We have discovered a novel viral-like signaling pathway associated with extracellular vesicles (EV). We found the Drosophila homolog of ARC (Actin-Regulated Cytoskeleton-associated protein) (darc1), is present in EVs as both an mRNA and protein. ARC is a master regulator of synaptic plasticity in the nervous system of mammals and is crucial for learning and memory. dArc1 bears a domain resembling retroviral/retrotransposon Gag-like proteins that multimerizes into a capsid that packages viral RNA. Our work shows dARC1 forms a capsid, associates with its own RNA, and then transports the darc1 transcript across the synapse. The transfer of dArc1 is needed for activity-dependent plasticity at the fly neuromuscular junction (NMJ). Besides dArc1, it is unknown whether other genes are in this viral-like pathway. We address this uncertainty in Aim 1. Here we describe our plan to identify other Gag-like proteins in EVs, and we have already found another Gag protein enriched in EVs, that is encoded by the retrotransposon Copia. We have found that Copia transfers across the synaptic bouton. When copia is knocked down at the NMJ this strikingly leads to increased plasticity. This is the opposite of darc1, where we reported a decrease in plasticity.
In Aim 2 we focus on what cargoes are co-transferring with dArc1 and Copia. We have identified through co-immunoprecipitation, mRNAs and proteins that associate with dArc1 and Copia. As to how the transfer of Arc occurs, we have found that the dArc1 3?untranslated region (UTR) is necessary and sufficient for the transfer of dArc1 across synaptic boutons. We are now testing if the dArc1 3?UTR directs the loading of dArc1 into EVs. As well, we propose experiments to understand how darc1 and copia mediate synaptic plasticity. We have co-immunoprecipitated dArc1 and Copia to identify potential interactors, and we will take a candidate approach to find genetic interactors. In preliminary work we found that dArc1 is needed for proper WNT pathway signaling at the NMJ. Additionally, we observe that Copia and dArc1 bind to some of the same proteins and mRNAs, suggesting that they may be antagonistic to each other, thus potentially explaining their seemingly opposite roles in mediating plasticity. Through this grant we will expand our understanding of EV trafficking and synaptic plasticity, while describing a novel physiological function of a retrotransposon in neuronal communication.

Public Health Relevance

Neurons communicate with muscles through a connection known as a neuromuscular junctions (NMJs). If NMJs do not form normally or are damaged this leads to many neuromuscular degenerative diseases. Recently we have discovered that neurons package RNA into a protein shell that travels to the muscle in a fashion that is very similar to a virus moving from one cell to another. Without this viral-like pathway NMJs do not form normally, through this work we hope to learn how NMJs form and come up with therapies for neuromuscular diseases.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Research Project (R01)
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Synapses, Cytoskeleton and Trafficking Study Section (SYN)
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Miller, Daniel L
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University of Massachusetts Medical School Worcester
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United States
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