Intranuclear Inclusions and the Ubiquitin–Proteasome Pathway: Digestion of a Red Herring?

Neuronal intranuclear inclusions (NIs) are ubiquitin-positive protein aggregates that have recently been described as a common ultrastructural feature of several neurodegenerative polyglutamine repeat expansion disorders, including Huntington's disease and the spinocerebellar ataxias (SCAs). Some early reports speculated that formation of such inclusions might be the key toxic event in this group of disorders. However, the pathological role of NIs has been controversial. Are these inclusions a proximate cause of cellular pathology, a protective reaction to it, or entirely beside the point? New work, including a paper by Cummings et al. 1999xCummings, C.J., Reinstein, E., Sun, Y., Antalffy, B., Jiang, Y.-H., Ciechanover, A., Orr, H.T., Beaudet, A.L., and Zoghbi, H.Y. Neuron. 1999; 24: 879–892Abstract | Full Text | Full Text PDF | PubMedSee all ReferencesCummings et al. 1999 in this issue of Neuron, shows that large inclusions are at least not required for expanded polyglutamine pathology and raise new questions about pathological mechanisms in this group of disorders.Recent evidence suggests that large inclusions are not required for toxicity in polyglutamine repeat expansions. First, cells from SCA2 patients reportedly lack such inclusions (Huynh et al. 1999xHuynh, D.P., Del Bigio, M.R., Ho, D.H., and Pulst, S.M. Ann. Neurol. 1999; 45: 232–241Crossref | PubMed | Scopus (114)See all ReferencesHuynh et al. 1999). Second, preventing inclusions through protein engineering does not block polyglutamine-dependent pathology induced by expanded ataxin-1 in mice (Klement et al. 1998xKlement, I.A., Skinner, P.A., Kaytor, M.D., Yi, H., Hersch, S.M., Clark, H.B., Zoghbi, H.Y., and Orr, H.T. Cell. 1998; 95: 41–53Abstract | Full Text | Full Text PDF | PubMed | Scopus (771)See all ReferencesKlement et al. 1998) or expanded huntingtin in transfected neuronal cultures (Saudou et al. 1998xSaudou, F., Finkbeiner, S., Devys, D., and Greenberg, M.E. Cell. 1998; 95: 55–66Abstract | Full Text | Full Text PDF | PubMed | Scopus (1162)See all ReferencesSaudou et al. 1998). Both normal huntingtin and ataxin-1 are degraded by a ubiquitin–proteasome pathway, in which addition of ubiquitin by specific ligases marks the protein for degradation by proteasome. NIs from both patient materials and animal models show high ubiquitin content and colocalization of proteasomes. Cummings et al. 1999xCummings, C.J., Reinstein, E., Sun, Y., Antalffy, B., Jiang, Y.-H., Ciechanover, A., Orr, H.T., Beaudet, A.L., and Zoghbi, H.Y. Neuron. 1999; 24: 879–892Abstract | Full Text | Full Text PDF | PubMedSee all ReferencesCummings et al. 1999 show that although wild-type and expanded ataxin-1 are similarly ubiquitinated, the expanded protein is selectively resistant to degradation and forms ubiquitin-positive, detergent-insoluble aggregates. Inhibition of proteasome activity with β-lactone increases the frequency of ubiquitin-positive NIs in HeLa cells transfected with expanded ataxin-1, suggesting that proteasomes either degrade the expanded ataxin-1 monomers before they form large aggregates or degrade the inclusions themselves. Strikingly, genetic loss of an E3 ubiquitin ligase (E6-AP) blocks formation of NIs but accelerates the pathology associated with expanded ataxin-1.The genetic interaction between expanded ataxin-1 and E6-AP raises interesting questions and places new constraints on models for the mechanism of polyglutamine-induced neurodegeneration. E6-AP is encoded by an imprinted gene, Ube3a, implicated in the mental retardation and other neurological features of Angelman syndrome. Loss of the maternal copy of Ube3a in mice results in developmental and cognitive defects reminiscent of some features of Angelman syndrome and altered turnover of p53 protein, but no overt cellular pathology (Jiang et al. 1998xJiang, Y.H., Armstrong, D., Albrecht, U., Atkins, C.M., Noebels, J.L., Eichele, G., Sweatt, J.D., and Beaudet, A.L. Neuron. 1998; 21: 799–811Abstract | Full Text | Full Text PDF | PubMed | Scopus (438)See all ReferencesJiang et al. 1998). In Cummings et al. 1999xCummings, C.J., Reinstein, E., Sun, Y., Antalffy, B., Jiang, Y.-H., Ciechanover, A., Orr, H.T., Beaudet, A.L., and Zoghbi, H.Y. Neuron. 1999; 24: 879–892Abstract | Full Text | Full Text PDF | PubMedSee all ReferencesCummings et al. 1999, transgenic mice expressing an expanded allele of ataxin-1 (line B05) that lack maternal Ube3a expression (Ube3a(m−/p+)) show much more rapid onset of SCA1-like pathology than transgenic mice that express Ube3a. However, it is unclear whether ataxin-1 is a direct target of E6-AP. Indeed, ataxin-1 is polyubiquitinated in Ube3a(m−/p+) mice. These results could be interpreted several ways. The least interesting interpretation is that E6-AP deficiency may simply lower the threshold for cellular pathology initiated by the expanded ataxin-1 in a relatively nonspecific manner: sick plus sick equals sicker. Alternatively, E6-AP may directly ubiquitinate ataxin-1 or an essential cofactor for initiating pathology. Ataxin-1 or its cofactor may be ubiquitinated by alternative ligases in the absence of E6-AP, but the alternative use of isozymes may alter the kinetics of ubiquitination or its coupling (by spatial or association constraints) to proteasomal degradation.Most of the recent data, including those in Cummings et al. 1999xCummings, C.J., Reinstein, E., Sun, Y., Antalffy, B., Jiang, Y.-H., Ciechanover, A., Orr, H.T., Beaudet, A.L., and Zoghbi, H.Y. Neuron. 1999; 24: 879–892Abstract | Full Text | Full Text PDF | PubMedSee all ReferencesCummings et al. 1999, argue that formation of large intranuclear inclusions is not the main cellular insult in expanded polyglutamine repeat diseases. Rather, the expanded polyglutamine tracts themselves are toxic and their incorporation into inclusions may, if anything, reduce the cellular impact by sequestering them away from other cellular components. However, a role for smaller, submicroscopic aggregates remains possible. This leaves open the question of how polyglutamine repeat expansions cause disease. Two general models now seem the most plausible (however, this riddle may have more than one right answer!). A synthetic framework encompassing both models is shown in the figurefigure.In Model 1, expanded polyglutamine-containing protein (working alone or in aggregates) associates with the proteasome but resists degradation; this may tie up proteasome activity nonproductively, decreasing its availability to degrade other key targets and thus disrupting the normal catabolism of one or more important regulatory proteins. For example, the best-characterized target of E6-AP is p53. p53 is proapoptotic, and cell death in several polyglutamine expansion models is caspase dependent (7xOna, V.O., Li, M., Vonsattel, J.P.G., Andrews, L.J., Khan, S.Q., Chung, W.M., Frey, A.S., Menon, A.S., Li, X., Stieg, P.E. et al. Nature. 1999; 399: 263–267Crossref | PubMed | Scopus (496)See all References, 9xSanchez, I., Xu, C., Juo, P., Akira, A., Bienis, J., and Yuan, J. Neuron. 1999; 22: 623–633Abstract | Full Text | Full Text PDF | PubMedSee all References).In Model 2, expanded polyglutamine proteins bind other proteins necessary for cell function into a nonproductive complex, disrupting the balance of key cell regulators. Polyglutamine has been described as a transcriptional modulator (Gerber et al. 1994xGerber, H.P., Seipel, K., Georgiev, O., Hofferer, M., Hug, M., Rusconi, S., and Schaffner, W. Science. 1994; 263: 808–811Crossref | PubMedSee all ReferencesGerber et al. 1994), and several nuclear proteins with regulatory functions (CBP, TATA-BP, and EYA) have been observed in coaggregates with expanded polyglutamines in model systems (8xPerez, M.K., Paulson, H.L., Pendse, S.J., Saionz, S.J., Bonini, N.M., and Pittman, R.N. J. Cell Biol. 1998; 143: 1457–1470Crossref | PubMed | Scopus (245)See all References, 5xKazantsev, A., Preisinger, E., Dranovsky, A., Goldgaber, D., and Housman, D. Proc. Natl. Acad. Sci. USA. 1999; 96: 11404–11409Crossref | PubMed | Scopus (342)See all References). Ubiquitin-mediated degradation (or self-aggregation) of expanded ataxin-1 could limit its ability to bind such proteins.A feature of both models is that neurotoxicity should roughly correspond to the available surface area of the misfolded and/or aggregated protein. This agrees well with the observation that disruption of NIs can increase toxicity. Thus, any treatment that partially disrupts the formation or maintenance of inclusions without reducing the mass of constituent protein would be expected to exacerbate the disease. The most promising therapeutic targets might therefore be misfolded protein or components of regulatory pathways disrupted by expanded polyglutamine repeats rather than the protein aggregates themselves. However, it remains to be seen whether such regulatory pathways can be identified and whether this kettle of herring will at last satisfy the palate.Figure 101Hypothetical Framework for Ataxin-1 Polyglutamine Toxicity, Degradation, and Aggregate FormationPotential toxic effects of expanded polyglutamine alleles include coopting regulatory factors into insoluble aggregates (which could be smaller and more diffuse than classical NIs under some conditions) and dysregulation of regulatory factors—such as p53—by occupying proteasomes with inefficient degradation. Inhibitory interactions are shown in red. Inhibition of NI formation in Ube3a maternal deficiency shown as dashed line to suggest an indirect effect, while potential for a direct effect is shown as E6-AP mediation of aggregate formation.View Large Image | View Hi-Res Image | Download PowerPoint Slide