The calcium-permeable transient receptor potential M2 (TRPM2) ion channel is activated following oxidative stress and has been implicated in ischemic damage; however, little experimental evidence exists linking TRPM2 channel activation to damage following cerebral ischemia. We directly assessed the involvement of TRPM2 channels in ischemic brain injury using pharmacological inhibitors and short-hairpin RNA (shRNA)-mediated knockdown of TRPM2 expression. Each of the four TRPM2 inhibitors tested provided significant protection to male neurons following in vitro ischemia (oxygen-glucose deprivation, OGD), while having no effect in female neurons. Similarly, TRPM2 knockdown by TRPM2 shRNA resulted in significantly reduced neuronal cell death following OGD only in male neurons. The TRPM2 inhibitor clotrimazole reduced infarct volume in male mice, while having no effect on female infarct volume. Finally, intrastriatal injection of lentivirus expressing shRNA against TRPM2 resulted in significantly smaller striatal infarcts only in male mice following middle cerebral artery occlusion, having no significant effect in female mice. Data presented in the current study demonstrate that TRPM2 inhibition and knockdown preferentially protects male neurons and brain against ischemia in vitro and in vivo, indicating that TRPM2 inhibitors may provide a new therapeutic approach to the treatment of stroke in men.
SummaryBackground and PurposeControl of nematode parasite infections relies largely on anthelmintic drugs, several of which act on nicotinic ACh receptors (nAChRs), and there are concerns about the development of resistance. There is an urgent need for development of new compounds to overcome resistance and novel anthelmintic drug targets. We describe the functional expression and pharmacological characterization of a homomeric nAChR, ACR‐16, from a nematode parasite.Experimental ApproachUsing RT‐PCR, molecular cloning and two‐electrode voltage clamp electrophysiology, we localized acr‐16 mRNA in Ascaris suum (Asu) and then cloned and expressed acr‐16 cRNA in Xenopus oocytes. Sensitivity of these receptors to cholinergic anthelmintics and a range of nicotinic agonists was tested.Key ResultsAmino acid sequence comparison with vertebrate nAChR subunits revealed ACR‐16 to be most closely related to α7 receptors, but with some striking distinctions. acr‐16 mRNA was recovered from Asu somatic muscle, pharynx, ovijector, head and intestine. In electrophysiological experiments, the existing cholinergic anthelmintic agonists (morantel, levamisole, methyridine, thenium, bephenium, tribendimidine and pyrantel) did not activate Asu‐ACR‐16 (except for a small response to oxantel). Other nAChR agonists: nicotine, ACh, cytisine, 3‐bromocytisine and epibatidine, produced robust current responses which desensitized at a rate varying with the agonists. Unlike α7, Asu‐ACR‐16 was insensitive to α‐bungarotoxin and did not respond to genistein or other α7 positive allosteric modulators. Asu‐ACR‐16 had lower calcium permeability than α7 receptors.Conclusions and ImplicationsWe suggest that ACR‐16 has diverse tissue‐dependent functions in nematode parasites and is a suitable drug target for development of novel anthelmintic compounds.
Cocaine- and amphetamine-regulated transcript (CART) is a hypothalamic neuropeptide implicated in both metabolic and reproductive regulation, raising the possibility that CART plays a role in reproductive inhibition during negative metabolic conditions. The current study characterized CART's regulatory influence on GnRH and kisspeptin (Kiss1) cells and determined the sensitivity of different CART populations to negative energy balance. CART fibers made close appositions to 60% of GnRH cells, with the majority of the fibers (>80%) originating from the arcuate nucleus (ARH) CART/pro-opiomelanocortin population. Electrophysiological recordings in GnRH-green fluorescent protein rats demonstrated that CART postsynaptically depolarizes GnRH cells. CART fibers from the ARH were also observed in close contact with Kiss1 cells in the ARH and anteroventral periventricular nucleus (AVPV). Recordings in Kiss1-GFP mice demonstrated CART also postsynaptically depolarizes ARH Kiss1 cells, suggesting CART may act directly and indirectly, via Kiss1 populations, to stimulate GnRH neurons. CART protein and mRNA levels were analyzed in 2 models of negative energy balance: caloric restriction (CR) and lactation. Both CART mRNA levels and the number of CART-immunoreactive cells were suppressed in the ARH during CR but not during lactation. AVPV CART mRNA was suppressed during CR, but not during lactation when there was a dramatic increase in CART-immunoreactive cells. These data suggest differing regulatory signals of CART between the models. In conclusion, both morphological and electrophysiological methods identify CART as a novel and potent stimulator of Kiss1 and GnRH neurons and suppression of CART expression during negative metabolic conditions could contribute to inhibition of the reproductive axis.
Hippocampal CA1 neurons are particularly sensitive to ischemic damage, such as experienced following cardiac arrest and cardiopulmonary resuscitation. In recent years transient receptor potential M2 (TRPM2) channels have been identified as mediators of ischemic damage. We previously demonstrated that neuroprotective strategies targeting TRPM2 channels preferentially protect male cortical neurons from ischemic injury both in vitro and in vivo. It is important to determine the role of TRPM2 in ischemic injury of hippocampal neurons as this population of neurons are particularly sensitive to ischemic injury and are therapeutic targets. Here we report significantly decreased neuronal cell death following in vitro ischemia preferentially in male hippocampal neurons using TRPM2 inhibitors or knockdown of TRPM2 expression. Electrophysiological characterization of sex-stratified cultures shows similar levels of functional TRPM2 channel expression in male and female hippocampal neurons under basal conditions. In contrast, recordings made during reperfusion following in vitro ischemia revealed that TRPM2 channels are activated only in male neurons, resulting in rapid and complete depolarization. These findings provide strong evidence for TRPM2 as a target for protection against cerebral ischemia in male brain and helps define a molecular cell death pathway that is differentially engaged in male and female neurons.
Here we review recent studies on the mode of action of the cholinergic anthelmintics (levamisole, pyrantel etc.). We also include material from studies on the free living nematode Caenorhabditis elegans. The initial notion that these drugs act on a single receptor population, while attractive, has proven to be an oversimplification. In both free living and parasitic nematodes there are multiple types of nicotinic acetylcholine receptor (nAChR) on the somatic musculature. Each type has different (sometimes subtly so) pharmacological properties. The implications of these findings are: (1) combinations of anthelmintic that preferentially activate a broad range of nAChR types would be predicted to be more effective; (2) in resistant isolates of parasite where a subtype has been lost, other cholinergic anthelmintics may remain effective. Not only are there multiple types of nAChR, but relatively recent research has shown these receptors can be modulated; it is possible to increase the response of a parasite to a fixed concentration of drug by altering the receptor properties (e.g. phosphorylation state). These findings offer a potential means of increasing efficacy of existing compounds as an alternative to the costly and time consuming development of new anthelmintic agents.
Many techniques for studying functional genomics of important target sites of anthelmintics have been restricted to Caenorhabditis elegans because they have failed when applied to animal parasites. To overcome these limitations, we have focused our research on the human nematode parasite Brugia malayi, which causes elephantiasis. Here, we combine single-cell PCR, whole muscle cell patch clamp, motility phenotyping (Worminator), and dsRNA for RNAi for functional genomic studies that have revealed, in vivo, four different muscle nAChRs (M-, L-, P-, and N-). The cholinergic anthelmintics had different selectivities for these receptors. We show that motility and patch-clamp responses to levamisole and pyrantel, but not morantel or nicotine, require the unc-38 and/or unc-29 genes. Derquantel behaved as a competitive antagonist and distinguished M-nAChRs activated by morantel (K b 13.9 nM), P-nAChRs activated by pyrantel (K b 126 nM), and L-nAChRs activated by levamisole (K b 0.96 μM) and bephenium. Derquantel was a noncompetitive antagonist of nicotine, revealing N-type nAChRs. The presence of four diverse nAChRs on muscle is perhaps surprising and not predicted from the C. elegans model. The diverse nAChRs represent distinguishable drug targets with different functions: Knockdown of unc-38+unc-29 (L-and/or P-receptors) inhibited motility but knockdown of acr-16+acr-26 (M-and/or N-receptors) did not.T he neglected tropical diseases caused by nematode parasites affect some 2 billion people globally, and effective vaccines are not available. Although these parasites by themselves do not produce high mortality, they produce significant morbidity that affects worker productivity and increase susceptibility to AIDS and malaria, and they are strongly associated with poverty in endemic countries (1-4). There are only a limited number of classes of anthelmintic drugs that are used to treat these diseases, and there are concerns about the development of resistance (5). There is a pressing need to advance the study of anthelmintic drugs.Many useful studies on modes of action of anthelmintic drugs have been conducted on the model nematode, Caenorhabditis elegans, but this is not a parasite: It is separated evolutionarily by millions of years from parasitic nematodes and it does not possess "parasitism genes" (6). This separation increases the impetus for anthelmintic studies using real parasites, but until now many techniques developed in C. elegans have not have been tractable when applied to animal parasites (7).The neuromuscular systems of nematodes, which control motility and support feeding, growth, and reproduction, are governed by significant parts of their gene pools (8, 9). Their neuromuscular systems are unlike those of vertebrates because nematode muscles send processes to the motor neurons to form synapses, rather than the other way around. Membrane ion-channels, which regulate nematode neuromuscular systems, are target sites of major classes of anthelmintic drugs, including the cholinergic anthelmintics levamisole,...
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