Studying polyglutamine diseases in Drosophila

Abstract

Polyglutamine (polyQ) diseases are a family of dominantly transmitted neurodegenerative disorders caused by an abnormal expansion of CAG trinucleotide repeats in the protein-coding regions of the respective disease-causing genes. Despite their simple genetic basis, the etiology of these diseases is far from clear. Over the past two decades, Drosophila has proven to be successful in modeling this family of neurodegenerative disorders, including the faithful recapitulation of pathological features such as polyQ length-dependent formation of protein aggregates and progressive neuronal degeneration. Additionally, it has been valuable in probing the pathogenic mechanisms, in identifying and evaluating disease modifiers, and in helping elucidate the normal functions of disease-causing genes. Knowledge learned from this simple invertebrate organism has had a large impact on our understanding of these devastating brain diseases.

Keywords: Atrophin-1; DRPLA; Dentatorubral-pallidoluysian atrophy; Drosophila model; HD; HTT; Huntingtin; Huntington's disease; Machado–Joseph disease; PolyQ diseases; Polyglutamine diseases; SBMA; SCA1; Spinobulbar muscular atrophy; Spinocerebellar ataxia.

Figure 1. HTT proteins and HD mutation

(A) Schematic illustration of the structure of amino acid glutamine (Q), which is encoded by the tri-nucleotide CAG. (B) HD is caused by an abnormal expansion of the glutamine tract (polyQ) located near the N-terminus of HTT protein. (C) Schematics of predicted secondary structures of human and Drosophila HTT proteins. Both are composed mainly of HEAT repeat (represented as cylinder boxes in the diagram, also see D). (D) Illustration of the proposed structure of the HEAT repeat, a ~40 amino acid long hairpin-like protein motif.

Figure 2. The wildtype Drosophila eye structure

(A) Scanning electron micrograph of a wild-type adult eye. (B) Tangential section of one ommatidium unit. High-magnification view. Neuronal photoreceptor cells (black) are surrounded by pigment cells (red). (C) Illustration of an ommatidium structure. The identity of each photoreceptor cell (black) is labeled. Pigment cells are painted in red.

Figure 3. Progressive neurodegeneration in a Drosophila HD model and its suppression by a modifier gene

(A-C) 30-day-old adult fly eyes. (A) GMR-Gal4 control. (B and C) HD model that expresses HTT exon 1 (HTTex1)-Q93 together with (B) a LacZ control or (C) wildtype dHsp110 protein. Note the dramatic de-pigmentation of the eye in (B), indicating the significant loss of underlying eye tissues, which is clearly suppressed by the co-expression of dHsp110 (C) but not LacZ (B). (D-G) Examination of photoreceptor cells in 7-day-old adult eyes, (D and E) visualized after dissection and immunofluorescent staining for F-Actin, or (F and G) visualized directly using pseudopupil technique without dissection. The seven well-organized photoreceptors in (D, F) wildtype (WT) were partially lost in (E, G) HTTex1-Q93 (HD) flies. Note that the pseudopupil method (F and G) produces comparable resolution for photoreceptor cells in the eye as that obtained by the more tedious dissection and staining approach (D and E).

Figure 4. Mutant HTT protein forms age-dependent aggregates in the fly brain

Confocal images of adult brains expressing HTTex1 with a 46 glutamine tract (HTTex1-Q46) at (A) day 2 and (B) day 30. (C and D) High-magnification views of the regions highlighted above. HTTex1-Q46 protein is evenly dispersed in mushroom bodies and other structures in young brains but forms prominent aggregates by day 30.

Figure 5. Aggregate formation by mutant HTT in Drosophila

Formation of aggregates by mutant HTT protein can be modeled and studied in (A) cultured Drosophila cells and (B-D) adult fly eyes. In these studies, mutant HTT exon 1 fragment is revealed by eGFP tag fused in frame at its C-terminus. (A) A double-labeling image of cultured Drosophila cells that express HTTex1-Q46. Aggregates (bright dots in top picture) are evident in some of the cells. The overall morphology of these cells are marked by staining for cytoskeletal protein F-actin (bottom picture in red), which reveals the sequestration of F-actin in these aggregates (bright dots in bottom picture). (B-D) Images of same adult fly eyes illuminated by (top panels) bright light to show the overall eye morphology and by (bottom panels) fluorescent light to reveal the presence of eGFP-label HTTex1 aggregates, respectively. (B) No fluorescent signal in the eye of a wildtype control fly (normal) that did not express human HTT protein. (C) No clear aggregates in the eye of a transgenic fly that expressed HTTex1 with 23 glutamine (HTTex1-Q23). (D) Numerous aggregates (bright dots) in the eye of a transgenic fly that expressed mutant HTTex1 with 103 glutamine (HTTex1-Q103).