- Research article
- Open Access
Oxidative stress promotes autophagic cell death in human neuroblastoma cells with ectopic transfer of mitochondrial PPP2R2B (Bβ2)
- Wan-Ting Cheng†1,
- Zhi-Xuan Guo†1,
- Chia-An Lin1,
- Ming-Yi Lin1,
- Li-Chu Tung1 and
- Kang Fang1Email author
© Cheng et al; licensee BioMed Central Ltd. 2009
- Received: 30 April 2009
- Accepted: 18 December 2009
- Published: 18 December 2009
The multifunctional protein phosphatase 2A (PP2A) is a heterotrimeric serine/threonine protein phosphatase composed of a scaffolding, catalytic and regulatory subunits. By modifying various downstream signal transducers, the aberrant expression of the brain-targeted regulatory subunit PPP2R2B is associated with the onset of a panel of neuronal disorders. The alternatively splicing of PPP2R2B encodes two regulatory subunit isoforms that determine cellular distribution of the neuron-specific holoenzyme to mitochondria (Bβ2) and cytoplasm (Bβ1), respectively.
Human neuroblastoma cells were transfected with PPP2R2B constructs encoding the complete sequences of Bβ2 and Bβ1, respectively. The colonies with antibiotic resistance were selected as stable cell lines. Both ectopic Bβ1 and Bβ2 clones exhibited characteristics of autophagy. To test how cells respond to reactive oxygen species generators, the cells were treated with either hydrogen peroxide or t-butyl hydroperoxide and Bβ2 clones induced cell death. Suppression of autophagy using either RNA interference of the essential autophagy gene or pharmacological inhibitor rescued cell death caused by oxidative stress.
Cells with ectopically expressed mitochondria-targeted regulatory subunit PPP2R2B of the holoenzyme PP2A were shown predisposed to autophagy and oxidative stress induced cell death that is related to apoptosis. The results promised a model for studying the mechanism and function of aberrant PPP2R2B expression in neuronal cells. The work provided a new target for understanding and prevention of neuropathogenesis.
- Spinocerebellar Ataxia
- Human Neuroblastoma Cell
- Autophagic Cell Death
- Autophagosome Formation
Protein phosphatase 2A (PP2A) regulates cellular proliferation and tissue growth by antagonizing protein kinases and plays an important role in controlling cell cycle checkpoints, regulating nuclear telomerase activity and modulating dephosphorylation of neurofilaments [1–5]. It is believed that the enzyme flexibility and the substrate specificity are determined by the regulatory subunit (B) that binds to the ubiquitous catalytic (C) and scaffolding (A) subunits  to form the heterotrimeric PP2A.
The regulatory B subunits can be categorized into three distinct families based on their homology, namely B (B55 or PR55), B' (B56 or PR61), B"(PR48/59/72/130) and B' " (PR93/110). Furthermore, each B family is composed of several isoforms from different genes [7, 8]. There is no sequence or structural similarities among the three gene families. The brain-specific regulatory B subunit, PPP2R2B (Bβ), is widely expressed in the neurons of brain and cerebellum and consists of tryptophan-glutamate repeat-containing β-propeller proteins. The various N termini of B subunit determine enzyme activity, subcellular localizations and neuronal functions of PP2A [3, 9, 10]. Mainly expressed in Purkinje cells of the cerebellar cortex [5, 11, 12] and in tissues of spinocerebellar ataxia [13, 14], PPP2R2B is also known in mediating PP2A-regulated dephosphorylation of several substrates; they include vimentin  and histone-1  as well as microtube-associated protein in neuron cells .
The alternative splicing of PPP2R2B generates two major isoforms. They include Bβ1 and Bβ2 with two distinct N terminal tails . The different N termini with 21 and 24 residues of Bβ1 and Bβ2 each encode distinctive subcellular signals that target PPP2R2B isoforms to cytoplasm and mitochondria, respectively, and affect cellular phosphatase activity . The up-regulated mitochondrial Bβ2 was reported promoting apoptosis in response to deprived growth factors , while an elevated expression of cytosolic Bβ1 due to CAG repeat expansion at the 5' end of the gene causes autosomal dominant disease of spinocerebellar ataxia type 12 (SCA12) .
The aberrant expression of brain-specific PPP2R2B affecting PP2A activity has been implicated in cerebellar atrophy . Translocation of PPP2R2B to mitochondria promotes mitochondria fission, apoptosis  and neuronal differentiation . Being implicated in neurodegenerative diseases, oxidative stress causes neuron cell damage and reduction of viable cells. How cells with overexpressed PPP2R2B react to environmental stress remains unclear. Mitochondria are the principal targets for reactive oxygen species (ROS). To address this, the work began with generation of stable cell lines by transferring Bβ1 and Bβ2, respectively, into human neuroblastoma SK-N-SH cells. The stable clones targeting ectopic PPP2R2B to mitochondria or cytoplasm exhibit characteristics of autophagy that is distinct from the parental cells. Furthermore, only Bβ2 clones were sensitive to ROS treatment and susceptible to autophagy-mediated cell death. The resulting apoptosis can be suppressed by chemical inhibitor and RNA interference of the related autophagy gene, and thereby preventing cells from ROS damage-induced cell death. The work illustrates the significance of autophagy in maintaining viabilities of neuronal cells with ectopically transferred PPP2R2B as well as in accelerating pathogenesis when encountered with oxidative stress.
The establishment of clones targeting Bβ 2 to mitochondria and Bβ 1 cytoplasma, respectively
The enhanced autophagosome formation in clones with ectopic PPP2R2B
Sensitivity of peroxide-induced apoptotic cell death is associated with ectopic Bβ 2
ROS-induced apoptosis in Bβ 2 clones can be rescued by autophagy inhibitor, 3-MA and ATG7 siRNA
PP2A deregulation has been vastly implicated in the onset of neurodegenerative diseases . The mechanisms of PP2A activity responsible for the disrupted neuron function remain unclear and are far from being understood . Intervention of normal brain or cerebellar PP2A function is crucial in the developing neurodegenerative pathology . In an effort to learn how the increased expression of PPP2R2B isoforms affects cell growth, genes encoding proteins encoding different targeting sequences were transferred into human SK-N-SH cells. The clones with ectopically expressed PPP2R2B can be propagated as stable cell lines. Our work showed that discrete antisera cross-reactivity, in which mitochondria can be stained by Bβ1 and cytoplasm by Bβ2, respectively. To understand the function of the ectopic gene in the stable clones, it will be important to further analyze the fractions at subcellular levels.
The stable clones forming aggregation in culture exhibited autophagy characteristics that are distinct from the parental cells. Autophagy is a lysosome-dependent degradation pathway regulated by extracellular nutrients or trophic factors . Cells exhibiting basal rate of autophagy maintain homeostasis and survival of neurons under stress that protects them from pathogenesis . Despite their relatively slow growth rate compared to the parental cells (Fig 7A), clones with ectopic brain-specific subunit PPP2R2B were able to sustain minor increases of autophagosome formation that maintains cell viable. The formation of autophagosome in both Bβ1 and Bβ2 clones (Fig 6) demonstrated how cells survive when encountered with stress conditions. While heterotrimeric PP2A holoenzyme containing all three regulatory subunit families can affect events ranging from the initiation of DNA replication to vertebrate axis formation to apoptosis , the up-regulated mitochondrial PPP2R2B putatively serves to deregulate protein kinases cascades, thereby making cells autophagic as seen in some neuron disorders [21, 33].
Numerous studies suggested a close relationship between dysfunctional mitochondria and autophagy. Mitochondrial perturbation is one of the sources to induce autophagic cell death . The disturbance of autophagy-lysosome balance has been recognized as a major cause in numerous neurodegenerative disorders . While low levels of autophagy maintains cell viabilities in both stable Bβ1 and Bβ2 clones, only the latter was sensitive to peroxide injury by initiating apoptosis (Fig. 5) with concomitant increase of AVO' s (Fig. 6). Inhibition of autophagy by the chemical inhibitor, 3-MA, (Fig. 7) and RNA interference-mediated knockdown of ATG7 (Fig. 8) reverted the process. Thus, oxidative stress-induced cell death in Bβ2 cells as shown in annexin V staining and growth assays demonstrated that the progression of autophagy lead Bβ2 cells to apoptosis.
Although autophagy is capable of keeping cells from being damaged, the work provided evidence that, in addition to being protective, the increased autophagy as a result of peroxide injury can be pathogenic resulting from apoptosis. Our work showed that the attenuated autophagy prolonged cell survival by reducing formation of the accumulated autophagosome following peroxide injury. Given the fact that oxidative stress is an origin of disturbing cell homeostasis, it is conceivable that the reduced autophagic source may be an alternative approach to ameliorate harmful effects to neuronal system . Being necessary at basal level for maintaining normal axonal and synaptic structures, particularly in Purkinje neurons, excessive autophagy can be neurotoxic and inhibition by chemical regulators prevents the onset of neurodegeneration . Further investigation on how excessive autophagy affects cell signaling and growth under oxidative stress will provide more targets to understand the basis of the diseases.
More evidence indicated that PP2A activity can be regulated by extracellular signals and cell cycle . How altered cell signaling in organelles, particularly those in mitochondria that impinge viable neuron cells as a result of increased expression of the regulatory subunit PPP2R2B, provides an interesting topic for developing more effective preventive measures in development of neuron disorders.
The peptides derived from the N terminus of mitochondria-specific Bβ2 (CFSRYLPYIFRPPNT) and cytoplasm-specific Bβ1(MKCFSRYLPYIFRPPNTILSSSCH) were coupled to keyhole limpet hemocyanin, respectively, via the sulfhydryl group of the N-terminal cysteine; the polyclonal antibody sera were generated in rabbits and purified by standard affinity techniques.
Cytomegalovirus promoter-driven expression constructs containing Bβ1 or Bβ2 cDNA and establishment of stable clones from SK-N-SH cells and cell culturing
The Bβ1 cDNA was restriction-digested by Xba I from the Bβ1 phagemid (provided by BA Hemmings, Katholieke Universiteit te Leuven, Belgium) containing N-terminus cytoplasm-specific sequence of PPP2R2B and ligated into Xba I site of pcDNA3. The Bβ2 cDNA containing mitochondria-specific sequence of PPP2R2B was restriction-digested from Bβ2 cDNA library clone by Xho I and Spe I from the plasmid (provided by S Strack, University of Iowa, Iowa City, Iowa) and ligated into Xho I and Spe I sites of pcDNA3. Each of the recombinant plasmids was confirmed of their DNA sequences by DNA sequencing from both ends.
Human neuroblastoma cell line SK-N-SH was acquired from American Type Tissue Collection (Rockville, MD) and grown in DMEM (Invitrogen, Grands Islands, NY). Medium was changed three times a week. Cells were observed with a phase-contrast microscope (Nikon Diaphot-300). SK-N-SH cells were cultured every 5 or 7 days using standard trypsinization procedures to maintain the cell line. All cultured cells were supplemented with L-glutamine, sodium pyruvate, and supplemented with 10% heat-inactivated FCS in the humidified atmosphere of 5% CO2 at 37°C. All cell lines were examined and found to be free of mycoplasma contamination using a MycoTect kit (Invitrogen, Grands Islands, NY). Both hydrogen peroxide and t-butyl hydroperoxide were from Sigma (St. Louis, MO).
A total of 5 × 105 exponentially growing SK-N-SH cells were cultured in 60-cm2 flasks and transfected with one microgram of either recombinant Bβ 1, Bβ 2 construct or the empty vector by Lipofectamine (Invitrogen) in serum-free media following the manufacturer's protocols. After three weeks of culturing and selection with 200 μg/ml of G418 (Invitrogen), the antibiotic-resistant colonies were harvested, and, after limited dilution, the subclones carrying overexpressed Bβ1 or Bβ2 isolated. The established clones can be continuously propagated as immortalized cell lines in 10% FCS-supplemented DMEM.
Determination for cell growth and apoptosis
Cell growth was measured by trypan blue exclusion assay. Briefly, cells were plated in 12-well culture plates (1 × 105 cells/well). After incubation for the time specified, the cells were treated with different concentrations of peroxides for the times as specified. The trypsinized cells suspended in PBS were mixed with trypan blue solution in a 1:1 ratio and the cell numbers counted by a hemocytometer.
The annexin V-positive cells were determined using Annexin V-FITC Apoptosis Detection Kit (Roche Diagnostics, Indianapolis, IN) according to the supplier's instructions. Each cell line was tested at least three times and apoptotic cells determined quantitatively by flow cytometry.
Flow cytometry of cell cycle analysis by propidium iodide staining
To determine phase distribution of DNA content, propidium iodide (PI) staining was performed. Briefly, 3 × 105 cells collected were washed once and fixed in 70% ethanol overnight. After centrifugation at 700 rpm for 5 min at 4°C, cell pellet was stained with 5 μg/ml PI (Sigma, St. Louis, MO) plus 0.5 mg/ml RNaseA in PBS buffer for 15 min at room temperature in the dark. The analysis was performed with FACScan flow cytometer (Becton-Dickinson, Mansfield, MA). Cell cycle distributions were analyzed by Cell-Quest and Modfit software (Becton-Dickinson, Mansfield, MA). The statistics of cell distributions were calculated from three individual experiments.
PP2A activity assay determination
The phosphatase activity was conducted by modifying the published work . Briefly, cell lysates were immune-precipitated with antiserum against β2, β1 or pre-serum depending on the type of clones used. Following precipitation with protein (A/G)-agarose and resuspension in the assay buffer, the collected supernatants were reacted with p-nitrobenzyl phosphate substrate. The optical readings of the assay as determined by spectrophotometer for each cells were subtracted from those by precipitation with pre-serum. Data were converted into percentage by comparing with those of the control parental cells, SK-N-SH with the empty vector.
Cells were seeded on poly-D-lysine-coated, chambered glasses (20-mm2chamber, Nalge Nunc), fixed with 4% paraformaldehyde for 20 min. The cells were incubated with the primary Bβ1 or Bβ2 antiserum followed by TRITC-conjugated secondary anti-rabbit antibody. A concentration 10 nM mitotracker green (Molecular Probes) was added to counterstain mitochondria. Pictures of fixed cells were taken on a Zeiss LSM 510 Meta laser scanning confocal microscope at the Optical Molecular Imaging Microscopy Core Facility, National Taiwan University.
Transfection of the GFP-LC3 construct and examination of puncta fluorescence
The stables clones Bβ2 and Bβ1 derived from SK-N-SH cells were grown on sterile histologic slides, and, after 24 h, transfected with GFP-LC3 construct (gifted by Dr. Wei-Pang Huang, Department of Life Science, National Taiwan University) using a mixture of Lipofectamine (Invitrogen) and plasmid in Opti-MEM medium (Invitrogen) at a ratio of 5 μL Lipofectamine per milliliter of medium per 1 μL plasmid. After 6 h of incubation, cells were placed in regular complete medium and cultured for 1 day. The slides were washed with PBS, and cells fixed in cold methanol. Cells were then washed in PBS twice, and coverslips mounted with glycerol/PBS (3:1) solution. Slides were examined under a fluorescent microscope (Leica). Autophagy was quantified based on the mean numbers of puncta displaying intense staining for three fields (containing at least 50 cells per field) for each experimental condition. The amount of GFP-LC3 dots, each dot signal was detected by eye and the area was measured using the Photoshop 7.0 software (Adobe).
Detection of AVO' s by acridine orange staining
Cells were cultured with or without peroxides for 48 hours, washed with PBS, stained with medium containing 0.5 μg/ml of acridine orange for 15 minutes. The media was removed, the cells were washed and fluorescent micrographs taken using an inverted fluorescent microscope.
Western blot analysis
Cells cultured in 0.5% serum-supplemented media were washed with PBS and scraped in lysis buffer containing 1% triton X-100, 150 mM NaCl, 5 mM EDTA, 1% aprotonin, 5 mM PMSF and 10 μg/ml leupeptin in 20 mM sodium phosphate. Protein concentration was determined by the BCA assay (Pierce Biotechnology, Rockford, IL) and 20 μg of total protein was performed for Western blots analysis. Protein samples were electrophoresed on SDS-polyacrylamide gels, transferred to nitrocellulose filters). The blots wee incubated in fresh blocking solution and probed for 1 h with 1:3,000 dilution of Bβ1 and Bβ2 antisera, respectively. Blots were washed twice in PBS-T and then incubated with a 1:4,000 dilution of peroxidase-conjugated secondary antibody (Kirkegaard and Perry Laboratories, Inc., Gaithersburg, MD) in PBS-T for 1 h at 22°C. Blots were then again washed twice for 10 min in PBS-T and then detected by ECL illumination system (Amersham).
For siRNA transfection, 1 × 106 cells were transfected by nucleofection (Applied Amaxa Biosystems) and cell suspensions mixed with 1 or 5 μg of siRNA for Bβ2 cells. The cells were diluted in 2 ml DMEM (supplemented with 10% FBS, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 2 mM L-glutamine) at 37°C. The siRNA corresponding to the human cDNA sequence for ATG7 (5'-CAGTGGATCTAAATCTCAAACTGAT-3') was acquired from Sigma-Aldrich. The control siRNAs (SO) were acquired from Ambion. The ATG7 signal was verified by Western blotting with the anti-ATG7 rabbit polyclonal antibody (Cell Signalling Technology).
Unless otherwise indicated, results were expressed as compiled means ± S.E. from at least three independent experiments. Values of p < 0.05 were considered significant.
This work was supported by grant (ORD94-3) from National Taiwan Normal University.
- Mayer RE, Hendrix P, Cron P, Matthies R, Stone SR, Goris J, Merlevede W, Hofsteenge J, Hemmings BA: Structure of the 55-kDa regulatory subunit of protein phosphatase 2A: evidence for a neuronal-specific isoform. Biochemistry. 1991, 30 (15): 3589-3597. 10.1021/bi00229a001.View ArticlePubMedGoogle Scholar
- Price NE, Mumby MC: Brain protein serine/threonine phosphatases. Curr Opin Neurobiol. 1999, 9 (3): 336-342. 10.1016/S0959-4388(99)80049-X.View ArticlePubMedGoogle Scholar
- Strack S: Overexpression of the protein phosphatase 2A regulatory subunit Bgamma promotes neuronal differentiation by activating the MAP kinase (MAPK) cascade. J Biol Chem. 2002, 277 (44): 41525-41532. 10.1074/jbc.M203767200.View ArticlePubMedGoogle Scholar
- Strack S, Westphal RS, Colbran RJ, Ebner FF, Wadzinski BE: Protein serine/threonine phosphatase 1 and 2A associate with and dephosphorylate neurofilaments. Brain Res Mol Brain Res. 1997, 49 (1-2): 15-28. 10.1016/S0169-328X(97)00117-4.View ArticlePubMedGoogle Scholar
- Strack S, Zaucha JA, Ebner FF, Colbran RJ, Wadzinski BE: Brain protein phosphatase 2A: developmental regulation and distinct cellular and subcellular localization by B subunits. J Comp Neurol. 1998, 392 (4): 515-527. 10.1002/(SICI)1096-9861(19980323)392:4<515::AID-CNE8>3.0.CO;2-3.View ArticlePubMedGoogle Scholar
- Strack S, Chang D, Zaucha JA, Colbran RJ, Wadzinski BE: Cloning and characterization of B delta, a novel regulatory subunit of protein phosphatase 2A. FEBS Lett. 1999, 460 (3): 462-466. 10.1016/S0014-5793(99)01377-0.View ArticlePubMedGoogle Scholar
- McCright B, Rivers AM, Audlin S, Virshup DM: The B56 family of protein phosphatase 2A (PP2A) regulatory subunits encodes differentiation-induced phosphoproteins that target PP2A to both nucleus and cytoplasm. J Biol Chem. 1996, 271 (36): 22081-22089. 10.1074/jbc.271.36.22081.View ArticlePubMedGoogle Scholar
- Virshup DM: Protein phosphatase 2A: a panoply of enzymes. Curr Opin Cell Biol. 2000, 12 (2): 180-185. 10.1016/S0955-0674(99)00074-5.View ArticlePubMedGoogle Scholar
- Griswold-Prenner I, Kamibayashi C, Maruoka EM, Mumby MC, Derynck R: Physical and functional interactions between type I transforming growth factor beta receptors and B alpha, a WD-40 repeat subunit of phosphatase 2A. Mol Cell Biol. 1998, 18 (11): 6595-6604.PubMed CentralView ArticlePubMedGoogle Scholar
- Sontag E, Luangpirom A, Hladik C, Mudrak I, Ogris E, Speciale S, White CL: Altered expression levels of the protein phosphatase 2A ABalphaC enzyme are associated with Alzheimer disease pathology. J Neuropathol Exp Neurol. 2004, 63 (4): 287-301.PubMedGoogle Scholar
- Holmes SE, Hearn EO, Ross CA, Margolis RL: SCA12: an unusual mutation leads to an unusual spinocerebellar ataxia. Brain Res Bull. 2001, 56 (3-4): 397-403. 10.1016/S0361-9230(01)00596-2.View ArticlePubMedGoogle Scholar
- Holmes SE, O'Hearn E, Margolis RL: Why is SCA12 different from other SCAs?. Cytogenet Genome Res. 2003, 100 (1-4): 189-197. 10.1159/000072854.View ArticlePubMedGoogle Scholar
- HolmesE SEFH, O'Hearn E: Spinocerebellar ataxia type 12 (SCA12): additional evidence for a causative role of the CAG repeat expansion in PPP2R2B. Abstract Am J Hum Genet. 2000, 67: 1074-Google Scholar
- Holmes SE, O'Hearn EE, McInnis MG, Gorelick-Feldman DA, Kleiderlein JJ, Callahan C, Kwak NG, Ingersoll-Ashworth RG, Sherr M, Sumner AJ, Sharp AH, Ananth U, Seltzer WK, Boss MA, Vieria-Saecker AM, Epplen JT, Riess O, Ross CA, Margolis RL: Expansion of a novel CAG trinucleotide repeat in the 5' region of PPP2R2B is associated with SCA12. Nat Genet. 1999, 23 (4): 391-392. 10.1038/70493.View ArticlePubMedGoogle Scholar
- Turowski P, Myles T, Hemmings BA, Fernandez A, Lamb NJ: Vimentin dephosphorylation by protein phosphatase 2A is modulated by the targeting subunit B55. Mol Biol Cell. 1999, 10 (6): 1997-2015.PubMed CentralView ArticlePubMedGoogle Scholar
- Mayer-Jaekel RE, Ohkura H, Ferrigno P, Andjelkovic N, Shiomi K, Uemura T, Glover DM, Hemmings BA: Drosophila mutants in the 55 kDa regulatory subunit of protein phosphatase 2A show strongly reduced ability to dephosphorylate substrates of p34cdc2. J Cell Sci. 1994, 107 (Pt 9): 2609-2616.PubMedGoogle Scholar
- Sontag E, Fedorov S, Kamibayashi C, Robbins D, Cobb M, Mumby M: The interaction of SV40 small tumor antigen with protein phosphatase 2A stimulates the map kinase pathway and induces cell proliferation. Cell. 1993, 75 (5): 887-897. 10.1016/0092-8674(93)90533-V.View ArticlePubMedGoogle Scholar
- Van Kanegan MJ, Adams DG, Wadzinski BE, Strack S: Distinct protein phosphatase 2A heterotrimers modulate growth factor signaling to extracellular signal-regulated kinases and Akt. J Biol Chem. 2005, 280 (43): 36029-36036. 10.1074/jbc.M506986200.View ArticlePubMedGoogle Scholar
- Dagda RK, Zaucha JA, Wadzinski BE, Strack S: A developmentally regulated, neuron-specific splice variant of the variable subunit Bbeta targets protein phosphatase 2A to mitochondria and modulates apoptosis. J Biol Chem. 2003, 278 (27): 24976-24985. 10.1074/jbc.M302832200.View ArticlePubMedGoogle Scholar
- Cholfin JA, Sobrido MJ, Perlman S, Pulst SM, Geschwind DH: The SCA12 mutation as a rare cause of spinocerebellar ataxia. Arch Neurol. 2001, 58 (11): 1833-1835. 10.1001/archneur.58.11.1833.View ArticlePubMedGoogle Scholar
- Dagda RK, Merrill RA, Cribbs JT, Chen Y, Hell J, Usachev YM, Strack S: The spinocerebellar ataxia 12 gene product and protein phosphatase 2A regulatory subunit Bbeta2 antagonizes neuronal survival by promoting mitochondrial fission. J Biol Chem. 2008, 283 (52): 36241-36248. 10.1074/jbc.M800989200.PubMed CentralView ArticlePubMedGoogle Scholar
- Chu CT: Autophagic stress in neuronal injury and disease. J Neuropathol Exp Neurol. 2006, 65 (5): 423-432. 10.1097/01.jnen.0000229233.75253.be.PubMed CentralView ArticlePubMedGoogle Scholar
- Dagda RK, Zhu J, Kulich SM, Chu CT: Mitochondrially localized ERK2 regulates mitophagy and autophagic cell stress: implications for Parkinson's disease. Autophagy. 2008, 4 (6): 770-782.PubMed CentralView ArticlePubMedGoogle Scholar
- Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T, Kominami E, Ohsumi Y, Yoshimori T: LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J. 2000, 19 (21): 5720-5728. 10.1093/emboj/19.21.5720.PubMed CentralView ArticlePubMedGoogle Scholar
- Paglin S, Hollister T, Delohery T, Hackett N, McMahill M, Sphicas E, Domingo D, Yahalom J: A novel response of cancer cells to radiation involves autophagy and formation of acidic vesicles. Cancer Res. 2001, 61 (2): 439-444.PubMedGoogle Scholar
- Seglen PO, Gordon PB: 3-Methyladenine: Specific inhibitor of autophagic/lysosomal protein degradation in isolated rat hepatocytes. Proceedings of the National Academy of Sciences USA. 1892, 79 (6): 1889-1982. 10.1073/pnas.79.6.1889.View ArticleGoogle Scholar
- Komatsu M, Wang QJ, Holstein GR, Friedrich VL, Iwata J-i, Kominami E, Chait BT, Tanaka K, Yue Z: Essential role for autophagy protein Atg7 in the maintenance of axonal homeostasis and the prevention of axonal degeneration. Proceedings of the National Academy of Sciences USA. 2007, 104 (36): 14489-14494. 10.1073/pnas.0701311104.View ArticleGoogle Scholar
- Goedert M, Satumtira S, Jakes R, Smith MJ, Kamibayashi C, White CL, Sontag E: Reduced binding of protein phosphatase 2A to tau protein with frontotemporal dementia and Parkinsonism linked to chromosome 17 mutations. J Neurochem. 2000, 75 (5): 2155-2162. 10.1046/j.1471-4159.2000.0752155.x.View ArticlePubMedGoogle Scholar
- Avila J, Lucas JJ, Perez MAR, Hernandez F: Role of tau protein in both physiological and pathological conditions. Physiol Rev. 2004, 84 (2): 361-384. 10.1152/physrev.00024.2003.View ArticlePubMedGoogle Scholar
- Duenas AM, Goold R, Giunti P: Molecular pathogenesis of spinocerebellar ataxias. Brain. 2006, 129 (Pt 6): 1357-1370. 10.1093/brain/awl081.View ArticlePubMedGoogle Scholar
- Codogno P, Meijer AJ: Autophagy and signaling: their role in cell survival and cell death. Cell Death Differ. 2005, 12 (Suppl 2): 1509-1518. 10.1038/sj.cdd.4401751.View ArticlePubMedGoogle Scholar
- Komatsu M, Waguri S, Chiba T, Murata S, Iwata J, Tanida I, Ueno T, Koike M, Uchiyama Y, Kominami E, Tanaka K: Loss of autophagy in the central nervous system causes neurodegeneration in mice. Nature. 2006, 441 (7095): 880-884. 10.1038/nature04723.View ArticlePubMedGoogle Scholar
- Holmes S, Hearn E, Ross C, Margolis R: SCA12: an unusual mutation leads to an unusual spinocerebellar ataxia. Brain Res Bull. 2001, 56 (3): 397-403. 10.1016/S0361-9230(01)00596-2.View ArticlePubMedGoogle Scholar
- Pan T, Kondo S, Le W, Jankovic J: The role of autophagy-lysosome pathway in neurodegeneration associated with Parkinson's disease. Brain. 2008, 131 (8): 1969-1978. 10.1093/brain/awm318.View ArticlePubMedGoogle Scholar
- Strack S, Cribbs JT, Gomez L: Critical role for protein phosphatase 2A heterotrimers in mammalian cell survival. J Biol Chem. 2004, 279 (46): 47732-47739. 10.1074/jbc.M408015200.View ArticlePubMedGoogle Scholar
- Ikehara T, Imamura S, Oshiro N, Ikehara S, Shinjo F, Yasumoto T: A protein phosphatase 2A (PP2A) inhibition assay using a recombinant enzyme for rapid detection of microcystins. Toxicon. 2008, 51 (8): 1368-1373. 10.1016/j.toxicon.2008.03.003.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.