Translation dysregulation in neurodegeneration

Amyotrophic lateral sclerosis (ALS) and fronto-temporal dementia (FTD) comprise a spectrum disorder characterized by progressive neuronal degeneration. A pathological hallmark of ALS/FTD is the presence of cytoplasmic aggregates containing the RNA binding protein TDP-43 (97% of ALS and 45% of FTD cases). To elucidate the molecular underpinnings of ALS/FTD, we developed Drosophila models of TDP-43 proteinopathy based on overexpression of human TDP-43 in motor neurons (ALS) or mushroom body (MB) neurons (FTD). These models exhibit remarkable similarities to the human disease, including cytoplasmic aggregates (ALS/FTD), neuromuscular deficits accompanied by locomotor dysfunction and reduced lifespan (ALS) as well as memory and sleep deficits (FTD). Using ALS fly models we identified phenotypes that were subsequently validated in patient derived iPSC motor neurons (iPSC MNs) or spinal cords, including alterations in the microtubule associated protein Futsch/MAP1B and the synaptic vesicle chaperone Hsc70-4/HSPA8. These findings support the ribostasis hypothesis, which posits that in disease, TDP-43 mis-localizes to the cytoplasm where it sequesters RNAs rendering them unavailable to ribosomes for translation. To further probe this hypothesis we used a combination of RNA ImmunoPrecipitations (RIP) and Tagged Ribosome Affinity Purifications (TRAP) in fly models of TDP-43 proteinopathy. Functional annotation tools highlight multiple targets and pathways with altered ribostasis in TDP-43 proteinopathy, including dally-like protein (Dlp/GPC6), a regulator of trans-synaptic signaling and plasticity at the neuromuscular junction (NMJ) and a risk factor for Alzheimer’s Disease. We found that Dlp/GPC6 is reduced at the fly NMJ but accumulates in the ventral nerve cord in flies, and ALS patient iPSC MNs and spinal cords. Interestingly, Dlp is significantly reduced in Drosophila MBs in an age dependent manner. Taken together, our published work and these findings led us to hypothesize that TDP- 43 toxicity in neurons is caused in part by translation dysregulation of specific mRNA targets including Dlp/GPC6. To test this hypothesis we will use genetic and molecular approaches in flies to elucidate the mechanism by which Dlp/GPC6 contributes to TDP-43 dependent, neuronal and synaptic dysfunction in ALS. These findings will be validated in patient derived iPSC MNs, NMJs and tissues (Aim 1). We will also establish the contribution of Dlp/GPC6 to FTD using flies and patient derived cortical neurons (CNs), organoids and tissues (Aim 2). Finally, we will use Non-Canonical Aminoacid Tagging (NCAT) in vivo, in flies to identify specific, TDP- 43 dependent changes in new protein synthesis that will be validated in iPSC NMJs and organoids. These studies in ALS and FTD relevant models are expected to fill a critical gap of knowledge, on TDP-43’s role in translation, to mechanistically determine the basis of axonal and synaptic dysfunction in TDP-43 proteinopathy, to provide an opportunity for probing neuronal specific vulnerabilities and may uncover novel molecular targets and pathways with therapeutic potential for ALS/FTD.