Accepted

More than twelve morphologically and physiologically distinct subtypes of primary somatosensory neuron report salient features of our internal and external environments. It is unclear how specialized gene expression programs emerge during development to endow these subtypes with their unique properties. To assess the developmental progression of transcriptional maturation of each subtype of principal somatosensory neuron, we generated a transcriptomic atlas of cells traversing the primary somatosensory neuron lineage in mice. Here we show that somatosensory neurogenesis gives rise to neurons in a transcriptionally unspecialized state, characterized by co-expression of transcription factors that become restricted to select subtypes as development proceeds. Single-cell transcriptomic analyses of sensory neurons from mutant mice lacking transcription factors suggest that these broad-to-restricted transcription factors coordinate subtype-specific gene expression programs in subtypes in which their expression is maintained. We also show that neuronal targets are involved in this process; disruption of the prototypic target-derived neurotrophic factor NGF leads to aberrant subtype-restricted patterns of transcription factor expression. Our findings support a model in which cues that emanate from intermediate and final target fields promote neuronal diversification in part by transitioning cells from a transcriptionally unspecialized state to transcriptionally distinct subtypes by modulating the selection of subtype-restricted transcription factors.

Our previous studies implicated glycosylation of the Ca3.2 isoform of T-type Ca channels (T-channels) in the development of Type 2 painful peripheral diabetic neuropathy (PDN). Here we investigated biophysical mechanisms underlying the modulation of recombinant Ca3.2 channel by de-glycosylation enzymes such as neuraminidase (NEU) and PNGase-F (PNG), as well as their behavioral and biochemical effects in painful PDN Type 1. In our study we used whole-cell recordings of current-voltage relationships to confirm that Ca3.2 current densities were decreased ~2-fold after de-glycosylation. Furthermore, de-glycosylation induced a significant depolarizing shift in the steady-state relationships for activation and inactivation while producing little effects on the kinetics of current deactivation and recovery from inactivation. PDN was induced by injections of streptozotocin (STZ) in adult female C57Bl/6j wild type (WT) mice, adult female Sprague Dawley rats and Ca3.2 knock-out (KO mice). Either NEU or vehicle (saline) were locally injected into the right hind paws or intrathecally. We found that injections of NEU, but not vehicle, completely reversed thermal and mechanical hyperalgesia in diabetic WT rats and mice. In contrast, NEU did not alter baseline thermal and mechanical sensitivity in the Ca3.2 KO mice which also failed to develop painful PDN. Finally, we used biochemical methods with gel-shift analysis to directly demonstrate that N-terminal fragments of native Ca3.2 channels in the dorsal root ganglia (DRG) are glycosylated in both healthy and diabetic animals. Our results demonstrate that in sensory neurons glycosylation-induced alterations in Ca3.2 channels directly enhance diabetic hyperalgesia, and that glycosylation inhibitors can be used to ameliorate painful symptoms in Type 1 diabetes. We expect that our studies may lead to a better understanding of the molecular mechanisms underlying painful PDN in an effort to facilitate the discovery of novel treatments for this intractable disease.

Epigenetic modulation by DNA methylation is associated with aberrant gene expression in sensory neurons, which consequently leads to pathological pain responses. In this study, we sought to investigate whether peripheral inflammation alters global DNA methylation in trigeminal ganglia (TG) and results in abnormal expression of pro-nociceptive genes. Our results show that peripheral inflammation remotely reduced the level of global DNA methylation in rat TG with a concurrent reduction in and expression. Using unbiased steps, we selected the following pro-nociceptive candidate genes that are potentially regulated by DNA methylation: , and . Inhibition of DNMT with 5-Aza-dC in dissociated TG cells produced dose-dependent upregulation of , and . Systemic treatment of animals with 5-Aza-dC significantly increased the expression of , and in TG. Furthermore, the overexpression of DNMT3a, as delivered by a lentiviral vector, significantly downregulated and expression and also reliably decreased and transcripts. MeDIP revealed that this overexpression also significantly enhanced methylation of CGIs associated with and . In addition, bisulfite sequencing data indicated that the CGI associated with was methylated in a pattern catalyzed by DNMT3a. Taken together, our results show that all 4 pro-nociceptive genes are subject to epigenetic modulation via DNA methylation, likely via DNMT3a under inflammatory conditions. These findings provide the first evidence for the functional importance of DNA methylation as an epigenetic factor in the transcription of pro-nociceptive genes in TG that are implicated in pathological orofacial pain responses.