Expression of apoptosis inhibitor protein Mcl1 linked to neuroprotection in CNS neurons.

Mcl1 is a Bcl2-related antiapoptotic protein originally isolated from human myeloid leukemia cells. Unlike Bcl2, expression has not been reported in CNS neurons. We isolated Mcl1 in a direct screen for candidate modifier genes of neuronal vulnerability by differential display of mRNAs upregulated following prolonged seizures in two mouse strains with contrasting levels of hippocampal cell death. Mcl1 is widely expressed in neurons, and transcription is rapidly induced in both strains. In resistant C57Bl/6J mice, Mcl1 protein levels remain persistently elevated in hippocampal pyramidal neurons after seizures, but fall rapidly in C3H/HeJ hippocampus, coinciding with extensive neuronal apoptosis. DNA damage and caspase-mediated cell death were strikingly increased in Mcl1-deficient mice when compared to +/+ littermates after similar seizures. We identify Mcl1 as a neuronal gene responsive to excitotoxic insult in the brain, and link relative levels of Mcl1 expression to inherited differences in neuronal thresholds for apoptosis.

A cluster of three novel Ca2+ channel gamma subunit genes on chromosome 19q13.4: evolution and expression profile of the gamma subunit gene family.

The CACNG1 gene on chromosome 17q24 encodes an integral membrane protein that was originally isolated as the regulatory gamma subunit of voltage-dependent Ca2+ channels from skeletal muscle. The existence of an extended family of gamma subunits was subsequently demonstrated upon identification of CACNG2 (22q13), CACNG3 (16p12-p13), and CACNG4 and CACNG5 (17q24). In this study, we describe a cluster of three novel gamma subunit genes, CACNG6, CACNG7, and CACNG8, located in a tandem array on 19q13.4. Phylogenetic analysis indicates that this array is paralogous to the cluster containing CACNG1, CACNG5, and CACNG4, respectively, on chromosome 17q24. We developed sensitive RT-PCR assays and examined the expression profile of each member of the gamma subunit gene family, CACNG1-CACNG8. Analysis of 24 human tissues plus 3 dissected brain regions revealed that CACNG1 through CACNG8 are all coexpressed in fetal and adult brain and differentially transcribed among a wide variety of other tissues. The expression of distinct complements of gamma subunit isoforms in different cell types may be an important mechanism for regulating Ca2+ channel function.

Identification of three novel Ca(2+) channel gamma subunit genes reveals molecular diversification by tandem and chromosome duplication.

Gene duplication is believed to be an important evolutionary mechanism for generating functional diversity within genomes. The accumulated products of ancient duplication events can be readily observed among the genes encoding voltage-dependent Ca(2+) ion channels. Ten paralogous genes have been identified that encode isoforms of the alpha(1) subunit, four that encode beta subunits, and three that encode alpha(2)delta subunits. Until recently, only a single gene encoding a muscle-specific isoform of the Ca(2+) channel gamma subunit (CACNG1) was known. Expression of a distantly related gene in the brain was subsequently demonstrated upon isolation of the Cacng2 gene, which is mutated in the mouse neurological mutant stargazer (stg). In this study, we sought to identify additional genes that encoded gamma subunits. Because gene duplication often generates paralogs that remain in close syntenic proximity (tandem duplication) or are copied onto related daughter chromosomes (chromosome or whole-genome duplication), we hypothesized that the known positions of CACNG1 and CACNG2 could be used to predict the likely locations of additional gamma subunit genes. Low-stringency genomic sequence analysis of targeted regions led to the identification of three novel Ca(2+) channel gamma subunit genes, CACNG3, CACNG4, and CACNG5, on chromosomes 16 and 17. These results demonstrate the value of genome evolution models for the identification of distantly related members of gene families.

Neurulation abnormalities secondary to altered gene expression in neural tube defect susceptible Splotch embryos.

The murine mutant Splotch (Sp) is a well-established model for studying neural tube closure defects. In the current investigation, the progression through neural tube closure (NTC) as well as the expression patterns of 12 developmentally regulated genes were examined in the neural tissue of wildtype (+/+), Splotch heterozygous (Sp/+), and Splotch homozygous (Sp/Sp) embryos during neurulation. The overall growth of the embryos, as measured by the number of somite pairs, did not differ significantly between the three genotypes at any of the collection time-points. There was, however, a significant delay in the progression through NTC for both the Sp/+ and Sp/Sp embryos. A univariate analysis on the expression of the 12 candidate genes (bcl-2, FBP-2, Hmx-2, Msx-3, N-cam, N-cad, noggin, p53, Pax-3, Shh, Wee-1, wnt-1) revealed that although 11 were statistically altered, across time or by genotype, there were no significant interactions between gestation age and genotype for any of these genes during NTC. However, a multivariate statistical analysis on the simultaneous expression of these genes revealed interactions at both gestation day (GD) 8:12 (day:hour) and 9:00 among Pax-3, N-cam, N-cad, bcl-2, p53, and Wee-1 that could potentially explain the aberrant NTC. The data from these studies suggest that a disruption in the genes that govern the cell cycle or extracellular matrices of the developing neural tube might play a critical role in the occurrence of the NTDs observed in Splotch embryos.