Protein Damage and Repair in the Brain

描述(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疾病中的潜在作用。异天冬氨酸位点的积累是蛋白质损伤的一种主要形式，通常由蛋白质L-异天冬氨酸甲基转移酶(PIMT)控制，这是一种在大脑中高度丰富的修复酶。PIMT基因敲除小鼠积累高水平的isAsp损伤蛋白，尤其是在脑和睾丸中，其表型主要是神经系统；出生后4-10周，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，以及致命性癫痫。IsoAsp的形成可以扰乱蛋白质的功能，可以诱导自身免疫，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isAsp位点的修复效率低下是导致老年和某些脑部疾病的神经退行性变的重要原因。我们最近的发现还表明，等天冬氨酸的形成可能是一种新的蛋白质聚集形式的基础，这种聚集涉及共价交联。我们建议通过以下四个具体目标来探索这些想法。目的1比较PIMT-/-小鼠和野生型仔鼠的脑提取物，以了解这种关键修复酶的完全丧失是否导致(A)在突触核蛋白和tau中的isAsp积聚，这两种蛋白质参与了几种形式的神经退行性变，据报道，这些蛋白质对isAsp的形成高度敏感，(B)突触核蛋白、tau和崩塌反应介质蛋白2(CRMP2)的聚集，以及(C)蛋白质的普遍过度磷酸化，以及tau和CRMP2的位置特异性过度磷酸化。目的2将PIMT小鼠(表达正常PIMT活性的50%-55%)与野生型小鼠进行比较，以了解体内PIMT活性的适度降低如何改变随着年龄的变化而产生的isAsp积累、蛋白质聚集和蛋白质过度磷酸化(如目标1所示)。AIMS 3源于我们最近的发现，重组小鼠CRMP2在pH 7.4和37℃的体外老化时，伴随着不溶于十二烷基硫酸钠的聚集体的形成而形成等天冬氨酸。我们将进行一系列研究，以确定是否如我们从最近的数据所怀疑的那样，这种聚集涉及共价交联，并与isAsp的形成有机械上的联系。目的4将PIMT-/-小鼠与野生型小鼠进行比较，以寻找体内积累isAsp的神经元酶的功能变化。我们首先将重点放在肌酸激酶B(脑特异性异构体)和70 kDa热休克同源蛋白(HSC70)，这是我们在PIMT-/-小鼠大脑中发现的22种蛋白质中积累了高水平的isAsp。如果指导这四个目标的假设被证明是正确的，这将需要药物或遗传干预，以促进PIMT修复系统，以帮助延缓人类认知功能和神经状态的下降，这种情况发生在老年。公共卫生相关性：蛋白质结构异常在人类神经系统退行性变中起主要作用，这种退行性变发生在老年以及阿尔茨海默病、帕金森氏病和肌萎缩侧索硬化症(ALS)等疾病中。这项研究将探索年龄和疾病相关认知下降的严重程度和时间进程是否受到状态的显着影响，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，从而为缓解或推迟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.