Elsevier

Neuroscience Letters

Volume 454, Issue 3, 1 May 2009, Pages 203-208
Neuroscience Letters

Role of autophagy and proteasome degradation pathways in apoptosis of PC12 cells overexpressing human α-synuclein

https://doi.org/10.1016/j.neulet.2009.03.027Get rights and content

Abstract

Parkinson's disease is a common neurodegenerative disease in the elderly. Its causes and mechanisms are not clearly understood. To explore the specific role of autophagy and the ubiquitin-proteasome pathway in apoptosis, a specific proteasome inhibitor and macroautophagy inhibitor and stimulator were selected to investigate pheochromocytoma (PC12) cell lines transfected with human mutant (A30P) and wild-type (WT) α-synuclein. The apoptosis ratio was assessed by flow cytometry. LC3, heat shock protein 70 (hsp70) and caspase-3 expression in cell culture were determined by Western blot. The hallmarks of apoptosis and autophagy were assessed with transmission electron microscopy. Compared to the control group or the rapamycin (autophagy stimulator) group, the apoptosis ratio in A30P and WT cells was significantly higher after treatment with inhibitors of the proteasome and macroautophagy. The results of Western blots for caspase-3 expression were similar to those of flow cytometry; hsp70 protein was significantly higher in the proteasome inhibitor group than in control, but in the autophagy inhibitor and stimulator groups, hsp70 was similar to control. These findings show that inhibition of the proteasome and autophagy promotes apoptosis, and the macroautophagy stimulator rapamycin reduces the apoptosis ratio. And inhibiting or stimulating autophagy has less impact on hsp70 than the proteasome pathway.

Section snippets

Acknowledgements

We are grateful to Dr. Ping Feng and Bingyan Li of Soochow University for their advice, generosity and extraordinary support with facilities and reagents. We also thank Professor IC Bruce for critical reading of the manuscript. Supported by grants from the National Natural Science Foundation of China (number 30870869) and the Jiangsu Province Foundation of China (07KJB320109).

References (44)

  • L.K. Nutt et al.

    Metabolic regulation of oocyte cell death through the CaMKII-mediated phosphorylation of caspase-2

    Cell

    (2005)
  • S. Pattingre et al.

    Bcl-2antiapoptotic proteins inhibit Beclin 1-dependent autophagy

    Cell

    (2005)
  • J.J. Qian et al.

    Differential effects of overexpression of wild-type and mutant human α-synuclein on MPP+-induced neurotoxicity in PC12 cells

    Neurosci. Lett.

    (2008)
  • I. Tanida et al.

    HsAtg4B/HsApg4B/autophagin-1 cleaves the carboxyl termini of three human Atg8 homologues and delipidates microtubule-associated protein light chain 3- and GABA(A) receptor-associated protein–phospholipid conjugates

    J. Biol. Chem.

    (2004)
  • J.L. Webb et al.

    Alpha-synuclein is degraded by both autophagy and the proteasome

    J. Biol. Chem.

    (2003)
  • L. Xue et al.

    Autophagy is activated by apoptotic signalling in sympathetic neurons: an alternative mechanism of death execution

    Mol. Cell. Neurosci.

    (1999)
  • C.M. Ashish et al.

    Consequences of the selective blockage of chaperone-mediated autophagy

    PNAS

    (2006)
  • N.F. Bence et al.

    Impairment of the ubiquitin-proteasome system by protein aggregation

    Science

    (2001)
  • F. Blandini et al.

    Peripheral markers of apoptosis in Parkinson's disease: the effect of dopaminergic drugs

    Ann. N.Y. Acad. Sci.

    (2003)
  • P.B. Ciechanover

    The ubiquitin proteasome pathway: on protein death and cell life

    EMBO J.

    (1998)
  • A.M. Cuerco et al.

    Impaired degradation of mutant alpha-synuclein by chaperone-mediated autophagy

    Science

    (2004)
  • Q. Cui et al.

    Autophagy preceded apoptosis in oridonin-treated human breast cancer MCF-7 cells

    Biol. Pharm. Bull.

    (2007)
  • Cited by (32)

    • Therapeutic applications

      2021, Nanomaterials for Biocatalysis
    • Role of Mitochondrial Fission and Mitophagy in Parkinson's Disease

      2014, Autophagy: Cancer, Other Pathologies, Inflammation, Immunity, Infection, and Aging
    • Protein damage, repair and proteolysis

      2014, Molecular Aspects of Medicine
      Citation Excerpt :

      Expression of mutant LRRK2 (leucine rich repeated kinase 2) that is observed in familial PD patients, also increases autophagic vacuole and autophagy-dependent shortening of neurites (Plowey et al., 2008). Excessive autophagic induction that leads to neuronal loss has been associated with overexpression of A53T α-synuclein and pharmacological induction of PD with MPP+ (Choubey et al., 2011; Kirik et al., 2002; Stefanis et al., 2001; Xilouri et al., 2009; Yang et al., 2009). In accordance with the inhibition of autophagy through the 3-methylamphetamine (3-MA) inhibitor, inhibition or knock-down of ATG5 or ATG12 expression attenuates neurodegeneration (Wong et al., 2011).

    • Reynosin protects against neuronal toxicity in dopamine-induced SH-SY5Y cells and 6-hydroxydopamine-lesioned rats as models of Parkinson's disease: Reciprocal up-regulation of E6-AP and down-regulation of α-synuclein

      2013, Brain Research
      Citation Excerpt :

      Human neuroblastoma SH-SY5Y cells (ATCC no. CRL-2266) were cultured in DMEM supplemented with 10% heat-inactivated FBS and 1% penicillin/streptomycin (Meiji Seika, Tokyo, Japan), at 37 °C in a humidified 5% CO2 atmosphere. SH-SY5Y cells were cultured for 24 h and then treated with reynosin (0.08, 0.4, and 2 µM) or 2 µM of APO (a positive control compound) (Hara et al., 2006) for 24 h, followed by subsequent treatment with DA (600 μM) for an additional 24 h. Cell viability was analyzed by flow cytometry (FACS) (Calibur, Becton Dickinson, USA) using PI staining (Yang et al., 2009). Male Sprague-Dawley rats were purchased from Samtako Bio Korea (Osan, Korea) and housed in groups (2–3 rats/cage) at room temperature for 1 week prior to the experiments in a humidity-controlled environment with a 12/12-light-dark cycle and unlimited access to food and water.

    • Retinoic acid protects against proteasome inhibition associated cell death in SH-SY5Y cells via the AKT pathway

      2013, Neurochemistry International
      Citation Excerpt :

      It is now generally accepted that abnormal protein accumulation is central to the pathophysiology of AD and PD (Cook and Petrucelli, 2009; Keller and Markesbery, 2000; Lopez Salon et al., 2000; McNaught and Olanow, 2003). Recent reports have suggested the involvement of autophagy, either independently or in conjunction with apoptosis in PD (Cheung and Ip, 2009; Gonzalez-Polo et al., 2005; Yang et al., 2009a). To develop therapeutic strategies for these diseases, it will be important both to identify and characterize mechanisms that regulate the proteasome–ubiquitination system, and to find ways to prevent dysfunction and cell death where abnormal protein aggregation has occurred.

    • Apoptosis in Parkinson's disease: Is p53 the missing link between genetic and sporadic Parkinsonism?

      2011, Cellular Signalling
      Citation Excerpt :

      More recently α-synuclein was shown to be cleared by chaperone-mediated autophagy (CMA) and macroautophagy in several neuronal cell lines [23], a control abolished by either pathogenic mutations or by drugs such as dopamine able to promote α-synuclein aggregation [24]. α-Synuclein was shown to block the interaction of the transcription factor MEF2D (myocyte enhancer factor 2D) with the chaperone protein Hsc70 leading to increased cell death due to impairment of CMA [25]. Other PD-associated gene products were also shown to control type II PCD.

    View all citing articles on Scopus
    View full text