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• W2042340343 abstract "Proteasomal dysfunction may underlie certain neuro-degenerative conditions such as Parkinson disease. We have shown that pharmacological inhibition of the proteasome in cultured neuronal cells leads to apoptotic death and formation of cytoplasmic ubiquitinated inclusions. These inclusions stain for α-synuclein and assume a fibrillar structure, as assessed by thioflavine S staining, and therefore resemble Lewy bodies. α-Synuclein is thought to be a central component of Lewy bodies. Whether α-synuclein is required for inclusion formation or apoptotic death has not been formally assessed. The present study examines whether α-synuclein deficiency in neurons alters their sensitivity to proteasomal inhibition-induced apoptosis or inclusion formation. Cortical neurons derived from α-synuclein-null mice showed a similar sensitivity to death induced by the proteasomal inhibitor lactacystin compared with neurons derived from wild-type mice. Furthermore, the absence of α-synuclein did not influence the percentage of lactacystin-treated neurons harboring cytoplasmic ubiquitinated inclusions or alter the solubility of such inclusions. In contrast, however, ubiquitinated inclusions in α-synuclein-deficient neurons lacked amyloid-like fibrillization, as determined by thioflavine S staining. This indicates that although α-synuclein deficiency does not affect the formation of ubiquitinated inclusions, it does significantly alter their structure. The lack of effect on survival in α-synuclein knock-out cultures further suggests that the fibrillar nature of the inclusions does not contribute to neuronal degeneration in this model. Proteasomal dysfunction may underlie certain neuro-degenerative conditions such as Parkinson disease. We have shown that pharmacological inhibition of the proteasome in cultured neuronal cells leads to apoptotic death and formation of cytoplasmic ubiquitinated inclusions. These inclusions stain for α-synuclein and assume a fibrillar structure, as assessed by thioflavine S staining, and therefore resemble Lewy bodies. α-Synuclein is thought to be a central component of Lewy bodies. Whether α-synuclein is required for inclusion formation or apoptotic death has not been formally assessed. The present study examines whether α-synuclein deficiency in neurons alters their sensitivity to proteasomal inhibition-induced apoptosis or inclusion formation. Cortical neurons derived from α-synuclein-null mice showed a similar sensitivity to death induced by the proteasomal inhibitor lactacystin compared with neurons derived from wild-type mice. Furthermore, the absence of α-synuclein did not influence the percentage of lactacystin-treated neurons harboring cytoplasmic ubiquitinated inclusions or alter the solubility of such inclusions. In contrast, however, ubiquitinated inclusions in α-synuclein-deficient neurons lacked amyloid-like fibrillization, as determined by thioflavine S staining. This indicates that although α-synuclein deficiency does not affect the formation of ubiquitinated inclusions, it does significantly alter their structure. The lack of effect on survival in α-synuclein knock-out cultures further suggests that the fibrillar nature of the inclusions does not contribute to neuronal degeneration in this model. Since the identification of two α-synuclein mutations that are linked to the development of autosomal dominant Parkinson disease (PD) 1The abbreviations used are: PD, Parkinson disease; E3, ubiquitin-protein isopeptide ligase; LBs, Lewy bodies; WT, wild-type; DIV, days in vitro; PIPES, 1,4-piperazinediethanesulfonic acid; HMW, higher molecular weight; KO, knock-out.1The abbreviations used are: PD, Parkinson disease; E3, ubiquitin-protein isopeptide ligase; LBs, Lewy bodies; WT, wild-type; DIV, days in vitro; PIPES, 1,4-piperazinediethanesulfonic acid; HMW, higher molecular weight; KO, knock-out. (1Polymeropoulos 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: 2045-2047Crossref PubMed Scopus (6511) Google Scholar, 2Krueger R. Kuhn W. Muller T. Woitalla D. Graeber M. Kosel S. Przuntek H. Epplen J.T. Schols L. Riess O. Nat. Genet. 1998; 18: 106-108Crossref PubMed Scopus (3263) Google Scholar), considerable effort has been made to characterize both the normal function of this protein and its role in the pathogenesis of PD and related synucleinopathies. Additional genetic abnormalities have since been described in patients with PD, including mutations in the genes encoding for Parkin (3Kitada T. Asakawa S. Hattori N. Matsumine H. Yamamura Y. Minoshima S. Yokochi M. Mizuno Y. Shimizu N. Nature. 1998; 392: 605-608Crossref PubMed Scopus (4123) Google Scholar) and UCH-L1 (4Leroy E. Boyer R. Auburger G. Leube B. Ulm G. Mezey E. Harta G. Brownstein M.J. Jonnalagada S. Chernova T. Dehejia A. Lavedan C. Gasser T. Steinbach P.J. Wilkinson K.D. Polymeropoulos M.H. Nature. 1998; 395: 451-452Crossref PubMed Scopus (1372) Google Scholar). These two proteins are notable in that they are both involved in proteasomal-dependent degradation of ubiquitinated proteins, linking dysfunction of ubiquitin-dependent protein clearance to the pathogenesis of PD (5Lang-Rollin I. Rideout H. Stefanis L. Histol. Histopathol. 2003; 18: 509-517PubMed Google Scholar). Parkin has E3 ligase activity, which is sensitive to PD-linked mutations (6Shimura H. Hattori N. Kubo S. Mizuno Y. Asakawa S. Minoshima S. Shimizu N. Iwai K. Chiba T. Tanaka K. Suzuki T. Nat. Genet. 2000; 25: 302-305Crossref PubMed Scopus (1682) Google Scholar). UCH-L1 is a neuronal-specific protein that mediates the de-ubiquitination of proteins and the cleavage of pro-ubiquitin polypeptides into monomeric ubiquitin (4Leroy E. Boyer R. Auburger G. Leube B. Ulm G. Mezey E. Harta G. Brownstein M.J. Jonnalagada S. Chernova T. Dehejia A. Lavedan C. Gasser T. Steinbach P.J. Wilkinson K.D. Polymeropoulos M.H. Nature. 1998; 395: 451-452Crossref PubMed Scopus (1372) Google Scholar, 7Ciechanover A. EMBO J. 1998; 17: 7151-7160Crossref PubMed Scopus (1175) Google Scholar). More recent data suggest that the main function of UCH-L1 is to stabilize monomeric ubiquitin (8Osaka H. Wang Y.L. Takada K. Takizawa S. Setsuie R. Li H. Sato Y. Nishikawa K. Sun Y.J. Sakurai M. Harada T. Hara Y. Kimura I. Chiba S. Namikawa K. Kiyama H. Noda M. Aoki S. Wada K. Hum. Mol. Genet. 2003; 12: 1945-1958Crossref PubMed Scopus (318) Google Scholar). Another report suggests that UCH-L1 has an additional E3 ligase activity that may be more relevant to its relationship to PD (9Liu Y. Fallon L. Lashuel H.A. Liu Z. Lansbury Jr., P.T. Cell. 2002; 111: 209-218Abstract Full Text Full Text PDF PubMed Scopus (686) Google Scholar). In addition, α-synuclein may associate with the 19 S or the 20 S proteasome (10Ghee M. Fournier A. Mallet J. J. Neurochem. 2000; 75: 2221-2224Crossref PubMed Scopus (93) Google Scholar, 11Snyder H. Mensah K. Theisler C. Lee J. Matouschek A. Wolozin B. J. Biol. Chem. 2003; 278: 11753-11759Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 12Lindersson E. Beedholm R. Hojrup P. Moos T. Gai W. Hendil K.B. Jensen P.H. J. Biol. Chem. 2004; 279: 12924-12934Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar), an association that may be responsible for the reported effects of α-synuclein on proteasome function in cellular systems (13Tanaka Y. Engelender S. Igarashi S. Rao R.K. Wanner T. Tanzi R.E. Sawa A. Dawson V.L. Dawson T.M. Ross C.A. Hum. Mol. Genet. 2001; 10: 919-926Crossref PubMed Google Scholar, 14Stefanis L. Larsen K.E. Rideout H.J. Sulzer D. Greene L.A. J. Neurosci. 2001; 21: 9549-9560Crossref PubMed Google Scholar, 15Petrucelli L. O'Farrell C. Lockhart P.J. Baptista M. Kehoe K. Vink L. Choi P. Wolozin B. Farrer M. Hardy J. Cookson M.R. Neuron. 2002; 36: 1007-1019Abstract Full Text Full Text PDF PubMed Scopus (503) Google Scholar).α-Synuclein is an abundant protein of unclear function. It is a natively unfolded protein and aggregates in vitro following an ordered transition to an oligomeric protofibril and ultimately to a fibrillar β-pleated sheet structure. Regions within α-synuclein have been identified that mediate or are necessary for such oligomerization or aggregation of the protein (16Conway K.A. Harper J.D. Lansbury P.T. Nat. Med. 1998; 11: 1318-1320Crossref Scopus (1242) Google Scholar, 17Giasson B.I. Uryu K. Trojanowski J.Q. Lee V.M. J. Biol. Chem. 1999; 274: 7619-7622Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar, 18Vekrellis K. Rideout H.J. Stefanis L. Mol. Neurobiol. 2004; 30: 1-22Crossref PubMed Google Scholar). α-Synuclein is a major component of Lewy bodies (LBs) in PD, and purified LBs have been shown to contain aggregated α-synuclein (19Spillantini M.G. Schmidt M.L. Lee V.M. Trojanowski J.Q. Jakes R. Goedert M. Nature. 1997; 388: 839-840Crossref PubMed Scopus (5968) Google Scholar, 20Baba M. Nakajo S. Tu P.H. Tomita T. Nakaya K. Lee V.M. Trojanowski J.Q. Iwatsubo T. Am. J. Pathol. 1998; 154: 879-884Google Scholar). Despite its abundance within LBs, it is, however, not clear whether α-synuclein is required for their formation or is involved in mediating toxicity associated with these structures.The data concerning the role of α-synuclein in cell death are somewhat contradictory. Several groups have reported that overexpression of mutant and, in some cases, wild-type (WT) α-synuclein directly induces death of various cell types (e.g. see Refs. 21Ostrerova N. Petrucelli L. Farrer M. Mehta N. Choi P. Hardy J. Wolozin B. J. Neurosci. 1999; 19: 5782-5791Crossref PubMed Google Scholar, 22Saha A.R. Ninkina N.N. Hanger D.P. Anderton B.H. Davies A.M. Buchman V.L. Eur. J. Neurosci. 2000; 12: 3073-3077Crossref PubMed Scopus (145) Google Scholar, 23Xu J. Kao S.Y. Lee F.J. Song W. Jin L.W. Yankner B.A. Nat. Med. 2002; 8: 600-606Crossref PubMed Scopus (628) Google Scholar, 24Zhou W. Schaack J. Zawada W.M. Freed C.R. Brain Res. 2002; 926: 42-50Crossref PubMed Scopus (124) Google Scholar). In contrast, several reports suggest a survival-promoting activity of WT or even mutant α-synuclein (25Da Costa C.A. Ancolio K. Checler F. J. Biol. Chem. 2000; 275: 24065-24069Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, 26Da Costa C. Paitel E. Vincent B. Checler F. J. Biol. Chem. 2002; 277: 50980-50984Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 27Manning-Bog A.B. McCormack A.L. Purisai M.G. Bolin L.M. Di Monte D.A. J. Neurosci. 2003; 23: 3095-3099Crossref PubMed Google Scholar). Therefore, the potential role of α-synuclein in cell death path-ways remains unclear.In addition to genetic data, pathological evidence indicates that proteasomal dysfunction occurs in the substantia nigra of PD patients (28McNaught K.S. Jenner P. Neurosci. Lett. 2001; 297: 191-194Crossref PubMed Scopus (544) Google Scholar, 29Tofaris G.K. Razzaq A. Ghetti B. Lilley K.S. Spillantini M.G. J. Biol. Chem. 2003; 278: 44405-44411Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar). We have used pharmacological inhibitors of the proteasome to model such pathological features of PD. Proteasomal inhibition of cultured rat cortical neurons induces apoptotic death (30Qiu J.H. Asai A. Chi S. Saito N. Hamada H. Kirino T. J. Neurosci. 2000; 20: 259-265Crossref PubMed Google Scholar, 31Rideout H.J. Stefanis L. Mol. Cell. Neurosci. 2002; 21: 223-238Crossref PubMed Scopus (114) Google Scholar) and the formation of cytoplasmic inclusions that contain, among other proteins, ubiquitin and α-synuclein (31Rideout H.J. Stefanis L. Mol. Cell. Neurosci. 2002; 21: 223-238Crossref PubMed Scopus (114) Google Scholar). Cell death and inclusion formation have also been observed in dopaminergic PC12 cells (32Rideout H.J. Larsen K.L. Sulzer D. Stefanis L. J. Neurochem. 2001; 78: 899-908Crossref PubMed Scopus (251) Google Scholar) as well as cultured ventral midbrain neurons (33McNaught K.S. Mytilineou C. Jnobaptiste R. Yabut J. Shashidharan P. Jenner P. Olanow C.W. J. Neurochem. 2002; 81: 301-306Crossref PubMed Scopus (270) Google Scholar) and in vivo following infusion of the proteasomal inhibitor lactacystin into the substantia nigra (34McNaught K.S. Bjorklund L.M. Belizaire R. Isacson O. Jenner P. Olanow C.W. Neuroreport. 2002; 13: 1437-1441Crossref PubMed Scopus (231) Google Scholar). An important feature of the cellular inclusions induced by inhibition of the proteasome is that they possess a fibrillar structure reminiscent of LBs and other inclusions, as revealed by the fluorescent histochemical marker thioflavine S (31Rideout H.J. Stefanis L. Mol. Cell. Neurosci. 2002; 21: 223-238Crossref PubMed Scopus (114) Google Scholar). The formation of ubiquitinated inclusions in this model is a regulated process requiring ubiquitination of specific substrates and transcription (31Rideout H.J. Stefanis L. Mol. Cell. Neurosci. 2002; 21: 223-238Crossref PubMed Scopus (114) Google Scholar). However, as with LBs, it is not clear whether α-synuclein is required for their formation.In the present work, we address the following questions. 1) What is the conformation of α-synuclein within cortical neuronal inclusions following proteasome inhibition? 2) What is the effect of lack of α-synuclein on the formation, solubility, and structure of such inclusions? 3) Does lack of α-synuclein alter the sensitivity of cortical neurons to proteasomal inhibition-induced death?EXPERIMENTAL PROCEDURESCell Culture and Genotyping—Rat embryonic cortical neurons were prepared as described (31Rideout H.J. Stefanis L. Mol. Cell. Neurosci. 2002; 21: 223-238Crossref PubMed Scopus (114) Google Scholar, 35Stefanis L. Park D.S. Friedman W.J. Greene L.A. J. Neurosci. 1999; 19: 6235-6247Crossref PubMed Google Scholar). α-Synuclein +/+, +/–, or –/– neurons (36Dauer W.T. Kholodilov N. Vila M. Trillat A.C. Goodchild R. Larsen K.E. Staal R. Tieu K. Schmitz Y. Yuan C.A. Rocha M. Jackson-Lewis V. Hersch S. Sulzer D. Przedborski S. Burke R. Hen R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14524-14529Crossref PubMed Scopus (491) Google Scholar) were obtained from embryonic day 16 mouse embryos resulting from heterozygous matings as described (37Dietrich P. Rideout H.J. Wang Q. Stefanis L. Mol. Cell. Neurosci. 2003; 24: 430-441Crossref PubMed Scopus (37) Google Scholar). Genotyping was performed on each individual embryo by PCR from tail DNA. The primers used were as follows: 1) SYN6591, TCA CAC TTA CAC CAG GAC TTG G and 2) SYN7005, GTC CCT GTT TGT TTC TGA GAG C to detect the wild-type allele; and 3) synNEO, ATG GAA GGA TTG GAG CTA CGG G to detect the targeted allele.Induction of Neuronal Death—Cortical neurons were cultured for 3 or 10 days in vitro (DIV) before application of reagents. The reason these two time points were selected was that, as we have previously reported, there is a marked induction of the levels of α-synuclein during this period in rat cortical neuron cultures (38Rideout H.J. Dietrich P. Savalle M. Dauer W.T. Stefanis L. J. Neurochem. 2003; 84: 803-813Crossref PubMed Scopus (41) Google Scholar). A similar induction is seen between DIV 3 and DIV 10 in embryonic day 16 mouse cortical neurons (data not shown). On DIV 3 or 10, the inhibitor of the 20 S proteasome lactacystin (39Fenteany G. Schreiber S. J. Biol. Chem. 1998; 273: 8545-8548Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar) was added to the cultures. At indicated times following addition of lactacystin (10 μm) or camptothecin (10 μm), cells were lysed and neuronal viability was estimated using counts of intact nuclei as described previously. (35Stefanis L. Park D.S. Friedman W.J. Greene L.A. J. Neurosci. 1999; 19: 6235-6247Crossref PubMed Google Scholar). Total surviving neurons are expressed as the percentage of untreated cultures at the time of cell lysis. In parallel experiments, the percentage of apoptotic nuclei, as an index of neuronal cell death, was assessed using the nuclear dye Hoechst 33342 (Sigma, 1 μg/ml) as described previously (35Stefanis L. Park D.S. Friedman W.J. Greene L.A. J. Neurosci. 1999; 19: 6235-6247Crossref PubMed Google Scholar).Immunofluorescence—Neurons grown on glass coverslips were fixed in freshly prepared 3.7% formaldehyde for 25 min at 4 °C and then incubated with 10% normal goat serum with 0.4% Triton X-100 to block nonspecific binding, followed by incubation with the primary antibody for 1 h at room temperature. Specific antibodies used were rabbit anti-ubiquitin (1:100; Dako), mouse anti-polyubiquitin (1:200; Affinity), or mouse anti-synuclein-1 (1:50; Transduction Laboratories). Following incubation with fluorescent secondary antibodies (Cy2, 1:100 or Cy3, 1:250; Jackson ImmunoResearch), coverslips were placed on glass slides and visualized using standard epifluorescence or confocal microscopy (Ziess LSM410). For thioflavine S staining, freshly fixed cells were incubated with 0.05% thioflavine S in phosphate-buffered saline (Sigma), washed for 5 min three times with 80% EtOH, and then blocked for subsequent immunostaining.For counts of ubiquitin- and thioflavine S-positive cytoplasmic inclusions, stained coverslips were observed by a rater blinded to both the genotype and the experimental condition using standard epifluorescence (100× magnification). From three to four coverslips per individual embryo, 100 cells were observed each and assessed for the presence of discrete ubiquitin- or thioflavine S-positive cytoplasmic inclusions as described previously (31Rideout H.J. Stefanis L. Mol. Cell. Neurosci. 2002; 21: 223-238Crossref PubMed Scopus (114) Google Scholar). The data were pooled from embryos of the same genotype for analysis.For in situ extraction of soluble protein (31Rideout H.J. Stefanis L. Mol. Cell. Neurosci. 2002; 21: 223-238Crossref PubMed Scopus (114) Google Scholar), cells were incubated with detergent-containing extraction buffer (85 mm PIPES, pH 6.94, 10 mm EGTA, 1 mm MgCl2, and 0.1% Triton X-100, supplemented with protease inhibitor mixture (Roche)) for 10 min at room temperature, washed in phosphate-buffered saline, and fixed as above with 3.7% formaldehyde for 25 min at 4 °C. The fixed cells were then processed as described for anti-ubiquitin. We have previously shown that proteasome inhibitor-treated rat cortical neurons show insoluble ubiquitinated inclusions that are resistant to such extraction (31Rideout H.J. Stefanis L. Mol. Cell. Neurosci. 2002; 21: 223-238Crossref PubMed Scopus (114) Google Scholar).Western Immunoblotting—Rat cortical neurons exposed at DIV 3 or DIV 10 to lactacystin (10 μm) for 30 h were incubated, as described above, with detergent-containing extraction buffer at room temperature for 10 min. The soluble extracted material in the buffer was removed, and the remaining insoluble material was scraped in the same buffer and solubilized by sonication. The samples were diluted in SDS-sample buffer containing 5% β-mercaptoethanol and subjected to polyacrylamide gel electrophoresis. The separated proteins were transferred to nitrocellulose membranes and incubated with a mouse monoclonal anti-synuclein 1 antibody (BD Transduction Laboratories; 1:1000) or rabbit anti-α-synuclein (1:500; generously provided by Dr. Janetta Culvenor, University of Melbourne) (40Culvenor J.G. McLean C.A. Cutt S. Campbell B.C.V. Maher F. Jakala P. Hartmann T. Beyreuther K. Masters C.L. Li Q.-X. Am. J. Pathol. 1999; 155: 1173-1181Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar) overnight at 4 °C. The membranes were washed and incubated with horseradish peroxidase-conjugated secondary anti-mouse or rabbit antibodies (Pierce; 1:10000) and developed with SuperSignal West (Pierce). Equal protein loading was assessed by Ponceau S staining or stripping of the membranes and probing for mouse anti-β-actin (Sigma; 1:20,000).RESULTSα-Synuclein Accumulates within Insoluble Inclusions in Proteasome Inhibitor-treated Rat Cortical Neurons—We have previously demonstrated that the ubiquitinated inclusions formed following proteasomal inhibition resist in situ extraction by a buffer containing 0.1% Triton X-100 (31Rideout H.J. Stefanis L. Mol. Cell. Neurosci. 2002; 21: 223-238Crossref PubMed Scopus (114) Google Scholar). To investigate whether the α-synuclein that we had identified within such inclusions would also be detergent-insoluble we performed similar in situ extraction of embryonic day 18 rat cortical neurons cultured for 10 days (DIV 10) and then treated with lactacystin (10 μm, 30 h) or vehicle (control). Cultures were then fixed and immunostained for anti-ubiquitin and anti-α-synuclein. Control cultures of rat embryonic cortical neurons showed little cytoplasmic ubiquitin and α-synuclein immunostaining following in situ extraction (Fig. 1A, top panel). In contrast, lactacystin-treated cultures showed a dramatic accumulation of ubiquitin immunoreactivity distributed diffusely throughout the cytoplasm (not shown) or organized within discrete cytoplasmic inclusions (Fig. 1A, bottom panel, arrow) as we have reported previously (31Rideout H.J. Stefanis L. Mol. Cell. Neurosci. 2002; 21: 223-238Crossref PubMed Scopus (114) Google Scholar). Many of the ubiquitin-positive inclusions also showed clear α-synuclein co-localization (Fig. 1A, arrow). Similar results were obtained with DIV 3 cultures (data not shown).Oligomerization of Detergent-insoluble α-Synuclein following Inhibition of the Proteasome—The results presented above indicate that α-synuclein accumulates within detergent-resistant inclusions in proteasome inhibitor-treated neurons. To determine the nature of α-synuclein within such inclusions, we performed Western immunoblot analysis of the detergent-insoluble fractions. Using a mouse monoclonal antibody raised against synuclein-1, we observed a reduction in the 15-kDa α-synuclein monomer band (Fig. 1B, asterisk) in lactacystin-treated samples. Upon longer exposure of the film we observed two higher molecular weight (HMW) bands in lysates derived from lactacystin-treated neurons migrating at Mr ∼45,000–50,000 and 65,000–70,000. These bands are present in multiple independent samples derived from lactacystin-treated cultures (Fig. 1B, arrows). The sizes of the HMW α-synuclein immuno-reactive bands do not indicate ubiquitination, which would be represented by multiple bands separated by Mr ∼8,000. It is likely that the bands we have identified with this antibody represent oligomeric forms of α-synuclein, in particular a trimer and a tetramer. Similar findings were obtained with DIV 3 cortical neuronal cultures (data not shown).To confirm the formation of the HMW oligomeric α-synuclein species, we utilized a second antibody raised against the C terminus of human α-synuclein. The monomeric form of α-synuclein is indicated by an asterisk in Fig. 1C, whereas the HMW oligomeric species are indicated by brackets. This α-synuclein antibody provided an increased sensitivity in detecting the HMW oligomeric species compared with the mouse monoclonal antibody (compare Fig. 1B and 1C). There are three apparent clusters of HMW α-synuclein oligomers in lactacystin-treated detergent-insoluble fractions migrating at Mr ∼31,000–35,000, 43,000–49,000, and 65,000–70,000 (Fig. 1C). These clusters confirm the oligomerization of α-synuclein revealed by the mouse monoclonal synuclein-1 antibody. The additional bands detected by this antibody may represent other post-translational modifications such as phosphorylation. We conclude that α-synuclein is at least partially present in an oligomeric form within detergent-insoluble inclusions in neurons treated with lactacystin.Deletion of α-Synuclein Does Not Alter Neuronal Sensitivity to Apoptotic Stimuli—Given the relationship between α-synuclein and the proteasome as well as the reported effects of α-synuclein on the apoptotic pathway, we wished to determine whether lack of α-synuclein would have an effect on neuronal survival/death following exposure to lactacystin. To this end, we used cultures derived from WT or knock-out (KO) α-synuclein mice (36Dauer W.T. Kholodilov N. Vila M. Trillat A.C. Goodchild R. Larsen K.E. Staal R. Tieu K. Schmitz Y. Yuan C.A. Rocha M. Jackson-Lewis V. Hersch S. Sulzer D. Przedborski S. Burke R. Hen R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14524-14529Crossref PubMed Scopus (491) Google Scholar). There was no difference in survival, as assessed by counts of intact nuclei (Fig. 2A) or apoptosis, as assessed by counts of apoptotic nuclei (Fig. 2B) between WT and α-synuclein KO cultured cortical neurons.Fig. 2The absence of α-synuclein does not alter the sensitivity of cortical neurons to various cell death agents. Mouse embryonic cortical neurons were plated in 96-well plates for counts of intact nuclei (A) or on glass coverslips for counts of apoptotic nuclei (B). In A, neurons cultured for 3 or 10 DIV were exposed to the proteasome inhibitor lactacystin (10 μm, 24 h), the cells were lysed, and intact nuclei were counted. Survival was expressed relative to untreated control neurons within the same genotype. Other cells were exposed to the DNA-damaging agent camptothecin (10 μm, 12 h) and lysed and counted similarly. Note that there is no significant difference in survival in response to either agent between wild-type or α-synuclein knock-out cultures. The number above each bar represents the number of individual embryos that were assessed. In B, neurons were cultured for 3 or 10 DIV prior to exposure to lactacystin (10 μm) for 16 or 24 h, fixed, and stained with the nucleic acid dye Hoechst. The numbers represent the percentage of apoptotic nuclei. The number of individual embryos assessed is shown in the legend. Note that as with intact nuclear counts, there is no significant difference in the induction of apoptotic death in neurons from wild-type or α-synuclein knock-out embryos. In all cases, counts were performed by a rater blind to the genotype.View Large Image Figure ViewerDownload (PPT)To investigate whether the lack of effect of deleting α-synuclein was specific for the apoptotic stimulus of proteasomal inhibition, we also tested the effects of the DNA-damaging agent camptothecin on these cultures. We and others have previously reported that this agent induces apoptotic death of postmitotic neurons (35Stefanis L. Park D.S. Friedman W.J. Greene L.A. J. Neurosci. 1999; 19: 6235-6247Crossref PubMed Google Scholar, 41Morris E.J. Geller H.M. J. Cell Biol. 1996; 134: 757-770Crossref PubMed Scopus (283) Google Scholar). Again, there was no difference in survival or apoptosis between WT and α-synuclein KO neurons (Fig. 2, A and B). We conclude that deleting α-synuclein has no significant impact on survival of embryonic cortical neurons exposed to apoptotic stimuli and, in particular, to proteasomal inhibition.Absence of α-Synuclein Does Not Alter the Number or the Solubility of Ubiquitinated Inclusions Formed following Lactacystin Treatment—Given the correlation between α-synuclein and cytoplasmic inclusions, we wished to investigate whether its presence was required for inclusion formation in this model. We therefore treated α-synuclein WT and KO cortical neurons with lactacystin and 16 and 24 h later assessed the percentage of neurons that showed cytoplasmic ubiquitinated inclusions. There was no difference between the two genotypes (Fig. 3).Fig. 3The absence of α-synuclein does not affect the formation of ubiquitinated inclusions in cortical neurons exposed to proteasome inhibition. Embryonic cortical neurons from wild-type (filled bars) or α-synuclein knock-out (open bars) were cultured on glass coverslips for 3 or 10 DIV, exposed to lactacystin (10 μm) for 16 or 24 h, fixed, and immunostained for ubiquitin. The percentage of neurons harboring single cytoplasmic ubiquitin-positive inclusions was determined by a rater blind to the experimental conditions and genotype. The number of individual embryos assessed is indicated in the legend. Note that cortical neurons from wild-type and α-synuclein knock-out embryos show the same extent of ubiquitinated inclusion formation in response to inhibition of the proteasome with lactacystin.View Large Image Figure ViewerDownload (PPT)The possibility existed that α-synuclein may not alter the initial formation of the inclusions but may alter their subsequent solubility through its aggregating properties. Using the same in situ detergent extraction method as in Fig. 1, we were unable to find a significant difference between the two genotypes in terms of detergent-insoluble ubiquitinated inclusions (Fig. 4, A and B).Fig. 4The absence of α-synuclein does not affect the solubility of ubiquitinated inclusions formed in cortical neurons following proteasome inhibition. A, cortical neurons were prepared from wild-type or α-synuclein knock-out embryos and grown on glass coverslips for 3 DIV, exposed to lactacystin (10 μm, 24 h) extracted in situ with 0.1% Triton X-100 buffer, fixed, and immunostained for ubiquitin. Note the prominent insoluble ubiquitinated inclusion (arrows) in lactacystin-treated neurons from wild-type and α-synuclein knock-out embryos. B, stained coverslips were examined by a rater blind to the genotype and experimental condition to determine the percentage of neurons harboring detergent-insoluble ubiquitinated inclusions. The number of embryos assessed is shown above each bar. Presented is the percentage of neurons in lactacystin (10 μm, 24 h)-treated cultures from neurons at DIV 3. Note that there is no significant difference between wild-type and α-synuclein knock-out of the percentage of neurons harboring detergent-insoluble ubiquitinated inclusi" @default.
• W2042340343 created "2016-06-24" @default.
• W2042340343 creator A5004447815 @default.
• W2042340343 creator A5028502903 @default.
• W2042340343 creator A5062315427 @default.
• W2042340343 creator A5078662242 @default.
• W2042340343 creator A5089947731 @default.
• W2042340343 date "2004-11-01" @default.
• W2042340343 modified "2023-10-16" @default.
• W2042340343 title "α-Synuclein Is Required for the Fibrillar Nature of Ubiquitinated Inclusions Induced by Proteasomal Inhibition in Primary Neurons" @default.
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