Drosophila melanogaster as a model to study autophagy in neurodegenerative diseases induced by proteinopathies

Abstract

Proteinopathies are a large group of neurodegenerative diseases caused by both genetic and sporadic mutations in particular genes which can lead to alterations of the protein structure and to the formation of aggregates, especially toxic for neurons. Autophagy is a key mechanism for clearing those aggregates and its function has been strongly associated with the ubiquitin-proteasome system (UPS), hence mutations in both pathways have been associated with the onset of neurodegenerative diseases, particularly those induced by protein misfolding and accumulation of aggregates. Many crucial discoveries regarding the molecular and cellular events underlying the role of autophagy in these diseases have come from studies using Drosophila models. Indeed, despite the physiological and morphological differences between the fly and the human brain, most of the biochemical and molecular aspects regulating protein homeostasis, including autophagy, are conserved between the two species.In this review, we will provide an overview of the most common neurodegenerative proteinopathies, which include PolyQ diseases (Huntington's disease, Spinocerebellar ataxia 1, 2, and 3), Amyotrophic Lateral Sclerosis (C9orf72, SOD1, TDP-43, FUS), Alzheimer's disease (APP, Tau) Parkinson's disease (a-syn, parkin and PINK1, LRRK2) and prion diseases, highlighting the studies using Drosophila that have contributed to understanding the conserved mechanisms and elucidating the role of autophagy in these diseases.

Keywords: Drosophila melanogaster; animal model; autophagy; neurodegeneration; non-autonomous signaling; protein-aggregate; protein-misfolding; proteinopathies.

Figure 1

Scheme of a pipeline to characterize genes associated with proteinopathies and to perform High-throughput Screens with small chemical compounds, to develop new therapeutical strategies in humans. (A–C) From the identification of a gene related to a human disease to the generation of the transformants carrying the GOI. (D–F) The effect of the mutant-disease genes can be tested at cellular and behavioral levels. (D) For example, the expression of exon1 of the human mutant Huntingtin containing 93-CAGs (HTTQ93) in the retina using the GMR-Gal4 promoter leads to loss of the pigmentation in the ommatidia of the compound eye (as seen in the upper image obtained using a stereo microscope) and to retinal degeneration accompanied by defects in tissue morphology and neuronal death [outlined by the white spot of missing tissues visible by transmission electron microscopy (TEM) showed in the images below; (Vernizzi et al, 2020)]. (E) Expression of mutant HTT in neurons using ELAV-Gal4 induces neuronal defects that can be indirectly quantified by measuring the decline over time of the animal motility (negative geotaxis assay). (F) Using the Elav promoter we can express the human HTT exon-1 with 97 CAGs as a fusion protein with GFP (HTT-GFP), and show the formation of HTT-GFP aggregates in larval neurons already at 48-72 hours of age. Photo in panel F, to the right is shown a larval brain of Elav-LexOP-HTTGFP-LexA larvae at 72 hrs AEL, control is to the left. BLUE stains the nuclei (inset 63x). (G–K) Potential pipeline for a High throughput screen (HTS) to identify drugs that reduce the formation of toxic aggregates. (G) Drosophila cells, induced to express the HTTGFP construct are cultured in medium containing small chemical compounds (libraries); analysis of the changes in GFP expression can be quantified using a microplate reader (TECAN). Compounds capable of reducing GFP expression will highlight potential pathways that could be involved in the reduction of mHTT aggregates. (J) Drosophila HD models can be used to perform genetic screens to analyze in vivo the expression of components of these pathways; (K) Drosophila HD models will be fed with the small compounds identified to analyze their effect in ameliorating animal motility or in reducing the size of mHTT aggregates; analyzed directly by immunofluorescence in the brain or by filter-trap assays using either organs or from whole animals. (H) For better performance, chemical drugs can be modified using ligand and structure-based drug design to improve their characteristics and then they can be tested again in vivo in Drosophila HD models such as in K. (I) Finally, the candidate drugs will be tested using human cells differentiated to iPSCs and then to neurons of glia, or directing to neurons iNs (Hong and Do, 2019) starting from cells of patients or from cells from healthy donors in which the specific mutation is introduced using the CRISPR/CAS9 technique.

Figure 2

Overview of the autophagic flux. The induction of the autophagic process is regulated upstream by 5†’-AMP-activated Protein Kinase (AMPK) and Target of Rapamycin (TOR), that modulate the activation of the Unc51-like Kinase 1 (ULK1/Atg1) via phosphorylation and the formation of the initiator the ULK1-Complex composed by ULK1, Autophagy-related protein 13 (ATG13), ATG101 and FIP200. Upon activation, ULK1/Atg1 Complex in turn activates via phosphorylation the phosphatidylinositol-3-kinase III (PI3KIII) Complex, which comprises vacuolar protein sorting 34 (VPS34), VPS15, ATG14 and Beclin-1. This allows its translocation on the ER membrane and the production of an isolation membrane enriched in phosphatidylinositol (3)-phosphate (PI3P). PI3P induces the recruitment of the PI3P Binding Complex, consisting of ATG5, ATG12, ATG16L and WD Repeat Domain, Phosphoinositide Interacting 2 protein (WIPI2), on the growing autophagophore (omegasome) and the Double FYVE-containing protein 1 (DFCP1). This process enhances the ATG3-mediated binding of Microtubule-associated protein 1A/1B-light chain 3 (LC3) to phosphatidylethanolamine (PE) on the autophagosomal membrane where is lipidated. This leads to its cleavage from LC3-I/ATG8a-I to LC3-II/ATG8-II, which is considered a feature of an active autophagic flux and can be visualized and quantified by western blot analysis (panel A). Ubiquitin-tagged proteins are recognized by specific autophagic adaptors/receptors, such as p62/SQSTM1/Ref2(P), with a mechanism that is selective for each different organelles or cellular structure called selective autophagy. The cargo receptors bind LC3/ATG8a and transport the cargo into the autophagophore where its content is hydrolyzed upon fusion with lysosome. Autophagosome formation can be visualized in vivo by ectopic expression of LC3/Atg8a fused to GFP or mCherry fluorescent proteins that form small “puncta” on the autophagosome membrane, and fluorescence can be quantified using imaging processing programs. Panel B shows the UAS-Atg8a-mCherry staining pattern under physiological conditions analyzed in the calyx region of Drosophila larval brain, where neuronal cells are visualized by expressing UAS-GFP using the ELAV-Gal4 promoter. Co-localization of mCherry puntae within GFP is used to quantify the presence of the autophagosome in neurons.