Faculty Bibliography

OBJECTIVE:Determination of possible sex differences in mechanisms promoting migraine progression and the contribution of prolactin and the prolactin long (PRLR-L) and short (PRLR-S) receptor isoforms. BACKGROUND:The majority of patients with chronic migraine and medication overuse headache are female. Prolactin is present at higher levels in women and increases migraine. Prolactin signaling at the PRLR-S selectively sensitizes nociceptors in female rodents, while expression of the PRLR-L is protective. METHODS:Medication overuse headache was modeled by repeated sumatriptan administration in male and female mice. Periorbital and hindpaw cutaneous allodynia served as a surrogate of migraine-like pain. PRLR-L and PRLR-S isoforms were measured in the trigeminal ganglion with western blotting. Possible co-localization of PRLR with serotonin 5HT1B and 5HT1D receptors was determined with RNAscope. Cabergoline, a dopamine receptor agonist that inhibits circulating prolactin, was co-administered with sumatriptan. Nasal administration of CRISPR/Cas9 plasmid was used to edit expression of both PRLR isoforms. RESULTS:PRLR was co-localized with 5HT1B or 5HT1D receptors in the ophthalmic region of female trigeminal ganglion. A single injection of sumatriptan increased serum PRL levels in female mice. Repeated sumatriptan promoted cutaneous allodynia in both sexes but down-regulated trigeminal ganglion PRLR-L, without altering PRLR-S, only in females. Co-administration of sumatriptan with cabergoline prevented allodynia and down-regulation of PRLR-L only in females. CRISPR/Cas9 editing of both PRLR isoforms in the trigeminal ganglion prevented sumatriptan-induced periorbital allodynia in females. INTERPRETATION/CONCLUSIONS:We identified a sexually dimorphic mechanism of migraine chronification that involves down-regulation of PRLR-L and increased signaling of circulating prolactin at PRLR-S. These studies reveal a previously unrecognized neuroendocrine mechanism linking the hypothalamus to nociceptor sensitization that increases the risk of migraine pain in females and suggest opportunities for novel sex-specific therapies including gene editing through nasal delivery of CRISPR/Cas9 constructs.

BACKGROUND:Pharmacological management of migraine can be ineffective for some patients. We previously demonstrated that exposure to green light resulted in antinociception and reversal of thermal and mechanical hypersensitivity in rodent pain models. Given the safety of green light emitting diodes, we evaluated green light as a potential therapy in patients with episodic or chronic migraine. MATERIAL AND METHODS:We recruited (29 total) patients, of whom seven had episodic migraine and 22 had chronic migraine. We used a one-way cross-over design consisting of exposure for 1-2 hours daily to white light emitting diodes for 10 weeks, followed by a 2-week washout period followed by exposure for 1-2 hours daily to green light emitting diodes for 10 weeks. Patients were allowed to continue current therapies and to initiate new treatments as directed by their physicians. Outcomes consisted of patient-reported surveys. The primary outcome measure was the number of headache days per month. Secondary outcome measures included patient-reported changes in the intensity and frequency of the headaches over a two-week period and other quality of life measures including ability to fall and stay asleep, and ability to perform work. Changes in pain medications were obtained to assess potential reduction. RESULTS:When seven episodic migraine and 22 chronic migraine patients were analyzed as separate cohorts, white light emitting diodes produced no significant change in headache days in either episodic migraine or chronic migraine patients. Combining data from the episodic migraine and chronic migraine groups showed that white light emitting diodes produced a small, but statistically significant reduction in headache days from (days ± SEM) 18.2 ± 1.8 to 16.5 ± 2.01 days. Green light emitting diodes resulted in a significant decrease in headache days from 7.9 ± 1.6 to 2.4 ± 1.1 and from 22.3 ± 1.2 to 9.4 ± 1.6 in episodic migraine and chronic migraine patients, respectively. While some improvement in secondary outcomes was observed with white light emitting diodes, more secondary outcomes with significantly greater magnitude including assessments of quality of life, Short-Form McGill Pain Questionnaire, Headache Impact Test-6, and Five-level version of the EuroQol five-dimensional survey without reported side effects were observed with green light emitting diodes. Conclusions regarding pain medications reduction with green light emitting diode exposure were not possible. No side effects of light therapy were reported. None of the patients in the study reported initiation of new therapies. DISCUSSION:Green light emitting diodes significantly reduced the number of headache days in people with episodic migraine or chronic migraine. Additionally, green light emitting diodes significantly improved multiple secondary outcome measures including quality of life and intensity and duration of the headache attacks. As no adverse events were reported, green light emitting diodes may provide a treatment option for those patients who prefer non-pharmacological therapies or may be considered in complementing other treatment strategies. Limitations of this study are the small number of patients evaluated. The positive data obtained support implementation of larger clinical trials to determine possible effects of green light emitting diode therapy.This study is registered with clinicaltrials.gov under NCT03677206.

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The TATA-box binding protein associated factor 1 (TAF1) is part of the TFIID complex that plays a key role during the initiation of transcription. Variants of TAF1 are associated with neurodevelopmental disorders. Previously, we found that CRISPR/Cas9 based editing of the TAF1 gene disrupts the morphology of the cerebral cortex and blunts the expression as well as the function of the CaV3.1 (T-type) voltage gated calcium channel. Here, we tested the efficacy of SAK3 (ethyl 8'-methyl-2', 4-dioxo-2-(piperidin-1-yl)-2'H-spiro [cyclopentane-1, 3'-imidazo [1, 2-a] pyridine]-2-ene-3-carboxylate), a T-type calcium channel enhancer, in an animal model of TAF1 intellectual disability (ID) syndrome. At post-natal day 3, rat pups were subjected to intracerebroventricular (ICV) injection of either gRNA-control or gRNA-TAF1 CRISPR/Cas9 viruses. At post-natal day 21, the rat pups were given SAK3 (0.25 mg/kg, p.o.) or vehicle for 14 days (i.e. till post-natal day 35) and then subjected to behavioral, morphological, and molecular studies. Oral administration of SAK3 (0.25 mg/kg, p.o.) significantly rescued locomotion abnormalities associated with TAF1 gene editing. SAK3 treatment prevented the loss of cortical neurons and GFAP-positive astrocytes observed after TAF1 gene editing. In addition, SAK3 protected cells from apoptosis. SAK3 also restored the Brain-derived neurotrophic factor/protein kinase B/Glycogen Synthase Kinase 3 Beta (BDNF/AKT/GSK3β) signaling axis in TAF1 edited animals. Finally, SAK3 normalized the levels of three GSK3β substrates - CaV3.1, FOXP2, and CRMP2. We conclude that the T-type calcium channel enhancer SAK3 is beneficial against the deleterious effects of TAF1 gene-editing, in part, by stimulating the BDNF/AKT/GSK3β signaling pathway.

Inhalation of the fungus Alternaria alternata is associated with an increased risk of allergic asthma development and exacerbations. Recent work in acute exposure animal models suggests that A. alternata-induced asthma symptoms, which include inflammation, mucus overproduction and airway hyperresponsiveness, are due to A. alternata proteases that act via protease-activated receptor-2 (PAR2). However, because other active components present in A. alternata may be contributing to asthma pathophysiology through alternative signaling, the specific role PAR2 plays in asthma initiation and maintenance remains undefined. Airway epithelial cells provide the first encounter with A. alternata and are thought to play an important role in initiating the physiologic response. To better understand the role for PAR2 airway epithelial signaling we created a PAR2-deficient human bronchial epithelial cell line (16HBEPAR^-

Global spread of severe acute respiratory syndrome coronavirus 2 continues unabated. Binding of severe acute respiratory syndrome coronavirus 2's spike protein to host angiotensin-converting enzyme 2 triggers viral entry, but other proteins may participate, including the neuropilin-1 receptor (NRP-1). Because both spike protein and vascular endothelial growth factor-A (VEGF-A)-a pronociceptive and angiogenic factor, bind NRP-1, we tested whether spike could block VEGF-A/NRP-1 signaling. VEGF-A-triggered sensory neuron firing was blocked by spike protein and NRP-1 inhibitor EG00229. Pronociceptive behaviors of VEGF-A were similarly blocked through suppression of spontaneous spinal synaptic activity and reduction of electrogenic currents in sensory neurons. Remarkably, preventing VEGF-A/NRP-1 signaling was antiallodynic in a neuropathic pain model. A "silencing" of pain through subversion of VEGF-A/NRP-1 signaling may underlie increased disease transmission in asymptomatic individuals.

Voltage-gated sodium channels are key players in neuronal excitability and pain signaling. Functional expression of the voltage-gated sodium channel Na_V1.7 is under the control of SUMOylated collapsin response mediator protein 2 (CRMP2). When not SUMOylated, CRMP2 forms a complex with the endocytic proteins Numb, the epidermal growth factor receptor pathway substrate 15 (Eps15), and the E3 ubiquitin ligase Nedd4-2 to promote clathrin-mediated endocytosis of Na_V1.7. We recently reported that CRMP2 SUMO-null knock-in (CRMP2^K374A/K374A) female mice have reduced Na_V1.7 membrane localization and currents in their sensory neurons. Preventing CRMP2 SUMOylation was sufficient to reverse mechanical allodynia in CRMP2^K374A/K374A female mice with neuropathic pain. Here we report that inhibiting clathrin assembly in nerve-injured male CRMP2^K374A/K374A mice precipitated mechanical allodynia in mice otherwise resistant to developing persistent pain. Furthermore, Numb, Nedd4-2 and Eps15 expression was not modified in basal conditions in the dorsal root ganglia (DRG) of male and female CRMP2^K374A/K374A mice. Finally, silencing these proteins in DRG neurons from female CRMP2^K374A/K374A mice, restored the loss of sodium currents. Our study shows that the endocytic complex composed of Numb, Nedd4-2 and Eps15, is necessary for non-SUMOylated CRMP2-mediated internalization of sodium channels in vivo.

Benefits of phototherapy were characterized in multiple diseases including depression, circadian rhythm disruptions, and neurodegeneration. Studies on migraine and fibromyalgia patients revealed that green light-emitting diodes (GLED) exposure provides a pragmatic and safe therapy to manage chronic pain. In rodents, GLED reversed hypersensitivity related to neuropathic pain. However, little is known about the underlying mechanisms of GLED efficacy. Here, we sought to understand how green light modulates the endogenous opioid system. We first characterized how exposure to GLED stimulates release of β-endorphin and proenkephalin in the central nervous system of male rats. Moreover, by individually editing each of the receptors, we found that µ- and δ-opioid receptors are required for green light's antinociceptive effect in naïve rats and a model of HIV-induced peripheral neuropathy. We investigated how GLED could increase pain thresholds, and explored its potential in reversing hypersensitivity in a model of HIV-related neuropathy. Through behavioral and gene editing approaches, we identified that green light provides antinociception via modulation of the endogenous opioid system in the spinal cord. This work identifies a previously unknown mechanism by which GLED can improve pain management. Clinical translation of these results will advance the development of an innovative therapy devoid of adverse effects. PERSPECTIVE: Development of new pain management therapies, especially for HIV patients, is crucial as long-term opioid prescription is not recommended due to adverse side effects. Green light addresses this necessity. Characterizing the underlying mechanisms of this potentially groundbreaking and safe antinociceptive therapy will advance its clinical translation.

OBJECTIVE:Fibromyalgia is a functional pain disorder in which patients suffer from widespread pain and poor quality of life. Fibromyalgia pain and its impact on quality of life are not effectively managed with current therapeutics. Previously, in a preclinical rat study, we demonstrated that exposure to green light-emitting diodes (GLED) for 8 hours/day for 5 days resulted in antinociception and reversal of thermal and mechanical hypersensitivity associated with models of injury-related pain. Given the safety of GLED and the ease of its use, our objective is to administer GLED as a potential therapy to patients with fibromyalgia. DESIGN:One-way crossover clinical trial. SETTING:United States. METHOD:We enrolled 21 adult patients with fibromyalgia recruited from the University of Arizona chronic pain clinic who were initially exposed to white light-emitting diodes and then were crossed over to GLED for 1 to 2 hours daily for 10 weeks. Data were collected by using paper surveys. RESULTS:When patients were exposed to GLED, but not white light-emitting diodes, they reported a significant reduction in average pain intensity on the 10-point numeric pain scale. Secondary outcomes were assessed by using the EQ-5D-5L survey, Short-Form McGill Pain Questionnaire, and Fibromyalgia Impact Questionnaire and were also significantly improved in patients exposed to GLED. GLED therapy was not associated with any measured side effects in these patients. CONCLUSION:Although the mechanism by which GLED elicits pain reduction is currently being studied, these results supporting its efficacy and safety merit a larger clinical trial.

The collapsin response mediator protein (CRMP) family proteins are intracellular mediators of neurotrophic factors regulating neurite structure/spine formation and are essential for dendrite patterning and directional axonal pathfinding during brain developmental processes. Among this family, CRMP5/DPYSL5 plays a significant role in neuronal migration, axonal guidance, dendrite outgrowth, and synapse formation by interacting with microtubules. Here, we report the identification of missense mutations in DPYSL5 in nine individuals with brain malformations, including corpus callosum agenesis and/or posterior fossa abnormalities, associated with variable degrees of intellectual disability. A recurrent de novo p.Glu41Lys variant was found in eight unrelated patients, and a p.Gly47Arg variant was identified in one individual from the first family reported with Ritscher-Schinzel syndrome. Functional analyses of the two missense mutations revealed impaired dendritic outgrowth processes in young developing hippocampal primary neuronal cultures. We further demonstrated that these mutations, both located in the same loop on the surface of DPYSL5 monomers and oligomers, reduced the interaction of DPYSL5 with neuronal cytoskeleton-associated proteins MAP2 and βIII-tubulin. Our findings collectively indicate that the p.Glu41Lys and p.Gly47Arg variants impair DPYSL5 function on dendritic outgrowth regulation by preventing the formation of the ternary complex with MAP2 and βIII-tubulin, ultimately leading to abnormal brain development. This study adds DPYSL5 to the list of genes implicated in brain malformation and in neurodevelopmental disorders.