Energy-based cold tolerance in genetically tractable eukaryotic systems

DESCRIPTION (provided by applicant): Cold-adapted organisms ranging from psychrophilic bacteria to glacier ice worms respond to declining temperature by raising intracellular energy levels (i.e., ATP;5'adenosine triphosphate), in an apparent mechanism to off-set inherent reductions in molecular motion (and thus lethargy and death) associated with cold temperature. This response is distinctively opposite to that observed in mesophilic animals, including mammals, which rapidly lose adenylates as temperatures fall making them vulnerable to irreversible cellular damage. Cold tolerance in disparate cells (e.g., hibernating animals, organs acclimated to low physiological temperatures) has been correlated with the maintenance of adenylate nucleotides, and recently we engineered a genetically modified bacterial strain with ~30% higher ATP levels than wild type that displayed enhanced cold tolerance. This proposal seeks to investigate the protective role of elevated ATP levels in tractable eukaryotic systems, namely the fruit fly, Drosophila melanogaster, and cultured mammalian cells (e.g., HeLa, NIH 3T3). We will target two AMP degradative enzymes, AMP phosphatase (5'-nucleotidase) and AMP deaminase, by siRNA and genetic knockout approaches, and introduce a panel of non-hydrolyzable AMP analogs in a collective effort to increase steady-state intracellular ATP levels. Experimental cells will be tested for their ability to survive at low physiological temperatures (0-4[unreadable]C) as a function of time. Based on circumstantial evidence throughout the literature and our own experimental results, we propose that cells/animals whose ATP "thermostat" is shifted to sustain higher intracellular energy levels will be significantly more cold tolerant, thus offering potential to enhance organ storage/transplantation capabilities. PUBLIC HEALTH RELEVANCE: The ability to store mammalian cells and tissues over long time periods (i.e., days to weeks) offers great potential for treating human disease, and facilitating cell and organ transplantation. This research will investigate the acquired ability of diverse metazoan cells to resist cold temperature exposure after manipulating intracellular energy levels. Knowledge gained from this study will be inherently applicable to translational research in mammals.