Imbalances in Calcium Homeostasis: Mechanism Behind Ethanol-Related Dendritic Deg

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DESCRIPTION (provided by applicant): Consistent, albeit moderate alcohol consumption in human adults results in permanent alterations in the cerebellum that discretely disrupt motor functions and become more pronounced with age. This is a major health concern as the aging population continues to escalate. In aging rats, chronic ethanol consumption results in regression of Purkinje neuron (PN) dendrites, a process that surely alters cerebellar function as PN are the sole output neurons from the cerebellum. Ethanol-related dendritic regression is coupled with other degenerative changes in dendrites such as dilation of the smooth endoplasmic reticulum (SER), the major calcium storage component of PN dendrites. Dilation of the SER in PN dendrites is strongly suggestive of increases in intradendritic calcium, a second messenger signal for a myriad of neuronal pathways. Ethanol-related dilation of the SER also suggests that ethanol-related alterations may have occurred in the sarcoplasmic reticulum calcium ATPase (SERCA) pump that resequesters calcium into the SER lumen following calcium signaling. The overall goal of this proposal is to show that ethanol-related increases in intracellular calcium levels and declines in the levels and/ or function of the SERCA 2b calcium ATPase pump may be the driving force that culminates in PN dendritic degeneration in aging and alcohol-fed rats. Calreticulin, a calcium binding protein that interacts with the SERCA pump will also be examined. To achieve this goal, 12 mo old F344 male rats will be fed the AIN-93M ethanol (EF rats), AIN-93M control (PF) rats, or rat chow (CF rats) for a period of weeks. Young control rats will also be included in the analysis. To measure calcium within dendrites and specific dendritic components, acute cerebellar slices will be made following 10, 20, or 40 weeks of ethanol treatment, electroporated to facilitate Fura-2 entry into PN, and ratiometric calcium measures made on the slices. Levels of the SERCA 2b ATPase pump and calreticulin will be assessed with confocal microscopy, and fluorescence intensity correlation after treatment durations of 10, 20 or 40 weeks in proximal and distal dendritic branches, spines, and branchpoints. SERCA 2b pump function will be assessed by observing the ability of the PN to handle large calcium transients when calcium sequestration systems other than the SERCA pump are blocked and by assessing whether ethanol predisposes the neuron to thapsigargin-induced expression of caspase-12.and GRP78 It is predicted that chronic ethanol consumption will result in increases in intracellular calcium ions that will be related to ethanol-related declines in the function or expression of the SERCA 2b pump. Results from this study will provide a basis for future studies of the actions of ethanol on intracellular calcium homeostasis and the mechanisms that result in PN dendritic degeneration. PUBLIC HEALTH RELEVANCE: The elderly U.S. population will double by 2050, an escalation accompanied by a tremendous increase in the number of elderly lifetime alcohol consumers. Recent studies show that consistent, albeit moderate alcohol consumption in human adults results in permanent alterations in cerebellar- based motor coordination and also in the cognitive activities ascribed to the cerebellum's connections with the frontal lobe. Permanent deficits result from these alterations that become more prevalent with age and predispose our increasing elderly population to falls, accidents, and cognitive dysfunctions.