May 21, 2018Basic Biology
How squirrel tissues adapt to the cold and metabolic stress has confounded researchers for years.
A structure in cells known to be vulnerable to cold is the microtubule cytoskeleton. This network of small tubes within a cell provides structural support and acts as a kind of inner cellular railway system, transporting organelles and molecular complexes vital for a cell's survival.
According to an article published on line the journal Cell (3 May 2018), cellular mechanisms were identified during hibernation that helped 13-lined ground squirrel survive near freezing temperatures, by dramatically slowing their heart rate and respiration. The findings could be a step to extending storage of human donor tissues awaiting transplantation and protecting traumatic brain injury patients who undergo induced hypothermia.
In a series of experiments, the research team compared cells from non-hibernators to cells from the ground squirrel to determine differences in their response to cold. Results showed that in ground squirrel neurons, the microtubule cytoskeleton remains intact, while it deteriorates in the neurons of humans and other non-hibernating animals, including rats.
To investigate the biological factors supporting the squirrel's cold adaptation, the authors created hibernation in a dish. They took cells from a newborn ground squirrel and reprogrammed them to become stem cells, which are undifferentiated cells capable of becoming any type of tissue in the body. Importantly, these lab-made cells, also known as induced pluripotent stem cells (iPSCs), retained the intrinsic cold-adaptive characteristics of the adult squirrel's cells, thus providing a type of platform for studying how various kinds of the rodent's cells adapt to the cold. Next, the authors compared gene expression of stem cell-derived neurons from ground squirrels and humans. Cold exposure revealed distinct differences in the reaction of mitochondria, organelles that provide energy to the cell in the form of adenosine, triphosphate (ATP). It was found that cold-exposed human stem cell-derived neurons tended to overproduce a byproduct of metabolism known as reactive oxygen species (ROS). The overabundance of ROS in human neurons appeared to cause proteins along the microtubules to oxidize, wreaking havoc with the microtubule structure. By comparison, ground squirrel ROS levels remained relatively low and their microtubules remained intact.
Cold exposure also interfered with the human stem cell-derived neurons' ability to dispose of the toxic oxidized proteins via its protein quality control system. Under normal conditions, lysosomes envelop oxidized proteins and digest them via enzymes called proteases, but in the cold-exposed human neurons, the proteases leaked from the lysosomes and digested nearby microtubules. The authors then treated non-hibernating cells prior to cold exposure with two drugs to alter the course of the cold-induced damage. One of the drugs, BAM15, inhibits the production of ATP, which reduces the production of ROS. The second drug inhibited protease activity. After bathing a variety of cell types from non-hibernators in both drugs, the research team exposed them to 4-degrees Celsius for four to 24 hours. The drug combination preserved microtubule structure in human stem cell-derived neurons, and rat retina - the light sensitive tissue at the back of the eye. Subsequent tests showed that the rat retina also remained functional. The authors also found that the drug combination also preserved nonneural tissue. Microtubules in renal cells from mouse kidneys showed improved structural integrity after cooling and rewarming.
In addition to the implications for organ transplantation, these findings pave the way for future studies looking at possible therapeutic applications. For example, inducing hypothermia is a commonly used strategy to protect the brain following a traumatic injury, but the potential benefits are weighed against the potential harm from cold-induced cellular damage. According to the authors, by understanding the biology of cold adaptation in hibernation, it may be possible to improve and broaden the applications of induced hypothermia in the future, and perhaps prolong the viability of organs prior to transplantation. For example, kidneys are typically stored for no more than 30 hours. After that, the tissue starts to deteriorate, impairing the organ's ability to function properly after its been rewarmed and reperfused. Heart, lungs and livers have an even shorter shelf life. The findings also suggest that stem cell-derived neurons from the ground squirrel can serve as a platform for studying other aspects of hibernation adaption, a field of research that has been limited by a lack of transgenic animal models and the inability to induce hibernation in the animal.