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.