Drosophila <em>couch potato</em> Mutants Exhibit Complex Neurological Abnormalities Including Epilepsy Phenotypes
Abstract
RNA-binding proteins play critical roles in regulation of gene expression, and impairment can have severe phenotypic consequences on nervous system function. We report here the discovery of several complex neurological phenotypes associated with mutations of couch potato (cpo), which encodes a Drosophila RNA-binding protein. We show that mutation of cpo leads to bang-sensitive paralysis, seizure susceptibility, and synaptic transmission defects. A new cpo allele called cpoEG1 was identified on the basis of a bang-sensitive paralytic mutant phenotype in a sensitized genetic background (sda/+). In heteroallelic combinations with other cpo alleles, cpoEG1 shows an incompletely penetrant bang-sensitive phenotype with ∼30% of flies becoming paralyzed. In response to electroconvulsive shock, heteroallelic combinations with cpoEG1 exhibit seizure thresholds less than half that of wild-type flies. Finally, cpo flies display several neurocircuit abnormalities in the giant fiber (GF) system. The TTM muscles of cpo mutants exhibit long latency responses coupled with decreased following frequency. DLM muscles in cpo mutants show drastic reductions in following frequency despite exhibiting normal latency relationships. The labile sites appear to be the electrochemical GF-TTMn synapse and the chemical PSI-DLMn synapses. These complex neurological phenotypes of cpo mutants support an important role for cpo in regulating proper nervous system function, including seizure susceptibility.
RNA-BINDING proteins perform myriad crucial roles throughout the life of an RNA molecule in eukaryotes. Subsequent to their genesis in the nucleus during transcription, pre-messenger RNAs (pre-mRNA's) are bound by RNA-binding proteins, which mediate their maturation into mRNA's via processing reactions, such as splicing, editing, capping, and polyadenylation. RNA-binding proteins then assist in transporting mRNA's to the cytoplasm where they are instrumental in regulating the translation, stability, and localization of the transcripts. In addition to translated RNAs, the discovery of regulatory nontranslated RNA genes, termed micro RNA's because of their minuscule size (<100 nucleotides) suggests additional functions for RNA-binding proteins (Ambros 2001). Thus, RNA-binding proteins serve a most critical role in the control of gene expression, especially in the nervous system where extensive alternative splicing occurs and aberrations frequently result in neurological disease.
Many neurological disorders result when the performance of RNA-binding proteins goes awry, highlighting their importance in the maintenance of fundamental neuronal processes. For example, in the human neurological condition paraneoplastic opsoclonus myoclonus ataxia (POMA), patients lose inhibitory control of motor neurons in spinal cord and brainstem. POMA is associated with the ectopic expression of the NOVA family of RNA-binding proteins, which regulate neuron-specific alternative splicing (Jensenet al. 2000a,b). In humans with fragile X syndrome, impaired expression of the cytoplasmic RNA-binding protein, FMRP, leads to mental retardation, likely resulting from misregulation of mRNA transport or translation (Perrone-Bizzozero and Bolognani 2002).
Several neurological disorders that have been characterized in animal models with defective RNA-binding proteins include jerky and quaking in mice and pumilio in Drosophila. The jerky mice exhibit temporal lobe epilepsy analogous to the most common seizure disorder in human adults. The jerky gene encodes an RNA-binding protein postulated to regulate mRNA usage in neurons, which is inactivated in the mutant (Liuet al. 2002). The quaking mice exhibit tonic-clonic seizures and hypomyelination. An RNA-binding protein involved with mRNA nuclear export appears responsible for the “quaking” defects (Larocqueet al. 2002). In Drosophila, pumilio mutants show defects in embryonic development and maintenance of neuronal excitability. The pumilio mutant exhibits increased rates of long-term facilitation at the larval neuromuscular junction (Schweerset al. 2002). The pumilio gene has been shown to encode an RNA-binding protein that acts as a translational repressor (Wredenet al. 1997).
The work presented here examines a Drosophila RNA-binding protein gene called couch potato (cpo) that, when impaired, causes several neurological abnormalities including epilepsy phenotypes. The cpo gene was originally identified in a screen for genes expressed in sensory organ precursor cells during peripheral nervous system (PNS) development (Bellenet al. 1992a). Cpo protein is localized to the nucleus and is expressed in the PNS and central nervous system (CNS) of embryos, larvae, and adults, as well as other tissues such as midgut, glia, and salivary glands (Bellenet al. 1992b). The protein contains an RNA recognition motif (RRM) and a nuclear localization sequence (Bellenet al. 1992b). The RRM domain of cpo shows homology to the hermes gene of Mus musculus, Gallus gallus, and Xenopus laevis; the Caenorhabditis elegans gene mec-8; and the human gene RBP-MS (Lundquistet al. 1996; Shimamotoet al. 1996; Gerberet al. 1999). The cpo gene has also been linked to the human neurodegenerative disorder spinocerebellar ataxia type 1 (SCA1). The defective human SCA1 gene causes neurodegeneration when expressed in Drosophila; this phenotype is enhanced by overexpressing cpo (Fernandez-Funezet al. 2000). Partial loss-of-function mutations of Drosophila cpo cause a variety of behavioral phenotypes including overall sluggishness and abnormal phototaxis, geotaxis, flight ability, ether recovery, and mating vigor (Bellenet al. 1992a; Hall 1994).
In this article, we identify a new cpo allele, cpoEG1. Electrophysiological analysis shows that the cpoEG1 mutation contributes to numerous defects in the giant fiber (GF) neural circuit. Additionally, the cpoEG1 mutation contributes to increased seizure susceptibility manifested as seizure thresholds that are less than half that of wild-type flies. We have examined existing cpo alleles and have shown that they fail to complement the electrophysiological defects associated with cpoEG1. Taken together, these findings suggest a neurological basis for the complex cpo behavioral defects described previously. In addition to the previously postulated role that cpo plays in PNS differentiation and normal adult behavior, this work provides evidence that RNA-binding proteins are also essential for the proper functioning of synapses in the adult CNS and for regulating susceptibility to seizure.
For each genotype, n ≥ 6.
Acknowledgments
We thank Hugo Bellen for supplying alleles of cpo and for his insight and advice. We thank Rod Murphey for providing the A307 enhancer trap line. We thank Pejmun Haghighi for performing some crucial pilot experiments. We also thank fellow laboratory members for their guidance and wise counsel, especially Daria Hekmat-Scafe, Jeff Tan, Juan Song, and Sang-Ohk Shim. This work was supported by a National Institutes of Health research grant and an Epilepsy Foundation grant to M.A.T.