Franziska Kohl

Shotgun Proteomics Analysis of Hibernating Arctic Ground Squirrels

Mammalian hibernation involves complex mechanisms of metabolic reprogramming and tissue protection. Previous gene expression studies of hibernation have mainly focused on changes at the mRNA level. Large scale proteomics studies on hibernation have lagged behind largely because of the lack of an adequate protein database specific for hibernating species. We constructed a ground squirrel protein database for protein identification and used a label-free shotgun proteomics approach to analyze protein expression throughout the torpor-arousal cycle during hibernation in arctic ground squirrels (Urocitellus parryii). We identified more than 3,000 unique proteins from livers of arctic ground squirrels. Among them, 517 proteins showed significant differential expression comparing animals sampled after at least 8 days of continuous torpor (late torpid), within 5 h of a spontaneous arousal episode (early aroused), and 1–2 months after hibernation had ended (non-hibernating). Consistent with changes at the mRNA level shown in a previous study on the same tissue samples, proteins involved in glycolysis and fatty acid synthesis were significantly underexpressed at the protein level in both late torpid and early aroused animals compared with non-hibernating animals, whereas proteins involved in fatty acid catabolism were significantly overexpressed. On the other hand, when we compared late torpid and early aroused animals, there were discrepancies between mRNA and protein levels for a large number of genes. Proteins involved in protein translation and degradation, mRNA processing, and oxidative phosphorylation were significantly overexpressed in early aroused animals compared with late torpid animals, whereas no significant changes at the mRNA levels between these stages had been observed. Our results suggest that there is substantial post-transcriptional regulation of proteins during torpor-arousal cycles of hibernation.

Phenological variation in annual timing of hibernation and breeding in nearby populations of Arctic ground squirrels

Ecologists need an empirical understanding of physiological and behavioural adjustments that animals can make in response to seasonal and long-term variations in environmental conditions. Because many species experience trade-offs between timing and duration of one seasonal event versus another and because interacting species may also shift phenologies at different rates, it is possible that, in aggregate, phenological shifts could result in mismatches that disrupt ecological communities. We investigated the timing of seasonal events over 14 years in two Arctic ground squirrel populations living 20 km apart in Northern Alaska. At Atigun River, snow melt occurred 27 days earlier and snow cover began 17 days later than at Toolik Lake. This spatial differential was reflected in significant variation in the timing of most seasonal events in ground squirrels living at the two sites. Although reproductive males ended seasonal torpor on the same date at both sites, Atigun males emerged from hibernation 9 days earlier and entered hibernation 5 days later than Toolik males. Atigun females emerged and bred 13 days earlier and entered hibernation 9 days earlier than those at Toolik. We propose that this variation in phenology over a small spatial scale is likely generated by plasticity of physiological mechanisms that may also provide individuals the ability to respond to variation in environmental conditions over time.

The Physiological Link between Metabolic Rate Depression and Tau Phosphorylation in Mammalian Hibernation

Abnormal phosphorylation and aggregation of tau protein are hallmarks of a variety of neurological disorders, including Alzheimers disease (AD). Increased tau phosphorylation is assumed to represent an early event in pathogenesis and a pivotal aspect for aggregation and formation of neurofibrillary tangles. However, the regulation of tau phosphorylation in vivo and the causes for its increased stage of phosphorylation in AD are still not well understood, a fact that is primarily based on the lack of adequate animal models. Recently we described the reversible formation of highly phosphorylated tau protein in hibernating European ground squirrels. Hence, mammalian hibernation represents a model system very well suited to study molecular mechanisms of both tau phosphorylation and dephosphorylation under in vivo physiological conditions. Here, we analysed the extent and kinetics of hibernation-state dependent tau phosphorylation in various brain regions of three species of hibernating mammals: arctic ground squirrels, Syrian hamsters and black bears. Overall, tau protein was highly phosphorylated in torpor states and phosphorylation levels decreased after arousal in all species. Differences between brain regions, hibernation-states and phosphosites were observed with respect to degree and kinetics of tau phosphorylation. Furthermore, we tested the phosphate net turnover of tau protein to analyse potential alterations in kinase and/or phosphatase activities during hibernation. Our results demonstrate that the hibernation-state dependent phosphorylation of tau protein is specifically regulated but involves, in addition, passive, temperature driven regulatory mechanisms. By determining the activity-state profile for key enzymes of tau phosphorylation we could identify kinases potentially involved in the differentially regulated, reversible tau phosphorylation that occurs during hibernation. We show that in black bears hibernation is associated with conformational changes of highly phosphorylated tau protein that are typically related to neuropathological alterations. The particular hibernation characteristics of black bears with a continuous torpor period and an only slightly decreased body temperature, therefore, potentially reflects the limitations of this adaptive reaction pattern and, thus, might indicate a transitional state of a physiological process.

Hibernating above the permafrost: effects of ambient temperature and season on expression of metabolic genes in liver and brown adipose tissue of arctic ground squirrels

SUMMARY Hibernating arctic ground squirrels (Urocitellus parryii), overwintering in frozen soils, maintain large gradients between ambient temperature (Ta) and body temperature (Tb) by substantially increasing metabolic rate during torpor while maintaining a subzero Tb. We used quantitative reverse-transcription PCR (qRT-PCR) to determine how the expression of 56 metabolic genes was affected by season (active in summer vs hibernating), metabolic load during torpor (imposed by differences in Ta: +2 vs –10°C) and hibernation state (torpid vs after arousal). Compared with active ground squirrels sampled in summer, liver from hibernators showed increased expression of genes associated with fatty acid catabolism (CPT1A, FABP1 and ACAT1), ketogenesis (HMGCS2) and gluconeogenesis (PCK1) and decreased expression of genes associated with fatty acid synthesis (ACACB, SCD and ELOVL6), amino acid metabolism, the urea cycle (PAH, BCKDHA and OTC), glycolysis (PDK1 and PFKM) and lipid metabolism (ACAT2). Stage of hibernation (torpid vs aroused) had a much smaller effect, with only one gene associated with glycogen synthesis (GSY1) in liver showing consistent differences in expression levels between temperature treatments. Despite the more than eightfold increase in energetic demand associated with defending Tb during torpor at a Ta of –10 vs +2°C, transcript levels in liver and brown adipose tissue differed little. Our results are inconsistent with a hypothesized switch to use of non-lipid fuels when ambient temperatures drop below freezing.

Interrelationships Among Timing of Hibernation, Reproduction, and Warming Soil in Free-Living Female Arctic Ground Squirrels

The effects of climate change on hibernating species will depend, in part, on their responsiveness to environmental cues used to adjust the seasonal timing of annual events of hibernation and reproduction. Using long-term data collected from two arctic ground squirrel populations living 20 km apart in northern Alaska, we investigate the relationships between the timing of hibernation and reproduction and, in addition, the potential for change in soil temperatures to act as a proximate cue. Previously, we found that female ground squirrels living at the southern-most site, Atigun River, emerge from hibernation and give birth 13 days earlier than females at Toolik Lake. Here we show that timing of parturition was tightly linked to the termination of heterothermy and subsequent emergence from the hibernacula at both sites, whereas timing of entrance into hibernation was only weakly correlated with date of parturition in Toolik Lake females. Females ended heterothermy in spring coincident with rising soil temperatures from winter minima, but since average soil temperatures did not differ between the two sites, a single threshold in warming cannot explain the differences in timing of spring emergence and reproduction between the two populations. Earlier reproduction at Atigun is associated with earlier snowmelt, yet, how this is achieved and the relative importance of phenotypic plasticity versus genetic differences between the two populations will require further investigation.

Seasonal body composition, water turnover, and field metabolic rates in porcupines (Erethizon dorsatum) in Alaska

Abstract Winter extremes of temperature and food shortage limit the distribution of arctic animals. North American porcupines (Erethizon dorsatum) are one of the most widely distributed mammals in North America and range from deserts to arctic tundra. In Alaska porcupines remain active at low winter temperatures (i.e., −39°C) while consuming woody plants that are low in nitrogen (N) and high in both fiber and plant secondary metabolites. Porcupines conserved lean body mass in winter by using fat stores. Fat mass declined from 50% ± 3% to 27% ± 7% of body mass over winter. Animals with small fat stores might be more reliant on food intake during winter, because proportional fat loss was correlated positively with total fat mass at the start of winter. Fat losses were minimized by lowering rates of energy expenditure. Field metabolic rate was 440 ± 18 kJ kg−0.77 day−1. Water turnovers were slow at 26.62 ml kg−0.75 day−1 in wild porcupines. Body temperatures were not reduced to save energy; core temperatures were maintained at 37.3°C ± 0.1°C despite variation in ambient air temperature from +7°C to −38°C in captivity. Persistence of porcupines at the northern limits of their range is due to plasticity of food intake and tolerance of low-quality diets and low ambient temperatures. Minimal expenditures of energy and N in winter are combined with the conservation of lean mass. Porcupines rely on abundant summer forages to replenish their stores of fat and protein for reproduction and survival in the subsequent winter.

PHF-like tau phosphorylation in mammalian hibernation is not associated with p25-formation

In Alzheimer’s disease and related disorders, hyperphosphorylation of tau is associated with an increased activity of cyclin dependent kinase 5 (cdk5). Elevated cdk5 activity is thought to be due to the formation of p25 and thereby represents a critical element in the dysregulation of tau phosphorylation under pathological conditions. However, there is still a controversy regarding the correlation of p25 generation and tau pathology. Recently, we demonstrated physiological, paired helical filament-like tau phosphorylation that reversibly occurs in hibernating mammals. Here we used this model to test whether the tau phosphorylation in hibernation is associated with the formation of p25. Analysing brain material of arctic ground squirrels and Syrian hamsters we found no evidence for a hibernation dependent generation of p25. Hence, we suppose that phosphorylation of tau does not require the formation of p25. Instead we suggest that the truncation of p35 to p25 represents a characteristic of pathological alterations and may contribute to aggregation and deposition of hyperphosphorylated tau.

Hibernation strategies and patterns in sympatric arctic species, the Alaska marmot and the arctic ground squirrel

We compared patterns of core body temperature (Tb) change, including inter-individual synchrony, in 2 free-living arctic hibernators that differ in size and sociality, the Alaska marmot (Marmota broweri) and the arctic ground squirrel (Urocitellus parryii). We report overwinter Tb changes from 3 to 4 marmots from the same hibernaculum in each of 3 years and from 7 ground squirrels that hibernated at 2 nearby burrow sites in 1 year. Very close synchrony in the timing of torpor and arousal cycles in Alaska marmots indicates social hibernation and thermoregulation, while lack of synchrony in arctic ground squirrels further confirms solitary hibernation. The mean duration between the first and last marmot measured within the group to initiate an arousal was 3.7 ± 2.5 h and to recool to 30°C during torpor entrance was 5.7 ± 3.7 h. The minimum Tb recorded in marmots was 0.6°C and in ground squirrels was -2.0°C. Marmots entering torpor displayed an interrupted pattern of Tb change defined by 2 distinct rates of cooling, early and late during entry, that differed by 21-fold. Ground squirrels cooled in a continuous pattern, initially 3-fold slower than marmots during rapid cooling but 4-fold faster during slow cooling. Both species must minimize energy expenditure to survive long arctic winters; our results suggest that Alaska marmots do this through social thermoregulation, while arctic ground squirrels decrease Tb below freezing to minimize the difference between body and ambient temperatures.

Arctic Ground Squirrels Limit Bone Loss during the Prolonged Physical Inactivity Associated with Hibernation

Prolonged disuse (e.g., physical inactivity) typically results in increased bone porosity, decreased mineral density, and decreased bone strength, leading to increased fracture risk in many mammals. However, bears, marmots, and two species of ground squirrels have been shown to preserve macrostructural bone properties and bone strength during long seasons of hibernation while they remain mostly inactive. Some small hibernators (e.g., 13-lined ground squirrels) show microstructural bone loss (i.e., osteocytic osteolysis) during hibernation, which is not seen in larger hibernators (e.g., bears and marmots). Arctic ground squirrels (Urocitellus parryii) are intermediate in size between 13-lined ground squirrels and marmots and are perhaps the most extreme rodent hibernator, hibernating for up to 8 mo annually with body temperatures below freezing. The goal of this study was to quantify the effects of hibernation and inactivity on cortical and trabecular bone properties in arctic ground squirrels. Cortical bone geometrical properties (i.e., thickness, cross-sectional area, and moment of inertia) at the midshaft of the femur were not different in animals sampled over the hibernation and active seasons. Femoral ultimate stress tended to be lower in hibernators than in summer animals, but toughness was not affected by hibernation. The area of osteocyte lacunae was not different between active and hibernating animals. There was an increase in osteocytic lacunar porosity in the hibernation group due to increased lacunar density. Trabecular bone volume fraction in the proximal tibia was unexpectedly greater in the hibernation group than in the active group. This study shows that, similar to other hibernators, arctic ground squirrels are able to preserve many bone properties during hibernation despite being physically inactive for up to 8 mo.