The consequences of prenatal alcohol exposure for the immature central nervous system represents a devastating but likely underestimated public health hazard, producing a range of ethanol-induced developmental defects including cognitive impairments having long-term impacts on society. Damage to neurons also includes cells within the retina, producing visual system facets of these fetal alcohol spectrum disorders (FASD). Critical gaps in knowledge remain regarding the specific mechanisms underlying ethanol- mediated damage to neurons, and their intrinsic vulnerabilities and neuroprotective features, which might be monitored, enhanced and/or exploited in some way to rescue neurons from damage. One proposed cellular target for ethanol damage is disruption in neurons of critical protein homeostasis pathways including the ubiquitin system. Our long-term goal is develop an understanding of the molecules and mechanisms producing photoreceptor neuron dysfunction and degeneration, and to elucidate potential rescue strategies. The overall objective of the current proposal is to determine the contribution of ubiquitin-system impairment to tissue damage by ethanol in these specific neurons, and the feasibility of ubiquitin-system manipulations to rescue neuronal function. The central hypothesis of our proposal is that binge-like ethanol exposure disrupts fundamental protein turnover mechanisms in photoreceptor neurons, undermining normal cell proteostasis. Our hypothesis is based on preliminary data from our lab demonstrating that binge-like exposure to ethanol produces loss of visual function in larval zebrafish, an advantageous model for FASD, with partial rescue of function via pharmacological enhancement of autophagy and the ubiquitin-proteasome system; ubiquitin-system impairment in photoreceptor-derived cells and photoreceptor neurons after ethanol exposure; and previously reported evidence for ubiquitin-system changes in the brain with ethanol. We plan to test our central hypothesis and accomplish the main objective of this proposal by completing the following specific aims: (1) determine the effects on ubiquitin-system manipulations on cone photoreceptor neuron function and proteostasis in the context of damaging exposure to alcohol; and (2) interrogate the changing landscape of ubiquitin-modified proteins in cone photoreceptors following ethanol exposure. These studies will involve molecular manipulations, including cell-specific expression of reporter proteins for ubiquitin- system impairment and CRISPR/Cas9 gene disruption specifically in cone photoreceptors; biochemistry; visual performance assays; ERG recordings; and photoreceptor-specific ubiquitin proteomics. The expected outcomes of these studies are an enhanced understanding of the relationship between early alcohol exposure, neuronal damage, and the ubiquitin system, and a mechanistic foundation for the possible development of therapeutic approaches to mitigate neuronal damage caused by alcohol abuse.
The proposed work has relevance to human health because it will provide mechanistic insight into damage to neurons arising from alcohol abuse. Protein degradation pathways are essential for normal cellular homeostasis and to protect cells in the context of damaged or improperly folded proteins; disturbances of these pathways contribute to neurodegeneration. Work outlined in this proposal will provide detailed knowledge about how these key mechanisms are impacted by alcohol in photoreceptor neurons, and offer potential molecular manipulations to protect neurons from alcohol damage.