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年龄相关神经系统疾病中的潜在作用。isoAsp位点的积累是蛋白质损伤的主要形式，其通常由蛋白质L-异丙基甲基转移酶（PIMT）（一种在脑中高度富集的修复酶）保持检查。PIMT基因敲除小鼠会积累高水平的isoAsp损伤蛋白，尤其是在大脑和睾丸中，其表型主要是神经系统的;大脑体积增大，神经元生理和代谢信号通路异常，认知功能下降，出生后4-10周出现致命性癫痫。IsoAsp形成可破坏蛋白质功能，可引发自身免疫，并且通常伴随着体外蛋白质聚集体的形成。我们假设，在神经元isoAsp网站的修复效率低下，显着有助于发生在老年和某些脑部疾病的神经退行性变。我们最近的研究结果还表明，isoAsp的形成可能是一种新形式的蛋白质聚集，涉及共价交联的基础。我们建议通过以下四个具体目标探讨这些想法。目的1将比较PIMT -/-小鼠与野生型同窝出生小鼠的脑提取物，以观察这种关键修复酶的完全丧失是否导致（a）突触核蛋白和tau中的isoAsp积累，这两种充分研究的蛋白质涉及几种形式的神经变性，已被其他人报道为对isoAsp形成高度敏感，（B）突触核蛋白tau的聚集，和胰蛋白酶应答介体蛋白2（CRMP 2），和（c）一般蛋白质的过度磷酸化，以及tau和CRMP 2的位点特异性过度磷酸化。目的2将比较PIMT小鼠（其表达50-55%的正常PIMT活性）与野生型同窝仔，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所示）作为年龄的函数。目的3源于我们最近的发现，即重组小鼠CRMP 2在pH7.4和37 ℃下体外老化时，伴随着SDS不溶性聚集体的形成而经历isoAsp形成。我们将进行一系列的研究，以确定是否，因为我们怀疑从最近的数据，这种聚集涉及共价交联，是机械相关的isoAsp的形成。目的4将比较PIMT -/-小鼠与野生型同窝仔，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（脑特异性亚型）和70 kDa热休克同源蛋白（HSC 70），它们是我们发现的在PIMT -/-小鼠脑中积累高水平isoAsp的22种蛋白质之一。如果指导这4个目标的假设被证明是正确的，这将需要药理学或遗传干预，可以促进PIMT修复系统，以帮助避免老年人认知功能和神经状态的下降。公共卫生相关性：异常的蛋白质结构在发生于老年的人类神经系统的退化中以及在诸如阿尔茨海默病、帕金森病和肌萎缩侧索硬化（ALS）的疾病中起主要作用。这项研究将探索年龄和疾病相关的认知能力下降的严重程度和时间进程受到状态的显著影响的可能性，这是修复神经细胞中受损蛋白质的关键代谢途径。这项研究的结果应该有助于阐明痴呆症的分子基础，从而为缓解或延迟其发作的新疗法铺平道路。

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.