Search

Migraine is a neurological disorder that is far more than just a bad headache. A hallmark of migraine is altered sensory perception. A likely contributor to this altered perception is the neuropeptide calcitonin gene-related peptide (CGRP). Over the past decade, CGRP has become firmly established as a key player in migraine. Although the mechanisms and sites of action by which CGRP might trigger migraine remain speculative, recent advances with mouse models provide some hints. This brief review focuses on how CGRP might act as both a central and peripheral neuromodulator to contribute to the migraine-like symptom of light aversive behaviour in mice.

Objective The trigeminovascular system plays a central role in migraine, a condition in need of new treatments. The neuropeptide, calcitonin gene-related peptide (CGRP), is proposed as causative in migraine and is the subject of intensive drug discovery efforts. This study explores the expression and functionality of two CGRP receptor candidates in the sensory trigeminal system. Methods Receptor expression was determined using Taqman G protein-coupled receptor arrays and immunohistochemistry in trigeminal ganglia (TG) and the spinal trigeminal complex of the brainstem in rat and human. Receptor pharmacology was quantified using sensitive signaling assays in primary rat TG neurons. Results mRNA and histological expression analysis in rat and human samples revealed the presence of two CGRP-responsive receptors (AMY1: calcitonin receptor/receptor activity-modifying protein 1 [RAMP1]) and the CGRP receptor (calcitonin receptor-like receptor/RAMP1). In support of this finding, quantification of agonist and antagonist potencies revealed a dual population of functional CGRP-responsive receptors in primary rat TG neurons. Interpretation The unexpected presence of a functional non-canonical CGRP receptor (AMY1) at neural sites important for craniofacial pain has important implications for targeting the CGRP axis in migraine.

To examine calcitonin gene-related peptide (CGRP) gene expression under inflammatory conditions using trigeminal ganglia organ cultures as an experimental system. These cultures have increased proinflammatory signaling that may mimic neurogenic inflammation in the migraine state.The trigeminal nerve sends peripheral pain signals to the central nervous system during migraine. Understanding the dynamic processes that occur within the trigeminal nerve and ganglion may provide insights into events that contribute to migraine pain. A neuropeptide of particular interest is CGRP, which can be elevated and play a causal role in migraine. However, most studies have overlooked a second splice product of the Calca gene that encodes calcitonin (CT), a peptide hormone involved in calcium homeostasis. Importantly, a precursor form of CT called procalcitonin (proCT) can act as a partial agonist at the CGRP receptor and elevated proCT has recently been reported during migraine.We used a trigeminal ganglion whole organ explant model, which has previously been demonstrated to induce pro-inflammatory agents in vitro. Quantitative polymerase chain reaction and immunohistochemistry were used to evaluate changes in messenger ribonucleic acid (mRNA) and protein levels of CGRP and proCT.Whole mouse trigeminal ganglia cultured for 24 hours showed a 10-fold increase in CT mRNA, with no change in CGRP mRNA. A similar effect was observed in ganglia from adult rats. ProCT immunoreactivity was localized in glial cells. Cutting the tissue blunted the increase in CT, suggesting that induction required the close environment of the intact ganglia. Consistent with this prediction, there were increased reactive oxygen species in the ganglia, and the elevated CT mRNA was reduced by antioxidant treatment. Surprisingly, reactive oxygen species were increased in neurons, not glia.These results demonstrate that reactive oxygen species can activate proCT expression from the CGRP gene in trigeminal glia by a paracrine regulatory mechanism. We propose that this glial recruitment pathway may occur following cortical spreading depression and neurogenic inflammation to increase CGRP nociceptive actions in migraine.

Calcitonin gene-related peptide (CGRP) and nitric oxide are involved in the underlying pathophysiology of migraine and other diseases involving neurogenic inflammation. We have tested the hypothesis that nitric oxide might trigger signaling mechanisms within the trigeminal ganglia neurons that would coordinately stimulate CGRP synthesis and release. Treatment of primary trigeminal ganglia cultures with nitric oxide donors caused a greater than four-fold increase in CGRP release compared with unstimulated cultures. Similarly, CGRP promoter activity was also stimulated by nitric oxide donors and overexpression of inducible nitric oxide synthase (iNOS). Cotreatment with the antimigraine drug sumatriptan greatly repressed nitric oxide stimulation of CGRP promoter activity and secretion. Somewhat surprisingly, the mechanisms of nitric oxide stimulation of CGRP secretion did not require cGMP or PI3-kinase signaling pathways, but rather, nitric oxide action required extracellular calcium and likely involves T-type calcium channels. Furthermore, nitric oxide was shown to increase expression of the active forms of the mitogen-activated protein kinases Jun amino-terminal kinase and p38 but not extracellular signal-related kinase in trigeminal neurons. In summary, our results provide new insight into the cellular mechanisms by which nitric oxide induces CGRP synthesis and secretion from trigeminal neurons.

BACKGROUND: Mutations in the PITX2 homeobox gene are known to contribute to Axenfeld-Rieger syndrome (ARS), an autosomal-dominant developmental disorder. Although most mutations are in the homeodomain and result in a loss of function, there is a growing subset in the C-terminal domain that has not yet been characterized. These mutations are of particular interest because the C-terminus has both inhibitory and stimulatory activities. METHODS: In this study we used a combination of in vitro DNA binding and transfection reporter assays to investigate the fundamental issue of whether C-terminal mutations result in gain or loss of function at a cellular level. RESULTS: We report a new frameshift mutation in the PITX2 allele that predicts a truncated protein lacking most of the C-terminal domain (D122FS). This newly reported mutant and another ARS C-terminal mutant (W133Stop) both have greater binding than wild-type to the bicoid element. Of interest, the mutants yielded ! 5-fold greater activation of the prolactin promoter in CHO cells, even though the truncated proteins were expressed at lower levels than the wild-type protein. The truncated proteins also had greater than wild-type activity in 2 other cell lines, including the LS8 oral epithelial line that expresses the endogenous Pitx2 gene. CONCLUSIONS: The results indicate that the PITX2 C-terminal domain has inhibitory activity and support the notion that ARS may also be caused by gain-of-function mutations. Birth Defects Research (Part A) 76:175‐181, 2006. © 2006 Wiley-Liss, Inc.

We have previously demonstrated an augmentation of serotonergic activity within various brain areas following infusion of brain-derived neurotrophic factor (BDNF) into the midbrain near the periaqueductal gray and dorsal and median raphe nuclei (PAG/DR). However, the mechanism of this BDNF-induced modulatory effect on serotonergic systems was unclear. The aim of the present work was to study the regulation of tryptophan hydroxylase (TPH) mRNA levels after chronic BDNF administration in vivo. TPH mRNA levels were measured using a quantitative competitive reverse transcription polymerase chain reaction (RTPCR) assay. A significant increase in the expression of TPH mRNA (13-fold) was found within the PAG/DR as early as 24 hr after onset of BDNF infusion and was sustained throughout the duration of infusion (11 days). This was accompanied by increased serotonin (5-hydroxytryptamine, 5-HT) levels and decreased nociceptive responsiveness assessed by tail-flick latency. BDNF induction of TPH mRNA levels was also observed in a serotonergic cell line derived from raphe neurons, indicating that BDNF can directly regulate TPH mRNA levels. These results suggest that BDNF augments 5-HT synthesis in vivo by directly enhancing steady-state TPH mRNA levels, and subsequently leading to marked behavioral alterations. J. Neurosci. Res. 52:149‐158, 1998. r 1998 Wiley-Liss, Inc.

Thyroid C cells are neural crest-derived neuroendocrine cells that can acquire features similar to serotonergic neurons. Based on developmental and phenotypic markers, we have previously proposed that C cells and serotonergic enteric neurons arise from a common sympathoadrenal progenitor. In this report, we genetically examined this relationship using mice lacking the mammalian achaete-scute homologue 1 (MASH-1) transcription factor, since MASH-1 has recently been shown to be required for differentiation of serotonergic enteric neurons. We found that MASH-1 knockout mice have a greatly reduced number of C cells based on the lack of calcitonin and serotonin immunoreactivity. In contrast, calcitonin and serotonin were still expressed in cultured mature C cells that no longer express MASH-1, demonstrating that MASH-1 is not directly required for the expression of these two markers. Hence, MASH-1 is required to establish the C-cell phenotype and supports the model that C cells lie in the neuronal differentiation pathway of the sympathoadrenal neural crest. © 1998 John Wiley & Sons, Inc. J Neurobiol 34: 126–134, 1998

We have investigated the possibility of mutations in the calcitonin/calcitonin gene related peptide (CGRP) gene in children with Williams syndrome. Involvement of the calcitonin/CGRP gene in Williams syndrome is postulated on the basis that Williams syndrome children often have infantile hypercalcemia and deficient expression of calcitonin, a hormone that lowers serum calcium levels. To test the hypothesis that mutations in the calcitonin/CGRP gene might be responsible for the reduced calcitonin levels, we examined the calcitonin/CGRP gene structure in Williams syndrome children. Analysis of white blood cell DNA by Southern blot hybridizations in 5 individuals did not show any detectable large deletions or rearrangements in the calcitonin/CGRP gene locus. The possibility of small deletions or point mutations within the exon encoding the mature calcitonin hormone is unlikely based on ribonuclease protection assays with patient DNA amplified by the polymerase chain reaction (PCR) technique. These findings suggest that the calcitonin deficiency might be due either to mutations elsewhere in the gene or to defects in the cellular machinery needed for calcitonin synthesis and/or secretion.

Photophobia is a subjective experience that alters sensory per-ception of light. Patients often describe photophobia in differentways. For example, some will say “light hurts my eyes” or “lightmakes my headache worse,” while others will not report associ-ated pain or pain aggravation, but unpleasantness, and state “Icannot stand even low levels of light.” However, regardless of thesubjective experience reported, they all manifest a very consistentbehavior; they retreat to a dark place or protect their eyes fromlight.This variability in the subjective experience of photophobicpatients is not surprising, considering that many different disor-ders can result in photophobia.We would like to put out the idea that photophobia involvesnot only pain pathways, but also limbic system pathways thatsuperimpose an emotional processing of discomfort leading tolight avoidance. The interaction of these pathways could providethe drive to seek out darkness. This is especially important in thecontext of migraine, where photophobia may reflect an allodynicresponse when even very low levels of light become painful.However, this avoidance can also be protective. Everyone expe-riences the need to avoid exposure of the eyes to high lightintensity. For example, looking directly at the sun is unpleasant,and most people will squint or look away to avoid retinaldamage. Likewise, the glare of skiing on a sunny day withoutsunglasses is unpleasant no matter how good the snow is. Wesuggest that these protective pathways are likely to be the sameones activated by low light in migraine and other pathologiesassociated with photophobia.So, how might the brain integrate light with pain and emo-tional circuits to yield the phenomena of photophobia? In thepast few years, we have begun to get glimpses of potential path-ways and the direction of needed future research. The cascadebegins with light activation of non-image-forming melanopsinretinal ganglion cells.

Objectives To investigate the formulation of the peptide-based antagonist (34Pro,35Phe)CGRP27–37, of the human calcitonin gene-related peptide (CGRP) receptor as a potential nasally delivered migraine treatment. Methods Peptide sequences were prepared using automated methods and purified by preparative HPLC. Their structure and stability were determined by LC-MS. Antagonist potency was assessed by measuring CGRP-stimulated cAMP accumulation in SK-N-MC, cells and in CHO cells overexpressing the human CGRP receptor. In vivo activity was tested in plasma protein extravasation (PPE) studies using Evans blue dye accumulation. Peptide-containing chitosan microparticles were prepared by spray drying. Key findings (34Pro,35Phe)CGRP27–37 exhibited a 10-fold increased affinity compared to αCGRP27–37. Administration of (34Pro,35Phe)CGRP27–37 to mice led to a significant decrease in CGRP-induced PPE confirming antagonistic properties in vivo. There was no degradation of (34Pro,35Phe)CGRP27–37 and no loss of antagonist potency during formulation and release from chitosan microparticles. Conclusions (34Pro,35Phe)CGRP27–37 is a potent CGRP receptor antagonist both in vitro and in vivo, and it can be formulated as a dry powder with no loss of activity indicating its potential as a nasally formulated anti-migraine medicine.