Table_4_Brain cell type specific proteomics approach to discover pathological mechanisms in the childhood CNS disorder mucolipidosis type IV.XLSX

Mucolipidosis IV (MLIV) is an ultra-rare, recessively inherited lysosomal disorder resulting from inactivating mutations in MCOLN1, the gene encoding the lysosomal cation channel TRPML1. The disease primarily affects the central nervous system (CNS) and manifests in the first year with cognitive and motor developmental delay, followed by a gradual decline in neurological function across the second decade of life, blindness, and premature death in third or fourth decades. Brain pathology manifestations in MLIV are consistent with hypomyelinating leukodystrophy with brain iron accumulation. Presently, there are no approved or investigational therapies for MLIV, and pathogenic mechanisms remain largely unknown. The MLIV mouse model, Mcoln1−/− mice, recapitulates all major manifestations of the human disease. Here, to better understand the pathological mechanisms in the MLIV brain, we performed cell type specific LC–MS/MS proteomics analysis in the MLIV mouse model and reconstituted molecular signatures of the disease in either freshly isolated populations of neurons, astrocytes, oligodendrocytes, and neural stem cells, or whole tissue cortical homogenates from young adult symptomatic Mcoln1−/− mice. Our analysis confirmed on the molecular level major histopathological hallmarks of MLIV universally present in Mcoln1−/− tissue and brain cells, such as hypomyelination, lysosomal dysregulation, and impaired metabolism of lipids and polysaccharides. Importantly, pathway analysis in brain cells revealed mitochondria-related alterations in all Mcoln1−/− brain cells, except oligodendrocytes, that was not possible to resolve in whole tissue. We also report unique proteome signatures and dysregulated pathways for each brain cell population used in this study. These data shed new light on cell-intrinsic mechanisms of MLIV and provide new insights for biomarker discovery and validation to advance translational studies for this disease.IV型粘脂贮积症（Mucolipidosis IV, MLIV）是一种超罕见的隐性遗传性溶酶体疾病，由编码溶酶体阳离子通道TRPML1的MCOLN1基因发生失活突变所致。该疾病主要累及中枢神经系统（central nervous system, CNS），于出生后第一年发病，表现为认知与运动发育迟缓；随后在人生第二个十年逐渐出现神经功能衰退、失明，并于第三或第四个十年过早死亡。IV型粘脂贮积症的脑部病理表现与伴脑铁沉积的髓鞘形成低下性脑白质营养不良一致。目前，尚无获批或在研的IV型粘脂贮积症治疗方案，其致病机制仍未完全阐明。MLIV小鼠模型Mcoln1−/−小鼠可复现人类疾病的所有主要表型。为深入阐明IV型粘脂贮积症的脑部致病机制，本研究以该小鼠模型为对象，开展了细胞类型特异性LC-MS/MS蛋白质组学分析，并从年轻成年症状性Mcoln1−/−小鼠中新鲜分离神经元、星形胶质细胞、少突胶质细胞与神经干细胞，或获取完整组织皮层匀浆，重构了该疾病的分子特征。本研究的分析在分子层面证实了Mcoln1−/−小鼠组织与脑细胞中普遍存在的IV型粘脂贮积症主要病理标志，如髓鞘形成低下、溶酶体功能失调、脂质与多糖代谢受损。值得注意的是，对脑细胞进行的通路分析发现，除少突胶质细胞外，Mcoln1−/−小鼠所有脑细胞均存在线粒体相关的分子改变，这一现象在完整组织分析中无法被检测到。本研究还报道了本次实验所用的每一种脑细胞群体独特的蛋白质组特征与失调通路。这些数据为阐明IV型粘脂贮积症的细胞内在致病机制提供了新视角，并为该疾病的生物标志物发现与验证提供了新见解，以推动相关转化研究。