Differentially expressed genes in male and female DRG during pain and pain-relief; isoform specific cellular expression of RNAs - The objectives of the proposed research include 1) to define the differentially expressed RNA splice isoforms of ATF3 responsible for the sex-specific differences in pain and pain-relief and develop a model for their involvement in the associated molecular signaling, and 2) to provide rigorous training to undergraduate research scholars about pain research. Nerve injury creates a dynamic dialog between glia, infiltrating immune cells and neurons. In response to the milieu of cytokines, the peripheral neurons become hypersensitive, which the central nervous system interprets as pain. This environment causes changes in gene expression leading to chronic hypersensitivity, persisting even after healing. While acute hypersensitivity is an important component of the nociceptive signaling of injury, disruption of this process can cause protracted or debilitating pain, leading some patients to opioids for relief, and the possibility of experiencing opioid use disorder. Here, we propose to develop a more comprehensive understanding of the transcription factors that are differentially expressed in the rat dorsal root ganglia during neuroinflammation associated with hypersensitivity and pain, and that are differentially expressed between sexes during pain-relief. Rat neuropathic pain resulting from surgical chronic constriction of the sciatic nerve, causes hypersensitivity over the same time course and quantitatively to the same degree in both sexes. However, when treated with a single micro-dose of a nanotherapeutic containing 0.24 mg/kg of celecoxib, we find that males achieve multi-day pain relief equal to pre-surgical baseline, while females receiving the same treatment achieve only partial relief. This indicates that the underlying biology of the neuroinflammatory response that is reacting to the NSAID is different in males as compared to females. Using a higher single dose of celecoxib per nanodroplet (2.4 mg/kg), we have found that males and females can achieve the same relief. Under the conditions of equal relief, our NextSeq (Illumina) and separately Revio Long-Read (PacBio) RNA sequencing of the dorsal root ganglia revealed differential expression and alternative RNA splicing between the neuroinflammatory state and relief for males versus females. This collection of sexually dimorphic and differentially expressed genes includes genes of the Druggable Proteome. The primary target of our proposed analysis is ATF3, a cyclic AMP Response Element Binding protein (CREB) transcription factor responsive to nerve injury and nerve regeneration. Research by others show that ATF3 has distinct functions depending on the protein isoform produced by different mRNA splice variants. With teams of undergraduate research scholars, we propose to explore the cell specific and splice isoform specific expression of Atf3 mRNAs in the neurons, glia and infiltrating immune cells during pain, during pain relief and during tissue regeneration through the use of multi-color fluorescent in situ hybridization for both males and females. Other candidate genes that we will study include the transcription factors Fos and Nr1d1, and the dynamin-like GTPase MX1, each exhibiting sexually dimorphic and differential expression.
