Polyglutamine fibrillogenesis: the pathway unfolds.

N ine neurodegenerative diseases are caused by expanding CAG repeats coding for polyglutamine (polyGln) (1–4). These include Huntington’s disease, dentatorubral and pallidoluysian atrophy, several forms of spino-cerebellar ataxia, and spinal and bulbar muscular atrophy. Within the central nervous system, each disease has a distinctive pattern of degeneration, with considerable overlap among the diseases (5, 6). The genes containing CAG repeats show no homology to each other outside of the glutamine repeats, and most are genes of unknown function. Thus, speculation concerning pathogenesis has focused on the polyGln expansion itself. For all of these diseases, there is a threshold of repeat length that causes disease. This threshold varies somewhat among the different diseases, but is generally in the range of 35–45 consecutive glutamines. In all polyGln diseases, the age of disease onset is strongly correlated with polyGln length, so that above the threshold, a longer repeat results in an earlier age of onset. A pathological hallmark of these diseases is the aggregation of mutant polyGln protein, resulting in the formation of intranuclear inclusion bodies. In some of the diseases, inclusions have been observed in the cytoplasm, dendrites, and axonal processes. The inclusions are generally seen in affected areas of the brain (7, 8), though not limited to those neurons most likely to degenerate (9). Thus, whether inclusions are responsible for neurotoxicity has been controversial. Some studies have indicated a correlation between polyGln-containing inclusions and disease progression (10). However, in other studies, inclusion formation was dissociated from cytotoxicity (11, 12). In fact, inclusion formation may be, in part, a reflection of cellular protective mechanisms (13). Nevertheless, the inclusions are a useful marker for pathology and may provide clues to pathogenesis. Aggregation of mutant polyGln proteins can be observed biochemically using a filter trap assay (14, 15). Aggregation in vitro proceeds by means of a nucleation-dependent process and results in the accumulation of -sheet rich fibrillar structures detected by electron microscopy. Thus, the polyGln aggregation pathway appears to resemble that of A protein in Alzheimer’s disease and -synuclein in Parkinson’s disease, as well as other amyloidogenic proteins (16–18). Even if polyGln inclusions are not the major toxic species, the aggregation process appears linked to pathogenesis. Therefore, it is critical to understand the structure of both normal and mutant polyGln stretches. Detailed structural information on polyGln has been difficult to obtain, because both long and short stretches of synthetic polyGln peptides are quite insoluble. Nearly a decade ago, Max Perutz attempted to address this issue by using a Q15 peptide flanked by basic residues to improve its solubility. He found this peptide to adopt -structure, and constructed an atomic model of poly(L-glutamine) consisting of antiparallel -sheets held together by hydrogen bonds between main-chain and side-chain amides (Fig. 1a). This structure, described as a ‘‘polar zipper,’’ has been influential for studies of polyGln aggregation (14, 19, 20). Computer modeling studies have generated additional possible structures for expanded polyGln, such as parallel -sheets (21), -hairpins, and highly compact random coil (22) or -sheet structures (Fig. 1 b–e). Based on x-ray diffraction and electron microscopy data, Perutz and his group (23, 24) have recently suggested a polyGln -helix model with 20 residues per turn (Fig. 1f). Biophysical analysis of synthetic or recombinant polyGln peptides with stretches containing 5–44 consecutive glutamines have demonstrated that monomeric polyGln is unstructured (25– 27). In contrast, expanded polyGln aggregates derived from isolated polyGln peptides or from recombinant polyGlncontaining proteins adopt -sheet structure, as shown by x-ray fiber diffraction studies, circular dichroism, Fourier transform infrared spectroscopy, and other methods (20, 25, 28, 29). Thus, it is likely that expanded polyGln sequences in the aggregated state are -sheets, though detailed structural information is currently not available. In a recent issue of PNAS, Thakur and Wetzel (30) provide a mutational analysis to address the question of polyGln aggregate structure. Previously, the Wetzel laboratory demonstrated that polyGln peptides have aggregation properties similar to exon-1 huntingtin (25). In the current study, they used an ingenious strategy of inducing -turns in synthetic peptides with Pro–Gly pairs at different intervals within a long polyGln stretch. To enhance the solubility of their peptides, they incorporated charged residues at both ends of the polyGln peptides, as introduced by Max Perutz and colleagues. Thakur and Wetzel investigated the influence of Pro–Gly