Mitochondrial Hydrogen Peroxide Activates PTEN and Inactivates Akt Leading To Autophagy Inhibition Dependent Cell Death in Neuronal Models of Parkinson's Disease

Molecular Neurobiology (2023) 60:3345–3364
https://doi.org/10.1007/s12035-023-03286-y
Mitochondrial Hydrogen Peroxide Activates PTEN and Inactivates Akt

Leading to Autophagy Inhibition‑Dependent Cell Death in Neuronal
Models of Parkinson’s Disease
Qianyun Yu1,2 · Ruijie Zhang1,3 · Tianjing Li1 · Liu Yang1 · Zhihan Zhou1 · Long Hou1 · Wen Wu1 · Rui Zhao1 ·
Xiaoling Chen1 · Yajie Yao1 · Shile Huang4,5,6 · Long Chen1
Received: 12 October 2022 / Accepted: 3 February 2023 / Published online: 28 February 2023
© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2023
Abstract
Defective autophagy relates to the pathogenesis of Parkinson’s disease (PD), a typical neurodegenerative disease. Our recent
study has demonstrated that PD toxins (6-OHDA, MPP+, or rotenone) induce neuronal apoptosis by impeding the AMPK/
Akt-mTOR signaling. Here, we show that treatment with 6-OHDA, MPP+, or rotenone triggered decreases of ATG5/LC3-II
and autophagosome formation with a concomitant increase of p62 in PC12, SH-SY5Y cells, and primary neurons, suggesting
inhibition of autophagy. Interestingly, overexpression of wild-type ATG5 attenuated the inhibitory effect of PD toxins on
autophagy, reducing neuronal apoptosis. The effects of PD toxins on autophagy and apoptosis were found to be associated
with activation of PTEN and inactivation of Akt. Overexpression of dominant negative PTEN, constitutively active Akt and/
or pretreatment with rapamycin rescued the cells from PD toxins-induced downregulation of ATG5/LC3-II and upregula-
tion of p62, as well as consequential autophagosome diminishment and apoptosis in the cells. The effects of PD toxins on
autophagy and apoptosis linked to excessive intracellular and mitochondrial hydrogen peroxide (H2O2) production, as evi-
denced by using a H2O2-scavenging enzyme catalase, a mitochondrial superoxide indicator MitoSOX and a mitochondria-
selective superoxide scavenger Mito-TEMPO. Furthermore, we observed that treatment with PD toxins reduced the protein
level of Parkin in the cells. Knockdown of Parkin alleviated the effects of PD toxins on H2O2 production, PTEN/Akt activity,
autophagy, and apoptosis in the cells, whereas overexpression of wild-type Parkin exacerbated these effects of PD toxins,
implying the involvement of Parkin in the PD toxins-induced oxidative stress. Taken together, the results indicate that PD
toxins can elicit mitochondrial H2O2, which can activate PTEN and inactivate Akt leading to autophagy inhibition-dependent
neuronal apoptosis, and Parkin plays a critical role in this process. Our findings suggest that co-manipulation of the PTEN/
Akt/autophagy signaling by antioxidants may be exploited for the prevention of neuronal loss in PD.
Keywords H2O2 · PTEN · Akt · Autophagy · Parkin · Neuronal cells
Abbreviations
6-OHDA 6-Hydroxydopamine
AD Alzheimer disease
Qianyun Yu and Ruijie Zhang contributed equally to this work
3
* Shile Huang College of Life Sciences, Anhui Medical University,
shile.huang@lsuhs.edu Anhui 230032, People’s Republic of China
4
* Long Chen Department of Biochemistry and Molecular Biology,
lchen@njnu.edu.cn Louisiana State University Health Sciences Center, 1501
Kings Highway, Shreveport, LA 71130‑3932, USA
1
Jiangsu Key Laboratory for Molecular and Medical 5
Department of Hematology and Oncology, Louisiana
Biotechnology, College of Life Sciences, Nanjing
State University Health Sciences Center, Shreveport,
Normal University, 1 Wenyuan Road, Chixia District,
LA 71130‑3932, USA
Nanjing 210023, People’s Republic of China
6
2 Feist‑Weiller Cancer Center, Louisiana State University
Department of Biological Sciences, College of Science
Health Sciences Center, Shreveport, LA 71130‑3932, USA
and Technology, Xinyang University, Xinyang 464000,
People’s Republic of China
13
Vol.:(0123456789)
3346 Molecular Neurobiology (2023) 60:3345–3364
AMPK AMP-activated protein kinase [10, 12, 13]. Numerous reports have indicated that autophagy
Atg Autophagy-related plays a crucial role in maintaining cellular homeostasis and
CAT Catalase protects cells from varying insults, which is particularly piv-
DAPI 4′,6-Diamidino-2-phenylindole otal in neuronal survival [6, 14, 15]. However, autophagy is
DMEM Dulbecco’s modified Eagle’s medium in fact a double-edged sword in the pathogenesis of many
FBS Fetal bovine serum human diseases [14]. In many circumstances, autophagy
HA Hemagglutinin can be adaptive to stimuli and promote cell survival, yet
HD Huntington’s disease under certain conditions, it can also lead to cell death [14,
H2DCFDA 2′7′-Dichlorodihydrofluorescein diacetate 16]. Phosphatase and tensin homologue on chromosome
H2O2 Hydrogen peroxide 10 (PTEN), a lipid/protein dual phosphatase, antagonizes
IGF-1R Insulin-like growth factor-1 receptor the phosphatidylinositol 3-kinase (PI3K) and is a nega-
IOD Integral optical density tive regulator of Akt, controlling cell proliferation/growth,
LC3 Microtuble-associated protein 1 light chain 3 survival, and apoptosis in cells [17–20]. Though inhibition
MPP+ 1-Methyl-4-phenylpyridin-1-ium of the Akt pathway due to activation of PTEN promotes
mTOR Mammalian target of rapamycin autophagy in many cases [21, 22], inactivation of Akt can
PBS Phosphate-buffered saline also block autophagic process through impairing autophagic
PD Parkinson’s disease flux, thereby leading to cell death in skeletal myoblast cells
PDL Poly-D-lysine [23]. Of note, multiple studies have documented that exces-
PI3K Phosphatidylinositol 3-kinase sive reactive oxygen species (ROS)-induced neuronal apop-
PKB/Akt Protein kinase B tosis links to dysfunction of PTEN, Akt, and/or autophagy
PTEN Phosphatase and tensin homologue on chro- signaling [24–27]. For example, PTEN is involved in ROS
mosome 10 production and neuronal death in in vitro models of stroke
PINK1 PTEN-induced kinase 1 and PD [26]. ROS can activate Akt by inactivating PTEN,
PTP1B Protein tyrosine phosphatase 1B but ROS can also inhibit Akt at high concentrations [27].
ROS Reactive oxygen species Defective autophagy leads to oxidative stress and lysosomal
TUNEL The terminal deoxynucleotidyl transferase rupture, triggering different types of cell death [24]. Our
(TdT)-mediated deoxyuridine triphosphate recent studies have demonstrated that PD toxins (6-OHDA,
(dUTP) nick-end labeling MPP+ or rotenone) induce neuronal apoptosis by induc-
tion of hydrogen peroxide (H2O2), impeding the AMPK/
Akt-mTOR signaling [25]. Based on the above findings,
Introduction we hypothesized that PD stress may inhibit autophagy via
H2O2-mediated PTEN-Akt signaling pathway, thereby lead-
Parkinson’s disease (PD) is a neurodegenerative movement ing to neuronal apoptosis.
disorder characterized by the loss of dopaminergic neurons in Parkin, an E3 ubiquitin ligase mainly present in the
the substantia nigra pars compacta (SNpc) [1, 2]. To understand cytoplasm, is not only a stress-protective protein, but also
the molecular mechanism of neuronal cell death in PD for a stress-inducible protein [28]. Parkin can provide speci-
development of effective neuroprotective therapies, numerous ficity for degradation of accumulated proteins through the
studies have been carried out, using postmortem brains, classic ubiquitin proteasome system [29–32]. Parkin dys-
experimental cell/animal models of PD [3–5]. Increasing function will lose its E3 ubiquitin ligase activity, which
evidence has pointed to the association of neuronal loss with leads to abnormal accumulation of various proteins, and
apoptotic cell death triggered by oxidative stress, impairment of thus triggers neuronal cell death [33]. Merged data have
mitochondrial respiration, and abnormal protein aggregation in pointed out that loss-of-function of Parkin results in mito-
PD [6–9]. Especially, recent findings have shown that defective chondrial turnover/dysfunction and dopaminergic neuronal
autophagy is a hallmark of neurodegenerative diseases in which loss involved in oxidative stress, which is a core pathogenic
misfolded proteins or dysfunctional mitochondria accumulate process in PD [32, 34, 35]. Especially, Parkin mutations can
in neurons [10]. Defective autophagy relates to the pathogenesis cause defective autophagy on dysfunctional mitochondria,
of PD [2, 7, 10, 11]. However, how the defective autophagy known as mitophagy in PD [32, 34]. This prompted us to test
initiates and contributes to neuronal cell loss in the context of whether downregulation or upregulation of Parkin impedes
PD remains to be determined. H2O2-PTEN/Akt/autophagy signaling in PD stress.
Autophagy, as a highly conserved homeostasis mecha- Here we demonstrate that PD toxins-induced mitochondrial
nism from yeast to human, delivers cytoplasmic nonfunc- H 2O 2 activates PTEN and inactivates Akt leading to
tional or unwanted organelles and aggregate-prone and oxi- autophagy inhibition-dependent neuronal apoptosis, and
dized proteins to lysosomes for degradation and recycling Parkin plays a critical role in this process. The results provide
13
Molecular Neurobiology (2023) 60:3345–3364 3347
new insights into the mechanism behind the neuronal cell pre-coated with (for PC12) or without (for SH-SY5Y)
death in PD. Our findings suggest that co-manipulation of PDL (0.2 μg/ml). PC12 cells were cultured in antibiotic-
the PTEN/Akt/autophagy signaling by antioxidants may be free DMEM supplemented with 10% horse serum and 5%
exploited for the prevention of neuronal loss in PD. FBS, whereas SH-SY5Y cells were grown in antibiotic-free
DMEM supplemented with 10% FBS, in a humidified incu-
bator (37 °C, 5% C O2). Primary murine neurons were iso-
Materials and Methods lated from fetal mouse cerebral cortexes of 16–18 days of
gestation in female ICR mice as described [36], and seeded
Reagents in a PDL (10 μg/ml)-coated 6-well plate (5 × 105 cells/well)
or 96-well plate (1 × 104 cells/well) for experiments after
Rotenone (Cat. No. R8875), 6-hydroxydopamine (6-OHDA) 6 days of culture. The experiments involving animals in this
(Cat. No. 162957), 1-methyl-4-phenylpyridin-1-ium ( MPP+) study were approved by the Institutional Animal Care and
(Cat. No. M0896), 2′7′-dichlorodihydrofluorescein diacetate Use Committee of Nanjing Normal University (Certificate
(H2DCFDA) (Cat. No. D6883), catalase (CAT) (Cat. No. No. 200408), and were conducted in compliance with the
C9322), poly-D-lysine (PDL) (Cat. No. P6407), 4′,6-diami- guidelines set forth by the Guide for the Care and Use of
dino-2-phenylindole (DAPI) (Cat. No. D8417), and pro- Laboratory Animals.
tease inhibitor cocktail (Cat. No. P8340) were purchased
from Sigma (St Louis, MO, USA). Mito-TEMPO (Cat. No. Recombinant Adenoviral Constructs and Infection
ALX-430–171-M005) and rapamycin were from ALEXIS of Cells
Biochemicals Corporation (San Diego, CA, USA). Dulbec-
co’s modified Eagle’s medium (DMEM) (Cat. No. 30030), Recombinant adenovirus expressing human dominant
0.05% Trypsin–EDTA (Cat. No. 25300062), NEUROBA- negative PTEN (Ad-PTEN-C/S), and the control adenovi-
SAL™ Media (Cat. No. 25300062), and B27 Supplement rus expressing β-galactosidase (Ad-LacZ) were described
(Cat. No. 17504044) were from Invitrogen (Grand Island, previously [37, 38]. Recombinant adenovirus encoding
NY, USA). Fetal bovine serum (FBS) (Cat. No. SH30022) HA-tagged myristoylated, constitutively active Akt (Ad-
and horse serum were from Hyclone (Logan, UT, USA). myr-Akt) was generously provided by Dr. Kenneth Walsh
MitoSox (Cat. No. 40778ES) was bought from YEASEN (Boston University School of Medicine, Boston, MA, USA)
(Shanghai, China). CellTiter 96®AQueous One Solution (Cat. [39]. For experiments, PC12 cells were cultured in the
No. MR1007) Cell Proliferation Assay kit was from Pro- growth medium, and infected with the individual adenovirus
mega (Madison, WI, USA). Enhanced chemiluminescence for 24 h at 5 of multiplicity of infection (MOI = 5). Subse-
solution (Cat. No. MR1004) was from Sciben Biotech Com- quently, the cells were used for experiments and the cells
pany (Nanjing, China). The following antibodies were used: infected with Ad-LacZ alone served as a control. Expression
p-Akt (Thr308) (Cat. No. 9275), p-Akt (Ser473) (Cat. No. of HA-tagged myr-Akt was determined by Western blotting
9271), cleaved caspase-3 (Cat. No. 9664) (Cell Signaling with anti-HA antibody.
Technology, Beverly, MA, USA), p-PTEN (Thr366) (Cat.
No. 2195–1), PTEN (Cat. No. 5171–1) (Epitomics, Burl- Lentiviral Cloning, Production, and Infection
ingame, CA, USA), LC3B (Cat. No. L7543), ATG5 (Cat.
No. SAB5700062), SQSTM1/p62 (Cat. No. P0067) (Sigma), To generate lentiviral shRNA to Parkin, oligonucleo-
Akt (Cat. No. AT10694), Parkin (Cat. No. AT20255), tides containing the target sequences were synthesized,
β-tubulin (Cat. No. AT1001), HA (Cat. No. AT16321) (Sci- annealed, and inserted into FSIPPW lentiviral vector via
ben Biotech Company), goat anti-rabbit IgG-horseradish the EcoR1/BamH1 restriction enzymes [40]. Lentiviral
peroxidase (HRP) (Cat. No. 31402), and goat anti-mouse shRNA to GFP (for control) was produced as described
IgG-HRP (Cat. No. 31431) (Pierce, Rockford, IL, USA). [41]. To make FLAG-tagged wild-type Parkin (FLAG-
Other chemicals were from local commercial sources and Parkin) construct (for Parkin overexpression), wild-type
were of analytical grade, unless stated elsewhere. ATG5 (FLAG-ATG5) construct (for ATG5 overexpres-
sion) and EGFP construct (for control), PCR template for
Cell Culture Parkin, ATG5 or EGFP was from PC12 cells’ cDNA gen-
erated by RT-PCR using PrimeScript II 1st Strand cDNA
Rat pheochromocytoma (PC12) (Cat. No. CRL-1721) and Synthesis Kit (Takara Bio, Kusatsu, Japan) and plasmid
human neuroblastoma SH-SY5Y (Cat. No. CRL-2266) cell PX458 (Addgene, Cambridge, MA, USA), respectively.
lines were from American Type Culture Collection (ATCC) The PCR products of FLAG-Parkin, FLAG-ATG5, and
(Manassas, VA, USA), which were seeded in a 6-well plate EGFP were cloned into pSin4-EF2-IRES-Pur vector via
(5 × 105 cells/well) or 96-well plate (1 × 104 cells/well) EcoRI/BamHI double-digestion. The above primers used
13
Table 1 The sequences of oligonucleotides for Parkin, EGFP, FLAG-ATG5, and FLAG-Parkin
Name Sense Anti-sense
Parkin 5′-AATTCCCATCACCTGACAGTACAGAACTTGCAAGAG 5′-GATCCAAAAAATCACCTGACAGTACAGAACTTCTCT

AAGTTCTGTACTGTCAGGTGATTTTTTG-3′ TGCAAGTTCTGTACTGTCAGGTGATGGG-3′
EGFP 5′-CCGGAATTCATGGTGAGCAAGGGCGAGGAGCT-3′ 5′-CGCGGATCCGTTACTTGTACAGCTCGTCCATG-3′
FLAG-ATG5 5′-CGGAATTCATGGATTACAAGGATGACGACGATAAGA 5′-CGGGATCCGTTAGGAGATCTCCAAGGGTATG-3′
TGACAGATGACAAAGATGTGC-3′
FLAG-Parkin 5′-CCGGAATTCATGGATTACAAGGATGACGACGATAAG 5′-GGACTAGTCCCTACACGTCAAACCAGTGATCACCC-3′
ATGATAGTGTTTGTCAGGTT-3′
are listed in Table 1. To generate lentivirus, the above Cell Viability Assay
constructed plasmids were co-transfected together with
pMD2.G and psPAX2 (Addgene, Cambridge, MA, USA) The above indicated cells, respectively, were seeded and
to 293TD cells using MegaTran 1.0 reagent (OriGene cultured in a PDL-coated 96-well plate (1 × 10 4 cells/
Technologies, Rockville, MD, USA). Each supernatant well). Next day, cells were treated with/without 6-OHDA
containing viral particles was collected 48 h and 60 h (120 μM), MPP+ (1 mM) or rotenone (1 μM) for 24 h, or
post-transfection and filtered through a 0.45-μm filter, treated with/without 6-OHDA (120 μM), MPP+ (1 mM) or
and stored at − 80 °C. For use, a monolayer of PC12 cells, rotenone (1 μM) for 24 h following pre-incubation with/
when grown to about 70% confluency, were infected with without rapamycin (100 ng/ml) for 2 h, with 5 replicates
the corresponding lentivirus-containing medium in the of each treatment. Subsequently, MTS reagent (one solu-
presence of 8 μg/ml polybrene for 12 h, and reinfected tion reagent) (20 μl/well) was added and incubated for an
after 6 h. The transduced cells were selected by 48-h treat- additional 3 h. Finally, the values of optical density (OD)
ment with 2 μg/ml puromycin. After 5 days of culture, the at 490 nm were determined using a Victor X3 Light Plate
cells were used for experiments. Reader (PerkinElmer, Waltham, MA, USA).
DAPI and TUNEL Staining

GFP‑LC3 Assay
The above indicated cells, respectively, were seeded and cul-
PC12, SH-SY5Y cells and primary neurons, PC12 cells tured in a 6-well plate (5 × 105 cells/well) containing a PDL-
infected with lentiviral FLAG-Parkin, FLAG-ATG5 or coated or PDL-uncoated glass coverslip per well. Next day,
EGFP, or with lentiviral shRNA to Parkin or GFP, or PC12 cells were treated with/without 6-OHDA (120 μM), MPP+
cells infected with Ad-PTEN-C/S, Ad-myr-Akt, and/or (1 mM) or rotenone (1 μM) for 24 h, or treated with/with-
Ad-LacZ, respectively, were infected with Ad-GFP-LC3 out 6-OHDA (120 μM), MPP+ (1 mM) or rotenone (1 μM)
and seeded in a 6-well plate (5 × 105 cells/well) contain- for 24 h following pre-incubation with/without rapamycin
ing a PDL-coated or PDL-uncoated glass coverslip per (100 ng/ml) for 2 h, or CAT (350 U/ml) or Mito-TEMPO
well. Next day, cells were treated with/without 6-OHDA (10 μM) for 1 h, with 5 replicates of each treatment.
(30–240 or 120 μM), MPP+ (0.5 and/or 1 mM), or rote- Then, the apoptotic cells with fragmented and condensed
none (0.5 and/or 1 μM) for 24 h, or treated with/without nuclei were monitored using DAPI staining as described
6-OHDA (120 μM), M PP+ (1 mM) or rotenone (1 μM) [42]. For TUNEL staining, TUNEL reaction mixture (TdT
for 24 h following pre-incubation with/without rapamycin enzyme solution and labeling solution) was added accord-
(100 ng/ml) for 2 h, or a H2O2-scavenging enzyme CAT ing to the manufacturer’s protocol of In Situ Cell Death
(350 U/ml) or a mitochondria-targeted antioxidant Mito- Detection Kit® (Cat. No. A111-03) (Roche, Mannheim,
TEMPO (10 μM) for 1 h, with 5 replicates of each treat- Germany). Finally, slides were mounted in glycerol/phos-
ment. Afterwards, the cells on the coverslips were fixed phate-buffered saline (PBS) (1:1, v/v) containing 2.5%
with 4% paraformaldehyde in PBS for 30 min at 4 °C and 1,4-diazabiclo-(2,2,2) octane. Photographs were taken under
then washed 3 times with PBS, followed by photographing a fluorescence microscope (200 ×) (Leica DMi8, Wetzlar,
under a fluorescence microscope (Leica DMi8, Wetzlar, Germany) equipped with a digital camera. For quantitative
Germany) equipped with a digital camera and counting the analysis of the fluorescence intensity using TUNEL stain-
numbers of GFP-LC3 puncta (green) per cell to estimate ing, the integral optical density (IOD) for 200–300 cells
autophagosome formation. At least 50 cells were scored per graph was determined by Image-Pro Plus 6.0 software
in each experiment. (Media Cybernetics Inc., Newburyport, MA, USA).
13
Immunofluorescence and Imaging Statistical Analysis
PC12, SH-SY5Y cells, and primary neurons, respectively, All results were expressed as mean values ± standard error
were seeded in a 6-well plate (5 × 105 cells/well) contain- (mean ± SE). Student’s t-test for non-paired replicates was
ing a PDL-coated or PDL-uncoated glass coverslip per used to identify differences between treatment means. Group
well. Next day, cells were treated with/without 6-OHDA variability and interaction were compared using either one-
(30–240 μM), MPP+ (0.5 and 1 mM), or rotenone (0.5 and way or two-way ANOVA followed by Bonferroni’s post-tests
1 μM) for 24 h, with 5 replicates of each treatment. Then, to compare replicate means. The criterion for the statistical
the cells on the coverslips were fixed with 4% paraformal- significance was p < 0.05.
dehyde and blocked with 3% normal goat serum with 0.3%
Triton X-100 for 1 h, incubated with anti-phospho-PTEN Results
(Ser366) antibody (1:50, diluted in PBS containing 1%
BSA) overnight at 4 °C. After incubation, coverslips were PD Toxins Induce Decreases of ATG5/LC3‑II,
washed 3 × 5 min with PBS and then incubated with FITC- Autophagosome Formation, and Parkin
conjugated goat anti-rabbit IgG (Santa Cruz Biotechnol- with a Concomitant Increase of p62 in Neuronal
ogy, Dallas, TX, USA, 1:500, diluted in PBS containing Cells
1% BSA) for 1 h at room temperature. After three rinses,
slides were fixed and imaged as described [43]. The IOD ATG5 and LC3 are two essential components for the canoni-
for fluorescence intensity was quantitatively analyzed as cal autophagy [45, 46]. The conversion of LC3-I to LC3-II of
described above. LC3 is a protein marker for autophagosome formation [45].
Additionally, p62 protein (a substrate that is degraded by
autophagy), also called sequestosome 1 (SQSTM1), is com-
Intracellular H2O2 and Mitochondrial ROS Imaging monly used as a marker for execution of autophagy [47]. To
test autophagic manifestation in PD toxins-exposed neuronal
According to the information provided by the supplier, cells, PC12, SH-SY5Y cells, and primary neurons were treated
H2DCFDA and MitoSOX are able to trace intracellular with/without 6-OHDA (30–240 μM), MPP+ (0.5 and 1 mM)
H2O2 and mitochondrial superoxide levels, respectively. or rotenone (0.5 and 1 μM) for 24 h, followed by analyzing the
H2DCFDA is a stable non-fluorescent probe with peroxide- cellular protein levels of ATG5, LC3-II, and p62 using West-
selective dye that can passively diffuse into the intracellu- ern blotting. The results showed that treatment with 6-OHDA,
lar matrix of cells, where it is sheared by esterase and oxi- MPP+, or rotenone triggered decreases of ATG5 and LC3-II
dized by H2O2, forming fluorescent DCF [44]. MitoSOX is in a concentration-dependent manner in the cells (Fig. 1A–D).
a superoxide indicator dye that can specifically recognize Interestingly, the toxins elicited robust expression of p62
mitochondrial superoxide and produce red fluorescence in protein dose-dependently in the cells as well (Fig. 1A–D).
live cells. In brief, PC12, SH-SY5Ycells and primary neu- Sequentially, we monitored neuronal autophagic vacuoles
rons, or PC12 cells infected with lentiviral shRNA to Par- with GFP-LC3 localization. When PC12, SH-SY5Y cells, and
kin or GFP or with lentiviral FLAG-Parkin or EGFP, after primary neurons, infected with Ad-GFP-LC3, were exposed
treatment, were loaded with H 2DCFDA (20 μM) for 1 h or to the toxins for 24 h, autophagic vacuoles with GFP-LC3
with MitoSOX (5 μM) for 10 min at 37 ℃. Subsequently, (in green) significantly decreased in a concentration-depend-
all stained specimens were rinsed 3 times with PBS, fol- ent fashion (Fig. 1E–H), implying that PD toxins impaired
lowed by imaging under a fluorescence microscope, and autophagosome formation. Of note, treatment with 6-OHDA,
quantitatively measuring IOD of the fluorescence intensity MPP+, or rotenone also reduced the protein level of Parkin
as described above. dose-dependently in the cells (Fig. 1A and B). Collectively,
these results indicate that treatment with PD toxins induces
decreases of Atg5/LC3-II, autophagosome formation, and
Western Blot Analysis Parkin with a concomitant increase of p62 in neuronal cells,
suggesting inhibition of autophagy.
The indicated cells, after treatments, were briefly washed
with cold PBS, and then on ice, lysed in the radioimmu- Overexpression of ATG5 Attenuates PD
noprecipitation assay buffer. Afterwards, Western blotting Toxins‑Induced Autophagy Inhibition and Apoptosis
was performed as described previously [36], and the blots in Neuronal Cells
for detected proteins were semi-quantified using NIH
ImageJ software (National Institutes of Health, Bethesda, Deletion of ATG5 can completely inhibit autophagy [46].
MD, USA). We have observed that ATG5 protein level decreased in
13
PD toxins-induced neuronal cells (Fig. 1). To validate the lentiviral EGFP (control), resulted in a robust expression of
importance of ATG5 in PD toxins-induced autophagy inhi- FLAG-tagged ATG5, regardless of absence or presence of
bition and neuronal apoptosis, ATG5 in PC12 cells was 6-OHDA, MPP+, or rotenone. Interestingly, overexpression
overexpressed. As shown in Fig. 2A, infection with lentivi- of ATG5 conferred high resistance to decreases of LC3-II
ral FLAG-tagged wild-type ATG5 (FLAG-ATG5), but not and Parkin, as well as increases of p62 and cleaved caspase-3
13
◂Fig. 1 PD toxins-induced decreases of ATG5/LC3-II, autophagosome showed that decreased p-PTEN (Thr366) in the cells was
formation, and Parkin with a concomitant increase of p62 in neuronal seen starting in 6–12 h of postexposure to 6-OHDA, MPP+,
cells. PC12, SH-SY5Y cells, and primary neurons infected with/with-
out Ad-GFP-LC3, respectively, were treated with/without 6-OHDA
or rotenone and became more profound in 24 h, whereas
(30–240 μM), MPP+ (0.5 and 1 mM) or rotenone (0.5 and 1 μM) for decreased p-Akt (Ser473 and Thr308)/ATG5/LC3-II and
24 h. A, C Total cell lysates were subjected to Western blotting with increased p62 were observed in 12–24 h of postexposure of
indicated antibodies. The blots were probed for β-tubulin as a load- the cells to the PD toxins (Fig. 3I and J), clearly indicating
ing control. Similar results were obtained in at least five independent
experiments. B, D The relative densities for ATG5, LC3-II, p62, Par-
that p-PTEN changes earlier than p-Akt and the autophagy
kin to β-tubulin were semi-quantified using NIH ImageJ. E, G Repre- flux do, and PTEN is the upstream factor. Taken together, the
sentative GFP-LC3 puncta imaging (in green) in the cells was shown findings imply that PD toxins activate PTEN and inactivate
by using GFP-LC3 assay. Scale bar: 2 μm. F, H The number of GFP- Akt in neuronal cells.
LC3 puncta per cell was quantified. At least 50 cells were scored in
each experiment. All data were expressed as mean ± SE (n = 3 for B,
Next, PC12 cells, infected with Ad-PTEN-C/S, Ad-
D; n = 5 for F, H). Using one-way ANOVA, *p < 0.05, **p < 0.01, dif- PTEN-C/S/Ad-myr-Akt, Ad-LacZ (as control), and/or
ference vs control group Ad-GFP-LC3, were exposed to 6-OHDA (120 μM), M PP+
(1 mM), or rotenone (1 μM) for 24 h. We showed that infec-
tion with Ad-PTEN-C/S and Ad-myr-Akt, but not Ad-LacZ,
in PC12 cells treated with 6-OHDA, M PP+, or rotenone increased the total protein levels of PTEN and Akt, respec-
(Fig. 2 A and B). Consistent with this, overexpression of tively (Fig. 4A). Overexpression of PTEN-C/S in PC12 cells
ATG5 potently blocked the toxins-induced autophagosome increased the basal level of p-Akt and rendered remarkable
decline, cell viability reduction and apoptosis in the cells, resistance to 6-OHDA, MPP+ or rotenone-induced dephos-
as evidenced by GFP-LC3 assay (Fig. 2C), MTS assay phorylation of Akt, decreases of ATG3/LC3-II/autophago-
(Fig. 2D), DAPI staining (Fig. 2E and F), and TUNEL stain- somes, increases of p62 and cleaved caspase-3 (Fig. 4A–C,
ing (Fig. 2G and H). The results demonstrate that PD tox- Fig. S1A), as well as cell viability reduction and apoptosis
ins-downregulated ATG5 contributes to PD toxins-induced (Fig. 4D and E). Of importance, the toxins-triggered events
autophagy inhibition-dependent apoptosis in neuronal cells. were ameliorated in the cells co-infected with Ad-PTEN-
C/S/Ad-myr-Akt more potently than those infected with
PD Toxins Activates PTEN and Inactivates Akt, Ad-PTEN-C/S (Fig. 4A–E, Fig. S1A) or Ad-myr-Akt (data
Resulting in Autophagy Inhibition and Apoptosis not shown) alone. The results suggest that the PTEN/Akt
in Neuronal Cells pathway mediates PD toxin-induced neuronal autophagy
impairment and death. However, overexpression of PTEN-
It is well known that PTEN antagonizes PI3K by catalyzing C/S and/or myr-Akt failed to rescue PD toxins-induced
phosphatidylinositol (3,4,5)-trisphosphate (PIP3) to phos- decrease of Parkin in PC12 cells (Fig. 4A and B), implying
phatidylinositol 4,5-bisphosphate (PIP2), thereby inhibiting that the PTEN/Akt signaling does not mediate the expression
Akt, regulating cell proliferation/growth, survival, and apop- of Parkin in PD toxins-induced neuronal cells.
tosis [17–20]. Akt inactivation can block autophagic process To define the role of Akt in PD toxins-induced
via impairing autophagic flux, causing cell death [23]. Our autophagy inhibition and apoptosis in neuronal cells,
previous studies have found that PD toxins inactivate Akt PC12 cells, infected with Ad-myr-Akt, Ad-LacZ, and/
signaling, leading to neuronal apoptosis [48]. Therefore, we or Ad-GFP-LC3, were exposed to 6-OHDA (120 μM),
reasoned that PTEN might be involved in PD toxins-induced M PP + (1 mM) or rotenone (1 μM) for 24 h following
Akt inactivation, autophagy inhibition, and apoptosis in neu- pre-incubation with/without rapamycin (100 ng/ml), a
ronal cells. To this end, we checked the phosphorylation sta- known autophagy inducer [49], for 2 h. As shown in
tus of PTEN and Akt in PC12, SH-SY5Y cells, and primary Fig. 4E, expression of high HA-tagged Akt mutant was
neurons exposed to 6-OHDA (30–240 μM), MPP+ (0.5 and seen in PC12 cells infected with Ad-myr-Akt, but not in
1 mM), or rotenone (0.5 and 1 μM) for 24 h. Western blot the cells infected with Ad-LacZ (control virus). Overex-
analysis showed that treatment with these toxins repressed pression of myr-Akt significantly prevented 6-OHDA-,
the phosphorylation of PTEN (Thr366) and Akt (Thr308 and M PP+-, or rotenone-induced dephosphorylation of Akt,
Ser473) in the cells dose-dependently (Fig. 3A–D). Simi- decreases of ATG3/LC3-II/autophagosomes, increases
larly, our immunofluorescence staining also showed a dose- of p62 and cleavage of caspase-3 (Fig. 4F–H, Fig. S1B),
dependent decrease in p-PTEN (Thr366) (in green) in the as well as cell viability reduction and apoptosis (Fig. 4I
cells treated with 6-OHDA, M PP+, or rotenone (Fig. 3E–H). and J), but not decrease of Parkin (Fig. 4F and G).
Additionally, an experiment of different time points (0, 6, 12, Intriguingly, pretreatment of the cells with rapamycin
and 24 h) after the PD toxin treatment was conducted to see possessed more powerful inhibitory effects on the PD
time-dependent manifestations of PTEN, Akt, ATG5, LC3- toxins-induced events than ectopic expression of myr-Akt
II, and p62 in PC12 cells and primary neurons. The results alone (Fig. 4F–J, Fig. S1B). Of interest, pretreatment
13
with rapamycin rescued the cells from decrease of Par- together, our findings underscore the concept that PD
kin in response to 6-OHDA, MPP+, or rotenone (Fig. 4F toxins induce autophagy inhibition and consequential
and G), implying that Parkin protein level is regulated apoptosis in neuronal cells, in part, by activation of
through an autophagy-dependent mechanism. Taken PTEN and inactivation of Akt.
13
◂Fig. 2 Overexpression of ATG5 prevents PD toxins from induc- attenuated the PD toxins-induced generation of H2O2 in the
ing autophagy inhibition and apoptosis in neuronal cells. PC12 cells (Fig. 6A and Fig. S3A). MitoSOX, a mitochondrial
cells, infected with lentiviral FLAG-ATG5 or EGFP (as control) and
infected with/without Ad-GFP-LC3, respectively, were treated with/
superoxide indicator, was used to define mitochondrial ROS,
without 6-OHDA (120 μM), M PP+ (1 mM), or rotenone (1 μM) for exhibiting that the increases of MitoSOX red fluorescence
24 h. A Total cell lysates were subjected to Western blotting with were obviously diminished by Mito-TEMPO in the cells
indicated antibodies. The blots were probed for β-tubulin as a load- (Fig. 6B and C), clearly indicating the PD toxins’ induc-
ing control. Similar results were obtained in at least five independ-
ent experiments. B The relative densities for ATG5, LC3-II, p62,
tion of mitochondrial ROS. Consistently, Mito-TEMPO
Parkin, cleaved caspase-3 to β-tubulin were semi-quantified using substantially reversed the PD toxins-triggered decreases of
NIH ImageJ. C The number of GFP-LC3 puncta per cell was quanti- p-PTEN, p-Akt, ATG5/LC3-II/autophagosomes and Parkin
fied by GFP-LC3 assay. At least 50 cells were scored in each experi- and increase of p62, as well as cleavage of caspase-3 and
ment. D The relative cell viability was determined by the MTS assay.
E, G Apoptotic cells were evaluated by using DAPI staining for
apoptosis in the cells (Fig. 6D–G, Fig. S3B). The results
nuclear fragmentation and condensation (arrows) and TUNEL stain- indicate that the PD toxins indeed evoke mitochondrial
ing for fragmented DNA (in green), respectively. Scale bar: 20 µm. H2O2, which inhibits autophagy contributing to apoptosis
F, H The percentage of cells with fragmented nuclei and IOD val- by impairing the PTEN/Akt signaling in neuronal cells.
ues of TUNEL-positive cells were quantified. All data were expressed
as mean ± SE (n = 3 for B; n = 5 for C, D, F, H). Using one-way
ANOVA or Student’s t-test, ap < 0.05, difference vs control group; Parkin Exerts a Critical Role for PD Toxins‑Induced
b
p < 0.05, FLAG-ATG5 group vs EGFP control group H2O2 Production, PTEN Activation/Akt Inactivation,
Autophagy Inhibition, and Apoptosis in Neuronal
Cells
PD Toxins‑Induced Intracellular and Mitochondrial
H2O2 Activates PTEN and Inactivates Akt Parkin is not only a stress-protective protein but also a stress-
Contributing to Autophagy Inhibition and Apoptosis inducible protein [28], which is an essential protein for deg-
in Neuronal Cells radation of accumulated proteins through the classic ubiqui-
tin proteasome pathway [29–32]. Having observed that PD
Our group has recently found that PD toxins’ induction of toxins-induced autophagy inhibition and apoptosis related
H2O2 impedes the AMPK/Akt-mTOR signaling pathway to declined Parkin (Fig. 1 and Fig. 2), we postulated that
contributing to cell death in neuronal cells [25]. To validate silencing Parkin might potentiate PD toxins-elicited neu-
whether PD toxins-impaired PTEN/Akt signaling leading ronal apoptosis. To test this, PC12 cells, infected with len-
to autophagy inhibition and neuronal apoptosis is due to tiviral shRNA to Parkin or GFP and/or Ad-GFP-LC3, were
induction of intracellular H
2O2, we used catalase (CAT), a exposed to 6-OHDA (120 μM), MPP+ (1 mM), or rotenone
H2O2-scavenging enzyme. The results showed that pretreat- (1 μM) for 24 h. As shown in Fig. 7A, lentiviral shRNA to
ment with CAT markedly mitigated the toxins-induced H 2O2 Parkin, but not GFP, downregulated the protein expression
production in PC12, SH-SY5Y cells and primary neurons, of Parkin by ~ 90% in PC12 cells, as detected by Western
as evidenced by using a peroxide-selective probe H2DCFDA blotting. To our surprise, knockdown of Parkin substantially
for imaging and quantifying (Fig. 5A and B). Of importance, elevated the basal and/or the PD toxins-reduced p-PTEN
CAT rescued the cells from PD toxins-elicited decreases in (Thr366), p-Akt (Thr308 and Ser473), ATG5 and LC3-II
p-PTEN, p-Akt, ATG5, LC3-II, and Parkin and increase (Fig. 7A and B), and suppressed the PD toxins-induced
in p62 (Fig. 5C and D). CAT also profoundly blocked the increase of p62 and cleavage of caspase-3 (Fig. 7A and B).
diminishment of autophagosomes, the activation of cas- Silencing Parkin conferred significant resistance to the PD
pase-3 and the increase of fragmented nuclei in the cells toxins-evoked decrease of autophagosomes and increase of
exposed to the PD toxins (Fig. 5C–F, Fig. S2). The findings H2O2 production (Fig. 7C and D, Fig. 4S), and attenuated
support that the PD toxins induce intracellular H2O2, which the PD toxins-triggered cell viability reduction and apop-
mediates autophagy inhibition leading to apoptosis by acti- tosis (Fig. 7E–G) in the cells as well. The results imply an
vating PTEN and inactivating Akt in neuronal cells. important role of Parkin in PD toxins-induced decrease of
Next, we investigated whether the effects of PD toxins autophagosome formation and increase of apoptosis in neu-
on PTEN/Akt signaling leading to autophagy inhibition ronal cells, and that depletion of Parkin attenuated the effects
and neuronal apoptosis are associated with excessive H2O2 of PD toxins on the PTEN/Akt signaling.
production in the mitochondria of neuronal cells. For this, To substantiate the role of Parkin in PD toxins-
PC12, SH-SY5Y cells, and primary neurons were pretreated induced H2O2 induction, PTEN activation/Akt inactiva-
with/without a mitochondria-targeted antioxidant Mito- tion, autophagy inhibition, and neuronal apoptosis, we
TEMPO (10 μM) for 1 h, and then exposed to 6-OHDA further constructed lentivirus FLAG-tagged wild-type
(120 μM), MPP+ (1 mM), or rotenone (1 μM) for 24 h. Parkin (FLAG-Parkin) to overexpress Parkin. As shown
We found that pretreatment with Mito-TEMPO obviously in Fig. 8A and B, overexpression of Parkin in PC12 cells
13
Fig. 3 PD toxins induce activation of PTEN and subsequent inac- p-Akt (Thr308), ATG5, LC3-II, p62 to β-tubulin were semi-quantified
tivation of Akt/inhibition of autophagy in neuronal cells. PC12, using NIH ImageJ. E, G Expression of p-PTEN (Thr366) was stained
SH-SY5Y cells, and/or primary neurons, respectively, were treated and imaged using immunofluorescence, showing that treatment
with/without 6-OHDA (30–240 μM or 120 μM), MPP+ (0.5 and/or of the cells with PD toxins for 24 h caused lower p-PTEN expres-
1 mM), or rotenone (0.5 and/or 1 μM) for 6, 12, and/or 24 h. A, C, I sion (in green). Scale bar: 20 µm. F, H IOD for fluorescence inten-
Total cell lysates were subjected to Western blotting with indicated sity of p-PTEN expression was determined. All data were expressed
antibodies. The blots were probed for β-tubulin as a loading control. as mean ± SE (n = 3 for B, D, J; n = 5 for F, H). Using one-way
Similar results were obtained in at least five independent experiments. ANOVA, *p < 0.05, **p < 0.01, difference vs control group
B, D, J The relative densities for p-PTEN (Thr366), p-Akt (Ser473),
strengthened the basal and/or 6-OHDA-, M PP + -, or Discussion

rotenone-induced decreases of p-PTEN, p-Akt, ATG5,
and LC3-II and increases of p62 and cleaved caspase-3 PD is a typical neurodegenerative disease characterized by
(Fig. 8A and B). Consistently, overexpression of Parkin the loss of dopaminergic nigrostriatal neurons [1, 2]. Deep
also reinforced the basal and/or the PD toxins-induced brain stimulation in PD onsets represents a paradigmatic
autophagosomes’ loss, excessive H 2O 2 generation, cell cross-talk between mammalian disease models and clinical
viability reduction, and apoptosis in the cells (Fig. 8C–F, evidence in humans [50]. The cellular and rodent models
Fig. 5S). Taken together, the results support the notion for PD toxins (6-OHDA, MPTP/MPP+, and/or rotenone)
that Parkin plays a critical role for PD toxins-induced have contributed to understanding of the PD pathology
H 2 O 2 production, PTEN activation/Akt inactivation, [25, 48, 50, 51]. Data show that PD patients suffer only a
autophagy inhibition, and apoptosis in neuronal cells. 60–70% loss of SNc neuropils [50]. PD early stages are
13
Fig. 4 PD toxins-induced activation of PTEN and inactivation of using NIH ImageJ. C, H The number of GFP-LC3 puncta per cell
Akt contribute to autophagy inhibition and apoptosis in neuronal was quantified by GFP-LC3 assay. At least 50 cells were scored in
cells. PC12 cells, infected with Ad-PTEN-C/S, Ad-myr-Akt, and/ each experiment. D, I The relative cell viability was determined by
or Ad-LacZ (as control) and infected with/without Ad-GFP-LC3, the MTS assay. E, J Apoptotic cells were evaluated by nuclear frag-
respectively, were treated with/without 6-OHDA (120 μM), MPP+ mentation and condensation using DAPI staining. All data were
(1 mM), or rotenone (1 μM) for 24 h, or pretreated with/without rapa- expressed as mean ± SE (n = 3 for B, G; n = 5 for C, D, H–J). Using
mycin (100 ng/ml) for 2 h and then treated with/without 6-OHDA, one-way ANOVA or Student’s t-test, ap < 0.05, difference vs control
MPP+ or rotenone for 24 h. A, F Total cell lysates were subjected to group; bp < 0.05, Ad-PTEN-C/S group or Ad-PTEN-C/S + Ad-myr-
Western blotting with indicated antibodies. The blots were probed Akt group vs Ad-LacZ group; cp < 0.05, Ad-PTEN-C/S + Ad-myr-Akt
for β-tubulin as a loading control. Similar results were obtained in grouo vs Ad-PTEN-C/S group; dp < 0.05, Ad-myr Akt group or Ad-
at least five independent experiments. B, G The relative densities myr-Akt + Rapamycin group vs Ad-LacZ group; ep < 0.05, Ad-myr-
for p-PTEN (Thr366), p-Akt (Ser473), p-Akt (Thr308), ATG5, LC3- Akt + Rapamycin group vs Ad-myr-Akt group
II, p62, Parkin, cleaved caspase-3 to β-tubulin were semi-quantified
probably dominated by the subtle impairment of synaptic and contributes to neuronal cell loss in the context of PD.
transmission and the derangement of α-synuclein oligom- Recently, our group has shown that 6-OHDA, MPP+, or
ers [50, 52]. In agreement with these findings, during the rotenone induces neuronal apoptosis by induction of H2O2,
course of this research, we also noticed increased levels of impeding the AMPK/Akt-mTOR signaling [25]. Here, we
α-synuclein in PC12, SH-SY5Y cells, and primary neurons provide evidence that PD toxins induce excessive intra-
exposed to 6-OHDA, M PP+, or rotenone in a concentra- cellular and mitochondrial H 2O2, which elicits autophagy
tion- and time-dependent manner. Autophagy dysfunction inhibition contributing to neuronal apoptosis via activating
has been implicated as a hallmark of several neurodegen- PTEN and inactivating Akt. We also identified that Parkin
erative diseases [10]. Especially, numerous studies have plays a critical role in the process.
shown defective autophagy in PD [2, 7, 10, 11]. However, Autophagy has been considered as a double-edged
little is known about how the defective autophagy initiates sword for cells [14]. Autophagy plays an important role
13
Fig. 5 PD toxins elicit intracellular H

2O2, resulting in activation obtained in at least five independent experiments. D The relative den-
of PTEN and inactivation of Akt contributing to autophagy inhibi- sities for p-PTEN (Thr366), p-Akt (Ser473), p-Akt (Thr308), ATG5,
tion and apoptosis in neuronal cells. PC12, SH-SY5Y cells and pri- LC3-II, p62, Parkin, cleaved caspase-3 to β-tubulin were semi-quan-
mary neurons were pretreated with/without CAT (350 U/ml) for 1 h tified using NIH ImageJ. E The number of GFP-LC3 puncta per cell
and then exposed to 6-OHDA (120 μM), MPP+ (1 mM), or rotenone was quantified by GFP-LC3 assay. At least 50 cells were scored in
(1 μM) for 24 h. A Cell H 2O2 was imaged using a peroxide-selective each experiment. F Apoptotic cells were evaluated by nuclear frag-
probe H2DCFDA. Scale bar: 20 μm. B IOD for cell H2O2 fluores- mentation and condensation using DAPI staining. All data were
cence intensity was quantitatively analyzed. C Total cell lysates were expressed as mean ± SE (n = 3 for D; n = 5 for B, E, F). Using one-
subjected to Western blotting with indicated antibodies. The blots way ANOVA or Student’s t-test, ap < 0.05, difference vs control
were probed for β-tubulin as a loading control. Similar results were group; bp < 0.05, + CAT group vs—CAT group
in the removal of intracellular misfolded proteins and dam- reported that treatment with MPP+ (1 mM, 24 h) reduced
aged organelles to maintain the survival and homeostasis the levels of ATG5 and LC3-II, but increased the level of
of neuronal cells [6, 14, 15]. Yet, under stress state, it p62 in SH-SY5Y cells [53], and treatment with rotenone
can cause cell death as well [14, 16]. Studies have shown (1 μM, 24 h) decreased the level of LC3-II and increased
that ATG5 and LC3, as two essential components for the level of p62 in PC12 cells [54], suggesting inhibition
the canonical autophagy, are involved in the formation of autophagy, while other studies have shown that the PD
of autophagosomes [45, 46], and especially the conver- toxins may induce impaired autophagic flux in neuronal
sion of LC3-I to LC3-II of LC3 is a protein indicator for cells [55–57]. The discrepancy may be due to different
autophagic activity [45]. In addition, the p62 protein is experimental conditions used in these experiments. In
a marker for execution of autophagy [47]. It has been line with the above reports, in this study, we found that
13
Fig. 6 PD toxins evoke mitochondrial H 2O2/ROS, resulting in acti- lar results were obtained in at least five independent experiments. E
vation of PTEN and inactivation of Akt contributing to autophagy The relative densities for p-PTEN (Thr366), p-Akt (Ser473), p-Akt
inhibition and apoptosis in neuronal cells. PC12, SH-SY5Y cells (Thr308), ATG5, LC3-II, p62, Parkin, cleaved caspase-3 to β-tubulin
and primary neurons were pretreated with/without Mito-TEMPO were semi-quantified using NIH ImageJ. F The number of GFP-LC3
(10 μM) for 1 h and then exposed to 6-OHDA (120 μM), MPP+ puncta per cell was quantified by GFP-LC3 assay. At least 50 cells
(1 mM), or rotenone (1 μM) for 24 h. A Cell H 2O2 was imaged and were scored in each experiment. G Apoptotic cells were evaluated
quantified using a peroxide-selective probe H2DCFDA. B, C Mito- by nuclear fragmentation and condensation using DAPI staining. All
chondrial ROS (in red) were imaged and quantified using a mitochon- data were expressed as mean ± SE (n = 3 for D; n = 5 for A, C, F, G).
drial superoxide indicator MitoSOX. Scale bar: 20 μm. D Total cell Using one-way ANOVA or Student’s t-test, ap < 0.05, difference vs
lysates were subjected to Western blotting with indicated antibod- control group; bp < 0.05, + Mito-TEMPO group vs—Mito-TEMPO
ies. The blots were probed for β-tubulin as a loading control. Simi- group
treatment with 6-OHDA, M PP +, or rotenone triggered fashion in PC12, SH-SY5Y cells and primary neurons, as
decreases of ATG5/LC3-II and autophagosomes with a detected by Western blotting and GFP-LC3 assay (Fig. 1).
concomitant increase of p62 in a concentration-dependent Using genetic rescue experiments for ATG5, we showed
13
Fig. 7 Depletion of Parkin attenuates PD toxins-induced H2O2, PTEN ImageJ. C The number of GFP-LC3 puncta per cell was quantified
activation/Akt inactivation, autophagy inhibition, and apoptosis in by GFP-LC3 assay. At least 50 cells were scored in each experiment.
neuronal cells. PC12 cells, infected with lentiviral shRNA to Parkin D Cell H2O2 was imaged and quantified using a peroxide-selective
or GFP (as control) and infected with/without Ad-GFP-LC3, respec- probe H2DCFDA. E The relative cell viability was determined by the
tively, were treated with/without 6-OHDA (120 μM), M PP+ (1 mM) MTS assay. F Apoptotic cells were evaluated by using DAPI stain-
or rotenone (1 μM) for 24 h. A Total cell lysates were subjected to ing for nuclear fragmentation and condensation (arrows). Scale bar:
Western blotting with indicated antibodies. The blots were probed 20 µm. G The percentage of cells with fragmented nuclei was quan-
for β-tubulin as a loading control. Similar results were obtained in tified. All data were expressed as mean ± SE (n = 3 for B; n = 5 for
at least five independent experiments. B The relative densities for C–E, G). Using one-way ANOVA or Student’s t-test, ap < 0.05, dif-
p-PTEN (Thr366), p-Akt (Ser473), p-Akt (Thr308), ATG5, LC3-II, ference vs control group; bp < 0.05, Parkin shRNA group vs GFP
p62, cleaved caspase-3 to β-tubulin were semi-quantified using NIH shRNA group
13
Fig. 8 Overexpression of Parkin potentiates PD toxins-induced H2O2, ATG5, LC3-II, p62, cleaved caspase-3 to β-tubulin were semi-quan-
PTEN activation/Akt inactivation, autophagy inhibition, and apop- tified using NIH ImageJ. C The number of GFP-LC3 puncta per cell
tosis in neuronal cells. PC12 cells, infected with lentiviral FLAG- was quantified by GFP-LC3 assay. At least 50 cells were scored in
Parkin or EGFP (as control) and infected with/without Ad-GFP- each experiment. D Cell H 2O2 was imaged and quantified using a
LC3, respectively, were treated with/without 6-OHDA (120 μM), peroxide-selective probe H2DCFDA. E The relative cell viability was
MPP+ (1 mM), or rotenone (1 μM) for 24 h. A Total cell lysates determined by the MTS assay. F Apoptotic cells were evaluated by
were subjected to Western blotting with indicated antibodies. The nuclear fragmentation and condensation using DAPI staining. All
blots were probed for β-tubulin as a loading control. Similar results data were expressed as mean ± SE (n = 3 for B; n = 5 for C–F). Using
were obtained in at least five independent experiments. B The rela- one-way ANOVA or Student’s t-test, ap < 0.05, difference vs control
tive densities for p-PTEN (Thr366), p-Akt (Ser473), p-Akt (Thr308), group; bp < 0.05, FLAG-Parkin group vs EGFP control group
that overexpression of wild-type ATG5 substantially H) in PC12 cells. Collectively, these observations support
attenuated the inhibitory effect of PD toxins on autophagy that there exists ATG5 deficiency in PD toxins-induced
(Fig. 2A–C). Concurrently, overexpression of ATG5 con- oxidative stress, which causes autophagy inhibition-
ferred high resistance to PD toxins-induced apoptotic dependent neuronal cell death.
cell death, as evidenced by less cell viability reduction, PTEN is a negative regulator for Akt activity [17–20].
decreased percentages of cells with nuclear fragmentation In the current study, we observed that 6-OHDA, M PP+, or
and condensation (Fig. 2E and F), as well as the number of rotenone activated PTEN and inactivated Akt, as the tox-
TUNEL-positive cells with fragmented DNA (Fig. 2G and ins reduced the phosphorylation levels of PTEN and Akt in
13
PC12, SH-SY5Y cells, and primary neurons by using West- also observed that when PC12, SH-SY5Y cells, and primary
ern blotting and/or immunofluorescence staining (Fig. 3). neurons were exposed to 6-OHDA, MPP+, or rotenone for
In general, PTEN activation and Akt inactivation have 24 h, cellular H2O2 level was significantly elevated (Figs. 5
been thought to promote autophagy [21, 22]. For example, and 6). Importantly, we revealed that excessive intracellular
PTEN positively regulates macroautophagy by inhibiting and mitochondrial H 2O2 due to PD toxins activated PTEN
the PI3K/Akt pathway [21]. PTEN attenuates the PI3K/ and inactivated Akt contributing to autophagy inhibition and
Akt/mTOR signaling pathway to enhance autophagy [22]. apoptosis in the cells. This is supported by the observations
However, under certain circumstances, inactivation of Akt that pretreatment with CAT, a H2O2-scavenging enzyme, and
can result in impaired autophagic flux leading to cell death Mito-TEMPO, a mitochondria-selective superoxide scaven-
[23]. Accordingly, we reasoned that there may exist a cross- ger, markedly ameliorated 6-OHDA-, M PP+-, or rotenone-
talk between PTEN, Akt, and autophagy pathways in neu- induced generation of H2O2, decreases of p-PTEN, p-Akt,
ronal cells exposed to 6-OHDA, M PP+, or rotenone, i.e., the ATG5/LC3-II/autophagosomes, and increase of p62, as well
toxins-induced activation of PTEN and concurrent deactiva- as cleavage of caspase-3 and apoptosis in the cells (Figs. 5
tion of Akt may cause inhibition of autophagy. At first, we and 6, Fig. S2 and S3). Our observations are in agreement
noticed that the cells exhibited decreased p-PTEN (Thr366) with the above findings. Collectively, our results support
in 6–12 h, while decreased p-Akt (Ser473 and Thr308)/ that PD toxins-induced mitochondrial ROS may inhibit
ATG5/LC3-II and increased p62 in 12–24 h of post exposure autophagy contributing to apoptosis by activating PTEN
to 6-OHDA, M PP+, or rotenone (Fig. 3I and J), suggesting and inactivating Akt in neuronal cells. However, it is worth
that PTEN, as an upstream factor, regulates Akt activity and mentioning that some studies have shown that H2O2 can oxi-
autophagy in neuronal models of PD. Importantly, here, for dize PTEN resulting in PTEN inactivation [63, 64], which is
the first time, we present evidence that 6-OHDA, M PP+, or in contrast to our findings here. Likely, activation of PTEN
rotenone induced inhibition of autophagy pathway indeed is attributed to PD toxins-induced mitochondrial H2O2 in
by activation of PTEN and inactivation of Akt, resulting in this study. Further research is needed to address whether
neuronal apoptosis. This is strongly supported by the find- this is the case, or due to different cell lines used or other
ings that ectopic expression of dominant negative PTEN factors, and whether PD toxins-induced inhibition of Akt is
(PTEN-C/S) and/or myr-Akt, or myr-Akt and/or pretreat- a consequence of activating PTEN alone, activating PTEN/
ment with rapamycin dramatically rescued the cells from inactivating Akt jointly, or more mechanisms involved.
the toxins-induced downregulation of ATG5/LC3-II and Studies have demonstrated a close relationship of Par-
autophagosome formation and upregulation of p62, as well kin dysfunction, as a critical etiological factor of neuronal
as consequential apoptosis in PC12 cells (Fig. 4, Fig. S1). loss, to pathogenic process of PD [32, 34, 35]. Parkin muta-
Our findings underscore that PD toxins induce activation of tions can impact autophagy in PD [32, 34] and especially,
PTEN and inactivation of Akt, leading to autophagy inhibi- PTEN-induced kinase 1 (PINK1)/Parkin mitophagy is a key
tion and eventually apoptosis in neuronal cells. mechanism to contribute mitochondrial quality control, and
Many studies have shown that ROS can alter the struc- the defects are thought to be a cause of those PD onsets
tures and functions of cellular proteins, and also activate [65]. Akt is significantly decreased in Parkin-knockout
or inhibit related signaling pathways, leading to defects in compared with wild-type synaptosomes in mouse brain
their physiological function and subsequently more ROS [66]. These facts prompted us to search for the logic that
production and ultimately SNpc neuronal cell death/loss ties Parkin, PTEN/Akt and autophagy together. In this study,
in PD [58–60]. Excessive ROS-induced neuronal apoptosis we observed that PD toxins (6-OHDA, MPP+ or rotenone)
links to dysfunction of PTEN, Akt, and/or autophagy signal- concentration-dependently elicited decreases of Parkin in
ing [24–27]. Oxidation by H2O2 can result in inhibition of PC12, SH-SY5Y cells, and primary neurons (Fig. 1A–D).
protein tyrosine phosphatase 1B (PTP1B) and PTEN [61], This is in agreement with the report that L-DOPA-induced
but accumulation of mitochondrial superoxide anions can phospho-ubiquitin formation causes Parkin degradation
activate PTP1B and PTEN, leading to inhibition of insulin- [67]. Interestingly, overexpressing ATG5, or pretreatment
like growth factor-1 receptor (IGF-1R) and Akt in murine with CAT or Mito-TEMPO to scavenge H2O2 conferred
dermal fibroblasts [62]. Of note, treatment with rotenone great resistance to PD toxins-induced reduction of Parkin
(500 μM, 3 h, inducing mitochondrial superoxide anions) (Figs. 2, 5, and 6), implying that the protein level of Parkin
did not obviously alter the levels of PTEN and PTP1B, but is regulated by H2O2 and autophagy. To gain more insights
significantly increased the activity of the two phosphatases, into the role and significance of Parkin in PD toxins-evoked
resulting in inhibition of IGF-1R and Akt in murine der- H2O2-mediated PTEN/Akt signaling, autophagy, and apop-
mal fibroblasts [62]. Our recent studies have demonstrated tosis, we extended our experiments using lentiviral shRNA
that 6-OHDA, MPP+, or rotenone induces intracellular and to Parkin for knockdown of Parkin, or lentiviral FLAG-
mitochondrial H2O2 overproduction [25]. In this study, we tagged wild-type Parkin (FLAG-Parkin) for overexpression
13
Fig. 9 A schematic model

of how PD toxins (6-OHDA/
MPP+/rotenone) inhibit
autophagy leading to neuronal
apoptosis. PD toxins evoke
intracellular/mitochondrial
H2O2. This causes activation
of PTEN and inactivation of
Akt, convergently inhibiting
autophagy contributing to apop-
tosis in neuronal cells. Parkin is
essential for PD toxins-induced
H2O2-PTEN/Akt-autophagy
pathway and apoptosis in neu-
ronal cells
of Parkin. Surprisingly, knockdown of Parkin markedly sporadic PD [69]. Although the data support our obser-
weakened rather than strengthened PD toxins-induced H2O2 vations, further research in vitro and in vivo is necessary
production, PTEN activation/Akt inactivation, autophagy to establish stronger links between PD toxins-induced
inhibition and apoptosis in PC12 cells (Fig. 7, Fig. S4), yet oxidative stress-PTEN/Akt-autophagy-Parkin signaling
overexpression of Parkin potentiated the above effects of PD pathways and human PD.
toxins (Fig. 8, Fig. S5). These results suggest the involve- It would be interesting to explore the role and
ment of Parkin in the PD toxins-induced oxidative stress and significance of Parkin in the transcriptome level in a PD
a feedback mechanism for loss of Parkin involved. Thus, model. The latest research shows that Parkin participates
we tentatively propose that though PD toxins downregu- in the assembly of inf lammatory body NLRP3 and
late Parkin expression, a certain level of Parkin is required triggers downstream inflammatory reaction, leading to
for PD toxins to exert the action on H
2O2-mediated PTEN/ the death of dopaminergic neurons in a PD model [68].
Akt signaling, autophagy and apoptosis in neuronal cells. In the follow-up research, we will investigate the relevant
Undoubtedly, more studies are needed to further understand mechanism in the PD animal model and Parkin-knockout
the mechanism in depth. mice. We will detect the autophagy of dopaminergic
In this study, we have demonstrated that abnormal neurons by injecting 6-OHDA into the substantia nigra
H 2 O 2 -PTEN/Akt signaling leads to autophagy inhibi- to further understand the molecular mechanism of Parkin
tion-dependent cell death in neuronal models of PD, and in the PD model.
Parkin plays an essential role in this process. Because In conclusion, we have shown that PD toxins evoke
the physiological and pathophysiological status in the ATG5 deficiency, contributing to autophagy inhibition-
context of in vivo PD is more complex, this might be dependent apoptosis in neuronal cells. PD toxins elevate
very different from the models used in this study. It has the level of intracellular/mitochondrial H2O2, which causes
been reported that inactivation of Parkin from dopamine activation of PTEN and inactivation of Akt, convergently
neurons results in activation of the neuroinflammation, inhibiting autophagy leading to apoptosis in neuronal cells.
contributing to the death of dopamine neurons and neuro- We have also identified that Parkin is essential for PD
degeneration in PD, and loss of Parkin causes the death of toxins-mediated H2O2-PTEN/Akt-autophagy pathway and
dopamine neurons, accompanied by the increase of ROS apoptosis in neuronal cells (Fig. 9). Our findings suggest
in a mouse model of PD [68]. Decreased Parkin solubility that co-manipulation of the PTEN/Akt/autophagy signaling
reflects diminished Parkin function, which is associated by antioxidants may be exploited for the prevention of
with impairment of autophagy in the nigrostriatum of neuronal loss in PD.
13
Supplementary Information The online version contains supplementary autophagy, and cell protection in familial forms of Parkinson’s
material available at https://doi.org/10.1007/s12035-023-03286-y. disease. FEBS J 289(3):699–711. https://doi.org/10.1111/febs.16198
7. Zhang H, Duan C, Yang H (2015) Defective autophagy in Par-
Author Contribution LC and SH conceived the project. QY, SH, and LC kinson’s disease: lessons from genetics. Mol Neurobiol 51(1):89–
designed the experiments. QY, RZ, TL, and LY performed the experiments. 104. https://doi.org/10.1007/s12035-014-8787-5
QY, RZ, SH, and LC analyzed the data. ZZ, LH, WW, RZ, XC, and YY 8. van der Merwe C, van Dyk HC, Engelbrecht L, van der West-
contributed reagents/materials/analysis tools. QY, RZ, SH, and LC wrote huizen FH, Kinnear C, Loos B, Bardien S (2017) Curcumin
the paper. All authors read and approved the final manuscript. rescues a PINK1 knock down SH-SY5Y cellular model of
Parkinson’s disease from mitochondrial dysfunction and cell
Funding This work was supported in part by the grants from the death. Mol Neurobiol 54(4):2752–2762. https://doi.org/10.1007/
National Natural Science Foundation of China (Nos. 81873781, s12035-016-9843-0
81271416, 82101337), National Institutes of Health (CA115414), 9. Henchcliffe C, Beal MF (2008) Mitochondrial biology and oxi-
Project for the Priority Academic Program Development of Jiangsu dative stress in Parkinson disease pathogenesis. Nat Clin Pract
Higher Education Institutions of China (PAPD-14KJB180010), BSKY Neurol 4(11):600–609. https://doi.org/10.1038/ncpneuro0924
Scientific Research from Anhui Medical University (XJ201813), and 10. Corti O, Blomgren K, Poletti A, Beart PM (2020) Autophagy
American Cancer Society (RSG-08–135-01-CNE). in neurodegeneration: new insights underpinning therapy for
neurological diseases. J Neurochem 154(4):354–371. https://doi.
Data Availability The data used to support the findings of this study org/10.1111/jnc.15002
are available from the corresponding author upon reasonable request. 11. Pan PY, Yue Z (2014) Genetic causes of Parkinson’s disease and their
links to autophagy regulation. Parkinsonism Relat Disord 20(Suppl
Declarations 1):S154-157. https://doi.org/10.1016/S1353-8020(13)70037-3
12. Damme M, Suntio T, Saftig P, Eskelinen EL (2015) Autophagy in
Ethical Approval The experiments involving animals in this study were neuronal cells: general principles and physiological and pathologi-
handled in accordance with the guidelines issued by the animal ethics cal functions. Acta Neuropathol 129(3):337–362. https://doi.org/
committee (IACUC Certificate No. 200408), and were in compliance 10.1007/s00401-014-1361-4
with the guidelines set forth by the Guide for the Care and Use of 13. Feng Y, He D, Yao Z, Klionsky DJ (2014) The machinery of
Laboratory Animals. macroautophagy. Cell Res 24(1):24–41. https://doi.org/10.1038/
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Consent to Participate Not applicable. 14. Zhang Z, Miah M, Culbreth M, Aschner M (2016) Autophagy in
neurodegenerative diseases and metal neurotoxicity. Neurochem
Consent for Publication Not applicable. Res 41(1–2):409–422. https://d oi.o rg/1 0.1 007/s 11064-0 16-1 844-x
15. Suresh SN, Verma V, Sateesh S, Clement JP, Manjithaya R (2018)
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Mitochondrial Hydrogen Peroxide Activates PTEN and Inactivates Akt Leading To Autophagy Inhibition Dependent Cell Death in Neuronal Models of Parkinson's Disease

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Molecular Neurobiology (2023) 60:3345–3364