Cell death pathways differ in several mouse models with motoneurone disease: analysis of pure motoneurone populations at a presymptomatic age

To identify candidate genes that are responsible for motoneurone degeneration, we combined laser capture microdissection with microarray technology. We analysed gene expression in pure motoneurones from two mouse mutants that develop motoneurone degeneration, progressive motor neuronopathy and wobbler. At a presymptomatic age, there was a significant differential expression of a restricted number of genes (25 and 72 in progressive motor neuronopathy and wobbler respectively, of 22 600 transcripts screened). We compared these results to our previous analyses in the copper-zinc superoxide dismutase mutant mouse (SOD1(G93A)) in which we observed a de-regulation of 27 genes. Some of these genes were de-regulated uniquely in one mouse mutant and some have already been identified in cell death pathways implicated in amyotrophic lateral sclerosis and animal models of motoneurone degeneration (i.e. de-regulation of intermediate filaments, axonal transport, the ubiquitin-proteasome system and excitotoxicity). One gene, vimentin, was differentially up-regulated in all mouse mutants; this main candidate gene has been confirmed by in situ hybridization and immunohistochemistry to be expressed in motoneurones in all mouse mutants. Furthermore, vimentin expression correlated with the state of motoneurone degeneration. These results identify early molecular changes that may be involved in the pathogenesis of motoneurones leading to cell death and favour a complex multipathway induction of the disease; surprisingly, there was no important modification in cell death-associated genes. This is the first study to show a clear difference in the genes that are de-regulated at an early stage in three different mouse models of motoneurone disease

Action of HMX1 on the <i>EPHA6</i> promoter.

<p>Schematic representation of the subcloned fragment of the <i>EPHA6</i> promoter with the three binding sites (red arrows and black characters), and the mutated sequences (red characters) (A). Luciferase assay on the wt <i>EPHA6</i> promoter with HMX1, HMX1 del SD1, HMX1 del SD2 and HMX1 del HD. HMX1 inhibits the promoter by 42%, HMX1 del SD1 and HMX1 del HD have no effect, and HMX1 del SD2 slightly activates the promoter (B). Chromatin immunoprecipitation on 2-week-old C57Bl/6J retinas demonstrated the physical interaction between HMX1 and the <i>EphA6</i> promoter. 5% BSA was added in the control conditions instead of the Hmx1 antibody. TIC = total input chromatin (C). Mutation of the <i>HMX1</i> binding sites attenuates the effect of HMX1 and HMX1 del SD2 but does not completely abolish it (D). Data points represent the mean of three experiments +/− SD. ** : P<0.01. * : P<0.05 (Student’s T-test).</p

Dimerization of HMX1 in HEK 293T cells.

<p>HMX1 is dimerizing as shown by the increasing BRET² ratio in presence of the two fusion proteins. Data points represent the mean of two experiments +/− SD (A). Co-immunoprecipitation was only observed in presence of the two HMX1 fusion proteins. IP was performed with anti-Renilla Luciferase antibody and WB with anti-GFP antibody (B). The size of the HMX1 band revealed with anti-HA-Tag (6E2) Mouse mAb was twice the size in non-denaturing conditions as in denaturing conditions. As no higher molecular weight bands were observed, it is unlikely that trimers were formed (C).</p

Regulation after <i>hmx1</i> misexpression in zebrafish.

<p>Expression of <i>hmx1</i> after heat shock in wt and tg (hsp70:hmx1) embryos (A, B). <i>Hmx1</i>, normally restricted to the nasal retina, lens and ear (arrows in A), was broadly expressed in the transgenic embryo (B). <i>Epha4b</i> expression after heat shock in wt and Tg (hsp70:hmx1) embryos (C–F). Dissected eye showed a strong reduction of <i>epha4b</i> expression in the temporal retina when <i>HMX1</i> was co-expressed (E, F). <i>Pax6</i> regulation after <i>HMX1</i> misexpression in zebrafish. Ocular expression of <i>pax6</i> in wt (G) was not modified by overexpression of <i>HMX1</i> in Tg(hsp70:hmx1) embryos (H). Scale bars: 100 µm.</p

Determination of the nature of the GFP-HMX1 del HD aggregates.

<p>GFP-HMX1 del HD aggregates do not colocalize with LC3B and are thus not included in autophagosomes (A–C). Cells expressing GFP-HMX1 del HD (green) present no increased autophagy, and HMX1 del HD aggregates do not colocalize with autophagosomes (LC3B staining in red, after 50 µM chloroquine treatment for 16 hrs). Likewise, p62 and ubiquitin levels were not increased, confirming that autophagy was not activated (D). Scale bars: 50 µm.</p

Identification of the domains of HMX1 needed for dimerization.

<p>Deletions of two of the conserved domains, the homeobox (black) and SD1 (blue) prevented dimerization. Deletion of the SD2 domain (red) had no effect (A). Deletion of the C-terminus of the protein does not prevent dimerization (B). Data points represent the mean of two experiments +/− SD.</p

Mutations in the SPARC-Related Modular Calcium-Binding Protein 1 Gene, SMOC1, Cause Waardenburg Anophthalmia Syndrome

Waardenburg anophthalmia syndrome, also known as microphthalmia with limb anomalies, ophthalmoacromelic syndrome, and anophthalmia-syndactyly, is a rare autosomal-recessive developmental disorder that has been mapped to 10p11.23. Here we show that this disease is heterogeneous by reporting on a consanguineous family, not linked to the 10p11.23 locus, whose two affected children have a homozygous mutation in SMOC1. Knockdown experiments of the zebrafish smoc1 revealed that smoc1 is important in eye development and that it is expressed in many organs, including brain and somites