Numerous neurological diseases and pathologies include, or are hypothesized to include, cellular injury and/or stress as part of the mechanism. Experimental approaches to examine these mechanisms, or even determine if they are indeed at play in certain conditions, are generally limited. They include post-mortem analyses of known stress-response genes/molecules, in vivo pharmacological manipulations which are often systemic, or functional assessments/manipulations which often cannot separate stressed from non-stressed neurons. Ideally, it would be possible to determine, a priori on a cell-by-cell basis, which neurons in a mixed population were exhibiting a cell-stress response. Further, since some stress responses, or at least some components of the stress response, are only transiently expressed even though the overall stress responses may have a cumulative effect on the cell, it would be highly useful to have an a priori indication of which neurons had exhibited a stress response at some point in the past. It would also be highly useful to be able to selectively assess the cellular and inter-cellular functionality of the stressed neurons. To these ends we propose to generate new functional-reporter transgenic mouse lines which will incorporate these characteristics. The reporters will be driven by the promoter region of Activating Transcription Factor 3 (ATF3), a "hub" protein involved in a variety of cellular stress responses. Generation of the mice will be via BAC-transgenes in order to preserve function of the native ATF3 alleles, which are necessary for certain cellular processes. The BAC transgene with the ATF3 locus will drive production of the light-gated ion channel channelrhodopsin-2 (ChR2) fused to a fluorescent protein. One model will have the reporters produced in an analogue fashion (i.e., to reflect native ATF3 expression). A second model will use an inducible Cre-recombinase system to permanently "switch-on" reporter production, thus allowing, at any later time, identification of neurons that previously expressed a stress response. These functional-reporter models will allow offline anatomical assessments, FACS or laser-capture separations, fate-tracing of previously-stressed neurons, and in vivo or in vitro functional assessments specifically of stressed neurons (i.e., those expressing ChR2).
This project is intended to generate research tools, specifically 2 new lines of transgenic mice that will produce functional and anatomical reporters in response to cellular stress. These lines would significantly enhance basic science approaches to understand neurological processes and conditions that have a cellular injury and/or stress component. They would provide capabilities that are currently either highly limited or entirely lacking.
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