Characterization of Fragile X Mental Retardation Protein expression in human nociceptors and their axonal projections to the spinal dorsal horn

Abstract

Fragile X Mental Retardation Protein (FMRP) regulates activity‐dependent RNA localization and local translation to modulate synaptic plasticity throughout the central nervous system. Mutations in the FMR1 gene that hinder or ablate FMRP function cause Fragile X Syndrome (FXS), a disorder associated with sensory processing dysfunction. FXS premutations are associated with increased FMRP expression and neurological impairments including sex dimorphic presentations of chronic pain. In mice, FMRP ablation causes dysregulated dorsal root ganglion (DRG) neuron excitability and synaptic vesicle exocytosis, spinal circuit activity, and decreased translation‐dependent nociceptive sensitization. Activity‐dependent, local translation is a key mechanism for enhancing primary nociceptor excitability that promotes pain in animals and humans. These works indicate that FMRP likely regulates nociception and pain at the level of the primary nociceptor or spinal cord. Therefore, we sought to better understand FMRP expression in the human DRG and spinal cord using immunostaining in organ donor tissues. We find that FMRP is highly expressed in DRG and spinal neuron subsets with substantia gelatinosa exhibiting the most abundant immunoreactivity in spinal synaptic fields. Here, it is expressed in nociceptor axons. FMRP puncta colocalized with Nav1.7 and TRPV1 receptor signals suggesting a pool of axoplasmic FMRP localizes to plasma membrane‐associated loci in these branches. Interestingly, FMRP puncta exhibited notable colocalization with calcitonin gene‐related peptide (CGRP) immunoreactivity selectively in female spinal cord. Our results support a regulatory role for FMRP in human nociceptor axons of the dorsal horn and implicate it in the sex dimorphic actions of CGRP signaling in nociceptive sensitization and chronic pain.

Interleukin-6 induces nascent protein synthesis in human DRG nociceptors via MNK-eIF4E signaling

“…Click chemistry reactions were carried out at room temperature while mixing for 20 to 24 hrs. Sections were then processed for immunofluorescence (IF) to detect either TRPV1 (488) or p-S209-eIF4E (488 or 555) and NeuN (488 or 555) (Shiers et al, 2021; Mitchell et al, 2023). All antibodies used in this study are listed in Table 3 .…”

“…For DRG neuronal somata, ROIs were identified in 12-bit images of TRPV1 IF (nociceptors) or NeuN IF (DRG neurons) channels. Somata were defined as having a minimum surface area of 350 µm 2 (Mitchell et al, 2023)1. Then, raw FUNCAT and/or p-eIF4E IF signals were measured per ROI, respectively.…”

“…Samples were treated with primary antibodies overnight at room temperature and incubated with secondary antibodies the next day for 1 hr before mounting. Lipofuscin, a structure inherent to human nervous tissues that is highly autofluorescent, was detected as described previously for subtraction (Mitchell et al, 2023). For explant fluorescence intensity approximations, FUNCAT signal was measured across the field of view for the entire section surface area for 4 donors.…”

“…Lipofuscin auto-signal was subtracted from each channel in 10x images using FIJI (NIH, Bethesda, MD). Auto-signal ROIs were identified using automated detection or manual tracing as described previously (Mitchell et al, 2023). Our automated macro parameters and codes are available upon request.…”

Case Series: Vestibular Migraines in Fragile X Premutation Carriers

“…Other manifestations of CN VIII toxicity in FXTAS include hearing loss, tinnitus, and imbalance, which are common [15,27,37]. A study by Mitchell et al [38] revealed that FMRP may be a modulator of calcitonin gene-related peptide (CGRP) signaling in nociceptive sensitization and chronic pain. CGRP signaling plays an important role in hearing and balance and the development of migraine [39]; therefore, altered levels of FMRP in premutation carriers could contribute to further dysfunction of CN VIII and increased risk of developing VM.…”