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In mammals, the molecular mechanisms underlying transgenerational inheritance of phenotypic traits in serial generations of progeny after ancestral environmental exposures, without variation in DNA sequence, remain elusive. We've recently described transmission of a beneficial trait in rats and mice, in which F0 supplementation of methyl donors, including folic acid, generates enhanced axon regeneration after sharp spinal cord injury in untreated F1 to F3 progeny linked to differential DNA methylation levels in spinal cord tissue. To test whether the transgenerational effect of folic acid is transmitted via the germline, we performed whole-genome methylation sequencing on sperm DNA from F0 mice treated with either folic acid or vehicle control, and their F1, F2, and F3 untreated progeny. Transgenerational differentially methylated regions (DMRs) are observed in each consecutive generation and distinguish folic acid from untreated lineages, predominate outside of CpG islands and in regions of the genome that regulate gene expression, including promoters, and overlap at both the differentially methylated position (DMP) and gene levels. These findings indicate that molecular changes between generations are caused by ancestral folate supplementation. In addition, 29,719 DMPs exhibit serial increases or decreases in DNA methylation levels in successive generations of untreated offspring, correlating with a serial increase in the phenotype across generations, consistent with a 'wash-in' effect. Sibship-specific DMPs annotate to genes that participate in axon- and synapse-related pathways.

While embryonic mammalian central nervous system (CNS) axons readily grow and differentiate, only a minority of fully differentiated mature CNS neurons are able to regenerate injured axons, leading to stunted functional recovery after injury and disease. To delineate DNA methylation changes specifically associated with axon regeneration, we used a Fluorescent-Activated Cell Sorting (FACS)-based methodology in a rat optic nerve transection model to segregate the injured retinal ganglion cells (RGCs) into regenerating and non-regenerating cell populations. Whole-genome DNA methylation profiling of these purified neurons revealed genes and pathways linked to mammalian RGC regeneration. Moreover, whole-methylome sequencing of purified uninjured adult and embryonic RGCs identified embryonic molecular profiles reactivated after injury in mature neurons, and others that correlate specifically with embryonic or adult axon growth, but not both. The results highlight the contribution to both embryonic growth and adult axon regeneration of subunits encoding the Na + /K + -ATPase. In turn, both biochemical and genetic inhibition of the Na + /K + -ATPase pump significantly reduced RGC axon regeneration. These data provide critical molecular insights into mammalian CNS axon regeneration, pinpoint the Na + /K + -ATPase as a key regulator of regeneration of injured mature CNS axons, and suggest that successful regeneration requires, in part, reactivation of embryonic signals., (© 2023. The Author(s).)

Alterations in environmentally sensitive epigenetic mechanisms ( e.g ., DNA methylation) influence axonal regeneration in the spinal cord following sharp injury. Conventional DNA methylation detection methods using sodium bisulphite treatment do not distinguish between methylated and hydroxymethylated forms of cytosine, meaning that past studies report a composite of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). To identify the distinct contributions of DNA methylation modifications to axonal regeneration, we collected spinal cord tissue after sharp injury from untreated adult F3 male rats with enhanced regeneration of injured spinal axons or controls, derived from folate- or water-treated F0 lineages, respectively. Genomic DNA was profiled for genome-wide 5hmC levels, revealing 658 differentially hydroxymethylated regions (DhMRs). Genomic profiling with whole genome bisulphite sequencing disclosed regeneration-related alterations in composite 5mC + 5hmC DNA methylation levels at 2,260 differentially methylated regions (DMRs). While pathway analyses revealed that differentially hydroxymethylated and methylated genes are linked to biologically relevant axon developmental pathways, only 22 genes harbour both DhMR and DMRs. Since these differential modifications were more than 60 kilobases on average away from each other, the large majority of differential hydroxymethylated and methylated regions are unique with distinct functions in the axonal regeneration phenotype. These data highlight the importance of distinguishing independent contributions of 5mC and 5hmC levels in the central nervous system, and denote discrete roles for DNA methylation modifications in spinal cord injury and regeneration in the context of transgenerational inheritance.