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Inbred strains of mice differ in their susceptibility to excitotoxin-induced cell death, but the genetic basis of individual variation in differential susceptibility is unknown. Previously, we identified a highly significant quantitative trait locus (QTL) on chromosome 18 that influenced susceptibility to kainic acid-induced cell death (Sicd1). Comparison of susceptibility to seizure-induced cell death between reciprocal congenic lines for Sicd1 and parental background mice indicates that genes influencing this trait were captured in both strains. Two positional gene candidates, Galr1 and Mbp, map to 55 cM, where the Sicd1 QTL had been previously mapped. Thus, this study was undertaken to determine if Galr1 and/or Mbp could be considered as candidate genes. Genomic sequence comparison of these two functional candidate genes from the C57BL/6J (resistant at Sicd1) and the FVB/NJ (susceptible at Sicd1) strains showed no single-nucleotide polymorphisms. However, expression studies confirmed that Galr1 shows significant differential expression in the congenic and parental inbred strains. Galr1 expression was downregulated in the hippocampus of C57BL/6J mice and FVB.B6-Sicd1 congenic mice when compared with FVB/NJ or B6.FVB-Sicd1 congenic mice. A survey of Galr1 expression among other inbred strains showed a significant effect such that ‘susceptible’ strains showed a reduction in Galr1 expression as compared with ‘resistant’ strains. In contrast, no differences in Mbp expression were observed. In summary, these results suggest that differential expression of Galr1 may contribute to the differences in susceptibility to seizure-induced cell death between cell death-resistant and cell death-susceptible strains.
Linkage studies using (C57BL/6J X FVB/NJ)N2 mice mapped three susceptibility loci, with the most significant locus, named seizure-induced cell death 1 (Sicd1), to the distal region of mouse chromosome 18 (Schauwecker et al. 2004). To confirm genetic linkage on distal chromosome 18, we created the congenic strain, FVB.B6-Sicd1, in which the relevant donor segment from the resistant C57BL/6J strain was placed on the susceptible FVB/NJ background. The presence of C57BL/6 chromosome 18 alleles on an FVB genetic background conferred protection against seizure-induced cell death, as compared with FVB/NJ parental controls (Lorenzana et al. 2007; Schauwecker et al. 2004). These results suggested that the causal gene(s) influencing susceptibility to seizure-induced cell death may reside in the Sicd1 locus.
As we verified that the Sicd1 locus significantly reduced susceptibility to kainic acid (KA)-induced excitotoxic cell death in the congenic FVB.B6-Sicd1 strain (Lorenzana et al. 2007; Schauwecker et al. 2004), we took a candidate gene approach to identify the causal susceptibility gene(s) responsible for the Sicd1 effect. The genetic variation underlying this quantitative trait locus (QTL) could consist of polymorphisms in either the coding region, thus altering the amino acid sequence of the translated protein, or the regulatory region, affecting expression of a gene. Among the many candidate genes present in the 12-Mb Sicd1 region, galanin receptor 1 (Galr1) and myelin basic protein (Mbp) are the most compelling. Both of these genes approximate the peak position of the Sicd1 QTL at 55 cM (Schauwecker et al. 2004) and have relevance to modulation of neuronal excitability and seizure threshold (Donarum et al. 2006; Jacoby et al. 2002; Mathis et al. 2000; Mazarati et al. 2000, 2006; McColl et al. 2006; Zini et al. 1993b).
In this paper, we describe a systematic gene identification strategy in which we conducted comparative genomic sequencing, expression analyses and comparative cDNA sequencing of two putative candidate genes, Galr1 and Mbp. To examine the possibility that variation in one of these genes could be involved in the susceptibility to seizure-induced excitotoxic cell death, we assessed the expression of mRNA for Galr1 and Mbp in the hippocampus of our two strains and between the FVB.B6-Sicd1 and the B6.FVB-Sicd1 congenic strains and their respective parental background strains, FVB/NJ or C57BL/6J. We also conducted a strain survey to examine the association between the Sicd1 genotype and the susceptibility to seizure-induced cell death among inbred strains of mice.
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- Materials and methods
Phenotyping studies of a new congenic strain developed in our laboratory have confirmed the significant genetic influence of a chromosome 18 QTL: Sicd1 for susceptibility to seizure-induced cell death (Schauwecker et al. 2004). To validate the Sicd1 effect and to move towards identification of the quantitative trait gene(s) for Sicd1, we generated reciprocal congenic lines of mice. These strains were differentially susceptible to seizure-induced cell death because they had received a chromosomal donor region from either FVB/NJ that contained the susceptible Sicd1 alleles (B6.FVB-Sicd1) or C57BL/6J that contained the resistant Sicd1 alleles (FVB.B6-Sicd1). These results give additional support for Sicd1 containing a gene (or a set of closely linked genes) contributing to seizure-induced cell death.
Because these congenic strains retained the phenotype of the original linked QTL, we hypothesized that candidate genes responsible for conferring susceptibility to seizure-induced excitotoxic cell death were present within the identified 12-Mb Sicd1 interval on chromosome 18. The mechanism(s) underlying phenotypic strain differences in susceptibility to seizure-induced cell death could involve genes affecting hippocampal excitability, hyperexcitability and glutamate release. Therefore, genes encoding proteins involved in these processes are likely candidates for modifying susceptibility to KA-induced excitotoxic cell death. Two such genes in the reduced Sicd1 interval satisfied this functional criteria, Galr1 and Mbp, and were subjected to candidate gene analysis. Combining the gene expression data generated in the congenic FVB.B6-Sicd1 strain with the association data of inbred strains of mice segregating for the trait of susceptibility to KA-induced excitotoxic cell death, we were able to identify Galr1 as a putative causal susceptibility gene for KA-induced excitotoxicity. In contrast, we did not find any evidence to support Mbp as a causal susceptibility gene for KA-induced excitotoxicity.
It was hypothesized that the gene(s) producing the difference in response to kainate-induced cell death between B6 and FVB mice would be differentially expressed in the hippocampus of untreated control mice. We were especially interested in the Galr1 and Mbp candidate genes because of their published associations with modifying hippocampal excitability and modifying hyperexcitability (Ben-Ari 1990; Donarum et al. 2006; Mazarati et al. 2000; Noveroske et al. 2005). To confirm the identity of any putative candidate genes, expression studies were performed using qRT-PCR, and any gene expression differences observed would presumably be the result of changes in allele frequencies because of selection rather than a response to kainate treatment. We found that B6 mice showed reduced levels of hippocampal Galr1 mRNA as compared with FVB mice. Second, the congenic FVB.B6-Sicd1 strain carrying the B6-derived resistant allele at the Sicd1 region, where Galr1 is located, on the FVB genetic background showed significantly decreased basal hippocampal Galr1 expression compared with the susceptible FVB control littermates. In contrast, the congenic B6.FVB-Sicd1 strain carrying the FVB-derived susceptible allele at the Sicd1 region on the B6 genetic background showed significantly enhanced basal hippocampal Galr1 expression compared with the resistant B6 control littermates.
We speculate that the downregulation of Galr1 observed in B6 and FVB.B6-Sicd1 mice supports its role as a modulator of neuronal excitability in the hippocampus (Ben-Ari 1990; Zini et al. 1993a,b). This speculation is supported by two lines of evidence suggesting that the neuropeptide galanin is a powerful regulator of seizure activity and neuronal excitability. Previous studies have showed that acute administration of galanin receptor agonists can effectively attenuate convulsive activity induced by the pro-convulsant picrotoxin (Mazarati & Lu 2005) and can delay kindling epileptogenesis (Mazarati et al. 2006). Second, loss-of-function experiments have showed enhanced susceptibility to excitotoxin-induced neuronal injury in Galr1 knockout mice (Mazarati et al. 2004), and a recent study by McColl et al. (2006) show Galr1 knockout mice exhibit spontaneous partial seizures with impaired synaptic inhibition in the hippocampus. In contrast, gain-of-function experiments have showed that galanin overexpression is known to decrease hippocampal neuronal injury resulting from limbic seizures (Haberman et al. 2003; Mazarati et al. 2000) presumably through Galr1 receptor modulation (Mazarati & Lu 2005). Furthermore, the fact that our reciprocal congenic strains were differentially susceptible to seizure-induced cell death would suggest that differences in the level of Galr1 expression alone may be sufficient to confer enhanced susceptibility to KA-induced neuronal death on a genetically resistant background, as was the case for the B6.FVB-Sicd1 congenic strain. Thus, taken together, our findings support the observation that Galr1 can regulate neuronal excitability in the hippocampus.
We predicted that the gene, or regulatory region of the gene, producing the difference in response to kainate between B6 and FVB mice would be polymorphic between these two strains. Thus, we documented the sequence variation between these strains for the putative candidate genes, Galr1 and Mbp. Sequence analysis showed no sequence variation in the promoter, 5′-UTR, coding exons, 3′-UTR or splice sites for Galr1 or Mbp between B6 and FVB strains. While sequence analysis of Galr1 detected no polymorphisms in the regions analyzed, it is important to note that SNPs in noncoding sequences may also affect the levels and forms of mRNA transcripts. However, based on a survey of SNPs identified in the Perlegan mouse database (http://mouse.perlegen.com/mouse/) in conjunction with the Center for Rodent genetics (http://www.niehs.nih.gov/crg/) and for intronic regions of Galr1 and Mbp, there is no evidence that an SNP in a noncoding region exists between FVB and B6. Second, while sequence analysis of Galr1 detected no polymorphisms, we cannot rule out Galr1 as a putative QTL gene. For example, Galr1 could be differentially regulated by an upstream mechanism. The downregulation of Galr1 expression in the hippocampus of B6 and FVB.B6-Sicd1 mice indicates a positive association between Galr1 expression and reduced susceptibility to excitatory amino acid-induced cell death. Whether or not changes in Galr1 activity are involved in producing the phenotype of reduced susceptibility remains to be determined. The mechanism by which Galr1 could influence susceptibility in this mouse model is not yet known, but could result from changes in the regulatory region of the gene, as suggested by the observed strain differences in expression.
While we did not find any evidence to support Mbp as a causal susceptibility gene for KA-induced excitotoxicity, there is still a possibility that other gene(s) within the Sicd1 region are involved as well. Of the 12 known genes in the region defined by the one LOD drop (79–84 Mb), five of these have been published on. For example, a nucleotide substitution in the gene encoding Ctdp1 results in an autosomal recessive disorder called congenital cataracts facial dysmorphism neuropathy (Varon et al. 2003). Nfatc1 is considered as the master transcription factor for osteoclasts – the bone resorbing cells that play a key role both in the normal bone remodeling and in the skeletal osteopenia of arthritis, osteoporosis, periodontal disease and certain malignancies (Sato et al. 2007; Zhao et al. 2007). Sall3 is a member of the Spalt gene family that encodes putative transcription factors. These Spalt homologues are widely expressed in neural and mesodermal tissues during early embryogenesis. Sall3 is required for the development of nerves that are derived from the hindbrain and for the formation of adjacent branchial arch derivatives (Parrish et al. 2004) and has also been showed to be an epigenetic hotspot of aberrant DNA methylation (Ohgane et al. 2004). Setbp1 is a protein that binds to the acute undifferentiated leukemia-associated protein, SET. SET is thought to play a key role in leukemogenesis by its nuclear localization and protein–protein interactions. While the function of Setbp1 remains unknown, it has been proposed to play a key role in the mechanism of SET-related leukemogenesis and tumorigenesis (Minakuchi et al. 2001). Lastly, Pard6g (Par6) is a key member of a multicomponent polarity complex that controls a variety of cellular processes, such as asymmetric cell division, establishment of epithelial apico-basal polarity and polarized cell migration (Bose & Wrana 2006; Yoshimura et al. 2006). While none of these genes, at present, is thought to play a relevant role in modulating neuronal excitability on its own, we cannot exclude that some of these genes, which play important roles in cellular-signaling pathways might be involved subsequently at some level. However, at present, we can only provide evidence that Galr1 is at least one of the causal susceptibility genes for KA-induced excitotoxic cell death in mice.
In summary, our results identify Galr1 as a promising candidate gene, but additional work is necessary to establish with certainty that Galr1 is a seizure-induced cell death susceptibility gene. Until then, it must be kept in mind that another gene within the QTL interval in linkage disequilibrium with Galr1 may ultimately be shown to contribute all or part of the QTL effect. Importantly, our congenic model and prospective sublines will be used to narrow and refine the differential locus on chromosome 18, as well as to examine gene interactions and subphenotypes in the control of excitatory amino acid-induced cell death susceptibility, with the ultimate goal of identifying the set of genes responsible for this complex trait. Validation of Galr1 as causal for the trait of susceptibility to seizure-induced cell death will involve the construction of animals that are genetically altered with respect to Galr1 activity followed by screens for variations in the trait of susceptibility to seizure-induced cell death.