commentaries Shock and awe: unleashing the heat shock response to treat Huntington disease Meredith E. Jackrel and James Shorter

Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

The heat shock response (HSR) is a highly conserved protective mechanism improving disease phenotypes in mouse that enables cells to withstand diverse environmental stressors that disrupt models of neurodegenerative disease (11). protein homeostasis (proteostasis) and promote protein misfolding. It has Therefore, it has been proposed that acti- been suggested that small-molecule drugs that elicit the HSR by activating vating the entire HSR, rather than over- the transcription factor heat shock factor 1 might help mitigate protein mis- expressing a single chaperone, may prove folding and aggregation in several devastating neurodegenerative disorders, more successful in remediating toxic pro- including Huntington disease (HD). In this issue of the JCI, Labbadia et al. tein conformations (12). Specifically, it has use a brain-penetrant Hsp90 inhibitor, HSP990, to induce the HSR in mouse been suggested that activation of the HSR models of HD. Unexpectedly, they observed that HSP990 confers only tran- via the transcription factor heat shock fac- sient amelioration of a subset of HD-related phenotypes, because alterations tor 1 (HSF1), a master regulator of the HSR in chromatin architecture impair the HSR upon disease progression. These in eukaryotes, may be beneficial in target- findings suggest that synergistic combination therapies that simultaneously ing neurodegenerative disorders (12). Con- unleash the HSR and prevent its impairment are likely to be needed to sistent with this proposal, a constitutively restore proteostasis in HD. active form of HSF1 antagonizes polyglu- tamine aggregation and extends lifespan in Proteins must fold into intricate 3-dimen- cells have evolved to counter the proteotoxic a mouse model of HD (13). Furthermore, sional structures to perform an extraordi- stress caused by potentially deleterious pro- HSF1 provides a protective function in nary variety of functions (1, 2). A complex tein-misfolding events (1, 7–9). In this issue mice inoculated with prions: Hsf1-knock- network of highly conserved molecular of the JCI, Labbadia and colleagues find that out mice succumb to prion disease 20% chaperones ensures successful protein fold- although one such approach ameliorates faster than control mice (14). Thus, strate- ing (1, 2). Aberrant protein folding that some disease phenotypes in a mouse model gies to pharmacologically activate the HSR overwhelms this chaperone network is of HD, the benefits are only transient (10), via HSF1 hold promise for the treatment closely linked to several devastating neu- which indicates that synergistic combina- of a broad spectrum of presently incurable rodegenerative disorders, including Hun- tion therapies are likely to be needed if such protein-misfolding disorders (1). tington disease (HD), Parkinson disease, strategies are to make it to the clinic. Under nonstressful physiological condi- and Alzheimer disease (1–3). There are no tions, HSF1 is unable to induce the tran- treatments for these disorders, all of which Inducing the heat shock response as scription of genes encoding HSR proteins are associated with the misfolding, oligo- a therapeutic strategy because it is held in a complex with Hsp90 merization, and aggregation of specific pro- In response to proteotoxic stress, cells (Figure 1). Thus, one pharmacological teins in the CNS (1–3). HD is a fatal, inher- maintain protein homeostasis (proteos- strategy to activate the HSR via HSF1 is ited, late-onset neurodegenerative disorder tasis) by rapidly adjusting the concentra- to use Hsp90 inhibitors, such as geldana- that afflicts approximately 1 individual tions of appropriate molecular chaperones mycin and 17-AAG, that dissociate the per 10,000 worldwide (4). It is caused by a (1, 2). They do this by rapidly activating Hsp90:HSF1 complex and enable HSF1 to CAG triplet repeat expansion in exon 1 of ancient and highly conserved pathways, stimulate heat shock gene expression. In the huntingtin (HTT) gene, which generates such as the heat shock response (HSR), fly and mammalian cell models of HD and an expanded polyglutamine tract (approxi- which responds to stress in the cytoplasm, Parkinson disease, treatment with geldana- mately 40 residues or longer) in the mutant and the unfolded protein response (UPR), mycin reduces aggregate load and toxicity huntingtin protein (4). This polyglutamine which responds to stress in the secretory (15, 16). However, the use of geldanamycin tract expansion increases the propensity pathway (1, 2). These pathways upregulate and 17-AAG in mammals is complicated of huntingtin protein to form a heteroge- the requisite components of the chaperone by their toxicity and inability to efficiently neous array of misfolded species, ranging network. Molecular chaperones can then cross the blood-brain barrier. from toxic oligomers to diverse aggregated buffer aberrant protein conformers either structures (5, 6). An increasing number of by returning them to their native form or HSP990: a brain-penetrant Hsp90 potential therapeutic strategies now focus by facilitating their degradation and elimi- inhibitor on unleashing the natural defenses that nation (1, 2). Indeed, the overexpression of The careful and elegant study of Labbadia a single chaperone, such as Hsp70, can sup- et al. assesses the utility of a brain-penetrant press neurodegeneration in Drosophila mod- Hsp90 inhibitor, HSP990, in activating the Conflict of interest: The authors have declared that no conflict of interest exists. els of polyglutamine toxicity (9). However, HSR as a potential therapy in a mouse model Citation for this article: J Clin Invest. 2011; increasing the level of expression of a single of HD (10). Using a well-tolerated chronic 121(8):2972–2975. doi:10.1172/JCI59190. chaperone has met with mixed success in dosing regimen, the authors administered

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Figure 1 HSR activation varies with disease stage in mice that model HD. Upon inhibition of Hsp90 by the small molecule HSP990, the HSR is activated via HSF1. HSF1 dissociates from its repressive complex with Hsp90, is hyperphosphorylated, and translocates to the nucleus in both early- and late- stage HD mice. As HD progresses and the mice age, histone H4 becomes hypoacetylated at heat shock (HS) gene promoters, preventing efficient transcription of the heat shock genes and impairing the HSR. While early-stage HD mice induce an HSR upon HSP990 treatment and reduce the aggregate burden, late-stage HD mice cannot induce the HSR, and the aggregate burden and toxicity increases. HSE, heat shock element.

HSP990 orally to WT mice and R6/2 trans- a 30% improvement in rotarod performance. induction was already apparent for the R6/2 genic mice, which express exon 1 of a mutant However, not all symptoms of HD were ame- mice, and this deterioration continued with human huntingtin gene with more than 150 liorated. HSP990 treatment did not improve age until the mice reached end-state disease CAG repeats. R6/2 mice model HD with an body weight, grip strength, or exploratory at 15 weeks (Figure 1 and ref. 10). In these early age of onset and rapid disease progres- activity in the R6/2 mice. It remains unclear late-stage disease R6/2 mice, treatment with sion. Importantly, HSP990 activated the why only a subset of HD-related phenotypes HSP990 failed to induce Hsp expression. HSR by liberating HSF1 from Hsp90, which was partially rescued. Thus, HSP990 efficacy declined sharply in in turn led to hyperphosphorylation and R6/2 mice in an age-dependent manner. activation of HSF1 (Figure 1 and ref. 10). As The HSR becomes impaired as Importantly, this impairment of the HSR a consequence, levels of Hsp70, Hsp40, and disease progresses was not an artifact of the R6/2 model of HD. Hsp25 were greatly increased in the brains Surprisingly, the benefits afforded by The HSR also became impaired in late-stage of mice. Treatment with HSP990 did not HSP990 were transient. Rotarod perfor- HdhQ150 knockin mice, which model late- alter the levels of UPR chaperones, indica- mance of R6/2 HD mice improved with onset HD and express full-length human tive of specific induction of the HSR. How- treatment at 8 and 10 weeks of age, but not huntingtin with a polyglutamine tract of ever, induction of the UPR might also hold at 14 weeks, which suggests that impairment 150 residues. Taken together, these data value for treating polyglutamine toxicity of the HSR might occur upon HD progres- suggest it will be important to determine (17). Promisingly, administration of HSP990 sion (10). This decline in behavior with HD whether the HSR also becomes impaired in to R6/2 mice resulted in a 20% decrease in progression was also reflected in Hsp expres- the brains of HD patients. aggregate load in brain tissues and was asso- sion levels. In young mice, HSP990-induced Further studies revealed that HSR ciated with improved brain weight (Figure 1 upregulation of Hsp70, Hsp40, and Hsp25 impairment occurred at the level of tran- and ref. 10). Additionally, a general improve- was comparable in WT and R6/2 mice. How- scription (10). This impaired transcription ment in disease phenotype was indicated by ever, by 8 weeks of age, impairment in Hsp could have several potential origins. To

The Journal of Clinical Investigation http://www.jci.org Volume 121 Number 8 August 2011 2973 commentaries induce Hsp expression, HSF1 must dis- longevity remains unknown. However, even such small molecule, HSF1A, has recently sociate from its repressive complex with a transient improvement of disease pheno- been isolated in a cunning yeast screen (23). Hsp90, translocate to the nucleus, and be type may bring an enhanced quality of life HSF1A increased the expression levels of hyperphosphorylated (Figure 1). Coim- to HD patients, regardless of any potential several chaperones and consequently dimin- munoprecipitation studies indicated that increase in lifespan. ished the toxicity of polyglutamine proteins HSP990 effectively dissociated Hsp90 and in mammalian cell culture and Drosophila HSF1 even in older R6/2 mice (10). Addi- Prospects for inducing the HSR as models (23). We suggest that brain-pene- tionally, after HSP990 treatment, HSF1 was an HD therapy trant variants of HSF1A and other potential hyperphosphorylated and localized to the Overall, the work of Labbadia and col- small molecules that activate HSF1 without nucleus in R6/2 brain tissue. Thus, HSF1 leagues (10) demonstrates that HSF1-medi- inhibiting Hsp90 should also be explored in is activated by HSP990 equally well in WT ated activation of the HSR via Hsp90 inhi- mouse models of HD. and R6/2 mice. However, the authors fur- bition may hold great therapeutic value. ther noted that alterations in chromatin However, it also emphasizes the complexi- Summary architecture in older R6/2 mice precluded ties of targeting the HSR pathway, because The work of Labbadia et al. (10) has HSF1 from engaging the promoters of heat impairment of the HSR was an unforeseen revealed important complications that shock genes. As the R6/2 mice aged, lower effect of HD progression. This unexpected must be addressed if the HSR response is to levels of HSF1 binding at various heat deterioration of the HSR indicates that be induced as a potential HD therapy. The shock gene promoters was observed upon simply developing therapeutics to activate way is now open to define how the HSR HSP990 treatment. Furthermore, chroma- this pathway might not be sufficient to becomes impaired and how this impair- tin immunoprecipitation using the Hsp70 remediate HD and perhaps other protein- ment might be prevented or reversed. Of promoter revealed lower levels of associat- misfolding disorders. Indeed, the HSR particular interest is the development of ed RNA polymerase II in R6/2 than in WT also becomes impaired in prion-infected small-molecule drugs that might antago- brain tissue, consistent with reduced tran- cells, although this deficit is rescued by gel- nize HSR impairment and synergize with scription. Additionally, hypoacetylation of danamycin (18). For HD, it may be neces- HSP990 to treat mice that model HD. We histone H4 was observed at various heat sary to combine approaches that activate suggest that specific histone deacetylase shock genes as disease progressed (10), HSF1 with approaches that circumvent (HDAC) inhibitors might be interesting which might reduce the ability of HSF1 to the impairment of the HSR, which might lead candidates to combine with HSP990. bind target promoters by reducing chroma- include methods to increase histone H4 In isolation, HDAC inhibitors have exhib- tin accessibility (Figure 1). However, addi- acetylation. Moving forward, it will be key ited therapeutic effects in mice that model tional experiments showed that the Hsp70 to define precisely how HD-induced altera- HD (4). Moreover, they could prevent the promoter region was equally accessible in tions in chromatin architecture preclude hypoacetylation of histone H4 that corre- both WT and R6/2 mice. Thus, accessibility HSF1 binding. More broadly, combination lates with HSR impairment (10) and there- per se does not appear to be the issue. therapies that synergize to target multiple by help maintain an active HSR. Labbadia et al. propose a model whereby aspects of proteostasis, including direct as HD progresses, histone H4 becomes interactions with the misfolded protein Acknowledgments hypoacetylated at heat shock gene promot- itself, are promising weapons against vari- The authors acknowledge support from ers, perhaps because polyglutamine aggre- ous protein-misfolding disorders (8, 19). an American Heart Association postdoc- gates sequester key histone acetyltransferas- Finally, it should be noted that eliciting toral fellowship (M.E. Jackrel), NIH grants es (10). Through an undefined mechanism, the HSR is not the only effect of inhibit- 1DP2OD002177-01 and 5R21NS067354-02 hypoacetylation precludes HSF1 binding ing Hsp90. Hsp90 controls the matu- (J. Shorter), an Ellison Medical Foundation to heat shock gene promoters. Thus, the ration of many key signal transducers, New Scholar in Aging Award (J. Shorter), a HSR is impaired (Figure 1). The discovery including kinases and transcription fac- Bill and Melinda Gates Grand Challenges that the HSR becomes impaired as disease tors (20). Consequently, Hsp90 is a potent Explorations Award (J. Shorter), and a grant progresses in HD model mice is perhaps phenotypic capacitor. Inhibition of Hsp90 from the Packard Center for ALS Research the authors’ most significant finding. The exposes cryptic genetic variation that can at Johns Hopkins (J. Shorter). reduced ability to launch the HSR is likely generate diverse, complex multigenic phe- to exacerbate HD progression by rendering notypes that are difficult to predict, depend Address correspondence to: James Shorter, cells incapable of responding appropriate- on the specific genetic background, and are Department of Biochemistry and Biophys- ly to environmental stressors. However, it not necessarily advantageous (20). Indeed, ics, Perelman School of Medicine, Univer- remains unknown whether similar defects inhibition of Hsp90 can yield highly stable sity of Pennsylvania, 422 Curie Boulevard, in the HSR occur in HD patients. Thus, it changes in chromatin states that can persist Philadelphia, Pennsylvania 19104, USA. is possible (although unlikely, in our view) in a heritable manner (21, 22). Thus, inhibit- Phone: 215.573.4256; Fax: 215.573.7290; that these deficits reflect events unique to ing Hsp90 might even accentuate the altera- E-mail: [email protected]. mouse models of HD that are unrelated tions in chromatin architecture that impair 1. Balch WE, Morimoto RI, Dillin A, Kelly JW. Adapt- to events in HD patients. It will be impor- the HSR in mice that model HD. In any case, ing proteostasis for disease intervention. Science. tant, although challenging, to corroborate it is probable that an HSR in which Hsp90 is 2008;319(5865):916–919. these findings in HD patient tissue or cell not inhibited will be a more effective means 2. Powers ET, Morimoto RI, Dillin A, Kelly JW, Balch lines. Of note, the authors did not gener- to restore proteostasis. Thus, small mole- WE. Biological and chemical approaches to dis- eases of proteostasis deficiency. Annu Rev Biochem. ate survival curves (10), so the therapeutic cules that activate HSF1 without inhibiting 2009;78:959–991. value of HSP990 treatment with regard to Hsp90 could be a critical innovation. One 3. Cushman M, Johnson BS, King OD, Gitler AD,

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Shorter J. Prion-like disorders: blurring the divide 1999;23(4):425–428. cell culture model of Huntington’s disease. Hum between transmissibility and infectivity. J Cell Sci. 10. Labbadia J, et al. Altered chromatin architecture Mol Gen. 2001;10(12):1307–1315. 2010;123(pt 8):1191–1201. underlies progressive impairment of the heat shock 17. Duennwald ML, Lindquist S. Impaired ERAD and 4. Crook ZR, Housman D. Huntington’s disease: response in mouse models of Huntington disease. ER stress are early and specific events in polygluta- can mice lead the way to treatment? Neuron. J Clin Invest. 2011;121(8):3306–3319. mine toxicity. Genes Dev. 2008;22(23):3308–3319. 2011;69(3):423–435. 11. Hay DG, et al. Progressive decrease in chaperone 18. Winklhofer KF, Reintjes A, Hoener MC, Voellmy 5. Scherzinger E, et al. Huntingtin-encoded poly- protein levels in a mouse model of Hunting- R, Tatzelt J. Geldanamycin restores a defective glutamine expansions form amyloid-like pro- ton’s disease and induction of stress proteins heat shock response in vivo. J Biol Chem. 2001; tein aggregates in vitro and in vivo. Cell. 1997; as a therapeutic approach. Hum Mol Gen. 2004; 276(48):45160–45167. 90(3):549–558. 13(13):1389–1405. 19. Shorter J. Emergence and natural selection of drug- 6. Nekooki-Machida Y, Kurosawa M, Nukina N, Ito 12. Westerheide SD, Morimoto RI. Heat shock resistant prions. Mol Biosyst. 2010;6(7):1115–1130. K, Oda T, Tanaka M. Distinct conformations of response modulators as therapeutic tools for 20. Jarosz DF, Taipale M, Lindquist S. Protein homeo- in vitro and in vivo amyloids of huntingtin-exon1 diseases of protein conformation. J Biol Chem. stasis and the phenotypic manifestation of genetic show different cytotoxicity. Proc Natl Acad Sci U S A. 2005;280(39):33097–33100. diversity: principles and mechanisms. Annu Rev 2009;106(24):9679–9684. 13. Fujimoto M, et al. Active HSF1 significantly sup- Genet. 2010;44:189–216. 7. Lo Bianco C, et al. Hsp104 antagonizes alpha- presses polyglutamine aggregate formation in 21. Sangster TA, Queitsch C, Lindquist S. Hsp90 synuclein aggregation and reduces dopaminergic cellular and mouse models. J Biol Chem. 2005; and chromatin: where is the link? Cell Cycle. 2003; degeneration in a rat model of Parkinson disease. 280(41):34908–34916. 2(3):166–168. J Clin Invest. 2008;118(9):3087–3097. 14. Steele AD, et al. Heat shock factor 1 regulates lifes- 22. Sollars V, Lu X, Xiao L, Wang X, Garfinkel MD, 8. Mu TW, et al. Chemical and biological approaches pan as distinct from disease onset in prion disease. Ruden DM. Evidence for an epigenetic mechanism synergize to ameliorate protein-folding diseases. Proc Natl Acad Sci U S A. 2008;105(36):13626–13631. by which Hsp90 acts as a capacitor for morphologi- Cell. 2008;134(5):769–781. 15. Auluck PK, Bonini NM. Pharmacological preven- cal evolution. Nat Genet. 2003;33(1):70–74. 9. Warrick JM, Chan HYE, Gray-Board GL, Chai Y, tion of Parkinson disease in Drosophila. Nat Med. 23. Neef DW, Turski ML, Thiele DJ. Modulation of Paulson HL, Bonini NM. Suppression of polyglu- 2002;8(11):1185–1186. heat shock transcription factor 1 as a therapeutic tamine-mediated neurodegeneration in Drosoph- 16. Sittler A, et al. Geldanamycin activates a heat shock target for small molecule intervention in neurode- ila by the molecular chaperone HSP70. Nat Genet. response and inhibits huntingtin aggregation in a generative disease. PLoS Biol. 2010;8(1):e1000291.

Oxidized CaMKII: a “heart stopper” for the sinus node? Sabine Huke and Björn C. Knollmann

Division of Clinical Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.

Each normal heart beat is triggered by an electrical impulse emitted from a pathology of an ischemic, inflammatory, or group of specialized cardiomyocytes that together form the sinoatrial node degenerative nature. SND also frequently (SAN). In this issue of the JCI, Swaminathan and colleagues demonstrate a occurs when an individual develops heart new molecular mechanism that can disrupt the normal beating of the heart: failure. Histological studies demonstrate angiotensin II — typically found in increased levels in heart failure and a loss of SAN cells and increased fibrosis hypertension — oxidizes and activates Ca2+/calmodulin-dependent kinase in SAN tissue obtained postmortem (2, II via NADPH oxidase activation, causing SAN cell death. The loss of SAN 3), suggesting that cell death and tissue cells produces an electrical imbalance termed the “source-sink mismatch,” remodeling importantly contribute to which may contribute to the SAN dysfunction that affects millions of people SND. In this issue of the JCI, Swaminathan later in life and complicates a number of heart diseases. and coworkers elegantly combine studies in mice and human tissue to demonstrate The electrical impulses that trigger the put is reduced during times of rest and a molecular chain reaction that can cause heart to beat originate from a group of increased during physical and emotional SND (4). The study demonstrates a here- specialized cardiomyocytes that together exercise. Not surprisingly, SAN dysfunc- tofore unrecognized molecular mechanism form the sinoatrial node (SAN). The SAN tion (SND) affects millions of individuals responsible for SND and provides a clear is a complex anatomical structure located later in life; it also complicates a number target for developing new treatments aimed in the wall of the right atrium, near the of heart diseases. While a large body of at preventing SND in the future. entrance to the superior vena cava. The work has elucidated the molecular signal- SAN triggers billions of heart beats during ing processes that regulate physiological Ang II–induced Ca2+/calmodulin- an individual’s lifetime. Neurohormonal pacemaking, much less is known about the dependent kinase II oxidation regulation of the SAN allows us to adapt molecular signaling that causes SND (1). causes SAN cell death and SND our cardiac output to precisely match life’s SND is characterized by physiologically Anderson and colleagues have shown pre- rapidly changing demands: cardiac out- inappropriate heart rates, most often sinus viously that Ang II induces myocardial bradycardia (a regular but abnormally slow dysfunction and heart failure at least in heart rate), and the only currently available part via myocyte apoptosis (5). They recog- Conflict of interest: The authors have declared that no 2+ conflict of interest exists. treatment option is implantation of an nized at that time that inhibition of Ca / Citation for this article: J Clin Invest. 2011; electrical pacemaker. The typical patient is calmodulin-dependent kinase II (CaMKII) 121(8):2975–2977. doi:10.1172/JCI58389. elderly and presents with additional cardiac provided protection against myocyte cell

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