Intranuclear Neuronal Inclusions: A Common Pathogenic Mechanism for Glutamine-Repeat Neurodegenerative Diseases?
Authors: Christopher A. Ross
Journal: Neuron (1997)

Eight progressive neurodegenerative disorders, including Huntington's disease (HD) and several forms of spinocerebellar ataxia (SCA; see Table 1), are currently known to be caused by expanding CAG-triplet repeats coding for polyglutamine (13Ross C.A. Neuron. 1995; 15: 493-496Abstract Full Text PDF PubMed Scopus (221) Google Scholar, 1Andrew S.E. Goldberg Y.P. Hayden M.R. Hum. Mol. Genet. 1997; 6: 2005-2010Crossref PubMed Scopus (117) Google Scholar, 7Gusella J.F. Persichetti F. MacDonald M.E. Mol. Med. 1997; 3: 238-246PubMed Google Scholar). The genes causing these degenerative disorders show no homology to each other outside of the glutamine repeat, and most are genes of unknown function. In each of these diseases, an overlapping but distinct set of cortical and subcortical regions is affected. However, although the genes for the disorders are expressed widely throughout the brain, and also in peripheral tissues, only certain populations of neurons degenerate. The mechanism for this selective neuronal death has remained puzzling.Table 1Polyglutamine Disease Genes and ProteinsDiseaseGeneNormal Repeats*Data on repeat lengths in part from Andrew et al. 1997.Expanded Repeats*Data on repeat lengths in part from Andrew et al. 1997.ProteinMolecular Weight (kDa)Nuclear TargettingHDHuntingtin (IT15)6-3536-121Huntingtin348−DRPLAAtrophin-13-3649-88Atrophin-1124−SCA-1Ataxin-16-3940-83Ataxin-187+SCA-2Ataxin-214-3233-77Ataxin-290−SCA-3Ataxin-312-4055-86Ataxin-342−SCA-7Ataxin-77-1738-130Ataxin-795+SBMAAndrogen receptor (AR)9-3638-62AR99+SCA-6**The lengths of the expanded repeats are considerably shorter for SCA-6 than for the other genes, and the pathogenic mechanism may be different.CACNL1A44-1821-30**The lengths of the expanded repeats are considerably shorter for SCA-6 than for the other genes, and the pathogenic mechanism may be different.P/Q Ca2+ channel(large)−* Data on repeat lengths in part from 1Andrew S.E. Goldberg Y.P. Hayden M.R. Hum. Mol. Genet. 1997; 6: 2005-2010Crossref PubMed Scopus (117) Google Scholar.** The lengths of the expanded repeats are considerably shorter for SCA-6 than for the other genes, and the pathogenic mechanism may be different. Open table in a new tab Genetic evidence, including the pattern of inheritance of the disorders in patients and evidence from animal models, has suggested that these disorders are caused by a genetic "gain-of-function" mechanism. This concept is supported by the observation that the lengths of the disease-causing repeats are similar for all of the genes and are longer than the corresponding normal alleles (1Andrew S.E. Goldberg Y.P. Hayden M.R. Hum. Mol. Genet. 1997; 6: 2005-2010Crossref PubMed Scopus (117) Google Scholar; the one apparent exception SCA-6, may involve a different pathogenic mechanism). In all of the disorders, longer repeats yield earlier ages of onset and more severe pathology. But how could polyglutamine cause brain pathology? Perutz has suggested that long stretches of polyglutamine might be able to form hydrogen bonds to other long stretches of polyglutamine in an antiparallel fashion (12Perutz M.F. Johnson T. Suzuki M. Finch J.T. Proc. Natl. Acad. Sci. USA. 1994; 91: 5355-5358Crossref PubMed Scopus (922) Google Scholar). This so-called "polar zipper" could form as a hairpin within a single polyglutamine stretch or could form between two molecules containing polyglutamine. Other molecules could then be recruited to form a stable β-pleated sheet, which may aggregate and precipitate within cells. Striking in vitro evidence for the formation of aggregates containing polyglutamine has recently been generated using a truncated piece of the huntingtin protein encoded by exon 1 of the HD gene (14Scherzinger E. Lurz R. Turmaine M. Mangiarini L. Hollenbach B. Hasenbank R. Bates G.P. Davies S.W. Lehrach H. Wanker E.E. Cell. 1997; 90: 549-558Abstract Full Text Full Text PDF PubMed Scopus (1051) Google Scholar). Polarized light microscopy revealed structural information consistent with the possibility of β sheet formation. Genetic manipulations have demonstrated that polyglutamine can lead to intranuclear protein aggregation and disease-like symptoms. Transgenic animals overexpressing the short exon 1 N-terminal fragment of the huntingtin protein with a very long glutamine repeat developed a progressive neurological phenotype with weight loss, involuntary movements, seizures, and early death. The brains were smaller overall. Postmortem examination of these animals revealed intranuclear inclusions when labeled with antibodies to the N terminus of huntingtin or to ubiquitin (4Davies S.W. Turmaine M. Cozens B.A. DiFiglia M. Sharp A.H. Ross C.A. Scherzinger E. Wanker E.E. Mangiarini L. Bates G.P. Cell. 1997; 90: 537-548Abstract Full Text Full Text PDF PubMed Scopus (1828) Google Scholar). The inclusions were most dense in the cortex and striatum, the areas affected in HD. The inclusions contained fibrillar and amorphous deposits of huntingtin, consistent with protein aggregation. Similarly, mice transgenic for ataxin-1 (the protein product of the SCA-1 gene) with expanded repeats under the control of a Purkinje cell–specific promotor show small intranuclear deposits in Purkinje cells. In this model, the cells degenerate, as they do in patients with SCA-1 (15Skinner P.J. Koshy B.T. Cummings C.J. Klement I.A. Helin K. Servadio A. Zoghbi H.Y. Orr H.T. Nature. 1997; 389: 971-974Crossref PubMed Scopus (484) Google Scholar). Cell models also provide complementary results. Cells transiently transfected with either ataxin-1 or ataxin-3 show small intranuclear accumulations of protein (11Paulson H.L. Perez M.K. Trottier Y. Trojanowski J.Q. Subramony S.H. Das S.S. Vig P. Mandel J.-L. Fischbeck K.H. Pittman R.N. Neuron. 1997; 19: 333-344Abstract Full Text Full Text PDF PubMed Scopus (698) Google Scholar, 15Skinner P.J. Koshy B.T. Cummings C.J. Klement I.A. Helin K. Servadio A. Zoghbi H.Y. Orr H.T. Nature. 1997; 389: 971-974Crossref PubMed Scopus (484) Google Scholar). Although these intranuclear inclusions are not identical to those in the transgenic animals, these data indicate that the polyglutamine expansion can yield nuclear aggregation. Despite decades of study of pathological material from human diseases, intranuclear inclusions had never been noted as pathognomonic for these disorders. Thus, it is remarkable that this year intranuclear inclusions have been identified in all four of the disorders in which they have been sought (2Becher M.W. Kotzuk J.A. Sharp A.H. Davies S.W. Bates G.P. Price D.L. Ross C.A. Neurobiol. Dis. 1997; 4 (in press)Google Scholar, 5DiFiglia M. Sapp E. Chase K.O. Davies S.W. Bates G.P. Vonsattel J.P. Aronin N. Science. 1997; 277: 1990-1993Crossref PubMed Scopus (2175) Google Scholar, 11Paulson H.L. Perez M.K. Trottier Y. Trojanowski J.Q. Subramony S.H. Das S.S. Vig P. Mandel J.-L. Fischbeck K.H. Pittman R.N. Neuron. 1997; 19: 333-344Abstract Full Text Full Text PDF PubMed Scopus (698) Google Scholar, 15Skinner P.J. Koshy B.T. Cummings C.J. Klement I.A. Helin K. Servadio A. Zoghbi H.Y. Orr H.T. Nature. 1997; 389: 971-974Crossref PubMed Scopus (484) Google Scholar). In each case, the inclusions have a similar appearance—well-demarcated structures ∼2 μm across without a surrounding membrane and distinct from the nucleolus. Some neurons may have two or three inclusions, but most neurons have only one. Furthermore, in HD, in which a sufficient number of cases have been studied, there is a clear correlation between increasing CAG-repeat length and increasing density of the inclusions (2Becher M.W. Kotzuk J.A. Sharp A.H. Davies S.W. Bates G.P. Price D.L. Ross C.A. Neurobiol. Dis. 1997; 4 (in press)Google Scholar). In all of these disorders, the inclusions can be labeled with antibodies to the disease protein product itself or with antibodies to ubiquitin. All of these results are consistent with the idea that expanded stretches of polyglutamine can be toxic. However, they do not rule out the possibility that other components of the disease gene products might be necessary. The question remains: what would happen if polyglutamine were expressed ectopically in the context of a nonpathogenic protein? This experiment has now been performed by Detloff and colleagues (10Ordway J.M. Tallaksen-Greene S. Gutekunst C.-A. Bernstein E.M. Cearley J.A. Wiener H.W. Dure IV, L.S. Lindsey R. Hersch S.M. Jope R.S. Albin R.L. Detloff P.J. Cell. 1997; 91: 753-763Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar). A long CAG repeat was inserted into the mouse hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene, which codes a metabolic enzyme. Mice with the CAG-repeat insertion developed a progressive late-onset behavioral phenotype, which included ataxia, seizures, and early death. Neuropathological examination of the mice with the ectopic CAG repeats in HPRT revealed no clear neuronal cell death, but intranuclear inclusions were evident when labeled with antibodies to the HPRT protein or ubiquitin. Thus, CAG repeats translated into polyglutamine, even in an ectopic protein without neurological function, can produce a progressive neurological phenotype accompanied by the formation of intranuclear inclusions. This confirms that polyglutamine can directly yield aggregation and supports the idea that intranuclear neuronal inclusions are pathogenic. When taken together, these results raise a number of questions. HPRT is a relatively small protein of ∼48 kDa, including the polyglutamine. Therefore10Ordway J.M. Tallaksen-Greene S. Gutekunst C.-A. Bernstein E.M. Cearley J.A. Wiener H.W. Dure IV, L.S. Lindsey R. Hersch S.M. Jope R.S. Albin R.L. Detloff P.J. Cell. 1997; 91: 753-763Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar propose that it enters the nucleus by diffusion, consistent with the cutoff of ∼50 kDa for passive exchange of proteins between nucleus and cytoplasm. It is also possible that passive diffusion accounts for the entry of ataxin-3 into the nucleus, though with the expanded CAG repeat it runs on a denaturing gel as a protein of nearly 70 kDa (11Paulson H.L. Perez M.K. Trottier Y. Trojanowski J.Q. Subramony S.H. Das S.S. Vig P. Mandel J.-L. Fischbeck K.H. Pittman R.N. Neuron. 1997; 19: 333-344Abstract Full Text Full Text PDF PubMed Scopus (698) Google Scholar). Presumably larger proteins would have to be translocated actively into the nucleus. Ataxin-1 has a nuclear import signal and is normally enriched in the nucleus—especially in cerebellar Purkinje cells, the cells most affected in SCA-1. Ataxin-7 is present in nuclear fractions and also has a nuclear localization signal. Similarly, the androgen receptor of spinal and bulbar muscular atrophy (SBMA) undergoes nuclear translocation on binding of androgen. However, the nuclear localization of the inclusions in HD and dentato-rubral and pallido-luysian atrophy (DRPLA) are not accounted for, since both huntingtin and atrophin 1 are large proteins without any nuclear localization signals. How might they enter the nucleus? For huntingtin, there is evidence that proteolytic processing may be involved. Huntingtin can be cleaved by caspase 3, one of the proteases involved in triggering apoptosis (6Goldberg Y.P. Nicholson D.W. Rasper D.M. Kalchman M.A. Koide H.B. Graham R.K. Bromm M. Kazemi-Esfarjani P. Thornberry N.A. Vaillancourt J.P. Hayden M.R. Nat. Genet. 1996; 13: 442-449Crossref PubMed Scopus (491) Google Scholar). It is possible that the other glutamine-repeat proteins may also be cleaved by this or related enzymes. This would fit with immunohistochemical studies of the nuclear inclusions in HD, which can identify inclusions with antibodies to the N terminus of huntingtin but not with antibodies directed against internal sequences (2Becher M.W. Kotzuk J.A. Sharp A.H. Davies S.W. Bates G.P. Price D.L. Ross C.A. Neurobiol. Dis. 1997; 4 (in press)Google Scholar, 5DiFiglia M. Sapp E. Chase K.O. Davies S.W. Bates G.P. Vonsattel J.P. Aronin N. Science. 1997; 277: 1990-1993Crossref PubMed Scopus (2175) Google Scholar). Furthermore, a ∼40 kDa huntingtin fragment recognized by the N-terminal antibody is enriched in nuclear fractions of brains from patients with HD (5DiFiglia M. Sapp E. Chase K.O. Davies S.W. Bates G.P. Vonsattel J.P. Aronin N. Science. 1997; 277: 1990-1993Crossref PubMed Scopus (2175) Google Scholar). Thus, it is quite likely that, at least for huntingtin, what accumulates is predominantly a small, truncated fragment of the protein. A necessary pathogenic event in HD therefore may be proteolytic cleavage by caspase, other proteolytic enzymes, or both. However, even if the small portion of huntingtin can enter the nucleus, why should it aggregate there? One must also ask why other proteins that are normally cytoplasmic aggregate in the nucleus. Recent data may provide an answer for SCA-1. Yeast two-hybrid methods were used to identify a nuclear protein, the leucine-rich acidic nuclear protein (LANP), which binds far more effectively to ataxin-1 with an expanded repeat than it does to ataxin-1 with a normal repeat (8Matilla A. Koshy B.T. Cummings C.J. Isobe T. Orr H.T. Zoghbi H.Y. Nature. 1997; 389: 974-978Crossref PubMed Scopus (228) Google Scholar). The two proteins colocalize in subdomains of the nucleus termed nuclear bodies (8Matilla A. Koshy B.T. Cummings C.J. Isobe T. Orr H.T. Zoghbi H.Y. Nature. 1997; 389: 974-978Crossref PubMed Scopus (228) Google Scholar). By contrast, a number of other proteins present in other subdomains of the nucleus did not colocalize with ataxin-1 (15Skinner P.J. Koshy B.T. Cummings C.J. Klement I.A. Helin K. Servadio A. Zoghbi H.Y. Orr H.T. Nature. 1997; 389: 971-974Crossref PubMed Scopus (484) Google Scholar). Thus, binding to ataxin-1 appears to alter the distribution of LANP within the nucleus and to make the two together bind tightly to the nuclear matrix. It is possible that other nuclear binding proteins, perhaps related to LANP, may aggregate with truncated portions of other glutamine-repeat disease proteins. However, it is unclear if such protein interactions could explain the preferential aggregation of the HPRT protein with its ectopically located glutamine repeat. At least for the interaction of ataxin-1 and LANP, other portions of the protein besides the glutamine repeat itself appear to be critically involved. How HPRT is recruited to nuclear inclusions, therefore, is uncertain. In addition to possibly affecting where protein aggregates, in some way the "context" provided by other portions of the disease proteins appears to influence the ability of the polyglutamine to aggregate. In in vitro aggregation experiments, the polyglutamine-containing HD exon-1 construct was fused to glutathione S-transferase (GST; 14Scherzinger E. Lurz R. Turmaine M. Mangiarini L. Hollenbach B. Hasenbank R. Bates G.P. Davies S.W. Lehrach H. Wanker E.E. Cell. 1997; 90: 549-558Abstract Full Text Full Text PDF PubMed Scopus (1051) Google Scholar). In this complex, the GST appeared to inhibit formation of aggregates. When the protein was cleaved from the GST, aggregate formation was enhanced. The differences in sensitivity to repeat number in the different diseases (7Gusella J.F. Persichetti F. MacDonald M.E. Mol. Med. 1997; 3: 238-246PubMed Google Scholar) could be due in part to a similar context effect. The intranuclear neuronal inclusions are now strong candidates for a central role in the pathogenesis of all of the glutamine-repeat diseases. Consistent with this is the distribution of the inclusions in brains of patients with the various disorders. For instance, in HD the inclusions are enriched in medium-sized neurons of the striatum and neurons of layers III, V, and VI of the cerebral cortex, which are all selectively vulnerable in HD. They are found less often in other brain regions and rarely, if at all, in nonneuronal cells (2Becher M.W. Kotzuk J.A. Sharp A.H. Davies S.W. Bates G.P. Price D.L. Ross C.A. Neurobiol. Dis. 1997; 4 (in press)Google Scholar, 5DiFiglia M. Sapp E. Chase K.O. Davies S.W. Bates G.P. Vonsattel J.P. Aronin N. Science. 1997; 277: 1990-1993Crossref PubMed Scopus (2175) Google Scholar). Similarly, in DRPLA and in SCA-1 and SCA-3, the inclusions appear to be enriched in the most vulnerable cells, although not as many cases have been published as for HD (2Becher M.W. Kotzuk J.A. Sharp A.H. Davies S.W. Bates G.P. Price D.L. Ross C.A. Neurobiol. Dis. 1997; 4 (in press)Google Scholar, 11Paulson H.L. Perez M.K. Trottier Y. Trojanowski J.Q. Subramony S.H. Das S.S. Vig P. Mandel J.-L. Fischbeck K.H. Pittman R.N. Neuron. 1997; 19: 333-344Abstract Full Text Full Text PDF PubMed Scopus (698) Google Scholar, 15Skinner P.J. Koshy B.T. Cummings C.J. Klement I.A. Helin K. Servadio A. Zoghbi H.Y. Orr H.T. Nature. 1997; 389: 971-974Crossref PubMed Scopus (484) Google Scholar). In transgenic mice, the data are also consistent with a role for the inclusions in the development of the phenotype. In the ataxin-1 transgenic mice, the inclusions are present before the onset of the phenotype, and the neurons later degenerate. Similarly, in the HD exon-1 transgenic mice, the inclusions are present before the onset of the earliest behavioral symptoms (4Davies S.W. Turmaine M. Cozens B.A. DiFiglia M. Sharp A.H. Ross C.A. Scherzinger E. Wanker E.E. Mangiarini L. Bates G.P. Cell. 1997; 90: 537-548Abstract Full Text Full Text PDF PubMed Scopus (1828) Google Scholar). In both the HD exon-1 model and in the HPRT model, inclusions are present in great number, but neuronal cell death cannot be demonstrated. It is possible that the inclusions in this case cause neuronal dysfunction long before neuronal death. The mouse model of SCA-1 clearly indicates that neuronal dysfunction long preceeds detectable neuronal death. Interestingly, occasional HD patients are reported who have had clear symptoms prior to death but have no qualitatively detectable neuronal loss in the striatum. Nevertheless, the arguments linking intranuclear inclusions to cell death rest on correlation. As 10Ordway J.M. Tallaksen-Greene S. Gutekunst C.-A. Bernstein E.M. Cearley J.A. Wiener H.W. Dure IV, L.S. Lindsey R. Hersch S.M. Jope R.S. Albin R.L. Detloff P.J. Cell. 1997; 91: 753-763Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar point out, it is equally conceivable that the inclusions are merely a marker for some other process that is actually responsible for the neuronal cell death. Conversely, the inclusions might be an attempt by the cell to defend against polyglutamine toxicity, and they might actually exercise a protective role. These issues could be addressed in cell-transfection experiments, by designing constructs with nuclear localization or nuclear export signals strategically placed to increase or decrease the concentration of polyglutamine repeats in the nucleus. Even if intranuclear inclusions were to have a causative role in neuronal cell death and dysfunction, the mechanism remains unclear. The inclusions appear to be relatively circumscribed and to exclude DNA (4Davies S.W. Turmaine M. Cozens B.A. DiFiglia M. Sharp A.H. Ross C.A. Scherzinger E. Wanker E.E. Mangiarini L. Bates G.P. Cell. 1997; 90: 537-548Abstract Full Text Full Text PDF PubMed Scopus (1828) Google Scholar), suggesting that they don't physically disrupt the chromatin in the nucleus. They may, however, have more subtle effects on gene transcription. For instance, it is conceivable that they could alter transcription of genes required for neuronal viability, perhaps including glutamate receptors, mitochondrial proteins, or other molecules previously implicated in neuronal vulnerability. Dystrophic neurites that label for the N-terminal huntingtin antibody have been observed in HD (5DiFiglia M. Sapp E. Chase K.O. Davies S.W. Bates G.P. Vonsattel J.P. Aronin N. Science. 1997; 277: 1990-1993Crossref PubMed Scopus (2175) Google Scholar). This raises the possibility that deposition of polyglutamine outside of the nucleus may also be important, at least in HD. Such deposition may take place in other diseases. 11Paulson H.L. Perez M.K. Trottier Y. Trojanowski J.Q. Subramony S.H. Das S.S. Vig P. Mandel J.-L. Fischbeck K.H. Pittman R.N. Neuron. 1997; 19: 333-344Abstract Full Text Full Text PDF PubMed Scopus (698) Google Scholar observed "deposits" in SCA-3. Similarly, a recent cell-transfection study (9Onodera O. Burke J.R. Miller S.E. Hester S. Tsuji S. Roses A.D. Strittmatter W.J. Biochem. Biophys. Res. Commun. 1997; 238: 599-605Crossref PubMed Scopus (58) Google Scholar) found that fusion proteins of polyglutamine with green fluorescent protein (GFP) could form cytoplasmic (and occasionally nuclear) aggregates, which disrupted the intermediate filament cytoskeleton. It was not clear whether or not this was toxic to the cells. In addition, there could be a role for other interactions in the cytoplasm. For instance, interactions of huntingtin with other proteins may conceivably alter cell signaling or other events within the cytoplasm (3Colomer V. Engelender S. Sharp A.H. Duan K. Cooper J.K. Lanahan A. Lyford G. Worley P. Ross C.A. Hum. Mol. Genet. 1997; 9: 1519-1525Crossref Scopus (105) Google Scholar). Whether this has any role in the pathogenesis of the disease is still speculative. As noted above, the distribution of intranuclear inclusions in brains from patients with several of the diseases appears to match the distribution of pathology in these diseases, despite the fact that the mutant proteins are widely expressed. How might this come about? One possibility is the involvement of interacting proteins. The distribution of expression of LANP in normal brain corresponds quite closely to the pattern of vulnerability in SCA-1 (8Matilla A. Koshy B.T. Cummings C.J. Isobe T. Orr H.T. Zoghbi H.Y. Nature. 1997; 389: 974-978Crossref PubMed Scopus (228) Google Scholar). This suggests a role for LANP in specifying neuronal vulnerability by forming a nidus for aggregation. A second possibility is that protein cleavage itself is a cell type–specific event. Experiments directed at this issue will be necessary to resolve the factors governing differential vulnerability of cells to protein aggregation. The unexpected discovery of inclusion bodies in the polyglutamine disorders, and the clear demonstration that polyglutamine-containing proteins can aggregate, indicate that these polyglutamine disorders are more similar to each other than was previously anticipated and may be related to other neurodegenerative disorders. As is described in the accompanying review (Lansbury, 1997 [this issue of Neuron]), protein aggregation is a common mechanism in other late-onset neurodegenerative diseases, including Alzheimer's disease, prion disease, and Parkinson's disease. Shared mechanisms or shared biological modifiers may underlie several of these diseases. Cross-fertilization among investigators working on these various disorders is bound to lead to rapid advances in our understanding of the pathogenesis of these diseases.