2003/06/29 by Julie L. Webb, Brinda Ravikumar, Jane Atkins +3 · 39 citations
Medicine · Neuroscience · #Parkinson's Disease Mechanisms and Treatments #Autophagy in Disease and Therapy #Nerve injury and regeneration
paper · pdf · doi:10.1074/jbc.m300227200
Parkinson's disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra and the formation of aggregates (Lewy bodies) in neurons. α-Synuclein is the major protein in Lewy bodies and rare mutations in α-synuclein cause early-onset PD. Consequently, α-synuclein is implicated in the pathogenesis of PD. Here, we have investigated the degradation pathways of α-synuclein, using a stable inducible PC12 cell model, where the expression of exogenous human wild-type, A30P, or A53T α-synuclein can be switched on and off. We have used a panel of inhibitors/stimulators of autophagy and proteasome function and followed α-synuclein degradation in these cells. We found that not only is α-synuclein degraded by the proteasome, but it is also degraded by autophagy. A role for autophagy was further supported by the presence of α-synuclein in organelles with the ultrastructural features of autophagic vesicles. Since rapamycin, a stimulator of autophagy, increased clearance of α-synuclein, it merits consideration as a potential therapeutic for Parkinsons disease, as it is designed for chronic use in humans. Parkinson's disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra and the formation of aggregates (Lewy bodies) in neurons. α-Synuclein is the major protein in Lewy bodies and rare mutations in α-synuclein cause early-onset PD. Consequently, α-synuclein is implicated in the pathogenesis of PD. Here, we have investigated the degradation pathways of α-synuclein, using a stable inducible PC12 cell model, where the expression of exogenous human wild-type, A30P, or A53T α-synuclein can be switched on and off. We have used a panel of inhibitors/stimulators of autophagy and proteasome function and followed α-synuclein degradation in these cells. We found that not only is α-synuclein degraded by the proteasome, but it is also degraded by autophagy. A role for autophagy was further supported by the presence of α-synuclein in organelles with the ultrastructural features of autophagic vesicles. Since rapamycin, a stimulator of autophagy, increased clearance of α-synuclein, it merits consideration as a potential therapeutic for Parkinsons disease, as it is designed for chronic use in humans. Parkinson's disease (PD) 1The abbreviations used are: PD, Parkinson's disease; HA, hemagglutinin; DAPI, 4′,6-diamidino-2-phenylindole; 3-MA, 3-methyladenine; PIPES, 1,4-piperazinediethanesulfonic acid; baf A1, bafilomycin A1. is caused by the degeneration of dopaminergic neurons in the substantia nigra. The pathogenic hallmark of PD is the accumulation and aggregation of α-synuclein in susceptible neurons. The cytoplasmic aggregates/inclusions characteristic of PD are called Lewy bodies and their major constituent is α-synuclein (1Kahle P.J. Haass C. Kretzschmer H.A. Neumann M. J. Neurochem. 2002; 82: 449-457Google Scholar). Lewy pathology is also found in dementia with Lewy bodies, the Lewy body variant of Alzheimer's disease, in neurodegeneration with brain iron accumulation type I and in glial cytoplasmic inclusions of multiple system atrophy. These diseases are collectively known as “α-synucleinopathies” (2Spillantini M.G. Schmidt M.L. Lee V.M. Trojanowski J.Q. Jakes R. Goedert M. Nature. 1997; 388: 839-840Google Scholar, 3Mezey E. Dehejia A.M. Harta G. Suchy S.F. Nussbaum R.L. Brownstein M.J. Polymeropoulos M.H. Mol. Psychiatry. 1998; 3: 493-499Google Scholar). α-Synuclein is likely to play a role in PD, since two missense mutations in α-synuclein (A53T and A30P) cause autosomal dominant, early-onset PD (4Polymeropoulos M.H. Lavedan C. Leroy E. Ide S.E. Dehejia A. Dutra A. Pike B. Root H. Rubenstein J. Boyer R. Stenroos E.S. Chandrasekharappa S. Athanassiadou A. Papapetropoulos T. Johnson W.G. Lazzarini A.M. Duvoisin R.C. Di Iorio G. Golbe L.I. Nussbaum R.L. Science. 1997; 276: 2045-2047Google Scholar, 5Krüger R. Kuhn W. Muller T. Woitalla D. Graeber M. Kosel S. Przuntek H. Epplen T.T. Schols L. Riess O. Nat. Genet. 1998; 18: 106-108Google Scholar), and animal models overexpressing α-synuclein develop a disease phenotype with Lewy body-like pathology and locomotor impairment (6Feany M.B. Bender W.W. Nature. 2000; 404: 394-398Google Scholar, 7Masliah E. Rockenstein E. Veinbergs I. Mallory M. Hashimoto M. Takeda A. Sagara Y. Sisk A. Mucke L. Science. 2000; 287: 1265-1269Google Scholar). However, while α-synuclein knock-out mice show some disruption in synaptic neurotransmission, they do not manifest obvious disease and develop normally, with a normal life span and behavior (8Abeliovich A. Schmitz Y. Farinas I. Choi-Lundberg D. Ho W.H. Castillo P.E. Shinsky N. Verdugo J.M. Armanini M. Ryan A. Hynes M. Phillips H. Sulzer D. Rosenthal A. Neuron. 2000; 25: 239-252Google Scholar, 9Chen P.E. Specht C.G. Morris R.G. Schoepfer R. Eur. J. Neurosci. 2002; 16: 154-158Google Scholar, 10Cabin D.E. Shimazu K. Murphy D. Cole N.B. Gottschalk W. McIlwain K.L. Orrison B. Chen A. Ellis C.E. Paylor R. Lu B. Nussbaum R.L. J. Neurosci. 2002; 22: 8797-8807Google Scholar). Two other forms of inherited PD are caused by mutations in Parkin (an E3 ubiquitin ligase) (11Kitada T. Asakawa S. Hattori N. Matsumine H. Yamamura Y. Minoshima S. Yokochi M. Mizuno Y. Shimizu N. Nature. 1998; 392: 605-608Google Scholar) and ubiquitin carboxyL-terminal hydrolase L1 (UCH-L1) (12Leroy E. Boyer R. Auburger G. Leube B. Ulm G. Mezey E. Harta G. Brownstein M.J. Jonnalagada S. Chernova T. Dehejia A. Lavedon C. Gasser T. Steinbach P.J. Wilkinson K.D. Polymeropoulos M.H. Nature. 1998; 395: 451-452Google Scholar). Both of these proteins are involved in the ubiquitin-dependent degradation of intracellular proteins (13Ciechanover A. EMBO J. 1998; 17: 7151-7160Google Scholar). This evidence and the identification of proteasome subunits in Lewy bodies (14Ii K. Ito H. Tanaka K. Hirano A. J. Neuropathol. Exp. Neurol. 1997; 56: 125-131Google Scholar) has led to the speculation that an impairment of the ubiquitin-proteasome system may contribute to the progression of PD. A number of studies have investigated the effect of proteasomal inhibition on α-synuclein metabolism, with conflicting results. Some groups reported that proteasomal inhibition caused an accumulation of α-synuclein, inclusion formation and increased cell death (15Bennett M.C. Bishop J.F. Leng Y. Chock P.B. Chase T.N. Mouradian M.M. J. Biol. Chem. 1999; 274: 33855-33858Google Scholar, 16McNaught K.St.P. Mytilineou C. JnoBaptiste R. Yabut J. Shashidharan P. Jenner P. Olanow C.W. J. Neurochem. 2002; 81: 301-306Google Scholar, 17Tofaris G.K. Layfield R. Spillantini M.G. FEBS Lett. 2001; 509: 22-26Google Scholar), while other studies suggested α-synuclein is not a proteasome substrate (18Rideout H.J. Larsen K.E. Sulzer D. Stefanis L. J. Neurochem. 2001; 78: 899-908Google Scholar, 19Ancolio K. Alves da Costa C. Ueda K. Checler F. Neurosci. Lett. 2000; 285: 79-82Google Scholar). Another pathway that may be relevant to α-synuclein clearance is autophagy, a process mediating bulk degradation of cytoplasmic proteins or organelles in the lytic compartment. Autophagy involves the formation of double-membrane structures called autophagosomes, which fuse with primary lysosomes to become an autophagolysosome where their content is degraded and then either disposed of or recycled back to the cell (20Klionsky D.J. Oshumi Y. Annu. Rev. Cell Dev. Biol. 1999; 15: 1-32Google Scholar). We have recently shown that aggregate-prone proteins with polyglutamine and polyalanine expansions are degraded by autophagy (21Ravikumar B. Duden R. Rubinsztein D.C. Hum. Mol. Gen. 2002; 11: 1107-1117Google Scholar). Since α-synuclein is also an aggregate-prone protein, we have tested whether it is degraded by this pathway. Our previous data suggested that aggregate-prone proteins were more likely to be cleared by autophagy than the more soluble species that did not have expanded polyglutamines or polyalanines. In the context of α-synuclein, this model predicts that the A53T mutation that forms aggregates most readily may be more dependent on autophagy than the wild-type protein or A30P mutation (that forms oligomers efficiently but not aggregates) (22Conway K.A. Harper J.D. Lansbury P.T. Nat. Med. 1998; 4: 1318-1320Google Scholar). In this study, we have used PC12 cells to establish stable, doxycycline-inducible lines as they are dopaminergic and can be differentiated into a neuron-like phenotype with nerve growth factor (23Greene L.A. Tischler A.S. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 2424-2428Google Scholar). We used the Tet-On system, where regulation of expression is achieved through a tetracycline-controlled transactivator. This allows us to turn on and off α-synuclein expression by adding and then removing doxycycline and therefore enables us to follow α-synuclein clearance. In our cell lines we found that α-synuclein was degraded by both proteasome and autophagy pathways. Establishment of Inducible PC12 Cell Lines—Human α-synuclein with a HA tag at the NH2 terminus and a His6 tag at the COOH terminus was amplified from pHM6α-synuclein constructs (24Furlong R.A. Narain Y. Rankin J. Wyttenbach A. Rubinsztein D.C. Biochem. J. 2000; 346: 577-581Google Scholar) and inserted into pTRE2hyg vector (Clontech) using NheI/SalI sites. Constructs were confirmed by sequencing before use. The pTRE2hygα-synuclein construct and pTet-tTS (Clontech) were co-transfected into PC12 Tet-On cells (Clontech), using LipofectAMINE (Invitrogen) and Plus Reagent (Invitrogen). Single colonies were isolated using cloning cylinders (Sigma), and cells were grown and tested for α-synuclein expression upon induction. Cell lines were subjected to a further round of purification from single colonies to ensure that lines were pure. Cell Culture—PC12 cells were grown in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% horse serum (Sigma), 5% fetal bovine serum (Sigma), 100 units/ml penicillin/streptomycin, 2 mm l-glutamine, 50 μg/ml G418 (Invitrogen), and 149 μg/ml hygromycin B (Calbiochem) at 37 °C, 10%CO2. To induce differentiation, cells were grown in media containing 1% horse serum and 100 ng/ml nerve growth factor (2.5 S, Upstate Biotechnology) and incubated for about 5 days. Cells were induced to express synuclein with 2 μg/ml doxycycline (Sigma). Immunofluorescence—Coverslips were placed in six-well dishes and coated with 0.01% poly-l-lysine (Sigma). Cells were seeded and induced with 2 μg/ml doxycycline and incubated as necessary. Cells were fixed with 4% paraformaldehyde (Sigma) for 30 min and then washed with PBS and permeabilized with 0.1% Triton-X-100 (Sigma) for 15 min. Cells were blocked in 10% fetal calf serum for at least 30 min. Anti-α-synuclein monoclonal antibody (BD Biosciences) was used at 1:200 for 2–16 h, cells were washed and 1:200 Cy3-conjugated anti-mouse antibody (Jackson ImmunoResearch Laboratories) was added for 1 h. This anti-α-synuclein antibody could detect both human and rat α-synuclein; therefore, uninduced controls were performed in parallel to compare endogenous α-synuclein levels. Staining was always considerably fainter in the uninduced controls. For double staining with LysoTracker Red (Molecular Probes) cells were incubated in Earle's balanced salts solution (Sigma) with 75 nm LysoTracker for 2 h at 37 °C. Cells were fixed and stained with anti-α-synuclein antibody and 1:200 fluorescein isothiocyanate-conjugated anti-mouse antibody (Jackson ImmunoResearch Laboratories) was used as secondary antibody. Slides were mounted in Citifluor (Citifluor Ltd.) with 3 μg/ml 4′,6-diamidino-2-phenylindole (DAPI, Sigma). Cells were visualized using a Zeiss LSM510 confocal microscope. Treatment with Autophagy/Proteasome Drugs—Cells were induced for 24 h and then washed twice with medium to remove doxycycline. Then cells were incubated in media containing either 10 mm 3-methyladenine (3-MA, Sigma), 200 nm bafilomycin A1 (Sigma), 0.2 mg/ml rapamycin (Sigma), 10 μm lactacystin (Sigma), 10 μm epoxomicin (Affinity Research Products Ltd.) or carrier controls (water or Me2SO (Sigma)). 3-MA and lactacystin were made up in water and rapamycin, epoxomicin, and bafilomycin were dissolved in Me2SO. After 24 h the medium was replaced with fresh medium plus drug, and after a further 24 h cell pellets were collected and stored at –80 °C until required. Western Blot Analysis—Cell pellets were collected and stored at –80 °C until needed. Cells were lysed on ice in lysis buffer: 1% Triton-X-100, 20 mm Tris, pH 7.5, 137 mm NaCl, 1 mm EGTA, 10% glycerol, 1.5 mm MgCl2, and protease inhibitor mixture (Complete, Roche Applied Science). Samples were then mixed with loading buffer: 62.5 mm Tris, pH 6.8, 2% SDS, 10% glycerol, 0.05% bromphenol blue, 100 mm dithiothreitol, and 700 mm β-mercaptoethanol and boiled before loading onto 14% denaturing polyacrylamide gels. Each lane was loaded with protein from a similar number of cells, based on cell counting at the time of seeding. Proteins were transferred onto Hybond ECL nitrocellulose membrane α-Synuclein was with an monoclonal antibody at with h anti-mouse antibody Biosciences) at was then added to and antibody was using ECL Biosciences) and ECL were and then for (Sigma) at for were induced with 2 μg/ml doxycycline for h. The cells were fixed in with 2% and 0.05% in and by The cells were incubated in containing 5% bovine serum and by at in a and the was of the cells were mounted onto and by into in After the cells were transferred into a in of containing were at °C for 24 h followed by 24 h at °C and 24 h at °C. were with 3 and by with for h. were using a and mounted on The were incubated in primary in at pH containing 0.1% 0.1% Triton-X-100, fetal calf and 10% normal The were washed in and incubated with anti-mouse to in the for the primary antibody at pH added serum for 1 h J. 2000; Scholar). were in and twice in water and stained with and before in a microscope. Establishment of Inducible α-Synuclein Cell stable, inducible lines for human wild-type, A30P, and A53T α-synuclein in PC12 cells using the Tet-On system, where of doxycycline on We two lines for of α-synuclein, on the of expression and and tested these lines in our We at α-synuclein in our induced cells. We found that α-synuclein was the cells, with a of staining The cytoplasmic and of α-synuclein was similar to previous with this protein in PC12 cells (18Rideout H.J. Larsen K.E. Sulzer D. Stefanis L. J. Neurochem. 2001; 78: 899-908Google Scholar). The shown is of α-synuclein in our cell lines wild-type, A30P, and A53T with either or differentiated cells, induced for or 10 days. the it was whether the structures were We did not aggregates characteristic of polyglutamine and polyalanine expansions Y. Wyttenbach A. Rankin J. R.A. Rubinsztein D.C. J. Med. Genet. 1999; Scholar) for either wild-type, A30P, or after expression of α-synuclein for 10 days. The staining for α-synuclein in uninduced cells was the of with confocal using the that for induced cells However, α-synuclein in the uninduced cells was the was this staining is likely to endogenous α-synuclein in the cells, since is of expression in uninduced cells We also cell death by cell and of after We did not a in of cells after of α-synuclein expression in of the lines wild-type, A30P, or A53T not In both and differentiated cells, cell death was at up to 10 of by staining for wild-type, A30P, or of α-synuclein after expression is switched off in the inducible cell Western of exogenous human α-synuclein in the inducible PC12 cell Cells were uninduced or induced with 2 μg/ml doxycycline for 24 h or induced for 24 h and then washed to remove doxycycline and for a further h in The show the α-synuclein with an and the show as a loading that where the uninduced and induced as these were from from the of from single where from uninduced and induced cells were were to In this study, we have α-synuclein by Western using an antibody to of the in this are and were performed at least twice with cell We used our cell model to on expression of α-synuclein with doxycycline and then off expression by removing doxycycline from the to follow α-synuclein of α-synuclein were h after expression was switched with the time at which doxycycline was α-Synuclein by the in Our Cell switched on α-synuclein expression for 24 h, then doxycycline and added the proteasome epoxomicin or lactacystin for h. We an in α-synuclein with with the proteasome epoxomicin or in both and differentiated cells in cell lines We used both as have shown that lactacystin also A of the and may not be H. C. S. S. L. T. K. P. J. Biochem. Cell Biol. 2000; Scholar, A. Eur. J. Cell Biol. Scholar). of these did not cause increased cell death or α-Synuclein by then tested whether α-synuclein is also degraded by autophagy using a panel of a we have used in the context of 3-MA is a inhibitor of autophagy (20Klionsky D.J. Oshumi Y. Annu. Rev. Cell Dev. Biol. 1999; 15: 1-32Google Scholar) autophagy at the where a double membrane forms a of the A1 is a inhibitor that with the A. Y. T. Y. R. Y. Cell 1998; Scholar), and rapamycin is an that autophagy. To the role of autophagy on α-synuclein clearance in our cell they were induced for 24 h, then doxycycline was and the were added for h. The we were similar in cells with for 24 and h not autophagy with 3-MA and baf A1 led to an obvious of α-synuclein in the A53T lines These only induced at in α-synuclein in wild-type and A30P lines 3-MA with which autophagy, increased clearance of wild-type, A30P, and A53T α-synuclein in cells In differentiated cells, the A53T lines to α-synuclein after with 3-MA and baf A1. Since the of these on the wild-type and A30P lines were these were to us to do We the of α-synuclein a function of for 3-MA and baf A1 to for cells and the in α-synuclein in the cells after Both on A30P accumulation 3-MA, in A30P baf A1, the for wild-type α-synuclein that these were the data did not 3-MA, wild-type baf A1, we autophagy in differentiated cells by adding rapamycin, we increased clearance of wild-type, A30P, and A53T α-synuclein α-Synuclein in with the of α-synuclein in our cell lines using We used an antibody to the on the α-synuclein to detect α-synuclein for α-synuclein was with autophagic A of is in these since are to of or in or in PC12 cells. the we with in previous studies of autophagy N. A. M. Y. Y. K. T. Y. T. J. Cell Biol. 2001; Scholar), disease M. E. C. N. M. J. Neurosci. 2000; Scholar), and α-synuclein L. Larsen K.E. H.J. Sulzer D. L.A. J. Neurosci. 2001; Scholar). these were as autophagic on similar in the of α-Synuclein was either or with bodies of 100 nm in were two or more these bodies, which could α-synuclein bodies were also in the with is that these bodies may be studies that α-synuclein A30P, and was using LysoTracker Red LysoTracker Red autophagic and that α-synuclein was not from these and of α-synuclein are these These data our studies that a role for the pathway for α-synuclein We have shown that α-synuclein is degraded by both the proteasome and autophagy in our inducible PC12 cell Our Western data autophagy as a clearance for this protein are supported by of wild-type, and A30P species in autophagic by This is the that autophagy as as the proteasome as a for α-synuclein of autophagy a more effect on the degradation of A53T α-synuclein, which may be to to with wild-type and A30P (22Conway K.A. Harper J.D. Lansbury P.T. Nat. Med. 1998; 4: 1318-1320Google Scholar). This be with the of K. M.G. Hum. Mol. Genet. 2002; 11: Scholar), that but not polyglutamine expansions could be degraded by the This is proteins the to the proteasome this is the then proteins be to the pathway for A model where soluble α-synuclein is cleared by the proteasome but α-synuclein is cleared by autophagy may for the conflicting previous data by the proteasome (15Bennett M.C. Bishop J.F. Leng Y. Chock P.B. Chase T.N. Mouradian M.M. J. Biol. Chem. 1999; 274: 33855-33858Google Scholar, 16McNaught K.St.P. Mytilineou C. JnoBaptiste R. Yabut J. Shashidharan P. Jenner P. Olanow C.W. J. Neurochem. 2002; 81: 301-306Google Scholar, 17Tofaris G.K. Layfield R. Spillantini M.G. FEBS Lett. 2001; 509: 22-26Google Scholar, H.J. Larsen K.E. Sulzer D. Stefanis L. J. Neurochem. 2001; 78: 899-908Google Scholar, 19Ancolio K. Alves da Costa C. Ueda K. Checler F. Neurosci. Lett. 2000; 285: 79-82Google Scholar). α-Synuclein expression and aggregation is likely to cell and with and this may the (15Bennett M.C. Bishop J.F. Leng Y. Chock P.B. Chase T.N. Mouradian M.M. J. Biol. Chem. 1999; 274: 33855-33858Google Scholar, 16McNaught K.St.P. Mytilineou C. JnoBaptiste R. Yabut J. Shashidharan P. Jenner P. Olanow C.W. J. Neurochem. 2002; 81: 301-306Google Scholar, 17Tofaris G.K. Layfield R. Spillantini M.G. FEBS Lett. 2001; 509: 22-26Google Scholar, H.J. Larsen K.E. Sulzer D. Stefanis L. J. Neurochem. 2001; 78: 899-908Google Scholar, 19Ancolio K. Alves da Costa C. Ueda K. Checler F. Neurosci. Lett. 2000; 285: 79-82Google Scholar). studies do a role for the clearance of forms of α-synuclein by the proteasome H. M.G. Hattori N. A. R. Mizuno Y. D.J. Science. 2001; Scholar, L. C. P.J. M. K. L. P. B. M. J. Neuron. 2002; Scholar). Our data that the of wild-type and A30P α-synuclein on autophagy for degradation is in differentiated cells. could that this may be the of cell in the differentiated cells may of our data is that proteasome were more than autophagy in For in bafilomycin A1 has rapamycin clearance of This may be with increased of the α-synuclein into an autophagy pathway that may be by the of this protein L. Larsen K.E. H.J. Sulzer D. L.A. J. Neurosci. 2001; Scholar). rapamycin autophagy 3-MA, the clearance of proteasomal E. M. S. EMBO J. 1999; 18: Scholar) or intracellular E.S. J. Biol. Chem. 2002; Scholar). However, for the of autophagy with proteasome on α-synuclein clearance may be that the autophagy are or that the of intracellular α-synuclein clearance through the proteasome is than through the autophagy this be with the that α-synuclein is cleared by autophagy, while soluble forms may be efficiently degraded by the We have shown that the autophagy rapamycin clearance of forms of This may have therapeutic potential for of Parkinson's disease as rapamycin is for use in A at of α-synuclein in may be as a of PD, of this protein as as