Transcription- and phosphorylation-dependent control of a functional interplay between XBP1s and PINK1 governs mitophagy and potentially impacts Parkinson disease pathophysiology

Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=kaup20 Autophagy ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/kaup20 Transcriptionand phosphorylation-dependent control of a functional interplay between XBP1s and PINK1 governs mitophagy and potentially impacts Parkinson disease pathophysiology Wejdane El Manaa, Eric Duplan, Thomas Goiran, Inger Lauritzen, Loan Vaillant Beuchot, Sandra Lacas-Gervais, Vanessa Alexandra Morais, Han You, Ling Qi, Mario Salazar, Umut Ozcan, Mounia Chami, Frédéric Checler & Cristine Alves da Costa To cite this article: Wejdane El Manaa, Eric Duplan, Thomas Goiran, Inger Lauritzen, Loan Vaillant Beuchot, Sandra Lacas-Gervais, Vanessa Alexandra Morais, Han You, Ling Qi, Mario Salazar, Umut Ozcan, Mounia Chami, Frédéric Checler & Cristine Alves da Costa (2021): Transcriptionand phosphorylation-dependent control of a functional interplay between XBP1s and PINK1 governs mitophagy and potentially impacts Parkinson disease pathophysiology, Autophagy, DOI: 10.1080/15548627.2021.1917129 To link to this article: https://doi.org/10.1080/15548627.2021.1917129 © 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. View supplementary material Published online: 24 May 2021. Submit your article to this journal Article views: 1036 View related articles View Crossmark data RESEARCH PAPER Transcriptionand phosphorylation-dependent control of a functional interplay between XBP1s and PINK1 governs mitophagy and potentially impacts Parkinson disease pathophysiology Wejdane El Manaa a , Eric Duplan a , Thomas Goiran a , Inger Lauritzen a , Loan Vaillant Beuchot a , Sandra Lacas-Gervais b , Vanessa Alexandra Morais c , Han You d , Ling Qi e , Mario Salazar f , Umut Ozcan f , Mounia Chami a , Frédéric Checler a , and Cristine Alves da Costa a a INSERM, CNRS, IPMC, Team Labeled “Laboratory of Excellence (LABEX) Distalz”, Sophia-Antipolis, Université Côte d’Azur, Valbonne, France; b Centre Commun de Microscopie Appliquée, Université Côte d’Azur, Nice, France; c Instituto de Medicina Molecular - João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal; d State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, Fujian, China; e Division of Metabolism, Endocrinology & Diabetes, University of Michigan, Ann Arbor, NY, USA; f Division of Endocrinology, Boston Children’s Hospital, Harvard Medical School, Boston, MA, USA ABSTRACT Parkinson disease (PD)-affected brains show consistent endoplasmic reticulum (ER) stress and mitophagic dysfunctions. The mechanisms underlying these perturbations and how they are directly linked remain a matter of questions. XBP1 is a transcription factor activated upon ER stress after unconventional splicing by the nuclease ERN1/IREα thereby yielding XBP1s, whereas PINK1 is a kinase considered as the sensor of mitochondrial physiology and a master gatekeeper of mitophagy process. We showed that XBP1s transactivates PINK1 in human cells, primary cultured neurons and mice brain, and triggered a pro-mitophagic phenotype that was fully dependent of endogenous PINK1. We also unraveled a PINK1-dependent phosphorylation of XBP1s that conditioned its nuclear localization and thereby, governed its transcriptional activity. PINK1-induced XBP1s phosphorylation occurred at residues reminiscent of, and correlated to, those phosphorylated in substantia nigra of sporadic PD-affected brains. Overall, our study delineated a functional loop between XBP1s and PINK1 governing mitophagy that was disrupted in PD condition. Abbreviations: 6OHDA: 6-hydroxydopamine; baf: bafilomycin A 1 ; BECN1: beclin 1; CALCOCO2/ NDP52: calcium binding and coiled-coil domain 2; CASP3: caspase 3; CCCP: carbonyl cyanide chlorophenylhydrazone; COX8A: cytochrome c oxidase subunit 8A; DDIT3/CHOP: DNA damage inducible transcript 3; EGFP: enhanced green fluorescent protein; ER: endoplasmic reticulum; ERN1/ IRE1α: endoplasmic reticulum to nucleus signaling 1; FACS: fluorescence-activated cell sorting; HSPD1/HSP60: heat shock protein family D (Hsp60) member 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MFN2: mitofusin 2; OPTN: optineurin; PD: Parkinson disease; PINK1: PTENinduced kinase 1; PCR: polymerase chain reaction:; PRKN: parkin RBR E3 ubiquitin protein ligase; XBP1s [p-S61A]: XBP1s phosphorylated at serine 61; XBP1s [p-T48A]: XBP1s phosphorylated at threonine 48; shRNA: short hairpin RNA, SQSTM1/p62: sequestosome 1; TIMM23: translocase of inner mitochondrial membrane 23; TM: tunicamycin; TMRM: tetramethyl rhodamine methylester; TOMM20: translocase of outer mitochondrial membrane 20; Toy: toyocamycin; TP: thapsigargin; UB: ubiquitin; UB (S65): ubiquitin phosphorylated at serine 65; UPR: unfolded protein response, XBP1: X-box binding protein 1; XBP1s: spliced X-box binding protein 1 ARTICLE HISTORY Received 16 July 2019 Revised 1 April 2021 Accepted 9 April 2021 KEYWORDS Mitophagy; Parkinson disease; phosphorylation; PINK1; transcription; unfolded protein response; XBP1 Introduction Parkinson disease (PD) is a movement disorder characterized by the degeneration of dopaminergic neurons. Various cellular dysfunctions including altered dopamine metabolism, increased oxidative stress, mitochondrial failure, altered calcium homeostasis, neuroinflammation, impaired autophagy and proteasome dysfunctions appear tightly linked to PDassociated neuronal loss [1]. Furthermore, numerous evidence indicate that the intracellular accumulation of misfolded proteins notably SNCA/alpha-synuclein [2,3] and endoplasmic reticulum (ER) overload could contribute to this pathology [1,4,5]. The ER overload-associated stress leads to the activation of the unfolded protein response (UPR). The UPR consists of a coordinated signaling response tailored to the stress severity that will either restore ER homeostasis and preserve the cell or trigger its elimination by apoptosis in case of exacerbated stress. The UPR response is elicited by three stress sensors located at the ER membrane, namely, EIF2AK3/PERK (eukaryotic translation initiation factor 2 alpha kinase 3), ATF6 (activating transcription factor 6) and ERN1/IRE1α (endoplasmic reticulum to nucleus signaling 1) [6,7]. Importantly, postmortem studies of PD-affected human brains [8–11] as well as toxin-induced [12–14] and genetically CONTACT Cristine Alves da Costa [email protected]; Frédéric Checler [email protected] CNRS, IPMC, Team Labeled “Laboratory of Excellence (LABEX) Distalz”, Sophia-Antipolis, Université Côte d’Azur, INSERM, 660 route des Lucioles, Valbonne 06560, France Supplemental data for this article can be accessed here. AUTOPHAGY https://doi.org/10.1080/15548627.2021.1917129 © 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way. designed cellular and animal models of PD converge to point to the key role of the UPR in PD [4,15]. ERN1 is the most evolutionary conserved ER stress transducer of the UPR that harbors a kinase and RNAse activity [16]. ERN1-mediated signaling is a positive regulator of cell survival that stops upon beyond repair ER stress resulting in apoptosis. It is responsible for the unconventional splicing of XBP1 (X box binding protein 1) [15–17] yielding its transcriptionally active form XBP1s (XBP1 spliced). XBP1s regulates genes involved in protein folding and ER-associated degradation (ERAD) and is frequently associated with a prosurvival phenotype in dopaminergic neurons [18–20]. Interestingly, XBP1s was shown to participate in the macroautophagy/autophagy-mediated degradation process [21,22]. The molecular mechanisms underlying this control remain poorly understood. It should be noted that specialized elimination of dysfunctional mitochondria by mitophagy requires the recruitment of PRKN/parkin by PINK1 (PTEN induced kinase 1), a kinase that is considered as the cellular sensor of mitochondrial health [23]. Noteworthy, PRKN has been shown to control XBP1s transcription [24] and to promote PSEN (presenilin)-dependent control of PINK1 [25]. Thus, we questioned whether XBP1s could be directly involved in the control of mitophagy and whether such phenotype could involve a PINK1-dependent process. Our work demonstrated that XBP1s controlled mitophagy via the transcriptional regulation of PINK1 in various cellular models as well as in vivo. Interestingly, we established that XBP1s underwent PINK1dependent phosphorylation at sites reminiscent of those hyperphosphorylated in sporadic PD-affected substantia nigra. This was the first demonstration of a direct role of XBP1s transcriptional factor function in the control of mitophagy and the occurrence of a functional XBP1s-PINK1 loop governing mitophagy that could be disrupted in pathological conditions. Results Stress activators and ERN1 blockers similarly affect XBP1s and PINK1 in dopaminergic and primary cultured neurons as well as in mice brain In SH-SY5Y dopaminergic neurons, the ER stress inducer thapsigargin (TP) similarly increased XBP1s and PINK1 protein expressions in a time-dependent manner (Fig. S1A and S1B). Accordingly, 8 h of TP treatment enhanced PINK1 promoter activity (Fig. S1C) and mRNA levels (Fig. S1D). We have chosen a treatment with TP for 8 h because it corresponded to the optimal time of induction for both PINK1 and XBP1s protein levels. It should be noted that in our experimental setting, after 8 h of TP treatment, SH-SY5Y cells were already committed to apoptotic program as illustrated by the increased expression of the ER stress-induced protein DDIT3/CHOP (DNA damage inducible transcript 3) (Fig. S1A). We examined whether ER stress-mediated enhancement of PINK1 transcriptional regulation could well be triggered by XBP1s. In order to address this question, we used toyocamycin (Toy), a specific blocker of ERN1 that is responsible for XBP1s functional activation after XBP1 splicing [26] and that displays few nonspecific effects on several other kinases [27–29]. As expected, Toy fully prevented the TP-induced increase of XBP1s expression (Figure 1A). Interestingly, Toy also reduced the basal protein (Figure 1A) and mRNA (Figure 1B) expressions of PINK1 but also fully blocked their TP-induced increases (Figure 1A, B). It should be noted that this data could indicate that even in basal non-stimulated condition, there exists a low but functional XBP1s production (poorly detectable by western blot but that can be unraveled upon long gel exposures) as we previously established by mRNA splicing analysis [24] that is prevented by Toy. Importantly, in order to rule out any bias linked to cell immortalization, we examined the influence of TP and another ER stress modulator tunicamycin (TM) on XBP1s and PINK1 regulation in rat primary cultured neurons. Both TP (Fig. S2A-C) and TM (Fig. S2D-F), enhanced XBP1s (Fig. S2A and S2D) and PINK1 (Fig. S2A, S2B, S2D, and S2E) protein expressions and Pink1 mRNA levels (Fig. S2C and S2F). Of importance, TM also increased XBP1s (Fig. S2G) and PINK1 (Fig. S2G and S2H) protein expressions and Pink1 mRNA levels (Fig. S2I) in 2-month-old treated wild-type mice. XBP1s modulates PINK1 expression at a transcriptional level Next, we examined the impact of XBP1s on PINK1 regulation by a genetic approach. We demonstrated that Xbp1s transient overexpression (see expression of XBP1s in Figure 1C, upper panel) increased PINK1 protein expression (Figure 1C) mRNA levels (Figure 1E) and promoter transactivation (Figure 1D). It should be noted that in agreement with its promitophagic role, XBP1s overexpression (Fig. S3A and S3C) led to an accumulation of PINK1 in the TIMM23 (translocase of inner mitochondrial membrane 23)-positive mitochondriaenriched compartment (Fig. S3A and S3B) similarly to control carbonyl cyanide m-chlorophenylhydrazone (CCCP) treatment known to stabilize PINK1 protein at the outer mitochondrial membrane. In order to confirm these data at an endogenous level, we examined the regulation of endogenous PINK1 in SH-SY5Y stably infected with lentiviral construct harboring a short hairpin RNA (shRNA) targeting XBP1. Fig. S4 showed infection efficacy by means of GFP imaging (Fig. S4A) that was correlated with a drastic reduction of XBP1s protein expression (Fig. S4B) and a 54% reduction in XBP1s mRNA levels (Fig. S4C). Genetic downregulation of XBP1 led to drastic reductions of PINK1 protein expression (Figure 1F), mRNA levels (Figure 1H) and promoter transactivation (Figure 1G). Interestingly, in primary cultured neurons, XBP1s overexpression (Fig. S5A-C) increased PINK1 protein expression (Fig. S5A), mRNA levels (Fig. S5C) and promoter activity (Fig. S5B) while conversely, Xbp1 knockdown (Xbp1 KD) (Fig. S5D-F) lowered PINK1 protein (Fig. S5D) and mRNA (Fig. S5F) expressions. XBP1s protein overexpression and mRNA depletion levels are illustrated in Fig. S5A and S5E, respectively. Moreover, we examined the influence of XBP1 depletion on PINK1 expression in differentiated dopaminergic SH-SY5Y cells (Fig. S5G). The efficacy of dopaminergic differentiation by retinoic acid and phorbol ester 2W. EL MANAA ET AL. Figure 1. ER stress modulators and XBP1s regulate PINK1 transcription. (A and B) SH-SY5Y cells were treated for 8 h with vehicle (Ct, DMSO), toyocamycin (Toy, 1 µM), thapsigargin (TP, 1 µM) or thapsigargin and toyocamycin (TP/Toy). In TP/Toy condition, cells were pretreated for 16 h with Toy then TP was added for 8 h. Then, PINK1 and XBP1s protein expressions (A, N = 12, One-way ANOVA, Tukey’s multiple comparisons test) and PINK1 mRNA levels (B, N = 9, One-way ANOVA, Tukey’s AUTOPHAGY 3 subsequent treatment was illustrated by the increased level of the canonical dopaminergic marker, tyrosine hydroxylase (TH). We showed that, as we observed in non-differentiated SH-SY5Y cells, the depletion of endogenous XBP1 led to decreased PINK1 protein levels in SH-SY5Y differentiated cells. The above set of data confirmed the ability of endogenous and overexpressed XBP1s to upregulate PINK1 in both dopaminergic, primary cultured neurons as well as in differentiated dopaminergic SH-SY5Y cells. The fact that XBP1s is a transcription factor and that PINK1 mRNA levels were modulated led us to examine whether XBP1s could act as a direct activator of Pink1 transcription. Interestingly, in silico analysis of Pink1 promoter region identified two putative XBP1s responsive elements (Figure 1I, black boxes in P2.0 construct). We carried out 5ʹ end deletion experiments of the full-length Pink1 promoter in frame with a luciferase gene reporter. First, we confirmed that Xbp1s expression (Figure 1J, upper panel) dramatically enhanced the transactivation of full-length Pink1 promoter (Figure 1J, P2.0). Truncation of the region harboring the most 5ʹ end responsive element (−2054/-2049, see Figure 1I, construct P1.3) did not affect the XBP1s-mediated transactivation of the Pink1 promoter construct (Figure 1J). Conversely, the deletion of the −1354/-854 region harboring the second responsive element (−1026/-1021, see Figure 1I construct P0.8) abolished the XBP1s-mediated Pink1 transactivation (Figure 1J). This was supported by the examination of the P0.4 construct (Figure 1I, J). We confirmed the functionality of this responsive element by site-directed deletion. We showed that partial removal of this responsive element (−1024/-1021) yielded a construct (P2.0∆, Figure 1K left panel) that remained unresponsive to XBP1s (Figure 1K, right panel). The direct interaction of XBP1s with PINK1 promoter was further demonstrated by gel shift analysis. In this experiment, recombinant XBP1s was incubated with biotinylated Pink1 probes encompassing the delineated functional 1025–1021 domain of Pink1 promoter. This bimolecular in vitro interaction allowed unraveling a direct physical interaction without the participation of any additional transcriptional cofactor. Figure 1L showed that, indeed, XBP1s interacted with Pink1 probe (Figure 1L compare lane 1 and 2), a label abolished by an excess of unlabeled probe (Figure 1L, compare lanes 2 and 3). Overall, this set of data indicated that XBP1s acted as a direct transcriptional activator of Pink1 gene and identified the responsive element mediating XBP1s-Pink1 promoter functional interaction. XBP1s modulates mitophagy and mitochondrial health We assessed the functional influence of XBP1s expression on mitochondrial physiology and mitophagy, two processes tightly controlled by PINK1. This was assessed by monitoring a panel of autophagy/mitophagy protein reporters, some of which were previously shown to be linked to PINK1 [30,31]. We measured: 1) the expression of BECN1 (beclin 1) that is a pro-autophagy protein implicated in autophagosome formation and maturation [32]; 2) the ratio of MAP1LC3-II/LC3-II (lipidated microtubule associated protein 1 light chain 3) vs. LC3-I expression, the modulation of which reflects autophagosome formation [33]; 3) SQSTM1/p62 (sequestosome 1), OPTN (optineurin) and CALCOCO2/NDP52 levels that are autophagic receptors that have been linked to PRKN-PINK1 mitophagy process [34]; 4) the expressions of TOMM20, TIMM23 and HSPD1/HSP60 (heat shock protein mitochondrial chaperone located at the mitochondrial matrix) that are biochemical markers of mitochondrial mass classically used to follow early and late mitophagic processes; 5) the levels of ubiquitin phosphorylated at serine 65 (UB [S65]) and PRKN, two substrates of PINK1 involved in the control of mitophagy [33]. First, we assessed the impact of XBP1s on autophagic flux. Xbp1s cDNA transfection led to an increased LC3-II:LC3-I ratio (Figure 2A, B) and reduced SQSTM1 (Figure 2A, C) expression. Importantly, bafilomycin A 1 (baf), which is classically used to trigger autolysosome acidification and to disrupt autophagosome-lysosome fusion [33], increased XBP1smediated modulations of LC3-II:LC3-I and SQSTM1 (Figure 2A, C) thereby confirming that XBP1s increased autophagic/ mitophagic flux. Next, we showed that Xbp1s transient multiple comparisons test) were analyzed as described in Materials and Methods. Data are expressed as percent of control DMSO-treated cells (taken as 100%) and are the means ± SEM of 3–4 independent experiments performed in triplicates. GAPDH expression (A) is provided as a control of protein load. (C-E) SH-SY5Y cells were transiently transfected with an empty pcDNA3 vector (Ev) or wild-type Xbp1s cDNA. Twenty-four hours after transfection, PINK1 protein expression (C, N = 12, analyzed by Student’s t test), promoter transactivation (D, N = 15, Student’s t test) and mRNA levels (E, N = 12, Student’s t test) were analyzed as described in Materials and Methods. SH-SY5Y cells treated with CCCP (CP, 10 µM for 6 h) were included as migration controls for PINK1. Data are expressed as percent of control Ev-transfected cells (taken as 100%) and are the means ± SEM of 4–5 independent experiments performed in triplicates. ACTB and XBP1s expressions are provided in (C) as a control of protein load and Xbp1s transfection efficiency. (F-H) SH-SY5Y stably expressing scrambled (SC) or shRNA-targeting XBP1 (XBP1 KD) were assessed for PINK1 protein expression (F, N = 9, analyzed by Student’s t test), PINK1 promoter transactivation (G, N = 15, Student’s t test) and PINK1 mRNA levels (H, N = 18, Student’s t test) as described in Methods. Data are expressed as percent of control SC cells (taken as 100%) and are the means ± SEM of 3–6 independent experiments performed in triplicates. ACTB expression is provided in (F) as a control of protein load. (I) The scheme represents the full-length (FL, P2.0) mouse Pink1 promoter region and 5ʹ end deletion constructs (P1.3, P0.8 and P0.4) in frame with luciferase. Black boxes on P2.0 construct correspond to two putative Xbp1s responsive elements. (J) Promoter constructs were then co-transfected in SH-SY5Y cells with the GLB1 (galactosidase beta 1) reporter gene (in order to normalize transfection efficiencies) and either empty vector (Ev, black bars) or Xbp1s (gray bars) cDNAs. Twenty-four hours after transfection, luciferase activity was measured (N = 12, analyzed by One-way ANOVA, Tukey’s multiple comparisons test) then expressions of XBP1s and ACTB were analyzed as described in Methods. Data are expressed as percent of control Ev/GLB1-transfected cells (taken as 100%) and are the means ± SEM of 4 independent experiments performed in triplicates. (K) The scheme (left panel) represents the PINK1 promoter construct (P2.0∆) lacking the −1024/-1021 Xbp1s-responsive element. P2.0 and P2.0∆ promoter constructs were co-transfected with the GLB1 reporter gene and either empty vector (Ev, black bars) or Xbp1s (gray bars) cDNAs in SH-SY5Y cells. Twenty-four hours after transfection, luciferase activity was measured (N = 9, analyzed by One-way ANOVA, Tukey’s multiple comparisons test) then expression of XBP1s and ACTB were analyzed as described in Methods (right panel). Data are expressed as percent of control Ev/GLB1-transfected cells (taken as 100%) and are the means ± SEM of 3 independent experiments performed in triplicates. Statistical significances are: **, P < 0.01, ****, P < 0.0001 and ns for non-significant. (L) EMSA analysis of the physical interaction of purified recombinant XBP1s and Pink1 biotinylated probes encompassing the −1024/-1021 sequence of the mouse promoter (see panel K) in absence (lane 2) or in the presence (lane 3) of an excess of unlabeled probe. Lane 1 corresponds to biotinylated probe alone. 4W. EL MANAA ET AL. Figure 2. Xbp1s overexpression leads to increased mitophagy in SH-SY5Y cells. (A-C) SH-SY5Y cells were transiently transfected with an empty vector (Ev) or Xbp1s cDNA treated or not with bafilomycin A1 (Baf, 100 nM) then analyzed by western blot for XBP1s (A, N = 15), LC3-II:LC3-I ratio (A and B, N = 12) and SQSTM1/p62 (A and C, N = 15) protein levels. Statistical significances were analyzed by ordinary one-way ANOVA followed by Sidak’s multiple comparison test, * P < 0.05, ** P < 0.01, *** P < 0.001, ****, P < 0.0001. (D-J) SHSY5Y cells were transiently transfected with an empty vector (Ev) or Xbp1s cDNA then analyzed for BECN1 (D and E, N = 9), OPTN (D and F, N = 15), TIMM23 (D and G, N = 12), TOMM20 (D and H, N = 9), UB (S65) (D and I, N = 12), PRKN (D and J, N = 12) and TUBB protein levels as described in Methods. (K) SH-SY5Y cells were transiently transfected with an empty vector (Ev) or Xbp1s cDNA then mitochondrial membrane potential was measured by cell imaging (upper panel, N = 100 cells, two independent experiments, IF in histogram) or flow cytometry (, N = 15, 5 independent experiments, FACS in histogram) by means of TMRM probe as detailed in the Methods. Values are expressed as percent of control Ev-transfected cells (taken as 100%) and correspond to the means ± SEM of 2–5 independent experiments. Statistical significances were analyzed by Mann-Whitney test, ****, P < 0.0001. (L) SH-SY5Y cells were transiently transfected with COX8A-EGFP-mCherry cDNA together with an empty vector (Ev) or Xbp1s cDNAs. Fragmented mitochondria visualized by red fluorescence punctate (left panel) were counted as described in Methods. The degree of mitophagy (right panel) was calculated by the increase of number of cells harboring red punctae. (M,) CASP3 activity fluorimetric assay was performed as described in Methods. Values are expressed as percent of control Ev-transfected cells (taken as 100%) and correspond to the means ± SEM of 4 independent experiments performed in triplicates. Statistical significances were analyzed by ordinary one-way ANOVA followed by Sidak’s multiple comparison test, ***, P < 0.001, ****, P < 0.0001. AUTOPHAGY 5 overexpression in dopaminergic neurons increased the protein levels of BECN1 (Figure 2D, E), OPTN (Figure 2D, F), UB (S65) (Figure 2D,Figure2I) and PRKN (Figure 2D, J). Conversely, Xbp1s overexpression reduced TIMM23 (Figure 2D, G) and TOMM20 (Figure 2D, H) protein expressions. Furthermore, we measured the mitochondrial membrane potential in living cells using Tetramethyl rhodamine methylester (TMRM) probe and quantified fluorescence signal by confocal imaging (Figure 2K, upper panel and IF in histogram) and fluorescence-activated cell sorting (FACS) (Figure 2K, FACS in histogram). We showed that Xbp1s overexpression prevented the disruption of mitochondrial membrane potential (Figure 2K) indicating that XBP1s controlled PINK1-mediated basal mitophagy. In order to analyze the impact of XBP1s on mitophagic flux in living cells, we used COX8A (cytochrome c oxidase subunit 8A)-EGFP (enhanced green fluorescent protein)-mCherry mitophagy reporter [35]. The degree of mitophagy was calculated by the increase of the number of cells harboring red fragmented mitochondria (engulfed in the lysosomal acidic compartment) (Figure 2L). Importantly, this data clearly indicated that Xbp1s transfection increased mitophagic flux corroborating the LC3-II:LC3-I and SQSTM1 biochemical data. Thus, XBP1s expression exacerbated a punctate COX8Alinked red fluorescence corresponding to fragmented mitochondria, clearance in the lysosomal compartment (Figure 2L). Of importance, transient overexpression of Xbp1s in primary cultured neurons fully reproduced the phenotypes observed above for Xbp1s overexpression in dopaminergic cells (Fig. S6A and S6L). Finally, Xbp1s overexpression (Figure 2M, lower panel) allowed us delineating a protective phenotype illustrated by a reduction of basal and TP-mediated activation of the pro-apoptotic protein CASP3 (caspase 3; Figure 2M, histogram). We performed similar extensive analysis of autophagic/ mitophagic reporters in XBP1 KD dopaminergic cells (Figure 3). First, in basal conditions, we observed decreased LC3-II: LC3-I ratios (Figure 3A, B) and augmented SQSTM1 expression (Figure 3A, C) associated with XBP1 knockdown. Of note, baf treatment time-dependently increased both basal and XBP1 KD -linked LC3-II:LC3-I ratios (Figure 3A, B) and SQSTM1 expressions (Figure 3A, C), confirming our previous conclusion (see Figure 2) that XBP1s controlled mitophagy flux at endogenous levels. Furthermore, we observed decreased expressions of BECN1 (Figure 3D, E), OPTN (Figure 3D, F), CALCOCO2 (Figure 3D, G), UB (S65) (Figure 3D, K) and PRKN (Figure 3D, L) while TIMM23 (Figure 3D, H), TOMM20 (Figure 3D, I) and HSPD1 (Figure 3D, J) levels were augmented. Of note, all above-described protein markers varied in a perfect opposite manner in Xbp1s-overexpressing dopaminergic cells (Figure 2A–J) and primary cultured neurons (Fig. S6A-K) compared to XBP1 KD (Figure 3A–L) cells. Further, opposite to overexpression of Xbp1s, reduction of endogenous levels of XBP1s also drastically reduced mitochondrial membrane potential (Figure 3M) and increased basal and TP-stimulated CASP3 activity (Figure 3N). The latter observation was corroborated by our observation of an activation of the apoptotic UPR illustrated by the increase of DDIT3 protein and mRNA levels (data not shown). Overall, the above data concurred to conclude that XBP1s modulated mitochondrial function and mitophagy. XBP1s-induced modulation of mitophagy and mitochondrial health is dependent on PINK1 Our data indicated that XBP1s-associated effects on mitochondrial and mitophagic processes strictly resembled those described for PINK1-associated phenotypes. Although we showed that XBP1s was a transcriptional activator of PINK1, it remained to definitely establish whether XBP1s-induced phenotype was fully or partly dependent on endogenous PINK1. To address this question, we have overexpressed Xbp1s in PINK1 knockdown (PINK1 KD) dopaminergic cells and assessed the influence of PINK1 reduction on the expression of the above-described panel of mitophagy/autophagy protein reporters as well as on the mitochondrial membrane potential (Figure 4). First and importantly, comparative analyses of PINK1 control (PINK1 CT) and PINK1 KD cells (compare lanes [-] of black and gray bars in Figure 4B–L) indicated that the PINK1 KD-associated mitophagy response perfectly mimicked XBP1 KD-induced phenotype (see Figure 3). Thus, PINK1 KD cells showed decreased levels of BECN1 (Figure 4A, B), LC3-II:LC3-I ratio (Figure 4A, C), OPTN (Figure 4A, E), CALCOCO2 (Figure 4A, F), UB (S65) (Figure 4A, 4J) and PRKN (Figure 4A, K) while SQSTM1 (Figure 4A, D), TIMM23 (Figure 4A, G), TOMM20 (Figure 4A, H), HSPD1 (Figure 4A, I) and MFN2 (mitofusin 2) levels (Figure 4A, L) were increased. Membrane mitochondria potential analysis (Figure 4M) also corroborated the protective role of endogenous PINK1 in the preservation of mitochondria function. Second, in this set of independent experiments, we fully reproduced the effects described in Figures 2, 3 and S6 concerning the impact of Xbp1s on the control of mitophagy in PINK1 CT cells. Thus, Xbp1s overexpression (compare black bars, lanes [-] and [+]) triggered an upregulation of BECN1 (Figure 4A, B), LC3-II:LC3-I ratio (Figure 4A, C), OPTN (Figure 4A, E), CALCOCO2 (Figure 4A, F), UB (S65) (Figure 4A, J) and PKKN (Figure 4A, K) while SQSTM1 (Figure 4A, D), TIMM23 (Figure 4A, G), TOMM20 (Figure 4A, H), HSPD1 (Figure 4A, I) and MFN2 (Figure 4A, L) levels were decreased. Membrane mitochondria potential analysis (Figure 4M) also emphasized the protective function of XBP1S in the preservation of mitochondria function. Finally, of utmost importance, all Xbp1s-linked effects on expressions of protein reporters and TMRM were fully abolished by PINK1 depletion (Figure 4A–M, compare [-] and [+] gray bars in PINK1 KD cells). Overall, our data clearly demonstrated that XBP1s-mediated control of mitochondrial physiology and mitophagy was fully PINK1-dependent. Pharmacological blockade of ERN1 reduces endogenous PINK1 expression and mimics PINK1-knockdownassociated phenotype in mice Our study showed that XBP1s was a transcriptional activator of PINK1 and that this accounted for all PINK1-dependent 6W. EL MANAA ET AL. Figure 3. Endogenous XBP1s lowers mitophagic response. (A-C) SH-SY5Y cells stably expressing either control (scrambled, SC, black bars) or Xbp1 shRNA (Xbp1KD, gray bars) were either treated or not with bafilomycin A1 (Baf 100 nM for the indicated times) then examined by western blot (see Methods) for LC3-II:LC3-I ratio (A and B, N = 9, one-way ANOVA, Tukey’s multiple comparison test), SQSTM1 (A and C, N = 6, Kruskal Wallis multiple comparison). (D-L) BECN1 (D and E, N = 6, MannWhitney test), OPTN (D and F, N = 9, Student’s t test), CALCOCO2 (D and G, N = 6, Mann-Whitney test), TIMM23 (D and H, N = 9, Student’s t test), TOMM20 (D and I, N = 12, Mann-Whitney test), HSPD1 (D and J, N = 9, Student’s t test), UB (S65) (D and K, N = 9, Student’s t test) and PRKN (D and L, N = 9, Student’s t test) protein expressions in SC or XBP1 KD cells. ACTB expressions are provided in (A and D) as controls of protein load. (M) Mitochondrial potentials of SC and XBP1 KD cells were analyzed by flow cytometry as described in Methods (N = 15, analyzed by Student’s t test). (N) CASP3 activity expression was measured in basal and TP-stimulated conditions as described in Methods (N = 12, analyzed by One-way ANOVA followed by Sidak’s multiple comparison test). (B-N) Data are expressed as percent of SC (CT) cells (taken as 100%) and are the means ± SEM of 2–5 experiments performed in triplicates. Statistical significances are * P < 0.05, ** P < 0.01, *** P < 0.001, ****, P < 0.0001. AUTOPHAGY 7 Figure 4. XBP1s-mediated control of mitophagy is fully PINK1-dependent. (A-L) Control (PINK1 CT) or PINK1 knocked-down (PINK1 KD) SH-SY5Y cells were transiently transfected with an empty vector (Ev) or with Xbp1s (Xbp1s) cDNA. Twenty-four hours after transfection, BECN1 (A and B, N = 8) LC3-II:LC3-I ratio (A and C, N = 8), SQSTM1 (A and D, N = 8), OPTN (A and E, N = 8), CALCOCO2 (A and F, N = 8), TIMM23 (A and G, N = 12), TOMM20 (A and H, N = 12), HSPD1 (A and I, N = 12), UB (S65) (A and J, N = 8), PRKN (A and K, N = 8) and MFN2 (A and L, N = 12) protein expressions were analyzed by western blot as described in Methods. ACTB expression is provided in (A) as a control of protein load. (M) PINK1 CT and PINK1 KD cells either empty vector (-) or Xbp1s (+) cDNA-transfected were analyzed by flow cytometry to measure mitochondrial membrane potential as described in Methods. (B-M) Data are expressed as percent of PINK1 CT non-transfected cells (taken as 100%) and are the means ± SEM. of 4–6 independent experiments performed in duplicates. Statistical analyses were performed by Kruskal Wallis multiple comparison test (B) and Sidak’s multiple comparisons test (C-M). Statistical significances: ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. 8W. EL MANAA ET AL. Figure 8. PINK1, XBP1s phosphorylation and mitophagic markers expressions are altered in sporadic Parkinson disease (PD)-affected brains. (A-J) PINK1 (A and B), XBP1s [p-S61A] (A and C), XBP1s [p-T48A] (A and D), TIMM23 (A and E), TOMM20 (A and F), UB (S65) (A and G), OPTN (A and H) and CASP3 (A and I) protein expressions in control (CT, N = 10) and PD, (N = 8) brains were analyzed by western blot as described in the Methods. Data are expressed as percent of CT brains (taken as 100%). Statistical significances were analyzed by Student’s t test: ns, non-significant, ** P < 0.01, *** P < 0.001. Correlations analyses of PINK1 and XBP1s [p-S61A] (J) and XBP1s [p-T48A] (K) protein expressions are illustrated in (J and K). PINK1 full gel illustrating the migration profile of full-length PINK1 in control (PINK1 CT) and shRNA-depleted PINK1 (PINK1 KD) samples is provided in (A). A representative ACTB gel is provided to illustrate equal protein load. AUTOPHAGY 15 in PD. Considering that MAPK14 and CDK5 kinases are affected in PD [71,72], it remains to be established if they independently or coordinately interact functionally with PINK1 to modulate XBP1s in PD. It should be noted that we have previously shown that nuclear TP53, a tumor suppressor that also controls autophagy [73], acts as a transcriptional repressor of PINK1 [52]. Further, we have established that TP53 also downregulates XBP1s expression [24]. Thus, these data fully agree with the present study and indicate that TP53 can repress autophagy by direct transcriptional repression of PINK1 or indirectly, via the downregulation of XBP1s. XBP1s elicited a pro-mitophagic response. This positive regulation agreed with the beneficial role of autophagy/mitophagy in physiological conditions or non-chronic stress conditions [74]. The protective role of autophagy was supported by the fact that the depletion of the key autophagy proteins ATG5 or ATG7 in vivo resulted in increased neurodegeneration and the presence of cytoplasmic inclusion bodies mainly composed of protein aggregates [75,76]. That said, the observation of an activation of an XBP1s-PINK1 axis and the delineated forward loop questioned whether this corresponded to a protective mechanism or if it accounted for ER-stress and mitochondrial defects observed in PD. Since XBP1s phosphorylation enhanced its nuclear localization and thus, its function, it could be envisioned that PINK1-induced phosphorylation of XBP1s is beneficial. This could be a transient adaptive response that does not last enough to circumvent chronic disease establishment. The transient expression and protective effect of XBP1s has been documented in another neurodegenerative disease, namely Alzheimer disease. Thus, we have shown that in AD-affected brains and AD animal models, XBP1s expression is transiently enhanced at early stages of the pathology and that this protects against Aβ oligomers-mediated EPHB2 (EPH receptor B2)-linked toxicity [77]. In the same line of reasoning, we have shown that Aβ oligomers enhance XBP1s levels leading to a decrease of BACE1 (beta secretase 1) activity, the enzyme responsible for Aβ production [78] as a protective response [79]. Thus, the protective response elicited by XBP1s is probably sufficient to delay the onset and even the progression of several neurodegenerative disorders including Alzheimer and Parkinson diseases at initial stages, but may become inefficient considering their chronic, long-lasting nature. According to this statement, it is worth noting that both the UPR and autophagy processes are characterized by an adaptation and an apoptotic phase [74,80]. Thus, one can envision that, even if the beneficial transient activation of the XBP1s-PINK1 axis occurs, the chronic activation of both UPR and autophagy responses may lead to the firing of apoptotic stigmata that characterize neurodegenerative diseases. Interestingly, we documented in this study a correlation between the XBP1s-PINK1 interplay and activation of apoptotic response (illustrated by CASP3 activation) in sporadic PDaffected brains suggesting that the protective effects are mediated by the interplay PINK1-XBP1s had been overcome by the sustained PINK1-mediated mitophagy response at late stage of the disease. Overall, our work is the first demonstration of a link between the UPR and mitophagy responses via a functional crosstalk between XBP1s and PINK1 and that this physiological signaling cascade may be disrupted in PD conditions. Materials and methods Constructs description and transfection approaches The mouse Xbp1s-Flag pcDNA3 has been designed by one of us and described in [81]. The XBP1 shRNA and scramble (SC) shRNA sequences have been cloned in the FUGW lentiviral vector (Addgene, 14883 [82]). The human PINK1 and mouse Pink1 promoters have been described in [51]. The pGL3 vector (Promega, U47295) containing the mouse Pink1 promoter served as a template to generate the promoter deleted of the 5′-CGAG-3′ nucleotides. This deleted motif constitutes part of the XBP1s putative binding site. This sequence is located from nucleotides −1026 to −1021 upstream of Pink1 ATG start codon. The primers used were forward: 5ʹGTGGATTTCTGAGTTGCCAGCCTGGTCTAC-3ʹ and reverse 5ʹ-GTAGACCAGGCTGGCAA CTCAGAAATCCAC-3ʹ. The generation of wild-type (WT PINK1) and mutant PINK1 K219 V5tagged of human PINK1 coding sequence in the mammalian expression vector pcDNA6 (Invitrogen, V220-20) has been described [38] and graciously provided by Dr. St Georges Hyslop (Tanz Center for Research in Neurodegenerative Diseases, Toronto, Canada). Wild-type and mutated Xbp1s coding sequences in pcDNA3.1 vector (mutants: Xbp1s [p-T48A], Xbp1s [p-S61A] and Xbp1s [p-T48A/S61A]) have been described [36]. Oligonucleotides containing an shRNA targeting human PINK1 (primer forward: 5ʹGATCCCCCCAAGCTGGTCTAGTAGATTTCAAGAGAATCTACTAGACCAGCTTGGTTTTTA-3ʹ and reverse: 5ʹAGCTTAAAAACCAAGCTGGTCTAGTAGATTCTCTTGAAATCTACTAG ACCAGCTTGGGGG-3ʹ) or a scrambled RNA (primer forward: 5ʹ-GATCCCCGAGTTACCCGC TAGATGTATTCAAGAGATACATCTAGCGGGTAACTCTTTTTA-3ʹ and reverse: 5ʹAGCTTAAAAAGAGTTACCCGCTAGATGTATCTCTTGAATACATCTAGCGGGTAACTCGGG-3ʹ) have been inserted in the pSUPER.neo+GFP (green fluorescent protein) vector according to the manufacturer’s instructions (Oligoengine, VEC-PBS -0006). All the constructs were verified by sequencing. Transient and stable transfections of SH-SY5Y (ATCC®, CRL-2266™) were carried by means of Lipofectamine 2000 (Invitrogen, 11668019) according to the manufacturer’s instructions. Lentivirus production and SH-SY5Y cells infection shRNA lentivirus production was performed as described in [77]. In brief, XBP1 depletion was performed by means of validated shRNA sequences targeting human and mouse XBP1 under the RNU6 promoter. Target sequence was 5′- GGTCTGCTGAGTCCGCAGCA-3ʹ [24,83]. The RNU6-shRNA expression cassette (pSilencer 2.1-U6 Neo; Ambion, Thermo Fisher Scientific, AM5764) was inserted in the PacI site of a modified FUGW lentiviral backbone, placing the shRNA cassette upstream of a UBC (ubiquitin C) promoter directing expression of enhanced GFP. A similar construct expressing a scrambled scRNA (5′- 16 W. EL MANAA ET AL. GCCCGTCTGCGTGGAGCTAA-3′) was used as a control. Viral titers were determined by p24 ELISA (Cell Biolabs, VPK-107). Concentrated lentiviruses (scramble RNA XBP1 = 3.39 x 10 10 Lentiviral Particles [LPS]/ml, shRNA XBP1 = 3.08 x 10 10 LPS/ml) were directly added to SH-SY5Y cells. Four days after infection, cells were re-plated in 100 mm diameter dishes in order to generate enough cells for either conservation at −150°C or analysis of the percentage of protein and mRNA XBP1s depletion in basal and TP conditions by western blot and real-time quantitative PCR analysis, respectively (see below). Electrophoretic mobility gel shift assay (EMSA) We performed EMSA by means of a commercial gel shift chemiluminescent EMSA assay kit (Promega, E3050). In brief, purified wild-type XBP1s recombinant protein (300 ng) or control nuclear extracts were pre-incubated in 1X Gel Shift Binding buffer (Promega, E3050) at 20°C for 10 min. When indicated, an excess (4 pmol) of unlabeled competitor oligonucleotides was added. Positive control of the experiment corresponds to a non-related labeled control DNA (20 fmol) added to the nuclear extract (data not shown). After this pre-incubation step, we added when indicated 20 fmol of double-stranded 5ʹ biotin end-labeled PINK1-derived oligonucleotides (forward: 5ʹGGATTTCTGAGTTCGAGGCCAGCCTGGTCT-3ʹ; reverse: 5ʹ-AGACCAGGCTGGCCTCGAACTCAGAAATCC-3ʹ) containing the delineated -1025-1021 XBP1s responsive element and incubated all the reactions at 20°C for 20 min. The samples were resolved by electrophoresis on a nondenaturing acrylamide gel (5%) at 4°C, transferred to a positively charged nylon membrane (Thermo Fisher Scientific, 77016), and after cross-linking with an UV-light cross-linker (equipped with a 254-nm bulb), revealed by means of streptavidin conjugated to horseradish peroxidase (HRP; Active Motif, 37341) and a chemiluminescent substrate. Cell models and pharmacological ER-stress modulation Most of the experiments were performed in nondifferentiated SH-SY5Y human neuroblastoma cells cultured at 37°C and 5% (vol:vol) CO 2 , in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco-Invitrogen, 41965–039) supplemented with 10% fetal calf serum (Dutscher, S1900-500) containing penicillin and streptomycin (100 U/ml; Gibco, 15140–122). These cells were routinely profiled and validated by Short Tandem Repeat profiling according to the manufacturer’s instructions (GenePrint® 10 System, Promega) and tested for mycoplasma contamination PCR according to a previously published paper [84]. When indicated, these cells were treated with 1 µM of thapsigargin (Sigma-Aldrich, T9033) or 10 µg/ml of tunicamycin (Sigma-Aldrich, T7765) for 8 and 6 h respectively. A pre-treatment with 1 µM toyocamycin (Sigma-Aldrich, T3580) for 16 h was performed in a subset of experiments in which cells were co-treated with thapsigargin and toyocamycin. For cell fractionation procedures, these cells were treated with 5 µM of CCCP (Sigma Aldrich, C2759) for 6 h. In a subset of experiments, cells were treated for 2 or 4 h with baf (100 nM, Enzo Life Sciences, BML-CM110-0100). SH-SY5Y cells stably overexpressing the shRNA targeting XBP1 or a control scrambled sequence were obtained by transduction approaches. SH-SY5Y cells overexpressing the shRNA targeting PINK1 or a control scrambled sequence were obtained by transfection approaches. Mouse embryonic fibroblasts (MEFs) control or invalidated for PINK1 were provided by Dr. B. De Strooper (VIBKU Leuven center for brain and disease research, Belgium) [85]. SH-SY5Y differentiation and treatments In the experiments described in Fig. S5G and S8 , we have differentiated stable naïve and XBP1-depleted SH-SY5Y neuroblastoma cells by means of a subsequent treatment with retinoic acid (Sigma-Aldrich, R2625) and 12-O-tetradecanoyl-phorbol -13-acetate (TPA, Sigma-Aldrich, P8139) according to an established protocol [86]. In brief, SH-SY5Y (2 x 10 5 cells) were plated on 6-well culture plates (Corning Costar, 3516) in normal medium containing DMEM/F12, 10% fetal bovine serum, penicillinstreptomycin and sodium pyruvate. Twenty-four hours after plating, SH-SY5Y cells were differentiated for 3 d by addition of retinoic acid (10 µM) in neurobasal media containing B-27 (2%) and L-glutamine (1%, Sigma-Aldrich, Milan, Italy), then the media were removed and replaced with neurobasal media containing TPA (80 nM), B-27 (2%) and L-glutamine (1%) for another 3 d. Differentiation state was confirmed by analyzing the protein levels of the dopaminergic marker TH (tyrosinehydroxylase) by western blot (antibody referenced in Table 1). In a subset of experiments, differentiated SH-SY5Y cells were treated for 24 h with either 6-hydroxydopamine (6OHDA; Sigma-Aldrich, H4381) at 10, 25 and 50 µM or with SNCA monomers or oligomers (2 µM for 8 h). As described previously [87], SNCA oligomers were produced by incubating recombinant human SNCA (Anaspec, AS-55555) at 1 mg/ml (70 µM) with a 30:1 excess of 4-hydroxy-2-nonenal (HNE; SigmaAldrich, H9538) overnight at 37°C. After incubation, the reaction was centrifuged by using an Amicon 3-kDa cutoff ultracentrifugal unit (Millipore, UFC500324) for 10 min at 14 000 x g to remove unbound aldehyde. Mouse and human brains description and processing for analysis Brains from pink1 knockout (pink1 −/- ) male mice have been kindly provided by Dr. J. Shen and have been extensively described [88]. Control or pink1 −/- brains of 2–4 or 6–10month-old were transferred to green bead tubes (MagNA Lyser Green beads; Roche, 03358941001) containing 1 ml of lysis buffer (10 mM, Tris-HCl, pH 7.5 supplemented with a protease inhibitors cocktail [Sigma-Aldrich, P2714], and phosphatase inhibitors [1 mM sodium orthovanadate, 5 µM sodium fluoride]). Mouse brains were subsequently beadbeaten for 45 s at 4,700 x g in a MagNA Lyser instrument (Roche). Homogenates were then sonicated on ice before western blot analysis. AUTOPHAGY 17 Human substantia nigra samples were obtained from brains collected in a Brain Donation Program of the Brain Bank “Neuro-CEB” run by a consortium of patients Associations: CSC (cerebellar ataxias), Fondation ARSEP (research on multiple sclerosis), France Parkinson, “Vaincre Alzheimer Fondation”. The consents were signed by the patients themselves or their next of kin in their name, in accordance with the French Bioethical Laws. The Brain Bank Neuro-CEB (BB-0033-00011) has been declared at the Ministry of Higher Education and Research and has received approval to distribute samples (agreement AC-2013-1887). These samples include 10 controls and 8 PD patients. Controls include four amyotrophic lateral sclerosis (ALS) patients (control non-PD-associated pathology): 1811 (male, 55 years-old), 1821 (female, 68 years-old), 1822 (male, 64 years-old), 1823 (female, 62 years-old), one sample from an aged-matched healthy patient 3659 (male, 61 years-old), five samples from healthy patients with Alzheimer like lesions: 8730 (male, 82 years-old), 6283 (female, 83 years-old), 6658 (female, 93 years-old), 7024 (female, 76 years-old), 7197 (male, 85 years old). Samples from PD patients include: 3605 (male, 64 years-old), 4489 (male, 75 years-old), 4513 (female, 77 years-old), 5193 (male, 75 years-old), and 8460 (male, 66 years-old), 4291 (female, 72 years-old), 8418 (male, 82 years-old) and 7743 (male, 72 years-old). The last three cases of PD patients have Alzheimer-like lesions. The mean postmortem delay was 28.2 ± 15.3 h. The samples were homogenized and lysed as done for mice brain samples. Primary cultured neurons (pharmacology, transfection and transduction) Primary cultures of rat cortical neurons were performed as extensively described in [77]. In brief, cortical neurons from rat pups (P0) (http://www.criver.com/products-services/basicresearch/find-a-model/spraguedawley-rat) were obtained after trypsin dissociation. Cells were plated in polylysine-coated wells and maintained in serum-free neurobasal medium (Gibco, 12348–017) supplemented with B27 (Gibco, 17504044) and antibiotics (Thermo Fisher Scientific, 15070–063). Half of the medium was changed after 1 d in culture. Cells were used after 9 d in culture. Neuronal cultures were either submitted to ER stress by pharmacological treatments (see above) or transiently transfected with empty or Xbp1s cDNA or with scramble or Xbp1s shRNA expressing plasmids with lipofectamine according to manufacturer conditions. After treatment and transfection, cells were harvested and frozen at −80°C for subsequent determination of either proteins or mRNA levels and analysis of promoter activity. Western blot (cells and mouse brains) Mouse brains and cells were resuspended in lysis buffer (10 mM Tris-HCl, pH 7.5, containing a protease inhibitors cocktail and phosphatase inhibitors (1 mM sodium orthovanadate, 5 µM sodium fluoride) and then sonicated before western blot analysis. Expressions of proteins were analyzed with 50 µg of cell lines or mouse brain homogenates loaded on 10–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and semi-dry transferred for 10 min by means of the ready to use transfer kit nitrocellulose (Bio-Rad, 1704271) and the TransBlot® Turbo™ Transfer System (pre-programmed Bio-Rad protocol for 2 mini gels of 1.5 mm). Transferred proteins were then immunoblotted using the antibodies listed in Table 1. The full gels of PINK1 containing migration controls were provided for each cell and tissue type (first time appearance) to illustrate antibody specificity. Immunological complexes were revealed with adequate anti-rabbit or anti-mouse IgG-coupled peroxidase antibodies (Jackson ImmunoResearch, 111–036-045) by the electrochemiluminescence detection method (Roche Diagnostics S.A.S). Chemiluminescence was recorded using a luminescence image analyzer LAS-4000 (Raytest, Fuji) and quantifications of non-saturated images were performed with the FUJI Film Multi Gauge image analyzer software. Table 1. List of antibodies used in western blot analysis. ANTIBODIES SPECIES DILUTION Company (catalog #) PINK1 Rabbit 1: 1000 Abiocode (R3173-2) XBP1 Rabbit 1: 1000 Santa Cruz Biotechnology Inc. (sc-8015) LC3 Rabbit 1: 2000 Novus Biologicals (NB100-2220) SQSTM1/p62 Rabbit 1: 2000 Novus Biologicals (NBP1 – 49956) OPTN Mouse 1: 1000 Santa Cruz Biotechnology Inc. (sc-166576) CALCOCO2/NDP52 Mouse 1: 500 Santa Cruz Biotechnology Inc. (sc-376540) TIMM23 Mouse 1: 1000 BD Biosciences (611222) TOMM20 Mouse 1: 1000 BD Biosciences (612278) H2AZ1 Rabbit 1: 1000 GeneTex (GTX10823S) Phospho-ubiquitin S65 UB (S65) Rabbit 1: 1000 Boston Biochem (A110) Ubiquitin Rabbit 1: 1000 Novus Biologicals (NB300-129) PRKN Mouse 1: 2000 Merck Millipore (MAB5512) MFN2 Mouse 1: 1000 Abcam (ab56889) HSPD1/HSP60 Mouse 1: 1000 Santa Cruz Biotechnology Inc. (sc-59567) XBP1s [p-S61A] Rabbit 1: 10 000 U.Ozcan XBP1s [p-T48A] Rabbit 1: 10 000 U.Ozcan GAPDH Mouse 1: 5000 EMD Millipore (MAB374) ACTB/actin Mouse 1: 5000 Sigma-Aldrich (A5316) TUBB/tubulin Mouse 1:2000 Sigma-Aldrich (T5168) TH (tyrosine hydroxylase) Rabbit 1: 1000 GeneTex (GTX113016) SNCA Mouse 1: 1000 BD Biosciences (610787) BECN1 Rabbit 1: 1000 Enzo Life Sciences (ADI-905-721-100) 18 W. EL MANAA ET AL. mRNA analysis (cells and mouse brains) RNA from cells and mouse brains (one hemisphere per mouse was stabilized in RNAlater [RNA stabilization reagent] Qiagen, 76104) were extracted and treated with DNAse using RNeasy or RNeasy Plus Universal mini kits respectively following manufacturer’s instructions (Qiagen, 74106 and 73404, respectively). Two µg of total RNA were reverse transcribed (GoScript Reverse Transcriptase; Promega, A5002) using oligo-dT priming then samples were subjected to real-time PCR by means of a RotorGene 6000 apparatus (Qiagen), using the SYBR Green detection protocol (Roche Life Science). Specific primers (Eurogentec) for human, mouse and rat PINK1/Pink1, XBP1s/Xbp1s and housekeeping genes were designed with the Universal Probe Library Assay Design Center software (Roche Applied Science) and are listed in Table S1. Promoter activity Mouse/human full-length and 5′ end-truncated Pink1/PINK1 promoter-luciferase constructs have been previously described [51]. The transcriptional regulation of Pink1/PINK1 promoters was measured after co-transfection of 1 µg of the above cDNA and 1 µg of GLB1 (galactosidase beta) cDNA (in order to normalize for transfection efficiencies) by means of luciferase reporter gene and GLB1 kits according to the manufacturer’s instructions (Promega). In a subset of experiments, 1 µg of empty pcDNA3.1 or wild-type Xbp1s cDNA were cotransfected with 0.5 µg of GLB1 and 0.5 µg of Pink1 promoter cDNAs. CASP3 activity measurement SH-SY5Y cells overexpressing or not XBP1s were plated and grown in 6-well plates for 24 h and treated for 8 h without or with thapsigargin (1 µM). CASP3-like enzymatic activity was fluorimetrically recorded on a spectral scanning multimode reader (Varioscan, Thermo Fisher Scientific) as described in [89]. Mitophagy flux analysis pCLBW COX8-EGFP-mCherry plasmid was a kind gift from David Chan (Addgene, 78520 [35]). Mitophagic flux was performed as described in [35]. This probe allows the detection of mitophagy flux thanks to differences in pKa of enhanced green fluorescent protein (EGFP), and mCherry protein expressed in tandem with the mitochondrial localization signal of COX8. In neutral compartment (pH7) the probe fluoresces yellow (merge of green and red signals). During mitophagy, fragmented mitochondria are delivered to lysosomes where the low pH quenches the EGFP signal. The result is that a portion of mitochondria forms punctae structures and fluorescence is red only. Images of live cells were acquired 48 h post-transfection with Zeiss LSM 780 and 63X objective. The quantification was performed on different fields of view obtained in three independent experiments. Data show the percentage of cells undergoing mitophagy. A threshold of a single or more red-alone punctae per cell was applied to all cells expressing human COX8-EGFPmCherry probe. Mitochondrial potential disruption analysis Mitochondrial membrane potential (Δψm) was accessed using live imaging analysis of TMRM probe, a fluorescent cation that distributes into the mitochondrial matrix of active mitochondria following the electrochemical gradient as detailed in [25]. Cells spotted on 25 mm cover slips were loaded with 10 nM TMRM in cell culture medium at 37°C for 30 min. Images were acquired (excitation: 559 nm, emission: 575–675 nm) on a LEICA TCS SP5 confocal microscope (Leica Microsystems) at 37°C. To obtain normalized TMRM fluorescence signal, Z-stack images were acquired before and after application of the mitochondrial uncoupler carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP, 5 mM, SigmaAldrich, C2920). To demonstrate specific TMRM binding, measurements were corrected for residual TMRM fluorescence after full Δψm collapse with FCCP [90]. TMRM intensity was quantified on Z-stack maximal projection images after thresholding, using ImageJ software (NIH) [91]. The TMRM signal was also analyzed using the Novocyte TM flow cytometer (ACEA Biosciences Inc.). TMRM was excited with the 543 nm laser line, and emission was performed at 600 ± 10 nm. SH-SY5Y neuroblastoma cells and primary cultured neurons were loaded for 30 min at 37°C with TMRM (5 nM). TMRM fluorescence from 10,000 cells was acquired and the median value was obtained using the NovoExpress TM software (ACEA Biosciences Inc.). Cellular gating was set the same way in all measurements. Unstainedand FCCP-treated cells were used as controls. Phosphorylation in vitro XBP1s phosphorylation by PINK1 was analyzed as described [92]. In brief, recombinant XBP1s (1 µg) and ubiquitin (positive control, Boston Biochem, U100H, 1 µg) were incubated with recombinant WT PINK1 or PINK1 D359A (Ubiquigent, 66–0043-050 and 66–0044-050 respectively, 1 µg) in a final volume of 20 µL of kinase assay buffer (20 mM HEPES [Sigma-Aldrich, H3375], pH 7.4, 10 mM dithiothreitol [DTT, Sigma Aldrich, 20–265], 0.1 mM EGTA [SigmaAldrich, E4378], 10 mM MgCl 2 , 1 mM ATP) for 2 h at 37° C. The reactions were terminated by addition of 5X SDS (Sigma-Aldrich, L3771) loading buffer (375 mM Tris, pH 6.8, 9% [w:v] SDS, 50% [v:v] glycerol [Prolabo VWR,24388.295] 9% [v:v] beta-mercaptoethanol [SigmaAldrich, M3148], 0.03% (w:v) bromophenol blue [SERVA, 15375]), incubated for 15 min at 56°C to prevent spontaneous ubiquitin dimers formation, then 200 ng of proteins were analyzed by western blot using phospho-specific XBP1s and ubiquitin antibodies as described in Table 1. Cell fractionation Cells were harvested, pelleted by centrifugation at 1,000 x g for 3 min at 4°C, lysed in 300 µl of homogenization buffer AUTOPHAGY 19 (20 mM HEPES pH 7.4, 1.5 mM MgCl 2 , 1 mM EDTA, 1 mM EGTA, 1 mM DTT, protease inhibitors cocktail) and homogenized with a syringe (Agani Terumo 26 G needles). Homogenates were left in ice during 15 min then centrifuged at 850 x g for 5 min at 4°C in order to recover the nuclear fraction (pellet). Pellets were homogenized in RIPA buffer (50 mM Tris pH 7.4 containing 150 mM NaCl, 1 mM EDTA, 1% Triton X-100 [Sigma-Aldrich, X100], 0.5% deoxycholate [Sigma-Aldrich, D6750], 0.1% SDS complemented with a cocktail of protease inhibitors) then sonicated twice for 15 s on ice. Supernatant was centrifuged at 20,000 x g for 1 h 30 min at 4°C to obtain the cytosolic fraction. Both nuclear and cytosolic fractions were submitted to western blot analysis. To isolate a crude mitochondrial fraction from cultured cells (one 6-well plate per condition), we removed the media, washed cells once with PBS, added 1 mL per well of 1X PBS (Euromedex, ET330-A), 5 mM EDTA. Detached cells were pelleted by centrifugation at 1,000 x g for 3 min at 4°C. After rinsing in PBS, another centrifugation was performed then pellets were resuspended and incubated for 15 min in isotonic buffer (250 mM mannitol [SigmaAldrich, M9647], 5 mM HEPES, 500 µM EGTA, 0.01 g BSA [Sigma-Aldrich, A9647]) supplemented with a protease inhibitors cocktail in ice. Swollen cells were manually disrupted with a Dounce homogenizer (hundred up and downs). Lysates were centrifuged at 1,000 x g for 5 min at 4°C. Supernatants were collected and centrifugated again at 1,000 x g for 5 min at 4°C. Supernatants were carefully collected and submitted to centrifugation for 10 min at 8000 x g at 4°C. The supernatants (containing the cytosolic fraction, plasma membrane, lysosomes and microsomes) and the mitochondrial pellets resuspended in 35 µL of isotonic buffer were both kept for further western blots analyses. Electron microscopy Electronic microscopy was performed as previously described [93]. In brief, mice treated with toyocamycin or its vehicle received a lethal dose of a combination of ketamine (120 mg/ kg) and xylasine (24 mg/kg) and transcardially perfused with ice-cold physiological saline followed by 2.5% glutaraldehyde, 0.1 M cacodylate buffer, pH 7.4. Brains were sliced (200 μm) on a vibratome and 2 mm cubes from the cortices were microdissected under binoculars and post-fixed in osmium tetroxide (1% in 0.1 M cacodylate buffer). The tissues were embedded in EPON resin (EMS, 14120) and 70 nm ultrathin sections were contrasted with uranyl acetate and lead citrate and visualized using a JEM 1400 electron microscope operating at 100 kV equipped with a Morada SIS camera. We analyzed mitochondrial area and perimeter by the software Image J (http://rsb.info.nih.gov/ij/). In vivo studies Wild-type mice purchased from Charles River were housed with a 12:12 h light/dark cycle and were given free access to food and water. All experimental procedures were in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) and local French legislation. Adult wild-type males (C57BL6) aged 2 months were used. Mice were intraperitoneally injected with either vehicle (0.03% DMSO, 0.02 M Na 2 HPO 4 , pH 7.4 buffer containing 150 mM dextrose [Sigma-Aldrich, D9434]) or tunicamycin (1 mg/kg; Merck, 504570) dissolved in the same vehicle [94]. Mice were intraperitoneally injected with either vehicle (0.0125% DMSO, 0.02 M Na 2 HPO 4 buffer containing 150 mM dextrose) or toyocamycin (1 mg/kg) dissolved in the same vehicle [26]. Animals were sacrificed 72 h after injection. Mice brains were recovered and one hemisphere was immediately frozen for ulterior proteins analysis while the other was stabilized with RNAlater for mRNA analysis by real-time PCR. Statistical analysis Statistical analyses were performed with GraphPad Prism software (San Diego, California USA). The choice of parametric versus non-parametric test was established after assessment of the normality test (D’Agostino-Pearse omnibus Normality test) to assure Gaussian distribution of values. Two groups of variables that have passed the normality test were analyzed by unpaired Student’s t-test while two groups of variables that have not passed the normality test were analyzed by the Mann-Whitney test. Analysis of more than two groups of variables that have passed the normality test was performed by ordinary One-way ANOVA while analysis of more than two groups of variables that have not passed the normality test were analyzed by Kruskal-Wallis test. Grouped analysis of one or more groups was performed by two-way ANOVA followed by a Tukey’s multiple comparisons test. Data correlation analyses were performed by either Pearson or Sperman tests after the evaluation of the Gaussian distribution of values. All tests are two-sided; the mean was defined as the center value and error bars correspond to SEM. The number of samples, replication of experiments and the p values (stars) are provided in figure legends. Significant differences are: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 and ns = nonsignificant. Acknowledgments We wish to thank Drs. T. Kawarai and P. St-Georges Hyslop for providing PINK1 constructs. We sincerely thank Dr. B. de Strooper for providing us with PINK1-depleted cells and Dr. J.Shen for providing us control and PINK1 knockout mice brains. We thank the Neuro-CEB (PitiéSalpêtrière Hospital) for providing us with human brain samples. Disclosure statement No potential conflict of interest was reported by the author(s). Funding This work was supported by the Labex DISTALZ and by the University Hospital Federation (FHU OncoAge). WEM was granted by the ANR and France Parkinson and TG by the Ligue Nationale Contre le Cancer. 20 W. EL MANAA ET AL. ORCID Ling Qi http://orcid.org/0000-0001-8229-0184 Mounia Chami http://orcid.org/0000-0003-1498-7187 References [1] Zeng XS, Geng WS, Jia JJ, et al. Cellular and molecular basis of neurodegeneration in Parkinson disease. Front Aging Neurosci. 2018;10:109. [2] Cooper AA, Gitler AD, Cashikar A, et al. Alpha-synuclein blocks ER-Golgi traffic and Rab1 rescues neuron loss in Parkinson’s models. Science. 2006;313:324–328. [3] Sugeno N, Takeda A, Hasegawa T, et al. Serine 129 phosphorylation of alpha-synuclein induces unfolded protein response-mediated cell death. J Biol Chem. 2008;283:23179–23188. [4] Hetz C, Saxena S. 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