FUS/TLS mutations disrupt axonal transport, synaptic development, and synaptic function: a screen for genetic modifiers. James B. Machamer¹, Thomas Lloyd^1,2. 1) Dept of Neurology, JHMI, Baltimore, MD; 2) Dept of Neuroscience, JHMI, Baltimore, MD.

   FUS is an RNA binding protein that has been implicated in the pathogenesis of both familial and sporadic Amyotrophic Lateral Sclerosis (ALS). FUS regulates RNA metabolism in both the nucleus and in the cytoplasm, and the majority of ALS-causing mutations lie within the nuclear localization sequence (NLS) of FUS. These mutations result in the formation of cytoplasmic aggregates and loss of protein function in the nucleus. However, it remains unclear whether FUS-mediated ALS is due to a gain of toxic function or a loss of FUS protein, and little is known about the mechanism leading to neuronal dysfunction. In this study, we analyze FUS overexpression in larvae to investigate the earliest changes in motoneuron function. We first investigated the effect of mutant FUS on axonal transport and found decreased processivity of cargo transport. We next measured the level and localization of essential synaptic proteins in the larval neuromuscular junction (NMJ) and find that FUS expression in the motoneurons reduces the number of presynaptic active zones and postsynaptic levels of Discs large (DLG), a scaffolding protein that localizes glutamate receptors to the active zone. Consequently, we find reduced amplitude of synaptic transmission at the NMJ due to decreased quantal content. Interestingly, overexpression of mutant forms of the Drosophila homolog of FUS, Cabeza (Caz) also disrupt normal synaptic transmission, but as a result of reduced quantal size. Finally, we screened for modifiers of FUS-mediated rough eye and motoneuron phenotypes using 2nd chromosome deficiencies followed by RNAi and identified multiple splicesome subunits, suggesting that FUS-mediated alterations in RNA splicing underlie neuronal toxicity. Thus, in this fly model of ALS, we find significant early changes in mutant FUS-expressing motoneurons including disruption of axonal transport, synapse development, and synapse function. Furthermore, the identification of splicesomal proteins as genetic modifiers suggests that these changes may be a consequence of disrupted nuclear RNA processing.