Intra- and intergenerational changes in the cortical DNA methylome in response to therapeutic intermittent hypoxia in mice

Abstract

Recent evidence from our laboratory documents functional resilience to retinal ischemic injury in untreated mice derived from parents exposed to repetitive hypoxic conditioning (RHC) before breeding. To begin to understand the epigenetic basis of this intergenerational protection, we used methylated DNA immunoprecipitation and sequencing to identify genes with differentially methylated promoters (DMGPs) in the prefrontal cortex of mice treated directly with the same RHC stimulus (F0-RHC) and in the prefrontal cortex of their untreated F1-generation offspring (F1-*RHC). Subsequent bioinformatic analyses provided key mechanistic insights into how changes in gene expression secondary to promoter hypo- and hypermethylation might afford such protection within and across generations. We found extensive changes in DNA methylation in both generations consistent with the expression of many survival-promoting genes, with twice the number of DMGPs in the cortex of F1*RHC mice relative to their F0 parents that were directly exposed to RHC. In contrast to our hypothesis that similar epigenetic modifications would be realized in the cortices of both F0-RHC and F1-*RHC mice, we instead found relatively few DMGPs common to both generations; in fact, each generation manifested expected injury resilience via distinctly unique gene expression profiles. Whereas in the cortex of F0-RHC mice, predicted protein-protein interactions reflected activation of an anti-ischemic phenotype, networks activated in F1-*RHC cortex comprised networks indicative of a much broader cytoprotective phenotype. Altogether, our results suggest that the intergenerational transfer of an acquired phenotype to offspring does not necessarily require the faithful recapitulation of the conditioning-modified DNA methylome of the parent.

Epigenetic Conditioning Induces Intergenerational Resilience to Dementia in a Mouse Model of Vascular Cognitive Impairment

“…While extended periods of hypoxia can be pathologic, appropriately titrated intermittent hypoxia shows efficacy as a therapeutic for a number of diseases, without evidence of injury (Navarrette-Opazo 2014; Burtscher et al, 2021). In fact, we showed in our methylome study that even 4 months of RHC – twice the duration as used herein – did not adversely affect the viability of CA1 pyramidal cells, known to be the most sensitive neurons in the brain to hypoxia (Belmonte et al, 2020). In the present study, we found no change in white matter myelin density in RHC-treated mice, again suggesting that our epigenetic stimulus was nonharmful, and yet robustly efficacious in triggering gene expression changes sufficient to induce resilience to memory loss in the face of chronic hypoperfusion.…”

“…In an earlier genome-wide methylome analysis of the adult mouse brain prefrontal cortex, we found that genes that become differentially methylated in response to RHC are largely distinct from the differentially-methylated genes that define the cortex of their untreated adult progeny. To our surprise, bioinformatic analyses of these methylomes implicated gene expression changes across a much larger network of broad-based, multifunctional cytoprotective genes in the F1 generation, relative to a smaller, ischemia-protective network of genes in the F0 generation, suggesting that intergenerational transfer of an acquired phenotype to offspring does not necessarily require the faithful recapitulation of the conditioning-modified DNA methylome of the parent (Belmonte et al, 2020). Ultimately, uncovering the proteomic, lipidomic, metabolomic, and other cellular- and regional (e.g., prefrontal cortex, hippocampus)- ‘omic features that define the dementia-resilient phenotype will be critical to the identification of specific downstream therapeutic effectors that protect against memory loss; that said, systemic treatments like RHC and RLIC that trigger multiple responses in tissues may be considered a ‘cocktail’ therapeutic that will not likely be mimicked by single molecule-targeted therapeutics.…”

Intermittent Hypoxia Promotes Functional Neuroprotection from Retinal Ischemia in Untreated First-Generation Offspring: Proteomic Mechanistic Insights

“…In additional cohorts, we also found no quantitative differences in hippocampal CA1 pyramidal cell density between F0 parental groups, as reported previously. 26 Fecundity was also unaffected by the 16-week RHC treatment; the F0 normoxic control breeders gave birth to as many litters as the F0 RHC-treated breeder pairs, and neither litter size nor male/female distributions varied between groups. In random, intermittent inspections prior to weaning at 21 days of age, we did not observe any differences in maternal/paternal care behaviors of the RHC-treated F0 parents and the untreated control F0 parents.…”

Epigenetic conditioning induces intergenerational resilience to dementia in a mouse model of vascular cognitive impairment

“…While extended periods of hypoxia can be pathologic, appropriately titrated, intermittent exposures to hypoxia show therapeutic efficacy in a number of disease states, without evidence of injury by the hypoxia itself 31–33 . We previously demonstrated that 4 months of RHC did not affect the viability of hippocampal CA1 pyramidal cells, known to be highly sensitive to hypoxia 34 . Herein, neither mice treated directly with 2 months of RHC, nor their adult offspring, showed changes in white matter myelin density, neurocognitive function, or synaptic plasticity.…”

“…Resilience may also be afforded by direct pro‐survival adaptations that RHC induces in cells of the neurovascular unit, rendering them more resistant to the pathological consequences of CCH. The relative contributions of these yet‐to‐be‐identified mechanisms may show generation‐dependence as well, given that a previous genome‐wide methylome analysis of DNA promoters in the prefrontal cortices of F0 mice treated with RHC, and F1 mice derived from F0 parents treated with RHC, revealed largely distinct methylomes relative to their respective generation‐matched controls 34 . It is likely that all three primary epigenetic regulatory mechanisms—DNA methylation, histone post‐translational modifications, and long‐noncoding RNAs—are involved in establishing and maintaining disease‐resilient phenotypes in the somatic cells of the F0 mice, as well as laying down the differentiation blueprint in F0 germ cells such that their F1 progeny recapitulate these disease‐resilient phenotypes throughout adulthood 61,62 .…”