Abstract
BackgroundNiemann-Pick disease Type C1 (NPC1) is a rare progressive neurodegenerative disorder caused by mutations in the NPC1 gene. The pathological mechanisms, underlying NPC1 are not yet completely understood. Especially the contribution of glial cells and gliosis to the progression of NPC1, are controversially discussed. As an analysis of affected cells is unfeasible in NPC1-patients, we recently developed an in vitro model system, based on cells derived from NPC1-patient specific iPSCs. Here, we asked if this model system recapitulates gliosis, observed in non-human model systems and NPC1 patient post mortem biopsies. We determined the amount of reactive astrocytes and the regulation of the intermediate filaments GFAP and vimentin, all indicating gliosis. Furthermore, we were interested in the assembly and phosphorylation of these intermediate filaments and finally the impact of the activation of protein kinase C (PKC), which is described to ameliorate the pathogenic phenotype of NPC1-deficient fibroblasts, including hypo-phosphorylation of vimentin and cholesterol accumulation.MethodsWe analysed glial cells derived from NPC1 patient specific induced pluripotent stem cells, carrying different NPC1 mutations. The amount of reactive astrocytes was determined by means of immuncytochemical stainings and FACS-analysis. Semi-quantitative western blot was used to determine the amount of phosphorylated GFAP and vimentin. Cholesterol accumulation was analysed by Filipin staining and quantified by Amplex Red Assay. U18666A was used to induce NPC1 phenotype in unaffected cells of the control cell line. Phorbol 12-myristate 13-acetate (PMA) was used to activate PKC.ResultsImmunocytochemical detection of GFAP, vimentin and Ki67 revealed that NPC1 mutant glial cells undergo gliosis. We found hypo-phosphorylation of the intermediate filaments GFAP and vimentin and alterations in the assembly of these intermediate filaments in NPC1 mutant cells. The application of U18666A induced not only NPC1 phenotypical accumulation of cholesterol, but characteristics of gliosis in glial cells derived from unaffected control cells. The application of phorbol 12-myristate 13-acetate, an activator of protein kinase C resulted in a significantly reduced number of reactive astrocytes and further characteristics of gliosis in NPC1-deficient cells. Furthermore, it triggered a restoration of cholesterol amounts to level of control cells.ConclusionOur data demonstrate that glial cells derived from NPC1-patient specific iPSCs undergo gliosis. The application of U18666A induced comparable characteristics in un-affected control cells, suggesting that gliosis is triggered by hampered function of NPC1 protein. The activation of protein kinase C induced an amelioration of gliosis, as well as a reduction of cholesterol amount. These results provide further support for the line of evidence that gliosis might not be only a secondary reaction to the loss of neurons, but might be a direct consequence of a reduced PKC activity due to the phenotypical cholesterol accumulation observed in NPC1. In addition, our data support the involvement of PKCs in NPC1 disease pathogenesis and suggest that PKCs may be targeted in future efforts to develop therapeutics for NPC1 disease.
Highlights
Niemann-Pick disease Type C1 (NPC1) is a rare progressive neurodegenerative disorder caused by mutations in the NPC1 gene
These results provide further support for the line of evidence that gliosis might not be only a secondary reaction to the loss of neurons, but might be a direct consequence of a reduced protein kinase C (PKC) activity due to the phenotypical cholesterol accumulation observed in NPC1
Rescue from gliosis and NPC1 phenotypical cholesterol accumulation As we have demonstrated an altered assembly of glial fibrillary acidic protein (GFAP) and vimentin, as well as an altered amount of Phosphorylated GFAP (p-GFAP) and p-vimentin in cells carrying a NPC1 mutation and in unaffected control cells treated with U18666A, we asked if the activation of PKC leads to a rescue of the observed gliosis as well as of the NPC1 phenotypical accumulation of cholesterol
Summary
Niemann-Pick disease Type C1 (NPC1) is a rare progressive neurodegenerative disorder caused by mutations in the NPC1 gene. The contribution of glial cells and gliosis to the progression of NPC1, are controversially discussed. Niemann-Pick type C1 is a rare lysosomal storage disorder with an incidence of 1.12:100.000 live births [1] The phenotype of this lipidosis exhibit various symptoms ranging from hepatosplenomegaly, motor dysfunctions, cerebellar ataxia and seizures to dementia, whereby the age of onset varies from early infantile to adult onset forms. In regards of the neurological symptoms, like cerebellar ataxia, based on a progressive loss of cerebellar Purkinje cells, the pathological mechanism is not yet understood. Gliosis is a certainly proved pathological feature of NPC1 the role of glial cells in the progression of the disease is controversially discussed
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