RNAi for the large non-coding hsrω transcripts suppresses polyglutamine pathogenesis inDrosophilamodels

Abstract

Polyglutamine diseases are a class of inherited neurodegenerative disorders, characterized by expansion of CAG trinucleotide repeats translated into elongated glutamine tracts within the mutant proteins. Overexpression of the non-coding hsromega transcripts has been shown to dominantly enhance polyQ induced cytotoxicity in Drosophila. In the present study we demonstrate that RNA interference mediated downregulation of hsromega-n transcripts is sufficient to suppress pathogenesis in several Drosophila models of human polyQ neurodegenerative diseases. Loss of hsromega-n RNA not only suppresses the eye-specific degeneration mediated by GMR-GAL4 driven expression of the 127Q or MJDtr-Q78 or ataxin1 82Q or httex1p Q93 transgene, but also rescues premature death of flies expressing the expanded polyQ proteins pan-neuronally using the elav-GAL4 driver. We further demonstrate that the morphological and functional rescue of polyQ toxicity observed upon hsromega-n RNAi is associated with substantial reduction of polyQ protein aggregation without affecting transcription of the 127Q transgene. Unlike in the polyQ expressing cells, co-expression of hsromega-n RNAi also abolishes the induction of Hsp70. These results suggest that the hsromega transcripts have a role in early stages of polyQ aggregate formation. Interestingly, hsromega-RNAi has, at best, only a marginal effect on neuropathy following overexpression of normal or mutant tau protein in flies. Functional analogues of the large non-coding hsromega transcripts in human thus appear to be promising candidates as therapeutic targets for the polyQ-mediated neurodegenerative diseases.

The Developmentally Active and Stress-Inducible Noncoding hsrω Gene Is a Novel Regulator of Apoptosis in Drosophila

“…Initial studies showed that RNAi-mediated eye-specific depletion of hsrv transcripts rescued the retinal damage seen in flies with two copies of the GMR-GAL4 transgene. Furthermore, other studies in our laboratory demonstrated that misexpression of the hsrv transcripts modulates poly(Q)-induced neurodegeneration in fly models (Sengupta and Lakhotia 2006;Mallik and Lakhotia 2009). Since eye degeneration in GMR-GAL4 homozygous individuals is due to an elevated incidence of apoptosis (Kramer and Staveley 2003) and since proteins with expanded poly(Q) also trigger apoptosis (Sanchez et al 1999;Evert et al 2000;Gunawardena et al 2003), we undertook the present study to examine if the hsrv transcripts play a role in the cell death pathway(s).…”

“…The following transgenic lines were used: y w; 1/1; GMR-rpr/TM6 (White et al 1996) y w 67c23 ; GMR-hid/CyO; 1/1 (Grether et al 1995) y w; 1/1; GMR-grim/TM6B (Hawkins et al 2000) w; 1/1; UAS-pro-dronc W /TM3, Ser (Meier et al 2000) w; 1/1; UAS-pro-dronc S /TM3, Ser (Meier et al 2000) w; 1/1; UAS-DN dronc/TM3, Ser (Meier et al 2000) y w; 1/1; GMR-DN-dcp-1/GMR-DN-dcp-1 (Song et al 2000) w; GMR-GAL4 UAS-DIAP1-RNAi/CyO; 1/1 (Leulier et al 2006) w 1118 ; UAS-egr/CyO; 1/1 (Igaki et al ) w 1118 ; UAS-dTAK1/UAS-dTAK1; 1/1 (Takatsu et al 2000) w; EP0578 (DTRAF1)/EP0578 (DTRAF1); 1/1 (Cha et al 2003) ; GMR-argos/GMR-argos (Sawamoto et al 1998) w; GMR-GAL4 UAS-DN DER/CyO; 1/1 (Freeman 1996) w/w; UAS-hsrv-RNAi Of the three hsrv-RNAi transgenic lines available with us, the hsrv-RNAi 3 transgenic line was used, unless otherwise mentioned, in most of the present studies. The hsrv-RNAi transgene from the desired transgenic line or the EP93D or EP3037 alleles (Mallik and Lakhotia 2009) were introgressed or recombined, as required, with the GMR-GAL4 driver and used in various crosses to generate the desired genotypes. Most of the genetic interactions were carried out with single copies of hsrv-RNAi, EP93D, or EP3037, unless stated otherwise.…”

“…All Drosophila species show a functional homolog of this gene (Lakhotia and Singh 1982; Mutt and S. C. Lakhotia, unpublished data). To understand physiological roles of the noncoding hsrv gene, we established EP and RNAi lines (Mallik and Lakhotia 2009) designed to, respectively, overexpress or deplete hsrv transcript levels using the UAS/GAL4 expression system (Brand and Perrimon 1993). Initial studies showed that RNAi-mediated eye-specific depletion of hsrv transcripts rescued the retinal damage seen in flies with two copies of the GMR-GAL4 transgene.…”

“…We used GAL4-driven expression of the hsrvRNAi transgenes (Mallik and Lakhotia 2009) or of the overexpressing EP3037 and EP93D alleles of hsrv (Sengupta and Lakhotia 2006;Mallik and Lakhotia 2009) to examine effects of altered levels of the hsrv transcripts on apoptotic cell death brought about by coexpression of the various transgenes that induce or inhibit apoptosis. Our results show that while depletion of hsrv transcripts through conditional RNAi blocked caspase-mediated induced apoptosis, overexpression significantly enhanced the cell death phenotypes.…”

DNApol‐ϵ gene is indispensable for the survival and growth of Drosophila melanogaster

“…This effect may be due to the observed over-expression of the hsrx transcripts in pl10R mutant cells (Fig. 1) because the enhanced levels of these transcripts are known to enhance polyQ based neurodegeneration (Mallik and Lakhotia, 2009). Additionally, the pl10R allele may affect the chromatin remodeling role of DNApol-e, which in turn can exaggerate the polyQ phenotype since compromised chromatin remodeling activity also enhances the polyQ damage (Mallik and Lakhotia, 2010).…”