Accelerated accumulation of misfolded prion protein and spongiform degeneration in a Drosophila model of Gerstmann-Sträussler-Scheinker syndrome - PubMed (original) (raw)

Comparative Study

Accelerated accumulation of misfolded prion protein and spongiform degeneration in a Drosophila model of Gerstmann-Sträussler-Scheinker syndrome

Brendan A Gavin et al. J Neurosci. 2006.

Abstract

Prion diseases are CNS disorders that can occur in sporadic, infectious, and inherited forms. Although all forms of prion disease are associated with the accumulation of pathogenic conformers of the prion protein, collectively termed PrP(Sc), the mechanisms by which PrP(Sc) molecules form and cause neuronal degeneration are unknown. Using the bipartite galactosidase-4-upstream activating sequence expression system, we generated transgenic Drosophila melanogaster heterologously expressing either wild-type (WT) or mutant, disease-associated (P101L) mouse PrP molecules in cholinergic neurons. Transgenic flies expressing neuronal P101L PrP molecules exhibited severe locomotor dysfunction and premature death as larvae and adults. These striking clinical abnormalities were accompanied by age-dependent accumulation of misfolded PrP molecules, intracellular PrP aggregates, and neuronal vacuoles. In contrast, transgenic flies expressing comparable levels of WT PrP displayed no clinical, pathological, or biochemical abnormalities. These results indicate that transgenic Drosophila expressing neuronal P101L PrP specifically exhibit several hallmark features of human Gerstmann-Sträussler-Scheinker (GSS) syndrome. Because the rates of abnormal PrP accumulation and clinical progression are highly accelerated in Drosophila compared with the rates of these processes in rodents or humans, the P101L mutant may be used for future genetic and pharmacologic studies as a novel invertebrate model of GSS.

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Figures

Figure 1.

Figure 1.

WT and P101L prion protein expression in the brains of transgenic Drosophila. Western blot of head homogenates from transgenic flies expressing either WT PrP or P101L in cholinergic neurons is shown, using D13 Fab to probe PrP levels. A, GAL4-Cha2, PRNP, and P101LA are second chromosomal inserts; GAL4-Cha3 and P101LD inserts are on the third chromosome. All flies were ∼1 week old at the time of sample preparation. B, The GAL4-Ddc insert is on the third chromosome. C, Two copies of P101L, but only one copy of GAL4-Cha, are required for P101L expression. Flies were ∼3 d old at the time of sample preparation. PRNP, P element containing a wild-type copy of mouse PRNP gene fused to the yeast UAS enhancer; P101L, P element containing a mutant copy of mouse PRNP gene, with proline to leucine substitution at codon 101, fused to the yeast UAS enhancer. Heterozygotes are indicated as transgene/+. Molecular weight bands indicate 40, 25, and 20 kDa.

Figure 2.

Figure 2.

P101L Accumulates as aggregates in the brains of transgenic larvae. Immunological staining and ultrastructural analysis of larval brain sections is shown. A, Whole-mount third-instar larval brain samples were prepared, fixed, and stained with AH6 Mab, as described in methods. P101L larval brains were homozygous for the P101L insert, but may have carried either one or two copies of the GAL4-Cha2 driver. All PRNP flies were heterozygous for the PRNP insert and for the GAL4-Cha2 driver. Green fluorescence is caused by the expression of GFP in cholinergic neurons, whereas Texas Red fluorescence indicates PrP staining. PrP (Texas Red) is displayed as magenta to be red/green color blind compatible. The colocalization of GFP (green) and Texas Red appears as white. Higher magnification of GAL4-Cha2;P101L samples is shown in three far right panels. Arrows indicate aggregated PrP both in the cell periphery and cytoplasm. B, WT or P101L PrP-expressing third-instar larval brain samples stained with toluidine blue. P101L (left) shows a healthy brain section, whereas in P101L (right) dark punctate staining is observed (arrows). Scale bars: A, 10 μm; B, 20 μm.

Figure 3.

Figure 3.

Climbing activity of adult P101L and WT PrP expressing transgenic flies. The climbing assay was measured as described in Materials and Methods using flies that were <1 d old. Thirty flies from each group were examined in 10 consecutive climbing assay tests. Each bar represents the percentage of flies from each group that failed to climb >4 cm in the climbing chamber within 30 s. Asterisks indicate a significant difference between nontransgenic controls and PrP-expressing flies. PRNP;Cha3 and PRNP;Ddc were both heterozygous for PRNP. The χ2 value (df = 1) = 41.71 with p < 0.0001 using a contingency table analysis. NT, Nontransgenic controls; Cha2, GAL4-Cha2; Cha3, GAL4-Cha3; Ddc, GAL4-Ddc. Error bars indicate SEM.

Figure 4.

Figure 4.

Survival curve of P101L and WT PrP-expressing transgenic flies. Flies from each genotype were maintained at 25°C in fresh food vials changed every other day for the duration of this study. The graph reflects the percentage of surviving animals with dead flies recorded daily. The PRNP/+;GAL4-Ddc and PRNP;Cha3 survival assays are ongoing experiments.

Figure 5.

Figure 5.

Neuropathology of adult P101L and WT PrP-expressing transgenic flies. A–C, Hematoxylin- and eosin-stained frontal sections from 1-d-old GAL4-Cha2 flies expressing WT PrP (A) show minimal vacuolation (arrow). One-day-old GAL4-Cha2 flies expressing P101L (B) are also histologically normal. Twenty-eight-day-old flies expressing P101L show prominent vacuolar pathology (C, arrows). D–G, Flies expressing WT PrP show accumulation of prion protein from day 1 (D) to day 28 (E). Flies expressing P101L also accumulate prion protein from day 1 (F) to day 28 (G) and in addition show a more punctate pattern of immunoreactivity in both the neuropil and cell cortex. Note also the optic lobe vacuole in the aged P101L animal (G, arrow). H, At higher magnification, P101L flies show prominent intracellular inclusion formation in neuronal cell bodies (arrows) and punctate neuropil structures (arrowhead). Scale bars: (in G) A–G, 50 μm; H, 10 μm. I, Average number of vacuoles observed in hemibrain sections from adult flies. Black bars, PRNP/GAL4-Cha2; red bars, GAL4-Cha2;P101L. Asterisks indicate significance for number of vacuoles; ***p < 0.005 and was determined using Excel software (Microsoft, Redmond, WA) with supplementary Student's t test. Error bars indicate SD.

Figure 6.

Figure 6.

Transgenic flies accumulate misfolded P101L PrP in an age-dependent manner. Biochemical analysis of brain homogenate samples from transgenic GAL4-Cha2 flies is shown. All flies used in the experiments designed for the biochemical analysis of P101L PrP, unless stated otherwise, were at least 3 weeks in age. Molecular weight bands shown indicate 40, 25, and 20 kDa. A, For each sample indicated, 80 fly heads were homogenized to generate each sample, and brain homogenates were subjected to 15B3 immunoprecipitation according to the protocol described in Materials and Methods. The input reflects one-fourth of the total amount of protein loaded in the immunoprecipitation experiment. The Western blots were probed with a 6H4-HRP conjugate. B, For each sample indicated, 40 fly heads were homogenized and subjected to immunoprecipitation using the 15B3 antibody. The Western blot was probed with a 6H4-HRP conjugate. C, Head homogenates were solubilized in 2% Sarkosyl for 1 h followed by a 1 h 100,000 × g centrifugation, and samples were analyzed by Western blot. T, Total (original sample); S, soluble; P, pellet. D, Head homogenates were treated with or without 10 μg/ml PK for 1 h at 4°C and subsequently analyzed by Western blot using Fab D13.

Comment in

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