Purified regenerating retinal neurons reveal regulatory role of DNA methylation-mediated Na+/K+-ATPase in murine axon regeneration

Abstract

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.

Contribution of the neuron-specific ATP1A3 to embryonic spinal circuit emergence

“…In vitro, DIN axon extension was not impaired by ouabain application, consistent with studies using DRG neurons 72 and iPS cells defective for ATP1A3 73 . This contrasts with axon growth during regeneration, found to depend on ATP1A1 and ATP1A2 and possibly ATP1A3 72,74,75 . It might rely on differences between mature and immature neurons and/or on processes specific to regeneration such as membrane resealing of injured axons thought to require Na + /K + pumps to restore Na+ levels 76 .…”

Folate-mediated transgenerational inheritance of sperm DNA methylation patterns correlate with spinal axon regeneration

“…DNA methylation levels between the 3 generations of untreated progeny derived from F0 animals treated with folic acid revealed over 29,000 CpGs that exhibit progressively increasing or decreasing DNA methylation from F1 to F3, an effect sometimes referred to as ‘wash-in’ [ 10 ]. Genes annotated to incremental DMP levels between generations in the present study were previously linked to axonal regeneration, including subunits of the Na + /K + -ATPase [ 35 ]. The top terms from ontological analyses showed a high association with axonogenesis and synapse organization.…”