Lysosomal Function of Progranulin and Neurodegeneration

颗粒蛋白前体的溶酶体功能与神经退行性变 颗粒蛋白（GRN）基因突变导致颗粒蛋白前体（PGRN）蛋白单倍不足，这是一种遗传学上的突变。 额颞叶变性（FTLD）的主要原因。PGRN由7.5个颗粒蛋白重复序列组成， 在溶酶体中加工成单个颗粒蛋白肽。PGRN和颗粒蛋白肽对于适当的 溶酶体活动和调节许多小胶质细胞介导的功能。然而，颗粒蛋白肽如何发挥作用， 在溶酶体中的作用仍然未知。使用我们新产生的针对单个颗粒蛋白的抗体，我们 显示单个颗粒蛋白肽的水平可以被差异调节。一种颗粒蛋白肽，颗粒蛋白 E与CD 68相互作用，CD 68是一种在小胶质细胞中高度表达的溶酶体膜蛋白。我们发现 CD 68特异性影响颗粒蛋白E的水平，但没有其他颗粒蛋白和PGRN缺乏导致 CD 68水平和分子量的变化，表明颗粒蛋白E和CD 68相互调节， 溶酶体内的稳态。我们还发现PGRN与α-N- 乙酰氨基半乳糖苷酶（那牙），一种溶酶体糖苷酶，通过颗粒蛋白F结构域，并调节那牙 活动基于这些初步数据，我们假设单个颗粒蛋白肽具有独特的 通过与特定的溶酶体蛋白质结合而在溶酶体中发挥性质和功能。三个具体目标 提出进一步剖析颗粒蛋白肽在溶酶体中的性质和功能。在AIM 1中，我们 将通过测量颗粒蛋白肽的水平和半- 单个颗粒蛋白与全长PGRN在神经元与小胶质细胞中的寿命，这两种主要的细胞类型表达 PGRN在大脑中将在老化过程中测定单个颗粒蛋白水平的变化， 神经变性将阐明CD 68调节颗粒蛋白E水平的机制。在 目的2，我们将剖析单个颗粒蛋白在溶酶体中的功能。颗粒蛋白 将分别研究肽E和F调节CD 68和那牙功能。的结合配偶体 其它颗粒蛋白肽将通过蛋白质组学筛选鉴定并表征。在目标3中，我们 研究颗粒蛋白在维持溶酶体膜完整性和转录程序中的作用 参与PGRN缺失后的小胶质细胞活化，因为我们发现PGRN缺乏导致受损的 溶酶体膜的完整性和溶酶体蛋白和炎症标志物的表达增强 小胶质细胞这些研究将有助于阐明颗粒蛋白肽在机体中的生理功能。 溶酶体和溶酶体功能障碍和小胶质细胞活化之间的联系。我们的工作也将 促进FTLD和其他破坏性神经退行性疾病的治疗开发。

Lysosomal function of progranulin and neurodegeneration Mutations in the granulin (GRN) gene, resulting in haploinsufficiency of the progranulin (PGRN) protein, are a main cause of frontotemporal lobar degeneration (FTLD). PGRN is comprised of 7.5 granulin repeats and processed into individual granulin peptides in the lysosome. PGRN and granulin peptides are critical for proper lysosome activities and regulate many microglia mediated functions. However, how granulin peptides function in the lysosome is still unknown. Using our newly generated antibodies against individual granulins, we have shown that levels of individual granulin peptides can be differentially regulated. One granulin peptide, granulin E, interacts with CD68, a lysosomal membrane protein highly expressed in microglia. We have found that loss of CD68 specifically affects the levels of granulin E but no other granulins and PGRN deficiency leads to changes in CD68 levels and molecular weight, indicating that granulin E and CD68 regulate each other’s homeostasis in the lysosome. We have also discovered that PGRN interacts with alpha-N- acetylgalactosaminidase (NAGA), a lysosomal glycosidase, via the granulin F domain, and regulates NAGA activities. Based on these preliminary data, we hypothesize that individual granulin peptides have unique properties and functions in the lysosome through binding to specific lysosome protein(s). Three specific aims are proposed to further dissect the properties and function of granulin peptides in the lysosome. In Aim1, we will determine the processing and stability of individual granulin peptides, by measuring the levels and half- lives of individual granulins vs full length PGRN in neurons vs microglia, the two main cell types that express PGRN in the brain. Changes in the levels of individual granulins will be assayed during aging and neurodegeneration. The mechanism by which CD68 regulates the levels of granulin E will be elucidated. In Aim2, we will dissect the functions of individual granulins in the lysosome. The mechanisms by which granulin peptides E and F regulate CD68 and NAGA functions will be investigated, respectively. Binding partners for other granulin peptides will be identified through proteomic screens and characterized. In Aim 3, we will investigate the role of granulins in maintaining lysosomal membrane integrity and the transcriptional program involved in microglial activation upon PGRN loss, since we have found that PGRN deficiency leads to impaired lysosomal membrane integrity and enhanced expression of lysosomal proteins and inflammation markers in microglia. The proposed studies will shed light on the physiological functions of granulin peptides in the lysosome and the connection between lysosomal dysfunction and microglial activation. Our work will also facilitate therapeutic development for FTLD and other devastating neurodegenerative diseases.