13 December 2004. The link between protein aggregation and neurodegenerative diseases is certainly a hot topic these days, awash with questions over whether reducing aggregation is a good idea, and if it is, what aggregates should be targeted. Perhaps just the small-scale or nonfibrillar oligomers are the bad seeds, and the larger, or fibrillar ones, protect the nervous system (see ARF related news story). If so, then how can we target the toxic forms without also attacking the good guys? Two recent Huntington disease-related studies weigh in with different approaches to interfering with the aggregation of mutant huntingtin protein.
(Heat) Shocking Treatment
Writing in the December issue of Nature Structural and Molecular Biology, Paul Muchowski and colleagues at the University of Washington, Seattle, report taming the aggregation of mutant huntingtin with heat shock proteins, and also confirm the kinship of polyglutamine repeat huntingtin with aggregating proteins from AD and PD. First author Jennifer Wacker and colleagues first used atomic force microscopy to get a close-up look at the aggregates that mutant huntingtin (mhtt) forms in vitro. They noted that HD53Q, containing 53 polyQ repeats, assembled into spherical and annular oligomers—"similar in size and morphology to those formed by Aβ and α-synuclein"—at concentrations too low to allow formation of fibrillar aggregates.
But when the authors added heat shock proteins to the mix, a different picture emerged. The molecular chaperone heat shock protein 70 (Hsp70) and its compadre Hsp40 work together to enhance proper folding of proteins, and Hsp70 has proven neuroprotective in experimental models of HD and PD (for review see Muchowski, 2002. According to Wacker and colleagues, the chaperones may exert this benefit in HD by diverting mhtt from forming potentially toxic oligomers. When the authors added Hsp70 and Hsp40, in the presence of ATP, to mhtt at concentrations that would otherwise yield only nonfibrillar oligomers, they found that mhtt fibrils formed, with concomitant decreases in nonfibrillar oligomers.
Does this mean that some species of mhtt oligomer—typically classed as spherical, annular, or amorphous—are intermediates on the path to fibrillar and toxic aggregates? A recent study suggests that spherical oligomers might be such an intermediate (Shorter and Lindquist, 2004), but the data from Wacker and colleagues do not fit well with this model. When the researchers added the heat shock proteins an hour after the mhtt incubation was begun, the chaperones were unable to drive any of the oligomers to form fibrils. "These results indicate that Hsp70 and Hsp40 act on a conformation of HD53Q that precedes or is independent of the formation of spherical structures, and cannot disassemble, preformed spherical structures," write the authors.
In concluding, Wacker and colleagues discuss the possibility that fibrillar mhtt protects neurons, and that the amorphous, spherical, and/or annular oligomers are the neurotoxic species that we should be worried about. "Indeed, we propose that the ability of Hsp70 and Hsp40 to attenuate formation of these assemblies may account for their protective effects in animal models of neurodegenerative disease," they write.
Antibodies slipped into neurons via viral vectors—so-called "intrabodies"—would seem to be a good approach to interfering with mhtt—in fact, with any protein prone to aggregation. And, if causal links between abnormal protein aggregates and neurodegenerative diseases are confirmed (see, however, ARF related news story), intrabodies may prove an effective therapy for some diseases. Indeed, there have been some experimental successes already with this approach in an HD model (see ARF related news story).
But first, there are technical hurdles that need to be overcome, including the need to develop small, simple antibodies, which, according to Dane Wittrup at MIT in Cambridge, would have fewer epitopes that might be immunogenic, would put less stress on the cell to synthesize the protein, and would fold more simply and efficiently. To that end, Wittrup's group recently reported that they had developed just such an antibody—a single variable light-chain (VL) domain targeted against the N-terminal 20 amino acids of htt (Colby et al., 2004). But even a minimal antibody like this has its problems; the disulfide bonds that antibodies employ to enhance their stability only form under oxidizing conditions, whereas the cytoplasm is a reducing environment. But in a study reported online last week in PNAS, Wittrup, first author David Colby, and colleagues report that they have made an intrabody whose stability and activity does not depend on the local redox potential.
To generate a redox-insensitive intrabody, Colby and colleagues replaced the two cysteine residues in their VL htt antibody with valine and alanine. Surprisingly, these mutations did not alter the stability of the protein, but they did reduce its affinity for the htt peptide fragment—by 2-3 orders of magnitude—suggesting that disulfide bonds are important in maintaining the structure of the antigen binding site, if not the antibody as a whole.
To improve the affinity of the cysteine-free antibody, Colby and colleagues subjected the gene for the VL intrabody to three rounds of random mutagenesis, generating over 10 million different antibodies. From this pool they found an intrabody with four mutations—designated VL12.3—that had significantly higher binding affinity for the htt fragment. Colby and colleagues then put the new intrabody through its paces in cell models of HD. When they coexpressed intrabody and mhtt—at a five-to-one ratio—in ST14A cells (derived from embryonic rat striatum) the new and improved VL12.3 virtually eliminated aggregates, as determined by fluorescence microscopy or Western blot. Even when the ratio of intrabody to mhtt plasmid was reduced to 1:1—a ratio at which earlier varieties of intrabodies had been completely ineffective—the VL12.3 intrabody reduced aggregates by nearly 80 percent. “Given the relative inefficiency of viral gene delivery to the CNS, it is essential that in any proposed gene therapy, the therapeutic protein whose gene is delivered should work as efficiently as possible. For this reason, VL12.3 may prove useful in treating HD through gene therapy, in addition to use as a research tool in further studies…,” write the authors.—Hakon Heimer.
Colby DW, Chu YJ, Cassady J, Duennwald M, Zazulak H, Webster JM, Messer A, Lindquist S, Ingram VM, Wittrup KD. Potent inhibition of huntingtin aggregation and cytotoxicity by a disulfide bond-free single-domain intracellular antibody. PNAS Early Edition. DOI 10.1073/pnas.0408134101. Abstract
Wacker JL, Zareie MH, Fong H, Sarikaya M, Muchowski PJ. Hsp70 and Hsp40 attenuate formation of spherical and annular polyglutamine oligomers by partitioning monomer. Nat Struct Mol Biol. 2004 Dec;11(12):1215-22. Epub 2004 Nov 14. Abstract