Cytoplasmic aggregates trap polyglutamine-containing proteins and block axonal transport in a Drosophila model of Huntington's disease

Abstract

Huntington's disease is an autosomal dominant neurodegenerative disorder caused by expansion of a polyglutamine tract in the huntingtin protein that results in intracellular aggregate formation and neurodegeneration. Pathways leading from polyglutamine tract expansion to disease pathogenesis remain obscure. To elucidate how polyglutamine expansion causes neuronal dysfunction, we generated Drosophila transgenic strains expressing human huntingtin cDNAs encoding pathogenic (Htt-Q128) or nonpathogenic proteins (Htt-Q0). Whereas expression of Htt-Q0 has no discernible effect on behavior, lifespan, or neuronal morphology, pan-neuronal expression of Htt-Q128 leads to progressive loss of motor coordination, decreased lifespan, and time-dependent formation of huntingtin aggregates specifically in the cytoplasm and neurites. Huntingtin aggregates sequester other expanded polyglutamine proteins in the cytoplasm and lead to disruption of axonal transport and accumulation of aggregates at synapses. In contrast, Drosophila expressing an expanded polyglutamine tract alone, or an expanded polyglutamine tract in the context of the spinocerebellar ataxia type 3 protein, display only nuclear aggregates and do not disrupt axonal trafficking. Our findings indicate that nonnuclear events induced by cytoplasmic huntingtin aggregation play a central role in the progressive neurodegeneration observed in Huntington's disease.

Figures

Fig. 1.

Generation of a Drosophila transgenic model of HD. (A) Domain structure of the human and Drosophila Htt homologs with predicted Huntington elongation factor 3 protein phosphatase 2A Tor1 (HEAT)-like motifs indicated. The 548-aa N terminus of human Htt used for transgenic construction is indicated. (B) Heat-shock induction of Htt in Q0 and Q128 pHS strains. Western blotting was performed with an antibody generated to the N terminus of human Htt that recognizes both the Q0 and Q128 variants. (C and D) External morphology and pseudopupil analysis of transgenic Drosophila expressing either Htt-Q0 (C) or Htt-Q128 (D) driven by the eye-specific GMR-GAL4 driver. Flies were aged for 2-4 days at 25°C before analysis. Htt-Q128 causes a rough-eye phenotype with loss of pigmentation, abnormal bristle pattern, and photoreceptor degeneration.

Fig. 2.

Physiological and behavioral analysis of Htt-Q128-expressing transgenic Drosophila. (A) Both UAS-Htt-Q128/GMR-GAL4 and pHS-Htt-Q128 exhibit reduced photoreceptor depolarization and abolished synaptic transmission, as indicated by loss of the on/off transients (arrows) in electroretinogram recordings. (B) Extracellular recordings from the DLM flight muscles of Htt-Q0 and Htt-Q128 Drosophila are shown. A developmental heat-shock paradigm that induces Htt-Q128 expression results in abnormal seizure activity in the flight circuit at 38°C. (A and B) Drosophila were aged for 2-4 days at 25°C. (C) Quantitative analysis of wandering third-instar larval crawling behavior indicates that pan-neuronal expression of Htt-Q128 disrupts motor pattern generation, resulting in a significant decrease in locomotor speed. Thirty larvae were analyzed for each genotype. (D) Viability analysis of Drosophila maintained at 25°C indicates that pan-neuronal expression of Htt-Q128 with a second chromosome elav-GAL4 driver results in decreased lifespan compared with control strains. Expression of Htt-Q128 with the stronger X-chromosome elav-GAL4 driver C155 leads to 100% pharate adult lethality.

Fig. 3.

Cytoplasmic aggregation of Htt-Q128 in neuronal and nonneuronal tissues. (A) Immunocytochemical detection of Htt (red) and neuronal membranes by anti-horseradish peroxidase (green) in multidendritic neurons of Htt-Q0-expressing third-instar larva. Htt-Q0 is found diffusely throughout the cytoplasm. (B) Immunocytochemical detection of Htt (red) and horseradish peroxidase (green) in multidendritic neurons of Htt-Q128-expressing third-instar larva. Unlike Htt-Q0, Htt-Q128 is found in cytoplasmic aggregates throughout the cell body and neurites. (C-E) Expression of UAS-Htt-Q128 (red) and UAS-GFP-nls (green) by tubP-GAL4 in the CNS (C), gut (D), salivary gland (E), trachea (F), muscle (G), and epidermis (H) of third-instar larvae. In all cases, cytoplasmic aggregates are observed. However, muscle and epidermis form far fewer aggregates than other tissues. In polarized cells like the gut, basolateral transport of Htt aggregates is observed, with an absence of aggregates in the apical domain. The nucleus is indicated by N, and Htt aggregates are indicated by arrows.

Fig. 4.

Protein context is important for polyQ-mediated aggregation and aggregate localization. (A-D) Immunolocalization of aggregates in third-instar larvae expressing Htt-Q128 (A), Q127 (B), SCA3-Q78 (C), and Dsh-Q108 (D) in multidendritic neurons with the C155 elav-GAL4 driver. Htt-Q128 aggregates are exclusive to the cytoplasm, whereas Q127 and SCA3-Q78 aggregates are found only in the nucleus. Dsh-Q108 is mostly diffuse in the cytoplasm, with few aggregates visible. The nucleus is represented by N. (E and F) Double transgenic third-instar larvae expressing Htt-Q128 and Q127 (E) or Htt-Q128 and SCA3-Q78 (F) with the C155 elav-GAL4 driver were dissected and immunostained for Htt (red) and SCA3/Q127 (green). Multidendritic sensory neurons were identified as in A-D and assayed for aggregate colocalization. No colocalization of aggregates is observed, indicating distinct nuclear and cytoplasmic aggregation pathways. Similar results are observed in the CNS (H) and in nonneuronal tissues such as the salivary glands (G). (I) In contrast, in double transgenic larvae expressing Htt-Q128 and Dsh-Q108 with C155 elav-GAL4, Htt-Q128 (red) is able to completely sequester Dsh-Q108 (green) into aggregates, as indicated by colocalization (yellow) in Right.

Fig. 5.

Htt-Q128 aggregates block axonal transport. (A and B) Expression of Htt-Q128 by GMR-GAL4 results in axonal transport of Htt aggregates (arrows) in the developing visual system. (C and D) Expression of Htt-Q128 (D), but not Htt-Q0 (C), by C155 elav-GAL4 results in axonal transport and synaptic accumulation of Htt aggregates at neuromuscular junctions in third-instar larvae. Axonal transport of aggregates is abundant in Htt-Q128-expressing third-instar larvae (F) but absent in animals expressing Htt-Q0 (E), Q127 (G), or Dsh-Q108 (H). Nerves are stained green by anti-horseradish peroxidase, and polyQ proteins are visualized in red. (I) Consistent with the trapping of Dsh-Q108 by Htt-Q128 aggregates in the cell bodies of multidendritic sensory neurons, Htt-Q128 also traps and transports Dsh-Q108 in peripheral nerves. (J and K) Expression of Htt-Q128 (K), but not Htt-Q0 (J), results in an accumulation of the synaptic protein synaptotagmin I in sites of axonal swelling, colocalizing with large accumulations of Htt-Q128 aggregates. Synaptotagmin I is not trapped in Htt-Q128 aggregates, as indicated by the lack of protein colocalization in smaller Htt-Q128 aggregates, but rather concentrates specifically at sites where larger aggregate accumulations result in swollen axons, indicating blockage of axonal transport.