Elucidating molecular mechanisms of lysosomal dysfunction underlying progranulin deficiency

项目总结 原颗粒蛋白(PGRN)由grn基因编码，是一种分泌型多效性蛋白，参与多种过程 包括炎症、细胞外信号和神经元存活。杂合性、功能丧失的GRN 突变会导致神经退行性疾病额颞叶痴呆(FTD)。GRN中的遗传变异 循环中PGRN水平的降低也会增加患阿尔茨海默病或帕金森氏症的风险 疾病。这些基因发现证明了PGRN对神经元健康很重要，但尚不清楚。 为什么PGRN的缺失会导致神经退变。有趣的是，最近发现了纯合的GRN突变 导致神经元蜡样脂褐素沉着症(CLN11)。这组溶酶体储存障碍(LSD)表现为 多个组织中的神经变性、认知退化和脂褐素沉积症。发现这一点 GRN纯合子突变导致LSD结合FTD-GRN患者共享CLN11的观察 病理特征，强烈提示溶酶体功能障碍可能是这两种疾病的基础。这项提议将 有助于阐明原颗粒在溶酶体内神经退行性变潜在机制中的分子作用。 PGRN运输到溶酶体，在那里它被加工成亚单位颗粒。有人建议， 颗粒蛋白具有神经毒性和促炎作用，但库卡实验室和其他机构最近的研究表明， 颗粒蛋白在溶酶体中起着稳态作用。数据显示颗粒在溶酶体中是稳定的 与全长PGRN相比，人FTD-GRN细胞和脑组织中的颗粒减少。 此外，PGRN和颗粒蛋白的完全丧失会导致神经酰胺在大脑中积累。这些 数据表明，颗粒可能是参与脂肪降解的溶酶体功能的生物活性成分， 然而，个体颗粒改善体内PGRN缺乏症表型的能力尚不清楚。目标1将 评估单个颗粒的表达是否足以挽救失调的溶酶体功能 Grn-/-小鼠的炎症反应。原颗粒蛋白与炎症反应有关，高度依赖于 在小胶质细胞中表达，小胶质细胞是大脑的常驻免疫细胞。PGRN参与小胶质细胞的激活、吞噬、 迁移和突触修剪。此外，PGRN的丢失已被证明是导致早期和选择性的 小鼠小胶质细胞溶酶体组织蛋白成熟的损伤和脂滴的积累 提示小胶质细胞溶酶体可能特别容易受到PGRN丢失的影响。动态平衡的小胶质细胞 功能对大脑健康至关重要，然而PGRN缺乏对人类小胶质细胞的影响尚不清楚。目标 2将评估PGRN缺乏对人类小胶质细胞的功能影响，利用一个新的患者来源 由库卡实验室开发的ipsc品系和等基因对照。这些实验将是第一个直接评估 颗粒对PGRN缺乏系统的分子和病理表型的作用。评估 PGRN和颗粒蛋白的生物活性将阐明与疾病相关的分子途径，并导致 发展有效和精确的疗法来治疗由PGRN/颗粒蛋白缺乏引起的疾病。

PROJECT SUMMARY Progranulin (PGRN) encoded by the gene GRN is a secreted pleiotropic protein implicated in several processes including inflammation, extracellular signaling, and neuronal survival. Heterozygous, loss of function GRN mutations cause the neurodegenerative disorder frontotemporal dementia (FTD). Genetic variants in GRN that decrease circulating levels of PGRN also increase the risk of developing Alzheimer’s disease or Parkinson’s disease. These genetic discoveries demonstrate that PGRN is important for neuronal health, but it is unclear why the loss of PGRN leads to neurodegeneration. Interestingly, recently identified homozygous GRN mutations cause neuronal ceroid lipofuscinosis (CLN11). This group of lysosomal storage disorders (LSD) presents with neurodegeneration, cognitive deterioration, and lipofuscinosis in multiple tissues. The discovery that that homozygous GRN mutations cause an LSD combined with the observation that FTD-GRN patients share CLN11 pathological features, strongly suggests lysosomal dysfunction may underlie both disorders. This proposal will help elucidate the molecular role of Progranulin in the lysosome underlying mechanisms of neurodegeneration. PGRN traffics to the lysosome where it is processed into subunit granulins. It has been suggested that granulins are neurotoxic and pro-inflammatory, but recent work from the Kukar lab and others indicates that granulins play a homeostatic role in the lysosome. Data shows that granulins are stable in the lysosome compared to full length PGRN, and that granulins are decreased in human FTD-GRN cells and brain tissue. Furthermore, the complete loss of PGRN and granulins leads to accumulation of ceramides in the brain. These data suggest that granulins may be a bioactive component of lysosomal function involved in lipid degradation, yet the ability of individual granulins to ameliorate phenotypes of PGRN deficiency in vivo in unknown. Aim 1 will assess whether the expression of individual granulins is sufficient to rescue dysregulated lysosome function, and inflammation in Grn-/- mice. Progranulin has been implicated in the inflammatory response and is highly expressed in microglia, the brain’s resident immune cells. PGRN is involved in microglia activation, phagocytosis, migration, and synapse pruning. Moreover, the loss of PGRN has been shown to cause early and selective impairments in the maturation of lysosomal cathepsins, and accumulation of lipid droplets in murine microglia suggesting that microglial lysosomes may be particularly vulnerable to the loss of PGRN. Homeostatic microglial function is critical for brain health, however the impact of PGRN deficiency on human microglia is unknown. Aim 2 will assess the functional effects of PGRN deficiency on human microglia leveraging a novel patient derived iPSC line and isogenic controls developed by the Kukar lab. These experiments will be the first to directly assess the role of granulins on the molecular, and pathological, phenotypes of PGRN-deficient systems. Evaluating the bioactivity of PGRN and granulins will elucidate disease-relevant molecular pathways, and lead to the development of effective and precise therapeutics to treat diseases caused by PGRN/granulin deficiency.