Pain is the most common and debilitating medical problem for which patients seek medical care. Opioids remain the gold standard in the treatment of pain but are limited by poor side effect profiles such as emesis, constipation, dependence/addiction and respiratory depression. Despite a myriad of analgesic compounds on the market, tri-cyclic antidepressants, opioids, anticonvulsants, non-steroidal anti-inflammatory agents and combinations thereof, nearly two thirds of the chronic pain patients report inadequate pain relief; therefore, a new approach in the development of pain management is necessary. In recent years, the N-Type voltage gated calcium channel (CaV2.2) has become an attractive target in the treatment of chronic pain. Ziconotide, a selective CaV2.2 blocker, has been FDA approved in the United States for the treatment of severe chronic pain that is refractory to other treatments, but due to its profound side effect profile (nausea/vomiting, somnolence, vertigo, muscle spasms, myalgia, insomnia, anxiety, tremor, memory impairment and induced psychiatric disorders), the use of Ziconotide is severely limited. Mapping of the CaV2.2 interactome led to the identification of novel regulatory proteins, including collapsin response mediator protein 2 (CRMP2). Initially identified as an intracellular protein in the specification of axon/dendrite fate and axonal outgrowth, it is now known that this protein can regulate the activity of CaV2.2 and hence may be a critical regulatory node in pain modulation. Here, I describe a novel peptide aptamer derived from CRMP2, designated CaV2.2 binding domain 3 (CBD3), which when fused with the HIV transactivator of transcription protein (TAT), created tat-CBD3, which was able to significantly reverse thermal and mechanical hypersensitivity induced by the surgical incision on the plantar surface of the left hind paw in rats, a pre-clinical model of post-operative pain. Additionally, tat-CBD3 significantly attenuated thermal hypersensitivity induced via intraplantar injection of carrageenan, a model of acute inflammatory pain. Furthermore, the administration of tat-CBD3 did not produce any rewarding behaviors as measured by the conditioned placed preference (CPP) paradigm, nor did the administration of tat-CBD3 produced any motor coordination deficits measured using the rotarod performance test. Moreover, the addition of a 14-carbon myristate (myr) group to the parent peptide, myr-tat-CBD3, had increased efficacy in the attenuation of paw incision and carrageenan induced thermal/mechanical hypersensitivities when compared to the parent peptide (tat-CBD3). These types of novel compounds that lack unwanted side effects and addiction propensities are urgently needed to relieve individuals suffering from chronic pain.

Yes / O-GlcNAcylation is an abundant post-translational modification in the nervous system, linked to both neurodevelopmental and neurodegenerative disease. However, the mechanistic links between these phenotypes and site-specific O-GlcNAcylation remain largely unexplored. Here, we show that Ser517 O-GlcNAcylation of the microtubule-binding protein Collapsin Response Mediator Protein-2 (CRMP2) increases with age. By generating and characterizing a Crmp2S517A knock-in mouse model, we demonstrate that loss of O-GlcNAcylation leads to a small decrease in body weight and mild memory impairment, suggesting that Ser517 O-GlcNAcylation has a small but detectable impact on mouse physiology and cognitive function. / a Wellcome Trust Senior Research Fellowship (WT087590MA) to D.M.F.v.A., an ARUK Pilot Project grant to R.W., and support from Tenovus Scotland to V.M. The phosphoproteomics mass spectrometry work was supported by the Horizon 2020 program INFRAIA project Epic-XS (project 823839) to A.J.R.H.

Le mélanome uvéal (MU) est une tumeur rare, affectant 500 à 600 nouveaux cas par an en France, mais il s’agit de la tumeur intraoculaire la plus fréquente chez l’adulte. Cette tumeur est due à la transformation des mélanocytes dérivés de la crête neurale et localisés dans l’uvée (choroïde, corps ciliaire et iris). C’est une tumeur très agressive puisque, malgré le traitement de la tumeur primaire, jusqu’à 50% des patients développeront des métastases hépatiques. En effet, malgré les nombreux efforts fournis pour le développement de nouvelles thérapies, celles-ci se sont révélées jusqu’à présent peu efficaces. Ainsi, une meilleure compréhension du processus métastatique et l’identification des gènes impliqués dans celui-ci sont des enjeux importants pour permettre le développement de nouvelles thérapies. Une analyse transcriptomique réalisé dans le laboratoire a permis d’identifier la phosphatase PRL-3 dont la surexpression est en corrélation avec le développement métastatique et le mauvais pronostic de survie des patients. Des études fonctionnelles ont montré que les cellules de MU exprimant PRL-3 avaient une capacité de migration et d’invasion accrue par rapport aux cellules exprimant une forme mutante catalytiquement inactive de la phosphatase. L’implication de PRL-3 dans le développement tumoral et le processus métastatique a été très largement décrite dans de nombreux cancers et son mécanisme d’action et ses cibles sont en cours d’investigation. De façon intéressante, certaines cibles de PRL-3 sont des protéines liées au cytosquelette. Dans le but d’identifier de nouvelles cibles de PRL-3 dans le MU, nous avons réalisé une analyse phosphoprotéomique qui nous a permis de mettre en évidence la protéine CRMP2. CRMP2 est une protéine liée au cytosquelette dont le rôle a majoritairement été décrit dans le système nerveux. CRMP2 joue un rôle dans le guidage axonal, l’extensition des neurites, la dynamique des microtubules et le trafic vésiculaire. Au cours de ma thèse, j’ai pu confirmer que dans les cellules de MU, PRL-3 modifie l’état de phosphorylation de CRMP2 en particulier sur les résidus T514 et S522. De plus, CRMP2 et PRL-3 interagisse et cette interaction aboutit à une diminution de la phosphorylation de CRMP2 sur le T514, ce qui suggère que CRMP2 est une cible directe de PRL-3. Des études fonctionnelles ont montré que l’extinction de CRMP2 par ARN interférent augmente la vitesse de migration et l’invasion des cellules de MU qui expriment PRL-3. CRMP2 est donc un frein à la migration et l’invasion des cellules de MU. La migration et l’invasion accrue dans ces cellules sont corrélées à un réarrangement du réseau d’actine avec une diminution des fibres de stress. L’étude des propriétés micro-rhéologiques des cellules de MU montre également que l’expression de PRL-3 et/ou l’extinction de CRMP2 augmente la viscosité, l’élasticité et la dureté du cytoplasme. L’extinction de CRMP2 ne modifie pas ces propriétés dans les cellules exprimant déjà PRL-3 ce qui montre que l’effet de l’extinction de CRMP2 est équivalent à celui de déphosphorylation. Enfin, dans les tumeurs l’expression de CRMP2 est associée à un bon pronostic de survie. Au cours de ma thèse, j’ai également participé à la création d’une carte du réseau d’interaction des molécules et des voies de signalisation impliquant PRL-3 dans le développement du cancer dans le cadre du projet NaviCell. Cette carte a été intégrée à la base de donnée ACSN. / Uveal melanoma (UM) is a rare tumor (500 to 600 new cases by year in France) but this is the most common intraocular cancer in adult. This tumor is due to melanocytes transformation derived from neural crest and localized in uvea (choroid, ciliary body and iris). Despite the primary tumor treatment, up to 50% of patients will develop metastases mostly localized in liver within the years following diagnosis. The median survival rate of metastasis formation is only six months. Indeed, there are currently no effective treatments against metastasis. Therefore, a better comprehension of UM metastatic process and identification of the genes involved are major issues for the development of new therapies. A transcriptomic analysis done in our laboratory allows to identify the phosphatase PRL-3 whose overexpression is correlated with metastatic development and patients poor prognosis. Besides being a bad prognosis marker, functional studies showed that UM cells expressing PRL-3 migrate faster and are more invasive than cells expressing the catalytic dead mutant of the phosphatase. The role of PRL-3 in tumorigenesis and metastatic development is well described, and numerous studies investigate his mechanism of action and his intracellular substrates. Interestingly, some of the identified PRL-3’s targets are cytoskeletal proteins. In order to identify new PRL-3’s targets in UM, we realized a phosphoproteomic analysis that allows us to identify CRMP2. CRMP2 is a cytoskeletal protein that has been mostly described in the nervous system. CRMP2 plays a role in axonal guidance, neuritis extension, microtubules dynamics and vesicular trafficking. During my PhD, I confirmed that in UM cells, PRL-3 expression modify the phosphorylation state of CRMP2 in particular on T514 and S522 residues. Moreover, CRMP2 and PRL-3 interact together and CRMP2 is less phosphorylated on the T514 following the interaction, which suggests that CRMP2 is a target of PRL-3. Furthermore, we showed that CRMP2 KO by shRNA increases UM cells velocity and invasion in cells expressing PRL-3. So CRMP2 is a brake to the migration and invasion process in UM cells. These observations are correlated with a reorganization of actin cytoskeleton with a reduction of stress fibers. The study of the microrheological properties of UM cells showed that PRL-3 expression and/or CRMP2 KO increases the viscosity, elasticity and stiffness of the cytoplasm. CRMP2 KO changes these properties only when PRL-3 is not functional which suggests that concerning the micro-rheology, CRMP2 KO is equivalent the its dephosphorylation by PRL-3. Finally, in UM tumors, CRMP2 expression are correlated with good survival prognosis. My PhD also gave me the opportunity to participate to the creation of a map about interactions and signaling pathways involving PRL-3 in cancers as part of the NaviCell project. This map was integrated in ASCN database.

Chronic pain affects the life of millions of people. Current treatments have deleterious side effects. We have advanced a strategy for targeting protein interactions which regulate the N-type voltage-gated calcium (CaV2.2) channel as an alternative to direct channel block. Peptides uncoupling CaV2.2 interactions with the axonal collapsin response mediator protein 2 (CRMP2) were antinociceptive without effects on memory, depression, and reward/addiction. A search for small molecules that could recapitulate uncoupling of the CaV2.2-CRMP2 interaction identified (S)-lacosamide [(S)-LCM], the inactive enantiomer of the Food and Drug Administration-approved antiepileptic drug (R)-lacosamide [(R)-LCM, Vimpat]. We show that (S)-LCM, but not (R)-LCM, inhibits CRMP2 phosphorylation by cyclin dependent kinase 5, a step necessary for driving CaV2.2 activity, in sensory neurons. (S)-lacosamide inhibited depolarization-induced Ca influx with a low micromolar IC50. Voltage-clamp electrophysiology experiments demonstrated a commensurate reduction in Ca currents in sensory neurons after an acute application of (S)-LCM. Using constellation pharmacology, a recently described high content phenotypic screening platform for functional fingerprinting of neurons that uses subtype-selective pharmacological agents to elucidate cell-specific combinations (constellations) of key signaling proteins that define specific cell types, we investigated if (S)-LCM preferentially acts on certain types of neurons. (S)-lacosamide decreased the dorsal root ganglion neurons responding to mustard oil, and increased the number of cells responding to menthol. Finally, (S)-LCM reversed thermal hypersensitivity and mechanical allodynia in a model of postoperative pain, and 2 models of neuropathic pain. Thus, using (S)-LCM to inhibit CRMP2 phosphorylation is a novel and efficient strategy to treat pain, which works by targeting specific sensory neuron populations.

No description available.

Neurons are generated from unspecialized neural precursor cells (NPCs) in a process termed neurogenesis. This neuronal development continues throughout life in the ventricular zone (VZ) of the lateral ventricles, and the subgranular zone (SGZ) of the dentate gyrus in the hippocampus. NPCs undergo a complex and highly regulated set of behaviours in order to ultimately integrate into the existing brain circuitry as fully functional neurons. Recently the pannexin (Panx) large-pore channel proteins were discovered. One family member, Panx1 is expressed in the nervous system in mature neurons, and acts as an ATP release channel in various cell types throughout the body. Post-natal NPCs are responsive to ATP via activation of purinergic receptors, which modulate a variety of NPC behaviours. I therefore investigated the hypothesis that Panx1 was expressed in post-natal VZ NPCs, where it functioned as an ATP release channel and regulated neuronal development. In the course of my studies, I found that Panx1 positively regulated NPC proliferation and migration, and negatively regulated neurite outgrowth in vitro. Using an NPC-specific Panx1 knock-out strategy, I showed that Panx1 expression was required for maintenance of a consistent population of VZ NPCs in vivo in both healthy and injured brain. Together these data indicated that Panx1 directed NPC behaviours associated with neuronal development both in vitro and in vivo. To further understand the molecular underpinnings of this regulation, I examined the Panx1 interactome, and uncovered a novel association with collapsin response mediator protein 2 (Crmp2). Functional studies suggested that this interaction likely was at least in part responsible for Panx1’s negative impact on neurite outgrowth. Overall, my results represent important novel findings that contribute to our understanding of post-natal neuronal development and the molecular function of Panx1 within the brain. / Graduate / 0317 / 0379 / leighws@uvic.ca

Regulation of axon guidance and pruning of inappropriate synapses is key to development of neural circuits. Secreted semaphorins are integral part of both processes. Collapsin response mediator protein 2 (CRMP2) has been shown to regulate axon guidance by mediating Semaphorin 3A (Sema3A) signaling, however, nothing is known about its role in the synapse pruning. Similarly, it is also not known if CRMP2 mediates signals from other semaphorins. We herein studied CRMP2 protein and revealed its role in growth and pruning of selected axons and dendrites. In newly generated crmp2-/- and crmp2a-/- mice we demonstrate that CRMP2 has a moderate effect on Sema3A-dependent axon guidance in vivo, and its deficiency leads to a mild defect in axon guidance in peripheral nerves and corpus callosum. CRMP2A isoform is specifically involved in development of callosal axons. Surprisingly, we show that crmp2-/- mice display prominent defects in stereotyped axon pruning in hippocampus and visual cortex and altered dendritic spine remodeling, which are consistent with impaired Sema3F signaling and with models of autism spectrum disorder (ASD). Indeed, we demonstrate that CRMP2 mediates Sema3F signaling in primary neurons and that crmp2-/- mice display ASD-related social behavior changes in early postnatal period as well...

Indiana University-Purdue University Indianapolis (IUPUI) / The voltage-gated sodium channel Nav1.7 has gained traction as a pain target with recognition that loss-of-function mutations in SCN9A, the gene encoding Nav1.7, are associated with congenital insensitivity to pain, whereas gain-of-function mutations produce distinct pain syndromes due to increased Nav1.7 activity. Selective inhibition of Nav1.7 is fundamental to modulating pain via this channel. Understanding the regulation of Nav1.7 at the cellular and molecular level is critical for advancing better therapeutics for pain. Although trafficking of Nav1.7 remains poorly understood, recent studies have begun to investigate post-translational modifications of Navs and/or auxiliary subunits as well as protein-protein interactions as Nav-trafficking mechanisms. Here, I tested if post-translational modifications of a novel Nav1.7-interacting protein, the axonal collapsin response mediator protein 2 (CRMP2) by small ubiquitin-like modifier (SUMO) and phosphorylation could affect Nav trafficking and function. Expression of a CRMP2 SUMOylation incompetent mutant (CRMP2-K374A) in neuronal model CAD cells, which express predominantly Nav1.7 currents, led to a significant reduction in huwentoxin-IV-sensitive Nav1.7 currents. Increasing deSUMOylation with sentrin/SUMO-specific protease SENP1 or SENP2 in wildtype CRMP2-expressing CAD cells decreased Nav1.7 currents. Consistent with reduced current density, biotinylation revealed significant reduction in surface Nav1.7 levels of CAD cells expressing CRMP2-K374A or SENP proteins. Diminution of Nav1.7 sodium current was recapitulated in sensory neurons expressing CRMP2-K374A. Because CRMP2 functions are regulated by its phosphorylation state, I next investigated possible interplay between phosphorylation and SUMOylation of CRMP2 on Nav1.7. Phosphorylation of CRMP2 by cyclin dependent kinase 5 (Cdk5) was necessary for maintaining Nav1.7 surface expression and current density whereas phosphorylation by Fyn kinase reduced CRMP2 SUMOylation and Nav1.7 current density. Binding to Nav1.7 was decreased following (i) loss of CRMP2 SUMOylation, (ii) loss of CRMP2 phosphorylation by Cdk5, or (iii) gain of CRMP2 phosphorylation by Fyn. Altering CRMP2 modification events simultaneously was not synergistic in reducing Nav1.7 currents, suggesting that Nav1.7 co-opts multiple CRMP2 modifications for regulatory control of this channel. Loss of either CRMP2 SUMOylation or Cdk5 phosphorylation triggered Nav1.7 internalization involving E3 ubiquitin ligase Nedd4-2 as well as endocytosis adaptor proteins Numb and Eps15. Collectively, my findings identify a novel mechanism for regulation of Nav1.7.

Indiana University-Purdue University Indianapolis (IUPUI) / Posttraumatic epilepsy, the development of temporal lobe epilepsy (TLE) following traumatic brain injury, accounts for 20% of symptomatic epilepsy. Reorganization of mossy fibers within the hippocampus is a common pathological finding of TLE. Normal mossy fibers project into the CA3 region of the hippocampus where they form synapses with pyramidal cells. During TLE, mossy fibers are observed to innervate the inner molecular layer where they synapse onto the dendrites of other dentate granule cells, leading to the formation of recurrent excitatory circuits. To date, the molecular mechanisms contributing to mossy fiber sprouting are relatively unknown. Recent focus has centered on the involvement of tropomycin-related kinase receptor B (TrkB), which culminates in glycogen synthase kinase 3β (GSK3β) inactivation. As the neurite outgrowth promoting collapsin response mediator protein 2 (CRMP2) is rendered inactive by GSK3β phosphorylation, events leading to inactivation of GSK3β should therefore increase CRMP2 activity. To determine the involvement of CRMP2 in mossy fiber sprouting, I developed a novel tool ((S)-LCM) for selectively targeting the ability of CRMP2 to enhance tubulin polymerization. Using (S)-LCM, it was demonstrated that increased neurite outgrowth following GSK3β inactivation is CRMP2 dependent. Importantly, TBI led to a decrease in GSK3β-phosphorylated CRMP2 within 24 hours which was secondary to the inactivation of GSK3β. The loss of GSK3β-phosphorylated CRMP2 was maintained even at 4 weeks post-injury, despite the transience of GSK3β-inactivation. Based on previous work, it was hypothesized that activity-dependent mechanisms may be responsible for the sustained loss of CRMP2 phosphorylation. Activity-dependent regulation of GSK3β-phosphorylated CRMP2 levels was observed that was attributed to a loss of priming by cyclin dependent kinase 5 (CDK5), which is required for subsequent phosphorylation by GSK3β. It was confirmed that the loss of GSK3β-phosphorylated CRMP2 at 4 weeks post-injury was likely due to decreased phosphorylation by CDK5. As TBI resulted in a sustained increase in CRMP2 activity, I attempted to prevent mossy fiber sprouting by targeting CRMP2 in vivo following TBI. While (S)-LCM treatment dramatically reduced mossy fiber sprouting following TBI, it did not differ significantly from vehicle-treated animals. Therefore, the necessity of CRMP2 in mossy fiber sprouting following TBI remains unknown.