Drosophila melanogaster in the study of human neurodegeneration

Abstract

Human neurodegenerative diseases are devastating illnesses that predominantly affect elderly people. The majority of the diseases are associated with pathogenic oligomers from misfolded proteins, eventually causing the formation of aggregates and the progressive loss of neurons in the brain and nervous system. Several of these proteinopathies are sporadic and the cause of pathogenesis remains elusive. Heritable forms are associated with genetic defects, suggesting that the affected protein is causally related to disease formation and/or progression. The limitations of human genetics, however, make it necessary to use model systems to analyse affected genes and pathways in more detail. During the last two decades, research using the genetically amenable fruitfly has established Drosophila melanogaster as a valuable model system in the study of human neurodegeneration. These studies offer reliable models for Alzheimer's, Parkinson's, and motor neuron diseases, as well as models for trinucleotide repeat expansion diseases, including ataxias and Huntington's disease. As a result of these studies, several signalling pathways including phosphatidylinositol 3-kinase (PI3K)/Akt and target of rapamycin (TOR), c-Jun N-terminal kinase (JNK) and bone morphogenetic protein (BMP) signalling, have been shown to be deregulated in models of proteinopathies, suggesting that two or more initiating events may trigger disease formation in an age-related manner. Moreover, these studies also demonstrate that the fruitfly can be used to screen chemical compounds for their potential to prevent or ameliorate the disease, which in turn can directly guide clinical research and the development of novel therapeutic strategies for the treatment of human neurodegenerative diseases.

Fig. (1)

Drosophila as a model organism in the study of age-related neurodegeneration. (a) The arthropod Drosophila melanogaster belongs to a subspecies of the Drosophilidae, dipteran insects that fit on a pencil tip and can be easily kept en masse in the laboratory. Their anatomy displays characteristic features such as compound eyes, wings and bristles that can be used as phenotypes to study neurodegeneration without affecting the survival of the fly. (b) The lifespan of Drosophila depends on diet and stress and varies between 40-120 days. The more rigid a diet (e.g. cornmeal), the longer a fly can live, whereas increase in carbohydrates and cholesterol (e.g. 15% sugar/yeast) can lower life expectancy. These similarities to human ageing and lifespan, together with a highly conserved genetic makeup, make Drosophila a powerful model system in the study of adult-onset, age-related neurodegeneration.

Fig. (2)

Adult brain of Drosophila. (a) Confocal image of a parafin cross-section through the adult Drosophila head; auto-immunofluorescence visualises the ommatidia of the compound eye (CE), the optic lobe (OL) and the central brain (CB). Note that cell bodies (arrowheads) are topologically separated from axonal extensions which make up the neuropil. (b) Confocal image of a whole mount adult brain immunolabelled with anti-nc82 which recognises the Bruchpilot protein that is specifically enriched in active zones of synaptic terminals. This allows the visualisation of cortical areas in the fly brain, including optic lobes (OL), antennal lobes (AL), superior protocerebrum (SP), lateral protocerebrum (LP), mushroom bodies (MB), deuterocerebrum (D), and subesophageal ganglion (SG). (c) Optical cross-section of a whole-mount adult brain of a transgenic Drosophila immunolabeled with anti-nc82; tyrosine hydroxylase (TH)-specific Gal4 drives UAS-mCD8:GFP expression, a membrane-tagged GFP (TH>mGFP). Because TH is the rate-limiting enzyme of dopamine synthesis, this transgenic Gal4/UAS combination visualises dopaminergic neurons and their axonal extensions (white/light grey). Based on this method, dopaminergic neurons can not only be monitored, but also manipulated, and cell numbers as well as axonal projections can be used as phenotypic read-out parameters to study parkinsonism in Drosophila. Scale bar: 50 µm.

Fig. (3)

Drug treatment in Drosophila. (a) Four vials with flies are kept on cornmeal food each of which has been supplemented with a different concentration of the same drug. The applied drug is screened for its potential to either enhance or suppress a given neurodegeneration phenotype that has been caused by targeted genetic manipulation, such as rough eyes caused by mis-expression of human tau, a movement disorder caused by dysfunction of Drosophila TDP-43, or reduced lifespan caused by mis-expression of human ASYN. In this way, Drosophila models of neurodegeneration can be used to screen compound collections for their potential to prevent or ameliorate a specific neurodegenerative “disease”. (b) Three different concentrations of a drug (10 µM, 100 µM, and 1 mM) are chronically applied to ageing Drosophila, as compared to vehicle treated flies. The resulting effects on locomotion are quantified using a negative geotaxis assay: flies are shaken to the bottom of a vial/cylinder; their innate behaviour triggers them to move upwards (against geotaxis) and the time it takes them to reach the top is scored for a cohort of flies and multiple replicates. A calculus then determines the relative performance of these flies exposed to a given drug concentration and at a specific day. The graph shows that the geotaxis performance inversely correlates with drug concentration and age, suggesting that this drug enhances a movement disorder in a concentration-dependant and age-related manner.

Fig. (4)

Experimental study of Drosophila locomotor behaviour. (a) An adult wild-type fly (wt, arrow) is kept in an arena which can be a converted petri dish. The fly’s activity and movement is recorded with a high-speed video camera and a computer programme tracks the resulting trajectory during a given time-window (30sec). (b) 3min movement trajectory of a wt fly; (c) 3min trajectory of a mutant fly (ko) revealing motor deficits. Video-assisted movement tracking records locomotor behaviour and in turn allows the quantification of parameters that can be used to describe it, including walking activity, velocity, and distance travelled. By applying this method to Drosophila models of neurodegeneration, it is possible to mimic adult-onset neurodegenerative movement disorders including Parkinson’s disease, trinucleotide repeat expansion diseases, and motor neuron diseases, and to monitor their effect on neural circuits and behaviour, which in turn allows genetic dissection of the underlying pathogenic mechanism(s).