The threshold for polyglutamine-expansion protein aggregation and cellular toxicity is dynamic and influenced by aging in Caenorhabditis elegans - PubMed (original) (raw)

The threshold for polyglutamine-expansion protein aggregation and cellular toxicity is dynamic and influenced by aging in Caenorhabditis elegans

James F Morley et al. Proc Natl Acad Sci U S A. 2002.

Abstract

Studies of the mutant gene in Huntington's disease, and for eight related neurodegenerative disorders, have identified polyglutamine (polyQ) expansions as a basis for cellular toxicity. This finding has led to a disease hypothesis that protein aggregation and cellular dysfunction can occur at a threshold of approximately 40 glutamine residues. Here, we test this hypothesis by expression of fluorescently tagged polyQ proteins (Q29, Q33, Q35, Q40, and Q44) in the body wall muscle cells of Caenorhabditis elegans and show that young adults exhibit a sharp boundary at 35-40 glutamines associated with the appearance of protein aggregates and loss of motility. Surprisingly, genetically identical animals expressing near-threshold polyQ repeats exhibited a high degree of variation in the appearance of protein aggregates and cellular toxicity that was dependent on repeat length and exacerbated during aging. The role of genetically determined aging pathways in the progression of age-dependent polyQ-mediated aggregation and cellular toxicity was tested by expressing Q82 in the background of age-1 mutant animals that exhibit an extended lifespan. We observed a dramatic delay of polyQ toxicity and appearance of protein aggregates. These data provide experimental support for the threshold hypothesis of polyQ-mediated toxicity in an experimental organism and emphasize the importance of the threshold as a point at which genetic modifiers and aging influence biochemical environment and protein homeostasis in the cell.

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Figures

Fig 1.

Fig 1.

Length-dependent aggregation of polyQ-YFP fusion proteins in C. elegans. Epifluoresence micrographs of 3- to 4-day-old C. elegans expressing different lengths of polyQ-YFP (Q0, Q19, Q29, Q33, Q35, Q40, Q44, Q64, Q82). (Bar = 0.1 mm.)

Fig 2.

Fig 2.

Determination of polyQ-YFP solubility in living animals by using FRAP. (Left: A, D, G, J, M) Merged phase-contrast and fluorescence images (pseudocolored yellow). (Center: B, E, H, K, N) Fluorescence images of the same region before photobleaching (prebleach). Boxes indicate the area that was subjected to photobleaching. (Right: C, F, I, L, O) Fluorescence images of recovery at the indicated times after photobleaching. The earliest time point possible to assess recovery of the chimeric YFP signal was at 3 s. (Bars = 3 μm.)

Fig 3.

Fig 3.

Expression of polyQ expansions in C. elegans muscle results in a motility defect that directly corresponds to aggregate formation. (A) Time-lapse micrographs illustrating tracks left by 5-day-old wild-type (N2) and Q82 animals 2 and 30 min after being placed at the position marked by the red arrow. (B) Quantitation of motility index for 4- to 5-day-old Q0, Q19, Q29, Q35, Q40, Q82, and unc-54(r293) animals. Data are mean ± SD for at least 50 animals of each type as a percentage of N2 motility. (C_–_E) Epifluoresence micrographs of late larval/young adult Q40 animals illustrating various numbers of aggregates in different animals. (Bar = 0.1 mm.) (F) Comparison of motility and aggregate number in adult Q40 animals (squares) and nontransgenic siblings (circles, no aggregates). Aggregate number is representative of number of muscle cells affected as the body-wall muscle cells had, on average, 1.3 ± 0.6 aggregates per cell (n = 212).

Fig 4.

Fig 4.

Influence of aging on polyQ aggregation and toxicity. (A) Accumulation of aggregates in Q82 (○), Q40 (•), Q35 (□), Q33 (▪), Q29 (▵), and Q0 (▴) during aging. Data are mean ± SEM. Twenty-four animals of each type are represented at day 1. Cohort sizes decreased as animals died during the experiment, but each data point represents at least five animals. (B) Motility index as a function of age for the same cohorts of animals described in A. Data are mean ± SD as a percentage of age-matched Q0 animals. (C_–_E) Epifluorescence micrographs of the head of an individual Q35 animal at 4 (C), 7 (D), and 10 (E) days of age, illustrating age-dependent accumulation of aggregates. Arrowheads indicate positions of the same aggregates on different days. In E, the animal is rotated slightly relative to its position in D.

Fig 5.

Fig 5.

An extended lifespan mutation delays polyQ aggregate accumulation and onset of toxicity. (A) Differential interference contrast (Left) and epifluorescence (Right) micrographs showing embryos expressing Q82 in wild-type (Top), age-1(hx546) (Middle), or age-1(hx546);daf-16(RNAi) (Bottom) genetic backgrounds. (Bars = 5 μm.) (B) Aggregate accumulation in larval animals expressing Q40 or Q82 in the indicated genetic backgrounds relative to aggregate accumulation in wild-type background. Mean ± SEM. (C) Motility index for animals expressing Q40 or Q82 in the indicated genetic backgrounds. Data are mean ± SEM for 30 animals of each type. Motility of nontransgenic wild-type and age-1 animals was similar to that of wild-type (N2).

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