Investigating contributions of late endosomal and lysosomal chloride/proton antiporter dysfunction to neuronal storage

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Project Summary/Abstract Progressive neurodegenerative disorders such as Alzheimer’s and Parkinson’s are characterized by inappropriate accumulation of proteinaceous matter within neurons, and are accompanied by a constellation of symptoms, including gradual degeneration of movement and dementia. Accumulation of matter in lysosomes, an intracellular compartment that degrades obsolete protein, also characterizes lysosomal storage disorders (LSDs). The CLCN6 and CLCN7 genes encode CLC-6 and CLC-7. CLC-6, and CLC-7 in combination with an accessory subunit, OSTM1 (CLC-7/OSTM1), are proteins that function to transport chloride and protons across membranes defining contiguous intracellular compartments—late endosomes and lysosomes, respectively. Variants causing CLC-6 and CLC-7 loss of function (LoF) associate with accumulation of matter within these compartments, characteristic of LSDs. The LSDs associated with CLC-6 and CLC-7/OSTM1 LoF have distinct differences, in onset as well as disease severity. Interestingly, variants producing CLC-6 and CLC-7/OSTM1 gain of function (GoF) associate with distinct profound neurodegenerative disease. Presently, knowledge regarding their mechanism of function points to general similarities, as well as subtle regulatory differences. Greater understanding about the precise transport mechanism of wild type transporters, as well as both LoF and GoF disease-associated variants, is important for understanding disease etiologies, but also requires additional and deeper functional analysis. To gain this needed insight, Aim 1 combines biophysical, electrophysiological, and computational modeling approaches to extract and compare the detailed biophysical parameters regulating function of wild-type and disease variants of CLC-6 and CLC-7. Aim 2 proposes initially to generate human iPSC- derived CLCN6 gene-edited, differentiated neuronal lines to study early changes in compartmental homeostasis hypothesized to precipitate disease, and aims to monitor progressive dysfunction. Time-permitting, similar development of CLCN7 gene-edited lines can proceed. In summary, measurement, analysis, and detailed modeling of CLC-6 and CLC-7/OSTM1 transport function offer needed insight into functional disruption in disease-associated, transporter variants. Moreover, the proposed work provides unique neuronal cell models for monitoring disease progression at refined resolution. Forthcoming foundational knowledge informs strategies toward treatment of a plethora of devastating neurodegenerative diseases, thereby exerting sustained impact.