Chair of Metabolic Biochemistry, Biomedical Center (BMC), Faculty of Medicine, Ludwig-Maximilians-Universität München, 81377, Munich, Germany.
German Center for Neurodegenerative Diseases (DZNE) Munich, 81377, Munich, Germany.
Mol Neurodegener. 2018 Sep 4;13(1):48. doi: 10.1186/s13024-018-0281-5.
Heterozygous loss-of-function mutations in the progranulin gene (GRN) lead to frontotemporal lobar degeneration (FTLD) while the complete loss of progranulin (PGRN) function results in neuronal ceroid lipofuscinosis (NCL), a lysosomal storage disease. Thus the growth factor-like protein PGRN may play an important role in lysosomal degradation. In line with a potential lysosomal function, PGRN is partially localized and processed in lysosomes. In the central nervous system (CNS), PGRN is like other lysosomal proteins highly expressed in microglia, further supporting an important role in protein degradation. We have previously reported that cathepsin (Cat) D is elevated in GRN-associated FTLD patients and Grn knockout mice. However, the primary mechanism that causes impaired protein degradation and elevated CatD levels upon PGRN deficiency in NCL and FTLD remains unclear.
mRNA expression analysis of selected lysosomal hydrolases, lysosomal membrane proteins and autophagy-related genes was performed by NanoString nCounter panel. Protein expression, maturation and in vitro activity of Cat D, B and L in mouse embryonic fibroblasts (MEF) and brains of Grn knockout mice were investigated. To selectively characterize microglial and non-microglial brain cells, an acutely isolated microglia fraction using MACS microbeads (Miltenyi Biotec) conjugated with CD11b antibody and a microglia-depleted fraction were analyzed for protein expression and maturation of selected cathepsins.
We demonstrate that loss of PGRN results in enhanced expression, maturation and in vitro activity of Cat D, B and L in mouse embryonic fibroblasts and brain extracts of aged Grn knockout mice. Consistent with an overall enhanced expression and activity of lysosomal proteases in brain of Grn knockout mice, we observed an age-dependent transcriptional upregulation of certain lysosomal proteases. Thus, lysosomal dysfunction is not reflected by transcriptional downregulation of lysosomal proteases but rather by the upregulation of certain lysosomal proteases in an age-dependent manner. Surprisingly, cell specific analyses identified early lysosomal deficits in microglia before enhanced cathepsin levels could be detected in other brain cells, suggesting different functional consequences on lysosomal homeostasis in microglia and other brain cells upon lack of PGRN.
The present study uncovers early and selective lysosomal dysfunctions in Grn knockout microglia/macrophages. Dysregulated lysosomal homeostasis in microglia might trigger compensatory lysosomal changes in other brain cells.
颗粒体蛋白基因（GRN）杂合功能丧失突变导致额颞叶痴呆（FTLD），而颗粒体蛋白（PGRN）功能完全丧失则导致神经元蜡样脂褐质沉积症（NCL），即溶酶体贮积症。因此，生长因子样蛋白 PGRN 可能在溶酶体降解中发挥重要作用。与潜在的溶酶体功能一致，PGRN 部分定位于溶酶体并在溶酶体中加工。在中枢神经系统（CNS）中，PGRN 与其他溶酶体蛋白一样在小胶质细胞中高表达，进一步支持其在蛋白质降解中的重要作用。我们之前报道过，在 GRN 相关 FTLD 患者和 Grn 基因敲除小鼠中，组织蛋白酶 D（CatD）升高。然而，在 NCL 和 FTLD 中，由于 PGRN 缺乏导致蛋白质降解受损和 CatD 水平升高的主要机制仍不清楚。
通过 NanoString nCounter 面板对选定的溶酶体水解酶、溶酶体膜蛋白和自噬相关基因的 mRNA 表达进行分析。在鼠胚胎成纤维细胞（MEF）和 Grn 基因敲除小鼠的大脑中研究组织蛋白酶 D、B 和 L 的蛋白表达、成熟和体外活性。为了选择性地描述小胶质细胞和非小胶质细胞，使用与 CD11b 抗体偶联的 MACS 微珠（Miltenyi Biotec）从急性分离的小胶质细胞级分中分离并分析了选定的组织蛋白酶的蛋白表达和成熟。
我们证明 PGRN 缺失导致鼠胚胎成纤维细胞和年老 Grn 基因敲除小鼠大脑提取物中 Cat D、B 和 L 的表达、成熟和体外活性增强。与 Grn 基因敲除小鼠大脑中溶酶体蛋白酶整体表达和活性增强一致，我们观察到某些溶酶体蛋白酶的转录上调呈年龄依赖性。因此，溶酶体功能障碍不是通过溶酶体蛋白酶的转录下调来反映，而是通过某些溶酶体蛋白酶的年龄依赖性上调来反映。令人惊讶的是，细胞特异性分析发现，在其他脑细胞中可以检测到增强的组织蛋白酶水平之前，Grn 基因敲除小鼠的小胶质细胞中存在早期溶酶体缺陷，这表明在缺乏 PGRN 的情况下，小胶质细胞和其他脑细胞中的溶酶体稳态存在不同的功能后果。
本研究揭示了 Grn 基因敲除小胶质细胞/巨噬细胞中的早期和选择性溶酶体功能障碍。小胶质细胞中溶酶体动态平衡的失调可能触发其他脑细胞中的代偿性溶酶体变化。
