Huntington disease (HD) is a neurodegenerative disorder caused by the repetition of a cytosine-adenine-guanine (CAG) trinucleotide coding for the amino acid glutamine. The repeat occurs in the huntingtin gene and results in an expanded huntingtin (Htt) protein containing a polyglutamine (polyQ) tract. Though expanded huntingtin accumulates in neurons, there is continuing debate over whether these intracellular inclusions are damaging or protective (see ARF related news story), and as yet no consensus has emerged as to how mutant Htt causes neurodegeneration. Nonetheless, three recent papers show how RNAi, resveratrol, and inhibitors of kyneurenine-3-monooxygenase may offer hope as therapeutics for HD and other polyglutamine disorders.
Reporting in the April Nature Genetics, Christian Neri and colleagues at INSERM in Paris, France, show that resveratrol can attenuate polyQ huntingtin-mediated toxicity in worms and mammalian neurons. The polyphenol, found in grapes and wine, has gotten a lot of press since researchers found it dramatically increases lifespan in yeast, worms, and flies (see, for example, Howitz et al., 2003 and that it can protect axons against degeneration (see ARF related news story). These actions are attributed to activation of sirtuins, a family of NAD-dependent histone deacetylases that includes SIRT1, which may extend the life of mammalian cells (see ARF related news story).
First author Alex Parker and colleagues found that resveratrol improved HD-like pathology in the roundworm Caenorhabditis elegans. Though some would call these creatures primitive, they do have motor neuron function of a sort, which the authors tapped into by measuring the response of the animals’ tails to mechanical stimuli. Parker found that tail mechanosensitivity jumped 20 percent in worms fed resveratrol. Htt aggregation was unchanged, however, which supports suggestions that aggregates per se are not the bad guys in HD. Worms given another sirtuin activator, fisetin, were similarly feisty, as were those overexpressing sir-2.1, the worm homolog of SIRT1. The sirtuin activators did not work in worms expressing mutated, loss-of-function sir-2.1.
Worms, of course, may not represent a great model for human disease, but Parker and colleagues obtained similar results when they used striatal cells from mice that express Hdh109Q, the expanded mouse homolog of the human gene. In these cells, resveratrol rescued dystrophic processes by about 35 percent, again, only if active sir-2 was present. The polyphenol also reduced cell mortality by about 40 percent, but had no effect on Hdh expression or aggregation.
The effect of resveratrol on these HD models might be related to apoptosis. Sirtuins are thought to attenuate apoptotic signals through their actions on the proapoptotic transcription factors forkhead (see Motta et al., 2004 and Bax (see ARF related news story), and Parker found that loss-of-function mutations in forkhead family member daf16 also rescued tail sensitivity in worms by about 15 percent. Sirtuins are also activated when animals are placed on a caloric restriction diet. In this regard, it is telling that a loss-of-function mutation in the gene age-1, coding for a kinase that mediates insulin-like signaling, also rescued tail sensitivity by about 10 percent. Though it is unclear whether resveratrol’s effects are mediated by any of these signaling pathways, the authors conclude that sirtuin activators “may be useful in the development of therapeutic strategies for Huntington disease.”
However, things are never simple. The second Nature Genetics paper, this one from Paul Muchowski and colleagues at the University of Seattle, Washington, and the University of Maryland School of Medicine, Baltimore, suggests that activating histone deacetylases might have the opposite effect, exacerbating polyQ toxicity. First author Flaviano Giorgini and colleagues conducted screens of over 4,000 strains of yeast for gene deletions that suppress huntingtin toxicity. They found that absence of 28 proteins makes the yeast grow stronger. Twenty-four of these proteins were of known function and two, Ume1 and Rxt3, are members of the yeast Rpd3 histone deacetylase complex. Because Ume1 is required for full activity of the complex, this data indicates histone deacetylases may actually contribute to huntingtin toxicity, a suggestion that has much support. For example, inhibitors of histone deacetylases slow neurodegeneration in a fly model of Huntington disease (see ARF related news story), while lack of histone acetylases has indirectly linked deacetylation to huntingtin toxicity (see ARF related news story).
So how can both activation and inhibition of histone deacetylases protect against huntingtin toxicity? Perhaps the answer lies in substrate specificity. It is possible that NAD-dependent and NAD-independent deacetylases can have opposite effects on polyQ pathology, for example. But what is also intriguing is that Muchowski’s group found that deletion of kyneurenine-3-monooxygenase (KMO), an enzyme involved in NAD synthesis, strongly suppressed Htt128Q toxicity in yeast. Does this suggest that the NAD-dependent sirtuins also contribute to huntingtin toxicity, which would be at total odds with the data from Neri’s group? Perhaps not. Because there are alternative pathways for NAD synthesis, it is unclear if sirtuin activity is affected in KMO knockout mice. Instead, Muchowski and colleagues suggest a different reason for the increased Htt toxicity found in yeast with KMO—oxidative damage.
Two intermediaries, quinolinic acid and 3-hydroxykynurenine (3HK), which lie downstream of KMO in the NAD synthesis pathway, produce reactive oxygen species (ROS). They are also known to cause neurotoxicity in animals and are elevated in early-stage Huntington disease (see Guidetti et al., 2000 and Guidetti et al., 2004). Giorgini and colleagues found that both compounds are absent from KMO-negative yeast, and that that ROS levels were normal in these strains, even when they expressed Htt128Q. In contrast, the authors found that wild-type yeast expressing the expanded huntingtin had ROS levels eightfold higher than controls. But, when Girogini added the KMO inhibitor Ro 61-8048 to these sickly yeast, ROS were reduced by about twofold and growth was partially rescued. The author found these results so promising that “…preclinical trials with the compound are currently underway using mouse models of HD,” they write.
An alternative to tackling the toxicity of expanded huntingtin is to target production of the protein. In this week’s PNAS, Beverly Davidson and colleagues at the University of Iowa, Iowa City, and the NIH at Bethesda, Maryland, report that they have used RNA interference (RNAi) to ablate huntingtin mRNA in a mouse model of HD.
First author Scott Harper and colleagues used adenoassociated viral particles to infect the brains of transgenic mice (HD-N171-82Q) with an expression system that produces short hairpin RNAs (shRNAs) that target exon 2 of the huntingtin transcript (see ARF related news story). About three weeks after infection, Harper and colleagues detected robust levels of 50- and 21-nucleotide-long shRNAs in the brains of treated mice. They also found that the shRNA reduced expression of mutant Htt by over twofold, and almost completely eliminated Htt inclusions in cells where Htt expression was silenced—the system used to drive shRNA production also expresses enhanced green fluorescent protein, enabling the authors to see exactly which cells produce the interfering RNA.
But it was in behavioral tests where the system came through with flying colors. Huntington disease affects the motor neurons, leading to involuntary movements, balance problems, and muscle weakness. Transgenic mice expressing the mutant huntingtin are no different; they, too, have trouble walking and maintaining balance. But Harper found that animals treated with RNAi had dramatically improved performance. On the rotarod, for example, which is the mouse equivalent of the gymnast’s balance beam, treated animals learned to hang on for almost 350 seconds; 250 seconds was all that untreated mice could muster. Harper and colleagues also found that stride length was significantly improved in the treated animals, and the researchers concluded that the “data suggest the feasibility of treating HD by directly reducing mutant Htt gene expression by using RNAi and support its general applicability to treating other dominant neurodegenerative disorders.”—Tom Fagan
- New Microscope Resolves Role of Huntington Inclusions—Neuroprotection
- The Wine and Wherefore of Wallerian Degeneration
- Who Says Chivalry is Dead?—Sir2 Fights Against Aging in Mammals
- Drugs Slow Neurodegeneration in Fly Model of Huntington's
- Modeling Polyglutamine Diseases in Yeast Provides Support for Histone Deacetylase Connection
- RNA Interference in Vivo—Viral Vectors Deliver
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