The Social Amoeba Dictyostelium discoideum Is Highly Resistant to Polyglutamine Aggregation

Abstract

The expression, misfolding, and aggregation of long repetitive amino acid tracts are a major contributing factor in a number of neurodegenerative diseases, including C9ORF72 amyotrophic lateral sclerosis/frontotemporal dementia, fragile X tremor ataxia syndrome, myotonic dystrophy type 1, spinocerebellar ataxia type 8, and the nine polyglutamine diseases. Protein aggregation is a hallmark of each of these diseases. In model organisms, including yeast, worms, flies, mice, rats, and human cells, expression of proteins with the long repetitive amino acid tracts associated with these diseases recapitulates the protein aggregation that occurs in human disease. Here we show that the model organism Dictyostelium discoideum has evolved to normally encode long polyglutamine tracts and express these proteins in a soluble form. We also show that Dictyostelium has the capacity to suppress aggregation of a polyglutamine-expanded Huntingtin construct that aggregates in other model organisms tested. Together, these data identify Dictyostelium as a novel model organism with the capacity to suppress aggregation of proteins with long polyglutamine tracts.

Keywords: Dictyostelium; neurodegenerative disease; polyglutamine; protein aggregation; protein folding.

FIGURE 1.

Dictyostelium have multiple α-polyQ-reactive bands. A, Dictyostelium lysate contains multiple 1C2 (α-polyQ)-reactive bands. To obtain equal loading, 40 μg of protein from each organism were separated by SDS-PAGE, transferred to PVDF, and probed with α-polyQ antibody (43). HEK293 cells transfected with either GFP or ^GFPataxin-1^Q82 were used as negative and positive controls. B, Ponceau stain of protein loading in A. Samples from A were analyzed by Ponceau stain prior to Western blotting.

FIGURE 2.

Dictyostelium express α-polyQ-reactive bands throughout the developmental cycle. A, diagram of the life cycle of Dictyostelium. Dictyostelium exist as a free-living amoebae (0 h) until experiencing stress (e.g. starvation) at which point they aggregate (6 h) and proceed through a developmental process (12, 16, and 18 h) culminating in the formation of a fruiting body (24 h) that can release spores, allowing for transportation to new feeding sites. B, Dictyostelium express polyglutamine proteins throughout their developmental process. Dictyostelium cells were starved (0 h), and cells were collected at the indicated time points. Time points correlate with the different developmental stages outlined in A. C, Direct Blue staining of protein loading in B. Samples from B were analyzed by Direct Blue prior to Western blotting.

FIGURE 3.

Dictyostelium polyglutamine proteins are soluble. A, differential centrifugation reveals that endogenous α-polyQ-reactive bands are present in the soluble fraction. Dictyostelium lysate and HEK293 cells transfected with either GFP or ^GFPataxin-1^Q82 were separated into supernatant (S) and pellet (P) fractions. Samples were probed with α-polyQ antibody. B, filter trap analysis fails to identify α-polyQ-reactive bands in Dictyostelium. Either Dictyostelium cell lysate or lysate from HEK293 cells transfected with GFP, ^GFPataxin-1^Q30, or ^GFPataxin-1^Q82 was analyzed by filter trap assay.

FIGURE 4.

Dictyostelium are resistant to aggregation of a long polyglutamine tract. A, GFP-expanded Htt exon 1 aggregates in yeast in a polyglutamine length-dependent manner. Yeast were transformed with either ^GFPHtt^Q25 or ^GFPHtt^Q103 under the control of the GAL1 promoter. Changing the medium from glucose- to galactose-containing medium for 3 h induced expression. Cells were then analyzed by confocal microscopy. B, a GFP-Htt exon 1 construct aggregates in human cells in a polyglutamine length-dependent manner. HEK293 cells were transfected with either ^GFPHtt^Q23or ^GFPHtt^Q73 for 48 h prior to analysis by confocal microscopy. White arrowheads indicate aggregates. C, polyglutamine expanded Htt exon 1 does not form aggregates in Dictyostelium. Dictyostelium were selected with G418 for 4 days prior to analysis by confocal microscopy. D, biochemical analysis of polyglutamine length-dependent aggregation in yeast. Yeast expressing either ^GFPHtt^Q25 or ^GFPHtt^Q103 were analyzed by differential centrifugation. E, biochemical analysis of polyglutamine length-dependent aggregation in human cells. HEK293 cells expressing either GFP, ^GFPHtt^Q23, or ^GFPHtt^Q74 were analyzed by differential centrifugation. F, biochemical analysis of ^GFPHtt^Q25 or ^GFPHtt^Q103 aggregation in Dictyostelium. GFP, ^GFPHtt^Q25, or ^GFPHtt^Q103 was transformed into Dictyostelium, and lysates were analyzed by differential centrifugation. G, filter trap analysis of Dictyostelium expressing ^GFPHtt^Q103 fails to detect aggregates. Aggregation of normal and expanded polyglutamine Htt exon 1 was compared in yeast, HEK293 cells, and Dictyostelium by filter trap analysis. H, GFP is not cleaved from ^GFPHtt^Q25/103 in Dictyostelium. Dictyostelium expressing GFP, ^GFPHtt^Q25, or ^GFPHtt^Q103 were collected, and 25 μg of lysate were analyzed to ensure that GFP was not being cleaved from the ^GFPHtt^Q25/103 proteins. * indicates a cross-reacting band. I, ^GFPHtt^Q74/103 is expressed to similar levels in Dictyostelium and human cells. Twenty-five micrograms of cell lysate from Dictyostelium and human cells expressing ^GFPHtt^Q103 and ^GFPHtt^Q74, respectively, were analyzed by Western blotting. Direct Blue staining was performed to visualize the total protein loaded. Scale bars, 5 μm in A and 25 μm in B and C.

FIGURE 5.

^GFPHtt^Q103 aggregates can be detected in aged Dictyostelium. A, rare GPF-positive puncta can be detected in aged Dictyostelium cultures. Freshly thawed Dictyostelium cells were transformed with ^GFPHtt^Q25 or ^GFPHtt^Q103 and maintained for 5 weeks. White arrowheads indicate aggregates. Scale bars, 200 μm. B, ^GFPHtt^Q103 puncta are insoluble. A representative image of the FRAP analysis is shown. Boxed area indicates the region of photobleaching. C, FRAP analysis was performed, and samples were allowed to recover for the indicated times. Error bars represent S.E. D, filter trap analysis of Dictyostelium expressing ^GFPHtt^Q103 fails to detect aggregates in aged cells. Lysate from aged Dictyostelium expressing ^GFPHtt^Q25 or ^GFPHtt^Q103 was analyzed by filter trap assay to detect ^GFPHtt^Q103. Lysates from yeast expressing either ^GFPHtt^Q25 or ^GFPHtt^Q103 were used as negative and positive controls.

FIGURE 6.

Dictyostelium encode a large number of small heat shock proteins. Bioinformatics analysis of the number of HSP70, HSP90, and sHSP homologs in humans, yeast, and Dictyostelium was performed.