Heat-induced seizures, premature mortality, and hyperactivity in a novel Scn1a nonsense model for Dravet syndrome

Dravet syndrome (Dravet) is a severe congenital developmental genetic epilepsy caused by de novo mutations in the SCN1A gene. Nonsense mutations are found in ∼20% of the patients, and the R613X mutation was identified in multiple patients. Here we characterized the epileptic and non-epileptic phenotypes of a novel preclinical Dravet mouse model harboring the R613X nonsense Scn1a mutation. Scn1aWT/R613X mice, on a mixed C57BL/6J:129S1/SvImJ background, exhibited spontaneous seizures, susceptibility to heat-induced seizures, and premature mortality, recapitulating the core epileptic phenotypes of Dravet. In addition, these mice, available as an open-access model, demonstrated increased locomotor activity in the open-field test, modeling some non-epileptic Dravet-associated phenotypes. Conversely, Scn1aWT/R613X mice, on the pure 129S1/SvImJ background, had a normal life span and were easy to breed. Homozygous Scn1aR613X/R613X mice (pure 129S1/SvImJ background) died before P16. Our molecular analyses of hippocampal and cortical expression demonstrated that the premature stop codon induced by the R613X mutation reduced Scn1a mRNA and NaV1.1 protein levels to ∼50% in heterozygous Scn1aWT/R613X mice (on either genetic background), with marginal expression in homozygous Scn1aR613X/R613X mice. Together, we introduce a novel Dravet model carrying the R613X Scn1a nonsense mutation that can be used to study the molecular and neuronal basis of Dravet, as well as the development of new therapies associated with SCN1A nonsense mutations in Dravet

Extensions of MADM (Mosaic Analysis with Double Markers) in Mice

Mosaic Analysis with Double Markers (MADM) is a method for generating genetically mosaic mice, in which sibling mutant and wild-type cells are labeled with different fluorescent markers. It is a powerful tool that enables analysis of gene function at the single cell level in vivo. It requires transgenic cassettes to be located between the centromere and the mutation in the gene of interest on the same chromosome. Here we compare procedures for introduction of MADM cassettes into new loci in the mouse genome, and describe new approaches for expanding the utility of MADM. We show that: 1) Targeted homologous recombination outperforms random transgenesis in generation of reliably expressed MADM cassettes, 2) MADM cassettes in new genomic loci need to be validated for biallelic and ubiquitous expression, 3) Recombination between MADM cassettes on different chromosomes can be used to study reciprocal chromosomal deletions/duplications, and 4) MADM can be modified to permit transgene expression by combining it with a binary expression system. The advances described in this study expand current, and enable new and more versatile applications of MADM

Transsynaptic Teneurin Signaling in Neuromuscular Synapse Organization and Target Choice. Nature. 2012; (this issue

Synapse assembly requires trans-synaptic signals between the preand postsynapse 1 , but our understanding of the essential organizational molecules involved in this process remains incomplete 2 . Teneurin proteins are conserved, epidermal growth factor (EGF)-repeat-containing transmembrane proteins with large extracellular domains Vertebrate teneurins are enriched in the developing brain Both Ten-m and Ten-a were enriched at the larval NMJ ( The localization of Ten-a and Ten-m suggested their trans-synaptic interaction. To examine this, we co-expressed Myc-tagged Ten-a in nerves using the Q system 14 and haemagglutinin (HA)-tagged Ten-m in muscles using GAL4. Muscle Ten-m was able to coimmunoprecipitate nerve Ten-a from larval synaptosomes To determine Teneurin function at the NMJ, we examined the ten-a null allele and larvae with neuron or muscle RNAi of ten-a and/or ten-m. Following such perturbations, bouton number and size were altered: the quantity was reduced by 55

Structural consequences of replacement of an α- helical pro residue in Escherichia coli thioredoxin

While it is well known that introduction of Pro residues into the interior of protein α -helices is destabilizing, there have been few studies that have examined the structural and thermodynamic effects of the replacement of a Pro residue in the interior of a protein α -helix. We have previously reported an increase in stability in the P40S mutant of Escherichia coli thioredoxin of 1-1.5 kcal/mol in the temperature range 280-330 K. This paper describes the structure of the P40S mutant at a resolution of 1.8 Å . In wild-type thioredoxin, P40 is located in the interior of helix two, a long a-helix that extends from residues 32 to 49 with a kink at residue 40. Structural differences between the wild-type and P40S are largely localized to the above helix. In the P40S mutant, there is an expected additional hydrogen bond formed between the amide of S40 and the carbonyl of residue K36 and also additional hydrogen bonds between the side chain of S40 and the carbonyl of K36. The helix remains kinked. In the wild-type, main chain hydrogen bonds exist between the amide of 44 and carbonyl of 40 and between the amide of 43 and carbonyl of 39. However, these are absent in P40S. Instead, these main chain atoms are hydrogen bonded to water molecules. The increased stability of P40S is likely to be due to the net increase in the number of hydrogen bonds in helix two of E.coli thioredoxin

Structural consequences of replacement of an \alpha-helical Pro residue in Escherichia coli thioredoxin

While it is well known that introduction of Pro residues into the interior of protein $\alpha$-helices is destabilizing, there have been few studies that have examined the structural and thermodynamic effects of the replacement of a Pro residue in the interior of a protein $\alpha$-helix. We have previously reported an increase in stability in the P40S mutant of Escherichia coli thioredoxin of 1–1.5 kcal/mol in the temperature range 280–330 K. This paper describes the structure of the P40S mutant at a resolution of 1.8 \AA. In wild-type thioredoxin, P40 is located in the interior of helix two, a long $\alpha$-helix that extends from residues 32 to 49 with a kink at residue 40. Structural differences between the wild-type and P40S are largely localized to the above helix. In the P40S mutant, there is an expected additional hydrogen bond formed between the amide of S40 and the carbonyl of residue K36 and also additional hydrogen bonds between the side chain of S40 and the carbonyl of K36. The helix remains kinked. In the wild-type, main chain hydrogen bonds exist between the amide of 44 and carbonyl of 40 and between the amide of 43 and carbonyl of 39. However, these are absent in P40S. Instead, these main chain atoms are hydrogen bonded to water molecules. The increased stability of P40S is likely to be due to the net increase in the number of hydrogen bonds in helix two of E.coli thioredoxin

Targeted knock-in approach to create new <i>Rosa26</i> MADM with <i>GT</i> and <i>TG</i> cassettes.

<p><b>A</b>) Schematic representation of new alleles: <i>R26^GT</i> and <i>R26^TG</i>. <b>B), C) and D)</b> Representative confocal images from tissues indicated on the bottom and genotypes indicated on top. Expected labeling was observed only when Cre was present (compare <b>B</b> with <b>C</b> and <b>D</b>). Bright cellular labeling observed in <b>C</b> and <b>D</b> originates from native tdT and GFP fluorescence (no additional immunostaining was performed). Some sections were stained with DAPI to label nuclei (blue). Scale bars, 50 µm.</p

Cells with translocations and aneuploidy generated and labeled by MADM <i>in vivo</i>.

<p><b>A</b>) Schematic representation of cellular genotypes generated by interchromosomal recombination between non-homologous Chr. 6 and Chr. 10. In both chromosomes, the MADM cassettes are oriented in the telomere-to-centromere fashion. Each double-labeled cell contains the same reciprocal translocation, resulting in no net loss or gain of DNA. Single-labeled (green and red) cells exhibit abnormal copy numbers for parts of the chromosomes distal to the <i>loxP</i> sites: red cells are monosomic for the Chr. 6 portion and trisomic for the Chr. 10 portion; green cells have the reciprocal trisomy/monosomy. <b>B</b>) Representative confocal images of tissue sections obtained from <i>R26^GT/+;M10^TG/+;Hprt^Cre/Y</i> mice. The sections were unstained or stained only with DAPI to label nuclei (blue, in the olfactory epithelium panel). The insets within the olfactory epithelium panel show examples of twin-spot labeling where red and green cells are located in close proximity. Due to the overall low frequency of labeling, each twin-spot labeling most likely originated from a single mitotic recombination event. Scale bars, panels: 100 µm, insets: 25 µm. <b>C</b>) Schematic representation of cellular genotypes generated by interchromosomal recombination between non-homologous Chr. 10 and Chr. 11. The MADM cassettes are oriented differently in the two chromosomes with respect to the corresponding centromeres. Each double-labeled cell contains the reciprocal translocation, resulting in one acentric and one dicentric chromosome. Single-labeled cells contain a dicentric or an acentric chromosome, and also exhibit abnormal copy numbers; the red cells are trisomic for Chr. 11 portion distal to <i>loxP</i> and monosomic for Chr. 10 portion proximal to <i>loxP</i>; the green cells are monosomic for Chr. 11 portion distal to <i>loxP</i> and trisomic for Chr. 10 portion proximal to <i>loxP</i>. <b>D</b>) Representative confocal images of unstained tissue sections obtained from <i>M10^TG/+;H11^GT/+;Hprt^Cre/Y</i> mice. Scale bars, 100 µm.</p