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Feb 22, 2012

HDA3 Inhibitors May Help Slow TNR Expansions and Huntington Disease Progression

  • Scientists suggest that inhibiting the histone deacetylase enzyme HDA3 may provide a new approach to slowing the process of trinucleotide repeat (TNR) expansion that is responsible for Huntington disease (HD). Studies by a team at the National University of Ireland Galway’s Center for Chromosome Biology showed that chemical or genetic inhibition of Rpd3L or Hda1 in yeast, and HDA3 in human cells, suppressed up to 90% of expansions. 

    The investigators say their findings indicate that HDAC 3 inhibitors being developed for other expansion-associated diseases, such as Friedreich ataxia, may have secondary utility in suppressing somatic expansions contributing to HD. Robert S. Lahue, Ph.D., and colleagues, describe their results in PLoS Biology in an paper titled “Histone Deacetylase Complexes Promote Trinucleotide Repeat Expansions.”

    The expansion of TNRs causes neurodegenerative disorders including HD, myotonic dystrophy type 1 (DM1), and at least 15 other inherited neurological disorders. In HD, expansions occur in a CAG triplet repeat stretch within the huntingtin gene. TNR expansions are believed to arise due to the activity of proteins that actively promote lengthening of the repeat, rather than absence of proteins that prevent it, the Galway team explains. Moreover, previous work in yeast and human cells indicates that TNR expansion increases sharply once a threshold repeat length is crossed, leading to critical initiation mutations that enhance instability and lead to disease. Building on these previous observations, the CCB team set out to identify novel factors in yeast and human cells that promote expansions of TNR alleles near the threshold.

    The researchers carried out a large-scale yeast mutant screen to identify mutants with reduced expansion rates. Of 11 mutants that demonstrated consistently reduced TNR expansions, three demonstrated changes in genes encoding subunits of the histone deacetylase enzymes Rpd3L and Hda1, providing an initial indication of a causal relationship between specific HDACs and TNR expansion.

    Active knockdown of these three genes, sin3, pho23, and hda3, in yeast cells led to a deficiency in RPD3L and Hda1, and a more than 1,000-fold reduction in expansion rates. Reduced expansion rates could also be effected by treating wild-type cells with trichostatin A (TSA), which inhibits a number of HDACs. In fact mutation or chemical inhibition of yeast Rpd3L and Hda1 suppressed CTG repeat expansions by 50%–90% or more, supporting a mechanistic link between triplet repeat expansions and the yeast HDACs Rpd3L and Hda1, the researchers report.

    Class one human HDACs are the homologs of yeast Rpd3, so to see whether these HDACs promote expansion in human cells, the team treated human astrocyte lines with a small molecule HDAC inhibitor 4b, which is selective for the class I enzyme HDAC3. Glial cells such as astrocytes show somatic expansions in HD patients, and the HDAC inhibitor 4b has previously been shown to reverse FXN gene silencing in primary cells from Friedreich ataxia patients, and improve disease phenotype and transcriptional aberrations in HD transgenic mice. The assays showed that 4b efficiently suppressed the frequency of TNR expansions in SVG-A cells, in a dose-dependent manner, and at well-tolerated doses. Knockdown of HDAC3 also resulted in 76% reduction in expansion frequencies. Interestingly, it was the frequency, rather than the length of TNR expansions or cell viability that was reduced on 4b administration. Conversely, administration of an siRNA against the histone acetyltransferase CREB-binding protein (CBP) and p300 stimulated expansions.

    Genetic and molecular analysis both indicated that HDACs act at a distance from the triplet repeat to promote expansions, rather than at the site itself. Further studies in demonstrated that one of the downstream deacetylation targets of Rpd3L and Hda1 is the nuclease Sae2. This nuclease, along with the Mre11/Rad50/Xrs2 complex, is known to process hairpin DNA in vivo and in vitro, and, significantly, TNR expansions are thought to involve structured intermediates such as a hairpin.

    Based on both their latest data and that of previous studies, the authors have proposed a model whereby Rpd3L and Hda1 positively regulate Sae2 by stabilizing it, enabling Sae2 and Mre11 to function together as nucleases to promote expansions. Indeed, their studies confirmed that expansion frequencies increased in sae2 or mre11 yeast mutants.

    HDAC inhibitors are currently being evaluated as therapies to treat the transcriptional defects in several TNR expansion diseases, and just last month Italian regulators granted approval for the start of a Phase I trial with Repligen’s HDAC inhibitor RG2833. “Our work implies these inhibitors may have a second, beneficial effect of suppressing somatic expansions that contribute to disease progression,” the investigators conclude. 


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