Uncovering the mechanism of small molecule RAA-47 in ALS

Kulkarni, Yash

Uncovering the mechanism of small molecule RAA-47 in ALS

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Author:: Kulkarni, Yash
Graduate Program:: Neuroscience
Degree:: Master of Science
Document Type:: Master Thesis
Date of Defense:: June 20, 2024
Committee Members:: Daniela Zarnescu, Thesis Advisor/Co-Advisor
Anirban Paul, Committee Member
Alistair Barber, Program Head/Chair
Wesley M Raup-Konsavage, Committee Member
Keywords:: ALS
TDP-43
small molecule
Spermidine synthase
stress granule
Drosophila melanogaster
Abstract:: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder with a prevalence rate of 7 cases per 100,000 people worldwide. Cytoplasmic inclusions of hyperphosphorylated and ubiquitinated TAR DNA binding protein (TDP-43) are found in 97% of ALS cases and thus represent a major pathological hallmark of ALS. The current FDA approved therapeutics: Riluzole, Edaravone, and Tofersen focus on mitigating glutamate toxicity, oxidative stress, and reducing mutant SOD1 levels, respectively. Additional known pathologies include mitochondrial dysfunction, glial dysfunction, dysregulated nucleocytoplasmic transport. RAA-47 a small molecule was synthesized by our collaborator, Dr. Jon Njardarson (University of Arizona) was identified in a phenotypic screen for improved TDP-43 associated locomotor and survival phenotypes in Drosophila models of ALS. Here, we used label-free proteomics on Drosophila heads expressing TDP-43 in neurons to identify TDP-43 changes in the neuronal proteome reversed by RAA-47 and gain insights into the mechanism of action for this small molecule. We identified 60 candidates [log2FC>1 or log2FC<-1 and p-value < 0.05] that were significantly altered by RAA-47 in TDP-43 (WT or G298S mutant) expressing flies. Through pathway enrichment analysis we found that RNA processing, metabolic, microtubule and oxidative stress pathways were significantly altered. We chose to focus on Spermidine synthase (SpdS) which was increased by RAA-47 in TDP-43G298S samples (log2FC = 0.26 & p-value=0.031), as a first candidate target because of its previous links to Parkinson’s disease and aging. Western blots experiments showed that SpdS is increased in patient samples, where it may reflect a compensatory mechanism. However, spermidine treatment did not improve survival in Drosophila models of ALS, suggesting that increasing SpdS is likely not the mechanism by which RAA-47 is protective. Furthermore, using SH-SY5Y cell line and two different stressors (sorbitol and arsenite) we examined the effects of JTN-1 and RAA-47 on stress granule (SG) dynamics and TDP-43 mislocalization. These experiments show a downward trend in SG formation in the context of arsenite stress suggesting that RAA-47 could potentially mitigate oxidative stress in the context of TDP-43 proteinopathies.

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