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"Neurodegenerative diseases, including the prevalent Alzheimer’s disease and Parkinson’ disease, virtually always feature inclusion of aggregated proteins within susceptible brain neurons. Propagation of aggregated protein species by cell-to-cell transfer underlies the spread of pathology, the mechanisms that regulate and enact aggregate transfer remain very poorly understood (not unexpectedly, as the mammalian brain is quite a complex and inaccessible tissue). We model neuronal aggregate spreading biology in a simple, 959-celled genetically amenable animal model called C. elegans, in which we can visualize aggregate transfer events from single neurons in vivo.
A former undergraduate in our lab made a major discovery: under proteostress conditions, C. elegans neurons can extrude very large extracellular vesicles that selectively remove aggregated proteins and damaged organelles from neurons. We named these large vesicle extrusions “exophers”, and we published in Nature that the exopher extrusions can help “cure” proteostressed neurons of toxic consequences of aggregate accumulation.
The project we propose will capitalize on the ease of genetic manipulation in C. elegans to identify genetic pathways that regulate and promote exopher production. Our model is well suited to undergraduate experimentation, and involves targeted disruption of specific genes with cell biological observation of the impact of specific genetic disruption on exophers, all within the physiological context of the C. elegans body.
Exopher-like extrusions are seen in humans, including neurodegenerative disease, and we expect that our capacity to define genes that modulate exopher production will be relevant both to disease understanding and to targeting pathological processes in the clinic. As has often been the case, we anticipate that undergraduate researchers will make critical contributions to publishable science.
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In brief on specific sub projects open to undergraduates, with mentors ready and proposing projects for students (students can choose):
1) Pursuing specific genes identified as part of a whole-genome RNAi screen for exopher suppressors. Several genes have been validated; new students can drill down to determine autonomy vs. non-autonomy, subcellular localization, position in genetic pathway, testing of human homologs for similar function.
2) Investigating the potential role of non-canonical secretion autophagy in exopher-genesis. We previously discovered that gene ATG-16.2 is required for exopher formation in a way distinct from other autophagy proteins, and contains a WD40 domain that is required for this unexpected function in specialized secretion. We have now used CRISPR to engineer substitutions in ATG-16.2 that permit only autophagy function or only the non-canonical function to define mechanism and firmly establish how ATG-16.2WD40 might drive a novel activity under high proteostress.
3) Linking mechanically-induced neuronal calcium spikes to exopher initiation. To make a long story short, there is a wave of exophergenesis in early adult life that we hypothesize helps optimize neuronal function for the reproductive phase of life, by clearing cellular debris. In the touch neurons, the pressure of fertilized eggs in the young adult uterus elevates exopher production—this seems to be also associated with transient calcium influx, which we can measure at the single cell level using gCaMP and related reporters. We will combine imaging and genetic manipulations to quantitate and evaluate correlation.
4) Testing the hypothesis that tethering of healthy organelles is critical in exopher cargo sorting. Only select material enters exophers, including aggregates and organelles such as mitochondria and ER. A pressing question is how bad cargo might be distinguished from healthy entities that should be retained in the neuron. Although not our first hypothesis, evidence is now converging to support the hypothesis that healthy and valuable material is tethered to the cytoskeleton and retained, whereas protein aggregates and dysfunctional organelles are not strongly attached to the cytoskeleton. We will test this model by disrupting tethering molecules and cytoskeleton anchors, and tracking distribution of fluorescently tagged exopher contents.
Progress on any one of these fronts will generate novel insights into exopher biology at the same time that answers to the questions will be foundational for the next hypotheses to be tested. "
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