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

Abstract

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.

A blood-brain barrier-penetrant AAV gene therapy improves neurological function in symptomatic mucolipidosis IV mice

“…We have recently reported broad upregulation of lysosomal proteins as a hallmark of pathological molecular changes in the brain of Mcoln1 −/− mice. 29 A similar increase of lysosomal proteins was also reported in the single MLIV brain autopsy proteome, indicating the conservative nature of this phenomenon across species. 40 Additionally, the upregulation of lysosomal proteins was commonly reported in other lysosomal storage disorders, such as NPC, Gaucher, mucopolysaccharidoses, and others, and is thought to represent a compensatory mechanism for impaired lysosomal function.…”

“…Another major brain pathology feature in MLIV discovered by the proteomics data in the present and our previous studies was broad downregulation of the protein signature related to oligodendroglial cell lineage and myelination. 29 It is noteworthy that early intervention via ICV administration of AAV9-MCOLN1 in neonatal Mcoln1 −/− in our previous work resulted in an improvement of myelination in young adult Mcoln1 −/− mice at 2 months. However, CPP16-mediated MCOLN1 gene transfer in young adult symptomatic mice in the present study did not correct myelination, as shown by the lack of correction of myelination/oligodendrocyte signature in the brain proteomics dataset.…”

“…An increased abundance of the lysosomal proteins and decreased levels of oligodendrocyte and myelin-related proteins are molecular hallmarks in the brain of symptomatic Mcoln1 −/− mice. 29 To assess if administration of CPP16 - MCOLN1 rescued brain pathology in Mcoln1 −/− mice, we next performed LC-MS/MS proteomics analysis using cortical tissues of 5-month-old female Mcoln1 −/− mice as well as WT control mice. In accordance with previous findings, we observed a remarkably similar broad upregulation of the lysosomal proteins and downregulation of myelination and oligodendrocyte protein signature in the Mcoln1 −/− cortex ( Figure 4 A).…”

A blood-brain-barrier penetrant AAV gene therapy rescues neurological deficits in mucolipidosis IV mice

“…An increased abundance of the lysosomal proteins and decreased levels of oligodendrocyte and myelin related proteins are molecular hallmarks in the brain of symptomatic Mcoln1 -/- mice (29). To assess if administration of CPP16 -MCOLN1 rescued brain pathology in Mcoln1 -/- mice, we next performed LC-MS/MS proteomics analysis using cortical tissues of 5-month-old female Mcoln1 -/- mice as well as wild-type control mice.…”

“…Protein extraction from cerebral cortex, TMT labeling and LC-MS/MS analysis was performed as previously described (29). Raw data were submited for analysis in Proteome Discoverer 3.0.1.23 (Thermo Scientific) software with Chimerys (MSAID, Germany).…”