Caractérisation de l’ubiquitin-fold modifier (UFM1) dans un modèle C. elegans

L’ubiquitin-fold modifier (UFM1) fait partie de la classe 1 de la famille de protéine ubiquitin-like (Ubl). UFM1 et Ub ont très peu d’homologie de séquence, mais partagent des similarités remarquables au niveau de leur structure tertiaire. Tout comme l’Ub et la majorité des autres Ubls, UFM1 se lie de façon covalente à ses substrats par l’intermédiaire d’une cascade enzymatique. Il est de plus en plus fréquemment rapporté que les protéines Ubls sont impliquées dans des maladies humaines. Le gène Ufm1 est surexprimé chez des souris de type MCP développant une ischémie myocardique et dans les îlots de Langerhans de patients atteints du diabète de type 2. UFM1 et ses enzymes spécifiques, UBA5, UFL1 et UFC1, sont conservés chez les métazoaires et les plantes suggérant un rôle important pour les organismes multicellulaires. Le Caenorhabditis elegans est le modèle animal le plus simple utilisé en biologie. Sa morphologie, ses phénotypes visibles et ses lignées cellulaires ont été décrits de façon détaillée. De plus, son cycle de vie court permet de rapidement observer les effets de certains gènes sur la longévité. Ce modèle nous permet de facilement manipuler l’expression du gène Ufm1 et de mieux connaître ses fonctions. En diminuant l’expression du gène ufm-1 chez le C.elegans, par la technique de l’ARN interférence par alimentation, nous n’avons observé aucun problème morphologique grave. Les vers ressemblaient aux vers sauvages et possédaient un nombre de progéniture normal. Cependant, les vers sauvage exposés à l’ARNi d’ufm-1 vivent significativement moins longtemps que les contrôles et ce, de façon indépendante de la voie de signalisation de l’insuline/IGF. Chez le C. elegans la longévité et la résistance au stress cellulaire sont intimement liées. Nous n’avons remarqué aucun effet d’ufm-1 sur le stress thermal, osmotique ou oxydatif, mais il est requis pour la protection contre le stress protéotoxique. Il est également nécessaire au maintien de l’intégrité neuronale au cours du vieillissement des animaux. L’ensemble de nos données nous renseigne sur les fonctions putatives du gène Ufm1.The ubiquitin-fold modifier (UFM1) is part of the type 1 class of the family of ubiquitin-like protein (Ubl). UFM1 and Ub have very little sequence homology but share remarkable similarities in their tertiary structure. Like Ub and most other UBLS, UFM1 binds covalently to its substrates through an enzymatic cascade. It is frequently reported that UBLs are involved in human diseases. UFM-1 is overexpressed in mice developing a myocardial ischemia and in the islets of patients suffering from type 2 diabetes. UFM1 and its specific enzymes, UBA5, UFL1, and UFC1 are conserved in metazoans and plants suggesting an important role in multicellular organisms. Caenorhabditis elegans is one of the the simplest animal models used in biology. Some features such as morphology, visible phenotypes and cell lineage have completely been described. The short lifecycle of C. elegans makes it easy to observe gene effects on longevity. This model allows us to easily manipulate the expression of the Ufm1 gene and learn more about its putative functions. To study putative functions of Ufm1, we decreased the expression of ufm-1 using RNA interference introduces through feeding. No gross morphological disturbances were observed; worms resembled wild type and had a normal brood size. However, worms exposed to ufm-1 RNAi had a significantly shorter lifespan than the controls. This effect is independent of the insulin/IGF pathway, which is a major axis of longevity genetics. In C. elegans longevity and cellular stress resistance are intimately linked. We have observed no effect of ufm-1 on thermal, osmotic or oxidative stress, but it is required for protection against proteotoxic stress. It is also necessary to maintain neuronal integrity during aging. Together, our results shed light on putative functions of Ufm1 gene

Cucurbitacin E has neuroprotective properties and autophagic modulating activities on dopaminergic neurons

Natural molecules are under intensive study for their potential as preventive and/or adjuvant therapies for neurodegenerative disorders such as Parkinson’s disease (PD). We evaluated the neuroprotective potential of cucurbitacin E (CuE), a tetracyclic triterpenoid phytosterol extracted from the Ecballium elaterium (Cucurbitaceae), using a known cellular model of PD, NGF-differentiated PC12. In our postmitotic experimental paradigm, neuronal cells were treated with the parkinsonian toxin 1-methyl-4-phenylpyridinium (MPP+) to provoke significant cellular damage and apoptosis or with the potent N,N-diethyldithiocarbamate (DDC) to induce superoxide () production, and CuE was administered prior to and during the neurotoxic treatment. We measured cellular death and reactive oxygen species to evaluate the antioxidant and antiapoptotic properties of CuE. In addition, we analyzed cellular macroautophagy, a bulk degradation process involving the lysosomal pathway. CuE showed neuroprotective effects on MPP+-induced cell death. However, CuE failed to rescue neuronal cells from oxidative stress induced by MPP+ or DDC. Microscopy and western blot data show an intriguing involvement of CuE in maintaining lysosomal distribution and decreasing autophagy flux. Altogether, these data indicate that CuE decreases neuronal death and autophagic flux in a postmitotic cellular model of PD.peer-reviewe

Mutations in the Mitochondrial Methionyl-tRNA Synthetase Cause a Neurodegenerative Phenotype in Flies and a Recessive Ataxia (ARSAL) in Humans

The study of Drosophila neurodegenerative mutants combined with genetic and biochemical analyses lead to the identification of multiple complex mutations in 60 patients with a novel form of ataxia/leukoencephalopathy

Retinal degeneration and lifespan of <i>Aats-met</i> mutants.

<p>(A) TEM of a single ommatidium from a control 1-d-old fly eye, showing the characteristic seven dark rhabdomeres in the center. (B) TEM of a single ommatidium from the eye of a 1-d-old <i>HV/FB</i> escaper fly, showing no obvious defects. (C) TEM of the eye of a 1-d-old fly containing homozygous clones of a <i>PB</i> allele. (D) TEM of the eye of a 2-wk-old <i>HV/FB</i> escaper fly, showing the beginning of a neurodegenerative process, with a degenerating rhabdomere (arrowhead) and enlarged mitochondria (arrow). (E) TEM of the eye of a 3-wk-old escaper. (F) A neurodegenerative process is evident in clones of the <i>PB</i> allele in a 2-wk-old fly. Arrows indicate lipid droplets in pigment cells (arrowheads). (G) Quantification of 100 retinal photoreceptor rhabdomeres for the control, <i>HV/FB</i> escapers, and <i>PB</i> clone-containing mutants at different ages. (H) Quantification of the total mitochondrial area as a percentage of the retinal area: <i>HV/FB</i> mutants clearly have a higher mitochondrial content. (I) Quantification of average mitochondrial size, showing the mitochondrial number of the <i>HV/FB</i> mutant retinas (<i>n</i> = 50). (J) Graph showing the shortened lifespans of 100–200 <i>HV/FB</i> and <i>HV/HV</i> escapers of each gender compared to controls, with males denoted in blue and females in pink. Scale bars: 1 µm.</p

<i>Aats-met</i> mutants have reduced cell proliferation.

<p>(A–B) Brains of late 3^rd instar control and <i>HV/Df</i> larvae stained with Rhodamine-Phalloidin. (C–D) Wing discs of a late 3^rd instar control and mutant larvae stained with Rhodamine-Phalloidin. (E–F) Control and mutant pupae are shown. (G) Quantification of pupal length is shown. (H) Wing disc containing wild-type (outlined in yellow) and mutant clones (outlined in red) are seen. (I) Wild-type clones are significantly larger than mutant clones, quantified in 16 to 20 pairs of clones. (J–K) Cells in mutant clones in wing discs, stained with anti-Dlg, to mark the cell membrane, are similar in size to wild-type cells. (L) PH3-staining cells in mutant versus neighboring heterozygous tissue is quantified for five wing discs, indicating that there is less cell proliferation in mutant clones. Data are mean ± s.e.m. Scale bars for (A–D) and (H) are 100 microns, (E–F) are 0.3 mm, and (J–K) are 5 microns.</p

Identification/mapping of the <i>Aats-met</i> gene.

<p>(A) ERG of the control (<i>y w</i>; <i>FRT82B iso</i>). The black and white arrowheads indicate the “on” and “off” transients, respectively. The double-pointed arrow indicates the amplitude. (B–C) ERGs of homozygous <i>HV</i> clone-containing flies at 1 d and 4 wk after eclosion. (D–E) ERGs of homozygous <i>FB</i> clone-containing flies at 1 d and 4 wk after eclosion. (F) ERG of a 1-d-old <i>HV/FB</i> escaper. (G) ERG of a 3-wk-old <i>HV/FB</i> escaper. (H) ERG of a 2-wk-old <i>HV/Df</i> fly rescued with <i>actin-Gal4</i> and <i>UAS-Aats-met</i>. (I) ERG of a 2-wk-old <i>HV/Df</i> fly rescued with <i>actin-Gal4</i> and <i>UAS-HMARS2</i>. (J) ERG of a 2-wk-old otherwise wild-type fly expressing HMARS2-FLAG driven by tub-Gal4. (K) Lethal stages of homozygous and transheretozygous allelic combinations reveal an allelic series: <i>Df>PB>FB>HV</i>. (L) The Aats-met protein's predicted domains are shown (drawn to scale), with position of mutations and percentage identity compared to human MARS2 shown. (M) The <i>Drosophila Aats-met</i> gene is homologous to the mitochondrial methionyl-tRNA synthetase genes of <i>S. cerevisiae</i>, <i>C. elegans</i>, <i>M. musculus</i>, and <i>H. sapiens</i>. (N) Colocalization of the Flag-tagged human MARS2 protein with Mito-GFP in the cell body of a neuron in the ventral nerve cord, driven by the D42-Gal4 driver, is shown.</p

The human <i>MARS2</i> mutations.

<p>(A) PCR amplification products of <i>MARS2</i> encompassing a portion of the coding sequence revealed the presence of a 268 bp deletion mutation segregating in ARSAL Family E but not in Family B. This truncated product is indicated by an arrow. The normal PCR product is around 500 bp. Segregation of the deletion is shown in Family E; brothers E10 and E11 carry the mutation. Their unaffected father E9 is also a carrier. The determined genotypes for the patients shown (summarized in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001288#pbio.1001288.s012" target="_blank">Table S5</a> for all patients) are shown above the PCR bands. (B) Wild-type sequence of <i>MARS2</i> PCR products. (C) DNA sequencing of the deletion (c.681Δ268bpfx236X). (D–E) Nonrecurrent rearrangements involving the <i>MARS2</i> gene was confirmed by the oligonucleotide custom aCGH. In patients E10 and E11, the array discriminated the presence of the duplication as well as the deletion (see arrows) as depicted by the lower band detecting only one additional copy. (F) PCR amplification products of <i>MARS2</i> encompassing the coding sequence revealed the presence of a ∼300 bp insertion mutation segregating in ARSAL family members C6 and C8 but not in Family B. This larger amplification product is indicated by an arrow. The normal amplicon size is about 800 bp. C5 is the unaffected father of C6 and C8 and also carries the mutation. (G) Wild-type sequence of <i>MARS2</i>. (H) DNA sequencing of the heterozygous case C6 corresponding to the insertion revealed parts of the <i>MARS2</i> duplication mutation. Rearrangement was confirmed by oligonucleotide custom aCGH. Note that the array data of C6, a compound heterozygote (<i>Dup2/Dup2</i>), demonstrates the presence of a potentially larger duplication while not showing the 300 bp insertion, the array not having been designed to include its sequence. (I) In homozygous patient B4 (<i>Dup1/Dup1</i>), the array suggests that the duplication has identical distal and proximal breakpoint junctions with the other ARSAL cases.</p

MARS2 mRNA expression, protein levels, mitochondrial protein translation, Complex I, aconitase activity, and cell proliferation.

<p>(A) Quantification of MARS2 mRNA expression levels was performed on six ARSAL cases and two control lymphoblast cell lines. Relative expression levels were normalized to GAPDH levels. ARSAL patients expressed up to 3× higher MARS2 mRNA levels compared to controls. (B) Mitochondrial protein synthesis was measured in lymphoblasts and fibroblasts from three controls and six ARSAL patients by pulse-labeling mitochondrial translation products with ³⁵S-methionine for 1 h in the presence of emetine, followed by electrophoresis on a 15%–20% linear-gradient polyacrylamide gel. The 13 mitochondrial products are identified at the left of the figure. A generalized mitochondrial translation deficiency is observed in three of the six ARSAL patients tested. ANOVA analysis revealed significance for three of the patient's mitochondrial translation levels: Ctrl 1-B4: **, Ctrl 1-B5: n.s., Ctrl 1-P24: n.s., Ctrl 2-B4: ***, Ctrl 2-B5: n.s., Ctrl 2-P24: *, Ctrl 3-B4: ***, Ctrl 3-B5: *, Ctrl 3-P24: ***. (C) Immunoblotting analysis was performed with antibodies against the proteins indicated at the left of the panel. MARS2 was visualized using a polyclonal antibody. For case E10 carrying the heterozygous deletion (c.681Δ268bpfx236X), the truncated product is detected at the estimated size of 24 kDa (arrow); ARSAL patients (B4, EE41, P24, B5, AA35, and E10) show decreased levels of MARS2 protein at the estimated normal size of MARS2 (67 kDa). The 130 kDa LRPPRC and the 12 kDa SLIRP were used as loading controls. (D) Each patient's MARS2 protein-level intensity from the Western Blot shown in (C) was quantified using ImageJ and divided by the protein-level intensities of LRRPRC and SLIRP. The results were then graphed for the controls and the patients, respectively. (E) Respiratory chain activity for Complex I was measured from patient fibroblast-derived disrupted mitochondria. Mutant mitochondria exhibit deficiency of complex I. Data are expressed as percentage control activity (mean ± s.e.m.). (F) Quantification of native and reactivated aconitase activity for ARSAL patient and control immortalized fibroblasts. Three controls and 6 ARSAL patients were used for the analysis. (G) Quantification of the proliferation rate for the same above-mentioned fibroblasts. (H) Graph showing the average age of onset for the three different genotypes involved.</p

<i>Aats-met</i> mutants exhibit a complex I deficiency and phenotypes can be suppressed with antioxidants.

<p>(A) Polarography (measurement of substrate-dependent O₂ consumption of isolated 3^rd instar larvae-derived mitochondria given needed substrates) was performed in the presence of Complex I–specific substrates or Complex II–specific substrate. State III is the ADP-stimulated oxygen consumption rate; state IV is the ADP-limited oxygen consumption rate; UC is the oxygen-consumption rate in the presence of an uncoupler; RCR is the Respiratory Control Ratio (state III rate/state IV rate). (B) Individual respiratory chain activities were measured from disrupted mitochondria. Mutant mitochondria exhibit partial deficiency of complex I as well as an increase in CS activity. Data are expressed as percentage control activity (mean ± s.e.m.). (C) Purified disrupted mitochondrial extracts from control 3^rd instar, <i>HV/Df</i>, and <i>FB/Df</i> larvae were quantified for aconitase activity, showing a significant decrease resulting from oxidation in the mutants. Treatment with reducing agent resulted in normal activity levels, indicating that the difference was not due to lower levels of aconitase but from increased oxidized aconitase. (D–E) <i>Aats-met^HV</i> eyes often exhibit glossy areas in the middle of large clones (arrow). In addition, the eyes are typically smaller. With 20 µg/ml Vitamin E, there is significant improvement in eye morphology and size (<i>p</i><0.001). (F) Mutant escaper rates are increased for females supplemented with antioxidants. Male escaper rates are already high, even without antioxidants. Three different drug supplementation regimens were used. For the female escaper rate, the last two drug regimens produced a significant improvement. Data are mean ± s.e.m.</p