Inhibition of specific HDACs and sirtuins suppresses pathogenesis in a Drosophila model of Huntington's disease

Abstract

Huntington's disease (HD) is associated with transcriptional dysregulation, and multiple studies with histone deacetylase (HDAC) inhibitors suggest that global approaches for restoring transcriptional balance and appropriate protein acetylation are therapeutically promising. To determine whether more targeted approaches might be effective, we have tested the impact of all the HDACs in Drosophila on Huntingtin (Htt)-induced pathology. Among the zinc-dependent or 'classic' HDACs, we find that neurodegeneration is most sensitive to levels of Rpd3. We also find that among the NAD(+)-dependent class III deacetylases, genetic or pharmacological reduction of either Sir2 or Sirt2 provides neuroprotection to Htt-challenged animals and that even greater neuroprotection is achieved when Rpd3 and Sir2 are simultaneously reduced. Our experiments suggest that longevity promoting strategies may be distinct from those that protect against neurodegeneration in Drosophila challenged with mutant human Htt. These results highlight a novel therapeutic approach for HD in the form of Sir2 inhibition and possible combinatorial inhibition of Sir2 and Rpd3.

Figure 1.

Rpd3 is unique among the classic deacetylases in impacting degeneration: (A) heterozygous reduction of Rpd3 improves survival and (B) reduces neurodegeneration of flies challenged with Httex1p Q93; (C) the effect of heterozygous loss of each of the HDACs in Drosophila on survival to eclosion of Httex1p Q93-challenged animals was determined using multiple heterozygous mutations and/or shRNA constructs (one allele from each is shown here, for the full set of allele data see Supplementary Material, Fig. S1A and B). Only reduction of Rpd3 led to an increase in survival. Relative survival was calculated as (HDACHtt/HDACwt)/(ctrlHtt/ctrlwt), where ‘HDACHtt’ and ‘ctrlHtt’ is the number of eclosed elav > Httex1p Q93 flies with or without the HDAC mutation, and ‘HDACwt’ and ‘ctrlwt’ the eclosion number of the Htt non-expressing siblings with or without the HDAC mutation; (D) the effect of reduced HDAC levels on survival of neurons in Httex1p Q93-challenged animals. The results are shown as the difference in photoreceptor number compared with internal controls with a normal genetic background. Only Rpd3 leads to improved neuronal survival. The null allele of Rpd3 shown here, Rpd3[m5-5], reduces the extent of neuronal loss even more than a weaker hypomorphic allele (see panel B and Supplementary Material, Fig. S1B).

Figure 2.

Reduced sirtuin levels are neuroprotective: A 50% reduction of Sir2 (i.e. heterozygous null) leads to (A) greater survival to adulthood and (B) an increased number of photoreceptor neurons of Httex1p Q93-expressing flies; (C) heterozygous loss of Sirt2 also reduces photoreceptor neuron survival; (D) sirtinol, (E) nicotinamide and (F) niacin administration result in reduced neuronal death; (G) pseudopupil images of a normal eye and 7-day-old Httex1p Q93 flies is shown; niacin reduces the rhabdomere loss in Htt flies.

Figure 3.

Combinatorial reduction of Rpd3 and Sir2 shows increased neuroprotection in Httex1p Q93 flies: (A) genetic reduction of either Rpd3 or Sir2 in heterozygotes is protective. The survival to adulthood of control flies (Httex1p Q93 alone) is increased when double heterozygous for Rpd3 and Sir2; (B) the level of neuroprotection of Httex1p Q93-challenged flies is also significantly increased by concurrent reduction of both Rpd3 and Sir2 compared with either alone; (C) pharmacological treatment using butyrate (B) and nicotinamide (N) is additive. Although each of these compounds can be neuroprotective at 10 times higher concentrations, here we fed animals low levels of nicotinamide (2 mm) and butyrate (10 mm) and combinatorial synergy is observed leading to neuroprotection; (D) reduced levels of Rpd3 do not lead to increased levels of Sir2 expression. Animals of the genotype Rpd3[04556]/TM3 were crossed to either wild-type (Ore-R) or w flies (to control for background effects if present), RNA was extracted from whole males and real time quantitative RT–PCR used to evaluate the levels of Sir2 expression.

Figure 4.

Relationship between lifespan altering treatments and neurodegeneration: (A) reduced Rpd3 does not extend the lifespan of Htt-challenged flies (solid lines) but does extend the lifespan of control flies (dashed lines). Black lines are flies with reduced Rpd3, gray lines are controls with normal Rpd3 levels; (B) the reduction in Sir2 levels does not alter the longevity of either the Htt-challenged animals (solid lines – black: reduced Sir2; gray: control) or Htt non-expressing siblings (dashed lines – black: reduced Sir2; gray: control); (C and D) caloric restriction (CR) does not increase longevity of Httex1p Q93-expressing animals (solid lines) (C) nor is it neuroprotective (D). control animals not expressing Htt reared on the calorie-restricted diet (dashed black line) showed an increase in lifespan compared with fully fed (FF) siblings (dashed gray line) as has been reported; (E–G) overexpression of Sir2 (oe) does not increase the percent of animals surviving to adulthood (E) nor does it alter the survival of photoreceptor neurons (F). As previously described, overexpression of Sir2 (G) does increase the longevity of normal flies (dashed lines – black: overexpression; gray: controls) and the longevity of diseased flies is slightly increased by elevated Sir2 (solid lines – black: overexpression; gray: normal Sir2 controls).

Figure 5.

Resveratrol feeding: (A) resveratrol exhibits a dose-dependent increase in neuronal survival of Httex1p Q93 animals; (B) 10 µm resveratrol does not rescue the early death phenotype of Htt-challenged flies; (C) 10 µM resveratrol improves the survival of neurons in Sir2 homozygous null Httex1p Q93 flies.