细胞中观察到磷酸化的Tau，并且广泛分布于各个脑区中，包括皮层、黑质、纹状体、丘脑和脑干被盖部。然而，进行性核上性麻痹tau蛋白过度磷酸化的机制尚不明确。Hyperphosphorylation of Tau protein is the main pathological phenotype of tauopathies. For patients with progressive supranuclear palsy, phosphorylated Tau can be observed in neurons, astrocytes and oligodendrocytes, and is widely Distributed in various brain regions, including the cortex, substantia nigra, striatum, thalamus, and tegmentum of the brainstem. However, the mechanism of tau hyperphosphorylation in progressive supranuclear palsy remains unclear.
蛋白激酶和磷酸酶对蛋白质磷酸化与去磷酸化的调控普遍存在于神经系统的多个生物过程中，多数蛋白磷酸酶及蛋白激酶是通过调控Tau蛋白过度磷酸化的过程以调节阿尔茨海默病的疾病进展。事实上，人类基因组编码超过500种蛋白激酶，然而蛋白磷酸酶的种类却少于200个，蛋白磷酸酶数量明显少于蛋白激酶数量的现象提示：在蛋白磷酸化和去磷酸化的修饰平衡中，蛋白磷酸酶的调控可能不仅仅依赖于其催化亚基的去磷酸化功能，复合体中的调节亚基同样发挥着重要作用。因此，探索蛋白磷酸酶家族成员调节亚基在Tau蛋白病中的作用有重要的理论和临床意义。The regulation of protein phosphorylation and dephosphorylation by protein kinases and phosphatases is ubiquitous in multiple biological processes in the nervous system. Most protein phosphatases and protein kinases regulate Alzheimer's disease by regulating the hyperphosphorylation process of Tau protein. disease progression. In fact, the human genome encodes more than 500 protein kinases, but the number of protein phosphatases is less than 200. The phenomenon that the number of protein phosphatases is significantly less than the number of protein kinases suggests that: in the modification balance of protein phosphorylation and dephosphorylation , the regulation of protein phosphatase may not only depend on the dephosphorylation function of its catalytic subunit, but the regulatory subunit in the complex also plays an important role. Therefore, exploring the role of regulatory subunits of protein phosphatase family members in tauopathies has important theoretical and clinical significance.
发明内容Contents of the invention
基于上述现有技术研究现状，本发明提供PPP4r3a和/或Kif2a在进行性核上性麻痹诊治中的应用。本发明通过研究发现，蛋白磷酸酶4调节亚基R3a(PPP4r3a)在老年大脑中表达水平明显降低，在进行性核上性麻痹患者的血清中，Ppp4r3a的转录水平较正常对照下降。Ppp4r3a缺失被证明具有导致小鼠出现运动功能障碍及认知功能障碍，病理水平可以观察到大脑中多个脑区有Tau蛋白的过度磷酸化。此外，本发明研究发现Ppp4r3a可通过调节Kif2a/AKT/GSK3β介导Tau蛋白磷酸化水平。通过促进Ppp4r3a的表达、或者抑制微管解聚蛋白Kif2a的表达，可以改善神经元内Tau蛋白的磷酸化水平，起到保护、治疗的作用。基于上述研究成果，从而完成本发明。Based on the research status of the above prior art, the present invention provides the application of PPP4r3a and/or Kif2a in the diagnosis and treatment of progressive supranuclear palsy. The present invention finds through research that the expression level of
具体的，本发明技术方案如下：Specifically, the technical scheme of the present invention is as follows:
本发明的第一个方面，提供检测PPP4r3a编码基因和/或Kif2a编码基因及其表达产物的物质在制备进行性核上性麻痹相关检测产品中的应用。The first aspect of the present invention provides the application of substances for detecting PPP4r3a encoding gene and/or Kif2a encoding gene and its expression products in the preparation of detection products related to progressive supranuclear palsy.
其中，所述进行性核上性麻痹相关检测产品为具有筛查、(辅助)诊断、监测和预测进行性核上性麻痹进展功能的产品。Wherein, the detection products related to progressive supranuclear palsy are products with the functions of screening, (auxiliary) diagnosis, monitoring and predicting the progress of progressive supranuclear palsy.
所述进行性核上性麻痹的病程进展包括但并不限于运动功能障碍、执行功能障碍、认知功能障碍以及Tau蛋白磷酸化水平增高。The progression of the progressive supranuclear palsy includes, but is not limited to, motor dysfunction, executive dysfunction, cognitive dysfunction and increased phosphorylation of Tau protein.
所述PPP4r3a编码基因的表达产物显然可以是PPP4r3a蛋白，其为蛋白磷酸酶4(PP4)的调节亚基。同理，所述Kif2a编码基因的表达产物可以为Kif2a蛋白，其是一种微管解聚酶驱动蛋白。The expression product of the gene encoding PPP4r3a can obviously be PPP4r3a protein, which is the regulatory subunit of protein phosphatase 4 (PP4). Similarly, the expression product of the gene encoding Kif2a may be Kif2a protein, which is a microtubule depolymerase kinesin.
本发明的第二个方面，提供一种用于筛查、(辅助)诊断、监测和预测进行性核上性麻痹进展的产品，所述产品其包含基于测序方法和/或基于定量PCR方法和/或基于探针杂交方法检测PPP4r3a编码基因和/或Kif2a编码基因转录的物质；或基于免疫检测方法检测PPP4r3a蛋白和/或Kif2a蛋白表达情况的物质。In a second aspect of the present invention, a product for screening, (aided) diagnosis, monitoring and prediction of progressive supranuclear palsy is provided, which comprises a sequencing-based method and/or a quantitative PCR-based method and /or substances for detecting PPP4r3a-encoding gene and/or Kif2a-encoding gene transcription based on probe hybridization method; or substances for detecting the expression of PPP4r3a protein and/or Kif2a protein based on immunoassay method.
本发明的第三个方面，提供一种用于筛查、(辅助)诊断、监测和预测进行性核上性麻痹进展的系统，所述系统包括：In a third aspect of the present invention, there is provided a system for screening, (aiding) diagnosis, monitoring and predicting the progression of progressive supranuclear palsy, said system comprising:
i)分析模块，所述分析模块包含：用于确定受试者的待测样品中选自PPP4r3a和/或Kif2a表达水平的检测物质，以及；i) an analysis module, which includes: a detection substance selected from the expression level of PPP4r3a and/or Kif2a in the sample to be tested for determining the subject, and;
ii)评估模块，所述评估模块包含：根据i)中确定的所述PPP4r3a和/或Kif2a表达水平判断所述受试者的患病情况。ii) an assessment module, the assessment module comprising: judging the subject's disease status according to the expression level of PPP4r3a and/or Kif2a determined in i).
本发明的第四个方面，提供上述PPP4r3a和/或Kif2a作为靶点在制备和/或筛选进行性核上性麻痹药物中的应用。The fourth aspect of the present invention provides the application of the above-mentioned PPP4r3a and/or Kif2a as targets in the preparation and/or screening of drugs for progressive supranuclear palsy.
本发明的第五个方面，提供促进PPP4r3a编码基因及其表达产物表达的物质和/或抑制Kif2a编码基因及其表达产物表达的物质在如下任意一种或多种中的应用：In a fifth aspect of the present invention, the application of substances that promote the expression of the PPP4r3a-encoded gene and its expression products and/or substances that inhibit the expression of the Kif2a-encoded gene and its expression products in any one or more of the following:
a1)制备改善因进行性核上性麻痹介导的运动及认知功能障碍的产品；a1) Preparation of products that improve motor and cognitive dysfunction mediated by progressive supranuclear palsy;
a2)制备减轻因进行性核上性麻痹介导的神经元、星形胶质细胞及少突胶质细胞内Tau蛋白的磷酸化水平的产品。a2) Prepare a product that reduces the phosphorylation level of Tau protein in neurons, astrocytes and oligodendrocytes mediated by progressive supranuclear palsy.
a3)制备防治进行性核上性麻痹的产品。a3) Prepare products for preventing and treating progressive supranuclear palsy.
所述产品可以为药物或实验试剂，所述实验试剂可供基础研究使用，诸如构建进行性核上性麻痹相关细胞或动物模型，从而对进行性核上性麻痹等相关疾病的发生发展机制等进行研究。The product can be a drug or an experimental reagent, and the experimental reagent can be used for basic research, such as the construction of cells or animal models related to progressive supranuclear palsy, so as to understand the mechanism of occurrence and development of related diseases such as progressive supranuclear palsy, etc. research.
上述一个或多个技术方案的有益技术效果：Beneficial technical effects of the above-mentioned one or more technical solutions:
上述技术方案首次公开了Ppp4r3a在老年大脑中表达水平明显降低，在进行性核上性麻痹患者的血清中，Ppp4r3a的转录水平较正常对照下降。Ppp4r3a缺失被证明具有导致小鼠出现运动功能障碍及认知功能障碍，病理水平可以观察到大脑中多个脑区有Tau蛋白的过度磷酸化。此外，研究发现Ppp4r3a可通过调节Kif2a/AKT/GSK3β介导Tau蛋白磷酸化水平。通过促进Ppp4r3a的表达、或者抑制Kif2a的表达，可以改善神经元内Tau蛋白的磷酸化水平，起到保护、治疗的作用。The above technical solution discloses for the first time that the expression level of Ppp4r3a is significantly reduced in the aged brain, and in the serum of patients with progressive supranuclear palsy, the transcription level of Ppp4r3a is lower than that of normal controls. Ppp4r3a deletion has been shown to cause motor dysfunction and cognitive dysfunction in mice, and hyperphosphorylation of Tau protein can be observed in multiple brain regions at the pathological level. In addition, the study found that Ppp4r3a can mediate the phosphorylation level of Tau protein by regulating Kif2a/AKT/GSK3β. By promoting the expression of Ppp4r3a or inhibiting the expression of Kif2a, the phosphorylation level of Tau protein in neurons can be improved to play a protective and therapeutic role.
综上，Ppp4r3a及Kif2a可作为进行性核上性麻痹的生物学标志物以及相关治疗靶点，因此具有良好的临床应用价值和社会效益。In summary, Ppp4r3a and Kif2a can be used as biological markers and related therapeutic targets of progressive supranuclear palsy, so they have good clinical application value and social benefits.
附图说明Description of drawings
构成本发明的一部分的说明书附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention, and the schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations to the present invention.
图1是本发明实施例中老年人脑组织中Ppp4r3a水平下降。其中，A为前额皮质转录组学测序数据，中青年组为20-44岁之间的组织样本，共15例，老年组为86-106岁之间，共14例；B为前海马转录组学测序数据，其中中青年组为20-45岁之间的组织样本，共15例，老年组为80-99岁之间，共16例；C为正常人(N＝276)、进行性核上性麻痹患者(N＝54)及皮质基底节变性患者(N＝36)血清PPP4R3A的转录测序结果；Fig. 1 shows the decrease of Ppp4r3a level in the brain tissue of the elderly in the embodiment of the present invention. Among them, A is the prefrontal cortex transcriptomics sequencing data, the young and middle-aged group is tissue samples between 20-44 years old, a total of 15 cases, and the elderly group is 86-106 years old, a total of 14 cases; B is the frontal hippocampal transcriptome Sequencing data, in which the young and middle-aged group were tissue samples between 20-45 years old, a total of 15 cases, and the elderly group were 80-99 years old, a total of 16 cases; C was normal people (N=276), progressive nuclear Transcript sequencing results of serum PPP4R3A in patients with superior paralysis (N=54) and patients with corticobasal degeneration (N=36);
图2是本发明实施例中Ppp4r3a敲除小鼠行为学改变，其中，A为旷场试验的小鼠运动轨迹(N＝5)，B为旷场试验小鼠运动距离(N＝5)，C为6月龄小鼠转棒试验(N＝5)，D为10月龄小鼠转棒试验(N＝5)，E为6月龄小鼠跑台试验(N＝5)，F图为小鼠筑巢试验(N＝5)，G为小鼠筑巢试验评分结果，H为小鼠T迷宫试验中学习阶段及逆向学习阶段的正确率；Fig. 2 is the behavioral change of the Ppp4r3a knockout mouse in the embodiment of the present invention, wherein, A is the mouse movement track (N=5) of the open field test, B is the movement distance of the mouse of the open field test (N=5), C is the rotarod test of 6-month-old mice (N=5), D is the rotarod test of 10-month-old mice (N=5), E is the treadmill test of 6-month-old mice (N=5), and Figure F Be the mouse nest test (N=5), G is the scoring result of the mouse nest test, and H is the correct rate of the learning stage and the reverse learning stage in the mouse T maze test;
图3是本发明实施例中小鼠脑组织中Tau蛋白过度磷酸化，其中，A、B、C分别为皮质、海马及纹状体AT8染色，D为海马p-Tau(T231)染色，E为皮层p-Tau(T231)染色，F为皮层p-Tau(S396)染色，G为皮层及海马组织蛋白质印迹；Figure 3 is the hyperphosphorylation of Tau protein in the brain tissue of the mouse in the embodiment of the present invention, wherein, A, B, and C are the AT8 staining of the cortex, hippocampus and striatum respectively, D is the p-Tau (T231) staining of the hippocampus, and E is Cortical p-Tau (T231) staining, F is cortical p-Tau (S396) staining, G is western blot of cortex and hippocampus;
图4是本发明实施例中Ppp4r3a与Kif2a相互作用图。其中，A为Kif2a与Ppp4r3a相互作用，B为敲除小鼠原代神经元中Kif2a的免疫荧光染色，C为敲除小鼠皮层神经元Kif2a免疫荧光染色，D为SH-SY5Y细胞中敲低Ppp4r3a后Kif2a亚细胞定位。Fig. 4 is a diagram of the interaction between Ppp4r3a and Kif2a in the embodiment of the present invention. Among them, A is the interaction between Kif2a and Ppp4r3a, B is the immunofluorescence staining of Kif2a in primary neurons of knockout mice, C is the immunofluorescence staining of Kif2a in knockout mouse cortical neurons, and D is knockdown in SH-SY5Y cells Subcellular localization of Kif2a after Ppp4r3a.
图5为本发明实施例中Ppp4r3a对神经元的拯救作用。其中，A为在SH-SY5Y细胞中高表达Ppp4r3a后的蛋白质印迹，B为在SH-SY5Y细胞中高表达Ppp4r3a后的α-tubinlin免疫荧光染色，C为在SH-SY5Y细胞中高表达Ppp4r3a后线粒体染色，D为在NHA、HMO6级SH-SY5Y细胞系中高表达Ppp4r3a后CCK8试验，E为SH-SY5Y细胞转染后蛋白质印迹，F为SH-SY5Y细胞转染后免疫荧光染色。G为SH-SY5Y细胞转染后CCK8试验。Fig. 5 shows the rescue effect of Ppp4r3a on neurons in the embodiment of the present invention. Among them, A is Western blot after high expression of Ppp4r3a in SH-SY5Y cells, B is α-tubinlin immunofluorescence staining after high expression of Ppp4r3a in SH-SY5Y cells, C is mitochondrial staining after high expression of Ppp4r3a in SH-SY5Y cells, D is CCK8 test after high expression of Ppp4r3a in NHA, HMO6 grade SH-SY5Y cell lines, E is Western blot after transfection of SH-SY5Y cells, F is immunofluorescence staining after transfection of SH-SY5Y cells. G is CCK8 test after transfection of SH-SY5Y cells.
具体实施方式Detailed ways
应该指出，以下详细说明都是示例性的，旨在对本发明提供进一步的说明。除非另有指明，本发明使用的所有技术和科学术语具有与本发明所属技术领域的普通技术人员通常理解的相同含义。It should be noted that the following detailed description is exemplary and intended to provide further explanation of the present 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.
需要注意的是，这里所使用的术语仅是为了描述具体实施方式，而非意图限制根据本发明申请的示例性实施方式。如在这里所使用的，除非上下文另外明确指出，否则单数形式也意图包括复数形式，此外，还应当理解的是，当在本说明书中使用术语“包含”和/或“包括”时，其指明存在特征、步骤、操作、器件、组件和/或它们的组合。下列具体实施方式中如果未注明具体条件的实验方法，通常按照本领域技术内的分子生物学的常规方法和条件，这种技术和条件在文献中有完整解释。参见例如Sambrook等人，《分子克隆：实验手册》中所述的技术和条件，或按照制造厂商所建议的条件。It should be noted that the terminology used here is only used to describe the specific implementation, and is not intended to limit the exemplary implementation according to the application of the present invention. As used herein, unless the context clearly dictates otherwise, the singular is intended to include the plural, and it should also be understood that when the terms "comprising" and/or "comprising" are used in this specification, they mean There are features, steps, operations, means, components and/or combinations thereof. In the following specific embodiments, if the experimental methods of specific conditions are not indicated, generally follow the conventional methods and conditions of molecular biology within the skill of the art, and such techniques and conditions are fully explained in the literature. See, eg, Sambrook et al., Molecular Cloning: A Laboratory Manual for techniques and conditions, or follow conditions suggested by the manufacturer.
本发明的一个典型具体实施方式中，提供检测PPP4r3a编码基因和/或Kif2a编码基因及其表达产物的物质在制备进行性核上性麻痹相关检测产品中的应用。In a typical embodiment of the present invention, the application of a substance for detecting PPP4r3a encoding gene and/or Kif2a encoding gene and its expression products in the preparation of progressive supranuclear palsy-related detection products is provided.
其中，所述进行性核上性麻痹相关检测产品为具有筛查、(辅助)诊断、监测和预测进行性核上性麻痹进展功能的产品。Wherein, the detection products related to progressive supranuclear palsy are products with the functions of screening, (auxiliary) diagnosis, monitoring and predicting the progress of progressive supranuclear palsy.
所述进行性核上性麻痹的病程进展包括但并不限于运动功能障碍、执行功能障碍、认知功能障碍以及Tau蛋白磷酸化水平增高。The progression of the progressive supranuclear palsy includes, but is not limited to, motor dysfunction, executive dysfunction, cognitive dysfunction and increased phosphorylation of Tau protein.
所述PPP4r3a编码基因的表达产物显然可以是PPP4r3a蛋白，其为蛋白磷酸酶4(PP4)的调节亚基。同理，所述Kif2a编码基因的表达产物可以为Kif2a蛋白，其是一种微管解聚酶驱动蛋白。The expression product of the gene encoding PPP4r3a can obviously be PPP4r3a protein, which is the regulatory subunit of protein phosphatase 4 (PP4). Similarly, the expression product of the gene encoding Kif2a may be Kif2a protein, which is a microtubule depolymerase kinesin.
本发明的一个或多个具体实施方式中，提供一种用于筛查、(辅助)诊断、监测和预测进行性核上性麻痹进展的产品，所述产品其包含基于高通量测序方法和/或基于定量PCR方法和/或基于探针杂交方法检测PPP4r3a编码基因和/或Kif2a编码基因转录的物质；或基于免疫检测方法检测PPP4r3a蛋白和/或Kif2a蛋白表达情况的物质。In one or more specific embodiments of the present invention, a product for screening, (aided) diagnosis, monitoring and prediction of progressive supranuclear palsy is provided, which comprises high-throughput sequencing-based methods and / or substances based on quantitative PCR method and / or based on probe hybridization method to detect the transcription of PPP4r3a encoding gene and / or Kif2a encoding gene; or based on immunoassay method to detect the expression of PPP4r3a protein and / or Kif2a protein.
本发明的一个或多个具体实施方式中，可以采用包括但不限于测序法、液相杂交、Northern杂交方法、miRNA表达谱芯片、核酶保护分析技术、RAKE法、原位杂交检测PPP4r3a编码基因和/或Kif2a编码基因的转录；采用包括但不限于ELISA、胶体金试纸条、蛋白芯片检测PPP4r3a蛋白和/或Kif2a蛋白的表达情况。In one or more specific embodiments of the present invention, methods including but not limited to sequencing, liquid phase hybridization, Northern hybridization, miRNA expression profile chip, ribozyme protection analysis technology, RAKE method, and in situ hybridization can be used to detect the PPP4r3a encoding gene And/or the transcription of the gene encoding Kif2a; use methods including but not limited to ELISA, colloidal gold test strips, and protein chips to detect the expression of PPP4r3a protein and/or Kif2a protein.
所述进行性核上性麻痹的病程进展包括但并不限于运动功能障碍、执行功能障碍、认知功能障碍以及Tau蛋白磷酸化水平增高。The progression of the progressive supranuclear palsy includes, but is not limited to, motor dysfunction, executive dysfunction, cognitive dysfunction and increased phosphorylation of Tau protein.
所述产品可以以引物、探针、核酸膜条、(基因或蛋白)芯片、制剂、试剂盒、检测装置和设备等任意现有已知形式存在。本领域技术人员可通过实际情况在不付出创造性劳动的前提下实现上述产品，因此上述产品均属于本申请的保护范围之内。The product can exist in any prior known form such as primers, probes, nucleic acid membrane strips, (gene or protein) chips, preparations, kits, detection devices and equipment. Those skilled in the art can realize the above-mentioned products through the actual situation without paying creative work, so the above-mentioned products all belong to the protection scope of the present application.
本发明的一个或多个具体实施方式中，提供一种用于筛查、(辅助)诊断、监测和预测进行性核上性麻痹进展的系统，所述系统包括：In one or more specific embodiments of the present invention, a system for screening, (assisting) diagnosis, monitoring and predicting the progression of progressive supranuclear palsy is provided, the system comprising:
i)分析模块，所述分析模块包含：用于确定受试者的待测样品中选自PPP4r3a和/或Kif2a表达水平的检测物质，以及；i) an analysis module, which includes: a detection substance selected from the expression level of PPP4r3a and/or Kif2a in the sample to be tested for determining the subject, and;
ii)评估模块，所述评估模块包含：根据i)中确定的所述PPP4r3a和/或Kif2a表达水平判断所述受试者的患病情况。ii) an assessment module, the assessment module comprising: judging the subject's disease status according to the expression level of PPP4r3a and/or Kif2a determined in i).
其中，所述受试者可以为人或非人哺乳动物(如小鼠等)，所述待测样品包括但不限于外周血以及脑相关组织及细胞(如神经元、星形胶质细胞及少突胶质细胞等)等。Wherein, the subject can be a human or a non-human mammal (such as a mouse, etc.), and the sample to be tested includes, but is not limited to, peripheral blood and brain-related tissues and cells (such as neurons, astrocytes, and glial cells, etc.), etc.
所述进行性核上性麻痹的病程进展包括但并不限于运动功能障碍、执行功能障碍、认知功能障碍以及Tau蛋白磷酸化水平增高。The progression of the progressive supranuclear palsy includes, but is not limited to, motor dysfunction, executive dysfunction, cognitive dysfunction and increased phosphorylation of Tau protein.
本发明的一个或多个具体实施方式中，提供上述PPP4r3a和/或Kif2a作为靶点在制备和/或筛选进行性核上性麻痹药物中的应用。In one or more specific embodiments of the present invention, the application of the above-mentioned PPP4r3a and/or Kif2a as targets in the preparation and/or screening of progressive supranuclear palsy drugs is provided.
其中，所述筛选进行性核上性麻痹药物的方法包括：Wherein, the method for screening progressive supranuclear palsy drugs comprises:
1)采用候选物质处理表达和/或含有所述PPP4r3a和/或Kif2a的体系；设置不采用候选物质处理的平行对照；1) using candidate substances to treat the system expressing and/or containing the PPP4r3a and/or Kif2a; setting a parallel control without using candidate substances;
2)完成步骤1)后，检测体系中所述PPP4r3a和/或Kif2a的表达水平；与平行对照相比，如果采用候选物质处理的体系中所述PPP4r3a的表达量显著提高和/或Kif2a的表达量显著降低，所述候选物质可作为候选的进行性核上性麻痹药物。2) After step 1), the expression level of PPP4r3a and/or Kif2a in the detection system is detected; compared with the parallel control, if the expression level of PPP4r3a and/or the expression of Kif2a in the system treated with the candidate substance is significantly improved The amount is significantly reduced, and the candidate substance can be used as a candidate drug for progressive supranuclear paralysis.
本发明的又一具体实施方式中，所述体系可为细胞体系、溶液体系、组织体系、器官体系或动物体系。In yet another specific embodiment of the present invention, the system can be a cell system, a solution system, a tissue system, an organ system or an animal system.
本发明的又一具体实施方式中，所述细胞体系中的细胞可以为神经元、星形胶质细胞及少突胶质细胞；In yet another specific embodiment of the present invention, the cells in the cell system may be neurons, astrocytes and oligodendrocytes;
本发明的又一具体实施方式中，所述组织体系中的组织可以为大脑皮层、黑质、纹状体、丘脑和脑干被盖部；In yet another specific embodiment of the present invention, the tissues in the tissue system may be cerebral cortex, substantia nigra, striatum, thalamus and brainstem tegmentum;
本发明的又一具体实施方式中，所述器官体系中的器官可以为大脑；In yet another specific embodiment of the present invention, the organ in the organ system may be the brain;
本发明的又一具体实施方式中，所述动物体系中的动物可以为哺乳动物，如大鼠、小鼠、豚鼠、兔、猴、人等。In yet another specific embodiment of the present invention, the animals in the animal system can be mammals, such as rats, mice, guinea pigs, rabbits, monkeys, humans, and the like.
本发明的一个或多个具体实施方式中，提供促进PPP4r3a编码基因及其表达产物表达的物质和/或抑制Kif2a编码基因及其表达产物表达的物质在如下任意一种或多种中的应用：In one or more specific embodiments of the present invention, the application of substances that promote the expression of the PPP4r3a-encoded gene and its expression products and/or the substances that inhibit the expression of the Kif2a-encoded gene and its expression products in any one or more of the following is provided:
a1)制备改善因进行性核上性麻痹介导的运动及认知功能障碍的产品；a1) Preparation of products that improve motor and cognitive dysfunction mediated by progressive supranuclear palsy;
a2)制备减轻因进行性核上性麻痹介导的神经元、星形胶质细胞及少突胶质细胞内Tau蛋白的磷酸化水平的产品。a2) Prepare a product that reduces the phosphorylation level of Tau protein in neurons, astrocytes and oligodendrocytes mediated by progressive supranuclear palsy.
a3)制备防治进行性核上性麻痹的产品。a3) Prepare products for preventing and treating progressive supranuclear palsy.
其中，所述促进PPP4r3a编码基因及其表达产物的物质包括但不限于采用基于基因特异性Mimics技术上调PPP4r3a表达和/或促进其活性的物质；如上调PPP4r3a表达的启动子或者慢病毒；同时也包括化合物类促进剂。Wherein, the substances that promote the PPP4r3a encoding gene and its expression products include but are not limited to substances that up-regulate the expression of PPP4r3a and/or promote its activity based on gene-specific Mimics technology; such as promoters or lentiviruses that up-regulate the expression of PPP4r3a; Includes compound accelerators.
所述抑制Kif2a编码基因及其表达产物的物质包括但不限于针对Kif2a的RNA干扰分子或反义寡核苷酸、小分子抑制剂、shRNA(小发夹RNA)、小干扰RNA(siRNA)，实施慢病毒感染或基因敲除的物质以及针对Kif2a蛋白本身或其上下游分子的特异性抗体，如抗Kif2aa抗体。The substances that inhibit the Kif2a encoding gene and its expression products include but are not limited to RNA interference molecules or antisense oligonucleotides, small molecule inhibitors, shRNA (small hairpin RNA), small interfering RNA (siRNA) against Kif2a, Substances for lentiviral infection or gene knockout and specific antibodies against the Kif2a protein itself or its upstream and downstream molecules, such as anti-Kif2aa antibodies.
所述产品可以为药物或实验试剂，所述实验试剂可供基础研究使用，诸如构建进行性核上性麻痹相关细胞或动物模型，从而对进行性核上性麻痹等相关疾病的发生发展机制等进行研究。The product can be a drug or an experimental reagent, and the experimental reagent can be used for basic research, such as the construction of cells or animal models related to progressive supranuclear palsy, so as to understand the mechanism of occurrence and development of related diseases such as progressive supranuclear palsy, etc. research.
根据本发明，当所述产品为药物时，所述药物还包括至少一种药物非活性成分。According to the invention, when the product is a medicament, the medicament further comprises at least one pharmaceutically inactive ingredient.
所述药物非活性成分可以是药学上通常使用的载体、赋形剂及稀释剂等。而且，根据通常的方法，可以制作成粉剂、颗粒剂、片剂、胶囊剂、混悬剂、乳剂、糖浆剂、喷雾剂等的口服剂、外用剂、栓剂及无菌注射溶液形式的剂型使用。The pharmaceutical inactive ingredients may be generally used pharmaceutical carriers, excipients, diluents and the like. Moreover, according to the usual method, it can be made into powder, granule, tablet, capsule, suspension, emulsion, syrup, spray, etc., in the form of oral preparations, external preparations, suppositories and sterile injection solutions. .
所述可以包含的载体、赋形剂及稀释剂等非药物活性成分在领域内是熟知的，本领域普通技术人员能够确定其符合临床标准。The non-pharmaceutical active ingredients such as carriers, excipients and diluents that may be included are well known in the art, and those of ordinary skill in the art can determine that they meet clinical standards.
本发明的又一具体实施方式中，所述载体、赋形剂及稀释剂包括但不限于有乳糖、葡萄糖、蔗糖、山梨糖醇、甘露醇、木糖醇、赤藓糖醇、麦芽糖醇、淀粉、阿拉伯胶、藻酸盐、明胶、磷酸钙、硅酸钙、纤维素、甲基纤维素、微晶纤维素、聚乙烯吡咯烷酮、水、羟基苯甲酸甲酯、羟基苯甲酸丙酯、滑石粉、硬脂酸镁和矿物油等。In yet another specific embodiment of the present invention, the carrier, excipient and diluent include but are not limited to lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, Starch, Gum Arabic, Alginate, Gelatin, Calcium Phosphate, Calcium Silicate, Cellulose, Methylcellulose, Microcrystalline Cellulose, Polyvinylpyrrolidone, Water, Methylparaben, Propylparaben, Talc Powder, Magnesium Stearate and Mineral Oil etc.
本发明的又一具体实施方式中，本发明的药物可通过已知的方式施用至体内。例如通过静脉全身递送或者局部注射递送到感兴趣组织中。可选地经由静脉内、经皮、鼻内、粘膜或其他递送方法进行施用。这样的施用可以经由单剂量或多剂量来进行。本领域技术人员理解的是，本发明中有待施用的实际剂量可以在很大程度上取决于多种因素而变化，如靶细胞、生物类型或其组织、待治疗受试者的一般状况、给药途径、给药方式等等。In yet another specific embodiment of the present invention, the drug of the present invention can be administered into the body by known means. For example, by intravenous systemic delivery or local injection into the tissue of interest. Administration is optionally via intravenous, transdermal, intranasal, mucosal or other delivery methods. Such administration can be via single dose or multiple doses. It will be appreciated by those skilled in the art that the actual dosage to be administered in the present invention may vary largely depending on various factors such as the target cell, the type of organism or its tissue, the general condition of the subject to be treated, the route of administration, method of administration, etc.
本发明的又一具体实施方式中，所述药物施用对象可以是人和非人哺乳动物，如小鼠、大鼠、豚鼠、兔、狗、猴、猩猩等。In yet another specific embodiment of the present invention, the subject of drug administration can be humans and non-human mammals, such as mice, rats, guinea pigs, rabbits, dogs, monkeys, orangutans, and the like.
以下通过实施例对本发明做进一步解释说明，但不构成对本发明的限制。应理解这些实施例仅用于说明本发明而不用于限制本发明的范围。The present invention is further explained and illustrated by the following examples, but does not constitute a limitation of the present invention. It should be understood that these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention.
实施例Example
实验方法：experimental method:
1.1细胞培养1.1 Cell culture
人神经母细胞瘤SH-SY5Y细胞系获自中国科学院细胞库。细胞在含10％胎牛血清的DMEM中培养，并加入GlutaMAX(1×)，青霉素/链霉素(1×)，在37℃下在含有5％CO2和95％空气的潮湿气氛中温育。The human neuroblastoma SH-SY5Y cell line was obtained from the Cell Bank of the Chinese Academy of Sciences. Cells were cultured in DMEM containing 10% fetal calf serum, GlutaMAX (1×), penicillin/streptomycin (1×), and incubated at 37°C in a humidified atmosphere containing 5% CO2 and 95% air.
1.2细胞转染1.2 Cell transfection
PPP4R3A过表达慢病毒，对照载体由吉凯基因提供。KIF2A抑制剂及对照siRNA由Genepharma提供，并转染至SH-SY5Y细胞中48小时。PPP4R3A was overexpressed with lentivirus, and the control vector was provided by Genesis. KIF2A inhibitor and control siRNA were provided by Genepharma and transfected into SH-SY5Y cells for 48 hours.
1.3蛋白印迹1.3 Western blot
将来自SH-SY5Y细胞的全细胞蛋白质提取物在裂解缓冲液中超声，并加入磷酸酶抑制剂。以12,000转/分离心后吸取上清液并进行蛋白质浓度测定。免疫印迹后将蛋白质转移至硝酸纤维素滤膜上并与特异性抗体孵育，在4℃冰箱中过夜。使用以下抗体：Whole-cell protein extracts from SH-SY5Y cells were sonicated in lysis buffer with the addition of phosphatase inhibitors. After centrifugation at 12,000 rpm, the supernatant was aspirated and assayed for protein concentration. After immunoblotting, proteins were transferred onto nitrocellulose filters and incubated with specific antibodies overnight in a 4°C refrigerator. Use the following antibodies:
将免疫复合物与荧光素缀合的第二抗体室温下孵育1小时，并与ECL发光液孵育进行荧光测定。The immune complex was incubated with fluorescein-conjugated secondary antibody for 1 hour at room temperature and incubated with ECL luminescent solution for fluorescence measurement.
1.4小鼠模型构建1.4 Mouse model construction
Ppp4r3a-floxed打靶小鼠由美国赛业生物科技有限公司构建，利用同源重组技术在Exon2两端插入loxP位点，将Ppp4r3afl/fl小鼠与Sox-Cre小鼠交配获得F1代杂合敲除小鼠，将杂合敲除小鼠自交获得纯合敲除小鼠。Ppp4r3a-floxed targeting mice were constructed by American Saiye Biotechnology Co., Ltd., using homologous recombination technology to insert loxP sites at both ends of Exon2, and mating Ppp4r3a fl/fl mice with Sox-Cre mice to obtain F1 hybrid knockout mice Homozygous knockout mice were obtained by selfing heterozygous knockout mice.
1.5小鼠行为学检测1.5 Behavioral testing of mice
在每次实验之前，将每只小鼠放置于单独的笼内，利用旷场试验、转棒试验、跑台试验、筑巢试验及T迷宫试验检测小鼠的运动功能、执行功能及认知功能。记录每只小鼠在相应试验中的数据。Before each experiment, each mouse was placed in a separate cage, and the motor function, executive function and cognition of the mice were detected by open field test, rotarod test, treadmill test, nesting test and T maze test Function. Record the data for each mouse in the corresponding experiment.
旷场试验：在一个黑暗安静的房间里，小鼠被轻轻放置在一个40×50×50厘米的甲基丙烯酸酯盒子里，并允许小鼠自由探索10分钟。一个摄像机安装在竞技场上方，自动记录老鼠的活动。Open field test: In a dark and quiet room, mice were gently placed in a 40×50×50 cm methacrylate box, and allowed to explore freely for 10 minutes. A video camera was installed above the arena to automatically record the activity of the rats.
转棒试验：首先将动物送去训练(连续2天，每天10转，600秒)。动物在旋转杆脱落时被放回。训练结束后，对动物进行连续6天的评估，速度从15转/分加速到40转/分，持续900秒。记录掉下旋转杆子的潜伏期。在被动旋转的情况下，将小鼠重新定位到旋转杆上。如果重复这种行为，我们认为这是下跌的时候。旋转超过900秒的动物记为900秒。Rotarod test: Animals were first sent to training (2 consecutive days, 10 rotations per day, 600 seconds). The animal is put back when the rotarod comes off. After training, the animals were evaluated for 6 consecutive days by accelerating from 15 to 40 rpm for 900 s. The latency to drop the rotating pole was recorded. With passive rotation, reposition the mouse onto the rotating rod. If this behavior repeats, we think it's time to drop. Animals that rotated for more than 900 seconds were counted as 900 seconds.
跑台试验：以9m/min和11m/min的速度训练小鼠10min(电流值1mA)，连续训练2天。实验阶段，小鼠以10m/min的初始速度(电流值1mA)置于跑步机带中。5min后，以1m/min的加速度提高速度。连续电击6秒的小鼠被视为“精疲力竭”，并记录精疲力竭的时间。Treadmill test: train mice at speeds of 9 m/min and 11 m/min for 10 min (
筑巢试验：将小鼠放置于黑暗中1h，将小鼠转移到含有2.5g压棉材料(50cm×60cm)的单笼中。24h后，按照Deacon的标准对巢的存在性和巢的质量进行1～5级的评定。1＝90％以上的压棉材料完整，2＝50％-90％的压棉材料完整，3＝压棉材料大部分被撕碎，但通常没有可识别的巢，4＝可识别但平坦的巢，5＝(接近)完美的巢。Nest-building test: the mice were placed in the dark for 1 hour, and then transferred to a single cage containing 2.5 g of pressed cotton material (50 cm×60 cm). After 24 hours, the presence of nests and the quality of nests were rated from 1 to 5 according to Deacon's standard. 1 = more than 90% of the pressed material is intact, 2 = 50%-90% of the pressed material is intact, 3 = the pressed material is mostly shredded, but usually no identifiable nest, 4 = identifiable but flat Nest, 5 = (close to) perfect nest.
T-迷宫：t型迷宫(长64厘米，臂长30厘米，宽12厘米，壁高16厘米)由透明有机玻璃制成，并充满水(23℃)。一个直径为10厘米的透明平台放置在水下1厘米的目标臂上。该方法由初始采集阶段和反转阶段组成。在第一阶段，平台被放置在一侧。老鼠被放置在开始区，面向后墙。小鼠有30秒的时间完成每个试验。如果没有找到平台，将小鼠放置在平台上10s。小鼠每天进行15次试验，持续3天。每只鼠标的正确选择都被记录下来。第3天，强化后20分钟，将平台切换到相反的手臂，并对小鼠进行逆转期测试。对于每一次追踪，无论是在初始捕获阶段还是反转阶段，如果老鼠直接游到平台上，就会记录下正确的选择。如果老鼠直接游到对面的手臂，则记录为不正确。如果鼠标进入目标臂，但在到达平台之前游回起点，则记录为“无选择”。T-maze: The t-maze (length 64 cm,
1.6小鼠原代神经元培养1.6 Mouse primary neuron culture
从E18.5胚胎中分离C57BL/6小鼠皮层和海马，仔细去除脑膜和血管后，用移液机械分离细胞。样品置于预冷的Hank’s平衡盐溶液(HBSS，赛默飞世尔)中，用0.05％胰蛋白酶消化(赛默飞世尔)。用含有10％胎牛血清(Gibco FBS,Thermo Fisher)的DMEM/F12和生长培养基(Neurobasal medium,Glutamax和B27补充剂，Thermo Fisher)重新悬浮细胞。将细胞离心(80g,8分钟)，在37℃和5％ CO2的条件下，以2×105个细胞/cm2的密度沉积在聚d-赖氨酸(Sigma-Aldrich)上。每三天更换一半的培养基。C57BL/6 mouse cortex and hippocampus were isolated from E18.5 embryos, and cells were mechanically dissociated by pipetting after careful removal of meninges and blood vessels. Samples were placed in pre-cooled Hank's Balanced Salt Solution (HBSS, Thermo Fisher), and digested with 0.05% trypsin (Thermo Fisher). Cells were resuspended with DMEM/F12 and growth medium (Neurobasal medium, Glutamax and B27 supplements, Thermo Fisher) containing 10% fetal bovine serum (Gibco FBS, Thermo Fisher). Cells were centrifuged (80 g, 8 min) and deposited on poly-d-lysine (Sigma-Aldrich) at a density of 2×10 5 cells/cm 2 at 37° C. and 5% CO 2 . Half of the medium was changed every three days.
1.7免疫荧光染色1.7 Immunofluorescence staining
用2％苯巴比妥麻醉小鼠，经心静脉灌注无菌生理盐水，然后再灌注4％多聚甲醛(PFA)。然后将解剖组织固定在4％的PFA中。使用以下抗体:PPP4R3A(Sigma-Aldrich,HPA002568)；p-Tau(Thr231)(Abways,CY5625)；p-Tau(Ser396)(Abways,CY5657)；AT8(ServiceBio,GB113883)；KIF2A(生工生物科技，D225969)。细胞核用DAPI(Abcam,ab104139)染色。Mice were anesthetized with 2% phenobarbital, perfused with sterile saline, and then reperfused with 4% paraformaldehyde (PFA). The dissected tissues were then fixed in 4% PFA. The following antibodies were used: PPP4R3A (Sigma-Aldrich, HPA002568); p-Tau (Thr231) (Abways, CY5625); p-Tau (Ser396) (Abways, CY5657); AT8 (ServiceBio, GB113883); , D225969). Nuclei were stained with DAPI (Abcam, ab104139).
实验结果：Experimental results:
我们分析了人前额叶及海马的mRNA表达数据。比较年轻(20-45岁)和老年(85-110岁)组发现，老年前额叶(图1A)和海马区(图1B)中Ppp4r3a下调。此外，我们发现在进行性核上性麻痹患者血清中PPP4R3A转录水平下降(图1C)，然而其他帕金森综合征患者(皮质基底节变性患者)血清中PPP4R3A并未下降。We analyzed mRNA expression data from the human prefrontal cortex and hippocampus. Comparing the young (20-45 years old) and old (85-110 years old) groups found that Ppp4r3a was downregulated in the prefrontal cortex (Fig. 1A) and hippocampus (Fig. 1B) of the old. Furthermore, we found that PPP4R3A transcript levels were decreased in the serum of patients with progressive supranuclear palsy (Fig. 1C), whereas PPP4R3A was not decreased in the serum of other Parkinsonian patients (corticobasal degeneration patients).
我们进行了一系列的行为测试来评估突变小鼠的状况。我们首先进行了旷场测试，以测量身体/运动活动。结果显示，8个月龄的Ppp4r3a-/-小鼠总行走距离减少，但4个月龄前的小鼠没有(图2A,B)。6个月龄(图2C)和10个月龄(图2D)纯合子小鼠旋转杆测试持续时间下降。值得注意的是，与野生型相比，从6个月到10个月的杂合小鼠运动耐力下降得更快。6个月大的雄性小鼠的跑台测试显示纯合子小鼠的运动耐受性较短(图3E)。根据8个月大的小鼠的Deacon评分16评估筑巢行为。野生型小鼠能够建立近乎完美的巢(4.5±0.29)，而杂合子(3.25±0.25)和纯合子(2.25±0.25)笼内的压棉被部分撕裂(图2F和G)。为了评估认知能力，对纯合子小鼠认知能力存在缺陷的老年小鼠(6月龄)进行T迷宫测试。在获得性学习阶段，Ppp4r3a-/+组和Ppp4r3a-/-组在第2天和第3天的正确选择比例较低。在逆向学习阶段，Ppp4r3a-/-组的正确选择比例也较低(图2H)。各组之间在达到获得标准所需的试验数量上无显著差异。然而，在第4天和第5天，Ppp4r3a-/-组有更多的尝试试验(图2I)。简而言之，在Ppp4r3a KO小鼠中，运动障碍和认知能力随着年龄的增长而下降，尤其是纯合子小鼠。We performed a series of behavioral tests to assess the condition of the mutant mice. We first performed an open field test to measure physical/motor activity. The results showed that the total walking distance was reduced in 8-month-old Ppp4r3a-/- mice, but not in 4-month-old mice (Fig. 2A,B). The duration of the rotarod test decreased in 6-month-old (Figure 2C) and 10-month-old (Figure 2D) homozygous mice. Notably, exercise tolerance declined more rapidly in heterozygous mice from 6 to 10 months of age compared with wild-type mice. Treadmill testing of 6-month-old male mice revealed shorter exercise tolerance in homozygous mice (Fig. 3E). Nesting behavior was assessed according to the Deacon score of 8-month-old mice. Wild-type mice were able to build near-perfect nests (4.5±0.29), whereas press quilts were partially torn in heterozygous (3.25±0.25) and homozygous (2.25±0.25) cages (Fig. 2F and G). To assess cognitive performance, a T-maze test was performed on aged mice (6 months old) homozygous for cognitive deficits. During the acquired learning phase, the Ppp4r3a-/+ group and the Ppp4r3a-/- group had lower proportions of correct choices on
接下来，我们研究了Ppp4r3a缺乏是否可以促进大脑中tau聚集物的形成。AT8免疫组化显示皮层、海马和纹状体中过度磷酸化神经元密度增加(图3A-C)。此外，在Ppp4r3a-/+和Ppp4r3a-/-的皮层和/或海马体中，p-Tau(T231)(图3D和E)和p-Tau(S396)(图3F)的沉积也有所增加。在皮层和海马中，Ppp4r3a-/-组p-pten、p-akt和p-GSK3β下调，p-tau(T231)过度磷酸化(图3G)。We next investigated whether Ppp4r3a deficiency could promote the formation of tau aggregates in the brain. AT8 immunohistochemistry revealed increased density of hyperphosphorylated neurons in the cortex, hippocampus, and striatum (Fig. 3A-C). Furthermore, deposition of p-Tau(T231) (Fig. 3D and E) and p-Tau(S396) (Fig. 3F) was also increased in the cortex and/or hippocampus of Ppp4r3a-/+ and Ppp4r3a-/-. In the cortex and hippocampus, p-pten, p-akt, and p-GSK3β were downregulated and p-tau(T231) was hyperphosphorylated in the Ppp4r3a-/- group (Fig. 3G).
免疫共沉淀(co-IP)实验揭示了Kif2a、RAN、PPP4C和Ppp4r3a之间的相互作用(图4A)。原代神经元中Ppp4r3a和Kif2a的共染色显示了Ppp4r3a-/-细胞质中Kif2a的增加(图4B)。在皮质中，我们观察到纯合子与野生型相比细胞质Kif2a的沉积(图4C)。有报道称，在有丝分裂细胞中，Kif2a在间期同时位于细胞质和细胞核中，并在有丝分裂前期至末期21逐渐聚集成细胞核中的梭形微管。当固定到中期时，与对照组相比，shPPP4R3A细胞的细胞质中Kif2a富集(图4D)Co-immunoprecipitation (co-IP) experiments revealed the interaction between Kif2a, RAN, PPP4C and Ppp4r3a (Fig. 4A). Co-staining of Ppp4r3a and Kif2a in primary neurons revealed an increase of Kif2a in Ppp4r3a-/- cytoplasm (Fig. 4B). In the cortex, we observed deposition of cytoplasmic Kif2a in homozygotes compared to wild type (Fig. 4C). It has been reported that in mitotic cells, Kif2a localizes in both the cytoplasm and nucleus during interphase and gradually aggregates into spindle-shaped microtubules in the nucleus from prophase to telophase. When fixed to metaphase, Kif2a was enriched in the cytoplasm of shPPP4R3A cells compared to controls (Fig. 4D)
为了说明Ppp4r3a的保护作用，我们在SH-SY5Y细胞中过表达Ppp4r3a。在PPP4R3A-oe中，p-PTEN、p-AKT和p-GSK3β上调，P-Tau(T231)水平下调(图5A)。在PPP4R3A过表达的细胞中，我们观察到延伸的神经突(图5B)和线粒体均匀地位于细胞质的近端和远端(图5C)。当Ppp4r3a过表达时，NHA和SH-SY5Y细胞中的CCK8检测显示细胞活力更高(图5D)。同时，我们也想验证过多的细胞质Kif2a是否与细胞毒性有关。当沉默shPPP4R3A细胞中的Kif2a时，western blot显示p-GSK3β水平升高，使GSK3β失活，从而抑制了由Ppp4r3a缺陷引起的tau磷酸化(图5E)。微管蛋白免疫染色显示siKIF2A shPPP4R3A细胞中保留微管(图5F)。对于线粒体功能，可以通过敲除Kif2a来挽救shPPP4R3A中降低的细胞活力(图5G)。这些结果共同证实了Kif2a通过调节AKT-GSK3β通路参与了与Ppp4r3a表达降低相关的Tau蛋白病理。To illustrate the protective role of Ppp4r3a, we overexpressed Ppp4r3a in SH-SY5Y cells. In PPP4R3A-oe, p-PTEN, p-AKT, and p-GSK3β were upregulated, and P-Tau(T231) levels were downregulated (Fig. 5A). In PPP4R3A-overexpressing cells, we observed extended neurites (Fig. 5B) and mitochondria evenly located proximal and distal to the cytoplasm (Fig. 5C). CCK8 detection in NHA and SH-SY5Y cells showed higher cell viability when Ppp4r3a was overexpressed (Fig. 5D). At the same time, we also wanted to verify whether excess cytoplasmic Kif2a is related to cytotoxicity. When Kif2a was silenced in shPPP4R3A cells, western blot showed increased p-GSK3β levels, which inactivated GSK3β and thereby suppressed tau phosphorylation caused by Ppp4r3a deficiency (Fig. 5E). Tubulin immunostaining revealed that microtubules were retained in siKIF2A shPPP4R3A cells (Fig. 5F). For mitochondrial function, reduced cell viability in shPPP4R3A could be rescued by knockdown of Kif2a (Fig. 5G). These results collectively confirm that Kif2a participates in Tau pathology associated with decreased Ppp4r3a expression by regulating the AKT-GSK3β pathway.
综上，在本发明中，我们报告了Ppp4r3a在老年人大脑中下降。为了分析Ppp4r3a缺失在tau病理进展中的功能，我们构建了Ppp4r3a敲除小鼠。在中年Ppp4r3a-/+和Ppp4r3a-/-小鼠中，我们观察到以运动和认知能力下降伴tau病理为特征的退行性表型，这些特征与进行性核上性麻痹的临床表型一致。在Ppp4r3a-/+和Ppp4r3a-/-小鼠的皮层、海马和纹状体中，检测到Tau蛋白AT8、pThr231和pSer396过度磷酸化。这些结果表明，即使没有tau突变的背景，Ppp4r3a也加速了tau的病理进展。In summary, in the present invention, we report that Ppp4r3a is decreased in aged brains. To analyze the function of Ppp4r3a deletion in the progression of tau pathology, we constructed Ppp4r3a knockout mice. In middle-aged Ppp4r3a-/+ and Ppp4r3a-/- mice, we observed degenerative phenotypes characterized by motor and cognitive decline with tau pathology that were consistent with the clinical phenotype of progressive supranuclear palsy unanimous. Hyperphosphorylation of Tau proteins AT8, pThr231 and pSer396 was detected in the cortex, hippocampus and striatum of Ppp4r3a-/+ and Ppp4r3a-/- mice. These results suggest that Ppp4r3a accelerates tau pathological progression even in the absence of tau mutation background.
Tau是CDK5和Gsk3β的底物研究表明，Kif2a可能通过β-Catenin-Akt通路调控GSK3β活性。我们在这里证明了Ppp4r3a缺失通过Akt通路激活Gsk3β激酶活性，导致tau蛋白过度磷酸化。敲除shPPP4R3A细胞中的Kif2a抑制了tau磷酸化水平，这意味着在Ppp4r3a缺陷细胞中增加的细胞质Kif2a可能是通过akt-gsk3β依赖方式导致tau过度磷酸化的原因。Tau is the substrate of CDK5 and Gsk3β. Studies have shown that Kif2a may regulate the activity of GSK3β through the β-Catenin-Akt pathway. We demonstrate here that loss of Ppp4r3a activates Gsk3β kinase activity through the Akt pathway, leading to tau hyperphosphorylation. Knockdown of Kif2a in shPPP4R3A cells suppressed tau phosphorylation levels, implying that increased cytoplasmic Kif2a in Ppp4r3a-deficient cells may be responsible for tau hyperphosphorylation in an akt-gsk3β-dependent manner.
总之，本发明证明了Ppp4r3a缺乏导致tau过度磷酸化和进行性核上性麻痹表型。Ppp4r3a通过蛋白结合促进Kif2a亚细胞定位，Kif2a在Ppp4r3a缺失细胞中的沉积通过介导AKT/GSK3β/tau信号通路导致tau蛋白过度磷酸化。本发明的结论是，Ppp4r3a对于维持神经元活力至关重要，因此，Ppp4r3a表达降低可能导致进行性核上性麻痹，并有可能成为进行性核上性麻痹的生物学标志物。In conclusion, the present invention demonstrates that Ppp4r3a deficiency results in tau hyperphosphorylation and a progressive supranuclear paralysis phenotype. Ppp4r3a promotes the subcellular localization of Kif2a through protein binding, and the deposition of Kif2a in Ppp4r3a-deficient cells leads to hyperphosphorylation of tau protein by mediating the AKT/GSK3β/tau signaling pathway. The conclusion of the present invention is that Ppp4r3a is essential for maintaining neuronal viability, therefore, decreased expression of Ppp4r3a may lead to progressive supranuclear palsy, and may become a biological marker of progressive supranuclear palsy.
最后应该说明的是，以上所述仅为本发明的优选实施例而已，并不用于限制本发明，尽管参照前述实施例对本发明进行了详细的说明，对于本领域的技术人员来说，其依然可以对前述实施例所记载的技术方案进行修改，或者对其中部分进行等同替换。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。上述虽然对本发明的具体实施方式进行了描述，但并非对本发明保护范围的限制，所属领域技术人员应该明白，在本发明的技术方案的基础上，本领域技术人员不需要付出创造性劳动即可做出的各种修改或变形仍在本发明的保护范围以内。Finally, it should be noted that the above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, for those skilled in the art, it is still The technical solutions described in the foregoing embodiments may be modified, or part of them may be equivalently replaced. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention. Although the specific implementation of the present invention has been described above, it is not a limitation to the protection scope of the present invention. Those skilled in the art should understand that on the basis of the technical solution of the present invention, those skilled in the art can do it without creative work. Various modifications or deformations are still within the protection scope of the present invention.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116949169A (en) * | 2023-07-31 | 2023-10-27 | 山东大学齐鲁医院 | Application of SMEK1 in diagnosis and treatment of cerebral arterial thrombosis |
| CN119881329A (en) * | 2025-01-14 | 2025-04-25 | 北京市神经外科研究所 | Use of Tau-carrying astrocyte-derived extracellular vesicles in aiding diagnosis of progressive supranuclear palsy |
| CN119985972A (en) * | 2025-01-14 | 2025-05-13 | 北京市神经外科研究所 | Biomarkers for assisting diagnosis of progressive supranuclear palsy and their application |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113564242A (en) * | 2021-07-28 | 2021-10-29 | 山东大学齐鲁医院 | Application of apoptosis-related genes in repeated implantation failure |
| JP2023005573A (en) * | 2021-06-29 | 2023-01-18 | 国立大学法人鳥取大学 | Diagnosis marker for progress property supranuclear paralysis |
-
2023
- 2023-03-13 CN CN202310251005.0A patent/CN116397021A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2023005573A (en) * | 2021-06-29 | 2023-01-18 | 国立大学法人鳥取大学 | Diagnosis marker for progress property supranuclear paralysis |
| CN113564242A (en) * | 2021-07-28 | 2021-10-29 | 山东大学齐鲁医院 | Application of apoptosis-related genes in repeated implantation failure |
Non-Patent Citations (3)
| Title |
|---|
| FEI GAO等: "Ppp4r3a deficiency leads to depression-like behaviors in mice by modulating the synthesis of synaptic proteins", 《DISEASE MODELS & MECHANISMS》, vol. 15, pages 527 - 12 * |
| 段若楠: "PADMAL变异型的致病基因验证以及SMEK1在中枢神经损伤中的病理机制研究", 《中国博士学位论文全文数据库医药卫生科技辑》, no. 11, pages 1 - 2 * |
| 陈向良等: "损伤杏仁核制备阳虚阿尔茨海默病大鼠模型", 《中国老年学杂志》, vol. 27, 31 January 2007 (2007-01-31), pages 139 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116949169A (en) * | 2023-07-31 | 2023-10-27 | 山东大学齐鲁医院 | Application of SMEK1 in diagnosis and treatment of cerebral arterial thrombosis |
| CN116949169B (en) * | 2023-07-31 | 2024-02-06 | 山东大学齐鲁医院 | Application of SMEK1 in the diagnosis and treatment of ischemic stroke |
| CN119881329A (en) * | 2025-01-14 | 2025-04-25 | 北京市神经外科研究所 | Use of Tau-carrying astrocyte-derived extracellular vesicles in aiding diagnosis of progressive supranuclear palsy |
| CN119985972A (en) * | 2025-01-14 | 2025-05-13 | 北京市神经外科研究所 | Biomarkers for assisting diagnosis of progressive supranuclear palsy and their application |
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