Regulation of SMAD transcription factors during freezing in the freeze tolerant wood frog, Rana sylvatica

Micromanaging Freeze Tolerance: The Biogenesis and Regulation of microRNAs in Frozen Frogs

“…The enhanced expression of miR-192 has been shown to be tightly associated with Smad3, where Smad3 is able to mediate TGF-β1-induced miR-192 overexpression by binding to, and activating the miR-192 promoter (Chung et al, 2010). Interestingly, this corroborates the aforementioned significant upregulation of p-Smad3 protein levels in R. sylvatica brains during both freezing, and to a larger extent during thawing (Aguilar et al, 2016;Chung et al, 2010). A known target of miR-192, that could be targeted in thawed brains, is Semaphorin 3A, the suggested miR-192 mediated silencing of Semaphorin 3A could be an ischemia protection mechanism.…”

“…Studies on diverse animal models have demonstrated that the brain is critical for coordinating environmental stimuli and for mounting a molecular stress responses. While global metabolic rate depression is one of the main characteristics of freeze tolerance, neuroprotective processes such as the promotion of tissue survival has been shown to be active and upregulated in frozen wood frog brains, emphasizing the importance of maintaining brain functionality (Aguilar et al, 2016). One of the main questions we sought to investigate is, what happens to the brains of freeze tolerant animals?…”

“…While it has been reported that frozen wood frog brains display no detectable brain activity and both visual observations and magnetic resonance imaging have shown ice accumulation in brain ventricles, little other physiological work has been done on freeze tolerant brains . Previous biochemical and molecular studies on frozen frog brains have revealed various cytoprotective "activations" of maintenance and freeze protective pathways that promote tissue survival (Aguilar et al, 2016;. The rapid, reversible, and energetically inexpensive nature of miRNA control renders these small molecules excellent candidates for coordinating the necessary neuroprotection of intricate neural networks and facilitating freeze survival.…”

“…The brain's persistent flow of information and adaptations to environmental stimuli plays a pivotal role in the facilitation of stress responses and prolonged hypometabolic periods . Despite the lack of any measurable brain activity or nerve conductance , biochemical studies on the brains of frozen R. sylvatica have revealed various neuroprotective aspects such as the increase in novel li16 and fr10 which have been linked with the mitigation of oxygen restriction damages and freeze protection, increased levels of c-FOS, and the elevation of phosphorylated Smad3 levels that has been implicated in the promotion of tissue survival (Aguilar et al, 2016;. Other neuronal "activations" found to occur in frozen wood frog brains include; the upregulation of protein kinase C phosphorylation status, the upregulation of the acidic ribosomal phosphoprotein P0, and increased levels of the large ribosomal subunit protein 7 (Dieni and Storey, 2014;Wu et al, 2008;).…”

“…Total protein extracts were prepared as previously described (Aguilar et al, 2016). Briefly, ~500 mg of brain tissue (n = 4 individual animals) were homogenized in 1:2 w:v chilled homogenization buffer (20 mM Hepes, 200 mM NaCl, 0.1 mM EDTA, 10 mM NaF, 1 mM Na3VO4, 10 mM β-glycerophosphate, pH 7.5) with a few crystals of phenylmethylsulfonyl fluoride (Bioshop) and 1 μL/mL of protease inhibitor (Cat# PIC002; Bioshop Canada Inc., Burlington, ON, Canada) using a Polytron PT10 homogenizer.…”