IRE1 promotes neurodegeneration through autophagy-dependent neuron death in the Drosophila model of Parkinson’s disease

Abnormal aggregation of misfolded pathological proteins in neurons is a prominent feature of neurodegenerative disorders including Parkinson’s disease (PD). Perturbations of proteostasis at the endoplasmic reticulum (ER) triggers ER stress, activating the unfolded protein response (UPR). Chronic ER stress is thought to underlie the death of neurons during the neurodegenerative progression, but the precise mechanism by which the UPR pathways regulate neuronal cell fate remains incompletely understood. Here we report a critical neurodegenerative role for inositol-requiring enzyme 1 (IRE1), the evolutionarily conserved ER stress sensor, in a Drosophila model of PD. We found that IRE1 was hyperactivated upon accumulation of α-synuclein in the fly photoreceptor neurons. Ectopic overexpression of IRE1 was sufficient to trigger autophagy-dependent neuron death in an XBP1-independent, JNK-dependent manner. Furthermore, IRE1 was able to promote dopaminergic neuron loss, progressive locomotor impairment, and shorter lifespan, whereas blocking IRE1 or ATG7 expression remarkably ameliorated the progression of α-synuclein-caused Parkinson’s disease. These results provide in vivo evidence demonstrating that the IRE1 pathway drives PD progression through coupling ER stress to autophagy-dependent neuron death.


Introduction
Neurodegenerative diseases share a prominent pathological feature of disturbed proteostasis, which is characterized by the accumulation and aggregation of specific misfolded proteins within the affected neurons 1 . These protein-misfolding disorders include Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD), prion-related diseases, and amyotrophic lateral sclerosis (ALS) 2 . PD is the second most common neurodegenerative disease with the hallmark of aggregation of α-synuclein in Lewy bodies, which is believed to cause selective death of dopaminergic neurons in the substantia nigra pars compacta [2][3][4] . Despite that several mechanisms such as mitochondrial dysfunction, oxidative stress, and defective intracellular Ca 2+ homeostasis have been implicated in dopaminergic (DA) neuron degeneration, no neuroprotective therapies are currently available owing to our limited understanding of whether a unifying mechanism is at work to drive the pathogenic progression of PD.
Emerging lines of evidence suggest a close association between chronic endoplasmic reticulum (ER) stress and neurodegenerative conditions, including PD 1,5-8 . Alphasynuclein, the key neurotoxic protein involved in PD, accumulates within the ER both in animal models of αsynucleinopathy and in human PD patients 9,10 . Perturbations of proteostasis at the ER, i.e. an overload of unfolded or misfolded proteins, cause ER stress and activate the adaptive unfolded protein response (UPR) 1 . In mammals, the UPR program is governed by three evolutionarily conserved ER transmembrane signal transducers, inositol-requiring enzyme 1 (IRE1), protein kinase RNAlike ER kinase (PERK), and activating transcription factor 6 (ATF6) 11,12 . As a homeostatic mechanism, the three UPR pathways cooperate to mitigate ER stress; but when ER homeostasis cannot be restored, cell death ensues 11,12 . However, the exact mechanism linking chronic activation of the UPR pathways to neuronal cell death remains largely elusive.
IRE1 is the most ancient ER stress sensor that is highly conserved from yeast to fruit fly and to mammals. It possesses both Ser/Thr protein kinase and endoribonuclease (RNase) activities in its cytoplasmic portion 11,13 . Upon ER stress, IRE1 is activated through transautophosphorylation and dimerization/oligomerization 13 , initiating a key branch of the UPR through catalyzing the unconventional splicing of X-box binding protein 1 (Xbp1) mRNA to generate XBP1s, the active spliced form of this transcription factor. Many studies have shown that IRE1 has a pivotal part in cell fate decision under ER stress conditions 13,14 . Whereas IRE1 is thought to promote cell survival by XBP1s-mediated enhancement of the ER's protein folding capacity, recent studies have indicated that IRE1 can control cell death through regulated IRE1dependent decay (RIDD) 15 of the mRNA encoding death receptor 5 (DR5) 16 or through cleavage of certain microRNA regulators of apoptosis [17][18][19] . Notably, the exact role of IRE1-XBP1 pathway in linking chronic ER stress to neuronal cell death appears to depend upon the disease context 1 . For instance, Valdés et al. showed that XBP1s exerted neuroprotective actions against a PD-inducing neurotoxin and promotes the survival of nigral DA neurons 20 . Casas-tinto et al. reported that XBP1 could suppress amyloid-beta neurotoxicity in a Drosophila AD model 21 . Similarly, reduction of Xbp1 gene dosage was shown to accelerate retinal degeneration in a Drosophila model for autosomal dominant retinitis pigmentosa 22 . In contrast, Vidal et al. and Hetz et al. reported that XBP1 deficiency resulted in protection against neurodegeneration in the transgenic mouse models of both HD 23 and ALS 24 , likely through enhancement of autophagy. Moreover, IRE1 was also suggested as a crucial mediator of ER stress-induced aggregation of mutant huntingtin via suppressing autophagy flux, thereby leading to its neuronal toxicity in HD 25 . Autophagy is a highly conserved catabolic process 26 and plays critical roles in proteostasis, tissue homeostasis and cell survival through lysosomal degradation of aggregate-prone proteins and intracellular organelles such as mitochondria and ER. Deregulation of the autophagic response may contribute to the development of neurodegenerative diseases 27,28 . Interestingly, reported studies indicated that the IRE1-JNK pathway might mediate autophagy activation and thus rendered cells more resistant to ER stress 29,30 . However, while being a topic of debate, emerging evidence also indicated that overactive autophagy might act as a lethal mechanism leading to "autophagy-dependent cell death" under certain physiological and pathological conditions [31][32][33][34][35][36][37][38][39] . Given their concurrent activation in the neurodegenerative states, it is of great significance to decipher whether the interplay of the IRE1 pathway and autophagy underlies the pathogenic progression of PD and other neurodegenerative disorders.
Here we investigated whether the IRE1 pathway links chronic ER stress and autophagy to autophagy-dependent neuron death in vivo. We utilized the well-established PD model in the fruit fly Drosophila melanogaster 40 via ectopically expressing human α-synuclein in the photoreceptor or DA neurons. We found that overexpression of wild type or missense mutant α-Syn in photoreceptor neurons induced the activation of IRE1. Chronic activation of IRE1 triggered strong autophagy and induced cell loss in photoreceptor neurons. Unexpectedly, inhibition of autophagy by knockdown of Atg genes, Atg7 or Atg8b, did prevent IRE1-caused neuron death. The autophagydependent neuron death induced by IRE1 was mediated by JNK signaling in an XBP1-independent manner. Our data demonstrate that in response to the accumulation of neurotoxic proteins, the IRE1 pathway serves as an unanticipated critical proteostatic "rheostat" to trigger autophagy-dependent neuron death, thereby driving the onset and progression of neurodegeneration in PD.

IRE1 activation is associated with α-synucleinopathy and promotes neuronal degeneration
As the most accessible organ of the nervous system, the fly eye is dispensable for life and has been widely used to model neurodegeneration 41 . We first tested whether the IRE1 pathway is activated upon α-synucleinopathy in the photoreceptor neurons of Drosophila. We specifically overexpressed the human wild-type (WT) or two missence mutant forms (A30P and A53T) of α-synuclein identified from familial PD 40,42 in the photoreceptor neurons. In 1-day old adult flies, histology analyses of the tangential sections showed normal retinal morphology and architecture, with well-organized R1-R7 photoreceptors observed in each ommatidium (Fig. S1a). By contrast, at 30 days of age, overt retinal degeneration, as manifested by the apparent loss of photoreceptor neurons along with vacuole formation, was observed in the GMR-Gal4 > α-Syn WT , GMR-Gal4 > α-Syn A30P and GMR-Gal4 > α-Syn A53T flies when compared to the GMR-Gal4 > + control line (Fig. S1a). Quantification of photoreceptor loss in tangential sections as analyzed by the percentage of intact ommatidia at each time point showed that ectopic expression of α-Syn WT , α-Syn A30P or α-Syn A53T resulted in progressive photoreceptor degeneration, with 45%, 55% and 47% of intact ommotidia observed, respectively, in GMR Gal4 > α-Syn WT , GMR Gal4 > α-Syn A30P and GMR Gal4 > α-Syn A53T flies relative to 94% in GMR Gal4 > + flies (Fig. S1b). No significant differences were found between flies expressing the WT and mutant α-synulein proteins. Subsequent immunoblot analyses of fly eyes revealed that this α-synucleinopathy was accompanied by elevated phosphorylation of IRE1 at Ser 703 (Fig. S1c), a conserved site that corresponds to Ser 724 within the activation loop of the kinase domain of murine IRE1α 43 . Notably, a higher extent of increase in IRE1 phosphorylation was detected in GMR-Gal4 > α-Sy n A30P and GMR-Gal4 > α-Syn A53T eyes than that in GMR-Gal4 > α-Syn WT counterparts (Fig. S1c). In accordance, elevations in Xbp1 mRNA splicing were also detected in fly eyes expressing α-synulein proteins (Fig. S1d), which indicates more severe ER stress induced by α-synuclein proteins. Moreover, elevations of IRE1 phosphorylation were paralleled by increased phosphorylation levels of c-Jun N-terminal kinase (JNK) (Fig. S1c), suggesting that αsynuclein-induced activation of IRE1 might be coupled to the JNK pathway during neuronal degeneration.
To determine if IRE1 is involved in such α-synucleinopathy, we inhibited its expression by RNAi. We found that IRE1 deficiency markedly rescued α-Syn-evoked retinal degeneration, as shown by 70%, 72%, and 62% of intact ommotidia, respectively, in retina from GMR-Gal4 > α-Syn WT ; Ire1-Ri, GMR-Gal4 > α-Syn A30P ; Ire1-Ri, and GMR-Gal4 > α-Syn A53T ; Ire1-Ri flies at 30 days of age ( Fig. S1a, b). Next, we wondered if hyperactivation of IRE1 is sufficient to mediate α-synuclein's neurotoxic effects. To test this idea, we generated transgenic flies with specific overexpression of V5-tagged Drosophila IRE1 in the photoreceptor neurons. Remarkably, overexpression of IRE1 caused large anomalies to the external eyes in comparison to those of GMR-Gal4 > + flies, and scanning electron microscopy (SEM) analysis revealed a glassy eye surface characterized by ommatidial disruption and loss of interommatidial bristles (Fig. 1a). Histological examination of the tangential sections also showed massive loss of photoreceptor neurons in IRE1-expressing eyes (Fig.  1a). To exclude the possible non-specific effects of IRE1 transgene insertion, we knocked down the expression of IRE1 by RNAi in the eyes of IRE1-expressing flies, and confirmed that the retinal neuron loss indeed resulted from IRE1 overexpression (Fig. 1a, b). We analyzed the mRNA abundance of Crc (the Drosophila homolog of ATF4, the downstream marker of the PERK pathway), Atf6 as well as PEK (the Drosophila homolog of PERK) in the adult head of GMR-Gal4 > IRE1 flies. No significant changes were observed in the expression of these UPR signaling genes (Fig S2), indicating that neither the PERK nor the ATF6 pathway was likely to have a critical role in IRE1-induced neuron loss.
To further quantitatively determine the extent of IRE1induced neuron loss, we used the mCD8-GFP reporter system 44 and found that IRE1 overexpression resulted iñ 75% loss of the retinal neurons in GMR-Gal4 > mCD8-GFP; IRE1 flies ( Fig. 1c). In addition, TUNEL analyses showed prominent IRE1-induced cell death in the eye imaginal discs of GMR-Gal4 > IRE1 larvae (Fig. 1d). These data demonstrated that IRE1 was sufficient to instigate neuronal cell death in Drosophila, which might mediate α-synuclein induction of neuronal degeneration.

IRE1 induces autophagy-dependent neuron death
To determine the cellular characteristics of IRE1-induced neuron death, we analyzed the tangential sections of fly eyes by transmission electron microscopy (TEM). GMR-Gal4 > + control flies had well-organized photoreceptors in each ommatidium, along with normal structural appearance of the ER and mitochondria (Fig. 2a). In contrast, GMR-Gal4 > IRE1 flies showed severe derangement or loss of the photoreceptor neurons, and TEM analyses revealed overt accumulation of autophagosomes/autolysosomes encircling mitochondria and amorphous structures which appeared to be the partially degraded cellular debris (Fig. 2a). This suggests that IRE1-induced neuronal loss was accompanied by activation of autophagy in the eyes of GMR-Gal4 > IRE1 flies. To further affirm the occurrence of autophagy, we used the dual-tagged GFP-mCherry-Atg8a reporter system 45 to enable the detection of autophagy flux. Indeed, as compared to the GMR-Gal4 > + control, marked increases of red mCherry-Atg8a-derived puncta were observed in GMR-Gal4 > IRE1 eye discs due to the quenching of the GFP signal under autophagy-associated acidic conditions (Fig. 2b). Pearson's coefficient is markedly decreased in GMR-Gal4 > IRE1 eye discs as compared to GMR-Gal4 > + control (Fig. 2b). To further affirm autophagy flux is functional, we examed the level of known autophagic substrate Ref(2)P, the fly homolog of the autophagy receptor p62, which degraded upon activation of autophagy flux 46,47 . Immunofluorescence staining of Ref(2)P on eye imaginal discs, as well as immune blot analysis, all showed dramatically decreased Ref(2)P in GMR-Gal4 > IRE1 flies relative to GMR-Gal4 > + control flies (Fig. S3a, b).
ATG (autophagy-related) proteins are essential for forming the double-membrane autophagosomal vesicles and for the execution of autophagy, among which ATG7 is a key component of the core autophagy machinery 26,48 . Given that the constituent ATG molecules are evolutionarily conserved from Drosophila to mammals, we first examined their mRNA levels in IRE1-expressing eyes. Notably, although the expression of Atg1, Atg9, Atg12, Atg6, and Atg8b was considerably upregulated, no significant alteration in the expression of Atg3, 18a, Atg5, or Atg7 was detected in GMR-Gal4 > IRE1 eyes (Fig. S4). Next, to determine whether the enhancement of autophagy mediated IRE1-induced loss of photoreceptor neurons, we performed in vivo screening of a number of the available Atg RNAi fly lines (Fig. S5). Remarkably, in GMR-Gal4 > IRE1; Atg7-Ri or GMR-Gal4 > IRE1; Atg8b-Ri flies, knockdown by~50% of the expression of Atg7 or Atg8b (Fig. S6a, b) almost completely or partially rescued, respectively, IRE1-evoked retinal structural derangement (~70% of GMR-Gal4 > IRE1; Atg8b-Ri flies showed phenotypical ameliorations), with restored organization of ommatidia and interommatidial bristles observed (Fig. 3a); whereas knockdown of Atg7 or Atg8b expression did not directly affect the morphology of GMR-Gal4 > Atg7-Ri or GMR-Gal4 > Atg8b-Ri eyes (Fig. 3a). TUNEL staining analyses of eye imaginal discs also revealed marked decreases in neuronal cell death (Fig. 3b), and TEM assessment showed significant reversal of IRE1-induced photoreceptor neuron loss, along with normalized structures of ER and mitochondria but no detectable accumulation of autophagosomes/autolysosomes in GMR-Gal4 > IRE1; Atg7-Ri or GMR-Gal4 > IRE1; Atg8b-Ri flies (Fig. 3c). Then we tested whether this autophagydependant cell death involves the action of caspases (cysteine aspartate-specific proteinases). Inhibition of caspases by overexpressing p35 49 , Dronc DN 50 or homozygous mutation for dronc I29 null allele 51 in GMR-Gal4 > b Immunoblot analysis of IRE1 expression from the head lysates of adult GMR-Gal4 > + versus GMR-Gal4 > IRE1 and GMR-Gal4 > IRE1; Ire1-Ri flies (n = 30 flies/genotype; representative of two independent experiments). Anti-V5 antibody was used. c Representative fluorescent microscopy images of heads of adult GMR-Gal4 > mCD8-GFP versus GMR-Gal4 > mCD8-GFP; IRE1 flies. Shown at bottom are the enlarged images of the individual ommatidium. Scale bar represents 50 µm. Fluorescence signals were quantified from three independent experiments and are presented as mean ± s.e.m. (n = 10 flies/ genotype). ***P < 0.001 by Student's t test. d Cell death analysis of eye discs from 3rd instar larvae of the indicated genotypes. Shown are representative images of TUNEL labeling along with IRE1 immunofluorescent staining with anti-V5 antibody with the enlarged regions indicated (n = 20 flies/genotype). Scale bar represents 30 µm IRE1; p35, GMR-Gal4 > IRE1; Dronc DN or GMR-Gal4 > IRE1; dronc I29 flies showed no rescuing effects upon IRE1evoked disorganization of ommatidia and bristles (Fig.  S7), indicating that caspase-dependent apoptosis may not have a prominent role in IRE1-induced neuron loss. Thus, these results suggest that IRE1-initiated activation of autophagy critically contributed to IRE1 promotion of neuronal cell death.

IRE1 drives neuronal cell death in an XBP1-independent fashion
Next, we asked if IRE1's downstream effector XBP1 is involved. As anticipated, overexpressed IRE1 manifested an automatic activation state in GMR-Gal4 > IRE1 eyes, in which increased Xbp1 mRNA splicing and elevated expression of XBP1s target genes, Bip and Edem1/2, were detected by quantitative RT-PCR (Fig. 4a). Further, when intercrossed to the Xbp1-EGFP reporter line in which EGFP expression is driven by Xbp1 mRNA splicing 22 , prominent Xbp1 mRNA splicing activity was detected in the eye discs of GMR-Gal4 > Xbp1-EGFP; IRE1 flies relative to the GMR-Gal4 > Xbp1-EGFP control line (Fig.  4b). Then, we examined if XBP1 could mediate IRE1induced neuronal cell death. Surprisingly, knockdown bỹ 40% of the expression of Xbp1 (Fig. S6c) had a more severe damaging effect upon the appearance of external eyes and the disorganization of ommatidia in GMR-Gal4 > IRE1; Xbp1-Ri flies, while overexpression of the spliced form of XBP1 (XBP1s, V5-tagged) appreciably attenuated these phenotypes in GMR-Gal4 > IRE1; XBP1s eyes (Fig. 4c). TUNEL analyses showed that suppression of Xbp1 expression exerted no effect upon IRE1-induced cell death in the imaginal discs of GMR-Gal4 > IRE1; Xbp1-Ri larvae (Fig. 4d), whereas overexpression of XBP1s considerably reduced it in GMR-Gal4 > IRE1; XBP1s discs. Notably, neither knockdown of Xbp1 expression nor XBP1s overexpression affected the fly eye morphology or cell viability in the absence of IRE1 overexpression (Fig. 4c, d). These results suggest that XBP1 does not mediate IRE1 promotion of neuronal loss; rather, XBP1s may function in a negative feedback loop to protect against IRE1-induced neuronal cell death. Therefore, IRE1 could drive neuronal cell death through a mechanism that is independent of XBP1 actions.
To investigate if RIDD has a role in mediating IRE1induced photoreceptor neuron loss, we first evaluated changes in the mRNA levels of a selective set of RIDD targets under IRE1-overexpressing conditions, including the fatty acid transport protein (Fatp) that has been shown to be implicated in IRE1 regulation of photoreceptor differentiation and survival 52,53 . Quantitative RT-PCR analyses revealed significant decreases in the mRNA abundance of Fatp, Cds and Indy by~50.8%,~31.0%, and 28.8%, respectively, in GMR-Gal4 > IRE1 eyes relative to their GMR-Gal4 > + controls (Fig. S7a). In addition, knockdown of the expression of IRE1 resulted in a trend of reversed increase in the mRNA levels of these RIDD targets (Fig. S8a). Despite that knockdown of the expression of Fatp, Cds or Indy did not affect the adult eye morphology (Fig. S8b), the possible contribution of these RIDD target genes remains to be dissected during autophagy-associated photoreceptor neuron loss in GMR-Gal4 > IRE1 flies.
IRE1 promotes autophagy-dependent neuron death through JNK activation IRE1 is known to be linked to JNK activation 54,55 . Given the association of α-synucleinopathy with phosphorylation activation of both IRE1 and JNK (Fig. S1b), we considered that IRE1 might promote autophagy-dependent neuron death through JNK signaling. Indeed, immunoblot analysis revealed increased JNK phosphorylation in the eyes of GMR-Gal4 > IRE1 flies (Fig. 5a). When intercrossed to the puc-lacZ reporter line for in vivo measurement of JNK activity 56 , markedly elevated activation of the IRE1-JNK pathway was observed in the imaginal discs of GMR-Gal4 > pucE69; IRE1 larvae (Fig. 5b). To determine the importance of JNK in IRE1 promotion of autophagy-dependent neuron death, we knocked down the expression of the JNK-encoding gene Basket (Bsk) in the eyes of GMR-Gal4 > IRE1; Bsk-Ri flies (Fig. 5c). While exerting no apparent effects upon the eyes of flies without IRE1 overexpression (Fig. 5d), knockdown of Bsk expression prominently alleviated IRE1-induced disruption of ommatidia (Fig. 5d) and significantly blocked IRE1-induced neuron death in the imaginal discs (Fig. 5e) in GMR-Gal4 > IRE1; Bsk-Ri flies. These data suggest that JNK acted as an important mediator in IRE1 promotion of autophagy-dependent neuron death.

IRE1 causes Parkinsonian neurodegeneration through autophagy-dependent dopaminergic neuron loss
To determine whether IRE1-induced neuron death underlies the neurodegenerative progression in the PD model, we first examined the effects of IRE1 or α-Syn A30P overexpression upon DA neurons in the brain using the Ddc-Gal4 > driver. Remarkably, Ddc-Gal4 > IRE1 flies, in which IRE1 was overexpressed in their DA neurons, phenotypically mimicked Ddc-Gal4 > α-Syn A30P flies, exhibiting similarly shorter lifespan (Fig. 6a) and agedependent impairment in their climbing ability relative to the control Ddc-Gal4 > + line (Fig. 6b). Furthermore, assessment of the integrity of DA neurons showed that, at (see figure on previous page) Fig. 3 Autophagy is required for IRE1-induced neuron death. a Representative light microscopy (top) and SEM (middle and bottom) images of external eyes from adult GMR-Gal4 > IRE1, GMR-Gal4 > IRE1;Atg7-Ri and GMR-Gal4 > IRE1;Atg8b-Ri flies versus GMR-Gal4 > +, GMR-Gal4 > Atg7-Ri and GMR-Gal4 > Atg8b-Ri flies (n = 5-8 flies/genotype). Scale bar represents 50 µm. b Cell death analysis of larval eye discs of the indicated lines. Shown are representative images of TUNEL and DAPI staining along with IRE1 immunostaining with the enlarged regions indicated (n = 20 flies/genotype). Scale bar represents 30 µm. c Representative light microscopy images of tangential sections of adult eyes stained with toluidine blue (top panels) and TEM micrographs of the tangential section of an ommatidium (middle and bottom; Scale bars, 2 µm, 0.5 µm, and 0.2 µm) from the indicated lines (n = 3-5 flies/genotype). ER endoplasmic reticulum. Arrows indicate autophagosomes/autolysosomes, which were quantified from two independent experiments and are shown as mean ± s.e.m. (n = 3-5 flies/genotype). *P < 0.05, **P < 0.01 by Student's t test. Scale bar represents 25 µm 1 day of age, IRE1 or α-Syn A30P overexpression had no apparent effect upon the number of DA neurons in the five indicated clusters (Fig. 6c), suggesting that IRE1 overexpression did not disrupt the development of DA neurons. However, at 40 days of age, Ddc-Gal4 > IRE1 flies exhibited significant loss of DA neurons in some of the clusters, similar to the extent of DA neuron loss observed in Ddc-Gal4 > α-Syn A30P flies (Fig. 6c). Therefore, chronic activation of IRE1 by its overexpression was sufficient to cause DA neuron degeneration.

Discussion
PD is a devastating neurodegenerative disease with an age-related decline of motor functions, largely arising from selective loss of DA neurons in the substantia nigra Dopaminergic neurons in the indicated clusters were quantified (n = 5 flies/genotype; two independent experiments). All data are shown as mean ± s.e.m. *P < 0.05, **P < 0.01 by one-way ANOVA. Scale bar represents 100 µm pars compacta region 57,58 . As a pathological hallmark of PD, α-synuclein aggregation in Lewy bodies may reflect the ultimate consequence of the cellular machinery that goes awry for disposal of misfolded proteins. In this scenario, the overload of misfolded α-synuclein can lead to activation of the ER stress pathways, which is implicated in promoting cell death under various stress conditions 13 . However, the precise contribution of the individual UPR signaling branches during α-synucleinopathy in DA neurons remains unclear. Our results demonstrate in vivo that the IRE1 pathway is critical in coupling neuronal ER stress to autophagy-dependent neuron death, thereby driving the Parkinsonian neurodegeneration. These findings suggest that targeted inhibition of the IRE1 pathway or the resultant autophagy-dependent neuron death may provide a valuable intervention strategy against PD.
An important notion from our findings is that IRE1induced autophagy-dependent neuron death may constitute a key component of the mechanism linking age-dependent accumulation of misfolded neurotoxic proteins to DA neuron loss and PD-like phenotypes. Autophagy is a cellular quality control mechanism for clearance of altered proteins and damaged organelles, and it is conceivable that dysfunction of autophagy in neurons can be associated with disruption of neuronal homeostasis 27 . Autophagy was also documented to be activated and exert cytoprotective effects during ER stress 29,[59][60][61] . Surprisingly, our results clearly suggest that chronic activation of the IRE1 branch of the UPR can direct autophagy to the route of cell death in DA neurons in response to accumulation of α-synuclein. It is indicating that autophagy can serve as a "double-sword" mechanism by triggering cell death under certain pathological conditions, particularly when cellular protein degradation machinery fails to clear up misfolded protein wastes derived from imbalanced proteostasis. In addition, such IRE1-induced autophagy-dependent cell death may not be a neuron-specific phenomenon, and it has yet to be Fig. 7 Knockdown of IRE1 or ATG7 expression reverses α-Synuclein-induced neurodegeneration. a Lifespan of Ddc-Gal4 > +, Ddc-Gal4 > α-Syn A30P , Ddc-Gal4 > α-Syn A30P ; Ire1-Ri, and Ddc-Gal4 > α-Syn A30P ; Atg7-Ri flies (n = 90 flies/genotype). b Climbing ability of the indicated genotype at 1, 3, or 5 weeks of age. Relative activities are shown as mean ± s.e.m. (n = 90 flies/genotype; two independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA. c Representative Z-stack confocal images of whole brains stained with anti-TH antibody for the indicated lines at 40 days of age. TH-positive DA neurons of the indicated clusters were quantified. PAL protocerebral anterior lateral, PPM protocerebral posterior medial, PPL protocerebral posterior lateral. The data are shown as mean ± s.e.m. (n = 5 flies/genotype; two independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA. Scale bar represents 100 µm further deciphered if this occurs in a tissue-or cellspecific and context-dependent manner.
The IRE1-XBP1 branch of the UPR has long been thought to promote cell survival during ER stress, but the exact role of IRE1 versus XBP1 during neurodegeneration has yet to be clearly defined. Mouse model studies indicated that XBP1s was able to exert neuroprotective effects upon DA neurons in PD 21,44 , whereas XBP1 deficiency might protect against neurodegeneration in HD via its action upon FOXO1-regulated autophagy 23 . Our results in the Drosophila model of PD indicated an appreciable protection effect of XBP1 upon neuronal survival, which is in accordance with the reported findings in the mouse PD models 20 . However, we observed an XBP1independent effect of IRE1 upon activation of neuronal autophagy in Drosophila. Based on the documented studies in mammalian cell lines 29 , IRE1 regulation of autophagy through the JNK pathway may operate as an evolutionarily conserved mechanism. Therefore, the regulatory actions of IRE1 versus XBP1 upon autophagy with regard to neuronal cell fate need to be dissected in the specific disease context.
In summary, our studies have uncovered a mechanism that the IRE1 pathway may act as a critical "rheostat" of proteostatic stress in the control of neuron cell fate in the context of α-synucleinopathy. IRE1 pathway-induced autophagy-dependent neuron death acts as a conserved pathogenic driver during PD progression, targeted modulation of IRE1 or autophgy in neurons may provide new avenues for developing therapeutics against this neurodegenerative disease.
The pUAS-IRE1 or pUAS-XBP1s, vector was used to generate two independent UAS-IRE1 orUAS-XBP1s transgenic lines following standard germline transformation procedures 62 at the Core Facility of Drosophila Resource and Technology, SIBCB, CAS. These lines were crossed with the special Drosophila line containing the double balancer (CyO/Bl; TM2, Ubx/TM6B, Tb) to identify the chromosome with the transgene insertion. The transgenic lines were backcrossed into the w 1118 background for over five generations before further genetic manipulations.
All the fly lines were raised on standard yeast-cornmealagar food and maintained in vials at 25°C with 50% humidity under a 12 h/12 h light/dark cycle.
Analysis of fly eyes and photoreceptor neurons. For analysis of external eyes by SEM, adult fly heads were dissected and directly examined by SEM. Images were taken at ×180 or ×800 magnification. For photoreceptor neuron analyses, fly heads were fixed in 4% formaldehyde for at least 24 h and embedded in Epon 812, followed by toluidine blue staining or TEM analysis. For toluidine blue staining, embedded fly eyes were semi-thin-sectioned at 500 μm and stained with toluidine blue as described 64 . For TEM analysis, the embedded eyes were ultrathinsectioned at 70 nm followed by staining with Reynold's lead citrate and 2% aqueous uranyl acetate. Retinal cells were subsequently analyzed and imaged using Tecnai G2 Spirit transmission electron microscope equipped with the GANTA-830 CCD camera.

Lifespan and locomotor activity
Cohorts of 90 flies for each genotype were monitored for survival. Mortality was scored every three days. Climbing ability of flies was measured as described 65 . Briefly, 90 flies of both sexes for each genotype were tapped to the bottom of a graduated cylinder (1.5 cm in diameter; 25 cm in length). Flies that could climb up to or above 5-cm from the bottom of the cylinder within 10 s were counted.

Quantification of DA neurons
Fly brains were dissected and subjected to whole-mount immunostaining using the anti-TH antibody as described previously 65 . Brains were examined by confocal laserscanning microscope (Olympus BX61), and the numbers of TH-positive neurons in all DA clusters within a half of the brain were counted, except those in the paired anterolateral medial (PAM) cluster in which the high fluorescent intensity did not allow for precise counting due to the high density of DA neurons.

Statistical analysis
All data are presented as the mean ± standard errors of the mean (s.e.m) from at least three independent experiments. Statistical analysis was performed using unpaired two-tailed Student's t test, one-way or two-way analysis of variance (ANOVA) followed by Bonferroni's post test with GraphPad Prism 5.0. P < 0.05 was considered to be statistically significant.