Project description:Hibernating mammals such as the thirteen-lined ground squirrel (Ictidomys tridecemlineatus) experience significant reductions in oxidative metabolism and body temperature when entering a state known as torpor. Animals entering or exiting torpor do not experience permanent loss of brain function or other injuries, and the processes that enable such neuroprotection are not well understood. To gain insight into changes in protein function that occur in dramatically different physiological states, we performed quantitative phosphoproteomics experiments on thirteen-lined ground squirrels that are summer active, winter torpid, and spring active. An important aspect of our approach was the use of focused microwave irradiation of the brain to sacrifice the animals and rapidly inactivate phosphatases and kinases to preserve the native phosphoproteome. Overall, our results showed pronounced changes in phosphorylated proteins for the transitions into and out of torpor, including for proteins involved in gene expression and DNA repair, cellular plasticity, and human disease. In contrast, the transition between active states showed minimal changes. This study offers valuable insight into the global changes of brain phosphorylation in hibernating mammals, the results of which may be relevant to the future therapeutic strategies for brain injury.
Project description:This study uses advanced proteogenomic approaches in a non-model organism to elucidate cardioprotective mechanisms used during mammalian hibernation. Mammalian hibernation is characterized by dramatic reductions in body temperature, heart rate, metabolism and oxygen consumption. These changes pose significant challenges to the physiology of hibernators, especially for the heart, which maintains function throughout extreme conditions resembling ischemia and reperfusion. To identify novel cardioadaptive strategies we merged large-scale RNA-seq data with large-scale iTRAQ-based proteomic data in heart tissue from thirteen-lined ground squirrels (Ictidomys tridecemlineatus) throughout the circannual cycle. Protein identification and data analysis were run through Galaxy-P, a new multi-omic data analysis platform enabling effective integration of RNA-seq and MS/MS proteomic data. Galaxy-P uses flexible, modular workflows that combine customized sequence database searching and iTRAQ quantification to identify novel ground squirrel-specific protein sequences and provide insight into molecular mechanisms of hibernation. This study allowed for the quantification of 2007 identified cardiac proteins, including over 350 peptide sequences derived from previously uncharacterized protein products. Identification of these peptides allows for improved genomic annotation of this non-model organism, as well as identification of potential splice variants, mutations, or genome re-organization that provide insights into novel cardioprotective mechanisms used during hibernation.
Project description:Water deprivation is a life-threatening condition that engages a protective physiological response to couple osmolyte retention with potentiation of thirst. This response, typical for most mammals, safeguards against short-term water deprivation, but fails in the long-term. Thirteen-lined ground squirrels (Ictidomys tridecemlineatus) employ the short-term response during summer, whereas during winter they lack thirst and survive without water for months. Here, we show that long-term thirst suppression occurs despite hormonal and behavioral signs of a dramatic fluid deficit and originates from hypoactivity of neurons in the circumventricular organs, which exhibit marked functional suppression during winter that blunts their sensitivity to thirst cues. Our work reveals a remarkable capacity of the evolutionarily conserved brain regions which control fluid homeostasis in mammals to enable long-term survival without water.
Project description:Mammalian hibernation is characterized by metabolic rate depression and a strong decrease in core body temperature that together create energy savings such that most species do not have to eat over the winter months. Brown adipose tissue (BAT), a thermogenic tissue that uses uncoupled mitochondrial respiration to generate heat instead of ATP, plays a major role in rewarming from deep torpor. The present study used label-free phosphoproteomics to investigate changes in BAT from thirteen-lined ground squirrels (Ictidomys tridecemlineatus), comparing euthermic squirrels with squirrels in deep torpor. Differential expression of mitochondrial proteins was also investigated. Surprisingly, mitochondrial membrane and matrix protein expression in BAT was largely constant between active euthermic squirrels and their hibernating counterparts. Validation by immunoblotting confirmed that the protein levels of mitochondrial respiratory chain complexes were largely unchanged. However, phosphoproteomics showed that pyruvate dehydrogenase (PDH) phosphorylation increased during ground squirrel hibernation, and this was confirmed by immunoblotting with phospho-specific antibodies. PDH phosphorylation leads to its inactivation, which suggests that BAT carbohydrate oxidation is inhibited during hibernation. Phosphorylation of hormone-sensitive lipase (HSL) also increased during hibernation, suggesting that HSL would be in a partly activated state in BAT to produce the fatty acids that are likely the primary fuel for thermogenesis during arousal.
Project description:Identify shifts in gene expression relevant to torpor phenotypes and recovery following torpor in five tissues of the 13-lined ground squirrel. Sampled tissues and time points overlap with prior hibernation RNA-seq studies in 13-lined ground squirrel and other species, allowing for the analysis of conserved gene expression patterns in torpor.