Novel treatments against migraine are an urgent medical requirement. The α6 subunit-containing GABA receptors (α6GABARs) are expressed in trigeminal ganglia (TG), the hub of the trigeminal vascular system (TGVS) that is involved in the pathogenesis of migraine. Here we reveal an unprecedented role of α6GABARs in ameliorating TGVS activation using several pharmacological approaches in an animal model mimicking pathological changes in migraine. TGVS activation was induced by intra-cisternal (i.c.) instillation of capsaicin in Wistar rats. Centrally, i.c. capsaicin activated the trigeminal cervical complex (TCC) measured by the increased number of c-Fos-immunoreactive (c-Fos-ir) TCC neurons. Peripherally, it elevated calcitonin gene-related peptide immunoreactivity (CGRP-ir) in TG and depleted CGRP-ir in the dura mater. Pharmacological approaches included a recently identified α6GABAR-selective positive allosteric modulator (PAM), the pyrazoloquinolinone Compound 6, two α6GABAR-active PAMs (Ro15-4513 and loreclezole), an α6GABAR-inactive benzodiazepine (diazepam), an α6GABAR-selective antagonist (furosemide), and a clinically effective antimigraine agent (topiramate). We examined effects of these compounds on both central and peripheral TGVS responses induced by i.c. capsaicin. Compound 6 (3-10 mg/kg, i.p.) significantly attenuated the TCC neuronal activation and TG CGRP-ir elevation, and dural CGRP depletion induced by capsaicin. All these effects of Compound 6 were mimicked by topiramate, Ro15-4513 and loreclezole, but not by diazepam. The brain-impermeable furosemide antagonized the peripheral, but not central, effects of Compound 6. These results suggest that the α6GABAR in TG is a novel drug target for TGVS activation and that α6GABAR-selective PAMs have the potential to be developed as a novel pharmacotherapy for migraine.

BackgroundThe roles of caspase 3 on the kainic acid-mediated neurodegeneration, dendritic plasticity alteration, neurogenesis, microglial activation and gliosis are not fully understood. Here, we investigate hippocampal changes using a mouse model that receive a single kainic acid-intracerebral ventricle injection. The effects of caspase 3 inhibition on these changes were detected during a period of 1 to 7 days post kainic acid injection.ResultNeurodegeneration was assessed by Fluoro-Jade B staining and neuronal nuclei protein (NeuN) immunostaining. Neurogenesis, gliosis, neuritic plasticity alteration and caspase 3 activation were examined using immunohistochemistry. Dendritic plasticity, cleavvage-dependent activation of calcineurin A and glial fibrillary acidic protein cleavage were analyzed by immunoblotting. We found that kainic acid not only induced neurodegeneration but also arouse several caspase 3-mediated molecular and cellular changes including dendritic plasticity, neurogenesis, and gliosis. The acute caspase 3 activation occurred in pyramidal neurons as well as in hilar interneurons. The delayed caspase 3 activation occurred in astrocytes. The co-injection of caspase 3 inhibitor did not rescue kainic acid-mediated neurodegeneration but seriously and reversibly disturb the structural integrity of axon and dendrite. The kainic acid-induced events include microglia activation, the proliferation of radial glial cells, neurogenesis, and calcineurin A cleavage were significantly inhibited by the co-injection of caspase 3 inhibitor, suggesting the direct involvement of caspase 3 in these events. Alternatively, the kainic acid-mediated astrogliosis is not caspase 3-dependent, although caspase 3 cleavage of glial fibrillary acidic protein occurred.ConclusionsOur results provide the first direct evidence of a causal role of caspase 3 activation in the cellular changes during kainic acid-mediated excitotoxicity. These findings may highlight novel pharmacological strategies to arrest disease progression and control seizures that are refractory to classical anticonvulsant treatment.

Chitin is an abundant biopolymer composed of units of N-acetyl-D-glucosamine linked by β-1,4 glycosidic bonds. Chitin is the main component of the shells of mollusks, the cell wall of fungi and yeast and of the exoskeleton of crustaceans and insects. The degradation of chitin is catalyzed by chitinases that occur in a wide range of organisms. Among them, the chitinases from microorganisms are extremely important for the degradation and recycling of the carbon and nitrogen trapped in the large amount of insoluble chitin in nature. Streptomyces sp. TH-11 was isolated from the sediment of the Tou-Chien River, Taiwan. The chitinolytic enzyme activities were detected using a rapid in-gel detection method from the cell-free preparation of the culture medium of TH-11. The chitinolytic enzyme activity during prolonged liquid culturing was also analyzed by direct measurement of the chitin consumption. Decomposition of the exoskeleton of shrimps was demonstrated using electron microscopy and atomic force microscopy.

Migraine is caused by hyperactivity of the trigeminovascular system, where trigeminal ganglia (TG) play an important role. This hyperactivity might originate from an underfunctional GABAergic system in TG. To investigate this possibility, we adapted a mouse model of migraine by inducing migraine-like grimaces in male mice via repeated injections of nitroglycerin (NTG, 10 mg/kg, i.p.), once every 2 days, for up to 5 sessions. Migraine-like facial pain scores were measured using the mouse grimace scale. Repeated NTG treatments in mice caused significant increases in migraine-like grimaces that were aborted and prevented by two anti-migraine agents sumatriptan and topiramate, respectively. After 5 sessions of NTG injections, the GABAsynthesizing enzyme, 65-kDa glutamate decarboxylase (GAD65), but not the GABA transporter 1 (GAT1) or the α6 subunitcontaining GABA A receptors (α6GABA A Rs), was downregulated in mouse TG tissues. Taking advantage of the unaffected TG α6GABA A R expression in NTG-treated mice, we demonstrated that an α6GABA A R-selective positive allosteric modulator (PAM), DK-I-56-1, exhibited both abortive and prophylactic effects, comparable to those of sumatriptan and topiramate, respectively, in this migraine-mimicking mouse model. The brain-impermeable furosemide significantly prevented the effects of DK-I-56-1, suggesting its peripheral site of action, likely via preventing α6GABA A R modulation in TG. Results suggest that a decreased GABA synthesis caused by the reduced GAD65 expression in TG contributes to the trigeminovascular activation in this repeated NTG-induced migraine-mimicking model and that the unaltered α6GABA A Rs in TG are potential targets for migraine treatment. Thus, α6GABA A R-selective PAMs are potential anti-migraine agents for both abortive and preventive therapies.Key Words Migraine . α6 subunit-containing GABA A receptor . trigeminal ganglia . nitroglycerin . mouse grimacescale . 65-kDa glutamate decarboxylase . GABA transporter 1.Hung-Ruei Tzeng and Ming Tatt Lee contributed equally to this work.

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