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. 2024 Aug 1;19(8):1828-1834.
doi: 10.4103/1673-5374.389356. Epub 2023 Nov 8.

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

Yongjiang Zhang  1 , Shiyi Yin  1 , Run Song  1 , Xiaoyi Lai  1 , Mengmeng Shen  1 , Jiannan Wu  1 , Junqiang Yan  1   2
Affiliations

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

Yongjiang Zhang et al. Neural Regen Res. .

Abstract

JOURNAL/nrgr/04.03/01300535-202408000-00037/figure1/v/2023-12-16T180322Z/r/image-tiff 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.

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Conflict of interest statement

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

Figures

Figure 1
Figure 1
Effect of MPP+ on PHB2 protein expression and mitochondrial function in SH-SY5Y cells. SH-SY5Y cells were treated with PHB2-shRNA and PHB2-Over Exp, and then induced with 1 mM MPP+ for 24 hours. (A, B) PHB2 protein concentration in SH-SY5Y cells after 1 mM MPP+ treatment for 6, 12, 24, and 48 hours. (A) Western blots showing PHB2 protein expression in MPP+-treated cells at different times. (B) Quantitative analysis of PHB2 protein expression. (C–G) Protein expression changes of Nrf2, HO-1, NQO-1, and PHB2. (C) Western blots showing Nrf2, HO-1, NQO-1, and PHB2 protein expression in MPP+-treated and PHB2-shRNA- or PHB2-Over Exp-treated cells. (D–G) Quantitative analysis of Nrf2, HO-1, NQO-1, and PHB2 protein expression. (H, I) The effect of PHB2 expression on mitochondrial membrane potential induced by MPP+. (H) JC-1 staining of SH-SY5Y cells treated with PHB2-shRNA and PHB2-Over Exp. Original magnification 10×, scale bar: 50 μm. (I) Quantitative analysis of JC-1 staining: the green/red ratio reflects changes in the mitochondrial membrane potential. (J, K) The effect of changes in PHB2 expression on ROS production under MPP+ induction. (J) Intracellular ROS fluorescence. SH-SY5Y cells were treated with PHB2-shRNA and PHB2-Over Exp and labeled with DCFH-DA (green fluorescence). Original magnification 4×, scale bar: 100 μm. (K) Quantitative analysis of ROS (DCFH-DA) in SH-SY5Y cells. (L) Electron microscopy images of morphological changes in mitochondria in PHB2-shRNA-treated and MPP+-induced cells. Red boxes show magnified mitochondria. Scale bars: 1 μm. Data are expressed as mean ± SEM (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, vs. control group; #P < 0.05,##P < 0.01, vs. MPP+ group (one-way analysis of variance with Tukey's multiple comparison test). DCFH-DA: 2′,7′-Dichlorodihydrofluorescein diacetate; HO-1: heme oxygenase-1; JC-1: 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide; MPP+: 1-methyl-4-phenylpyridinium; NQO-1: NAD(P)H quinone dehydrogenase 1; Nrf2: nuclear factor erythroid 2-related factor 2; Over Exp: overexpression; PHB2: prohibitin 2; ROS: reactive oxygen species; SH-SY5Y: human neuroblastoma cell line.
Figure 2
Figure 2
Effect of PHB2 on mitophagy in a PD cell model. (A–F) PHB2 and LC3II/LC3I protein expression in SH-SY5Y cells after treatment with PHB2-shRNA or PHB2-Over Exp and MPP+ (1 mM, 24 hours). (A) Protein expression levels of PHB2 and LC3 in SH-SY5Y cells treated with PHB2-shRNA under MPP+ induction. (B, C) Quantitative analysis of PHB2 and LC3II/LC3I protein expression. (D) Protein expression levels of PHB2 and LC3 in SH-SY5Y cells treated with PHB2-Over Exp under MPP+ induction. (E, F) Quantitative analysis of PHB2 and LC3II/LC3I protein expression. (G) Cellular localization of LC3 and TOM20 or TIM23 in SH-SY5Y cells treated with PHB2-shRNA under MPP+ induction. Immunofluorescence changes of LC3 and mitochondrial proteins (TOM20 and TIM23). Red fluorescence represents TOM20 and TIM23, green fluorescence represents LC3, and blue fluorescence represents DAPI. Original magnification 20×, Scale bars: 20 μm. Data are expressed as mean ± SEM (n = 3). **P < 0.01, ***P < 0.001, vs. control group (one-way analysis of variance with Tukey's multiple comparison test). DAPI: 4′,6-Diamidino-2-phenylindole; LC3: microtubule-associated protein 1 light chain 3; MPP+: 1-methyl-4-phenylpyridinium; Over Exp: overexpression; PHB2: prohibitin 2; SH-SY5Y: human neuroblastoma cell line; TIM23: translocase of inner mitochondrial membrane 23; TOM20: translocase of outer mitochondrial membrane 20.
Figure 3
Figure 3
Parkin increases PHB2/LC3-mediated mitophagy in SH-SY5Y cells. (A–D) Protein levels of Parkin, PHB2, and autophagy marker (LC3) in SH-SY5Y cells after treatment with Parkin-shRNA, Parkin-Over Exp, and MPP+ (1 mM, 24 hours). (A) Protein expression levels of Parkin, LC3, and PHB2 in SH-SY5Y cells treated with Parkin-shRNA or Parkin-Over Exp under MPP+ induction. (B–D) Quantitative analysis of Parkin, and LC3II/LC3I, PHB2 protein expression. (E) Immunofluorescence analysis of PHB2 and LC3. Cellular localization of LC3 and PHB2 in SH-SY5Y cells treated with Parkin-shRNA or Parkin-Over Exp under MPP+ induction. Red fluorescence represents PHB2, green fluorescence represents LC3, and blue fluorescence represents DAPI. Original magnification 40×, Scale bar: 20 μm. Data are expressed as mean ± SEM (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, vs. control group; #P < 0.05,###P < 0.001, vs. MPP+ group (one-way analysis of variance with Tukey's multiple comparison test). DAPI: 4′,6-Diamidino-2-phenylindole; LC3: microtubule-associated protein 1 light chain 3; MPP+: 1-methyl-4-phenylpyridinium; Over Exp: overexpression; Parkin: parkin RBR E3 ubiquitin-protein ligase; PHB2: prohibitin 2; SH-SY5Y: human neuroblastoma cell line.
Figure 4
Figure 4
Parkin regulates anti-oxidative stress protein expression via PHB2. (A–D) SH-SY5Y cells were treated with PHB2-shRNA, Parkin-Over Exp, and MPP+ (1 mM, 24 hours). Changes in Nrf2, HO-1, and NQO-1 protein levels and quantitative analysis. (A) Protein expression levels of Nrf2, HO-1, and NQO-1 in SH-SY5Y cells treated with PHB2-shRNA or Parkin-Over Exp under MPP+ induction. (B–D) Quantitative analysis of Nrf2, HO-1, and NQO-1 protein expression. Data are expressed as mean ± SEM (n = 3). ***P < 0.001, vs. control group; #P < 0.05,###P < 0.001, vs. MPP+ group (one-way analysis of variance with Tukey's multiple comparison test). HO-1: Heme oxygenase-1; MPP+: 1-methyl-4-phenylpyridinium; NQO-1: NAD(P)H quinone dehydrogenase 1; Nrf2: nuclear factor erythroid 2-related factor 2; Over Exp: overexpression; Parkin: parkin RBR E3 ubiquitin-protein ligase; PHB2: prohibitin 2; SH-SY5Y: human neuroblastoma cell line.
Figure 5
Figure 5
Phosphorylation of PERK (Thr980) increases Parkin protein expression in SH-SY5Y cells. (A–C) Changes and quantitative analysis of Parkin protein levels in SH-SY5Y cells after MPP+ and PERK-shRNA treatment. (A) Protein expression levels of Parkin, p-PERK, and PERK in SH-SY5Y cells treated with PERK-shRNA under MPP+ induction. (B, C) Quantitative analysis of Parkin, p-PERK, and PERK protein expression. Data are expressed as mean ± SEM (n = 3). **P < 0.01, ***P < 0.001, vs. control group; ##P < 0.01,###P < 0.001, vs. MPP+ group (one-way analysis of variance with Tukey's multiple comparison test). MPP+: 1-Methyl-4-phenylpyridinium; Parkin: parkin RBR E3 ubiquitin-protein ligase; PERK: protein kinase R-like endoplasmic reticulum kinase; SH-SY5Y: human neuroblastoma cell line.
Figure 6
Figure 6
Silencing PHB2 inhibits mitophagy and aggravates dopaminergic neuronal loss in PD mice. (A) Schematic of PHB2-shRNA and MPTP treatment in mice. (B, C) Quantitative analysis of PHB2 protein levels in the midbrain of C57BL/6J mice after PHB2-shRNA injection. (B) Protein expression levels of PHB2 in C57BL/6J mice treated with PHB2-shRNA. (C) Quantitative analysis of PHB2 protein expression. (D–F) An acute PD model was established by intraperitoneal injection of MPTP in C57BL/6J mice after PHB2-shRNA injection. Changes and quantitative analysis of LC3II/LC3I and PHB2 protein levels in the midbrain. (D) Protein expression levels of PHB2 and LC3 after MPTP injection in PD model mice. (E, F) Quantitative analysis of PHB2 and LC3II/LC3I protein expression. (G, H) TH immunofluorescence (Alexa Fluor 488, green fluorescence) shows dopaminergic neurons in the substantia nigra in PD mice with silenced PHB2. (G) Immunofluorescence of TH-positive neurons in the substantia nigra. Mice were treated with PHB2-shRNA and MPTP. Original magnification 10×, Scale bar: 200 μm. (H) Quantitative analysis of TH-positive neurons in the substantia nigra. (I) Fluorescence co-localization of LC3 and PHB2, TIM23, or TOM20 in the substantia nigra. Mice were treated with PHB2-shRNA and MPTP. Red fluorescence (Alexa Fluor 555): PHB2, TOM20, and TIM23, green fluorescence (Alexa Fluor 488): LC3, and blue fluorescence: DAPI. Original magnification 20×, Scale bars: 20 μm. Data are expressed as mean ± SEM (n = 3). *P < 0.05, ***P < 0.001, vs. control group; $P < 0.05, $$P < 0.01, $$$P < 0.001, vs. MPTP group (one-way analysis of variance with Tukey's multiple comparison test). LC3: Microtubule-associated protein 1 light chain 3; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; PD: Parkinson's disease; PHB2: Prohibitin 2; TH: tyrosine hydroxylase; TIM23: translocase of inner mitochondrial membrane 23; TOM20: translocase of outer mitochondrial membrane 20.
Figure 7
Figure 7
Silencing PHB2 reduces antioxidative stress protein expression and aggravates motor defecits in PD mice. (A–D) Changes and quantitative analysis of Nrf2, HO-1, and NQO-1 protein levels in the midbrain of PHB2-shRNA and MPTP-treated PD mice. (E) Quantitative analysis of mice in the tail suspension and rotarod tests. Data are expressed as mean ± SEM (n = 5). *P < 0.05, **P < 0.01, vs. control group; $P < 0.05, $$$P < 0.001, vs. MPTP group (one-way analysis of variance with Tukey's multiple comparison test). HO-1: Heme oxygenase-1; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; NQO-1: NAD(P)H quinone dehydrogenase 1; Nrf2: nuclear factor erythroid 2-related factor 2; PD: Parkinson's disease; PHB2: prohibitin 2.
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Grants and funding

Funding:This work was supported by the Key Science and Technology Research of Henan Province, No. 222102310351 (to JW); Luoyang 2022 Medical and Health Guiding Science and Technology Plan Project, No. 2022057Y (to JY); Henan Medical Science and Technology Research Program Province-Ministry Co-sponsorship, No. SBGJ202002099 (to JY).
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