SUMMARY More than 22,000 people each year die of alcoholic liver disease in the US alone with estimates as high as 3.3 million deaths each year globally (5.9% of all global deaths). So clearly, alcoholic liver disease is a major biomedical health concern world-wide. Because the liver is the major site of ethanol metabolism, it is the most susceptible organ to alcohol-induced injury. Although the progression of alcoholic liver disease is well-described clinically, the molecular basis for alcohol-induced liver injury is not understood. This proposal expands on our findings that microtubules are more highly acetylated and more stable in ethanol-treated WIF-B cells, liver slices, livers from ethanol-fed rats/mice - and from our preliminary data - also in human liver tissue. We have further shown that microtubule hyperacetylation directly explains alcohol-induced defects in protein trafficking and lipid droplet dynamics. In this proposal, we will test the broad hypothesis that ethanol-induced protein modification differentially disrupts microtubule-based protein/organelle motility that leads to peroxisome dysfunction and promotes alcoholic steatosis. We will also test the hypothesis that supplementation with caloric restriction mimetics protects against injury. Our findings that both microtubule acetylation and acetaldehyde adduction impair protein trafficking to similar extents suggest both modifications contribute to the impaired motility observed in ethanol-treated cells. We will examine how microtubule modifications (and modifications on other proteins) differentially impact protein and organelle dynamics in Aim 1. In Aim 2, we expand our studies on altered organelle motility to peroxisome dynamics. Despite their known role in regulating oxidative stress and fatty acid metabolism, peroxisomes are under-studied in their contribution to the progression of alcohol-induced steatosis. Emerging evidence indicates that microtubules and associated motors are important regulators of peroxisomal dynamics, and by extension, their function – a relationship we will explore in the context of alcohol- induced steatosis. Aim 3 takes us in an exciting direction where we expand on our studies with spermidine on its hepatoprotective effects against fibrosis. Spermidine and hydroxycitrate (caloric restriction mimetics) induce protein deacetylation (including microtubule deacetylation) by different mechanisms. Thus, in Aim 3 we propose that this enhanced protein deacetylation will counteract alcohol-induced global protein acetylation (and alcohol- induced microtubule-dependent protein trafficking) to confer hepatoprotection. We further propose that spermidine promotes cytoprotective autophagy thereby decreasing the levels of accumulated lipid droplets and dysfunctional mitochondria and peroxisomes. In general, studies will be initiated in polarized, hepatic WIF-B cells, confirmed in livers from ethanol/high fat diet-fed mice, and where possible, confirmed in human tis...