Faculty Bibliography

TAF1/MRSX33 intellectual disability syndrome is an X-linked disorder caused by loss-of-function mutations in the TAF1 gene. How these mutations cause dysmorphology, hypotonia, intellectual and motor defects is unknown. Mouse models which have embryonically targeted TAF1 have failed, possibly due to TAF1 being essential for viability, preferentially expressed in early brain development, and intolerant of mutation. Novel animal models are valuable tools for understanding neuronal pathology. Here, we report the development and characterization of a novel animal model for TAF1 ID syndrome in which the TAF1 gene is deleted in embryonic rats using clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9) technology and somatic brain transgenesis mediated by lentiviral transduction. Rat pups, post-natal day 3, were subjected to intracerebroventricular (ICV) injection of either gRNA-control or gRNA-TAF1 vectors. Rats were subjected to a battery of behavioral tests followed by histopathological analyses of brains at post-natal day 14 and day 35. TAF1-edited rats exhibited behavioral deficits at both the neonatal and juvenile stages of development. Deletion of TAF1 lead to a hypoplasia and loss of the Purkinje cells. We also observed a decreased in GFAP positive astrocytes and an increase in Iba1 positive microglia within the granular layer of the cerebellum in TAF1-edited animals. Immunostaining revealed a reduction in the expression of the CaV3.1 T-type calcium channel. Abnormal motor symptoms in TAF1-edited rats were associated with irregular cerebellar output caused by changes in the intrinsic activity of the Purkinje cells due to loss of pre-synaptic CaV3.1. This animal model provides a powerful new tool for studies of neuronal dysfunction in conditions associated with TAF1 abnormalities and should prove useful for developing therapeutic strategies to treat TAF1 ID syndrome.

Collapsin response mediator protein 2 (CRMP2),by regulating voltage-gated calcium channel activity, is a crucial regulator of neuronal excitability. Hyperphosphorylation of CRMP2 has been reported in brains of Alzheimer's disease (AD) patients and other neurodegenerative diseases. CRMP2 acting on N-methyl-d-aspartate receptors (NMDARs) may contribute to AD pathology. A short peptide from CRMP2, designated the Ca²⁺ channel-binding domain 3 (CBD3) peptide, has recently emerged as a Ca²⁺ channel blocker that exerts neuroprotective effects in traumatic brain injury and cerebral ischemia by disrupting pCRMP2/NMDAR interaction to inhibit calcium influx. ST2-104, a nona-arginine (R9)-conjugated CBD3 peptide derived from CRMP2, exerts a beneficial effect on neuropathic pain; however, the effect of ST2-104 on AD and its mechanism of action have not been studied. In this study we investigated the effects of ST2-104 on SH-SY5Y neuroblastoma cells stimulated by Aβ_25-35. To induce neurotoxicity, SH-SY5Y cells were incubated with Aβ_25-35, the shortest toxic fragment of Aβ. CRMP2 expression was manipulated by knockdown or overexpression of CRMP2 before ST2-104 treatment to further explore if the pCRMP2/NMDAR2B signaling pathway is involved in the action of the ST2-104 peptide. The results show that ST2-104 significantly enhanced cell viability, inhibited cell apoptosis, decreased LDH release, suppressed the expression of the pCRMP2 protein, disrupted pCRMP2/NMDAR2B interaction, inhibited Aβ_25-35-induced NMDAR currents, and decreased intracellular Ca²⁺ levels. The effects of ST2-104 was abolished by overexpression of CRMP2 and intensified by knockdown of CRMP2 in SH-SY5Y cells. Taken together, our results support ST2-104 as a possible biologic therapeutic in the face of Aβ_25-35-induced injury via the inhibition of the pCRMP2/NMDAR2B signaling pathway.

Neuropathic pain results from nerve injuries that cause ectopic firing and increased nociceptive signal transmission due to activation of key membrane receptors and channels. The dysregulation of trafficking of voltage-gated ion channels is an emerging mechanism in the etiology of neuropathic pain. We identify increased phosphorylation of collapsin response mediator protein 2 (CRMP2), a protein reported to regulate presynaptic voltage-gated calcium and sodium channels. A spared nerve injury (SNI) increased expression of a cyclin dependent kinase 5 (Cdk5)-phosphorylated form of CRMP2 in the dorsal horn of the spinal cord and the dorsal root ganglia (DRG) in the ipsilateral (injured) versus the contralateral (non-injured) sites. Biochemical fractionation of spinal cord from SNI rats revealed the increase in Cdk5-mediated CRMP2 phosphorylation to be enriched to pre-synaptic sites. CRMP2 has emerged as a central node in assembling nociceptive signaling complexes. Knockdown of CRMP2 using a small interfering RNA (siRNA) reversed SNI-induced mechanical allodynia implicating CRMP2 expression as necessary for neuropathic pain. Intrathecal expression of a CRMP2 resistant to phosphorylation by Cdk5 normalized SNI-induced mechanical allodynia, whereas mimicking constitutive phosphorylation of CRMP2 resulted in induction of mechanical allodynia in naïve rats. Collectively, these results demonstrate that Cdk5-mediated CRMP2 phosphorylation is both necessary and sufficient for peripheral neuropathic pain.

We report the development and characterization of a novel, injury-free rat model in which nociceptive sensitization after red light is observed in multiple body areas reminiscent of widespread pain in functional pain syndromes. Rats were exposed to red light-emitting diodes (RLED) (LEDs, 660 nm) at an intensity of 50 Lux for 8 hours daily for 5 days resulting in time- and dose-dependent thermal hyperalgesia and mechanical allodynia in both male and female rats. Females showed an earlier onset of mechanical allodynia than males. The pronociceptive effects of RLED were mediated through the visual system. RLED-induced thermal hyperalgesia and mechanical allodynia were reversed with medications commonly used for widespread pain, including gabapentin, tricyclic antidepressants, serotonin/norepinephrine reuptake inhibitors, and nonsteroidal anti-inflammatory drugs. Acetaminophen failed to reverse the RLED induced hypersensitivity. The hyperalgesic effects of RLED were blocked when bicuculline, a gamma-aminobutyric acid-A receptor antagonist, was administered into the rostral ventromedial medulla, suggesting a role for increased descending facilitation in the pain pathway. Key experiments were subjected to a replication study with randomization, investigator blinding, inclusion of all data, and high levels of statistical rigor. RLED-induced thermal hyperalgesia and mechanical allodynia without injury offers a novel injury-free rodent model useful for the study of functional pain syndromes with widespread pain. RLED exposure also emphasizes the different biological effects of different colors of light exposure. PERSPECTIVE: This study demonstrates the effect of light exposure on nociceptive thresholds. These biological effects of red LED add evidence to the emerging understanding of the biological effects of light of different colors in animals and humans. Understanding the underlying biology of red light-induced widespread pain may offer insights into functional pain states.

Amongst the regulators of voltage-gated ion channels is the collapsin response mediator protein 2 (CRMP2). CRMP2 regulation of the activity and trafficking of NaV1.7 voltage-gated sodium channels as well as the N-type (CaV2.2) voltage-gated calcium channel (VGCC) has been reported. On the other hand, CRMP2 does not appear to regulate L- (CaV1.x), P/Q- (CaV2.1), and R- (CaV2.3) type high VGCCs. Whether CRMP2 regulates low VGCCs remains an open question. Here, we asked if CRMP2 could regulate the low voltage-gated (T-type/CaV3.x) channels in sensory neurons. Reducing CRMP2 protein levels with short interfering RNAs yielded no change in macroscopic currents carried by T-type channels. No change in biophysical properties of the T-type currents was noted. Future studies pursuing CRMP2 druggability in neuropathic pain will benefit from the findings that CRMP2 regulates only the N-type (CaV2.2) calcium channels.

Neurofibromatosis type 1 (NF1) is the most common of a group of rare diseases known by the term, "Neurofibromatosis," affecting 1 in 3000 to 4000 people. NF1 patients present with, among other disease complications, café au lait patches, skin fold freckling, Lisch nodules, orthopedic complications, cutaneous neurofibromas, malignant peripheral nerve sheath tumors, cognitive impairment, and chronic pain. Although NF1 patients inevitably express pain as a debilitating symptom of the disease, not much is known about its manifestation in the NF1 disease, with most current information coming from sporadic case reports. Although these reports indicate the existence of pain, the molecular signaling underlying this symptom remains underexplored, and thus, we include a synopsis of the literature surrounding NF1 pain studies in 3 animal models: mouse, rat, and miniswine. We also highlight unexplored areas of NF1 pain research. As therapy for NF1 pain remains in various clinical and preclinical stages, we present current treatments available for patients and highlight the importance of future therapeutic development. Equally important, NF1 pain is accompanied by psychological complications in comorbidities with sleep, gastrointestinal complications, and overall quality of life, lending to the importance of investigation into this understudied phenomenon of NF1. In this review, we dissect the presence of pain in NF1 in terms of psychological implication, anatomical presence, and discuss mechanisms underlying the onset and potentiation of NF1 pain to evaluate current therapies and propose implications for treatment of this severely understudied, but prevalent symptom of this rare disease.

Naringenin (2S)-5,7-dihydroxy-2-(4-hydroxyphenyl)-3,4-dihydro-2H-1-benzopyran-4-one is a natural flavonoid found in fruits from the citrus family. Because (2S)-naringenin is known to racemize, its bioactivity might be related to one or both enantiomers. Computational studies predicted that (2R)-naringenin may act on voltage-gated ion channels, particularly the N-type calcium channel (CaV2.2) and the NaV1.7 sodium channel-both of which are key for pain signaling. Here we set out to identify the possible mechanism of action of naringenin. Naringenin inhibited depolarization-evoked Ca²⁺ influx in acetylcholine-, ATP-, and capsaicin-responding rat dorsal root ganglion (DRG) neurons. This was corroborated in electrophysiological recordings from DRG neurons. Pharmacological dissection of each of the voltage-gated Ca²⁺ channels subtypes could not pinpoint any selectivity of naringenin. Instead, naringenin inhibited NaV1.8-dependent and tetrodotoxin (TTX)-resistant while sparing tetrodotoxin sensitive (TTX-S) voltage-gated Na⁺ channels as evidenced by the lack of further inhibition by the NaV1.8 blocker A-803467. The effects of the natural flavonoid were validated ex vivo in spinal cord slices where naringenin decreased both the frequency and amplitude of sEPSC recorded in neurons within the substantia gelatinosa. The antinociceptive potential of naringenin was evaluated in male and female mice. Naringenin had no effect on the nociceptive thresholds evoked by heat. Naringenin's reversed allodynia was in mouse models of postsurgical and neuropathic pain. Here, driven by a call by the National Center for Complementary and Integrative Health's strategic plan to advance fundamental research into basic biological mechanisms of the action of natural products, we advance the antinociceptive potential of the flavonoid naringenin.

We have previously reported that the microtubule-associated collapsin response mediator protein 2 (CRMP2) is necessary for the expression of chronic pain. CRMP2 achieves this control of nociceptive signaling by virtue of its ability to regulate voltage-gated calcium and sodium channels. To date, however, no drugs exist that target CRMP2. Recently, the small molecule edonerpic maleate (1 -{3-[2-(1-benzothiophen-5-yl)ethoxy]propyl}azetidin-3-ol maleate), a candidate therapeutic for Alzheimer's disease was reported to be a novel CRMP2 binding compound with the potential to decrease its phosphorylation level in cortical tissues in vivo. Here we sought to determine the mechanism of action of edonerpic maleate and test its possible effect in a rodent model of chronic pain. We observed: (i) no binding between human CRMP2 and edonerpic maleate; (ii) edonerpic maleate had no effect on CRMP2 expression and phosphorylation in dorsal root ganglion (DRG) neurons; (iii) edonerpic maleate-decreased calcium but increased sodium current density in DRG neurons; and (iv) edonerpic maleate was ineffective in reversing post-surgical allodynia in male and female mice. Thus, while CRMP2 inhibiting compounds remain a viable strategy for developing new mechanism-based pain inhibitors, edonerpic maleate is an unlikely candidate.

The Federal Pain Research Strategy recommended development of nonopioid analgesics as a top priority in its strategic plan to address the significant public health crisis and individual burden of chronic pain faced by >100 million Americans. Motivated by this challenge, a natural product extracts library was screened and identified a plant extract that targets activity of voltage-gated calcium channels. This profile is of interest as a potential treatment for neuropathic pain. The active extract derived from the desert lavender plant native to southwestern United States, when subjected to bioassay-guided fractionation, afforded 3 compounds identified as pentacyclic triterpenoids, betulinic acid (BA), oleanolic acid, and ursolic acid. Betulinic acid inhibited depolarization-evoked calcium influx in dorsal root ganglion (DRG) neurons predominantly through targeting low-voltage-gated (Cav3 or T-type) and CaV2.2 (N-type) calcium channels. Voltage-clamp electrophysiology experiments revealed a reduction of Ca, but not Na, currents in sensory neurons after BA exposure. Betulinic acid inhibited spontaneous excitatory postsynaptic currents and depolarization-evoked release of calcitonin gene-related peptide from lumbar spinal cord slices. Notably, BA did not engage human mu, delta, or kappa opioid receptors. Intrathecal administration of BA reversed mechanical allodynia in rat models of chemotherapy-induced peripheral neuropathy and HIV-associated peripheral sensory neuropathy as well as a mouse model of partial sciatic nerve ligation without effects on locomotion. The broad-spectrum biological and medicinal properties reported, including anti-HIV and anticancer activities of BA and its derivatives, position this plant-derived small molecule natural product as a potential nonopioid therapy for management of chronic pain.

The peripherally expressed voltage-gated sodium Na_V1.7 (gene SCN9A) channel boosts small stimuli to initiate firing of pain-signaling dorsal root ganglia (DRG) neurons and facilitates neurotransmitter release at the first synapse within the spinal cord. Mutations in SCN9A produce distinct human pain syndromes. Widely acknowledged as a "gatekeeper" of pain, Na_V1.7 has been the focus of intense investigation but, to date, no Na_V1.7-selective drugs have reached the clinic. Elegant crystallographic studies have demonstrated the potential of designing highly potent and selective Na_V1.7 compounds but their therapeutic value remains untested. Transcriptional silencing of Na_V1.7 by a naturally expressed antisense transcript has been reported in rodents and humans but whether this represents a viable opportunity for designing Na_V1.7 therapeutics is currently unknown. The demonstration that loss of Na_V1.7 function is associated with upregulation of endogenous opioids and potentiation of mu- and delta-opioid receptor activities, suggests that targeting only Na_V1.7 may be insufficient for analgesia. However, the link between opioid-dependent analgesic mechanisms and function of sodium channels and intracellular sodium-dependent signaling remains controversial. Thus, additional new targets - regulators, modulators - are needed. In this context, we mine the literature for the known interactome of Na_V1.7 with a focus on protein interactors that affect the channel's trafficking or link it to opioid signaling. As a case study, we present antinociceptive evidence of allosteric regulation of Na_V1.7 by the cytosolic collapsin response mediator protein 2 (CRMP2). Throughout discussions of these possible new targets, we offer thoughts on the therapeutic implications of modulating Na_V1.7 function in chronic pain.