Dangerous liaisons: polyglutamine meets HMGB

NATURE CELL BIOLOGY VOLUME 9 | NUMBER 4 | APRIL 2007 359

N E W S A N D V I E W S

antigen-presentation capacity of dendritic cells. However, NOX2 is also expressed by other phagocytes. Neutrophils and activated mac-rophages both manufacture superoxide, and rely, in part, on this activity to kill invading microbes. Although most studies invoke the direct action of the reactive oxygen species on the invading microbe13, it has also been argued that NOX2 in neutrophils mediates release of elastase and cathepsin G from the neutrophil granules14, leading to an enhancement of pro-teolysis in the early phagosome. Moreover, it is accepted that activated macrophages generate superoxide, but their phagosomes show only minor differences in the kinetics of acidifica-tion15. Activated macrophages show a similar downregulation of their hydrolytic activity, which may maximize their antigen-presenting potential; however, this is achieved by differ-ential delivery of lysosomal hydrolases rather than regulating pH15. These data indicate that the effects induced by NOX2 activity vary between different phagocyte types and that the reduced phagosomal proteolysis may be achieved through different mechanisms.

Mutations in Rab27a lead to reduced anti-gen cross-presentation and are responsible for Griscelli syndrome in humans. Children with defective Rab27a genes develop unregulated T-lymphocyte and macrophage activation

known as haemaphagocytic lymphohistocyto-sis, which, without a bone-marrow transplant, will result in death8. The data in the current study provides new insight into the immuno-logical consequences of this syndrome. The authors hypothesize that this elevated autoim-mune response is a consequence of ineffec-tive thymic selection due to reduced antigen cross-presentation during development of the T cell repertoire, which enables preservation of harmful, auto-reactive lymphocytes5.

What are the practical implications of the authors observations? Clearly an improved understanding of immunogenicity at the cel-lular level has implications for vaccine design. The majority of published studies correlate reduced proteolysis to improved antigen pre-sention through MHC class I and class II mol-ecules to T cells in culture2–4. However, recently Delmarre et al. tested this hypothesis in vivo in mouse1. Following immunization of mice with two forms of a single antigen that differed only in their sensitivity to lysosomal proteolysis, they observed that the mice that received the resistant form of the antigen demonstrated a much more robust cellular and humoral immune response. Comparable data were also obtained with sta-ble and unstable forms of a second, unrelated antigen. In both cases, the immune responses recognized both forms of the antigen indicating

that the difference was, at least predominantly, one of degree and not specificity.

It is therefore clear that an appreciation of the proteolytic processing of antigens, and how the process is regulated by antigen-presenting cells such as dendritic cells, is crucial to enhancing the immunogenicity of modern vaccines.

COMPETING FINANCIAL INTERESTSThe authors declare that they have no competing financial interests.

1. Delamarre, L., Couture, R., Mellman, I. & Trombetta, E. S. J. Exp. Med. 203, 2049–2055 (2006).

2. Delamarre, L., Pack, M., Chang, H., Mellman, I. & Trombetta, E. S. Science 307, 1630–1634 (2005).

3. Lennon-Dumenil, A. M. et al. J. Exp. Med. 196, 529–540 (2002).

4. Moss, C. X., Villadangos, J. A. & Watts, C. Eur. J. Immunol. 35, 3442–3451 (2005).

5. Jancic, C. et al. Nature Cell Biol. 9, 367–378 (2007).

6. Yates, R. M., Hermetter, A. & Russell, D. G. Traffic 6, 413–420 (2005).

7. Savina, A. et al. Cell 126, 205–218 (2006).8. Menasche, G. et al. Nature Genet. 25, 173–176

(2000).9. Wilson, S. M. et al. Proc. Natl Acad. Sci. USA 97,

7933–7938 (2000).10. Ackerman, A. L., Kyritsis, C., Tampe, R. & Cresswell,

P. Proc. Natl Acad. Sci. USA 100, 12889–12894 (2003).

11. Guermonprez, P. & Amigorena, S. Springer Semin. Immunopathol. 26, 257–271 (2005).

12. Houde, M. et al. Nature 425, 402–406 (2003).13. Bryk, R., Griffin, P. & Nathan, C. Nature 407, 211–215

(2000).14. Reeves, E. P., Nagl, M., Godovac-Zimmermann, J. &

Segal, A. W. J. Med. Microbiol. 52, 643–651 (2003).15. Yates, R. M., Hermetter, A., Taylor, G. A. & Russell,

D. G. Traffic 8, 241–250 (2007).

Dangerous liaisons: polyglutamine meets HMGB Sokol V. Todi and Henry L. Paulson

Nine inherited neurodegenerative disorders are caused by polyglutamine (polyQ) expansions in diverse proteins. A study now suggests that polyQ-mediated depletion of nonhistone chromatin proteins enhances genotoxic stress induced by the disease protein.

Although much less common than Alzheimer’s and Parkinson’s diseases, the polyQ neurode-generative disorders have captured the attention of cell biologists. Expansion of polyQ repeats is a shared feature of all polyQ diseases and it is likely to offer important clues to the mecha-nisms by which misfolded proteins disrupt normal cellular pathways in age-related brain disorders. Precisely how expanded polyQ pro-

teins cause neurodegeneration remains a mys-tery. On page 402 of this issue, Qi et al. suggest new clues to the pathogenesis of polyQ expan-sion-based diseases. They find that expression of mutant polyQ protein leads to decreased lev-els of chromatin proteins that are implicated in diverse nuclear functions, including the cellular response to genotoxic stress1.

Inherited polyQ diseases are caused by CAG-repeat expansions that encode abnormally long polyQ tracts in otherwise dissimilar proteins2. Of the nine identified polyQ diseases, the best characterized are Spinocerebellar Ataxia Type 1

(SCA1) and Huntington’s disease, both of which are studied in the current report1. PolyQ expan-sion perturbs the native folding of disease pro-teins, increasing the probability that they will form abnormal oligomers, accumulate in the cell and associate aberrantly with other proteins, including their normal interacting partners and innocent bystanders. Intranuclear inclusions containing accumulated polyQ protein are a common pathological hallmark of neurons in diseased brain, although these inclusions are not thought to be directly pathogenic. Whatever the role of nuclear inclusions may be, the nucleus

Sokol V. Todi and Henry L. Paulson are in the Department of Neurology, University of Iowa College of Medicine, Iowa City, IA, USA.e-mail: [email protected]

N&V April.indd 359N&V April.indd 359 15/3/07 13:36:2715/3/07 13:36:27

360 NATURE CELL BIOLOGY VOLUME 9 | NUMBER 4 | APRIL 2007


is clearly a major site of polyQ toxicity. Nuclear localization of the disease protein is required for neurotoxicity in many of these disorders3. Most known polyQ disease proteins are either tran-scription factors (androgen receptor in spinob-ulbar muscular atrophy (SBMA) and TATA-box binding protein in SCA17), nuclear co-repres-sors or co-activators (ataxin-1 in SCA1, ataxin-7 in SCA7 and atrophin in dentatorubropallidoly-sian atrophy, DRPLA) or regulators of gene expression through other, indirect mechanisms (ataxin-3 in SCA3). In various model systems and disease tissue, transcription is perturbed resulting in greater gene repression, and a possi-ble route to therapy currently being tested is the use of histone deacetylase inhibitors to boost transcription4. A prevailing model of patho-genesis — but by no means the only one — is that expanded polyQ proteins bind aberrantly to components of transcriptional complexes, thereby disrupting the exquisite regulation of neuronal gene expression.

The current study provides further insight into the links between nuclear events and polyQ-mediated neurodegeneration1. To uncover polyQ-induced changes in nuclear

proteins, Qi et al. used a proteomics approach. They sought to identify soluble nuclear proteins whose levels are altered in neurons expressing mutant ataxin-1 or huntingtin (Htt), the SCA1 and Huntington’s disease proteins. Among the few altered proteins detected in this assay, HMGB1 and HMGB2 (High Mobility Group B proteins) stood out as being consistently reduced. HMGB1/2 are evolutionarily con-served, non-histone chromatin proteins that bind DNA and various transcription factors, including TATA-binding protein and p53. Through their dynamic interactions with DNA and nucleoprotein complexes, HMGB proteins participate in DNA recombination, replication and repair, regulation of gene expression and suppression of genotoxic stress signalling5–7. Highly mobile and abundant in the nucleus, HMGB proteins have even been described as chromatin chaperones that help to maintain chromatin in a fluidic state5.

Building on their initial results, the authors wanted to determine why soluble HMGB1/2 levels are reduced when mutant polyQ proteins are expressed1. They found that HMGB1/2 interacted with expanded, but not normal

Htt and ataxin-1, and that HMGB1/2 became sequestered in neuronal nuclear inclusions in mouse models of Huntington’s disease and SCA1. Importantly, however, the reduction in HMGB levels was not limited to cells contain-ing inclusions and occurred before mice devel-oped major disease manifestations, suggesting that changes in HMGB levels may represent a proximal event in disease pathogenesis rather than a secondary phenotype.

In neurons expressing mutant Htt or ataxin-1, expression of HMGB proteins did in fact suppress toxicity, while also correcting polyQ-induced transcriptional repression and loss of neuronal arborization. Genetic interaction studies in Drosophila further supported a role for HGMB proteins in counteracting polyQ toxicity as polyQ-mediated degeneration of the fly eye was rescued by coexpression of HMGB1 or HMGB2. These data suggest that the observed depletion of HMGB proteins may actually be important in the disease.

How might HMGB proteins suppress polyQ toxicity? Qi et al. asked whether they did so through their role in DNA stress signalling. This line of reasoning was suggested by the fact that two effectors of genotoxic stress, p73 (ref. 8) and p53 (ref. 9), are known to be acti-vated in neurons expressing expanded Htt. They found that expression of expanded Htt also leads to the phosphorylation of Chk1, a cell-cycle checkpoint kinase that signals DNA damage, and of H2AX, a histone that marks sites of DNA structural damage10. Importantly, both phosphorylation events were prevented by coexpression of HMGB1/2. In addition, siRNA-mediated knockdown of HMGB1 pro-moted H2AX phosphorylation and neuro-nal cell death, supporting the argument that HMGB proteins are important for normal neuronal physiology and that their reduction may enhance polyQ toxicity by a failure to sup-press stress-signalling pathways.

Taken together, these data suggest a disease model in which reduction in soluble HMGB proteins contributes to the neuronal toxicity of expanded polyQ proteins. In disease neurons, for example, HMGB1/2 levels might fall to a point where they can no longer counteract genotoxic stress induced by mutant polyQ protein, such as by suppressing Chk1 and H2AX phosphoryla-tion and downstream activation of p53 (Fig. 1). It is also possible that the reduction in HMGB proteins directly mediates genotoxic stress — in other words, represents an even more proximal, pivotal molecular event in the pathogenic cascade.

Expanded polyQ HMGB

Activation

Nuclearevents

Oth

er n

euro

toxi

c ev

ents

(nuc

lear

or

cyto

pla

smic

)

Activation, increaseDeactivation, decrease

DNAStress

CHK1 / H2AX pCHK1 / pH2AXGene

transcription

Activatedp53/p73

Celldeath

Figure 1 Schematic representation of a model for inhibiting polyQ-associated cell toxicity by HMGB proteins. HMGB proteins are DNA-binding proteins that participate in DNA recombination, replication and repair, regulation of gene expression and suppression of genotoxic stress signalling. PolyQ-expanded huntingtin and ataxin-1 lead to lower, soluble HMGB protein levels in the nucleus. Decreased HMGB levels may have detrimental effects on neurons due to alterations in gene transcription and/or failure to suppress genotoxic stress resulting from expanded polyQ proteins. This model acknowledges that expanded polyQ proteins may also cause neuronal dysfunction and cell death through other nuclear and extranuclear pathways.


NATURE CELL BIOLOGY VOLUME 9 | NUMBER 4 | APRIL 2007 361


Furthermore, depletion of HMGB proteins is pre-dicted to impair transcription independent of genotoxic stress, as these chromatin ‘chaperones’ normally enhance transcriptional activation. This prediction is consistent with the transcrip-tional dysregulation known to occur in this class of diseases3. However, the reduction in HMGB proteins could simply be one of many changes taking place in the nucleus during the course of disease, contributing only incrementally to a complex pathogenic cascade that still remains to be determined.

To their credit, the authors of this study began with a systematic, proteomics-based approach to an important biological problem. They assumed changes in soluble nuclear proteins would occur in disease, yet made no a priori assumptions about what they might be. No previous study had suggested the involvement of HMGB pro-teins in polyQ disease. However, HMGB pro-teins were detected in this proteomics screen, in part because they are abundant nuclear proteins. Despite its advantages, this screening method is likely to underestimate changes that may also occur in less abundant nuclear proteins that could potentially have major effects on the regulation of gene expression.

One question left unanswered is why the levels of HMGB proteins seem to be reduced in disease. The interaction of mutant polyQ pro-tein with HMGB proteins may promote their degradation by the proteasome or other clear-ance pathways, as suggested by the authors. Sequestration of these proteins into inclusions, as observed by the authors, is unlikely be the

whole answer, especially if inclusions have a protective role — as has been proposed.

Clearly more work needs to be done to strengthen the case that HMGB proteins are major players in disease pathogenesis. Analysis of human disease tissue to assess levels of HMGB proteins and the presence or absence of biomarkers of genotoxic stress should be a priority. Although plasma levels of 8-hydroxy-deoxyguanosine, a marker of oxidative DNA damage, are increased in the serum of Huntington’s disease patients11, little evidence to date shows that genotoxic stress is a proximal event in any polyQ disease. Although the overexpression studies show that HGMB proteins can counteract polyQ toxicity in cell models and Drosophila, it is unclear whether this represents a specific effect of these pro-teins on genotoxic stress, or a more nonspecific boosting of transcriptional activation, similar to the beneficial effect of histone deacetylase inhibitors4. Thus, it will be important to test the HMGB reduction hypothesis directly in mouse models of disease. Hmgb1-null mice unfortu-nately die at birth12, but mice in which Hmgb genes have been conditionally knocked out in brain or knocked down using RNAi technology should answer whether Hmgb protein reduc-tion promotes neurodegeneration in polyQ disease mouse models. Although a strength of the current study is that it tests two different polyQ proteins, it will be important to deter-mine whether these findings are applicable to the other seven polyQ diseases. Finally, any-time a gene is found to suppress polyQ toxic-

ity, it is incumbent on those of us in the field to think of ways this may lead to a therapy for these fatal disorders. One approach we can probably exclude is exogenous administra-tion of HMGB1 because this multi-functional protein also functions outside the nucleus as a cytokine in inflammatory events6.

Although this study does not address another major class of hereditary ataxias, the recessive ataxias, they deserve comment: because reces-sive ataxias so often involve defects in DNA repair and presumably genotoxic stress13, per-haps we now need to think about unanticipated connections between these disorders and the polyglutamine diseases.

COMPETING FINANCIAL INTERESTSThe authors declare that they have no competing financial interests.

1. Qi, M. et al. Nature Cell Biol 9, 402–414 (2007).2. Zoghbi, H. Y. & Orr, H. T. Annu. Rev. Neurosci. 23,

217–247 (2000).3. Riley, B. E. & Orr, H. T. Genes Dev. 20, 2183–2192

(2006).4. Butler, R. & Bates, G. P. Nature Rev. Neurosci. 7,

784–796 (2006).5. Agresti, A. & Bianchi, M. E. Curr. Opin. Genet. Dev. 13,

170–178 (2003).6. Lotze, M. T. & Tracey, K. J. Nature Rev. Immunol. 5,

331–342 (2005).7. Ulloa, L. & Messmer, D. Cytokine Growth Factor Rev.

17, 189–201 (2006).8. Hoshino, M. et al. J. Cell Biol. 172, 589–604

(2006).9. Bae, B. I. et al. Neuron 47, 29–41 (2005).10. Motoyama, N. & Naka, K. Curr. Opin. Genet. Dev. 14,

11–16 (2004).11. Browne, S. E., Ferrante, R. J. & Beal, M. F. Brain

Pathol. 9, 147–163 (1999).12. Calogero, S. et al. Nature Genet. 22, 276–280

(1999).13. Paulson, H. L. & Miller, V. M. Neuron 46, 845–848

(2005).

NF-κB links oestrogen receptor signalling and EMTDerek C. Radisky and Mina J. Bissell

The detrimental effects of nuclear factor-kappa B (NF-κB) signalling in cancer cells lacking the oestrogen receptor have now become clear, with the demonstration that increased NF-κB levels induce expression of Bcl-2 to both suppress apoptosis and induce epithelial–mesenchymal transitions (EMTs).

Originally identified for their role in immune cells, NF-κB transcription factors have since been identified in nearly every cell type and

have been found to mediate an incredibly diverse range of processes1. NF-κB factors are homo- and heterodimeric proteins composed of five homologous subunits: NF-κB1, NF-κB2, RelA/p65, RelB and c-Rel (Fig. 1). When local-ized to the nucleus, all the NF-κB complexes bind related DNA sequences to regulate gene expression, although each different complex

has distinct cell type-specific functions. NF-κB complexes are normally retained in the cyto-plasm in an inactive state through the action of IκBs, NF-κB inhibitors. The complex becomes active when IκB dissociates, allowing nuclear translocation of NF-κB. NF-κB is generally activated by either canonical or non-canoni-cal pathways: in the canonical pathway, the

Derek C. Radisky is at the Mayo Clinic Cancer Center, Jacksonville, FL 32224, USA. Mina J. Bissell is at the Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.e-mail: [email protected]


Documents

Dangerous liaisons: polyglutamine meets HMGB