Protein Phosphorylation And Regulation Of Cytoskeleton In Neuronal Systems

神经系统中蛋白质磷酸化和细胞骨架的调节

• 批准号: 8158171
• 负责人: HARISH C PANT
• 金额: $ 80.07万
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8158171
• 关键词: Cytoskeleton; Neurons; Phosphorylation; Regulation; System

In our previous studies it has been demonstrated that phosphorylation of the neuron specific intermediate filament proteins, such as neurofilaments (NFs) were tightly regulated developmentally and topographically, and generally confined to the axonal compartment and selectively occurs on proline directed serine (Ser) and threonine (Thr) residues. It was recognized that in neurodegenerative disorders such as Alzheimers disease (AD) and Amyotrophic lateral sclerosis (ALS), the pathology was characterized by an accumulation of aberrantly phosphorylated cytoskeletal proteins in perikarya on these residues, suggesting that topographic regulation had been compromised. Neurodegenerative diseases including AD are complex and chronic disorders that involve the disruption of the neuronal network in the human brain. We have made first time the comprehensive study of quantitative mass spectrometric analysis of human neuron specific major cytoskeletal proteins, medium (NF-M)and high (NF-H) molecular weight neurofilament proteins from control adult and AD matched brains. This study aimed to extend our knowledge of specific phosphorylation sites to provide insight into the role of kinases and phosphatases in the formation of neurofibrillary tangles (NFTs) and aggregates. The quantitative mass spectroscopic technique ( iTRAQ) used in this study identified specific phosphorylation sites among multiple lysine serine/threonine proline (KS/TP) repeats and non-S/TP sites in NF-M/H. For example, evidence of phosphorylation of proline-directed and non-proline-directed Ser residue suggests the involvement of both proline-directed and non-proline-directed kinases in AD.The iTRAQ analysis revealed primarily that specific Ser/Thr sites are phosphorylated at a higher fold in AD brain compared with control brain and that not all the neurofilament KSP sites are phosphorylated at similar levels in AD. Some of them are phosphorylated at a greater abundance compared with other sites. Hipocampal,CA1 pyramidal cells are known for susceptibility to neurofibrillary degeneration in AD. In human brain, these neurons also show an age-related perikaryal accumulation of NFs. Most of the phosphorylation sites in AD tangles are perikaryonal constituents. Normal perikarya do not contain phosphorylated neurofilament proteins.Our immunohistochemistry data suggest that phospho-NF is predominantly detected in intracellular NFTs and extracellular plaques in AD. The iTRAQ data obtained with phosphorylated NF-M from AD brain also show the higher abundance of phosphorylation of non-proline-directed Ser or Thr residues,suggesting the involvement of also non-proline-directed kinases in addition to proline - directed kinases in AD brain. This study first time demonstrates the importance of regulation of NF-M phosphorylation in physiology and pathology. It is found that KSP repeat phosphorylation of NF-M is significantly higher compared with NF-H, despite the greater number of these sites in NF-H. We were able to obtain the sequence coverage for 13/18 phosphorylation sites of NF-M; however, iTRAQ analysis resulted in the aberrant phosphorylation of 10/43 phosphorylation sites of NF-H in AD brain. Based on the stoichiometry, there is 2-fold higher KSP repeats phosphorylated in NF-M compared with NF-H; therefore, we believe that NF-M might contribute more to aberrant phosphorylation in AD compared with other cytoskeletal proteins. and also suggests that specific antibodies could be generated to the SP and non-SP sites that are phosphorylated at a greater abundance and can be used as bio-markers in AD and the specific inhibitors of these kinase can be used as therapeutic reagents for AD and other neurodegenerative diseases involved in aberrant neuronal cyytoskeletal protein phosphorylation. We have began to study the role of proein phosphatases to further understand the molecular basis of the phosphorylation during aging and associated neuropathology. We found that the protein phospatase activity declined relatively more, rather than activation of kinases, underlies aging-related neuronal cytoskeletal proten hyperphosphorylation. Purified protein phosphatase (PP2A) dephosphorylated the heavy neurofilament (NF-H) subunit or its extensively phorphorylated carboxyl-terminal domain in vitro. In cultured primary hippocampal neurons, inhibiting PP2A induced NF-H phosphorylation without activating known neurofilament kinases. Neurofilament phosphorylation in the mouse CNS, as reflected by levels of the RT-97 antibody phosphoepitope associated with late axon maturation, more than doubled during the 12-month period after the NF-H phosphorylation plateaued at P21. This was accompanied by declines in levels and activity of PP2A but not PP2B, and no rise in activities of neurofilament kinases (Erk1,2, cdk5 and JNK1,2). Inhibiting PP2A in mice in vivo restored brain RT-97 to levels seen in young mice. Declining PP2A activity, therefore, can account for rising neurofilament phosphorylation in maturing brain, potentially compounding similar changes associated with adult-onset neurodegenerative diseases.

在我们以前的研究中，已经证明了神经元特异性中间丝蛋白（如神经丝（NF））的磷酸化在发育和地形学上受到严格调控，并且通常局限于轴突隔室，并选择性地发生在脯氨酸指导的丝氨酸（Ser）和苏氨酸（Thr）残基上。人们认识到，在神经退行性疾病，如阿尔茨海默病（AD）和肌萎缩性侧索硬化症（ALS），病理学的特征是异常磷酸化的细胞骨架蛋白在这些残基上的胞体中的积累，表明地形调节已受到损害。包括AD在内的神经退行性疾病是复杂的慢性疾病，涉及人脑中神经元网络的破坏。本研究首次对正常成人和AD匹配脑中神经元特异性主要细胞骨架蛋白、中分子量（NF-M）和高分子量（NF-H）神经丝蛋白进行了全面的定量质谱分析研究。本研究旨在扩展我们对特定磷酸化位点的了解，以深入了解激酶和磷酸酶在神经元缠结（NFT）和聚集体形成中的作用。本研究中使用的定量质谱技术（iTRAQ）确定了NF-M/H中多个赖氨酸丝氨酸/苏氨酸脯氨酸（KS/TP）重复序列和非S/TP位点之间的特异性磷酸化位点。例如，脯氨酸定向和非脯氨酸定向Ser残基磷酸化的证据表明，AD中涉及脯氨酸定向和非脯氨酸定向激酶。iTRAQ分析主要揭示，与对照脑相比，AD脑中特定Ser/Thr位点以更高的倍数磷酸化，并且AD中并非所有神经丝KSP位点都以相似的水平磷酸化。与其他位点相比，它们中的一些以更高的丰度被磷酸化。海马CA 1区锥体细胞对AD的神经退行性变易感。在人脑中，这些神经元也显示出与年龄相关的核周NF积聚。AD缠结中的大多数磷酸化位点是核周成分。正常细胞核不含磷酸化的神经丝蛋白，我们的免疫组化数据表明，磷酸化NF主要在AD的细胞内NFT和细胞外斑块中检测到。用来自AD脑的磷酸化NF-M获得的iTRAQ数据还显示非脯氨酸定向的Ser或Thr残基的磷酸化的更高丰度，表明AD脑中除了脯氨酸定向的激酶之外还涉及非脯氨酸定向的激酶。本研究首次证实了NF-M磷酸化调控在生理和病理上的重要性。发现NF-M的KSP重复磷酸化显著高于NF-H，尽管NF-H中这些位点的数量更多。我们能够获得NF-M 13/18磷酸化位点的序列覆盖率;然而，iTRAQ分析导致AD大脑中NF-H 10/43磷酸化位点异常磷酸化。基于化学计量，有2倍高的KSP重复磷酸化NF-M相比，NF-H，因此，我们认为，NF-M可能有助于AD异常磷酸化相比，其他细胞骨架蛋白。 还表明可以针对更高丰度磷酸化的SP和非SP位点产生特异性抗体，并可用作AD的生物标记物，并且这些激酶的特异性抑制剂可用作AD和其他涉及异常神经元细胞骨架蛋白磷酸化的神经退行性疾病的治疗试剂。 我们已经开始研究蛋白磷酸酶的作用，以进一步了解衰老和相关神经病理学过程中磷酸化的分子基础。我们发现，蛋白磷酸酶活性下降相对较多，而不是激活的激酶，是衰老相关的神经元细胞骨架蛋白过度磷酸化的基础。纯化的蛋白磷酸酶（PP 2A）去磷酸化的重神经丝（NF-H）亚基或其广泛磷酸化的羧基末端结构域在体外。在培养的原代海马神经元中，抑制PP 2A诱导NF-H磷酸化而不激活已知的神经丝激酶。 小鼠CNS中的神经丝磷酸化，如与晚期轴突成熟相关的RT-97抗体磷酸化表位水平所反映的，在P21时NF-H磷酸化达到平台后的12个月期间增加了一倍以上。这伴随着PP 2A的水平和活性的下降，而不是PP 2B，并且神经丝激酶（Erk 1，2，cdk 5和JNK 1，2）的活性没有上升。在小鼠体内抑制PP 2A使脑RT-97恢复到在年轻小鼠中观察到的水平。因此，PP 2A活性的下降可以解释成熟大脑中神经丝磷酸化的上升，可能复合与成人发病的神经退行性疾病相关的类似变化。