(CCUG)n RNA toxicity in a Drosophila model of myotonic dystrophy type 2 (DM2) activates apoptosis

The myotonic dystrophies are prototypic toxic RNA gain-of-function diseases. Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are caused by different unstable, noncoding microsatellite repeat expansions - (CTG)DM1 in DMPK and (CCTG)DM2 in CNBP Although transcription of mutant repeats into (CUG)DM1 or (CCUG)DM2 appears to be necessary and sufficient to cause disease, their pathomechanisms remain incompletely understood. To study the mechanisms of (CCUG)DM2 toxicity and develop a convenient model for drug screening, we generated a transgenic DM2 model in the fruit fly Drosophila melanogaster with (CCUG)n repeats of variable length (n=16 and 106). Expression of noncoding (CCUG)106, but not (CCUG)16, in muscle and retinal cells led to the formation of ribonuclear foci and mis-splicing of genes implicated in DM pathology. Mis-splicing could be rescued by co-expression of human MBNL1, but not by CUGBP1 (CELF1) complementation. Flies with (CCUG)106 displayed strong disruption of external eye morphology and of the underlying retina. Furthermore, expression of (CCUG)106 in developing retinae caused a strong apoptotic response. Inhibition of apoptosis rescued the retinal disruption in (CCUG)106 flies. Finally, we tested two chemical compounds that have shown therapeutic potential in DM1 models. Whereas treatment of (CCUG)106 flies with pentamidine had no effect, treatment with a PKR inhibitor blocked both the formation of RNA foci and apoptosis in retinae of (CCUG)106 flies. Our data indicate that expression of expanded (CCUG)DM2 repeats is toxic, causing inappropriate cell death in affected fly eyes. Our Drosophila DM2 model might provide a convenient tool for in vivo drug screening

Common and Distinct Genetic Properties of ESCRT-II Components in Drosophila

BACKGROUND: Genetic studies in yeast have identified class E vps genes that form the ESCRT complexes required for protein sorting at the early endosome. In Drosophila, mutations of the ESCRT-II component vps25 cause endosomal defects leading to accumulation of Notch protein and increased Notch pathway activity. These endosomal and signaling defects are thought to account for several phenotypes. Depending on the developmental context, two different types of overgrowth can be detected. Tissue predominantly mutant for vps25 displays neoplastic tumor characteristics. In contrast, vps25 mutant clones in a wild-type background trigger hyperplastic overgrowth in a non-autonomous manner. In addition, vps25 mutant clones also promote apoptotic resistance in a non-autonomous manner. PRINCIPAL FINDINGS: Here, we genetically characterize the remaining ESCRT-II components vps22 and vps36. Like vps25, mutants of vps22 and vps36 display endosomal defects, accumulate Notch protein and--when the tissue is predominantly mutant--show neoplastic tumor characteristics. However, despite these common phenotypes, they have distinct non-autonomous phenotypes. While vps22 mutations cause strong non-autonomous overgrowth, they do not affect apoptotic resistance. In contrast, vps36 mutations increase apoptotic resistance, but have little effect on non-autonomous proliferation. Further characterization reveals that although all ESCRT-II mutants accumulate Notch protein, only vps22 and vps25 mutations trigger Notch activity. CONCLUSIONS/SIGNIFICANCE: The ESCRT-II components vps22, vps25 and vps36 display common and distinct genetic properties. Our data redefine the role of Notch for hyperplastic and neoplastic overgrowth in these mutants. While Notch is required for hyperplastic growth, it appears to be dispensable for neoplastic transformation

Inactivation of Effector Caspases through Nondegradative Polyubiquitylation

Ubiquitin-mediated inactivation of caspases has long been postulated to contribute to the regulation of apoptosis. However, detailed mechanisms and functional consequences of caspase ubiquitylation have not been demonstrated. Here we show that the Drosophila Inhibitor of Apoptosis 1, DIAP1, blocks effector caspases by targeting them for polyubiquitylation and nonproteasomal inactivation. We demonstrate that the conjugation of ubiquitin to drICE suppresses its catalytic potential in cleaving caspase substrates. Our data suggest that ubiquitin conjugation sterically interferes with substrate entry and reduces the caspase’s proteolytic velocity. Disruption of drICE ubiquitylation, either by mutation of DIAP1’s E3 activity or drICE’s ubiquitin-acceptor lysines, abrogates DIAP1’s ability to neutralize drICE and suppress apoptosis in vivo. We also show that DIAP1 rests in an “inactive” conformation that requires caspase-mediated cleavage to subsequently ubiquitylate caspases. Taken together, our findings demonstrate that effector caspases regulate their own inhibition through a negative feedback mechanism involving DIAP1 “activation” and nondegradative polyubiquitylation

Adult phenotypes of ESCRT-II mosaics.

<p><i>vps22</i> and <i>vps25</i> mosaics display strong overgrowth phenotypes of the adult eyes and heads, and the larval eye imaginal discs. In contrast, <i>vps36</i> mutants show no or only a mild proliferation phenotype and cause a roughening of the adult eye. (A–D) Side view of genetic eye mosaics of (A) control flies, (B) <i>vps22^5F8-3</i>, (C) <i>vps25^N55</i> and (D) <i>vps36^L5212</i> mutants. (E–H) Eye mosaics of (E) control (heterozygous <i>Notch</i>), (F) <i>vps22^5F8-3</i>, (G) <i>vps25^N55</i> and (H) <i>vps36^L5212</i> in heterozygous <i>Notch</i> (<i>N</i>) background. The Notch allele used is <i>N^264-39</i>. (I–L) Top view of genetic mosaics of (I) control flies, (J) <i>vps22^5F8-3</i>, (K) <i>vps25^N55</i> and (L) <i>vps36^L5212</i> mutants. (M–P) Head mosaics of (M) control (heterozygous <i>Notch</i>), (N) <i>vps22^5F8-3</i>, (O) <i>vps25^N55</i> and (P) <i>vps36^L5212</i> in heterozygous <i>Notch</i> (<i>N</i>) background. The Notch allele used is <i>N^264-39</i>. (Q–T) Size comparison of (Q) control, (R) <i>vps22^5F8-3</i>, (S) <i>vps25^N55</i> and (T) <i>vps36^L5212</i> mosaic eye imaginal discs. Green: GFP; red: BrdU labeling. The scale bars represent 100 µm. Genotypes: (A) <i>eyFlp</i> ; <i>FRT82B/FRT82B P[w⁺]</i>. (B,J) <i>eyFlp</i> ; <i>FRT82B vps22^5F8-3/FRT82B P[w⁺]</i>. (C,K) <i>eyFlp</i> ; <i>FRT42D vps25^N55/FRT42D P[w⁺]</i>. (D,L) <i>eyFlp</i> ; <i>vps36^L5212 FRT2A/P[w⁺] FRT2A</i>. (E,M) <i>N^264-39</i>/+. (F–H) and (N–P): same as (B–D) and (J–L) except they also carry <i>N^264-39</i> as heterozygous mutation. (Q–T) same as in corresponding panels A–D except they carry P[ubi-GFP] instead of P[w⁺].</p

Mutant clones of ESCRT-II components display endosomal defects and accumulate ubiquitinated proteins.

<p>Shown are eye imaginal discs of 3rd instar larvae mosaic for ESCRT-II mutants. Mutant clones are marked by the absence of GFP. Mutant clones of ESCRT-II components show abnormal accumulation of the early endosomal marker Hrs and accumulation of ubiquitin-conjugated proteins as visualized by the FK1 antibody. Hrs and ubiquitin-conjugated proteins accumulate in foci which frequently co-localize. Scale bars represent 50 µm. (A,B,C) GFP/Hrs/FK1 (green/red/blue) co-labelings of (A) <i>vps22^5F8-3</i>, (B) <i>vps25^N55</i> and (C) <i>vps36^L5212</i> eye mosaics. (A′,B′,C′) Hrs/FK1 (red/blue) co-labelings of (A′) <i>vps22^5F8-3</i>, (B′) <i>vps25^N55</i> and (C′) <i>vps36^L5212</i> eye mosaics. (A″,B″,C″) Hrs labeling of (A″) <i>vps22^5F8-3</i>, (B″) <i>vps25^N55</i> and (C″) <i>vps36^L5212</i> eye mosaics. (A′″,B′″,C′″) FK1 labeling of (A′″) <i>vps22^5F8-3</i>, (B′″) <i>vps25^N55</i> and (C′″) <i>vps36^L5212</i> eye mosaics. Genotypes: (A) <i>eyFlp</i> ; <i>FRT82B vps22^5F8-3</i>/<i>FRT82B</i> P[<i>ubi-GFP</i>]. (B) <i>eyFlp</i>; <i>FRT42D vps25^N55</i>/<i>FRT42D</i> P[<i>ubi-GFP</i>]. (C) <i>eyFlp</i> ; <i>vps36^L5212 FRT2A</i>/P[<i>ubi-GFP</i>] <i>FRT2A</i>.</p

Proliferation phenotype of ESCRT-II mosaics.

<p>Non-autonomous regulation of proliferation in <i>vps22</i> and <i>vps25</i> eye mosaics as depicted by BrdU incorporation (red). Arrows in (B) and (C) point to areas of increased BrdU density next to mutant clones. Compared to control discs, <i>vps36</i> mutations do not affect the proliferation pattern significantly. The scale bar represents 50 µm. (A–D) GFP/BrdU (green/red) co-labelings of (A) control, (B) <i>vps22^5F8-3</i>, (C) <i>vps25^N55</i> and (D) <i>vps36^L5212</i> eye mosaics. (A′–D′) BrdU labeling of (A′) control, (B′) <i>vps22^5F8-3</i>, (C′) <i>vps25^N55</i> and (D′) <i>vps36^L5212</i> eye mosaics. Genotypes: (A) <i>eyFlp</i>; <i>FRT42B/FRT42B P[ubi-GFP]</i>. (B) <i>eyFlp</i>; <i>FRT82B vps22^5F8-3/FRT82B P[ubi-GFP]</i>. (C) <i>eyFlp</i>; <i>FRT42D vps25^N55/FRT42D P[ubi-GFP]</i>. (D) <i>eyFlp</i>; <i>vps36^L5212 FRT2A/P[ubi-GFP] FRT2A</i>.</p

Suppression of the <i>GMR-hid</i>-eye ablation phenotype by ESCRT-II mosaics.

<p>(A) Expression of the pro-apoptotic gene <i>hid</i> under control of the eye-specific <i>GMR</i> enhancer (<i>GMR-hid</i>) gives rise to a strong eye ablation phenotype due to excessive apoptosis. (B–D) <i>vps25^N55</i> (C) and <i>vps36^L5212</i> (D) eye mosaics are strong suppressors of the <i>GMR-hid</i>-induced eye ablation phenotype in adult flies. <i>vps22^5F8-3</i> mosaics (B) do not suppress the <i>GMR-hid</i>-eye ablation phenotype. Genotypes: (A) <i>eyFlp ; GMR-hid</i>; <i>FRT82B/FRT82B P[w⁺]</i>. (B) <i>eyFlp</i>; <i>GMR-hid</i>; <i>FRT82B vps22^5F8-3/FRT82B P[w⁺]</i>. (C) <i>GMR-hid eyFlp</i>; <i>FRT42D vps25^N55/FRT42D P[w⁺]</i>. (D) <i>eyFlp</i>; <i>GMR-hid</i>; <i>vps36^L5212 FRT2A/P[w⁺] FRT2A</i>.</p