Huntington's disease (HD) is caused by polyglutamine (polyQ) expansion within the huntingtin protein (Htt), but the sequence of events leading to neurodegeneration remains unclear. Recent evidence has moved the spotlight away from the role of proteolytic fragments of huntingtin, see related news story, and now, findings from three independent labs build on the evidence for huntingtin's role in protein-protein interactions.
Last Tuesday's advanced online publication in Nature Cell Biology by Donald Nicholson at the Merck Frosst Centre for Therapeutic Research, Quebec, and colleagues, has turned that spotlight on the Htt-interacting protein, Hip-1, and its new partner Hippi. Hippi, for Hip-1 protein interactor, came up in a yeast two-hybrid screen using Hip-1 as bait. Both Hippi and Hip-1 contain pseudo death-effector domains (pDED) that are required for their interaction, though additional regions in the C-terminal of each protein appear to enhance their binding. Hippi also co-localizes with Hip-1 in the Golgi apparatus of brain neurons.
But how does Hippi fit into Huntington's disease? Knowing that polyQ expansion reduces the strength of Hip-1:huntingtin interaction, the authors looked at the effect of the mutation in cultured cells. They found that the amount of Hip-1 bound to Hippi was much higher in cells expressing Htt-Q(128) than in wild type cells (Htt-Q(15)). Hippi was found to enhance Hip-1-mediated apoptosis, and furthermore, immunoprecipitation experiments showed that Hippi, Hip-1, and the apoptotic protease caspase 8 all interact. The simplest model to explain this data is that polyQ expansion of huntingtin frees up Hip-1 to interact with Hippi and initiate apoptosis by activating the caspase 8 pathway, the authors write.
"Intrabodies" Manipulate Huntingtin Aggregation and Apoptosis
Meanwhile researchers in Paul Patterson's lab at the California Institute of Technology in Pasadena have used a different approach to probe huntingtin's role in the disease. They expressed, inside cultured cells, monoclonal antibodies raised against various epitopes of huntingtin to perturb its protein-protein interactions. Their results, published online on January 15 in PNAS, reveal two types of antibodies: those that stimulate aggregation of Htt and cellular apoptosis, and those with potential therapeutic value, which prevent aggregation and cell death.
The authors raised antibodies to the polyQ, polyP, and C terminus of exon 1 of Htt, which has been shown to be the major component of protein aggregates in HD. Expressed as single-chain variable regions (scFvs) in 293 cells, the antibodies had no effect on cell viability in the absence of huntingtin, though when co-expressed with Htt exon-1 the scFvs and the antigen co-localized and immunoprecipitated, indicating that the antibody and antigen interacted intracellularly.
Two different polyQ antibodies increased apoptosis, as judged by TUNEL staining, by 38 percent and 67 percent respectively. In contrast, a polyP antibody decreased apoptosis by 77 percent. The antibodies' effect on Htt- induced apoptosis may be related to their ability to perturb its aggregation, as more Htt was found in aggregates in a polyQ antibody background whereas no aggregation could be detected when the polyP antibody was present in the cells. These data fit with the suggested role of Htt in sequestering Hip-1. Competition for the polyQ site by the antibody would, presumably, free up more Hip-1 for interaction with Hippi.
Yeast Needs Microtubules to Make Inclusion Bodies
The cytoskeleton may also play a role in the etiology of Huntington's, as microtubules are known to bind huntingtin via the Htt-associated protein, HAP1. Scientists led by Stanley Fields at the University of Washington now have chemically or genetically disrupted the cytoskeleton and observed profound effects on the aggregation of huntingtin in yeast. Their work also appeared in the 15 January PNAS early online edition.
Expressing normal or expanded huntingtin exon 1 (53Q) in budding yeast, the authors found that expanded Htt led to the formation of SDS-insoluble inclusion bodies but did not affect cell viability. When the same cells were treated with the microtubule- depolymerizing drugs benomyl, cocodazole, or thiabendazole, the amount of aggregated Htt shrank to less than 2 percent. In contrast, the actin filament disrupters latrunculin A and cytochalasin B had little or no effect. In a strain made resistant to the benomyl-induced microtubule disruption by a single point mutation in the tubulin 2 gene, the drug had no effect on Htt aggregation, indicating that microtubules are required for the formation of Htt inclusions.
This suggests that formation of inclusion bodies does not happen by passive diffusion of huntingtin, and it lends support to the hypothesis that inclusion bodies result from an active cellular defense mechanism that is trying to sequester the mutant protein.—Tom Fagan
- Gervais FG, Singaraja R, Xanthoudakis S, Gutekunst CA, Leavitt BR, Metzler M, Hackam AS, Tam J, Vaillancourt JP, Houtzager V, Rasper DM, Roy S, Hayden MR, Nicholson DW. Recruitment and activation of caspase-8 by the Huntingtin-interacting protein Hip-1 and a novel partner Hippi. Nat Cell Biol. 2002 Feb;4(2):95-105. PubMed.
- Khoshnan A, Ko J, Patterson PH. Effects of intracellular expression of anti-huntingtin antibodies of various specificities on mutant huntingtin aggregation and toxicity. Proc Natl Acad Sci U S A. 2002 Jan 22;99(2):1002-7. PubMed.
- Muchowski PJ, Ning K, D'Souza-Schorey C, Fields S. Requirement of an intact microtubule cytoskeleton for aggregation and inclusion body formation by a mutant huntingtin fragment. Proc Natl Acad Sci U S A. 2002 Jan 22;99(2):727-32. PubMed.