Selenium is an essential micronutrient that functions as an antioxidant. Yet, at higher concentrations, selenium is pro-oxidant and toxic. In extreme cases, exposures to excess selenium can lead to death or selenosis, a syndrome characterized by teeth, hair and nail loss, and nervous system alterations. Recent interest in selenium as an anti- tumorigenic agent has reemphasized the need to understand the mechanisms underlying the cellular consequences of increased selenium exposure. We show here, that in the nematode, Caenorhabditis elegans, selenium has a concentration range in which it functions as an antioxidant, but beyond this range it exhibits a dose- and time-dependent lethality. Oxidation-induced fluorescence emitted by the dye, carboxy-H2DCFDA, indicative of reactive oxygen species formation was significantly higher in animals after a brief exposure to 5mM sodium selenite. Longer-term exposures lead to a progressive selenium-induced motility impairment that could be partially prevented by coincident exposure to the cellular antioxidant–reduced glutathione. The C elegans glrx-21 gene belongs to the family of glutaredoxins (glutathione-dependent oxidoreductases) and the glrx-21(tm2921) allele is a null mutation that renders animals hypersensitive for the selenium-induced motility impairment, but not lethality. In addition, the lethality of animals with the tm2921 mutation exposed to selenium was unaffected by the addition of reduced glutathione, suggesting that GLRX-21 is required for glutathione to moderate this selenium-induced lethality. Our findings provide the first description of selenium-induced toxicity in C elegans and support its use as a model for elucidating the mechanisms of selenium toxicity.

SUMMARYThe C. elegans daf-8 gene encodes an R-Smad that is expressed in a subset of head neurons, the intestine, gonadal distal tip cells and the excretory cell. We found that DAF-8, which inhibits the DAF-3 Co-Smad, is associated with DAF-3 and the DAF-14 Smad in vivo and in vitro. Overexpression of daf-8 conferred a dauer-defective phenotype and suppressed constitutive dauer formation in daf-8 and daf-14 mutants. In contrast to mammalian systems described thus far, active DAF-3 drives a feedback regulatory loop that represses transcription of daf-7 (a TGF ligand) and daf-8 by directly binding to their regulatory regions. Hence, DAF-8 and DAF-3 are mutually antagonistic. The feedback repression may reinforce the developmental switch by allowing DAF-3 to freely activate dauer transcription in target tissues, unless sufficiently inhibited by DAF-8 and DAF-14. In the adult, DAF-8 downregulates lag-2 expression in the distal tip cells, thus promoting germ line meiosis. This function does not involve DAF-3, thereby avoiding the feedback loop that functions in the dauer switch.

Migraine is an episodic pain disorder whose pathophysiology is related to deficiency of serotonin signaling and abnormal function of the P/Q-type calcium channel, CACNA1A. Because the relationship of the CACNA1A channel to serotonin signaling is unknown and potentially of therapeutic interest we have used genetic analysis of the Caenorhabditis elegans ortholog of this calcium channel, UNC-2, to help identify candidate downstream effectors of the human channel. By genetic dissection of the lethargic mutant phenotype of unc-2, we have established an epistasis pathway showing that UNC-2 function antagonizes a transforming growth factor (TGF)-b pathway influencing movement rate. This same UNC-2/TGF-b pathway is required for accumulation of normal serotonin levels and stress-induced modulation of tryptophan hydroxylase (tph) expression in the serotonergic chemosensory ADF neurons, but not the NSM neurons. We also show that transgenic expression of the migraine-associated Ca 2+ channel, CACNA1A, in unc-2 animals can functionally substitute for UNC-2 in stress-activated regulation of tph expression. The demonstration that these evolutionarily related channels share a conserved ability to modulate tph expression through their effects on TGF-b signaling provides the first specific example of how CACNA1A function may influence levels of the critical migraine neurotransmitter serotonin. Clinical observations such as relief of migraine attacks with serotonin (5HT)1D receptor agonists and migraine prevention with 5HT2 receptor antagonists suggest that serotonin regulation is central to the underlying pathophysiology of migraine. Biochemical assays showing altered serum serotonin levels during migraine headache (Ferrari et al. 1989) and studies showing hypermetabolism in the brainstem region of the serotonergic raphe nuclei during migraine attacks using [18F]-2-fluoro-2 deoxyglucose (FDG) positron emission tomography (Weiller et al. 1995) are also consistent with the importance of 5HT in this neurologic disorder. Human genetic studies have provided further insight into the pathophysiology of migraine through the discovery of dominant missense mutations in CACNA1A, an a1A-Ca 2+ channel subunit gene associated with a rare form of migraine called familial hemiplegic migraine (FHM) (Ophoff et al. 1996). Families with FHM have a severe migrainous aura including variable weakness on one side of the body, ataxia and also migraine headaches (Ophoff et al. 1996). As CACNA1A is one of the primary presynaptic voltage-gated calcium channels it plays an important part in the regulation of basal neuronal excitability, and in fact migraineurs display clinical symptoms of neuronal hyperexcitability called visual and sensory aura that can sometimes be suppressed by 5HT1B/1D receptor agonists (Klapper et al. 2001). However, no signaling connection between CACNA1A and the clinically demonstrated role of serotonin in migraine has yet been established.In mammals serotonin regulates cell excitability through activation of the ionotropic 5H...

Selenium is an essential micronutrient required for cellular antioxidant systems, yet at higher doses it induces oxidative stress. Additionally, in vertebrates environmental exposures to toxic levels of selenium can cause paralysis and death. Here we show that selenium-induced oxidative stress leads to decreased cholinergic signaling and degeneration of cholinergic neurons required for movement and egg-laying in Caenorhabditis elegans. Exposure to high levels of selenium leads to proteolysis of a soluble muscle protein through mechanisms suppressible by two pharmacological agents, levamisole and aldicarb which enhance cholinergic signaling in muscle. In addition, animals with reduction-of-function mutations in genes encoding post-synaptic levamisole-sensitive acetylcholine receptor subunits or the vesicular acetylcholine transporter developed impaired forward movement faster during selenium-exposure than normal animals, again confirming that selenium reduces cholinergic signaling. Finally, the antioxidant reduced glutathione, inhibits selenium-induced reductions in egg-laying through a cellular protective mechanism dependent on the C. elegans glutaredoxin, GLRX-21. These studies provide evidence that the environmental toxicant selenium induces neurodegeneration of cholinergic neurons through depletion of glutathione, a mechanism linked to the neuropathology of Alzheimer’s disease, amyotrophic lateral sclerosis, and Parkinson’s disease.

Proper calcium channel and insulin signaling are essential for normal brain development. Leaner mice with a mutation in the P/Q-type voltage-gated calcium channel, Cacna1a, develop cerebellar atrophy and mutations in the homologous human gene are associated with increased migraine and seizure tendency. Similarly, abnormalities in insulin signaling are associated with abnormal brain growth and migraine tendency. Previously, we have shown that in the ADF chemosensory neurons of Caenorhabditis elegans UNC-2/Ca(2+) channel function affects TGF-beta-dependent developmental regulation of tryptophan hydroxylase, the rate-limiting enzyme in serotonin synthesis. Here we show that developmental expression of a tryptophan hydroxylase: :GFP reporter construct is similarly decreased by reduction-of-function mutations in the daf-2/insulin receptor. This decreased expression of tryptophan hydroxylase observed in both the daf-2 and unc-2 mutant backgrounds is suppressible either genetically by reduction-of-function mutations in the daf-16/forkhead transcription factor, an effector of the DAF-2/insulin receptor, or pharmacologically by the serotonin receptor antagonist cyproheptadine. Overall, these data suggest that both UNC-2 and DAF-2 function are required in the developmental regulation of DAF-16 and serotonin-dependent inhibition of tryptophan hydroxylase expression.