Differential contributions of Caenorhabditis elegans histone deacetylases to huntingtin polyglutamine toxicity

Abstract

Expansion of a polyglutamine tract in the huntingtin protein causes neuronal degeneration and death in Huntington's disease patients, but the molecular mechanisms underlying polyglutamine-mediated cell death remain unclear. Previous studies suggest that expanded polyglutamine tracts alter transcription by sequestering glutamine rich transcriptional regulatory proteins, thereby perturbing their function. We tested this hypothesis in Caenorhabditis elegans neurons expressing a human huntingtin fragment with an expanded polyglutamine tract (Htn-Q150). Loss of function alleles and RNA interference (RNAi) were used to examine contributions of C. elegans cAMP response element-binding protein (CREB), CREB binding protein (CBP), and histone deacetylases (HDACs) to polyglutamine-induced neurodegeneration. Deletion of CREB (crh-1) or loss of one copy of CBP (cbp-1) enhanced polyglutamine toxicity in C. elegans neurons. Loss of function alleles and RNAi were then used to systematically reduce function of each C. elegans HDAC. Generally, knockdown of individual C. elegans HDACs enhanced Htn-Q150 toxicity, but knockdown of C. elegans hda-3 suppressed toxicity. Neuronal expression of hda-3 restored Htn-Q150 toxicity and suggested that C. elegans HDAC3 (HDA-3) acts within neurons to promote degeneration in response to Htn-Q150. Genetic epistasis experiments suggested that HDA-3 and CRH-1 (C. elegans CREB homolog) directly oppose each other in regulating transcription of genes involved in polyglutamine toxicity. hda-3 loss of function failed to suppress increased neurodegeneration in hda-1/+;Htn-Q150 animals, indicating that HDA-1 and HDA-3 have different targets with opposing effects on polyglutamine toxicity. Our results suggest that polyglutamine expansions perturb transcription of CREB/CBP targets and that specific targeting of HDACs will be useful in reducing associated neurodegeneration.

Figure 1.

cbp-1(ys2)/+, cbp-1(ys4)/+, and crh-1(3315) enhance Htn-Q150-induced degeneration. Sibling controls for cbp-1(ys2)/+; Htn-Q150, cbp-1(ys4)/+;Htn-Q150, and crh-1(e3315); Htn-Q150 were combined, because degeneration levels were not significantly different. No degeneration was detected in cbp-1(ys2)/+, cbp-1(ys4)/+, or crh-1 animals that did not express Htn-Q150. Error bars represent SD. ***p < 0.005 versus column 1. At least three trials were conducted for each experiment to total n > 150.

Figure 2.

A, hda-1(e1795)/+, hda-4, sir-2.1(ok434) enhance Htn-Q150 toxicity. sir-2.1 overexpression suppresses Htn-Q150 toxicity. sir-2.3 has no effect on Htn-Q150 toxicity. Controls were combined, because they are not significantly different. No degeneration was detected in hda-1(e1795)/+, hda-4, sir-2.1(ok434), or sir-2.3 animals without expression of Htn-Q150. At least three trials were conducted for each experiment to total n > 150. B, RNAi of class I HDACs, hda-1 and hda-2, enhances Htn-Q150 toxicity. RNAi of class I HDAC, hda-3, suppresses Htn-Q150 toxicity. RNAi of class II HDACs enhanced degeneration, as did RNAi of sir-2.2, a class III HDAC. Controls were combined, because they are not significantly different. At least three trials were conducted for each experiment to total n > 150. Error bars represent SD. *p < 0.05, **p < 0.005, ***p < 0.0005 in pairwise comparison with column 1 using a two-tailed t test.

Figure 3.

A, Expression of HDA-1 in ASH neurons restores Htn-Q150 toxicity to normal levels. Injection of equal concentrations of an empty srb-6 promoter plasmid and the transgenic marker fail to rescue the hda-1(e1795) enhancement of polyQ toxicity (29 ± 2%). B, In Htn-Q150 animals without the srb-6::hda-1 transgene, 47 ± 4% of the ASH neurons degenerate by day 8. Degeneration is decreased to 20 ± 4% by increased expression of HDA-1 (50 ng/μl) in Htn-Q150 ASH neurons. Expression of HDA-1 does not cause detectible degeneration of ASH neurons that do not express Htn-Q150 (2 ± 2%). No significant change in neurodegeneration is detectable in animals that carry an empty srb-6 promoter and the transgenic marker myo-2::GFP (50 ± 10%). ***p < 0.0005 compared with corresponding Htn-Q150 animals in column 1; ^###p < 0.0005 when comparing hda-1;Htn-Q150; srb-65::hda-1 with hda-1;Htn-Q150 in column 2 (to assess rescue) in A. Empty promoter transgenes did not significantly change polyglutamine toxicity. At least three trials were conducted for each experiment to total n > 150. Error bars represent SD.

Figure 4.

HDA-3 specifically rescues hda-3(tm1374) suppression of Htn-Q150 in ASH neurons. Degeneration is reduced from 27 ± 2% in rtIs11Htn-Q150 animals to 6 ± 6% in hda-3(tm1374); rtIs11(Htn-Q150) animals at day 7. Degeneration ranges from 14 to 63%, depending on the transgenic line (average, 45 ± 22%) in hda-3(tm1374); rtIs11(Htn-Q150); srb-6:hda-3 transgenic animals. As a control, a plasmid carrying the empty srb-6 promoter was injected along with transgenic markers. The empty promoter has no significant effect on polyQ toxicity (10 ± 3%). Overexpression of hda-3 in ASH using the srb-6 promoter increases degeneration from 24 ± 11 to 45 ± 13% (p = 0.03). ***p < 0.0005 compared with column 1 for each graph; ^###p < 0.0005 when comparing hda-3;Htn-Q150; srb-6::hda3 with hda-3;Htn-Q150. At least three trials were conducted for each experiment to total n > 150. Error bars represent SD.

Figure 5.

HDA-3 modulates Htn-Q150 toxicity via transcriptional regulation. Loss of hda-3 function suppresses the deleterious effects of loss of crh-1 function. ASH neuron degeneration is increased in crh-1;rtIs11[Htn-Q150] animals and decreased in hda-3(tm1374);rtIs11[Htn-Q150] animals compared with rtIs11[Htn-Q150]. ASH degeneration is suppressed in hda-3(tm1374);crh-1; rtIs11[Htn-Q150] animals and is not significantly different from degeneration levels in hda-3(tm1374);rtIs11[Htn-Q150] animals at day 7. Error bars represent SD. ***p < 0.0005 compared with column 1 of each graph. At least three trials were conducted for each determination to total n > 150.

Figure 6.

Normal hda-1 levels are critical for hda-3 modulation of polyglutamine toxicity. Loss of one copy of hda-1 eliminates the salubrious effects of hda-3 loss of function. ASH neuron degeneration is increased in hda-1(e1795)/+; rtIs14[Htn-Q150] animals and decreased in hda-3(tm1374);rtIs14[Htn-Q150i] animals compared with rtIs14[Htn-Q150] animals. Degeneration levels are increased in hda-3(tm1374);hda-1(e1795)/+; rtIs14[Htn-Q150] and are not significantly different from degeneration levels in hda-1(e1795)/+; rtIs14[Htn-Q150] animals. Error bars represent SD. ***p < 0.0005 compared with column 1. At least three trials were conducted for each determination to total n > 150.