Protein Damage and Repair in the Brain

描述（由申请人提供）：我们的长期目标是阐明异天冬氨酸（isoAsp）蛋白形成在脑老化和与年龄相关的神经系统疾病中的潜在作用。异asp位点的积累是蛋白质损伤的主要形式，通常由蛋白质l -异天冬氨酸甲基转移酶（PIMT）来控制，这是一种在大脑中高度富集的修复酶。ppimt敲除小鼠积累了高水平的isoasp损伤蛋白，特别是在大脑和睾丸中，其表型主要是神经学；出生后4-10周，脑体积增大，神经生理和代谢信号通路异常，认知功能下降，致命性癫痫。IsoAsp的形成可以破坏蛋白质功能，引发自身免疫，并且在体外经常伴随着蛋白质聚集体的形成。我们假设，神经元中isoAsp位点的修复效率低下，是老年和某些脑部疾病中发生的神经退行性变的重要原因。我们最近的研究结果还表明，isoAsp的形成可能是一种涉及共价交联的新型蛋白质聚集形式的基础。我们建议通过以下四个具体目标来探索这些想法。目的1将比较PIMT -/-小鼠的脑提取物与野生型小鼠的脑提取物，以观察这种关键修复酶的完全丧失是否会导致(a) synuclein和tau中isoAsp的积累，这两种蛋白质被研究得很好，涉及几种形式的神经退行性疾病，其他人已经报道过对isoAsp的形成高度敏感，(b) synuclein、tau和崩溃素反应介质蛋白2 （CRMP2）的聚集，以及(c)一般蛋白质的过度磷酸化。以及位点特异性的tau和CRMP2过度磷酸化。目的2将比较PIMT小鼠（表达正常PIMT活性的50-55%）与野生型幼崽，以了解体内PIMT活性的适度降低如何改变isoAsp积累，蛋白质聚集和蛋白质过度磷酸化（如目的1）作为年龄的函数。目的3源于我们最近的研究结果，重组小鼠CRMP2在pH 7.4和37℃的条件下体外老化时，在形成sds不溶性聚集体的同时发生isoAsp形成。我们将进行一系列的研究，以确定是否像我们从最近的数据中怀疑的那样，这种聚集涉及共价交联，并且与isoAsp的形成有机制关系。目的4将比较PIMT -/-小鼠与野生型幼崽，以寻找体内积累isoAsp的神经元酶的功能变化。我们将首先关注肌酸激酶B（脑特异性异构体）和70 kDa热休克同源蛋白（HSC70），这是我们发现的在PIMT -/-小鼠大脑中积累高水平isoAsp的22种蛋白之一。如果指导这四个目标的假设被证明是正确的，这将需要药理学或基因干预来促进PIMT修复系统，以帮助延缓人类认知功能和神经状态在老年时的下降。公共卫生相关性：异常蛋白质结构在老年和阿尔茨海默病、帕金森病和肌萎缩侧索硬化症（ALS）等疾病中发生的人类神经系统退化中起主要作用。本研究将探讨年龄和疾病相关认知能力下降的严重程度和时间进程是否受到神经细胞中修复受损蛋白质的关键代谢途径状态的显著影响。这项研究的结果应该有助于阐明痴呆症的分子基础，从而为减缓或延缓其发病的新疗法铺平道路。

DESCRIPTION (provided by applicant): Our long-range goal is to elucidate the potential role of isoaspartyl (isoAsp) protein formation in brain aging and age-related neurological disease. Accumulation of isoAsp sites is a major form of protein damage that is normally kept in check by protein L-isoaspartyl methyltransferase (PIMT), a repair enzyme that is highly enriched in brain. PIMT knockout mice accumulate high levels of isoAsp-damaged proteins, especially in the brain and testes, and their phenotype is mainly neurological; increased brain size, abnormal neuronal physiology and metabolic signaling pathways, decreased cognitive function, and fatal epilepsy at 4-10 weeks after birth. IsoAsp formation can disrupt protein function, can elicit auto-immunity, and is often accompanied by formation of protein aggregates in vitro. We hypothesize that inefficiencies in the repair of isoAsp sites in neurons contributes significantly to the neurodegeneration that occurs in advanced age and in certain brain diseases. Our recent findings also suggest that isoAsp formation may underlie a novel form of protein aggregation that involves covalent cross-linking. We propose to explore these ideas via the following four specific aims. Aim 1 will compare brain extracts of PIMT -/- mice with wild type littermates to see if complete loss of this key repair enzyme leads to (a) isoAsp accumulation in synuclein and tau, two well studied proteins involved in several forms of neurodegeneration that have been reported by others to be highly susceptible to isoAsp formation, (b) aggregation of synuclein, tau, and collapsin-response mediator protein 2 (CRMP2), and (c) hyperphosphorylation of proteins in general, and site-specific hyperphosphorylation of tau and CRMP2. Aim 2 will compare PIMT mice (which express 50-55% of normal PIMT activity) vs. wild type littermates to see how a moderate reduction of PIMT activity in vivo alters isoAsp accumulation, protein aggregation, and protein hyperphosphorylation (as in Aim 1) as a function of age. Aims 3 stems from our recent findings that recombinant mouse CRMP2 undergoes isoAsp formation concomitant with formation of SDS-insoluble aggregates when it is aged in vitro at pH 7.4 and 37degC. We will carry out a series of studies to determine if, as we suspect from recent data, this aggregation involves covalent cross-linking and is mechanistically related to isoAsp formation. Aim 4 will compare PIMT -/- mice with wild type littermates to look for functional changes in neuronal enzymes that accumulate isoAsp in vivo. We will initially focus on creatine kinase B (brain specific isoform) and the 70 kDa heat shock cognate protein (HSC70) which are among the 22 proteins we have found that accumulate high levels of isoAsp in the PIMT -/- mouse brain. If the hypotheses that guide these 4 aims are proven to be correct, this would call for pharmacological or genetic interventions that could boost the PIMT repair system to help stave off the decline in human cognitive function and neurological status that occurs in advanced age. PUBLIC HEALTH RELEVANCE: Abnormal protein structures play a major role in degeneration of the human nervous system that occurs in advanced age and in diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS). This research will explore the possibility that the severity and time course of age and disease-related cognitive decline is significantly influenced by the status a key metabolic pathway that repairs damaged proteins in nerve cells. The results of this research should help elucidate the molecular basis of dementias and thereby pave the way for new therapies that moderate or delay their onset.