Our long-term goals are to identify the genetic basis for disease traits in the lysosomal storage disorder Niemann-Pick Type C1 (NPC1), to study NPC1 disease pathogenesis, and to develop diagnostic and treatment paradigms for this disease. NPC1 is an autosomal recessive, neurovisceral lipid storage disorder that presents with variable hepatosplenomegaly, vertical supranuclear ophthalmoplegia, progressive ataxia, dystonia, and dementia. Our group has a long-term commitment to studying several aspects of this disease including those involving genetic diagnostic, prognostic and therapeutic approaches. The approaches we develop for assessing and treating NPC1 disease will also be used as a model for other rare human diseases. 1. Prognostics: The emergence of precision medicine brings the potential that individual variations of genomic sequence will allow prognosis of disease progression and direct decisions regarding appropriate therapeutic interventions. Earlier disease prediction is being facilitated by the identification of disease-causing mutations at younger ages, increasing the efficacy of genomic-directed treatment. However in many diseases such as NPC1, there is little concordance between the predicted functional consequences of identified coding mutations and clinically relevant parameters such as the time of onset or the severity of the disease. In these instances, new assays with predictive capacity need to be developed to allow improved care and treatment. Over the last year, we have developed such an assay, whereby we can potentially predict the time of onset of neurological symptoms in NPC1 disease by measuring the severity of lysosomal defects in fibroblasts from NPC1 patients. We utilized fibroblast cell lines from NPC1 patients enrolled in our Natural History Study (N=27;age of disease onset range = neonatal to 39 years) to quantitatively measure abnormal lysosomal accumulations. NPC1 patient fibroblasts exhibited a significantly increased mean fold-change in LysoTracker staining compared to control fibroblasts. Linear regression analyses of NPC1 patient LysoTracker staining indicated this measurement had significant predictive capacity for age of systemic disease onset (p = 0.0002;R2 = 0.42), age-adjusted clinical severity score (p = 0.0004;R2 = 0.4), and age of neurological symptom onset (p <0.0001;R2 = 0.61). This result derived from somatic cells was unexpected, as we and others have shown that the neurodegeneration in NPC1 disease is cell autonomous. This approach may now provide a novel prognostic assay for genetically characterized individuals with NPC1. As more individuals are diagnosed before the onset of neurological symptoms (for example either presenting with splenomegaly or having been identified in newborn genetic screening) the addition of this somatic cell assay could provide great assistance for patients and their families in preparation for future life and in the management of this disease by their physicians. Recently we have utilized this assay to undertake whole genome siRNA screens to identify modifier loci that contribute to NPC1 genetic variation and to identify potential treatment paradigms for testing in vitro and in vivo. We predict that this assay will have broader applications beyond NPC1 disease. Similar quantitative somatic cell alterations may also be prognostic for the >50 additional human lysosomal storage disorders that exhibit cholesterol and lipid accumulation in the lysosome. Furthermore, this novel ability to correlate a non-invasive assay on somatic cells with cell autonomous, CNS-associated neurological symptoms suggests this Lysotracker fibroblast assay could be extended to other neurodegenerative disorders. For example, recent links between Parkinson disease and lysosomal function indicate that assays such as this may be broadly applied to CNS diseases. Our study also provides a foundation for using this system to identify genetic components that contribute to time of onset and severity of neurological disease and to test possible pharmacological treatments. 2. Therapeutic assessments: We have an ongoing commitment to generating new models for assessing therapeutic interventions in mouse and human populations. We recently competed an assessment of the antioxidant N-acetylcysteine (NAC), as oxidative stress has been hypothesized to contribute to the NPC1 disease pathological cascade. To determine if treatments reducing oxidative stress could alleviate NPC1 disease phenotypes, the in vivo effects of the antioxidant NAC on two mouse models for NPC1 disease were studied. NAC was able to partially suppress phenotypes in both antisense-induced (NPC1ASO) and germline (Npc1-/-) knockout genetic mouse models, confirming the presence of an oxidative stress-related mechanism in progression of NPC1 phenotypes and suggesting NAC as a potential molecule for treatment. Gene expression analyses of NAC-treated NPC1ASO mice suggested NAC affects pathways distinct from those initially altered by Npc1 knockdown, data consistent with NAC achieving partial disease phenotype suppression. We also assessed pre-clinical findings in patient populations through a therapeutic trial of short-term NAC administration to NPC1 patients, but no significant effects on oxidative stress in these patients were identified other than moderate improvement of the fraction of reduced CoQ10, suggesting limited efficacy of NAC monotherapy. However, the mouse model data suggest that the distinct antioxidant effects of NAC could provide potential treatment of NPC1 disease, possibly in concert with other therapeutic molecules at earlier stages of disease progression. Our work also validated the NPC1ASO mouse as an efficient model for candidate NPC1 drug screening, and demonstrated similarities in hepatic phenotypes and genome-wide transcript expression patterns between the NPC1ASO and Npc1-/- models. We continue to assess other potential therapeutic paradigms in mouse and human populations.

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