Deciphering the Functions of Collapsin Response Mediator Protein 2 (CRMP2) in Nociceptive Spinal Neurotransmission

Abstract

Chronic neuropathic pain results from a malfunction of the sensory nervous system initially wired to perceive pain as a protective mechanism against tissue injury and damage. The first site of synaptic integration in the spinal dorsal horn is an important location where nociceptive signals can be curbed before they reach the brain. Previously, the Khanna laboratory identified the collapsin response mediator protein 2 (CRMP2) as a key protein controlling nociceptive signaling via dual regulation of voltage-gated sodium NaV1.7 and voltage-gated sodium CaV2.2 channels. However, the exact role of CRMP2 in spinal neurotransmission and in the chronification of pain had never been explored. My thesis focused on deciphering the function of CRMP2 in the dorsal horn of the spinal cord and identifying regulatory pathways upstream of CRMP2 that could regulate its functions.First, using siRNA and Cre-lox recombination to delete the spinal expression of CRMP2, we showed that CRMP2 exclusively regulates spinal glutamatergic neurotransmission while inhibitory neurotransmission was impervious to CRMP2 expression. Furthermore, we found that CRMP2 spinal expression is a major driver of pain signaling and is required for the transition from physiological to persistent pain after a nerve injury. Next, we explored if the canonical signaling pathway regulating CRMP2 during neuronal polarization establishment could also control CRMP2 spinal functions. The canonical pathway where the receptor Neuropilin 1 (NRP1) integrates several guidance signals such as Semaphorin3A (Sema3A) and vascular endothelial growth factor A (VEGFA, which reportedly contributes to different forms of neuropathic pain), leads to increased phosphorylation of CRMP2 by the cyclin dependent kinase 5 (Cdk5). We showed that activation of NRP1 by VEGFA enhances glutamatergic spinal neurotransmission and triggers acute nociceptive pain. We demonstrated via pharmacological and genetic (CRISPR/Cas9) inhibition of NRP1, that VEGFA-mediated hypersensitivity and increased spinal neurotransmission was via the engagement of the NRP1 pathway. In chronic neuropathic pain, Cdk5-mediated phosphorylation of CRMP2 is upregulated leading to strikingly similar outcomes than following NRP1 activation. Using an inhibitor of VEGFA binding to NRP1 developed in the laboratory, we confirmed that the VEGFA signaling via NRP1 increases CRMP2 phosphorylation by Cdk5. We then blocked CRMP2 phosphorylation by Cdk5 with (S)-Lacosamide, which showed that VEGFA/NRP1 mediated enhancement of nociceptor excitability required Cdk5 phosphorylated CRMP2. Finally, we took advantage of transgenic mice where CRMP2 is resistant to the phosphorylation by Cdk5 and used electrophysiological recordings from spinal cord slices and from cultured dorsal root ganglia (DRG). We found that sensory neurons prepared from this transgenic mouse line were resistant to VEGFA and that these animals failed to develop chronic mechanical allodynia following a spared nerve injury. Collectively, these results acquired during my thesis demonstrate that CRMP2 expression controls nociceptive spinal neurotransmission. I also found that in chronic neuropathic pain, VEGFA signaling via NRP1 increases CRMP2 phosphorylation by Cdk5 as an unknown mechanism essential for the transition to persistent pain. Additionally, I characterized a novel small molecule to target this previously unknown pathway in chronic pain which opens the path for a new class of therapeutics regulating CRMP2 for the treatment of chronic pain with the potential to be disease-modifying.