Intrastriatal sonic hedgehog injection increases Patched transcript levels in the adult rat subventricular zone.

The morphogen sonic hedgehog (Shh) is implicated in neural tissue patterning and the growth of brain structures during embryogenesis and postnatal development and is also present in the adult brain. Shh signals through interaction with the tumour suppressor Patched (Ptc). This receptor for Shh is associated with Smoothened (Smo), a protein with high homology to the G-protein coupled receptors. However, little is known about the transduction mechanisms implicated in Shh signalling in the adult brain. The study described here shows that injection of aminoterminal myristoylated Shh (myrShhN) into the adult rat striatum robustly increases the levels of Ptc transcripts in selective brain areas including the subventricular zone (SVZ). The adult SVZ contains cell progenitors, which can proliferate and differentiate into new neurons and glia. In the myrShhN injected animals, proliferation and differentiation of these SVZ precursor cells were not affected as demonstrated by BrdU incorporation and immunohistochemistry performed with specific antibodies for nestin (uncommitted neural progenitors), PSA-NCAM (migrating neuroblasts) or GFAP (astrocytes). Together with the presence of Smo expressing cells and amino-terminal Shh (ShhN) protein in SVZ area of untreated animals, the data presented here supports the hypothesis that the Shh pathway may be activated in the adult brain, and that a niche for Shh signalling exists within the adult SVZ

Generation and characterization of alpha-1 type VI collagen zebrafish mutant line

International audienc

A TALEN-Exon Skipping Design for a Bethlem Myopathy Model in Zebrafish.

International audiencePresently, human collagen VI-related diseases such as Ullrich congenital muscular dystrophy (UCMD) and Bethlem myopathy (BM) remain incurable, emphasizing the need to unravel their etiology and improve their treatments. In UCMD, symptom onset occurs early, and both diseases aggravate with ageing. In zebrafish fry, morpholinos reproduced early UCMD and BM symptoms but did not allow to study the late phenotype. Here, we produced the first zebrafish line with the human mutation frequently found in collagen VI-related disorders such as UCMD and BM. We used a transcription activator-like effector nuclease (TALEN) to design the col6a1ama605003-line with a mutation within an essential splice donor site, in intron 14 of the col6a1 gene, which provoke an in-frame skipping of exon 14 in the processed mRNA. This mutation at a splice donor site is the first example of a template-independent modification of splicing induced in zebrafish using a targetable nuclease. This technique is readily expandable to other organisms and can be instrumental in other disease studies. Histological and ultrastructural analyzes of homozygous and heterozygous mutant fry and 3 months post-fertilization (mpf) fish revealed co-dominantly inherited abnormal myofibers with disorganized myofibrils, enlarged sarcoplasmic reticulum, altered mitochondria and misaligned sarcomeres. Locomotion analyzes showed hypoxia-response behavior in 9 mpf col6a1 mutant unseen in 3 mpf fish. These symptoms worsened with ageing as described in patients with collagen VI deficiency. Thus, the col6a1ama605003-line is the first adult zebrafish model of collagen VI-related diseases; it will be instrumental both for basic research and drug discovery assays focusing on this type of disorders

Calpain is a major cell death effector in selective striatal degeneration induced in vivoin\ vivo by 3-nitropropionate: implications for Huntington's disease.

International audienceStriatal cell death in Huntington's Disease (HD) may involve mitochondrial defects, NMDA-mediated excitotoxicity, and activation of death effector proteases such as caspases and calpain. However, the precise contribution of mitochondrial defects in the activation of these proteases in HD is unknown. Here, we addressed this question by studying the mechanism of striatal cell death in rat models of HD using the mitochondrial complex II inhibitor 3-nitropropionic acid (3-NP). The neurotoxin was either given by intraperitoneal injections (acute model) or over 5 d by constant systemic infusion using osmotic pumps (chronic model) to produce either transient or sustained mitochondrial deficits. Caspase-9 activation preceded neurodegeneration in both cases. However, caspase-8 and caspase-3 were activated in the acute model, but not in the chronic model, showing that 3-NP does not require activation of these caspases to produce striatal degeneration. In contrast, activation of calpain was specifically detected in the striatum in both models and this was associated with a calpain-dependent cleavage of huntingtin. Finally, in the chronic model, which mimics a steady blockade of complex II activity reminiscent of HD, selective calpain inhibition prevented the abnormal calpain-dependent processing of huntingtin, reduced the size of the striatal lesions, and almost completely abolished the 3-NP-induced DNA fragmentation in striatal cells. The present results demonstrate that calpain is a predominant effector of striatal cell death associated with mitochondrial defects $in\ vivo$. This suggests that calpain may play an important role in HD pathogenesis and could be a potential therapeutic target to slow disease progression

In VivoIn\ Vivo Calpain/Caspase Cross-talk during 3-Nitropropionic Acid-induced Striatal Degeneration

International audienceThe role of caspases and calpains in neurodegeneration remains unclear. In this study, we focused on these proteases in a rat model of Huntington's disease using the mitochondrial toxin 3-nitropropionic acid (3NP). Results showed that 3NP-induced death of striatal neurons was preceded by cytochrome c redistribution, transient caspase-9 processing, and activation of calpain, whereas levels of the active/processed form of caspase-3 remained low and were even reduced as compared with control animals. We evidenced here that this decrease in active caspase-3 levels could be attributed to calpain activation. Several observations supported this conclusion. 1) Pharmacological blockade of calpain in 3NP-treated rats increased the levels of endogenous processed caspase-9 and caspase-3. 2) Cell-free extracts prepared from the striatum of 3NP-treated rats degraded $in\ vitro$ the p34 and p20 subunits of active recombinant caspase-9 and caspase-3, respectively. 3) This degradation of p34 and p20 could be mimicked by purified $\mu$-calpain and was prevented by calpain inhibitors. 4) $\mu$-Calpain produced a loss of the DEVDase (Asp-Glu-Val-Asp) activity of active caspase-3. 5) Western blot analysis and experiments with $^{35}$S-radiolabeled caspase-3 showed that $\mu$-calpain cleaved the p20 subunit of active caspase-3 near its catalytic site. 6) $\mu$-Calpain activity was selectively inhibited (IC$_{50}$ of 100 $\mu$M) by a 12 amino acid peptide corresponding to the C terminus of p20. Our results showed that calpain can down-regulate the caspase-9/caspase-3 cell death pathway during neurodegeneration due to chronic mitochondrial defects $in\ vivo$ and that this effect may involve, at least in part, direct cleavage of the caspase-3 p20 subunit

Neuroprotective effect of zVAD against the neurotoxin 3-nitropropionic acid involves inhibition of calpain.

The contribution of calpains and caspases to cell death has been widely studied using pharmacological inhibitors. Among them, the caspase inhibitor N-benzyloxycarbonyl-valyl-alanyl-aspartyl-fluoromethylketone (zVAD) has been used as a specific caspase inhibitor in nearly 1000 published studies. However, several studies showed that zVAD also behaves as a calpain inhibitor in peripheral cells. The effects of zVAD as a calpain inhibitor have never been assessed in neurodegeneration models. We examined here whether zVAD could reduce neurodegeneration in Huntington's disease models using the mitochondrial inhibitor 3-nitropropionic acid (3NP). In these models, 3NP toxicity has been shown to require calpain activation. In rats, intra-cerebro-ventricular infusion of zVAD significantly reduced 3NP-induced striatal degeneration, and decreased the 3NP-induced activation of calpain and calpain-dependent cleavage of fodrin. zVAD (100 microM) also blocked 3NP-induced death of cultured striatal neurons. In vitro, zVAD inhibited purified mu-calpain with high affinity (IC50=10 nM). The present data demonstrate that zVAD protects neurons against 3NP through calpain inhibition. This suggests that, in certain models of neuronal death where zVAD showed protective effects, caspases but also calpains may be involved.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Zebrafish midbrain slow-amplifying progenitors exhibit high levels of transcripts for nucleotide and ribosome biogenesis.

International audienceInvestigating neural stem cell (NSC) behaviour in vivo, which is a major area of research, requires NSC models to be developed. We carried out a multilevel characterisation of the zebrafish embryo peripheral midbrain layer (PML) and identified a unique vertebrate progenitor population. Located dorsally in the transparent embryo midbrain, these large slow-amplifying progenitors (SAPs) are accessible for long-term in vivo imaging. They form a neuroepithelial layer adjacent to the optic tectum, which has transitory fast-amplifying progenitors (FAPs) at its margin. The presence of these SAPs and FAPs in separate domains provided the opportunity to data mine the ZFIN expression pattern database for SAP markers, which are co-expressed in the retina. Most of them are involved in nucleotide synthesis, or encode nucleolar and ribosomal proteins. A mutant for the cad gene, which is strongly expressed in the PML, reveals severe midbrain defects with massive apoptosis and sustained proliferation. We discuss how fish midbrain and retina progenitors might derive from ancient sister cell types and have specific features that are not shared with other SAPs

Organization of the targeted locus and validation of the presence of alternatively spliced <i>col6a1</i> mRNA in the mutants.

<p>(A) Organization of the genomic locus surrounding the col6a1 exon 14. Scale in kb is shown on the left-hand side. Exons 10 to 19 are represented by vertical dashes. Introns are symbolized by a continuous line. Genotyping primers gp1 and gp2 are represented on top. (B) The mRNA structures of wild type (WT, 298 bp) and exon 14-skipped forms (244 bp) are represented. The scheme represents the mRNA with exon skipping of the mutant M2 (deleted bases are symbolized by stars). Exons are boxed. (C) Table of transmission of mutated alleles for the F0 founders 3, 4 and 10. The numbers of genotyped adult F1 fish, the number of times each different type of mutation (M1-M6) occurred and the counts for WT F1 fish are indicated. (D) Nucleotide sequence of the junction between exon and intron 14 and alignment of the mutations M1 to M6 targeted by the TALEN. Each deleted nucleotides is represented by a dash. The number of deleted bases is reported on the right-hand side of the alignment. M1 was an over-represented mutation (ORM) but was absent out of 5 males and 5 females wild type TU zebrafish (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133986#pone.0133986.s003" target="_blank">S3 Fig</a>). The <i>col6a1</i>^{<i>ama605003</i>} line was generated from the M3 mutation (star). (E) Results of RT-PCRs on fin clips of heterozygous F1 fish containing one of the corresponding mutations (as indicated). The 298 bp band corresponds to the wild-type allele of the mRNA, the 244 bp band to the mutated allele. RT-PCR from a wild type (WT) fish is shown on the right hand side.</p