In vivo characterization of the molecular drivers of biomolecular condensate formation in TDP-43 neuropathology

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Project Summary/Abstract (30 lines of text) Amyotrophic lateral sclerosis (ALS) is a motor neuron disease (MND) characterized by specific degeneration of upper and/or lower motor neurons. The key neuropathology, insoluble aggregates of a protein called TDP-43, is evident in almost all (~97%) ALS patients. TDP-43 aggregates are also present in several other neurodegenerative diseases, including Alzheimer’s disease (AD), dementias (FTLD), and limbic predominant age-related TDP-43 encephalopathy (LATE; found in about 25% of individuals over the age of 80). TDP-43 is a ubiquitous and predominately nuclear protein that is encoded by the TARDBP gene and mutations are a cause of familial ALS, in rare cases of ALS-FTLD or FTLD. TDP-43 is an RNA binding protein that regulates itself and thousands of mRNA transcripts via the modulation of numerous cellular processes (e.g. splicing, RNA transport, translation and more). TDP-43 aggregation is likely to serve as a convergence point during pathogenesis, despite potentially different etiologies and upstream mechanisms. However, identifying the origins and mechanisms that contribute to protein aggregation associated with neurodegeneration proves to be a major challenge in the field (Goedert and Spillantini 2006). Thus, the overarching aim of this study is to better understand the cellular and molecular mechanisms that drive biomolecular condensate (BMC) formation in a living cell, in real time, using our zebrafish model system. We will investigate fundamental molecular processes that regulate TDP-43 phase separation and therefore influence the cellular homeostasis that is affected in neurodegenerative diseases. Determining the processes that drive BMC formation, protein deficiencies, and pathology are imperative for the development of novel therapeutic avenues. In vitro evidence indicates that TDP-43 undergoes fluid de-mixing (liquid-liquid phase separation, LLPS) into BMCs to regulate its physiological levels and localization within a neuron (Tziortzouda, Van Den Bosch et al. 2021). However, in vivo evidence of this process is still lacking. In this proposal, we provide preliminary evidence of BMC characterization in the spinal cord of living zebrafish. We propose to further characterize this process in our fish using a suite of advanced molecular imaging tools, including Fluorescence Recovery After photobleaching (FRAP), Single Molecule Tracking (SMT), and optogenetics in combination with super-resolution microscopy approaches (dSTORM). We will assess how posttranslational modifications (PTMs) and single amino acid substitutions can affect BMC properties and lead to pathological mislocalization of TDP-43 in vivo (Buratti 2018). Taken together, this proposal will significantly accelerate the possibilities to monitor BMC formation in vivo by implementing novel technologies in our established zebrafish platform, and provide much needed insight on how BMC formation can affect a key pathology in a spectrum of neurodegenerative diseases.