Erik T. Dustrude

Inhibition of Transmitter Release and Attenuation of Anti-retroviral-associated and Tibial Nerve Injury-related Painful Peripheral Neuropathy by Novel Synthetic Ca2+ Channel Peptides

Background: N-type Ca2+ channels (CaV2.2) are clinically validated targets for chronic pain. Results: Two peptides from CaV2.2 and CaV1.2 perturb binding to a regulatory protein, CRMP2, inhibit calcium influx, and attenuate mechanical hyperalgesia in a rodent model of drug-induced chronic pain. Conclusion: Ca2+ channel peptides block drug- and nerve injury-induced chronic pain. Significance: Ca2+ channel peptide therapeutics can be useful in mitigating chronic pain. N-type Ca2+ channels (CaV2.2) are a nidus for neurotransmitter release and nociceptive transmission. However, the use of CaV2.2 blockers in pain therapeutics is limited by side effects resulting from inhibition of the physiological functions of CaV2.2 within the CNS. We identified an anti-nociceptive peptide (Brittain, J. M., Duarte, D. B., Wilson, S. M., Zhu, W., Ballard, C., Johnson, P. L., Liu, N., Xiong, W., Ripsch, M. S., Wang, Y., Fehrenbacher, J. C., Fitz, S. D., Khanna, M., Park, C. K., Schmutzler, B. S., Cheon, B. M., Due, M. R., Brustovetsky, T., Ashpole, N. M., Hudmon, A., Meroueh, S. O., Hingtgen, C. M., Brustovetsky, N., Ji, R. R., Hurley, J. H., Jin, X., Shekhar, A., Xu, X. M., Oxford, G. S., Vasko, M. R., White, F. A., and Khanna, R. (2011) Suppression of inflammatory and neuropathic pain by uncoupling CRMP2 from the presynaptic Ca2+ channel complex. Nat. Med. 17, 822–829) derived from the axonal collapsin response mediator protein 2 (CRMP2), a protein known to bind and enhance CaV2.2 activity. Using a peptide tiling array, we identified novel peptides within the first intracellular loop (CaV2.2(388–402), “L1”) and the distal C terminus (CaV1.2(2014–2028) “Ct-dis”) that bound CRMP2. Microscale thermophoresis demonstrated micromolar and nanomolar binding affinities between recombinant CRMP2 and synthetic L1 and Ct-dis peptides, respectively. Co-immunoprecipitation experiments showed that CRMP2 association with CaV2.2 was inhibited by L1 and Ct-dis peptides. L1 and Ct-dis, rendered cell-penetrant by fusion with the protein transduction domain of the human immunodeficiency virus TAT protein, were tested in in vitro and in vivo experiments. Depolarization-induced calcium influx in dorsal root ganglion (DRG) neurons was inhibited by both peptides. Ct-dis, but not L1, peptide inhibited depolarization-stimulated release of the neuropeptide transmitter calcitonin gene-related peptide in mouse DRG neurons. Similar results were obtained in DRGs from mice with a heterozygous mutation of Nf1 linked to neurofibromatosis type 1. Ct-dis peptide, administered intraperitoneally, exhibited antinociception in a zalcitabine (2′-3′-dideoxycytidine) model of AIDS therapy-induced and tibial nerve injury-related peripheral neuropathy. This study suggests that CaV peptides, by perturbing interactions with the neuromodulator CRMP2, contribute to suppression of neuronal hypersensitivity and nociception.

CRMP2 Protein SUMOylation Modulates NaV1.7 Channel Trafficking

Background: Post-translational modifications of CRMP2 protein direct its regulation of effector proteins. Results: Destruction of a CRMP2 SUMOylation site reduces surface expression and current density of sodium channel NaV1.7. Conclusion: CRMP2 SUMOylation choreographs NaV1.7, but not NaV1.1 or NaV1.3, trafficking. Significance: Learning how neuronal NaV1.7 trafficking is modulated by CRMP2 is important for understanding the mechanism of action of NaV-targeted anti-epileptic and anti-nociceptive drugs. Voltage-gated sodium channel (NaV) trafficking is incompletely understood. Post-translational modifications of NaVs and/or auxiliary subunits and protein-protein interactions have been posited as NaV-trafficking mechanisms. Here, we tested if modification of the axonal collapsin response mediator protein 2 (CRMP2) by a small ubiquitin-like modifier (SUMO) could affect NaV trafficking; CRMP2 alters the extent of NaV slow inactivation conferred by the anti-epileptic (R)-lacosamide, implying NaV-CRMP2 functional coupling. Expression of a CRMP2 SUMOylation-incompetent mutant (CRMP2-K374A) in neuronal model catecholamine A differentiated (CAD) cells did not alter lacosamide-induced NaV slow inactivation compared with CAD cells expressing wild type CRMP2. Like wild type CRMP2, CRMP2-K374A expressed robustly in CAD cells. Neurite outgrowth, a canonical CRMP2 function, was moderately reduced by the mutation but was still significantly higher than enhanced GFP-transfected cortical neurons. Notably, huwentoxin-IV-sensitive NaV1.7 currents, which predominate in CAD cells, were significantly reduced in CAD cells expressing CRMP2-K374A. Increasing deSUMOylation with sentrin/SUMO-specific protease SENP1 or SENP2 in wild type CRMP2-expressing CAD cells decreased NaV1.7 currents. Consistent with a reduction in current density, biotinylation revealed a significant reduction in surface NaV1.7 levels in CAD cells expressing CRMP2-K374A; surface NaV1.7 expression was also decreased by SENP1 + SENP2 overexpression. Currents in HEK293 cells stably expressing NaV1.7 were reduced by CRMP2-K374A in a manner dependent on the E2-conjugating enzyme Ubc9. No decrement in current density was observed in HEK293 cells co-expressing CRMP2-K374A and NaV1.1 or NaV1.3. Diminution of sodium currents, largely NaV1.7, was recapitulated in sensory neurons expressing CRMP2-K374A. Our study elucidates a novel regulatory mechanism that utilizes CRMP2 SUMOylation to choreograph NaV1.7 trafficking.

Hierarchical CRMP2 posttranslational modifications control NaV1.7 function.

Significance The voltage-gated sodium channel NaV1.7 is important for electrogenesis in sensory neurons. Insertion within the membrane is required for function of NaV1.7. However, the mechanisms determining how NaV1.7 is trafficked to neuronal cell membranes are poorly understood. Here, we elucidate a signaling program involving a complex and intriguing posttranslational modification regime of collapsin response mediator protein 2 (CRMP2), an NaV1.7-binding protein. NaV1.7 surface localization and currents are controlled by CRMP2 modifications. Activity of NaV1.7 is thought to modulate neuronal excitability that codes for several sensory modalities, including chronic pain, as inferred from human pain disorders caused by mutations in NaV1.7 channels. Understanding the role of cross-talk between CRMP2 modifications in modulation of NaV1.7 activity opens routes to exploit this system for pain. Voltage-gated sodium channels are crucial determinants of neuronal excitability and signaling. Trafficking of the voltage-gated sodium channel NaV1.7 is dysregulated in neuropathic pain. We identify a trafficking program for NaV1.7 driven by hierarchical interactions with posttranslationally modified versions of the binding partner collapsin response mediator protein 2 (CRMP2). The binding described between CRMP2 and NaV1.7 was enhanced by conjugation of CRMP2 with small ubiquitin-like modifier (SUMO) and further controlled by the phosphorylation status of CRMP2. We determined that CRMP2 SUMOylation is enhanced by prior phosphorylation by cyclin-dependent kinase 5 and antagonized by Fyn phosphorylation. As a consequence of CRMP2 loss of SUMOylation and binding to NaV1.7, the channel displays decreased membrane localization and current density, and reduces neuronal excitability. Preventing CRMP2 SUMOylation with a SUMO-impaired CRMP2-K374A mutant triggered NaV1.7 internalization in a clathrin-dependent manner involving the E3 ubiquitin ligase Nedd4-2 (neural precursor cell expressed developmentally down-regulated protein 4) and endocytosis adaptor proteins Numb and epidermal growth factor receptor pathway substrate 15. Collectively, our work shows that diverse modifications of CRMP2 cross-talk to control NaV1.7 activity and illustrate a general principle for regulation of NaV1.7.

Dissecting the role of the CRMP2-neurofibromin complex on pain behaviors.

Abstract Neurofibromatosis type 1 (NF1), a genetic disorder linked to inactivating mutations or a homozygous deletion of the Nf1 gene, is characterized by tumorigenesis, cognitive dysfunction, seizures, migraine, and pain. Omic studies on human NF1 tissues identified an increase in the expression of collapsin response mediator protein 2 (CRMP2), a cytosolic protein reported to regulate the trafficking and activity of presynaptic N-type voltage-gated calcium (Cav2.2) channels. Because neurofibromin, the protein product of the Nf1 gene, binds to and inhibits CRMP2, the neurofibromin–CRMP2 signaling cascade will likely affect Ca2+ channel activity and regulate nociceptive neurotransmission and in vivo responses to noxious stimulation. Here, we investigated the function of neurofibromin–CRMP2 interaction on Cav2.2. Mapping of >275 peptides between neurofibromin and CRMP2 identified a 15-amino acid CRMP2–derived peptide that, when fused to the tat transduction domain of HIV-1, inhibited Ca2+ influx in dorsal root ganglion neurons. This peptide mimics the negative regulation of CRMP2 activity by neurofibromin. Neurons treated with tat-CRMP2/neurofibromin regulating peptide 1 (t-CNRP1) exhibited a decreased Cav2.2 membrane localization, and uncoupling of neurofibromin–CRMP2 and CRMP2–Cav2.2 interactions. Proteomic analysis of a nanodisc-solubilized membrane protein library identified syntaxin 1A as a novel CRMP2-binding protein whose interaction with CRMP2 was strengthened in neurofibromin-depleted cells and reduced by t-CNRP1. Stimulus-evoked release of calcitonin gene–related peptide from lumbar spinal cord slices was inhibited by t-CNRP1. Intrathecal administration of t-CNRP1 was antinociceptive in experimental models of inflammatory, postsurgical, and neuropathic pain. Our results demonstrate the utility of t-CNRP1 to inhibit CRMP2 protein–protein interactions for the potential treatment of pain.

Blocking CRMP2 SUMOylation reverses neuropathic pain

Collapsin response mediator proteins 1−5 (CRMPs1−5) are a family of cytosolic proteins that coordinate neuronal migration, axonal guidance, dendritic organization, dendritic spine development and synaptic plasticity (reviewed in Khanna et al.1and Quach et al.2). Members of the CRMP family are reported to be involved in the pathogenesis of various neuronal disorders. For instance, proteomic, genomic and translational approaches linked the CRMP1 gene with chronic, negative symptoms of schizophrenia and severe major depression.3 Mice lacking CRMP1 manifest hyperactivity, impaired learning and memory, and impaired prepulse inhibition behavioral abnormalities related to schizophrenia.4 Genetic association and linkage studies pointed to CRMP2 as a liability gene for schizophrenia, autism, alcohol dependence, depression and bipolar disorders.5, 6, 7, 8, 9, 10 Mice with brain-specific Crmp2 deletion exhibited behavioral deficits in locomotor activity, sensorimotor gating, social behavior, and spatial learning and memory.11 Maternal autoantibodies against CRMP1 and CRMP2 were found in children with autism spectrum disorders that displayed core deficits in communication and reciprocal social interaction as well as repetitive or stereotypical behaviors.12 CRMP3-deficient mice display significant decreases in dendritic length and branching points, and an abnormal undulation of apical primary dendrites; these findings are recapitulated in the brain of Down syndrome where the expression of CRMP3 gene is also impaired.13 Little is known about the relationship between CRMP4 and neuropsychiatric disorders. However, mice lacking CRMP4 manifest impaired olfactory function and hyperactivity in the olfactory bulb and have increased levels of ionotropic glutamate receptors GluRs 1 and 2, which have been implicated in autism spectrum disorders and schizophrenia.14 CRMP5 knockout mice implicate this protein in dendritic development and synaptic plasticity in cerebellar purkinje cells,15 and CRMP5 autoantibodies were reported in patients with paraneoplastic neurological syndrome characterized by cerebellar ataxia and chorea. Therefore, understanding CRMP signaling has significant clinical implications.

Sensitization of Ion Channels Contributes to Central and Peripheral Dysfunction in Neurofibromatosis Type 1

Neurofibromatosis type 1 (Nf1) is a progressive, autosomal disorder with a large degree of variability and severity of manifestations including neurological, cutaneous, ocular/orbital, orthopedic, and vascular abnormalities. Nearly half of Nf1 patients presents with cognitive impairment, specifically spatial learning deficits. These clinical manifestations suggest a global impairment of both central and peripheral nervous system functions in neurofibromatosis. Nf1 encodes for neurofibromin, a Ras GTPase-activating protein (Ras GAP) that has been implicated in the regulation of long-term potentiation (LTP), Ras/ERK (extracellular signal-regulated kinase) signaling, and learning in mice. Over the last decades, mice with a targeted mutation in the Nf1 gene, Nf1−/− chimeric mice, Nf1 exon-specific knockout mice, and mice with tissue-specific inactivation of Nf1 have been generated to model the human Nf1 disease. These studies have implicated neurofibromin in regulation of the release of the inhibitory neurotransmitter γ-amino butyric acid (GABA) in the hippocampus and frontal lobe, which can regulate memory. Mutations in neurofibromin thus lead to perturbed ERK signaling, which alters GABA release, LTP, and subsequently leads to learning deficits. In addition to these cognitive deficits, Nf1 patients also have defects in fine and gross motor coordination as well as decreased muscle strength. Although the mechanisms underlying these motor deficits are unknown, deficits in GABAergic neurotransmission in both the motor cortex and cerebellum have been suggested. In this review, we present evidence to support the hypothesis that alterations of ion channel activity in Nf1 underscore the dysregulated neuronal communication in non-neuronal and neuronal cells that likely contributes to the clinical cornucopia of Nf1.

A single structurally conserved SUMOylation site in CRMP2 controls NaV1.7 function

ABSTRACT The neuronal collapsin response mediator protein 2 (CRMP2) undergoes several posttranslational modifications that codify its functions. Most recently, CRMP2 SUMOylation (addition of small ubiquitin like modifier (SUMO)) was identified as a key regulatory step within a modification program that codes for CRMP2 interaction with, and trafficking of, voltage-gated sodium channel NaV1.7. In this paper, we illustrate the utility of combining sequence alignment within protein families with structural analysis to identify, from several putative SUMOylation sites, those that are most likely to be biologically relevant. Co-opting this principle to CRMP2, we demonstrate that, of 3 sites predicted to be SUMOylated in CRMP2, only the lysine 374 site is a SUMOylation client. A reduction in NaV1.7 currents was the corollary of the loss of CRMP2 SUMOylation at this site. A 1.78-Å-resolution crystal structure of mouse CRMP2 was solved using X-ray crystallography, revealing lysine 374 as buried within the CRMP2 tetramer interface but exposed in the monomer. Since CRMP2 SUMOylation is dependent on phosphorylation, we postulate that this state forces CRMP2 toward a monomer, exposing the SUMO site and consequently, resulting in constitutive regulation of NaV1.7.

Carbamazepine potentiates the effectiveness of morphine in a rodent model of neuropathic pain.

Approximately 60% of morphine is glucuronidated to morphine-3-glucuronide (M3G) which may aggravate preexisting pain conditions. Accumulating evidence indicates that M3G signaling through neuronal Toll-like receptor 4 (TLR4) may be central to this proalgesic signaling event. These events are known to include elevated neuronal excitability, increased voltage-gated sodium (NaV) current, tactile allodynia and decreased opioid analgesic efficacy. Using an in vitro ratiometric-based calcium influx analysis of acutely dissociated small and medium-diameter neurons derived from lumbar dorsal root ganglion (DRG), we observed that M3G-sensitive neurons responded to lipopolysaccharide (LPS) and over 35% of these M3G/LPS-responsive cells exhibited sensitivity to capsaicin. In addition, M3G-exposed sensory neurons significantly increased excitatory activity and potentiated NaV current as measured by current and voltage clamp, when compared to baseline level measurements. The M3G-dependent excitability and potentiation of NaV current in these sensory neurons could be reversed by the addition of carbamazepine (CBZ), a known inhibitor of several NaV currents. We then compared the efficacy between CBZ and morphine as independent agents, to the combined treatment of both drugs simultaneously, in the tibial nerve injury (TNI) model of neuropathic pain. The potent anti-nociceptive effects of morphine (5 mg/kg, i.p.) were observed in TNI rodents at post-injury day (PID) 7–14 and absent at PID21–28, while administration of CBZ (10 mg/kg, i.p.) alone failed to produce anti-nociceptive effects at any time following TNI (PID 7–28). In contrast to either drug alone at PID28, the combination of morphine and CBZ completely attenuated tactile hyperalgesia in the rodent TNI model. The basis for the potentiation of morphine in combination with CBZ may be due to the effects of a latent upregulation of NaV1.7 in the DRG following TNI. Taken together, our observations demonstrate a potential therapeutic use of morphine and CBZ as a combinational treatment for neuropathic pain.

Homology‐guided mutational analysis reveals the functional requirements for antinociceptive specificity of collapsin response mediator protein 2‐derived peptides

N‐type voltage‐gated calcium (Cav2.2) channels are critical determinants of increased neuronal excitability and neurotransmission accompanying persistent neuropathic pain. Although Cav2.2 channel antagonists are recommended as first‐line treatment for neuropathic pain, calcium‐current blocking gabapentinoids inadequately alleviate chronic pain symptoms and often exhibit numerous side effects. Collapsin response mediator protein 2 (CRMP2) targets Cav2.2 channels to the sensory neuron membrane and allosterically modulates their function. A 15‐amino‐acid peptide (CBD3), derived from CRMP2, disrupts the functional protein–protein interaction between CRMP2 and Cav2.2 channels to inhibit calcium influx, transmitter release and acute, inflammatory and neuropathic pain. Here, we have mapped the minimal domain of CBD3 necessary for its antinociceptive potential.

Homology‐guided mutational analysis reveals the functional requirements for antinociceptive specificity of CRMP2‐derived peptides

N‐type voltage‐gated calcium (Cav2.2) channels are critical determinants of increased neuronal excitability and neurotransmission accompanying persistent neuropathic pain. Although Cav2.2 channel antagonists are recommended as first‐line treatment for neuropathic pain, calcium‐current blocking gabapentinoids inadequately alleviate chronic pain symptoms and often exhibit numerous side effects. Collapsin response mediator protein 2 (CRMP2) targets Cav2.2 channels to the sensory neuron membrane and allosterically modulates their function. A 15‐amino‐acid peptide (CBD3), derived from CRMP2, disrupts the functional protein–protein interaction between CRMP2 and Cav2.2 channels to inhibit calcium influx, transmitter release and acute, inflammatory and neuropathic pain. Here, we have mapped the minimal domain of CBD3 necessary for its antinociceptive potential.