A novel mechanism of PHB2-mediated mitophagy participating in the development of Parkinson’s disease

Yongjiang Zhang, Shiyi Yin, Run Song, Xiaoyi Lai, Mengmeng Shen, Jiannan Wu,*, Junqiang Yan,2,*

Abstract Endoplasmic reticulum stress and mitochondrial dysfunction play important roles in Parkinson’s disease, but the regulatory mechanism remains elusive.Prohibitin-2 (PHB2) is a newly discovered autophagy receptor in the mitochondrial inner membrane, and its role in Parkinson’s disease remains unclear.Protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) is a factor that regulates cell fate during endoplasmic reticulum stress.Parkin is regulated by PERK and is a target of the unfolded protein response.It is unclear whether PERK regulates PHB2-mediated mitophagy through Parkin.In this study,we established a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of Parkinson’s disease.We used adeno-associated virus to knockdown PHB2 expression.Our results showed that loss of dopaminergic neurons and motor deficits were aggravated in the MPTP-induced mouse model of Parkinson’s disease.Overexpression of PHB2 inhibited these abnormalities.We also established a 1-methyl-4-phenylpyridine (MPP+)-induced SH-SY5Y cell model of Parkinson’s disease.We found that overexpression of Parkin increased co-localization of PHB2 and microtubule-associated protein 1 light chain 3, and promoted mitophagy.In addition, MPP+ regulated Parkin involvement in PHB2-mediated mitophagy through phosphorylation of PERK.These findings suggest that PHB2 participates in the development of Parkinson’s disease by interacting with endoplasmic reticulum stress and Parkin.

Key Words: endoplasmic reticulum; dopaminergic neuron; microtubule-associated protein 1 light chain 3; mitophagy; oxidative stress; Parkin; Parkinson’s disease; PKR-like endoplasmic reticulum kinase; reactive oxygen species; prohibitin-2

Introduction

Parkinson’s disease (PD) is the second most common neurodegenerative disease, caused by the degeneration of dopaminergic neurons in the substantia nigra striatum (de Lau and Breteler, 2006; Chen et al., 2020; Liu et al., 2023; Yu et al., 2023).To date, there is no effective way to prevent the degeneration of dopaminergic neurons.Mitochondrial dysfunction is one of the cell death pathways of dopaminergic neurons and plays an important role in PD.As a neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP) acts on mitochondrial respiratory chain complex I and has selective cytotoxicity to dopaminergic neurons.1-Methyl-4-phenylpyridinium (MPP+) is generated by the oxidation of MPTP through the blood-brain barrier, and can accumulate in mitochondria leading to mitophagy (Perfeito et al., 2012).

Prohibitin-2 (PHB2) is a newly discovered autophagy receptor in the mitochondrial inner membrane, and is involved in microtubule-associated protein 1 light chain 3 (LC3)-mediated autophagy (Wei et al., 2017).PHB2 can inhibit the accumulation of fragmented mitochondria and plays a regulatory role in mitochondrial dynamics (Wang et al., 2014).When the mitochondrial outer membrane ruptures, PHB2 can bind to cytoplasmic LC3II and promote autophagy in damaged mitochondria.In a rat model of arachnoid hemorrhage, mitoquinone (a mitochondrial-targeted antioxidant) activated mitophagy through the PHB2 pathway, thereby inhibiting oxidative stressrelated neuronal death (Zhou et al., 2021).Knockdown of PHB2 reduced the level of mitophagy and increased cell death (Xu et al., 2019), while overexpression of PHB2 alleviated the inflammatory response of renal tubular epithelial cells by improving mitophagy (Yuk et al., 2020).However, whether PHB2-mediated mitophagy is involved in PD has not been reported.

A recent study found that the endoplasmic reticulum (ER) is in contact with mitochondria, with fundamental functions established in biosynthetic processes, cell signaling, cell execution, and mitochondrial dynamics (Csordás et al., 2018).The interaction between ER stress and mitophagy plays an important role in the pathogenesis of PD (Gómez-Suaga et al., 2018).The unfolded protein response (UPR) is essential for the survival of dopaminergic neurons (Zarate et al., 2021).ER stress can increase the folding capacity of the ER, reduce the load of unfolded proteins, and restore homeostasis of the ER (Wang and Kaufman, 2016).Excessive stress can lead to mitophagy or neuroinflammation, which ultimately leads to neuronal damage (Lizama and Chu, 2021; Qu et al., 2023).Protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) is a type I transmembrane protein kinase located on the ER membrane that controls the fate of cells under ER stress.Activation of PERK leads to phosphorylation of the eukaryotic initiation factor 2α and inhibits the synthesis of general proteins, a key upstream factor in the UPR.Parkin is regulated by PERK and is a target of the UPR (Almeida et al., 2022).It is unclear whether PERK regulates PHB2-mediated mitophagy through Parkin.In the present study, MPP+/MPTP was used to establish cell and animal models of PD.Protein expression levels of PERK, Parkin, PHB2, and autophagy proteins were observed after silencing/overexpression of PERK, Parkin,and PHB2, respectively.Co-localization of PHB2 and LC3 was detected by immunofluorescence to clarify the effect of PERK and Parkin on PHB2 binding to LC3.Finally, PHB2 was silenced in a MPTP-induced PD animal model to observe the effect of PHB2-mediated mitophagy on dopaminergic neurons and behavioral changes in mice.

Methods

Cell culture and treatment

SH-SY5Y cells (Stem Cell Bank, Chinese Academy of Sciences, Cat# SCSP-5014,RRID: CVCL_0019) were obtained from Sun Yat-sen University and cultured in Dulbecco’s modified Eagle medium (Corning, Corning, NY, USA, Cat# 10-092-CV) containing 10% fetal bovine serum in an incubator with 5% carbon dioxide at 37°C.Parkin-short hairpin RNA (sh-RNA) (Santa Cruz Biotechnology,Dallas, TX, USA, Cat# sc-42158-V) resistant to puromycin, PHB2-shRNA (Santa Cruz, Cat# sc-45849-V), and control shRNA (Santa Cruz Biotechnology, Cat#sc-108080) lentiviral particles were used.Overexpression lentiviruses were supplied by GeneChem (Genechem, Shanghai, China, Parkin, NM_004562,PHB2, NM_001144831, CV702).For knockdown and overexpression treatments, the relevant lentivirus (50 nM) was stably transfected into SHSY5Y cells (50% confluence) for 24 hours.After knockdown and overexpression treatment, cell culture medium containing 1 mM MPP+(Sigma-Aldrich, St.Louis, MO, USA, Cat# M7068) prepared in standard cell culture medium was applied to SH-SY5Y cells (60–70% confluence) for 24 hours.

Establishment of a stable PHB2-shRNA cell line

SH-SY5Y cells stably expressing PHB2-shRNA were established.Human PHB2 shRNA lentiviral particles (Santa Cruz Biotechnology, Cat# sc-45849-V)were used for viral infection.The cells were selected according to the manufacturer’s instructions.On the first day, SH-SY5Y cells were plated into a 12-well plate, 24 hours before viral infection.Dulbecco’s modified Eagle medium (1 mL) containing 10% fetal bovine serum was added to the SH-SY5Y cells overnight.The next day, cells at approximately 50% confluence were infected with lentiviral particles.The culture medium was removed from 12-well plates and replaced with 1 mL culture medium containing 5 μg/mL polybrene (Santa Cruz, Cat# sc-134220) per well.The cells were infected by adding 5 × 104U PHB2 shRNA lentivirus particles into the culture medium, and then the plate was incubated overnight with gentle shaking.On the third day,the medium was removed, and 1 mL of complete medium without polybrene was added.On the fourth day, to screen cell clones stably expressing shRNA,the cells were passaged at a ratio of 1:5 and cultured in complete medium for 24–48 hours.After 5–6 days of 10 μg/mL puromycin hydrochloride treatment,stable expression clones were generated.Selected cells were further cultured with medium containing 5 μg/mL puromycin for routine culture.

Mitochondrial function assay

When cell confluence reached approximately 50% after MPP+treatment,the cells were treated with 10 μg/mL JC-1 (Invitrogen, Los Angeles, CA, USA,T3168) and incubated at 37°C for 10 minutes.A fluorescence microscope(Olympus, Tokyo, Japan, BX-53) was used to observe green fluorescence(excitation at 490 nm and emission at 525 nm) and orange fluorescence(excitation at 555 nm and emission at 580 nm).

Transmission electron microscopy observation of mitochondria

More than 1 × 106SH-SY5Y cells were quickly scraped from a flask with a cell scraper and centrifuged at 1500–3000 ×gfor 5–10 minutes.Two or three samples were collected that were visible to the naked eye at the bottom of the tube (approximately the size of rice grains).The samples were treated with glutaraldehyde electron microscope fixative and embedded in resin.Ultrathin sections were then obtained, stained with lead citrate and uranium diacetate, and observed under a transmission electron microscope (Hitachi,Tokyo, Japan, ht7800).

In vivoexperimental design

Male C57BL/6J specific pathogen-free mice (approximately 25 g) aged 8 weeks were provided by Charles River Bioscience (Beijing, China; license No.SCXK (Zhe) 2019-0001).Mice were maintained in 23 ± 2°C, humidity 50–70%,light intensity 15–20 lx, alternating 12-hour light and dark cycles with free access to food and water.This study was approved by the Ethics Committee/Institutional Review Board of the First Affiliated Hospital of Henan University of Science and Technology on April 10, 2020.All animals were treated following the guidelines of the National Institute for Health’s Guide for the Care and Use of Laboratory Animals (National Research Council, 2011) and followed the guidelines of the International Association for the Study of Pain(IASP).

The experimental procedure was conducted as follows.Mice were acclimatized for 1 week and then randomly divided into four groups (n= 5/group): (1) sham, (2) MPTP, (3) PHB2-shRNA, and (4) MPTP + PHB2-shRNA.Eight weeks after adeno-associated virus (AAV)-9 PHB2-shRNA and AAV control-shRNA microinjection, MPTP was used for PD modeling.Seven days after the last MPTP injection, behavioral assessments were performed using the tail suspension test and rotarod test.When the behavioral experiments were completed, the mice were sacrificed for further study.Specific experimental details are described below.

Animal model of PD and PHB2 inhibition

First, the PHB2-shRNA mouse model was prepared by microinjection of AAV9 PHB2-shRNA.The mouse head was fixed in a stereotaxie apparatus(Shanghai Biowill Co., Ltd., Shanghai, China), followed by anesthetization with pentobarbital sodium (Sigma-Aldrich, Cat# P3761).Next, the scalp was shaved to expose the skull.Viral vector was injected into the striatum by microinjection (volume of 2 μL, rate of 0.2 μL/min).The injection point was 1.8 mm anterior to the bregma, 2.5 mm lateral from the midline, and 3.5 mm in depth (Antipova et al., 2018).After injection, the needle was kept in place for 10 minutes, and then slowly pulled out vertically.The scalp was sutured.Second, the PD animal model was produced by injection of MPTP 56 days after AAV injection.The mice were intraperitoneally injected with MPTP (20 mg/kg, Sigma-Aldrich, Cat# M0896) in normal saline solution, once every 2 hours for a total of four times.Behavioral experiments were then performed seven days after MPTP injection.

Tail suspension test

The tail suspension test is a behavioral despair model of depression used for evaluation of neuropsychiatric features (Cryan et al., 2005).The tail suspension test was performed using the SuperTst suspension tail experimental analysis system (Xinruan Information Technology Co., Ltd.,Shanghai, China), as previously described (Can et al., 2012).Immobility time was recorded for 6 minutes and analyzed as a correlate of depression-like behavior.

Rotarod test

The rotarod test was performed to assess motor dysfunction, as previously described (Shiotsuki et al., 2010).The 5 Station USB Rota Rod ENV-575MA(MED Associates Inc., Fairfax, VT, USA) was used for the evaluation of motor skill learning.Briefly, the mice were trained for 3–4 days on an accelerated rotarod to teach them to stay on the rotarod.The rod accelerated linearly from 3.5 to 35 r/min in 2 minutes.The time the mice stayed on the rod was measured within a 480-second test interval.

Western blotting

After pretreatment with MPP+alone or in combination with PHB2-shRNA,Parkin overexpression lentivirus, or Parkin-shRNA, cells and the midbrain of mice (removed following intraperitoneal injection of 2,2,2-tribromoethanol[Sigma-Aldrich, Cat# T48402], followed by quick cervical dislocation) were rinsed three times with ice-cold phosphate buffered saline (PBS) and incubated in ice-cold lysis buffer for 20 minutes on ice.Supernatants were then collected and subjected to a bicinchoninic acid assay to determine protein concentration.Samples were mixed with 5× loading buffer and heated for 5 minutes at 100°C.Next, aliquots with equivalent amounts of total protein (20 μg) were separated on 12% sodium dodecyl sulfatepolyacrylamide gel electrophoresis gels and transferred to polyvinylidene fluoride membranes (Millipore Immobilon-PSQ, Merck KGaA, Darmstadt,Germany, ISEQ00010).Membranes were incubated overnight at 4°C in solutions with primary antibodies against PHB2 (rabbit, 1:2000, Proteintech,Chicago, IL, USA, Cat# 12295-1-AP, RRID: AB_2164779), Parkin (mouse,1:2000, Abcam, Cambridge, UK, Cat# ab77924, RRID: AB_1566559), LC3B(rabbit, 1:2000, Abcam, Cat# ab192890, RRID: AB_2827794), p-PERK Thr980(rabbit, 1:2000, Cell Signaling Technology, Danvers, MA, USA, Cat# 3179,RRID: AB_2095853), nuclear factor erythroid 2-related factor (Nrf2; rabbit,1:2000, Cell Signaling Technology, Cat# 12721, RRID: AB_2715528), heme oxygenase 1 (HO-1; rabbit, 1:2000, Cell Signaling Technology, Cat# 43966,RRID: AB_2799254), quinone oxidoreductase 1 (NQO-1; mouse, 1:2000, Cell Signaling Technology, Cat #3187, RRID: AB_2154354), and β-actin (mouse,1:5000, CWBio, Beijing, China, Cat# CW0096, RRID: AB_2665433).The next day, membranes were washed three times and then incubated with horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit,1:5000, CWBio, Cat# CW0103, RRID: AB_2814709; or goat anti-mouse, 1:5000,CWBio, Cat# CW0102, RRID: AB_2814710) at 37°C for 1 hour.Negative controls were prepared by excluding primary antibodies.Images of labeled membranes were analyzed using ImageJ software (version 1.53f51; National Institutes of Health, Bethesda, MD, USA; Schneider et al., 2012).

Immunohistochemistry analysis

Mice were sacrificed by quick cervical dislocation after anesthesia with 2,2,2-tribromoethanol.The brain was post-fixed using 4% paraformaldehyde,cryoprotected in 30% sucrose, and processed for immunohistochemistry.Next, 40 μm thick coronal sections of the midbrain were cut and every fourth section analyzed.For tyrosine hydroxylase (TH) labeling, the sections were treated with anti-TH (rabbit, 1:1000, Proteintech, Cat# 25859-1-AP, RRID: AB_2716568) overnight at 4°C, then incubated with Alexa Fluor 488-conjugated secondary antibody (goat anti-rabbit Alexa Fluor 488,1:500, Thermo Fisher Scientific, Waltham, MA, USA, Cat# A-11008, RRID:AB_143165) at 37°C for 1 hour.Sections were imaged using an Olympus DP73 camera (Olympus).The number of TH-positive neurons was counted using a previously reported method (Yan et al., 2022).

SH-SY5Y cells were seeded onto a suitable treated circular coverslip, placed into a 12-well plate, and fixed with paraformaldehyde.The cells were then labeled overnight with primary antibodies (1:100) specific to PHB2 (rabbit,Proteintech, Cat# 12295-1-AP, RRID: AB_2164779), Parkin (mouse, Abcam,Cat# ab77924, RRID: AB_1566559), LC3B (rabbit, Abcam, Cat# ab232940),TOM20 (mouse, Santa Cruz Biotechnology, Cat# sc-17764, RRID:AB_628381),and TIM23 (mouse, Santa Cruz Biotechnology, Cat# sc-514463, RRID:AB_2923126) at 4°C.After incubation with secondary antibodies (1:500)(goat anti-rabbit Alexa Fluor 488, Thermo Fisher Scientific, Cat# A-11008,RRID: AB_143165; or goat anti-mouse Alexa Fluor 555, Cat# A-21422, RRID:AB_2535844) at 37°C for 1 hour, 4′,6-diamidino-2-phenylindole (DAPI; 1:1000,Beyotime Biotechnology, Shanghai, China, C1002) was used for nuclear analysis.Fluorescence distribution was observed with a LSM 780 confocal microscope (Zeiss, Jena, Germany).

Reactive oxygen species assay

Reactive oxygen species (ROS) were measured using the fluorescent dye 2,7-dichlorofluoresce diacetate (DCFH-DA) of the ROS assay kit (Beyotime Biotechnology).DCFH-DA solution was diluted with culture medium,and 2 μL dye solution added per mL of culture medium, according to the manufacturer’s instructions.Light was avoided during the experiment, and incubation was conducted in 5% CO2for 60 minutes in a 37°C incubator.Cells were treated with MPP+to induce ROS.Flow cytometry (BD Accuri C6,Franklin Lakes, NJ, USA) was used after 20 minutes of DCFH-DA treatment to analyze the fluorescence intensity of cells.

Statistical analysis

The number of experimental animals was based on a previous publication(Charan and Kantharia, 2013).The analyzer was blinded to the experimental treatment.GraphPad Prism 7.00 (GraphPad Software, San Diego, CA,USA, www.graphpad.com) was used to generate graphs and analyze data.Comparisons between groups were evaluated by one-way analysis of variance with Tukey’s multiple comparison test.Data are presented as mean ± standard error of mean (SEM).Differences between experimental conditions were considered to be statistically significant atPvalues < 0.05.

Results

Silencing PHB2 aggravates oxidative stress and mitochondrial damage in MPP+-treated SH-SY5Y cells

Human neuroblastoma SH-SY5Y cells are a dopaminergic neuronal cell line used as anin vitromodel for neurotoxicity experiments.MPP+inhibits the activity of oxidative respiratory chain complex I in mitochondria, which can cause oxidative stress injury in dopaminergic neurons and affect neuronal mitophagy (Ramsay et al., 1986).We treated SH-SY5Y cells with 1 mM MPP+,and found decreased PHB2 expression at 12 hours (Figure 1A and B).Next, we determined whether PHB2 affected the intracellular antioxidative response and mitochondrial function.Nrf2 is a nuclear factor erythroid 2-related factor that can translocate to the nucleus and recognize the enhancer sequence of antioxidant response elements.In turn, this increases expression of HO-1 and NQO-1 to resist various stress injuries (Hayes and Dinkova-Kostova, 2014).In our study, we found MPP+significantly reduced the expression of Nrf2, HO-1,and NQO-1.Moreover, overexpression of PHB2 increased protein expression of Nrf2, HO-1, and NQO-1, while silencing of PHB2 decreased levels of these anti-oxidative stress proteins under MPP+treatment (Figure 1C–G).We also examined the effect of PHB2 on JC-1 fluorescence (Figure 1H and I) and ROS accummulation (Figure 1J and K).We found that overexpression of PHB2 increased the mitochondrial membrane potential and reduced ROS levels in the presence of MPP+.Silencing PHB2 decreased the mitochondrial membrane potential and increased ROS levels.Finally, we observed the mitochondrial state of PHB2-shRNA-treated cells by electron microscopy (Figure 1L).Normal morphology of the mitochondrial inner ridge was observed in the control group.PHB2-shRNA treatment changed the morphology of the mitochondrial inner ridge, while MPP+treatment caused significant swelling of the inner ridge.

Interaction of PHB2 and LC3 increases mitophagy in a PD cell model

As PHB2 promotes mitophagy by recruiting LC3II (Wei et al., 2017), we further examined whether PHB2-mediated mitophagy is involved in PD.Changes in the LC3II/LC3I ratio were observed in MPP+-treated SH-SY5Y cells by silencing or overexpression of PHB2.The LC3II/LC3I ratio was lower after PHB2 silencing than in the control group (Figure 2A–C).The LC3II/LC3I ratio was higher following overexpression of PHB2 than in the control group (Figure 2D–F).We used the mitochondrial outer membrane protein, TOM20, and the mitochondrial inner membrane protein, TIM23, as mitochondrial localization markers.Silencing of PHB2 decreased colocalization of LC3 and TOM20 and TIM23 (Figure 2G).

Figure 1 ｜ Effect of MPP+ on PHB2 protein expression and mitochondrial function in SH-SY5Y cells.

Figure 2 ｜ Effect of PHB2 on mitophagy in a PD cell model.

Parkin increases protein expression and interaction of PHB2 and LC3 in MPP+-treated SH-SY5Y cells

Next, we investigated whether Parkin regulation affected protein expression and interaction of PHB2 and LC3.We found that Parkin overexpression increased protein expression of PHB2 and the LC3II/LC3I ratio.Silencing of Parkin decreased the LC3II/LC3I ratio and protein expression of PHB2 under MPP+treatment (Figure 3A–D).Immunofluorescence staining showed that silencing of Parkin decreased co-localization of PHB2 and LC3 under MPP+treatment, whereas overexpression of Parkin increased co-localization (Figure 3E).Taken together, these results suggest that Parkin affects mitophagy by regulating the expression and binding of PHB2 to LC3.

Parkin regulates anti-oxidative stress protein expression via PHB2

To further investigate whether Parkin affected oxidative stress through PHB2, we overexpressed Parkin while silencing PHB2 in MPP+-treated SHSY5Y cells.The results showed that overexpression of Parkin while silencing PHB2 decreased the expression of antioxidant stress proteins compared with overexpression of Parkin under MPP+treatment (Figure 4A–D).Taken together, these data suggested that Parkin regulated anti-oxidative stress protein expression via PHB2.

Phosphorylation of PERK (Thr980) increases Parkin protein expression in SH-SY5Y cells

Activation of PERK is associated with autophosphorylation of the cytoplasmic kinase domain, and phosphorylation of PERK at Thr980 is a hallmark of its activation state (Cui et al., 2011).Western blotting results showed that PERK silencing decreased expression of p-PERK (Thr980) and Parkin in MPP+-treated SH-SY5Y cells (Figure 5A–C).However, although PERK phosphorylation can up-regulate Parkin expression, the ability of MPP+to directly down-regulate Parkin expression was stronger than PERK phosphorylation.Therefore, the overall expression of Parkin in the PD cell model was reduced.

Silencing PHB2 inhibits mitophagy and aggravates the loss of dopaminergic neurons in PD mice

To observe the role of PHB2 in PD, we injected AAV9 PHB2-shRNA into the striatum of PD mice (Figure 6B and C).We found that silencing PHB2 decreased the LC3II/LC3I ratio (Figure 6D–F).TH immunofluorescence of substantia nigra neurons indicated that MPTP significantly reduced the number of TH-positive neurons, and silencing of PHB2 aggravated MPTPinduced loss of dopaminergic neurons (Figure 6G and H).Moreover, silencing of PHB2 under MPTP treatment reduced co-localization of LC3 with PHB2,TIM23, and TOM20 (Figure 6I).

Silencing PHB2 decreases antioxidant stress protein expression and aggravates motor defecits in PD mice

Our results showed significantly decreased protein expression of Nrf2, HO-1, and NQO-1 in PHB2-silenced PD mice compared with the MPTP group(Figure 7A–D).To further investigate the effect of PHB2 inhibition on behavior in PD mice, we evaluated grip strength and dynamic limb balance of mice by the classical tail suspension and rotarod tests.The results showed that PHB2 silencing increased immobility time of mice under MPTP treatment in the tail suspension test.Moreover, silencing of PHB2 in PD mice also decreased thetime that mice could stay on the roller in the rotarod test (Figure 7E).

Discussion

Herein, our major findings show that PHB2 is involved in the pathology and pathogenesis of PD by regulating LC3II to LC3I conversion and binding to LC3 to affect mitophagyin vivoandin vitro.Furthermore, we found that PHB2-mediated mitophagy and oxidative stress were regulated by the PERKParkin pathway.This study therefore reveals a new mitophagy pathway and regulatory mechanism for PD.

Our previous study found that PHB2-mediated mitophagy can be regulated by the tyrosine kinase, c-Abl (Zhang et al., 2022).The possible mechanism was that c-Abl phosphorylates the PHB2Y121 site to prevent PHB2 from binding to LC3 (Zhang et al., 2022).Furthermore, we found that rutin could increase mitophagy to improve oxidative stress injury in the PD cell model, with the key mechanism being that rutin rescued the decrease of PHB2 induced by MPP+(Lai et al., 2023).Here, we found that PHB2-mediated mitophagy can protect mice against MPTP-induced behavioral deficits and dopaminergic degeneration in the nigrostriatal system.Silencing of PHB2 reduced the antioxidant stress proteins Nrf2, HO-1, and NQO-1 inin vitroandin vivoPD models, further demonstrating the neuroprotective function of PHB2 in the dopaminergic system.

Figure 3 ｜ Parkin increases PHB2/LC3-mediated mitophagy in SH-SY5Y cells.

Figure 4 ｜Parkin regulates anti-oxidative stress protein expression via PHB2.

Figure 5 ｜Phosphorylation of PERK (Thr980) increases Parkin protein expression in SH-SY5Y cells.

PERK is a key upstream factor of the UPR.Phosphorylated PERK can be observed in substantia nigra neurons of Parkinson’s disease brain tissue, colocalized with α-synuclein, confirming that PERK is involved in the occurrence and progression of Parkinson’s disease as a key sensor of ER stress(Hoozemans et al., 2007).Parkin is a target regulated by the UPR through PERK/activating transcription factor 4 (ATF4) (Senft and Ronai, 2015).The up-regulation of Parkin induced by ER stress is mediated by ATF4.ATF4 is a transcription factor of UPR.When mitochondria are damaged, the PERK-ATF4 signaling pathway is initiated to up-regulate Parkin expression (Wu et al.,2014).Parkin silencing blocks the neuroprotective effect of ATF4.Silencing the downstream target, ATF4, can reduce Parkin expression in mouse primary cortical neurons and cause cell death.Parkin is a key molecule that bridges the UPR and autophagy in PD (Zhang et al., 2014).In conclusion,under conditions of ER stress, PERK activation induces the downstream mitochondrial quality control system, which directly stabilizes the ERmitochondrial axis through up-regulation of Parkin transcription, and has a resulting positive and protective effect on neurons (Rainbolt et al., 2014).Our study found that MPP+increased protein expression of phosphorylated PERK, while inhibiting PERK significantly reduced protein expression of Parkin.Phosphorylation of PERK induced by ER stress can increase protein expression of Parkin (Bouman et al., 2011).Indeed our results are consistent with these findings.Previous research found that Parkin overexpression can reduce MPP+-induced dopaminergic neuronal death, while silencing of Parkin can prevent the neuroprotective effect of PERK (Sun et al., 2013).We further confirmed that the neuroprotective mechanism of Parkin overexpression on dopaminergic neurons may be related to alleviation of oxidative stress injury by increasing PHB2-mediated mitophagy in PD.Mitochondria-associated ER membranes are direct contact sites between the ER and mitochondria that serve as platforms to coordinate fundamental cellular processes such as mitochondrial dynamics and bioenergetics, calcium and lipid homeostasis,autophagy, apoptosis, inflammation, and intracellular stress responses(Proulx et al., 2021).PD pathology is characterized by several mitochondriaassociated ER membrane-associated cellular processes including impaired autophagy, calcium homeostasis, lipid metabolism, ER stress, and mitochondrial dynamics (Rodríguez-Arribas et al., 2017; Rozzi et al., 2018).In our study, we found that PERK regulated PHB2-mediated mitophagy through Parkin.However, whether PERK directly affects PHB2-mediated mitophagy through mitochondria-associated ER membranes requires further investigation.Some limitations should be noted in our study.The interaction between Parkin and PHB2 has not been described and should be further verified at the gene and protein level, such as protein molecular docking.In summary,our findings are of great significance for understanding the role of ER stress and mitophagy in the degeneration of PD dopaminergic neurons.To the best of our knowledge, this is the first report in a preclinical model of PD showing that PHB2-mediated mitochondrial autophagy is involved in the regulation of PD oxidative damage.Therefore, the development of PHB2 agonists or upstream modulators of PHB2 in the PERK-Parkin pathway may be a new therapeutic strategy to slow or prevent the progression of dopaminergic neuron degeneration in PD.

Figure 6 ｜Silencing PHB2 inhibits mitophagy and aggravates dopaminergic neuronal loss in PD mice.

Figure 7 ｜Silencing PHB2 reduces antioxidative stress protein expression and aggravates motor defecits in PD mice.

Author contributions:Conception and design:JY,JW;western blot and confocal microscopy:SY;animal behavior experiments:MS,RS,XL;other experimental implementation and data analysis:YZ;manuscript draft:JY,YZ.All authors reviewed and edited the manuscript and approved the final version of the manuscript.

Conflicts of interest:The authors declare no competing financial interests.

Data availability statement:The original contributions presented in the study are included in the article material,further inquiries can be directed to the corresponding authors.

Open access statement:This is an open access journal,andarticles are distributed under the terms of the Creative Commons AttributionNonCommercial-ShareAlike 4.0 License,which allows others to remix,tweak,and build upon the work non-commercially,as long as appropriate credit is given and the new creations are licensed under the identical terms.

Open peer reviewers:Antonella Cardinale,Università Cattolica del Sacro Cuore,Italy.

Additional file:Open peer review report 1.