A specific function for Ataxin1 in controlling the autophagic pathway in flies has not been described, however TOR inhibitors in flies were shown to ameliorate the toxic effect of mutant Ataxin1 through the release of the inhibitory role or TORC1 on the lysosomal transcription factor Mitf (Drosophila homologue of human TFEB; Bouche et al., 2016). Drosophila harbors a gene encoding for Ataxin1 (dAtx) that lacks the polyQ domain, nevertheless its overexpression led to phenotypes like those obtained by human ATXN1 overexpression, but different from those observed upon overexpression of the polyQ alone. Genetic experiments showed that Atx1 interacts through its conserved AXH domain with the zinc-finger transcription factor Senseless (Sens). This mechanism is conserved for its mammal’s homolog, the growth factor independence-1 (Gfi-1) and proposed as a potential pathogenic mechanism for SCA1 (Tsuda et al., 2005).

Expression of the human mutant ATXN1 gene in Drosophila’s neurons leads to retinal degeneration and loss of axonal projections of the interneurons in the ventral nerve cord of the brains (Fernandez-Funez et al., 2000), expression of the pathogenic form of SCA1 or SCA3 in Drosophila larval dendritic neurons reduced their arborization with disruption of F-actin dendritic structures, defect that is partially rescued by Rac-PAK signaling activation (Lee et al., 2011). In vivo screens using the adult eye helped define the role of chaperones to proteins with an expanded polyQ. In particular, the chaperone-dependent E3 ubiquitin (Ub) ligase CHIP (carboxyl terminus of Hsp70-interacting protein) and the NAD synthase nicotinamide mononucleotide adenylyl transferase (NMNAT) together with Hsp70 mediate activation of the proteosome pathway (Zhai et al., 2008). Genetic experiments have delineated the role of transglutaminases (TGs), a class of enzymes that catalyzes the formation of cross-links between glutamine residues within proteins thus increasing their stiffness and insolubility (Muma, 2007). The relevance of these enzymes in proteotoxicity and autophagy was first demonstrated using a Drosophila model for PD and later also in HD, where TG2 phosphorylation by PINK blocked its degradation favoring proteotoxicity and mitochondria degradation. Further experiments, both in Drosophila and in human cells, showed that TG2 reduction ameliorates the defects in both PD and HD (Karpuj et al., 2002; McConoughey et al., 2010; Min et al., 2015). Similarly, a role for TG5 in enhancing the toxicity of mutant ATXN1 was shown in Drosophila models and in cells from SCA1-patients where TG5 was shown to colocalize with Ataxin1 (Lee et al., 2022b) further supporting a functional role for this enzymes in the disease. Genetic screen and chemical inhibitors revealed the RAS-MAPK–MSK1 as an important axis in the control of Ataxin1 aggregate formation (Park et al., 2013). Using small inhibitors of the MSK1/MAPK complex it has been demonstrated in mice and flies that the phosphorylation of Ataxin1 on Ser776 is relevant for its stability, while mutation of this residue in the wt protein reduced the level of Ataxin1-82Q in mice suggesting that Ser776 could represent a novel substrate for both drug and allele-specific therapies (Nitschke et al., 2021).

Not many studies ruled out the function of autophagy in SCA2 until recently, when it was shown that autophagy ameliorates mitochondrial dysfunction and oxidative stress in mice models for SCA2 and in cells from patients (Wardman et al., 2020). In addition, it was shown that cordycepin, a drug that activates AMPK and autophagy, reduces the abnormal accumulation of p62/SQSTM1 and of LC3 observed in cells form SCA2 patients, indicating a dysfunction in the autophagic flux in the disease (Marcelo et al., 2021). Drosophila possess one orthologue of the ATXN2 gene (Atx2) which encodes for a protein Atx2 that shares 23% of identity and 36% of similarity with the human Ataxin2 protein, but differently Drosophila Atx2 does not contain a long polyQ stretch but three short separate segments of glutamines. Structurally human Ataxin2 contains the Lilke-Sm (Lsm) and Lsm-AD domains found in RNA binding proteins (Satterfield and Pallanck, 2006), and further experiments in human and flies confirm that both Ataxin2 and Atx2 physically bind with the polyribosome suggesting that Ataxin2 may be involved in the control of translation (Satterfield et al., 2002).

Expression of human mutant ATXN2 in neurons resulted in the formation of aggregates leading to neuronal degeneration that (Lessing and Bonini, 2008). Further work demonstrated that overexpression of Drosophila Atx1 promotes the accumulation of human mutant Ataxin2 aggregates, indicating that the interaction between the two proteins could contribute to the pathogenesis of SCA1 and SCA2. This hypothesis is in line with the idea of a cooperative mechanism between Ataxin1 and Ataxin2, also suggested by the presence of Ataxin2-enriched aggregates in postmortem neurons of patients with SCA1 (Al-Ramahi et al., 2007). Further studies should be conducted using Drosophila’s model of SCA2 to better understand the physiological role of Ataxin2/Atx2 in the control of autophagy and how it could be perturbed in SCA2, to reveal novel mechanisms of intervention.

Autophagy is compromised by mutation of ATXN3 since the expression of the autophagic markers p62/SQSTM1 and LC3 was found abnormal in the brain of patients with SCA3. Furthermore, transgenic mice expressing ATXN3-71Q exhibit large aggregates that are reduced in the presence of the autophagy-promoting gene Beclin1 (Nascimento-Ferreira et al., 2011). Puromycin-sensitive aminopeptidase (PSA), a cytosolic enzyme able to digest polyQ sequences (Menzies et al., 2010) was shown to ameliorate also Ataxin3 phenotypes both in flies and mice and to induce autophagy (Menzies et al., 2010). Drosophila, like mammals has two isoforms of Atx3 proteins that contain either two or three ubiquitin interacting motifs (2UIM) with an additional at its C-terminus (3UIM; Johnson et al., 2019). Studies in mammals and flies highlighted the different ability of these isoforms to form aggregates when carrying the polyQ expansion. In particular, 2UIM is more prone to form aggregates than 3UIM, that is rapidly degraded mainly through the proteasome pathway (Harris et al., 2010). Further studies in flies showed that the UIMs motifs of Atx3 interact with the heat shock protein cognate-4 (Hsc70-4) to enhance Ataxin3 mutant aggregation and toxicity (Johnson et al., 2021). This data complement those showing that the auto-protective function of Drosophila endogenous Atx3 depends on its deubiquitinating catalytic activity that indirectly reduce the folding of the toxic polyQ present in mutant Ataxin3 proteins as in others polyQ proteins (Warrick et al., 2005), rather than on the activation of the proteasomal degradation pathway (Tsou et al., 2015).

The most widely used models for SCA3 express the C-terminal fragment of the human ATXN3 gene with 78CAGs or 82CAGs repeats, that in the retinal neurons cause abnormal eye morphology due to neuronal and tissues degeneration and motility defects (Warrick et al., 1998, 2005). Genetic screens for modifiers of the Ataxin3 mutant phenotypes led to the identification of several genes of the ubiquitin pathways, members of Hsp70/chaperone proteins, and potential regulators of RAN translation, a Repeat-associated non-AUG (RAN) translation that generates toxic dipeptide repeat proteins (DPRs) from pathological repeat RNA expansions hat do not contain the classical methionine start codon (Cleary et al., 2018), see also for C9orf72-ALS (Section 5.3.1). Genetic screen in flies, using the eye, identified members of the ubiquitin ligase family, such as Cullins and of Praja1, that protect against the effect of mutant Ataxin3 strongly supporting the relevance of the ubiquitin and of the DUBs in the disease (Chen et al., 2019; Ghosh et al., 2021). This screens also identified selective modifiers of Ataxin3 able to rescue also Tau-R406W-AD mutation (Section 5.4.2) supporting the existence of common pathways that converge and contribute to neuronal degeneration, controlled by the Ataxin3 DUB-activity (Bilen and Bonini, 2007). In addition, Ataxin3 DUB-activity controls parkin’s ubiquitination in the modulation of “physiological” mitophagy (Durcan and Fon, 2011), and ubiquitination of K63 in the formation of SOD1 aggregates (Wang et al., 2012). Treatment with antioxidant drugs such as the Nrf2-inducers caffeic acid and resveratrol have also been shown to indirectly reduce apoptosis and to induce autophagy, both in human cells and in Drosophila models of SCA3 (Wu et al., 2017).

The relationship between C9orf72 and autophagy is somewhat controversial, since some data showed that mutation in C9orf72 cause toxicity and alteration of the autophagy-lysosomal pathway (Beckers et al., 2021), others showed that C9orf72 acts as a negative regulator of autophagy as mice mutant for the C9orf72 gene showed reduction TOR activity with nuclear translocation of TFEB and activation of the autophagy flux (Ugolino et al., 2016). Moreover, DPRs induce dysfunctions of the autophagic-lysosomal pathway by synergizing with other mechanisms of toxicity increasing the pathogenesis of the disease (Beckers et al., 2021). Growth factors and insulin/IGF influences neurodegenerative diseases including AD, PD and ALS. indeed C9orf72-G₄C₂ mutation has been shown to downregulate insulin/IGF signaling in both fly and human cells while activating insulin/IGF signaling enhances the toxicity of poly-GR repeats (Atilano et al., 2021). IGFs also act as neurotrophic factors for the survival of motor neurons and have therapeutic effects in a mouse model for SOD1-ALS, and in the murine motoneurons NSC34 cells expressing mutant C9orf72-G₄C₂ (Kaspar et al., 2003; Stopford et al., 2017). Using high-throughput screen to identify chemical modulators of DPRs, we also found that cells expressing mutant C9orf72-G₄C₂ have increased cAMP levels resulting in protein kinase A (PKA) activation, event that was partially rescued by pharmacological or genetic inhibition of PKA (Licata et al., 2022). Since PKA is activated by growth factors, this observation raises the question of a connection between growth pathways and C9orf72 in ALS and, although the mechanisms are not yet fully understood, we presume that they may converge to the control of cellular proteostasis or autophagy.

Although Drosophila does not have a C9orf72 orthologue, transgenic models have been established and mimic the toxicity in humans due to its gain of function effect in flies (Mori et al., 2013; Xu et al., 2013; Mizielinska et al., 2014). The insertion of repeating sequences of GGGGCCs at different lengths was used to determine their different toxicity and ability to form structures called foci that include RNA-binding proteins and RNA (Mori et al., 2013; Mizielinska et al., 2014). RNA of the DPRs obtained by RAN translation was optimized in vitro to express as specific peptides (poly GA, GP, GR, PA, alone or fused with GFP or FLAG tags; Cleary et al., 2018). The difference in toxicity for each DPR is related to their different chemical composition and atomic charge. Indeed, highly arginine-rich GR and PR have been shown to alter the phase separation of LCD-containing proteins by changing the charge and structure of membrane organelles and their dynamics and functions (Wen et al., 2014; Lee et al., 2016). Works in flies evidenced a direct role of C9orf72-G₄C₂ in autophagy that led to the identification that its expression induced the accumulation of Ref(2)P and of poly-ubiquitinated proteins both in motor neurons and in whole larvae, due to defects in cargo protein degradation. These flies showed defects in lysosome formation, accompanied by reduced Mitf/TFEB nuclear localization (Cunningham et al., 2020). Recent work has demonstrated that nuclear transport of TFEB is mediated by nucleoporins (nuclear import proteins) and their expression is based on the molecular chaperone SIGMAR1/Sigma-1 (sigma-1 intracellular non-opioid receptor) mutated in ALS (Wang et al., 2022). Overexpression of SIGMAR1 in flies restores proper cellular transport, outlining the relevance of this chaperonin in nuclear import in this disease (Lee et al., 2020a).

wt SOD1 was found in a complex with Atg9/Beclin1 to control P62/SQSTM1 expression, indeed mice mutant for SOD1-ALS show reduced Atg9/Beclin1 and increased level of P62/SQSTM1 and of ubiquitinated proteins (Nassif et al., 2014). Work in Drosophila as well as in C. elegans, identified a role for lethal(2) malignant brain tumor (L3MBTL1) a histone methyl-lysine protein, and SET domain-containing protein-8 (SETD8) in the control protein misfolding and degradation as their reduction alleviate the phenotypes induced by SOD1. This mechanism is conserved also in mammals underlining the role of chromatin modification in the control of protein quality control (Lu et al., 2019). As we know the interplay of ubiquitin-mediated autophagy is controlled also by deubiquitinases (Clague et al., 2019) and studies in Drosophila, as in other invertebrate models, outlined the negative role of the DUB-USP7 that by reverting the ubiquitination of the E3-ligase NEDD4L, reduced autophagy and the clearance of misfolded SOD1 and TDP-43 proteins by reducing the SMAD2/TGF-beta pathway (Zhang et al., 2020).

Drosophila endogenous dSOD1 is necessary for neuronal health as its null mutation results in neuronal loss. Co-expression of a mutant dSOD1 allele, carrying specific structural LOF mutations, results in formation of unfunctional wt-SOD1/dSOD1 heterodimers indicating that functional dimers are necessary for the activity of the enzyme (Phillips et al., 1995). Studies in Drosophila and in mice demonstrated that expression of human SOD1 in motor neurons reduced ROS activity, leading to longevity (Parkes et al., 1998; Missirlis et al., 2001). Further studies in flies demonstrated that the enzymatic activity of human SOD1 is not required for its toxicity: In fact, homozygous inactive SOD1 mutants (G85R, H48R, and H71Y) expression in dSOD1 null flies results in neurodegeneration, motor defects, and shortened life span, suggesting that these phenotypes are associated to SOD1-mutations rather than SOD1 activity (Şahin et al., 2017; Agudelo et al., 2020). Expression of mutants SOD-1 results in deposition of protein aggregates visible in neurons, glia and in skeletal muscles in both mice-of ALS and cells from ALS patients (Dirren et al., 2015). Mutations in SOD1 lead to mitochondrial impairment, a defect that also occurs in Drosophila, in fact mutant SOD1 accumulates in the intermembrane space of the mitochondrial membranes modifying their structure, reducing the production of ATP and causing metabolic dysfunctions particularly relevant for the activity of motor neurons (Tafuri et al., 2015; Gallart-Palau et al., 2016). A distinctive characteristic of SOD1 mutations is the presence of non-autonomous signals from glia that induce lethality in neighboring cells. This effect was demonstrated in vivo in Drosophila and mice models for SOD1-ALS (Boillee et al., 2006; Watson et al., 2008) and supported by data showing that ablation of the glia ameliorate the disease in mice mutants for SOD1-ALS (Guttenplan et al., 2020). The importance of non-autonomous signaling has been strengthened by recent work demonstrating that motor neurons from SOD1-G93A mutation-bearing mice exhibit metabolic changes that affect surrounding myocytes (Peggion et al., 2022), and likewise between glia and neurons to induce survival of neurons (Chua et al., 2022). Understanding the nature of the non-autonomous signals is relevant to better define the mechanisms in the pathogenesis of ALS and to identify new components and modalities of therapeutic intervention. Thus, the design of Drosophila ALS models using the combination of the two binaries, LexA-LexOp with UAS-Gal4, may allow for the expression of SOD1 mutations in a specific tissue, for example motor neurons, and the concomitant manipulation of glia, using the UAS-Gal4 where many lines are available at stock centers. This would allow to rapidly identify genes that can interfere/suppress SOD1 neuron lethality similar to the approach used for identify mHTT non-autonomous interactors (Bason et al., 2019).

Autophagy plays an important role in the clearance of the cytoplasmic inclusions in fact, it has been shown that the TDP43 aggregates colocalize with markers of autophagy and inhibition of autophagy increases their aggregates formation (Brady et al., 2011). Moreover, TDP-43 itself modulates autophagy, therefore creating a complex scenario whose perturbation contributes to ALS (Huang et al., 2020). Despite the recognized importance of TDP-43 in ALS, it is still unclear whether the pathogenesis of ALS is related to its reduced physiological function, since wild-type TDP-43 is sequestered in inclusion bodies and unable to interact with its physiological targets, or if it is due to the formation of toxic aggregates containing TDP-43 (Scotter et al., 2015). Loss of TARDBP in mammals downregulates histone deacetylase 6 (HDAC6), an enzyme that controls ubiquitinated protein and autophagy (Fiesel et al., 2010). Controversial results have been obtained in TARDBP knockout cells, where although the lysosomal transcription factor TFEB is upregulated, accumulation of immature autophagic vesicles and reduced expression of Atg7 are observed, suggesting that reduction of TARDBP affects other signals/genes necessary for a functional autophagy (Xia et al., 2016).

Drosophila harbors a TARDBP orthologue, namely TBPH, and several groups have investigated its function to gain insights into ALS pathogenesis by performing loss of function studies. Interestingly, Atg7 overexpression suppresses the semi-lethality of TBPH null flies, ameliorating motility, lifespan and reducing the accumulation of Ref2(P)/P62 inclusions at the NMJ (Donde et al., 2020). Other studies in flies addressed the consequences of overexpressing wild-type or disease forms of TDP-43. Overexpression of a cytoplasmic wt-TDP-43 or of TDP-43-M337V mutant in the fly’s retina results in neurodegeneration that can be rescued by co-expression the heat shock protein HSP67Bc that promotes autophagy (Crippa et al., 2016). More recently, HEXA-018, a novel chemical compound that induces autophagy in a TOR-independent manner and ameliorates climbing activity and lifespan was identified as a suppressor of TDP-43 overexpression in flies (Lee et al., 2021).

Similarly, to TP-43, autophagy seems to play a crucial positive role in the elimination of toxic aggregates also in FUS-related ALS. Expression of wild-type FUS and ALS-related FUS mutations triggers mechanisms that lead the accumulation of toxic cytoplasmic aggregates and to inhibition of autophagy (Brunet et al., 2021). Furthermore, work in flies and human iPSCs demonstrated that the P525L-FUS mutation alters stress granule dynamics resulting in inhibition of PI3K/AKT/TOR signaling which indirectly increases autophagy by a yet unknown mechanism (Marrone et al., 2018).

Genetic screens using flies identified Mask, an Ankyrin-repeat containing protein, that promotes the expression of the proton-pumping vacuolar (V)-type ATPases, favoring the elimination of FUS aggregates via lysosomal autophagy (Zhu et al., 2017). Recently, attention has been placed on post translational modifications that interfere with the protein phase separation of FUS, indeed the enzyme glutathione transferase omega 2 (GstO2) was shown to reduce cytoplasmic FUS level and the formation of FUS aggregates both in Drosophila and in mouse neuronal cells overexpressing FUS. Glutathionylation of FUS promotes its separation into the liquid phase suggesting how an accurate analysis of the mechanism driving protein phase separation might be a promising target for novel therapeutic strategies (Cha et al., 2022).

Autophagy plays a complex and dual role in AD since it regulates both Aβ eneration and clearance (Mputhia et al., 2019). Several studies support a protective role: Atg5, Beclin1, and ULK1 were shown to be involved in Aβ degradation (Tian et al., 2011) and the adaptor protein AP2, a member of a complex responsible for the internalization of cargos in clathrin-mediated endocytosis, was shown to induce the degradation of the APP-βCTF (C-terminal fragment) via autophagy, affecting Aβ production (Tian et al., 2013). Moreover, autophagic degradation of APP-CTF and Aβ is promoted by NRBF2, a member of the class of III phosphatidylinositol 3-kinase (PtdIns3K) complex that regulates autophagosome maturation (Cai et al., 2021). On the other hand, activation of autophagy might play a detrimental role in AD depending on the context. For example, activation of TFEB, plays opposite effects on Aβ production in neurons depending on the expression level of APP or of the βCTF (Yamamoto et al., 2019; Song et al., 2020).

Drosophila harbors an orthologue of APP, namely APPL and since the Aβ sequence is not conserved Drosophila’ models of AD that contributed to gain insight into the role of autophagy were mostly obtained by overexpression of either human Aβ peptide or of human AD-associated genes. The protective action of autophagy has been proven by modulating the levels of autophagic activity in flies expressing human Aβ peptide. For example, lowering the expression of autophagy-related genes, such as Atg1, Atg8a and Atg18, strongly enhances the neuronal toxicity caused by Aβ expression (Omata et al., 2014). In line with this, Atg8a overexpression in an Alzheimer Drosophila model obtained by overexpression of human amyloid precursor (hAPP) and beta-secretase 1 (hBACE1), enhances stress-resistance, slows down neurodegeneration and improves lifespan (Tsakiri et al., 2021). In a similar AD model, promotion of autophagy by the Nicotinamide mononucleotide adenylyltransferase (NMNAT) reduced amyloid plaques formation (Zhu et al., 2022). In flies overexpressing Aβ42, the protective role of autophagy was demonstrated by the expression of Thioredoxin-80 (Trx80), which prevents the accumulation of the toxic peptides in the brain and rescues lifespan by inducing autophagosome formation (Gerenu et al., 2021). On the other hand, Ling and colleagues showed that dysfunctional AEL (autophagy-endosomal-lysosomal) plays a crucial role in Aβ42 accumulation. In fact, the abnormal accumulation of Aβ42 within AEL vesicles was reduced when the functional autophagy was decreased (modulation of Atg5 or Atg12 levels), suggesting that dysfunctional autophagy–endosomal–lysosomal may cause Amyloid-like plaque formation (Ling et al., 2014). Moreover, it was recently shown that ectopic APP expression is sufficient to trigger aberrant autophagy through its interaction with the carboxyl-terminus of Hsc70-interacting protein (CHIP), a U-box type chaperone associated E3 ubiquitin ligase via transcriptional upregulation of BACE1 and PSEN inducing an AD-like neurodegeneration (Zhuang et al., 2020).

Autophagy plays a protective role in tauopathies, as it favors the clearance of the soluble and the insoluble Tau form the last present in the aggregates. Furthermore, autophagy helps the movement of Tau through vesicles involved in its transport into neurons.

Drosophila possesses a tau gene with 46% identity and 66% similarity to the corresponding human Tau. Flies have been successfully used to pin down numerous pathways that control Tau toxicity via autophagy. For example, insulin signaling can induce abnormal Tau phosphorylation and accumulation, inhibiting its autophagic clearance and worsening the disease (Chatterjee et al., 2019). Silencing calpain, a family of proteases that inhibit autophagy, is protective against Tau toxicity in the Drosophila-eye, an effect that requires a functional autophagy since it was lost in Atg8a mutant flies (Menzies et al., 2015). A microRNAs screen aimed at identifying suppressors of Tau-mediated phenotypes, clarified that CG11070, a target of miR-9a mediates Tau ubiquitination and degradation primarily via the autophagy-lysosome system. Interestingly, this function is conserved for the human orthologue UBE4B in cells of neuroblastoma (Subramanian et al., 2021). In AD patients Tau protein is often hyper-phosphorylated and one of the kinases responsible is PTK2/FAK (focal adhesion kinase; Lee et al., 2022a). PTK2 plays a critical role in proteinopathies as it favors abnormal phosphorylation of proteins, including TDP-43 and p62/SQSTM1 leading to the formation of ubiquitinated aggregates and neurotoxicity induced by the dysregulation of the ubiquitin–proteasome system (UPS; Lee et al., 2020b). This mechanism is conserved for Tau (wt or mutant P301L) and also present in Drosophila where it was shown that attenuation of PTK2 expression reduces the phosphorylation of Ref2(P)/p62 ameliorating the motility of Tau expressing flies (Lee et al., 2022a). This also suggests the presence of a feed-back loop between PTK2 and p62 dysregulated in tauopathies.

Flies do not possess an orthologue of α-syn therefore models have been produced by ectopically expressing human mutants of α-syn. A large part of studies conducted in flies point to a dysfunction of autophagy in presence of α-syn mutation and to a positive role of autophagy in α-syn induced degeneration: ectopic expression of human α-syn leads to F-actin-mediated impairment in autophagic flux, accumulation of abnormal autophagosomes, impairment in mitophagy, leading to defects in cellular bioenergetics (Sarkar et al., 2021). Treatment of flies overexpressing α-syn with spermidine, a naturally occurring polyamine known to prolong lifespan by inducing autophagy, protects against α-syn induced neurotoxicity (Buttner et al., 2014). Overexpression of the human lysosomal membrane protein LAMP2A fully prevents the behavioral defects induced by neuronal expression of a PD-associated mutant form of α-syn (SNCA/A30P) as well as reduces the α-syn accumulation in older flies. Moreover, LAMP2A expression upregulates Atg5 that stimulates macroautophagy (Issa et al., 2018). Similarly, the progressive locomotor decline and the loss of dopaminergic (DA) neurons caused by the human α-syn-A30P variant is reduced by pan-neuronal overexpression of dDOR (orthologue of the human tumor protein 53-induced nuclear protein 1 (TP53INP1), able to activate autophagy and basal mitophagy (Dinh et al., 2021). Another group demonstrated that knockdown of inositol-requiring enzyme 1 (IRE1) or Atg7 reverses α-syn-A30P-induced neurodegeneration in terms of lifespan, locomotor activity, and DA neuron loss (Yan et al., 2019). This suggests that IRE1 and Atg7 may play different roles in the classical autophagy mechanism of survival and their loss activates protective mechanisms thereby explaining the reduction of toxic proteins and neuron survival in PD.

Parkin and Pink1 are involved in mitochondria quality control and turnover (Shimura et al., 2000), the recruitment of the E3 ubiquitin ligase Parkin on the damaged mitochondrial membrane by the kinase Pink1, controls mitochondrial turnover by inducing a physiological level of mitophagy (Truban et al., 2017). Drosophila harbors only one parkin and PINK1 orthologue (Clark et al., 2006) and their function is highly conserved. Several models have been created both using endogenous parkin and PINK1 null mutants and by overexpressing human genes harboring pathogenic mutations, that closely recapitulate many PD features including DA loss, decreased lifespan, and motor defects (Ishihara-Paul et al., 2008; Hewitt and Whitworth, 2017). In particular, these models have been fundamental to understand the molecular mechanisms linking parkin and Pink1 to mitophagy. Studies using flies’ flight muscles identified their relevance in the control physiological mitophagy (Greene et al., 2003), furthermore, new studies outline how the increase in mitophagy observed in aging depends on the parkin/Pink1 interaction and on the activity of two deubiquitinases USP15 and 30 (Cornelissen et al., 2018). The mitochondria phenotype observed in parkin null mutants recapitulates general autophagy inhibition obtained by loss of Atg7, supporting the physiological role of parkin in the activation of mitophagy as clearance pathway (Vincow et al., 2013). Many proteins have been identified that may affect parkin/Pink1 modulation of mitophagy: Fbxo7, whose gene PARK15 is mutated in early-onset autosomal recessive forms of Parkinson, directly interacts with parkin to control its translocation to the mitochondrial membrane and functionally interacts with Pink1 to regulate parkin-dependent mitophagy (Burchell et al., 2013). The mitochondrial protein BNIP3L, a BH3 protein member of the Bcl2 family, interacts with Pink1 to induce mitophagy, and work in flies’ muscles helped to understand the role of human BNIP3 as suppressor of Pink1-induced-mitophagy (Zhang et al., 2016). BNIP3L was identified as a substrate for PARKIN2, and this ubiquitination recruits the autophagic cargo receptor NBR1 to the mitochondria to induce PARKIN2-mediated mitophagy. Even if PARKIN2 is present only in mammals, it was demonstrated using Drosophila that ectopic expression of BNIP3L rescues the mitochondrial defects of PINK1 mutant but not the effect of parkin mutant, outlining that PARKIN2 is the principal substrate for BNIP3L and it is necessary for BNIP3L clearance of mitochondria (Gao et al., 2015). Using flies, it was also demonstrated how the reduction of the human orthologue of Mask (ANKHD1), a scaffolding protein that inhibits mitophagy, rescues parkin/PINK1 mutant defects, suggesting that the human ANKHD1 might represent an interesting novel therapeutic target for treating PD associated to parkin/Pink1 mutations (Zhu et al., 2015). A novel mechanistic link between mitophagy and translation lies in the evolutionarily conserved function of EFTU, a mitochondrial translation elongation factor, that acts as substrate and interacts with Pink1 independently of parkin. Pink1 regulates mitophagy by restraining EFTU in the cytosol after phosphorylation on Ser222, inhibiting mitophagy by interacting with the Atg5-Atg12 complex formation (Lin et al., 2020). Despite several studies strongly linking parkin/Pink1 to mitophagy, Lee et al. (2018) demonstrated that basal mitophagy is not strongly reduced in the muscles of parkin/PINK1 mutant animals, and similar results were obtained using mouse models for PD under conditions of high metabolic demand. A possible explanation could be that the metabolic state of cells could influence mitophagy, particularly in animals carrying parkin/PINK1 mutations (McWilliams et al., 2018).

Drosophila harbors a single LRRK2 orthologue gene (dLRKK) therefore it has been used to better understand the physiological functions of LRRK2 using loss of function models or overexpression of human LRRK2 variants responsible for PD (Hewitt and Whitworth, 2017; Seegobin et al., 2020). Overexpression of pathogenic mutant forms of LRRK2 causes defects in endolysosomal and autophagic assembly, and LRRK2-targeted therapies in PD are based on this molecular event. However, several studies in Drosophila have shown that dLRKK reduction affects lysosomal compartments and leads to the accumulation of dysfunctional autophagosomes, suggesting that proper expression of LRRK2 is required for functional signaling of autophagy and vesicle trafficking (Dodson et al., 2014; Seegobin et al., 2020). LRRK2 controls the phosphorylation of several small GTPases of the Ras-associated binding protein (Rab) family responsible for vesicle trafficking at the synapse level (Steger et al., 2016; Bellucci et al., 2022). Another relevant observation is the interaction of LRRK2 with other proteins leading to PD. Indeed, Ng and colleagues demonstrated that overexpression of the disease-associated LRRK2/G2019S mutant in flies flight muscles induces mitochondrial pathology and impairs locomotion by phenocopying the behavior of parkin-null flies, whereas in DA neurons it leads to significantly enlarged mitochondria. In both cases, the toxic effects of LRKK2 mutants were rescued by co-expression of parkin or activation of AMPK, known to reduce TOR signaling and to activate autophagy (Ng et al., 2012), suggesting a cooperative function in mitochondria between LRRK2 and parkin.