Translational control in the spinal cord regulates gene expression and pain hypersensitivity in the chronic phase of neuropathic pain

Sensitization of spinal nociceptive circuits plays a cardinal role in neuropathic pain. This sensitization depends on new gene expression that is primarily regulated via transcriptional and translational control mechanisms. The relative roles of these mechanisms in regulating gene expression in the clinically relevant chronic phase of neuropathic pain are not well understood. Here, we show that changes in gene expression in the spinal cord during the chronic phase of neuropathic pain are substantially regulated at the translational level. Downregulating spinal translation at the chronic phase alleviated pain hypersensitivity. Cell-type-specific profiling revealed that spinal inhibitory neurons exhibited greater changes in translation after peripheral nerve injury compared to excitatory neurons. Notably, increasing translation selectively in all inhibitory neurons or parvalbumin-positive (PV + ) interneurons, but not excitatory neurons, promoted mechanical pain hypersensitivity. Furthermore, increasing translation in PV + neurons decreased their intrinsic excitability and spiking activity, whereas reducing translation in spinal PV + neurons prevented the nerve injury-induced decrease in excitability. Thus, translational control mechanisms in the spinal cord, primarily in inhibitory neurons, play a critical role in mediating neuropathic pain hypersensitivity. Overall design: Translational regulation in chronic pain conditions was investigated at the tissue (exploratory stage) and cell level using two distinct workflows, namely Ribosome profiling and translating ribosome affinity purification (TRAP). First, Ribosome profiling was used to identify the genes that are translationally regulated in dorsal root ganglia (DRG) and spinal cord (SC) in early (day 4) and late (day 63) stages of neuropathic pain and at day 4 of inflammatory pain. Spared nerve injury (SNI) surgery was used to establish a mouse model of neuropathic pain, with sham surgery as a control, and Complete Freund's Adjuvant (CFA)injection was used to establish the mouse model for inflammatory pain, with vehicle injection as a control. Ribosome footprints (RFP, ~28 nucleotide fragment of mRNA where ribosomes were bound during translation) and bulk mRNA were sequenced from DRG and SC tissues of mice undergone either SNI/Sham surgery at day 4 or day 63 time points or CFA/vehicle injections at day 3 time point. Each condition had two biological replicates for both RFP and mRNA samples. Secondly, to identify the actively translating mRNAs in excitatory and inhibitory neuronal populations, we employed a translating ribosome affinity purification (TRAP) to study cell specific translational regulation. In TRAP, a ribosomal subunit L10a tagged with eGFP (L10a-eGFP) is expressed in specific cell types – GAD2 for inhibitory neurons and TAC1 for excitatory neurons. Immunoprecipitation (IP) with eGFP antibody was performed on cell lysates of spinal cord to capture the eGFP-tagged ribosomes and subsequent sequencing of immunoprecipitated ribosome-bound mRNAs was performed to identify actively translated mRNAs in the specific cell types. For each cell type, TRAP was performed on mice which underwent either SNI or sham surgery at two points (day 4 and day 60) post-surgery. For each condition the RNA obtained by immunoprecipitation (IP) and RNA extracted from a fraction of the input cell lysate (Input) were sequenced. The input sample was used to identify the mRNA and their abundance present in the tissue. Each of the IP and input samples had three biological replicates.