Mutations in a few C. elegans genes 1 can result in a convulsive phenotype marked by repeated end-to-end full body contractions caused by the simultaneous excitation of dorsal and ventral body wall muscles.
unc-43 encodes the only CaM Kinase II homologue in C. elegans .
unc-43(lf) worms have rare spontaneous convulsions that are intensified in frequency and severity by exposure to neurostimulants ( e.g., pentylenetetrazole [PTZ] and pilocarpine). To investigate the underlying molecular mechanisms of these convulsions, we are looking in detail at the neuronal requirement for UNC-43. Using neural subtype-specific promoters, we have delineated the requirement of UNC-43 to the motor neurons. In addition, we have demonstrated that this neural control of convulsions requires only that UNC-43 be present in the adult nervous system by using the heat-shock promoter
hsp16-2 . This result implies that the convulsion phenotype of
unc-43(lf) is separable from its defects in early neuron development 2 . These experiments show that UNC-43 expression in adult motor neurons is sufficient to suppress convulsions but we would like to address necessity as well. To these ends, we have devised the inverse experiments where we systematically remove UNC-43 function from certain neurons using the rat protein CaMKIIN, a CaMKII-specific inhibitor 3 . In controls where we drive CaMKIIN expression with the
unc-43 promoter in wild-type worms, we see a complete phenocopy of
unc-43(lf) including defects in locomotion and defecation. Other promoter constructs ( i.e. ,
hsp16-2,
unc-30 and
acr-2 ) are in progress and will be discussed. Finally, we have begun assessing the role of the various domains in the multifunctional UNC-43 protein. Transgenic worms containing a series of well-studied functionally modified rat CaMKII genes 4,5 are being made and the phenotypic outputs compared. Transgenic lines have verified that rCaMKII(+) rescues
unc-43(lf) defects fully. Additionally, the constitutively-active Ca2+/Calmodulin-independent rCaMKII (T286D) and (271trunc) behave like the other gain-of-function alleles of
unc-43 we have studied. We are presently assaying a kinase-dead variant rCaMKII(K42M) which will determine whether kinase function is necessary for convulsion mediation. These experiments characterize the role of UNC-43 in controlling simultaneous excitation of neuronal networks in C. elegans as phenotypically reported through repetitive contractions. Similar types of neuronal synchrony may cause human epilepsies, since a knockout of the a -CaM Kinase II 6 gene in mouse is susceptible to spontaneous and drug-induced seizures. We believe that our use of C. elegans , as a well-studied and tractable model organism combining sophisticated genetics and a OmappedO nervous system 7 may be a powerful way to characterize key molecular components controlling seizures. 1 See abstract McCormick, et al. (2004). Mutational analysis of genes disrupting neuronal synchrony in C. elegans , West Coast Worm Meeting 2 Rongo, C. and Kaplan, J. M. (1999). Nature 402:195-199 3 Chang, B.H. et al. (1998). PNAS 95:10890-10895 4 Hanson, P.I. et al. (1994). Neuron 12:943-956 5 Waldmann, R. et al. (1990). Biochemistry 29:1679-1684 6 Butler, L.S., et al. (1995). PNAS 92:6852-6855 7 White, J.G., et al. (1986). Phil. Trans. R. Soc. Lond. 314:1-340