(J-366) The RNA-binding protein CUGBP Elav-like family member 4 (CELF4) is a negative regulator of nociceptor excitability and hyperalgesia in mice

RNA binding proteins (RBPs) regulate gene function by controlling RNA processing, transport, stability, and translation. Dysfunctional RNA-protein interactions within the nervous system are a contributing factor to neurodegenerative disease and maladaptive pain. Computational analyses of binding sites on pro-nociceptive mRNAs revealed CUGBP Elav-like family member 4 (CELF4) as a candidate regulator of sensory neuron sensitivity. Previously, CELF4 expression and function have been characterized within the central nervous system where Celf4 deficient mice are reported to have abnormal excitatory neurotransmission that causes a complex seizure disorder ^[1]. Histological assessments of naïve rodent dorsal root ganglia (DRG) revealed that CELF4 is highly expressed in capsaicin sensitive (TRPV1+), small to medium diameter sensory neurons, likely nociceptors. Considering these findings, we sought to determine if CELF4 expression impacts the function of sensory neurons.

Tamoxifen-Inducible Celf4 Knockout: Conditional knockout of Celf4 from adult sensory neurons was performed in triple transgenic Celf4^fl/fl; Avil-creERT2+ ; TdTomato mice treated with i.p. administration of tamoxifen. Celf4^fl/fl ; Avil-creERT2- ; TdTomato and Celf4^wt/wt ; Avil-creERT2+ ; TdTomato mice were used as controls.

Confirmation of Celf4 Knockout: A previously characterized primary antibody highly specific for CELF4 was used to confirm CELF4 knockout in sensory neurons following tamoxifen treatment using immunohistochemistry and Western Blot protein assays.

Behavioral Assays: Both sexes were used for all behavioral assays. Behavioral testing was conducted 2 days after the final tamoxifen injection and mice were habituated to all testing equipment prior to experimentation. Von Frey filaments were used to determine tactile sensory thresholds at baseline and on day 7 using the up-down method of testing with a 0.4g starting filament. Hargreaves assay was used to assess thermal sensitivities following tamoxifen treatment by applying a light source at 20% active intensity to the hind paw and measuring thermal latencies.

Electrophysiology: Patch clamp recordings were performed on acutely dissociated adult DRG from male controls (Celf4^wt/wt ; Avil-creERT2+) and Celf4 knockout mice to assess changes in capsaicin-sensitive neuronal excitability.

Genetically modified mice were used to assess the phenotype associated with knockout (KO) of Celf4 from sensory neurons. Von Frey and Hargreaves behavioral assays were used to assess sensitivities to mechanical and warm thermal stimuli, respectively. As seen in Figure 1, von Frey and Hargreaves assays revealed that conditional KO of Celf4 from sensory neurons induces a robust behavioral hypersensitivity to mechanical and thermal stimuli in male and female mice. These hypersensitivities are represented as significant changes in tactile thresholds and thermal latencies between baseline measurements prior to tamoxifen treatment and behavioral measurements taken on day 7 following tamoxifen induced Celf4 KO. These reflexive behavioral assays showed no significant sex differences in mechanical or heat sensitivity. Future behavioral studies will focus on assessing sensitivities to cold stimuli using cold temperature assays and will utilize non-reflexive behavioral assays to further characterize this phenotype. In addition to behavioral assessments, patch-clamp recordings showed acutely dissociated, capsaicin sensitive (TRPV1+) dorsal root ganglia neurons become hyperexcitable following Celf4 KO. Together these data implicate CELF4 as a tonic suppressor of sensory neuron excitability and therefore a promising target for future therapeutic modulation of sensory neuron sensitivity in various neuropathic and inflammatory pain conditions.