Chaperones Join HDACs on Road to Neutralizing Poly-Q Toxicity

Histone deacetylase (HDAC) enzymes, best known as organizers of chromatin and regulators of gene expression, have been getting a lot of attention as targets for boosting neuronal survival and function. While the neuroprotective roles of HDACs have been mostly attributed to their effects on transcriptional regulation or DNA repair (see ARF related news story), that may not be the whole story. A paper out yesterday in Molecular Cell shows an unexpected link between HDAC4 and the toxicity of polyglutamate (poly-Q)-containing proteins, one that goes via chaperone proteins. In the work, Harm Kampinga and colleagues at the University of Groningen, The Netherlands, identify two heat shock proteins belonging to the DNAJ family of chaperones as potent protectors against the toxicity of poly-Q proteins. They then show that HDAC4 deacetylates and upregulates the chaperones’ activity. The results reveal a new activity for HDACs in protein homeostasis, and could open up new angles of attack on poly-Q diseases.

To identify chaperones that help cells withstand the insult of glutamine repeats, first authors Jurre Hageman and Maria Rujano systematically compared 22 different heat shock proteins (HSPs) for their ability to block aggregation of a 119-glutamine repeat-containing fragment of the huntingtin protein in cell overexpression experiments. They found that most of the chaperones of the Hsp70 family, which are good at protein refolding, were not very good at blocking aggregation. Instead, their survey revealed that two members of the Hsp40 family (DNAJB6b and DNAJB8) caused a dramatic reduction in aggregation based on protein solubility in gels, filter trap assay, or by confocal microscopy imaging of cytosolic inclusions. Along with that, the proteins were able to protect against poly-Q toxicity in cells in culture and in vivo in Xenopus tadpoles. Further, downregulation of endogenous DNAJB6b enhanced poly-Q aggregation and toxicity in cultured cells. The DNAJB6b and DNAJB8 proteins seemed to be general suppressors of poly-Q aggregations, since they worked against two other disease-causing poly-Q-expanded proteins, ataxin-3 and the androgen receptor. Their activity may also be highly conserved—some time ago the Drosophila ortholog of DNAJB6b was found to modify poly-Q toxicity in a fly model of Huntington disease (Kazemi-Esfarjani and Benzer, 2000).

How do the proteins work? The chaperones were unable to break up pre-formed aggregates, but seemed to prevent inclusions of newly expressed protein by forming large, disperse structures that included the poly-Q peptides. “We find they are very effective in the initial phase. As soon as aggregation starts, DNAJ proteins are there,” Kampinga told ARF.

In the canonical chaperone pathway, Hsp70 proteins serve as the refolding engine, and DNAJ proteins function to direct substrates to that engine. In this case, however, the activities of DNAJB6b and DNAJB8 largely bypass the classical folding pathway. Their activity did not require the induction of the heat shock response, and deletion of the N-terminal J domain, required for interaction with HSP70 and related proteins, only partly reduced their ability to prevent aggregation. However, a C-terminal region that functions in substrate binding was required for activity, and, in fact, was fully active on its own. That region, called the SSF-SST domain, was necessary for formation of the large chaperone complexes that associated with poly-Q peptides.

An Assist From Deacetylases
The researchers knew from previous work that the SSF-SST domain of DNAJB6b interacted with the histone deacetylase HDAC4 (Dai et al., 2005). In coexpression experiments, they confirmed the interaction and showed that the DNAJB6b and DNAJB8 proteins could bind two other deacetylases as well, HDAC6 and Sirt2. The association was functional, they showed, as the general HDAC inhibitor Trichostatin A blocked the anti-aggregation activity of DNAJB6b and DNAJB8. In addition, downregulation of HDAC4 was toxic to cells expressing the poly-Q protein, and DNAJB8 no longer could protect those cells.

Two conserved lysine residues in C-terminal region of DNAJB8 proved to be acetylated, and replacement of those sites with nonpolar alanine reduced deaggregation activity. The mutants could still bind substrate and form a poly-disperse network, suggesting that the lysine residues, and by extension HDAC4, were important not for binding, but for further processing of the peptide-chaperone complex.

Kampinga believes that the DNAJB6b and DNAJB8 serve a special function among chaperones, in that, rather than mediating protein refolding of full-length poly-Q-containing proteins, they act on poly-Q-containing peptides. “We think there is not a big problem in folding the full-length protein, but when it goes to get degraded, the proteasome spits out these poly-Q peptides because it cannot cleave them. This is controversial, but the idea is that those [peptides] may initiate the aggregation,” he said. The idea is that the DNAJB proteins would sequester those dangerous peptides, and Kampinga says he has some evidence to support that idea. “In collaboration with Eric Reits [Academic Medical Center, Amsterdam, The Netherlands], we found that exclusively DNAJB6b and DNAJB8 can prevent aggregation of synthetic poly-Q peptides,” he told ARF. “We really believe these are peptide scavengers.”

If that is the case, might the chaperones work for other toxic peptides, for example Aβ? Kampinga says they are working on this question, although it is not clear if the cytosolic chaperones would ever get at Aβ, which is mostly extracellular. “Although there are data now to suggest that extracellular Aβ needs to be internalized to become neurotoxic, we do not know whether, if that is true, the peptides need to be released from endosomes in order to become toxic,” Kampinga said. “Only in the latter case, one may envision that they then can be chaperoned and detoxified by the cytosolic DNAJB proteins we described.”

HDACs Galore
The results could explain the reported neuroprotective action of HDAC4 (Majdzadeh et al., 2008; Chen and Cepko, 2009), the authors say. However, with HDACs, things get complicated very quickly. HDAC inhibitors have been shown to be effective in fly and mouse models of Huntington disease (see ARF related news story on Steffan et al., 2001, and Thomas et al., 2008). HDAC inhibitors also increase memory function, possibly via HDAC2 (see ARF related news story on Guan et al., 2009). In contrast, other results suggest that inhibition of HDAC1 leads to neurodegeneration, at least in mice (see ARF related news story). Even as clinical work moves ahead on inhibitors (see ARF related news story), Kampinga’s work reminds us there is much more to learn about the substrate specificity and functional roles of the various HDACs that are becoming therapeutic targets.

A better alternative might be to target the chaperones directly, Kampinga says. HDAC effects are global, so he likes the idea of focusing on DNAJB6b or DNAJB8, rather than their modifiers. Their current work includes a study of the effects of boosting DNAJB6b expression in a Huntington’s mouse model, and screening for compounds that can increase the activity or level of chaperone proteins.—Pat McCaffrey

Comments

    • User Profile Image Ling Pan
      Massachusetts Institute of Technology
    • Posted:

    The authors demonstrated nicely that DNAJB6b and DNAJB8, members of DNAJB chaperone subfamily, can potently suppress poly-Q toxicity. The anti-aggregation activity is Hsp70-independent. Histone deacetylase 4 interacts with these DNAJBs and likely regulates their activity through deacetylation. The paper provided an intriguing mechanism for HDAC4 in suppressing cytotoxic protein aggregation. It would be interesting to test this mechanism in a more neuronal relevant system, such as a Huntington disease mouse model. This paper, together with other publications, also suggests that different HDACs could have either neuroprotective or neurotoxic roles, depending on the context. Collectively, these observations imply that development of isoform-selective HDAC inhibitors is necessary.

    View all comments by Ling Pan

  2. This paper by Jurre Hageman and colleagues provides more data suggesting that global histone deacetylase inhibition (HDACi) is fraught with potential complications, even though the integrated effect of HDACi is obviously beneficial for mice carrying polyQ expansions. It also raises the possibility that the induction of other Hsp chaperones by nuclear HDAC inhibition overcomes the downsides of HDAC4 inhibition.

    The field has long moved beyond global HDAC inhibition and has embraced the notion that selective HDAC isoforms, including HDAC2 (Guan et al., 2009), HDAC6 (Rivieccio et al., 2009), and HDAC1, according to a recent paper by Patricio Casaccia and colleagues (Kim et al., 2010), are the way to move forward therapeutically.

    HDAC4 is quite an interesting protein, and Santosh D'Mello, Eric Olson, and colleagues have demonstrated a pro-survival role of this molecule, with elegant in vitro and in vivo studies (see Majdzadeh et al., 2008). This function of HDAC4 appears not to depend on its deacylating activity and thus may not be fully abrogated by global HDAC inhibitors. Another very important paper that has been overlooked by the clinical neuroscience community is one by Jun Sadoshima and colleagues which shows that oxidation of HDAC4 moves it to the cytoplasm (Ago et al., 2008). We are currently working on a model by which HDAC4 movement from the nucleus to the cytoplasm could be a mechanism to de-repress or activate adaptive genes in the nucleus while placing HDAC4 in the cytoplasm to partner with DNAJB8 and DNAJB6b.

    The notion that small DNAJB proteins are involved in Huntington disease is not new, and elegant papers by Borrell-Pagés (see Borrell-Pagés et al., 2006) a few years ago and by Karpuj and Steinman (see Karpuj et al., 2002) suggest that HSJ1b is repressed transcriptionally by mutant huntingtin. We have a paper in revision at EMBO Molecular Medicine that shows that transglutaminase acts as a nuclear co-repressor for many genes including DNAJB2, the murine homolog of HSJ1b, and that transglutaminase inhibition, molecularly or pharmacologically, can normalize message levels for this important gene. Our findings, in the context of the studies described above, suggest that transglutaminase inhibition, selective HDAC inhibition (leaving HDAC4 unperturbed), or REST inhibition (see Rigamonte et al., 2009) may be viable strategies to optimize DNAJB8 levels in polyglutamine disorders and avoid the problems of global HDAC inhibition.

    DNAJB8 appears important not only to limit polyQ toxicity, as shown by this paper, but also to optimize trafficking of BDNF in HD (see Borrell-Pagés et al., 2006).

    References:

    Guan JS, Haggarty SJ, Giacometti E, Dannenberg JH, Joseph N, Gao J, Nieland TJ, Zhou Y, Wang X, Mazitschek R, Bradner JE, DePinho RA, Jaenisch R, Tsai LH. HDAC2 negatively regulates memory formation and synaptic plasticity. Nature. 2009 May 7;459(7243):55-60. PubMed.

    Rivieccio MA, Brochier C, Willis DE, Walker BA, D'Annibale MA, McLaughlin K, Siddiq A, Kozikowski AP, Jaffrey SR, Twiss JL, Ratan RR, Langley B. HDAC6 is a target for protection and regeneration following injury in the nervous system. Proc Natl Acad Sci U S A. 2009 Nov 17;106(46):19599-604. PubMed.

    Kim JY, Shen S, Dietz K, He Y, Howell O, Reynolds R, Casaccia P. HDAC1 nuclear export induced by pathological conditions is essential for the onset of axonal damage. Nat Neurosci. 2010 Feb;13(2):180-9. PubMed.

    Majdzadeh N, Wang L, Morrison BE, Bassel-Duby R, Olson EN, D'Mello SR. HDAC4 inhibits cell-cycle progression and protects neurons from cell death. Dev Neurobiol. 2008 Jul;68(8):1076-92. PubMed.

    Ago T, Liu T, Zhai P, Chen W, Li H, Molkentin JD, Vatner SF, Sadoshima J. A redox-dependent pathway for regulating class II HDACs and cardiac hypertrophy. Cell. 2008 Jun 13;133(6):978-93. PubMed.

    Borrell-Pagès M, Canals JM, Cordelières FP, Parker JA, Pineda JR, Grange G, Bryson EA, Guillermier M, Hirsch E, Hantraye P, Cheetham ME, Néri C, Alberch J, Brouillet E, Saudou F, Humbert S. Cystamine and cysteamine increase brain levels of BDNF in Huntington disease via HSJ1b and transglutaminase. J Clin Invest. 2006 May;116(5):1410-24. PubMed.

    Karpuj MV, Becher MW, Springer JE, Chabas D, Youssef S, Pedotti R, Mitchell D, Steinman L. Prolonged survival and decreased abnormal movements in transgenic model of Huntington disease, with administration of the transglutaminase inhibitor cystamine. Nat Med. 2002 Feb;8(2):143-9. PubMed.

    Rigamonti D, Mutti C, Zuccato C, Cattaneo E, Contini A. Turning REST/NRSF dysfunction in Huntington's disease into a pharmaceutical target. Curr Pharm Des. 2009;15(34):3958-67. PubMed.

    View all comments by Sama Sleiman

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References

News Citations

  1. Overworked HDACs Leave Transcriptional Posts to Push DNA Repair
  2. Drugs Slow Neurodegeneration in Fly Model of Huntington's
  3. It’s an HDAC2 Wrap— Memory-suppressing DNA Modifier Identified
  4. DC: Developing But Debatable—Deacetylase Inhibitors for CNS Disease?

Paper Citations

  1. Kazemi-Esfarjani P, Benzer S. Genetic suppression of polyglutamine toxicity in Drosophila. Science. 2000 Mar 10;287(5459):1837-40. PubMed.
  2. Dai YS, Xu J, Molkentin JD. The DnaJ-related factor Mrj interacts with nuclear factor of activated T cells c3 and mediates transcriptional repression through class II histone deacetylase recruitment. Mol Cell Biol. 2005 Nov;25(22):9936-48. PubMed.
  3. Majdzadeh N, Wang L, Morrison BE, Bassel-Duby R, Olson EN, D'Mello SR. HDAC4 inhibits cell-cycle progression and protects neurons from cell death. Dev Neurobiol. 2008 Jul;68(8):1076-92. PubMed.
  4. Chen B, Cepko CL. HDAC4 regulates neuronal survival in normal and diseased retinas. Science. 2009 Jan 9;323(5911):256-9. PubMed.
  5. Steffan JS, Bodai L, Pallos J, Poelman M, McCampbell A, Apostol BL, Kazantsev A, Schmidt E, Zhu YZ, Greenwald M, Kurokawa R, Housman DE, Jackson GR, Marsh JL, Thompson LM. Histone deacetylase inhibitors arrest polyglutamine-dependent neurodegeneration in Drosophila. Nature. 2001 Oct 18;413(6857):739-43. PubMed.
  6. Thomas EA, Coppola G, Desplats PA, Tang B, Soragni E, Burnett R, Gao F, Fitzgerald KM, Borok JF, Herman D, Geschwind DH, Gottesfeld JM. The HDAC inhibitor 4b ameliorates the disease phenotype and transcriptional abnormalities in Huntington's disease transgenic mice. Proc Natl Acad Sci U S A. 2008 Oct 7;105(40):15564-9. PubMed.
  7. Guan JS, Haggarty SJ, Giacometti E, Dannenberg JH, Joseph N, Gao J, Nieland TJ, Zhou Y, Wang X, Mazitschek R, Bradner JE, DePinho RA, Jaenisch R, Tsai LH. HDAC2 negatively regulates memory formation and synaptic plasticity. Nature. 2009 May 7;459(7243):55-60. PubMed.

Further Reading

Primary Papers

  1. Hageman J, Rujano MA, van Waarde MA, Kakkar V, Dirks RP, Govorukhina N, Oosterveld-Hut HM, Lubsen NH, Kampinga HH. A DNAJB chaperone subfamily with HDAC-dependent activities suppresses toxic protein aggregation. Mol Cell. 2010 Feb 12;37(3):355-69. PubMed.

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