Saturday, November 23, 2024
11/23/2024
Project Summary/Abstract The human SLC17A5 gene encodes a constituent of the lysosomal cell membrane of neurons, a proton- coupled transporter protein termed Sialin. This solute carrier protein has several defined activities, including the transport of the negatively charged sugars, glucuronic and free sialic acid, out of lysosomes for use in glycoprotein and glycolipid formation. Diseases associated with mutations in SLC17A5 are autosomal recessive neurodegenerative disorders that, depending on the mutation, manifest either as a severe infantile sialic acid storage disorder that leads to lethality in early childhood, or as Salla disease, a milder, slowly progressive adult form characterized by intellectual disability, ataxia, hypomyelination, hypotonia, epilepsy, and neurodegeneration. These are very rare and understudied conditions; it is estimated that there are less than 1,000 individuals with Salla disease in the United States. This R03 proposal is associated with RFA-TR-22- 030 for Pilot Projects to Investigate Understudied Proteins Associated with Rare Diseases. Here, we will model two prominent symptoms associated with Salla disease: epilepsy and neurodegeneration, using the nematode model organism, Caenorhabditis elegans, to circumvent the premature lethality resulting from SLC17A5 deletion in mice. The C. elegans slc-17.2 gene product shares over 55% similarity with human SLC17A5/Sialin and is widely expressed within neurons. In Aim 1, we will examine viable slc-17.2 CRISPR- generated null mutants for epileptic-like convulsions. C. elegans is well-studied for factors that influence neuronal excitability. Using two complimentary experimental paradigms, both involving a chemical-genetic approach to incite hyperexcitability in either cholinergic or GABAergic neurons, slc-17.2 mutants will be evaluated for evidence of convulsive behaviors with and without slc-17.2 expression. To reveal functional modifiers of a convulsive phenotype, we will perform a systematic genetic analysis of 10 conserved candidate targets corresponding to gene products listed as protein-protein interactors with human Sialin in the NIH Pharos database. Existing mutants for each of these candidates will be crossed to slc-17.2 knockout animals to screen for modifiers of convulsion phenotypes. In Aim 2, we will employ a complementary strategy to model the progressive nature of Salla disease by examining C. elegans slc-17.2 mutants for an impact on dopamine neuron degeneration as animals age. As in Aim 1, a systematic genetic analysis of the same 10 candidate interactors of SLC17A5/Sialin will be conducted to uncover modifiers of any SLC17A5 effects on C. elegans dopamine neurons. Candidate effectors will be parsed rigorous quantification of neurodegeneration, at the single neuron level, in isogenic populations, using different mutant backgrounds. This 1-year study will establish a platform for preclinical screening of factors that can correct or compensate for the lysosomal deficits caused by SLC17A5 mutation. Our approach exemplifies how intact invertebrate models can facilitate the ascription of functions to understudied proteins and accelerate mechanistic understanding of rare diseases.
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