4E-BP1-dependent translation in microglia controls mechanical hypersensitivity

Spinal microglia play a pivotal role in the development of neuropathic pain. Peripheral nerve injury induces changes in the transcriptional profile of microglia, including increased expression of components of translational machinery. Whether microglial protein synthesis is stimulated following nerve injury and has a functional role in mediating pain hypersensitivity is unknown. Here, we show that nascent protein synthesis is upregulated in spinal microglia following peripheral nerve injury. Stimulating mRNA translation in microglia, via selective ablation of the translational repressor, eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), promoted the transition of microglia to a reactive state and induced mechanical hypersensitivity. Conversely, inhibiting microglial translation by expressing mutant 4E-BP1 in microglia attenuated their peripheral nerve injury-induced activation and alleviated neuropathic pain. Thus, the stimulation of 4E-BP1-dependent translation promotes microglia reactivity and mechanical hypersensitivity, whereas its inhibition alleviates neuropathic pain. Ablation of 4E-BP1 stimulates translation of eIF4E-sensitive mRNAs, which are involved in immune responses, modulation of extracellular matrix, cell survival and growth. To better understand how 4E-BP1 ablation affects microglial functions and induces mechanical pain hypersensitivity, we studied changes in the translational landscape in microglia lacking 4E-BP1. To identify the actively translating mRNAs in microglia, we employed a translating ribosome affinity purification (TRAP) approach . In TRAP, a ribosomal subunit L10a tagged with eGFP (L10a-eGFP) is expressed in a specific cell type. Immunoprecipitation (IP) with eGFP antibody captures the eGFP-tagged ribosomes and subsequent sequencing of immunoprecipitated ribosome-bound mRNAs allows the identification of actively translated mRNAs in the specific cell type.