Szt2, a novel gene for seizure threshold in mice

Abstract

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.

Figure 1. Szt2^m1Frk homozygous mutants are susceptible to induced seizures

A. Stimulus (in mA) response curves for B6-Szt2^m1Frk homozygotes (“X’s”) and B6-+/+ control (triangles) male mice of approximately six to nine weeks of age (N=31; N=111, respectively). The dashed and solid lines represent the respective interpolated Probit curves for these responses. B. Mean latency (number of serial daily tests) to a partial seizure induced by corneal kindling in male B6-Szt2 homozygous vs. wild-type mice, approximately nine weeks of age. C. Mean latency (in minutes) to tonic-clonic seizures induced by acute pentylenetetrazole injection (PTZ, 40 mg/kg s.c.) in Szt2^m1Frk homozygous vs. heterozygous male mice from the mapping cross (B6-Szt2 x BALB/cByJ)F₁ X B6-Szt2, approximately eight weeks of age.

Figure 2. Genomic map and candidate gene testing of Szt2 locus on Chr 4

A. Interval map showing the respective LOD score curves based on the backcross population used initially to confirm linkage to Chr 4 (dashed curve) and that based on advanced backcross mice, mostly containing recombinations in the interval (solid curve). The relative marker distances are fixed to the physical map length, based on Ensembl build 36 of the C57BL/6J mouse genome sequence assembly. The stippled line shows the threshold for highly significant association, based on permutation tests. B. Genes corresponding to the indicated confidence interval (given as gene symbols or transcript or sequence identifiers from Ensembl build 36; number of exons and transcriptional orientation shown) were examined in homozygous Szt2^m1Frk mice vs. wild-type controls for either expression differences by quantitative real-time PCR (qPCR), Northern blot, or the corresponding coding exons were examined for structural differences by single stranded confirmation assay (SSCP), or by DNA sequencing. As described in the text, BC05982 is the sequence identifier for the eventual Szt2 gene. Genes shown in parentheses are no longer models in the current genome assembly.

Figure 3. Expression analysis of the Szt2 gene

A. Analysis of real-time PCR results for one of the three primer sets listed in Figure 2, showing a significant decrease in the number of PCR cycles required to amplify Szt2 in homozygotes compared with wild-type. Two mutant replicates, and a minimum of six technical replicates (triplicate amplification, two separate runs) were used for these experiments. B Northern blot of whole brain total RNA from Szt2^Gt(XH662)Byg (gene-trap) and Szt2^m1Frk homozygotes, heterozygous and wild-type mice. The positions of the respective transcripts are indicated at the right, and the position of size markers on the left – hybridization control (β-actin) shown at the bottom. C. Northern blot of poly-A+ RNA from various tissues of adult C57BL/6J wild-type male mice, showing the most abundant expression to be in brain and spinal cord. The graph below is a visualization of the Szt2/β-actin ratio in each sample. D. Northern blot of total brain RNA showing the developmental expression of Szt2 from embryonic day 17.5 through postnatal development, plus an older adult sample.

Figure 4. The mouse Szt2 gene and its two germ-line mutations

Shown is a graphic of the exons of Szt2 (blue boxes) and neighboring genes (red boxes), with the relative genomic positions based on Ensembl build 36 of the C57BL/6J mouse genome sequence assembly. Also indicated are the positions of the respective Szt2^Gt(XH662)Byg (“gene trap”) and Szt2^m1Frk (“ENU”) allelic mutations.

Figure 5. Expression and evolutionary conservation of the SZT2 protein

A. Sybro-ruby stained SDS-PAGE gel showing immunoprecipitation of pCMV-SZT2-GFP fusion protein (and CMV-GFP control) after transient transfection into COS-7 cells, indicating the position of SZT2 relative to size markers. The additional band represents co-precipitated immunoglobulins, present in both samples. B. MALDI-TOF profile of the SZT2 band, excised from the gel in panel A, with increasing mass (m/z) on the X-axis and arbitary units on the Y-axis. C. Conservation plot based on MAFFT protein sequence alignment of SZT2, in representative organisms from seven phyla, described in the text. The conservation score (Blosum 62 algorithm) for each residue was obtained using JalView Version 2 (Waterhouse et al., 2009). The highest scores represent the most conserved residues.

Figure 6. Genetic complementation analysis of the two Szt2 alleles

Shown at the top is the incidence of electroconvulsion (minimal clonic seizure response) in (B6 × 129S1)F₁ hybrids of Szt2^Gt(XH662)Byg (gene-trap, or Gt) and Szt2^m1Frk (ENU) male mice with compound and single heterozygous genotypes shown, at a stimulus level corresponding to the approximate 3% response level of wild-type F₁ hybrid mice. The p-values from Fisher exact test are shown at the bottom.