Molly Magarotto & Dr Anita Ho - Chen Lab

Molly Magarotto

TITLE: Transcriptomic profiling of familial ALS/FTD-associated RNA-binding deficient TDP-43 mutants reveals dysregulation of neuronal and synaptic genes in vitro

ABSTRACT:
TDP-43 is an RNA-binding protein constituting the pathological inclusions observed in ~95% of ALS and ~50% of FTD patients (Neumann et al., 2006). TDP-43 has vital roles in RNA metabolism and regulatory processes. TDP-43 pathology is predominantly characterised by its mislocalisation to the cytoplasm where it forms insoluble, hyperphosphorylated and ubiquitinated aggregates that enhance cytotoxicity and contribute to neurodegeneration. Despite its primary role as an RNA/DNA-binding protein, how RNA-binding deficiencies contribute to disease onset and progression are little understood. To date, only two ALS/FTD-associated mutations cause RNA-binding deficiency, K181E and K263E (Chen et al., 2019; Kovacs et al., 2009). In this study, we have used CRISPR/Cas9 to knock-in these mutations to SH-SY5Y cells, generating both homozygous and heterozygous versions of the genotypes that were confirmed as RNA-binding deficient. Using RNA-sequencing on the undifferentiated forms of these cells, we identified transcriptomic changes between K181E homozygous and heterozygous and the wild-type control. Interestingly, genes that were down-regulated in the homozygous were up-regulated in the heterozygous, and vice versa. The genes identified were significantly associated with neuronal and synaptic pathways. As SH-SY5Y cells can differentiate into a more neuronal phenotype, we investigated the consequence of these mutations on their ability to undergo this process. As expected, the K181E homozygous and K263E cells had a deficiency in their ability to differentiate and grow neuronal processes that correlated to the down-regulation of the identified neuronal genes. Conversely, K181E heterozygous saw an up-regulation in these same genes, and as such, were primed for differentiation.


Dr Anita Ho

Title: Identification of novel small molecule chaperone activators for neurodegenerative disease treatment
Abstract: A pathological hallmark of neurodegenerative disease is the accumulation of aberrant protein aggregates which contribute to the cytotoxicity and are therefore a target for new therapy development. One key machinery to manage cellular protein homeostasis is the chaperones proteins, the heat shock proteins (HSPs) which are known to target aberrant proteins including TDP-43, tau and amyloid and rescue neurodegeneration in various disease models. As an attempt to target HSP activation for neurodegeneration therapy, we develop a drug screening assay to search compounds that activate the master regulator of HSPs, the transcription factor heat shock factor 1 (HSF1). As HSF1 is bound by HSP90 which prevents its activation, we develop a NanoBRET assay, which allows us to monitor and quantify the HSF1-HSP90 interaction in living cells to screen for compounds disrupting this interaction and thereby releasing HSF1 for activation. After the assay was optimised and validated for its robustness and reliability, a two thousand compound library was screened which produced 10 hits including a couple of known HSP90 inhibitors. A follow-up functional study showed that one of the hit oxyphenbutazone (OPB) significantly reduces the accumulation of insoluble TDP-43 in a cell model, displaying no signs of stress or toxicity. This study demonstrates a viable strategy for new drug discovery in targeting aberrant proteins and identifies potential candidates for translation into neurodegenerative disease treatment.