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Huntingtin Fragments that Aggregate Go Their Separate Ways
Molecular Cell 224
Huntingtin Fragments that Aggregate Go Their Separate Ways N-terminal region of mutant huntingtin forms intranuclear and cytoplasmic aggregates in neurons that may contribute to neuronal death in Huntington’s disease. Lunkes et al. (2002) show that different endoprotease-cleaved huntingtin fragments form nuclear and cytoplasmic inclusions. A devastating disorder, Huntington’s disease (HD) attacks most victims in midlife, leaving them with progressive and severe motor and cognitive impairment; death ensues about 15 years after the onset of symptoms. No treatment exists, spurring researchers to search for underlying mechanisms for the disease. The mutation is inherited as autosomal dominant and causes expansion in a stretch of glutamines near the N terminus of huntingtin (The Huntington’s Disease Collaborative Group, 1993), a brain-enriched protein of uncertain function. Specific neurons in the brain within the striatum and the cortex are targeted for death, but the reason for the selective assault is obscure since huntingtin expression is widespread in most neurons and in other types of cells. In 1997, investigators got their first clue. Clumps of insoluble abnormal protein (aka inclusions or aggregates) conspicuously lodged in the nucleus and the cytoplasm of affected neurons in postmortem HD brain. Antisera to the N terminus but not to internal or C-terminal epitopes detected the inclusions, and purified nuclei from HD patients had an excess of N-terminal huntingtin fragments (DiFiglia et al., 1997). Transgenic mice made to express an arbitrarily truncated N-terminal fragment of mutant huntingtin expressed from the first exon, which encoded less than 3% of the protein, recapitulated the nuclear inclusions seen in the patient’s brain and with dire consequences for the mice—they developed severe motor deficits and died at 3 months of age (Davies et al., 1997). Analysis showed that the insoluble protein in the aggregates of the mice had an amyloidlike fibrillar structure (Scherzinger et al., 1997). A knowledge of how the N-terminal region of mutant huntingtin is processed is critical to understanding how neurons die. More than two dozen nuclear and cytoplasmic proteins interact with huntingtin; most of the interactions involve the N-terminal region of huntingtin and are altered by polyglutamine expansion. Huntingtin is a relatively large protein of 3144 amino acids. The polyglutamine tract begins with aa 18 (see Figure). Some sites of protease cleavage in the N-terminal region are known or estimated. Caspases cleave huntingtin at aa 513, 552, and 589 (Wellington et al., 2000), and calpain activity generates smaller fragments from cleavages at about aa 400–500 (Kim et al., 2001, Gafni and Ellerby, 2002). The cleavage sites for these proteases are C-terminal to the region in huntingtin found in inclusions, based on epitope mapping with different anti-huntingtin antisera. Therefore, smaller N-terminal mutant huntingtin fragments than those produced by caspases and calpains must form the inclusions. On
the other hand, a recent study proposed that mutant huntingtin is more resistant to proteolysis than wild-type huntingtin and that mutant fragments smaller than the caspase-cleaved ones are unlikely to occur in the HD brain (Dyer and McMurray, 2001). Lunkes et al. (2002), in a paper in this issue of Molecular Cell, embarked on a search for the potentially deadly and controversial N-terminal mutant huntingtin fragment. They used a panel of anti-huntingtin antibodies to mark reference points in the N-terminal region of huntingtin and performed immunohistofluorescence and biochemical analysis in cultured cells with stable or transient expression of the mutant protein. In addition, they introduced an HA sequence at a key location in the protein that could be tracked by anti-HA antisera. After cells were cultured for several weeks, the nuclear and cytoplasmic inclusions increased in number. The nuclear and cytoplasmic compartments were then separated from the harvested cells for biochemical analysis. Immunohistochemical study of inclusions in postmortem brain tissue of HD patients rounded out their analysis. What they concluded from this comprehensive evaluation was that not one but two insoluble-prone N-terminal mutant huntingtin fragments, simply termed cleavage products A and B (cp-A and cp-B), build inclusions; the smaller fragment, cp-A, appeared solely responsible for amassing nuclear aggregates. Cytoplasmic inclusions had fragments of different lengths, cp-A, cp-B, and larger huntingtin products. The exact mechanism for aggregation is unclear. Perutz proposed that a protein with an expanded polyglutamine tract has an altered structure that facilitates selfassociation (see review by Perutz, 1999). The mutant protein may become resistant to removal by the ubiquitin/proteasome system, the main proteolytic machinery that identifies and degrades misfolded proteins inside the cell. Indeed, Lunkes et al. (2002) showed that brief treatment of cultured cells with an inhibitor of proteasome activity greatly enhanced the accumulation of the fragments. But what about the protease generating the huntingtin fragments? The investigators found that the fragments formed if they allowed mutant huntingtin-enriched cell extracts to incubate in the absence of protease inhibitors, evidence an endogenous protease was at work. Adding back inhibitors of different proteases one at a time revealed one inhibitor to aspartyl proteases (pepstatin A) that was effective in blocking the formation of the fragments. Painstaking mapping by deleting or substituting residues allowed them to narrow the site of cleavage for cp-A to a 10 residue domain between aa 104–114, remarkably near the boundary of the product encoded by exon 1 (aa 89 in wild-type huntingtin). These findings leave little doubt that N-terminal proteolysis near the polyglutamine tract occurs in mutant huntingtin and requires proteasome and aspartyl protease activities. Many issues are yet to be resolved. Foremost is whether preventing production of cp-A or cp-B in fulllength mutant huntingtin or a large truncated fragment can delay the onset of symptoms in HD mice. The aspartyl protease(s) generating cp-A and cp-B needs to be identified. There could be two! This family of proteases includes cathepsin D, cathepsin E, and the notorious secretases and presenilins, which release -amyloid
Previews 225
Schematic of Full-Length Wild-Type Huntingtin with 23 Glutamines An enlargement of the N-terminal region shows the polyglutamine tract, the relative position of cleavage sites for cp-A, cp-B identified by Lunkes et al. (2002), and the fragments generated by calpain and caspase cleavage. Individuals with HD have 39 or more glutamines in huntingtin. Mice expressing the region encoded by exon 1 with an expanded polyglutamine tract develop a severe phenotype.
from amyloid precursor protein. Also worth considering is whether cp-A and cp-B engage in abnormal protein interactions or whether these interactions involve the participation of full-length mutant huntingtin or caspasecleaved or calpain-cleaved fragments before cp-A and cp-B are formed. How cp-A gets to the nucleus is unclear. Is it by passive diffusion or regulated transport? Finding where in the cell the protease activity that generates cp-A occurs may offer insights about the mechanism of pathogenesis. Is proteolytic processing critical for the formation of inclusions by other polyglutamine disease proteins? This may be so for mutant atrophin 1, which causes dentatorubral and pallidoluysian atrophy (DRPLA). In transgenic mice expressing the mutant fulllength atrophin-1, a fragment containing the expanded polyglutamine stretch accumulates in neuronal nuclei (Schilling et al., 1999). While much remains to be done, the work by Lunkes et al. greatly expands the possibilities for developing effective therapeutic targets to treat HD and gives hope to HD patients and their families. Marian DiFiglia Massachusetts General Hospital Department of Neurology 114 16th Street Charlestown, Massachusetts 02114
Selected Reading Davies, S.W., Trumaine, M., Cozens, B.A., DiFiglia, M., Sharp, A.H., Ross, C.A., Scherzinger, E., Wanker, E.E., Mangiarini, L., and Bates, G.P. (1997). Cell 90, 537–548. DiFiglia, M., Sapp, E., Chase, K.O., Davies, S.W., Bates, G.P., Von Sattel, J.P., Aronin, N. (1997). Science 277, 1990–1993. Dyer, R.B., and McMurray, C.T. (2001). Nat. Genet. 29, 270–278. Gafni, J., and Ellerby, L.M. (2002). J. Neurosci. 22, 4842–4849. Huntington’s Disease Collaborative Research Group (1993). Cell 72, 971–983. Kim, Y.J., Yi, Y., Sapp, E., Wang, Y., Cuiffo, B., Kegel, K.B., Qin, Z.-H., Aronin, N., and DiFiglia, M. (2001). Proc. Natl. Acad. Sci. USA 12784–12789. Lunkes, A., Lindenberg, K.S., Ben-Haiem, L., Weber, C., Devys, D., Landwehrmeyer, G.B., Mandel, J.L., and Trottier, Y. (2002). Mol. Cell 10, this issue, 259–269. Perutz, M. (1999). Trends Biochem. Sci. 24, 58–63. Schilling, G., Wood, J.D., Duan, K., Slunt, H.H., Gonzales, V., Yamada, M., Cooper, J.K., Margolis, R.L., Jenkins, N.A., Copeland, N.G., et al. (1999). Neuron 24, 275–286. Scherzinger, E., Lurz, R., Turmaine, M., Mangiarini, L., Hollenbach, B., Hasenbank, R., Bates, G.P., Davies, S.W., Lehrach, H., and Wanker, E.E. (1997). Cell 90, 549–558. Wellington, C.L., Singaraja, R., Ellerby, L., Savill, J., Roy, S., Leavitt, B., Cattaneo, E., Hackam, A., Sharp, A., Thornberry, N., Nicholson, D.W., Bredesen, D.E., and Hayden, M.R. (2000). J. Biol. Chem. 275, 19831–19838.
Huntingtin Fragments that Aggregate Go Their Separate Ways N-terminal region of mutant huntingtin forms intranuclear and cytoplasmic aggregates in neurons that may contribute to neuronal death in Huntington’s disease. Lunkes et al. (2002) show that different endoprotease-cleaved huntingtin fragments form nuclear and cytoplasmic inclusions. A devastating disorder, Huntington’s disease (HD) attacks most victims in midlife, leaving them with progressive and severe motor and cognitive impairment; death ensues about 15 years after the onset of symptoms. No treatment exists, spurring researchers to search for underlying mechanisms for the disease. The mutation is inherited as autosomal dominant and causes expansion in a stretch of glutamines near the N terminus of huntingtin (The Huntington’s Disease Collaborative Group, 1993), a brain-enriched protein of uncertain function. Specific neurons in the brain within the striatum and the cortex are targeted for death, but the reason for the selective assault is obscure since huntingtin expression is widespread in most neurons and in other types of cells. In 1997, investigators got their first clue. Clumps of insoluble abnormal protein (aka inclusions or aggregates) conspicuously lodged in the nucleus and the cytoplasm of affected neurons in postmortem HD brain. Antisera to the N terminus but not to internal or C-terminal epitopes detected the inclusions, and purified nuclei from HD patients had an excess of N-terminal huntingtin fragments (DiFiglia et al., 1997). Transgenic mice made to express an arbitrarily truncated N-terminal fragment of mutant huntingtin expressed from the first exon, which encoded less than 3% of the protein, recapitulated the nuclear inclusions seen in the patient’s brain and with dire consequences for the mice—they developed severe motor deficits and died at 3 months of age (Davies et al., 1997). Analysis showed that the insoluble protein in the aggregates of the mice had an amyloidlike fibrillar structure (Scherzinger et al., 1997). A knowledge of how the N-terminal region of mutant huntingtin is processed is critical to understanding how neurons die. More than two dozen nuclear and cytoplasmic proteins interact with huntingtin; most of the interactions involve the N-terminal region of huntingtin and are altered by polyglutamine expansion. Huntingtin is a relatively large protein of 3144 amino acids. The polyglutamine tract begins with aa 18 (see Figure). Some sites of protease cleavage in the N-terminal region are known or estimated. Caspases cleave huntingtin at aa 513, 552, and 589 (Wellington et al., 2000), and calpain activity generates smaller fragments from cleavages at about aa 400–500 (Kim et al., 2001, Gafni and Ellerby, 2002). The cleavage sites for these proteases are C-terminal to the region in huntingtin found in inclusions, based on epitope mapping with different anti-huntingtin antisera. Therefore, smaller N-terminal mutant huntingtin fragments than those produced by caspases and calpains must form the inclusions. On
the other hand, a recent study proposed that mutant huntingtin is more resistant to proteolysis than wild-type huntingtin and that mutant fragments smaller than the caspase-cleaved ones are unlikely to occur in the HD brain (Dyer and McMurray, 2001). Lunkes et al. (2002), in a paper in this issue of Molecular Cell, embarked on a search for the potentially deadly and controversial N-terminal mutant huntingtin fragment. They used a panel of anti-huntingtin antibodies to mark reference points in the N-terminal region of huntingtin and performed immunohistofluorescence and biochemical analysis in cultured cells with stable or transient expression of the mutant protein. In addition, they introduced an HA sequence at a key location in the protein that could be tracked by anti-HA antisera. After cells were cultured for several weeks, the nuclear and cytoplasmic inclusions increased in number. The nuclear and cytoplasmic compartments were then separated from the harvested cells for biochemical analysis. Immunohistochemical study of inclusions in postmortem brain tissue of HD patients rounded out their analysis. What they concluded from this comprehensive evaluation was that not one but two insoluble-prone N-terminal mutant huntingtin fragments, simply termed cleavage products A and B (cp-A and cp-B), build inclusions; the smaller fragment, cp-A, appeared solely responsible for amassing nuclear aggregates. Cytoplasmic inclusions had fragments of different lengths, cp-A, cp-B, and larger huntingtin products. The exact mechanism for aggregation is unclear. Perutz proposed that a protein with an expanded polyglutamine tract has an altered structure that facilitates selfassociation (see review by Perutz, 1999). The mutant protein may become resistant to removal by the ubiquitin/proteasome system, the main proteolytic machinery that identifies and degrades misfolded proteins inside the cell. Indeed, Lunkes et al. (2002) showed that brief treatment of cultured cells with an inhibitor of proteasome activity greatly enhanced the accumulation of the fragments. But what about the protease generating the huntingtin fragments? The investigators found that the fragments formed if they allowed mutant huntingtin-enriched cell extracts to incubate in the absence of protease inhibitors, evidence an endogenous protease was at work. Adding back inhibitors of different proteases one at a time revealed one inhibitor to aspartyl proteases (pepstatin A) that was effective in blocking the formation of the fragments. Painstaking mapping by deleting or substituting residues allowed them to narrow the site of cleavage for cp-A to a 10 residue domain between aa 104–114, remarkably near the boundary of the product encoded by exon 1 (aa 89 in wild-type huntingtin). These findings leave little doubt that N-terminal proteolysis near the polyglutamine tract occurs in mutant huntingtin and requires proteasome and aspartyl protease activities. Many issues are yet to be resolved. Foremost is whether preventing production of cp-A or cp-B in fulllength mutant huntingtin or a large truncated fragment can delay the onset of symptoms in HD mice. The aspartyl protease(s) generating cp-A and cp-B needs to be identified. There could be two! This family of proteases includes cathepsin D, cathepsin E, and the notorious secretases and presenilins, which release -amyloid
Previews 225
Schematic of Full-Length Wild-Type Huntingtin with 23 Glutamines An enlargement of the N-terminal region shows the polyglutamine tract, the relative position of cleavage sites for cp-A, cp-B identified by Lunkes et al. (2002), and the fragments generated by calpain and caspase cleavage. Individuals with HD have 39 or more glutamines in huntingtin. Mice expressing the region encoded by exon 1 with an expanded polyglutamine tract develop a severe phenotype.
from amyloid precursor protein. Also worth considering is whether cp-A and cp-B engage in abnormal protein interactions or whether these interactions involve the participation of full-length mutant huntingtin or caspasecleaved or calpain-cleaved fragments before cp-A and cp-B are formed. How cp-A gets to the nucleus is unclear. Is it by passive diffusion or regulated transport? Finding where in the cell the protease activity that generates cp-A occurs may offer insights about the mechanism of pathogenesis. Is proteolytic processing critical for the formation of inclusions by other polyglutamine disease proteins? This may be so for mutant atrophin 1, which causes dentatorubral and pallidoluysian atrophy (DRPLA). In transgenic mice expressing the mutant fulllength atrophin-1, a fragment containing the expanded polyglutamine stretch accumulates in neuronal nuclei (Schilling et al., 1999). While much remains to be done, the work by Lunkes et al. greatly expands the possibilities for developing effective therapeutic targets to treat HD and gives hope to HD patients and their families. Marian DiFiglia Massachusetts General Hospital Department of Neurology 114 16th Street Charlestown, Massachusetts 02114
Selected Reading Davies, S.W., Trumaine, M., Cozens, B.A., DiFiglia, M., Sharp, A.H., Ross, C.A., Scherzinger, E., Wanker, E.E., Mangiarini, L., and Bates, G.P. (1997). Cell 90, 537–548. DiFiglia, M., Sapp, E., Chase, K.O., Davies, S.W., Bates, G.P., Von Sattel, J.P., Aronin, N. (1997). Science 277, 1990–1993. Dyer, R.B., and McMurray, C.T. (2001). Nat. Genet. 29, 270–278. Gafni, J., and Ellerby, L.M. (2002). J. Neurosci. 22, 4842–4849. Huntington’s Disease Collaborative Research Group (1993). Cell 72, 971–983. Kim, Y.J., Yi, Y., Sapp, E., Wang, Y., Cuiffo, B., Kegel, K.B., Qin, Z.-H., Aronin, N., and DiFiglia, M. (2001). Proc. Natl. Acad. Sci. USA 12784–12789. Lunkes, A., Lindenberg, K.S., Ben-Haiem, L., Weber, C., Devys, D., Landwehrmeyer, G.B., Mandel, J.L., and Trottier, Y. (2002). Mol. Cell 10, this issue, 259–269. Perutz, M. (1999). Trends Biochem. Sci. 24, 58–63. Schilling, G., Wood, J.D., Duan, K., Slunt, H.H., Gonzales, V., Yamada, M., Cooper, J.K., Margolis, R.L., Jenkins, N.A., Copeland, N.G., et al. (1999). Neuron 24, 275–286. Scherzinger, E., Lurz, R., Turmaine, M., Mangiarini, L., Hollenbach, B., Hasenbank, R., Bates, G.P., Davies, S.W., Lehrach, H., and Wanker, E.E. (1997). Cell 90, 549–558. Wellington, C.L., Singaraja, R., Ellerby, L., Savill, J., Roy, S., Leavitt, B., Cattaneo, E., Hackam, A., Sharp, A., Thornberry, N., Nicholson, D.W., Bredesen, D.E., and Hayden, M.R. (2000). J. Biol. Chem. 275, 19831–19838.