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. 1999 Sep 28;96(20):11404-9.
doi: 10.1073/pnas.96.20.11404.

Insoluble detergent-resistant aggregates form between pathological and nonpathological lengths of polyglutamine in mammalian cells

A Kazantsev  1 , E Preisinger, A Dranovsky, D Goldgaber, D Housman
Affiliations

Insoluble detergent-resistant aggregates form between pathological and nonpathological lengths of polyglutamine in mammalian cells

A Kazantsev et al. Proc Natl Acad Sci U S A. .

Abstract

Pathological degeneration of neurons in Huntington's disease and associated neurodegenerative disorders is directly correlated with the expansion of CAG repeats encoding polyglutamines of extended length. The physical properties of extended polyglutamines and the intracellular consequences of expression of polyglutamine expansion have been the object of intensive investigation. We have extended the range of lengths of polyglutamine produced by recombinant DNA methodology by constructing a library of CAG/CAA repeats coding for a range of 25-300 glutamine residues. We have investigated the subcellular localization, interaction with other polyglutamine-containing polypeptides, and the physical properties of aggregated forms of polyglutamine in the cell. Extended polyQ aggregated in the cytoplasm and was only transported to the nucleus when a strong nuclear localization signal was present. Polyglutamine below pathological lengths could be captured in aggregates and transported to ectopic cell locations. The CREB-binding protein (CBP), containing a homopolymeric stretch of 19 glutamines, was likewise found to coaggregate in a polyglutamine-dependent manner, suggesting that pathology in polyglutamine disease may result from cellular depletion of normal proteins containing polyglutamine. We have observed a striking detergent resistance in aggregates produced from polyglutamine of pathological length. This observation has led to the development of a fluorescence-based assay exploiting the detergent resistance of polyglutamine aggregates that should facilitate high-throughput screening for agents that suppress polyglutamine aggregation in cells.

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Figures

Figure 1
Figure 1
Expression of normal length (25Q) and extended (104Q) polyglutamine constructs in single COS-1 cells shown at high magnification. (A) Normal length (25Q) naked construct shows diffuse cytoplasmic expression pattern. (B) Extended polyglutamine construct (104Q) aggregates in the cytoplasm. Diffuse, presumably soluble material is not seen in cells with aggregates. (C) 104Q/TBP shows multiple aggregates within the nucleus. (D) 104Q/nucleolin shows a single nucleolar aggregate.
Figure 2
Figure 2
Coexpression of 104Q/nucleolin/EGFP (green) and 104Q/c-myc (red) at high magnification. Extended polyglutamines are colocalized (yellow) in the nucleus (blue) and the cytoplasm (not stained) of a single cell. Interactions between polyglutamines are sufficiently strong to relocate 104Q/c-myc into the nucleus.
Figure 3
Figure 3
Cotransfection of polyglutamine/EGFP constructs (green) and CBP/c-myc (red) at high magnification. (A) 25Q/EGFP (green) and CBP/c-myc (red) show diffuse cytoplasmic colocalization (yellow). (B) 104Q/EGFP (green) and full-length CBP/c-myc (red) colocalized in a single cytoplasmic aggregate. (C) 104Q/TBP/EGFP (green) and CBP/c-myc (red) colocalized in nuclear aggregates (yellow). (D) CBP/c-myc (red) is not included in nucleolar aggregates formed by 104Q/nucleolin/EGFP (green). (E) Full-length CBP/c-myc (red) colocalized (yellow) with 104Q/EGFP (green). (F) CBP/c-myc (red) with deletion of 19 glutamines and surrounding glutamine-rich region fails to coaggregate with 104Q/EGFP (green). (G) CBP deletion construct shows greater than 8-fold reduction of coaggregation compared with full-length CBP.
Figure 4
Figure 4
In situ assay demonstrates detergent-resistant insolubility of extended polyQ aggregates and coaggregates. (A) Low-power view of 25Q/EGFP cotransfected with 104Q (not stained), demonstrating aggregation of normal length polyQ by extended polyQ. (B) 25Q/EGFP transfected alone and treated with 2.5% SDS/2.5% Triton X-100 for 2 min. Normal length polyQ alone does not aggregate and EGFP fluorescence is not preserved. (C) 25Q/EGFP cotransfected with 104Q (not stained), treated with 2.5% SDS/2.5% Triton X-100. Aggregates remained fluorescent for up to 24 hr of detergent treatment. (D) Lower-power view of stable inducible live culture expressing 104Q/EGFP (green) and forming aggregates. (E) Same field as A after 2-min treatment with 2.5% SDS/2.5% Triton X-100. Aggregates were similar in size, number, and fluorescent intensity even after 24 hr of treatment at 37°C. (F) Low-power view of stable inducible live culture expressing 25Q/EGFP. (G) Same field as C after 2-min treatment with detergent as above. No remaining fluorescence is detectable.
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