The nervous system is composed of vast neuronal circuits that are highly regulated to ensure proper synaptic transmission. Disruption of protein function at the synapse can impair communication between neurons and in some disease states cause neuronal cell death. In many of these neurodegenerative diseases, axon degeneration occurs prior to neuronal death, and neurodegenerative disease-associated mutations have been found in several endoplasmic reticulum (ER) resident proteins. In particular reticulon, atlastin and receptor accessory protein 1 (REEP1) are implicated in hereditary spastic paraplegia (HSP) and vesicle-associated membrane protein-associated protein-B (VAP-B) is implicated in amyotrophic lateral sclerosis (ALS). These proteins localize to contact sites between the ER and plasma membrane. ER-plasma membrane contact sites are specialized regions thought to be important in calcium signaling and lipid transport. However, not much is known about how contact proteins affect axonal plasma membrane integrity, synaptic function and axon degeneration. Extended Synaptotagmin-2 (ESYT-2) is an ER resident protein found at ER-plasma membrane contact sites. Mammals have three esyt genes whereas worms have a single esyt gene, making worms an excellent model system to characterize the role of this protein. Preliminary data using the loss of function allele of esyt-2 (tm5783) suggest that esyt-2 mutants have nervous system and lifespan defects. Imaging of single axons in esyt-2 mutants reveal accelerated changes in axon morphology that is reminiscent of known mutants of neuro- degeneration. Prior studies in yeast have demonstrated a major role for the yeast esyt-2 homolog (tricalbins) in establishing and maintaining ER-plasma membrane contact sites. Interestingly, expression of worm ESYT-2 in an ER-plasma membrane contact site deficient yeast can rescue contact sites suggesting its function is conserved. We will use Saccharomyces cerevisiae and Caenorhabditis elegans as model systems to examine if ESYT-2 regulates axonal membrane phospholipid and diacylglycerol levels and how dysregulation of these lipids perturbs axon integrity, shortens lifespan and ultimately causes neuro-degeneration.
Several forms of amyotrophic lateral sclerosis (ALS) and hereditary spastic paraplegia (HSP) begin with synapse loss and axon degeneration. Genetic studies of these neuro-degenerative diseases reveal several mutations associated with ER resident proteins that are found particularly at ER-plasma membrane contact sites. Currently, there is no information on why proteins found at these contact sites are important in maintaining neuronal health. This project will be the first in vivo characterization of an ER-plasma membrane contact site protein known as Extended Synaptotagmin-2 in an intact nervous system. Our research plan investigates how loss of ESYT-2 causes axonal lipid dysregulation, synaptic dysfunction and ultimately axon degeneration. Revealing the signaling pathway in which ESYT-2 functions may give insight into a general role for ER-plasma membrane contact sites. The knowledge could provide predictors of disease and development of new drug targets.
