Chronic treatment with the complex I inhibitor MPP+ depletes endogenous PTEN-induced kinase 1 (PINK1) via up-regulation of Bcl-2–associated athanogene 6 (BAG6)

Mitochondrial dysfunction is implicated in sporadic and familial Parkinson's disease (PD). However, the mechanisms that impair homeostatic responses to mitochondrial dysfunction remain unclear. Previously, we found that chronic, low-dose administration of the mitochondrial complex I inhibitor 1-methyl-4-phenylpyridinium (MPP+) dysregulates mitochondrial fission–fusion, mitophagy, and mitochondrial biogenesis. Given that PTEN-induced kinase 1 (PINK1) regulates mitochondrial function, dynamics, and turnover, we hypothesized that alterations in endogenous PINK1 levels contribute to depletion of mitochondria during chronic complex I injury. Here we found that chronic MPP+ treatment of differentiated SH-SY5Y neuronal cells significantly decreases PINK1 expression prior to reductions in other mitochondrial components. Furthermore, Bcl2-associated athanogene 6 (BAG6, BAT3, or Scythe), a protein involved in protein quality control and degradation, was highly up-regulated during the chronic MPP+ treatment. BAG6 interacted with PINK1, and BAG6 overexpression decreased the half-life of PINK1. Conversely, siRNA-mediated BAG6 knockdown prevented chronic MPP+ stress-induced loss of PINK1, reversed MPP+-provoked mitochondrial changes, increased cell viability, and prevented MPP+-induced dendrite shrinkage in primary neurons. These results indicate that BAG6 up-regulation during chronic complex I inhibition contributes to mitochondrial pathology by decreasing the levels of endogenous PINK1. Given that recessive mutations in PINK1 cause familial PD, the finding of accelerated PINK1 degradation in the chronic MPP+ model suggests that PINK1 loss of function represents a point of convergence between the neurotoxic and genetic causes of PD.

Parkinson's disease (PD) is a chronic, progressive movement disorder, characterized by degeneration of nigrostriatal dopaminergic neurons. Cells and brain tissue from PD patients exhibit reduced mitochondrial complex I activity (1), and exposure to complex I inhibitors such as 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP) and the pesticide rotenone cause parkinsonian neurodegeneration and may contribute to risk of PD (2,3). Although the majority of PD cases are sporadic, ϳ10% show a familial etiology. Genetic models based upon mutations in ␣-synuclein, Parkin, PINK1, LRRK2, and DJ-1 have further emphasized the role of mitochondrial dyshomeostasis in the pathogenesis of PD (1,4,5). Hence, understanding the mechanisms by which genetic and environmental susceptibilities may interact would provide valuable insights into PD pathogenesis.
The PTEN-induced putative kinase 1 (PINK1) is a ubiquitous serine-threonine kinase, localized in both cytosol and mitochondria (6 -9). A normal mitochondrial membrane potential allows full-length PINK1 to be imported into mitochondria, where it is cleaved by several mitochondrial proteases, including mitochondrial inner membrane presenilin-associated rhomboid-like protein (PARL), matrix-processing peptidase (MPP), or ATP-dependent Clp protease ATP-binding subunit (ClpX) (10,11). Processed PINK1 may regulate mitochondrial respiration, or it is exported to the cytosol (12), where its stability is regulated by complexing with chaperone proteins (7). PINK1 is implicated in maintaining mitochondrial function, reducing mitochondrial oxidative stress, regulating mitochondrial transport, fission-fusion, and autophagy (10,(13)(14)(15)(16). Loss of PINK1 function alters mitochondrial clearance, increases the production of reactive oxygen species (14,17,18), and renders cells more susceptible to neurotoxicity (19,20). Recently, PINK1 has been shown to regulate mitochondrial bioenergetics by phosphorylating a subunit of complex I, providing a possible link between the complex I dysfunction observed in PD and loss of PINK1 function (21). In addition to these mitochondrial functions, the cytosolic pool of PINK1 regulates neurite outgrowth and dendritic complexity (15) and is sufficient for protecting neurons against MPTP intoxication (8,15).
The BAG (Bcl-2-associated athanogene) proteins, BAG1-6, were initially identified because of their ability to interact with Bcl-2 (22). In recent years, it has been shown that BAG family members regulate not only cell death but also protein quality control by functioning as co-chaperones. BAG family members can interact with various chaperones including CHIP and the Hsp70/Hsc70 complex (23) through conserved C-terminal BAG domains. Members of the BAG family have been implicated in PD (24 -27), and overexpression of BAG2 (25,28) and BAG5 act to stabilize PINK1 (27). Other members of the BAG family act to negatively regulate HSP70 function (29,30).
Although it has been shown that engineered knockdown of PINK1 increases sensitivity to acute MPP ϩ and rotenone elic-This article contains supporting information. ‡ These authors contributed equally to this work. * For correspondence: Charleen T. Chu, ctc4@pitt.edu. cro ARTICLE ited apoptosis (8,18), chronic, low-dose treatment with complex I inhibitors elicit different mechanisms of injury (31). Here, we show that chronic MPP ϩ treatment elicits decreased expression of full-length and processed forms of endogenous PINK1 in both cytosolic and mitochondrial fractions. Mechanistically, there was a marked up-regulation of BAG6, which acts to accelerate PINK1 degradation. Furthermore, either restoration of PINK1 or knockdown of BAG6 conferred neuroprotection, reversing the pathological effects of MPP ϩ on cell viability, mitochondrial morphology, and neurite shortening/ dendritic retraction.

Chronic treatment with MPP ؉ in neuronally differentiated SH-SY5Y cells causes selective depletion of endogenous PINK1
In contrast to the mM doses of MPP ϩ commonly used to elicit acute injury in SH-SY5Y cells (32,33), we previously found that chronic, low-dose exposure reduces mitochondrial content through suppression of mitochondrial biogenesis in addition to mitophagy (31). Given that the low level complex I deficiency observed in Parkinson's patient cells is not associated with high levels of acute cell death, we selected a dose of MPP ϩ that was sublethal at 1 week of treatment with ϳ20 -25% toxicity at 2 weeks (31) for further study.
Chronic treatment of differentiated SH-SY5Y cells with 250 M MPP ϩ resulted in reduced expression of both full-length (FL) and processed (dN) forms of PINK1 normalized to GAPDH (Fig. 1, A and B, and Table 1). Loss of PINK1 was not due simply to mitophagy, because it occurred prior to the loss of other mitochondrial proteins (Fig. 1, B and C). Because PINK1 localizes to both mitochondria and cytosol, we further studied its alteration in these subcellular compartments. MPP ϩ elicited decreased PINK1 levels in both fractions, with a more striking percentage of decrease observed in the cytosol (Fig. 1D). There were no significant decreases in PINK1 mRNA levels, suggesting that the loss of PINK1 is due to enhanced degradation (Fig. 1E).

Restoration of PINK1 expression rescues MPP ؉ -induced neurodegenerative phenotypes
To determine whether loss of PINK1 played a pathogenic role in the chronic MPP ϩ model, we transfected neuronally differentiated SH-SY5Y cells with plasmids expressing either GFP or GFP-tagged WT PINK1. The cells were probed for the human mitochondrial p60 protein after treatment with 250 M MPP ϩ or vehicle for 2 weeks, and mitochondrial morphology was studied as previously described (14). As expected, chronic MPP ϩ treatment elicited mitochondrial fission with loss of interconnectivity (Fig. 2, B and C, and Table 2) and reduced mitochondrial content (Fig. 2D). Overexpression of PINK1 significantly restored each of these indices of mitochondrial injury (Fig. 2, A-D) and protected against chronic MPP ϩ -induced cell death (Fig. 2E).
In addition to mitochondrial injury, MPP ϩ is known to cause neurite retraction (34). Restoration of PINK1 expression reversed the effects of MPP ϩ on neurite shortening (Fig. 2, F  and G). These data suggest that the loss of PINK1 plays a pathogenic role in chronic MPP ϩ -induced neurodegeneration.

BCL2-associated athanogene (BAG6) is pathologically up-regulated during chronic MPP ؉ injury
PINK1 is known to be stabilized by the heat shock protein 90 chaperone system (9,35). However, we found no changes in the expression of HSP90, CDC37, or CHIP (Fig. S1). There were small decreases in BAG5 and BAG2 protein levels (Fig. 3, A-C), recently implicated in stabilizing PINK1 levels (25). However, the most striking change elicited by chronic MPP ϩ was a large up-regulation of BAG6 protein expression (Fig. 3, A and D), which was negatively correlated with PINK1 protein levels (Figs. 1, B, and F, and 3, A and E). The increase in BAG6 protein  Table 1 for details) followed by post hoc Bonferroni corrected t-tests.  There is a significant interaction between MPP ϩ treatment and PINK1 overexpression for all dependent variables analyzed (see Table 2).  Table 2 for analysis of main factors and interactions). Scale bars, 20 m in A and 50 m in F.

Stress-induced BAG6 modulates PINK1 expression
expression was linked to a small but statistically significant increase in mRNA expression (Fig. 3F).
To examine whether BAG6 up-regulation played a causal role in mediating PINK1 depletion during MPP ϩ toxicity, we applied siRNA targeting BAG6 2 days after initiating MPP ϩ intoxication and every 4 days until the end of the experiment. This procedure elicited a stable reduction in BAG6 expression (Fig. S2), which persisted in the presence of MPP ϩ (Fig. 4A). RNAi knockdown of BAG6 significantly reversed the changes in PINK1 expression elicited by 2 weeks of chronic MPP ϩ toxicity (Fig. 4, A-C). These results suggest that BAG6 plays an important role in down-regulating PINK1 expression under chronic stress.
Blocking the MPP ϩ -induced BAG6 up-regulation by siRNA also reversed indices of MPP ϩ -induced pathology (Fig. 5, A-D) and protected against cell death (Fig. 5E). Although knockdown of BAG6 by itself had little basal effect on neurite length, pos-sibly because BAG6 expression is very low under basal conditions (Fig. 3A), BAG6 knockdown in MPP ϩ -treated cells protected against neurite shortening (Fig. 5, F and G). Use of a second siRNA sequence against BAG6 showed similar protection (Fig. S3).
To determine whether BAG6 modulates PINK1 degradation, we co-expressed BAG6 or GFP with FLAG-tagged PINK1 in HEK293 cells and examined PINK1-FLAG levels after cycloheximide treatment to block new protein synthesis. Cells expressing BAG6 enhanced the degradation of both FL and dN forms of PINK1 compared with GFP ( Fig. 6, C and D), causing significant reductions in the half-lives (t1 ⁄ 2 ) of PINK1-FL and PINK1-dN (Fig. 6E). Elevating BAG6 expression also decreased steady-state levels of PINK1 in the absence of MPP ϩ (Fig. 6, F and G).

Increased BAG6 expression is associated with decreased PINK1 levels and dendritic shortening in primary mouse neurons
We next studied the effects of chronic, low-dose MPP ϩ exposure in primary mouse cortical neurons. Primary neurons are more sensitive to MPP ϩ , with an LD 50 of ϳ30 M (36). Under conditions of acute toxicity (25-50 M), BAG6 levels were decreased at 48 h (Fig. S4). Given that we were able to create a chronic model in SH-SY5Y cells by dropping to ϳ10% of the LD 50 , we studied the effects of chronic, repetitive treatment of primary neurons with 2.5-5 M of MPP ϩ . In contrast to acute toxicity, chronic low-dose exposure to MPP ϩ increased endogenous BAG6 expression accompanied by significantly decreased PINK1 expression (Fig. 7, A-C). As previously reported by others (37), antibodies that recognize mouse PINK1 are not robust, and we could only detect the stronger full-length band.
Using two distinct siRNA sequences (Fig. 7D), we studied the effects of mouse Bag6 knockdown on MPP ϩ -induced neuron injury. Both sequences conferred significant protection against MPP ϩ in primary neurons (Fig. 7E).

Discussion
Although acute treatment of cells with mitochondrial uncouplers results in accumulation of full-length PINK1 (1,4,16,31,38), the effect of less severe, chronic mitochondrial stress has not been previously delineated. MPP ϩ is the active metabolite of MPTP, which is recognized as an environmental cause of human parkinsonian injury, and these compounds are widely used in cell culture and rodent models of PD (31,39). Early studies using isolated brain mitochondria demonstrated that MPP ϩ binds specifically to complex I, inhibiting activity without eliciting irreversible damage (42). It is commonly accepted that MPP ϩ toxicity results from its ability to inhibit mitochon- A, control siRNA (siCtrl) and siBAG6 transfected cells were treated with 250 M MPP ϩ for 2 weeks as described under "Experimental procedures." B, PINK1-FL quantification. *, p ϭ 0.001 for siCtrl (MPP ϩ versus vehicle (Veh)); †, p ϭ 0.0113 for MPP ϩ (siBAG6 versus siCtrl). C, PINK1-dN quantification. *, p Ͻ 0.001 for siCtrl (MPP ϩ versus vehicle); †, p Ͻ 0.001 for MPP ϩ (siBAG6 versus siCtrl). Graphs represent means Ϯ S.D.; n ϭ 3 independent experiments; two-way ANOVA with post hoc t tests (see Table 2 for analysis of main factors and interactions).

Stress-induced BAG6 modulates PINK1 expression
drial complex I activity, as evidenced by the resistance of rho zero ( 0 ) cells to MPP ϩ -mediated cell death (32). Nevertheless, cytotoxic mechanisms that appear to be independent of complex I inhibitory effects have also been reported for MPP ϩ , rotenone, and paraquat (40,41). Indeed, metabolomic, transcriptomic, and proteomic studies demonstrate multiple effects of MPP ϩ treatment that may result directly or indirectly from complex I inhibition or occur independently of electron transport dysfunction (43,44). Although these studies confirm primarily mitochondrial or oxidative changes, other mechanisms may also contribute to MPP ϩ toxicity in acute or chronic settings.
Using a low-dose MPP ϩ model that elicits no cell death at 1 week and ϳ20% cell death at 2 weeks (31), we found that chronic complex I inhibition resulted in a significant decrease of endogenous PINK1 from both cytosolic and mitochondrial fractions of differentiated neuronal cells, with a more robust depletion noted in the cytosol. We also discovered that chronic MPP ϩ treatment robustly increased the expression of BAG6 protein. Similar effects of low-dose, chronic treatment were also observed in primary mouse neurons. Knockdown of BAG6 not only prevented PINK1 loss but also reduced the neurite shortening, mitochondrial fragmentation, and cell death induced by chronic MPP ϩ treatment, conferring protection against MPP ϩ -induced injury in primary neurons. Additionally, we found that BAG6 physically interacts with PINK1 and regulates PINK1 stability by increasing its turnover.

Stress-induced BAG6 modulates PINK1 expression
In healthy cells, PINK1 is continuously turned over with a half-life that varies from 0.45 to 2.3 h depending on the cell type (7, 26, 35, 58 -60). Uncoupling the mitochondria prevents its import, resulting in stabilization of full-length PINK1 at the mitochondrial surface; upon washout of CCCP, accumulated FL-PINK1 reverts to its original half-life (49). The variability in PINK1 half-lives may relate to cell type-and context-dependent differences in expression of chaperone proteins, because PINK1 mutants that are unable to bind HSP90 or CDC37 show a 2-4-fold increase in turnover (35,60).
Indeed, the PINK1 protein is known to be stabilized by chaperone proteins such as HSP90␣, CDC37, and TRAPs (9, 35), as well as BAG2 and BAG5 (25,27,28). Interestingly, there was also a mild decrease in expression of both BAG2 and BAG5. It is possible that loss of these chaperones also contributed to the loss of PINK1 in the chronic MPP ϩ model. The most striking change elicited by chronic, low-level complex I impairment was a large up-regulation of BAG6 expression. Given the effects on both full-length and processed PINK1, it is unclear whether BAG6 interacts with PINK1 at the mitochondrial surface or in the cytosol, because equilibrium changes in one compartment are likely to affect the other.
BAG6, also known as Bat3/Scythe, is a multifunctional protein involved in gene regulation, cell cycle, apoptosis, and protein quality control (61). Bag6 acts as a co-chaperone, negatively regulating HSP70's protein folding capacity (29), in opposition to BAG2 and BAG5. BAG6 acts to target misfolded

Stress-induced BAG6 modulates PINK1 expression
proteins for proteasomal degradation (62), regulates the distribution of damaged mitochondria (63), and stabilizes the apoptosis-inducing factor (64), which has been implicated in MPP ϩ toxicity (36). BAG6 knockout neurons are resistant to ER stress-induced cell death (66). Our current data demonstrate that accelerated degradation of the neuroprotective protein PINK1 also contributes to the detrimental effects of BAG6.
PINK1 plays an important role in the regulation of mitochondrial dynamics and function (14,(67)(68)(69)(70). There was increased mitochondrial fragmentation and swelling caused by chronic MPP ϩ treatment, accompanied by reduced mitochondrial content. Interestingly, siBAG6 transfection not only suppressed BAG6 up-regulation but also rescued the loss of PINK1 and prevented each of these chronic MPP ϩ -induced mitochon-drial changes. These data suggest that the up-regulation of BAG6 expression plays a central role in chronic MPP ϩ -induced mitochondrial pathology.
Neuritic shortening is a common pathological change in neurodegenerative diseases and has been reported in multiple PD models to include 6-hydroxydopamine, PINK1 knockdown, and mutant LRRK2 (15,30,(71)(72)(73). Here, we found that overexpression of PINK1 reversed the MPP ϩ -induced neurite shortening, consistent with the previously reported role of PINK1 in promoting dendrite outgrowth (15,52). These prodifferentiation effects of PINK1 may be mediated through indirect activation of Akt (53) or by the ability of PINK1 to phosphorylate and activate PKA (52). In addition, PINK1 may influence neurite outgrowth through its effects on mitochon-  Table 1 for details). D, mouse primary cortical neurons were either transfected with control siRNA (siCtrl) or 25 nM of siBAG6#1 or siBAG6#2 at DIV7. A second transfection with the same amount of siRNA was done at DIV11. The cells were harvested and lysed at DIV14 for Western blotting analysis (left panel) and densitometry (right panel) of Bag6 knockdown in mouse primary cortical neurons. *, p ϭ 0.01442 for siBAG6#1 versus siCtrl; *, p ϭ 0.0485 for siBAG6#2 versus siCtrl; Bonferroni-corrected two-tailed t test. E, mouse primary neurons plated on LabTek chamber slides were transfected with 25 nM of the indicated siRNA as described for D. The neurons were treated with 5 M of MPP ϩ as described for A. The dendritic arbor of transfected neurons was visualized by co-transfection with GFP (left panels). Summated dendrite length per neuron was quantified (right panel) as described under "Experimental procedures." *, p Ͻ 0.0001 for Ctrl (MPP ϩ versus vehicle); *, p ϭ 0.0002 for siCtrl (MPP ϩ versus vehicle); †, p ϭ 0.0170 for MPP ϩ (siBAG6#1 versus siCtrl); †, p ϭ 0.0354 for MPP ϩ (siBAG6#2 versus siCtrl). Scale bar, 100 m. The data were analyzed by two-way ANOVA with post hoc Bonferroni-corrected t test (see Table 2 for analysis of main factors and interactions). All graphs represent means Ϯ S.D. from three independent experiments.

Stress-induced BAG6 modulates PINK1 expression
drial trafficking (15), because the ability to deliver mitochondria plays a limiting role in synaptogenesis (65,74). Interestingly, siRNA against BAG6 not only prevented MPP ϩ -induced loss of endogenous PINK1 but also protected against neurite shortening in differentiated SH-SY5Y cells and dendritic shrinkage in primary mouse neurons.
In conclusion, our data indicate a novel role for BAG6 in regulating PINK1 levels under stress conditions. Under basal conditions, BAG6 is maintained at low levels. However, upon chronic MPP ϩ -induced stress, BAG6 levels are elevated, resulting in accelerated PINK1 degradation. Restoration of PINK1 levels through either overexpression or via BAG6 RNAi protected against chronic MPP ϩ -induced changes in mitochondrial structure, neurite shortening, and cell death. These data suggest a possible point of mechanistic convergence between neurotoxic/environmental and genetic causes of PD, which centers on the loss of PINK1 function.

Cell lines, primary neuron culture, and treatments
SH-SY5Y cells (ATCC, Manassas, VA) were maintained in antibiotic-free Advanced Dulbecco's modified Eagle's medium with 5% heat-inactivated fetal calf serum (BioWhittaker, Walkersville, MD), 2 mM glutamine, and 10 mM HEPES. HEK293 cells (ATCC) were maintained in antibiotic free DMEM (Bio-Whittaker) supplemented with 10% fetal bovine serum (Mediatech), 2 mM L-glutamine, and 10 mM HEPES in a humidified incubator at 37°C and 5% CO 2 . Timed pregnant female C57BL/6 mice were purchased from Charles Rivers Laboratories. All procedures for derivation of primary neuron cultures were approved by the University of Pittsburgh Institutional Animal Care and Use Committee. Primary cortical neurons from embryonic day 14 -16 male or female pups were isolated from cerebral cortices as described previously (15,30). Neurons were plated at 150,000 cells/cm 2 in LabTek II coverglass chamber slides coated with poly-L-lysine (0.1 mg/ml). They were maintained in antibiotic-free1 Neurobasal medium supplemented with 2% B27 and 2 mM GlutaMAX (Gibco, Bethesda, MD). Half of the medium was replaced with fresh medium every other day.
SH-SY5Y cells were plated in 6-well plates or LabTek II coverglass chamber slides (Thermo Fisher) and neuronally differentiated with 10 M retinoic acid (Sigma-Aldrich) for 72 h prior to and during each experiment. SH-SY5Y cells were treated with 250 M MPP ϩ three times per week for up to 2 weeks. Mouse primary cortical neurons were treated with 2.5 or 5 M MPP ϩ from DIV7 for 1 week with half medium change containing fresh toxin every 2 days. For cycloheximide experiments, HEK293 cells were transfected with the indicated plasmids for 48 h. Transfected cells were treated with 100 g/ml cycloheximide (Sigma-Aldrich) diluted in fresh DMEM from a 100 mg/ml stock. Cells were treated at staggered intervals every 30 min, and at all time points were quickly harvested and lysed after 2 h (30 min after the last set of cells was treated). Untreated transfected cells were used as the zero time point.

RNAi and DNA transfection
siRNAs targeting different portions of the BAG6 mRNA sequence were employed: assay HSS111844 (siBAG6#1), which targets human, mouse, and rat sequences, and assay s15467 (siBAG6#2), which targets human and mouse sequences (Thermo Fisher). SH-SY5Y cells were transfected with the siRNAs 2 days after the first dose of MPP treatment and again every 4 days, which results in persistent knockdown (Fig. S2). For DNA transfection in differentiated SH-SY5Y cells, the cells were transfected with 750 ng/well of either GFP-tagged WT PINK1, or control vector (Genecopoeia, Rockville, MD) 2 days before MPP ϩ treatment. HEK293 cells were co-transfected with either 2 g/well PINK1-FLAG and GFP or PINK1-FLAG and 1 g/well BAG6-V5 (HsCD00442162, DNASU plasmid repository) in 6-well plates at 2 days prior to cycloheximide chase experiments. For co-immunoprecipitation experiments, the cells were transfected with 2 g/well of PINK1-GFP or control vector and 1 g/well BAG6-V5 in a 6-well plate for 48 h. For immunoblotting experiments, mouse cortical neurons were transfected with 25 nM of the two siRNAs targeting different regions of Bag6 mRNA at 1 day after the start of MPP ϩ treatment and again at DIV 11. Mouse primary cortical neurons were transfected with 500 ng/well GFP-expressing plasmid 48 h before fixing the cells for total dendrite length analysis.

Immunoprecipitation
48 h after plasmid transfection, HEK293 cells were lysed in buffer containing 1% Triton X-100 and protease/phosphatase inhibitors. Protein concentration was determined by Coomassie Blue protein assay (Pierce). Equal amounts of protein (1 mg of total protein) were used for IP. For IP of GFP-tagged PINK1, the uMACS GFP isolation kit (catalog no. 130-091-125, Miltenyi Biotec, Gaithersburg, MD) was used according to the manufacturer-recommended protocol. For reverse IP, protein lysates were incubated with 2 g of mouse anti-V5 antibody (Santa Cruz), 50 l of protein G-agarose beads (catalog no. 16-266, EMD Millipore) overnight at 4°C. The beads were pelleted at 5000 ϫ g for 5 min, the supernatant was removed, and the beads were washed three times with wash buffer 1 and twice with wash buffer 2 from the GFP isolation kit. IPed complexes were eluted by boiling the beads in 50 l of elution buffer followed by centrifugation at 10,000 ϫ g for 5 min. The supernatant was used for SDS-PAGE.

Quantitative RT-PCR
Total RNA was extracted using RNeasy kit (Qiagen). A total of 1 g of RNA was used for reverse transcription reaction using the SuperScript IV one-step RT-PCR system (Life Technologies, Inc.). The cDNA was quantified by Q-PCR using PINK1 TaqMan probes (Hs00260868_m1, Life Technologies, Inc.) and BAG6 (Hs00190383_m1, Life technologies) and normalized to GAPDH mRNA (4333764F).

Stress-induced BAG6 modulates PINK1 expression
plemented with protease inhibitors as described previously (33). Equal amounts of protein as determined by Coomassie Plus protein assay (Thermo Scientific) were resolved on a 10% gel by SDS-PAGE and transferred to polyvinylidene difluoride membrane. The membranes were blocked with 5% nonfat milk and probed with antibodies as listed in Table 3 overnight with gentle agitation at 4°C. Immunoreactive bands were detected using anti-mouse or anti-rabbit horseradish peroxidaseconjugated secondary antibodies (GE Healthcare) followed by exposure to ECL solution. The images were acquired using the Odyssey Fc imaging system (Li-Cor) for densitometry using Image Studio software (Li-Cor). Densitometry data were normalized to loading control, and the half-lives of PINK1 isoforms were estimated by fitting a one-phase exponential decay curve using Prism 8 software (GraphPad, San Diego, CA).

Quantification of mitochondrial morphology and neurite length
The parameters of mitochondrial morphology including mitochondrial area, perimeter, and mitochondrial connectivity were analyzed using ImageJ software (National Institutes of Health). Mitochondrial connectivity was calculated as the square root of the perimeter/circularity ratio normalized by the minor elliptical axis of mitochondria as described previously with slight modification (12). The inverse circularity was used as a measure of mitochondrial elongation. Differentiated SH-SY5Y cells typically exhibit a small cell body and one major neurite projecting from the cell body. The longest neurite from the cell body was measured using the intensity-tracing algorithm in ImageJ (34). For neurite length and total dendritic length analysis, color images were extracted to 8-bit grayscale images for quantification using ImageJ supplemented with the NeuronJ plug-in.

Cell death assay and quantification
After 2 weeks of chronic MPP ϩ treatment, SH-SY5Y cells were gently washed once with warm DMEM and replaced with DMEM containing Hoechst (Thermo Scientific; 1:1000) and propidium iodide (Molecular Probes, Eugene, OR; 1:1000) and incubated for 20 min at 37°C. After that, the medium was replaced with dye free medium and incubated for 30 min at 37°C to remove extra dye. Before imaging, the cells were washed once with warm DMEM and imaged at room temperature using Olympus IX71 microscope using 10ϫ objective. Seven to nine random fields were imaged per condition. For quantification, the color images were background subtracted and converted to grayscale using ImageJ. After thresholding, the particle counting plugin was used to count total cells (Hoechst) and propidium iodide-positive cells. Percent dead cells was determined using the formula [(total propidium iodide Ϫ positive cells)/(total number of cells)] ϫ 100.

Statistics
All graphed data represent means Ϯ S.D. from independent, replicate experiments. Two group data were analyzed using two-tailed Student's t test. Multigroup data were analyzed using one-way or two-way ANOVA. If the null hypothesis was rejected, post hoc comparisons using Bonferroni-corrected two-tailed t tests were performed to test specific hypotheses in the context of each experiment. Values of p Ͻ 0.05 were considered significant.

Data availability
All data described are contained within the manuscript. Funding and additional information-This work was supported in part by National Institutes of Health Grants R01-AG026389, R01-NS065789, and R01-NS101628 (co-funded by NINDS/NIA). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Conflict of interest-The authors declare that they have no conflicts of interest with the contents of this article.