Etiology of a genetically complex seizure disorder in Celf4 mutant mice.

Mice deficient for the gene encoding the RNA-binding protein CELF4 (CUGBP, ELAV-like family member 4) have a complex seizure phenotype that includes both convulsive and non-convulsive seizures, depending upon gene dosage and strain background, modeling genetically complex epilepsy. Invertebrate CELF is associated with translational control in fruit fly ovary epithelium and with neurogenesis and neuronal function in the nematode. Mammalian CELF4 is expressed widely during early development, but is restricted to the central nervous system in adults. To better understand the etiology of the seizure disorder of Celf4 deficient mice, we studied seizure incidence with spatial and temporal conditional knockout Celf4 alleles. For convulsive seizure phenotypes, it is sufficient to delete Celf4 in adulthood at the age of 7 weeks. This timing is in contrast to absence-like non-convulsive seizures, which require deletion before the end of the first postnatal week. Interestingly, selective deletion of Celf4 from cerebral cortex and hippocampus excitatory neurons, but not from inhibitory neurons, is sufficient to lower seizure threshold and to promote spontaneous convulsions. Correspondingly, Celf4 deficient mice have altered excitatory, but not inhibitory, neurotransmission as measured by patch-clamp recordings of cortical layer V pyramidal neurons. Finally, immunostaining in conjunction with an inhibitory neuron-specific reporter shows that CELF4 is expressed predominantly in excitatory neurons. Our results suggest that CELF4 plays a specific role in regulating excitatory neurotransmission. We posit that altered excitatory neurotransmission resulting from Celf4 deficiency underlies the complex seizure disorder in Celf4 mutant mice.

Szt2, a novel gene for seizure threshold in mice.

In a chemical mutagenesis screen we identified Szt2 (seizure threshold 2) as a gene that confers low seizure threshold to mice and may also enhance epileptogenesis. The semidominant phenotype was mapped to Chromosome 4 and narrowed further to a critical interval of approximately 650 kb. A novel large (> 10 kb) transcript in the critical interval was found to have fourfold increased steady-state expression at the RNA level in Szt2 homozygous mutant brain. The corresponding 72 exon gene encodes a 378-kD protein with no significant or suggestive sequence similarities to any other protein. The mutant allele of Szt2 contains a splice donor mutation after exon 32, predicting transcriptional read-through, translational frameshift and premature stop. A second Szt2 allele, containing a gene-trap mutation in exon 21, also conferred a low seizure threshold and increased RNA expression, but unlike the original allele, some gene-trap homozygotes died embryonically. Szt2 is transcribed in many tissues, with the highest expression in brain, and it is also expressed during embryonic development. Szt2 is highly conserved in evolution, with a clear, single orthologue found in all land vertebrates and in many invertebrates. Interestingly, in mammals the Szt2 gene resides in a highly conserved head-to-head configuration with Med8 (which encodes a Mediator complex subunit), separated by only 91 nt. While the biological function of Szt2 remains unknown, its high conservation, unique structure and effect on seizure threshold suggest that it serves an important role in the central nervous system.

The mouse fidgetin gene defines a new role for AAA family proteins in mammalian development.

The mouse mutation fidget arose spontaneously in a heterogeneous albino stock. This mutant mouse is characterized by a side-to-side head-shaking and circling behaviour, due to reduced or absent semicircular canals. Fidget mice also have small eyes, associated with cell-cycle delay and insufficient growth of the retinal neural epithelium, and lower penetrance skeletal abnormalities, including pelvic girdle dysgenesis, skull bone fusions and polydactyly. By positional cloning, we found the gene mutated in fidget mice, fidgetin (Fign), which encodes a new member of the 'meiotic' or subfamily-7 (SF7; ref. 7) group of ATPases associated with diverse cellular activities (AAA proteins). We also discovered two closely related mammalian genes. AAA proteins are molecular chaperones that facilitate a variety of functions, including membrane fusion, proteolysis, peroxisome biogenesis, endosome sorting and meiotic spindle formation, but functions for the SF7 AAA proteins are largely unknown. Fidgetin is the first mutant AAA protein found in a mammalian developmental mutant, thus defining a new role for these proteins in embryonic development.

The mouse stargazer gene encodes a neuronal Ca2+-channel gamma subunit.

Stargazer mice have spike-wave seizures characteristic of absence epilepsy, with accompanying defects in the cerebellum and inner ear. We describe here a novel gene, Cacng2, whose expression is disrupted in two stargazer alleles. It encodes a 36-kD protein (stargazin) with structural similarity to the gamma subunit of skeletal muscle voltage-gated calcium (Ca2+) channels. Stargazin is brain-specific and, like other neuronal Ca2+-channel subunits, is enriched in synaptic plasma membranes. In vitro, stargazin increases steady-state inactivation of alpha1 class A Ca2+ channels. The anticipated effect in stargazer mutants, inappropriate Ca2+ entry, may contribute to their more pronounced seizure phenotype compared with other mouse absence models with Ca2+-channel defects. The discovery that the stargazer gene encodes a gamma subunit completes the identification of the major subunit types for neuronal Ca2+ channels, namely alpha1, alpha2delta, beta and gamma, providing a new opportunity to understand how these channels function in the mammalian brain and how they may be targeted in the treatment of neuroexcitability disorders.

Identification of the mouse neuromuscular degeneration gene and mapping of a second site suppressor allele.

The nmd mouse mutation causes progressive degeneration of spinal motor neurons and muscle atrophy. We identified the mutated gene as the putative transcriptional activator and ATPase/DNA helicase previously described as Smbp2, Rip1, Gf1, or Catf1. Mutations were found in two alleles-a single amino acid deletion in nmdJ and a splice donor mutation in nmd2J. The selective vulnerability of motor neurons is striking in view of the widespread expression of this gene, although the pattern of degeneration may reflect a specific threshold since neither allele is null. In addition, the severity of the nmd phenotype is attenuated in a semidominant fashion by a major genetic locus on chromosome (Chr) 13. The identification of the nmd gene and mapping of a major suppressor provide new opportunities for understanding mechanisms of motor neuron degeneration.

Intron/exon structure confirms that mouse Zfy1 and Zfy2 are members of the ZFY gene family.

Zfy1 and Zfy2 are homologous zinc finger genes on the mouse Y Chromosome. To ask whether these genes are properly classified as members of the ZFY family, we have characterized and compared their genomic organization to that of mouse Zfx, human ZFX, and human ZFY. We show that Zfy1 has 11 exons distributed across at least 56 kb, and Zfy2 has a minimum of 9 exons distributed across at least 52 kb. The Zfy2 locus contains regions similar in size and sequence to all 11 exons of Zfy1, plus an additional 5' UTR exon. All splice sites conform to the GT-AG rule. There are two instances of additional AG dinucleotides immediately 5' of 3' splice sites. Zfy1 and Zfy2 are homologous to other ZFY family members within the coding region, but the untranslated regions show no sequence similarity. Within the coding region, there is conservation of exon length and splice sites, with each splice preceding the second nucleotide of a codon. We conclude that Zfy1 and Zfy2 are indeed members of the ZFY family, which has evolved from a single common ancestral gene.