Golgi Fragmentation Occurs in the Cells with Prefibrillar α-Synuclein Aggregates and Precedes the Formation of Fibrillar Inclusion

Amyloid-like fibrillar aggregates of intracellular proteins are common pathological features of human neurodegenerative diseases. However, the nature of pathogenic aggregates and the biological consequences of their formation remain elusive. Here, we describe (i) a model cellular system in which prefibrillar α-synuclein aggregates and fibrillar inclusions are naturally formed in the cytoplasm with distinctive kinetics and (ii) a tight correlation between the presence of prefibrillar aggregates and the Golgi fragmentation. Consistent with the structural abnormality of Golgi apparatus, trafficking and maturation of dopamine transporter through the biosynthetic pathway were impaired in the presence of α-synuclein aggregates. Reduction in cell viability was also observed in the prefibrillar aggregate-forming condition and before the inclusion formation. The fibrillar inclusions, on the other hand, showed no correlation with Golgi fragmentation and were preceded by these events. Furthermore, at the early stage of inclusion formation, active lysosomes and mitochondria were enriched in the juxtanuclear area and co-aggregate into a compact inclusion body, suggesting that the fibrillar inclusions might be the consequence of an attempt of the cell to remove abnormal protein aggregates and damaged organelles. These results support the hypothesis that prefibrillar α-synuclein aggregates are the pathogenic species and suggest that Golgi fragmentation and subsequent trafficking impairment are the specific consequence of α-synuclein aggregation. Amyloid-like fibrillar aggregates of intracellular proteins are common pathological features of human neurodegenerative diseases. However, the nature of pathogenic aggregates and the biological consequences of their formation remain elusive. Here, we describe (i) a model cellular system in which prefibrillar α-synuclein aggregates and fibrillar inclusions are naturally formed in the cytoplasm with distinctive kinetics and (ii) a tight correlation between the presence of prefibrillar aggregates and the Golgi fragmentation. Consistent with the structural abnormality of Golgi apparatus, trafficking and maturation of dopamine transporter through the biosynthetic pathway were impaired in the presence of α-synuclein aggregates. Reduction in cell viability was also observed in the prefibrillar aggregate-forming condition and before the inclusion formation. The fibrillar inclusions, on the other hand, showed no correlation with Golgi fragmentation and were preceded by these events. Furthermore, at the early stage of inclusion formation, active lysosomes and mitochondria were enriched in the juxtanuclear area and co-aggregate into a compact inclusion body, suggesting that the fibrillar inclusions might be the consequence of an attempt of the cell to remove abnormal protein aggregates and damaged organelles. These results support the hypothesis that prefibrillar α-synuclein aggregates are the pathogenic species and suggest that Golgi fragmentation and subsequent trafficking impairment are the specific consequence of α-synuclein aggregation. Many human neurological disorders, including Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy, are characterized by amyloid-like fibrillar aggregates of α-synuclein, such as Lewy bodies (LBs) 1The abbreviations used are: LB, Lewy body; PBS, phosphate-buffered saline; m.o.i., multiplicity of infection; EM, electron microscopy; BSA, bovine serum albumin; GA, Golgi apparatus; AD, Alzheimer's disease; ALS, amyotrophic lateral sclerosis; CJD, Creutzfeldt-Jakob disease; MSA, multiple system atrophy; DAT, dopamine transporter; endoH, endoglycosidase H; ER, endoplasmic reticulum. and Lewy neurites (1Hardy J. Gwinn-Hardy K. Science. 1998; 282: 1075-1079Google Scholar, 2Trojanowski J.Q. Goedert M. Iwatsubo T. Lee V.M.-Y. Cell Death Differ. 1998; 5: 832-837Google Scholar). α-Synuclein is a 140-amino acid protein that is enriched in presynaptic terminals of neurons (3Goedert M. Nat. Rev. Neurosci. 2001; 2: 492-501Google Scholar). In the test tube, this protein forms fibrils, which resemble the ones isolated from postmortem brains with LB diseases (4Giasson B.I. Uryu K. Trojanowski J.Q. Lee V.M.-Y. J. Biol. Chem. 1999; 274: 7619-7622Google Scholar, 5Conway K.A. Harper J.D. Lansbury Jr., P.T. Biochemistry. 2000; 39: 2552-2563Google Scholar, 6Serpell L.C. Berriman J. Jakes R. Goedert M. Crowther R.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4897-4902Google Scholar). A causative role of fibrillar α-synuclein inclusions in neurodegeneration has been suggested in a mouse model in which formation of intracytoplasmic α-synuclein inclusions coincided with the severe motor impairment (7Giasson B.I. Duda J.E. Quinn S.M. Zhang B. Trojanowski J.Q. Lee V.M.-Y. Neuron. 2002; 34: 521-533Google Scholar). On the other hand, a study in a transgenic fly model that express human α-synuclein has shown that co-expression of molecular chaperone hsp70 alleviated the neurodegenerative phenotype but did not reduce the formation of α-synuclein-positive inclusion bodies (8Auluck P.K. Chan H.Y. Trojanowski J.Q. Lee V.M.-Y. Bonini N.M. Science. 2002; 295: 865-868Google Scholar). This result raised a question as to whether the fibrillar inclusions play a causative role in neurodegenerative process. Furthermore, recentin vitro studies revealed various non-fibrillar species during the course of fibrillation and suggested a possibility that these metastable intermediate species, not the fibrils themselves, might elicit cytotoxicity (9Conway K.A. Lee S.-J. Rochet J.C. Ding T.T. Williamson R.E. Lansbury Jr., P.T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 571-576Google Scholar, 10Conway K.A. Rochet J.C. Bieganski R.M. Lansbury Jr., P.T. Science. 2001; 294: 1346-1349Google Scholar). Elucidating which particular aggregate species possess the principal cytotoxic effect holds the key to understanding the etiologic role of protein aggregation in the disease pathogenesis. Study of this problem, however, has been hampered by the lack of an experimental system in which intermediates of the endogenous fibrillation process can be biochemically defined and analyzed. Here, we have established such a system and have assessed the effects of prefibrillar intermediates that are formed naturally in the cytoplasm. In this report, we refer to prefibrillar aggregates as non-fibrillar oligomeric assemblies that precede the formation of fibrils and to inclusions as large deposits of aggregates that are usually found in juxtanuclear location. Monoclonal anti-α-synuclein antibody, LB509, was purchased from Zymed Laboratories (South San Francisco, CA), and a polyclonal anti-α-synuclein serum, 7071, was provided by Peter Lansbury (Brigham and Women's Hospital, Boston, MA). Antibodies against GM130, TGN46, and calnexin were obtained from BD Biosciences (San Diego, CA), Serotec (Oxford, UK), and StressGen Biotechnologies Corp. (Victoria, BC, Canada), respectively. Antibodies for mannosidase II and DAT were purchased from Chemicon (Temecula, CA). All the fluorescently labeled secondary antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). 125I-Labeled anti-mouse IgG antibody was obtained from Amersham Biosciences (Piscataway, NJ). Goat anti-mouse IgG antibody, conjugated with a 10-nm gold particle, was obtained from Ted Pella Inc. (Redding, CA). Different amounts (see Fig. 1 legend) of adeno/α-syn (11Lee H.-J. Shin S.Y. Choi C. Lee Y.H. Lee S.-J. J. Biol. Chem. 2002; 277: 5411-5417Google Scholar) and empty viral vector were mixed to make the final number of viral particle be 1.5 × 108. COS-7 cells (1.5 × 106 cells) were infected with these mixtures as described previously (11Lee H.-J. Shin S.Y. Choi C. Lee Y.H. Lee S.-J. J. Biol. Chem. 2002; 277: 5411-5417Google Scholar). At day 3, the cells were extracted in phosphate-buffered saline (PBS) with 1% Triton X-100 and protease inhibitor mixture (Sigma), and the extracts were centrifuged at 16,000 × g for 5 min to separate detergent-soluble (supernatant) and insoluble (pellet) fractions. The pellets were resuspended in half the volume of 1× Laemmli sample buffer. Ten micrograms of supernatant and the equal volume of pellet were applied onto 12% SDS-polyacrylamide gel and subjected to Western blotting (12Lee S.-J. Liyanage U. Bickel P.E. Xia W. Lansbury Jr., P.T. Kosik K.S. Nat. Med. 1998; 4: 730-734Google Scholar). For the quantitative analysis, the proteins were visualized using125I-labeled secondary antibody, and the monomers and the aggregate smears (from 60 kDa to the top of the gel) were quantified by computer-assisted densitometry using ImageQuaNT software (Molecular Dynamics) under equal light and power settings. Three independent experiments were performed. For the Golgi analysis and cell viability assay, the cells were split onto cover-glasses into 12-well plates and into 35-mm dishes, respectively, at day 1 (the next day of infection) and incubated until the time of analysis, usually at day 3 or 4. We have noticed that the time course of the aggregation process changes slightly depending on the surface area of the tissue culture dish; i.e.aggregation is faster in 100-mm dishes than in 35-mm dishes. To normalize the effects of viral vector itself, the total amount of viral particles was adjusted to be equivalent with empty viral vector in all experiments. COS-7 cells were infected with adeno/α-syn at m.o.i. and the cells were split into 35-mm dishes at day 1 and incubated until 3 or 4. On the day of 5 1% Triton protease inhibitor was onto and incubated at for 5 The Triton supernatant was and with 1% Triton X-100 and protease inhibitor was to and was obtained by dishes and on for 5 the was centrifuged at × 5 at which condition the fibrillar inclusions to a pellet H.-J. Lee S.-J. J. Biol. Chem. 2002; 277: Scholar). The prefibrillar aggregates in the supernatant were by at 16,000 for including with was to the in Lee (11Lee H.-J. Shin S.Y. Choi C. Lee Y.H. Lee S.-J. J. Biol. Chem. 2002; 277: 5411-5417Google Scholar). For the of lysosomes and the cells were incubated with (Molecular and (Molecular respectively, in the for and and of was visualized using an antibody against GM130, TGN46, or mannosidase All the in this study were obtained with a For quantitative analysis, were obtained by the of area of and the number of cells with Golgi or fibrillar inclusions was in cells on and the was than Golgi fragmentation was defined as the of the cells with Golgi were the cells with Golgi were the obtained in this study is to the of Golgi of non-fibrillar aggregates the formation of fibrillar inclusions in changes in the of monomers and aggregates at the m.o.i. of were from detergent-soluble and insoluble respectively. The on the the gel the of cells that juxtanuclear inclusion bodies were at the time α-Synuclein was at m.o.i. of were obtained from to the of inclusion bodies that are in a fibrillar inclusions and prefibrillar aggregates were on their at 3 and and by Western The and the aggregates and respectively. The the gel For electron α-synuclein was in COS-7 cells at m.o.i. for The were as described in S. 2001; Scholar). the cells were in in on for the in 1% at for the cells were with 1% for 1 and with a of of the were in the were on and with 1% For the the were incubated in 1% by a with bovine serum the were incubated with antibody in and with 10-nm anti-mouse IgG The were with 1% and and observed with a electron were infected with various of α-synuclein and empty viral as described before and incubated until day For the of dopamine transporter the infected cells were with on the next and of cell surface proteins were to J. Neurosci. 1999; and all the were on the cells were with with and and incubated with in for min was with for and the cells were extracted in 1% Triton protease inhibitor mixture The cell extracts with the amounts of protein were incubated with at for min with The were with and with 1× Laemmli sample buffer. The were with acid and in 1× Laemmli sample buffer. The were onto and by Western to the of DAT to the cell surface DAT, of were The cells were and of cells were mixed with the equal volume of The number of cells that the was using a in The in Fig. were obtained from independent experiments. The of the cells were extracted and as described and the were subjected to Western blotting with To the aggregation process of α-synuclein in we various amounts of the into COS-7 cells using vector and the changes in the amounts of the monomers and aggregates. The of monomers with the of multiplicity of before a of total cellular the the the of aggregates in to m.o.i. This between the and the aggregation to the effects of the monomers or aggregates by the m.o.i. of α-synuclein aggregates were found by aggregates that are the cytoplasm and large juxtanuclear inclusion bodies H.-J. Lee S.-J. J. Biol. Chem. 2002; 277: Scholar). The large inclusions were with a specific to amyloid-like the aggregates were not H.-J. Lee S.-J. J. Biol. Chem. 2002; 277: Scholar). that the inclusion bodies are with with the of and were labeled with α-synuclein antibody The presence of α-synuclein fibrils in the inclusions was by of fibrils that were isolated from the inclusion study also the non-fibrillar nature of aggregates using the described H.-J. Lee S.-J. J. Biol. Chem. 2002; 277: Scholar). A amount of aggregates at day and at day 3 with a at day the this day 3, of the aggregates were non-fibrillar and than 1% of cells fibrillar inclusion However, during the formation of fibrillar inclusion To the time for the aggregate species, we have established a by which non-fibrillar aggregates and fibrillar inclusions can be by their and a of using and non-fibrillar aggregates and fibrillar inclusions are into the supernatant and respectively, by a at × g H.-J. Lee S.-J. J. Biol. Chem. 2002; 277: Scholar). this was applied to the we found that aggregates in the supernatant at day 3 and until day the inclusions in the pellet no than day that non-fibrillar aggregates before the formation of fibrillar the and the we that the aggregates are the of prefibrillar intermediates of To cellular consequences specific to α-synuclein we have the of Golgi for (i) α-synuclein aggregates aggregates in cell system in the S. Lee and S.-J. with the in that α-synuclein forms aggregates than the α-synuclein H.-J. Choi C. Lee S.-J. J. Biol. Chem. 2002; 277: (ii) fragmentation of has been found in neurodegenerative including Alzheimer's disease amyotrophic lateral Creutzfeldt-Jakob disease and multiple system A. Cell Biol. 1998; Scholar, A. K. K. 2000; Scholar, A. K. M. 2002; Scholar). the of is characterized by α-synuclein-positive inclusion bodies of neurons and K. S. A. M. M. 1998; Scholar, Crowther R.A. Jakes R. Goedert M. Neurosci. 1998; Scholar, W. J. M. R. M. T. J. A. C. R. S. J. 1999; Scholar). the of was with to the α-synuclein aggregation using an with the antibody against a protein In cells with α-synuclein the has compact the However, the cells with prefibrillar aggregates fragmentation and of the 3, and To Golgi fragmentation is an cells with Golgi were at m.o.i. the presence of a of α-synuclein, Golgi fragmentation at a m.o.i. of was not from the On the other hand, the Golgi fragmentation was prefibrillar aggregates formed with suggesting that of α-synuclein than the might be the of Golgi fragmentation. To the correlation between α-synuclein aggregation and Golgi an independent was to the Golgi fragmentation in a of cells that aggregates. Golgi fragmentation was found in the with the aggregates at m.o.i. and at m.o.i. than in the at m.o.i. and at m.o.i. In of protein to of total cellular protein did not changes in the Golgi not suggesting that Golgi fragmentation is not a consequence of of To the effects of α-synuclein aggregates on other Golgi we the of and using antibodies against TGN46, an of and mannosidase a that the Golgi and Furthermore, these proteins to the in these as in the compact that the was the fragmentation. the Golgi were for and mannosidase the of these proteins was also the However, the was not as as the of and TGN46, to their between the These results suggest that α-synuclein Golgi fragmentation in the Golgi of the through the Golgi and proteins a of such as and and the proteins are to their final To the consequence of the Golgi we the changes in the cell surface of a protein dopamine transporter The cells were infected with amounts of adeno/α-syn and with DAT on the of the cell surface proteins were labeled with and from the proteins using Western analysis of these that cell surface of DAT was in the prefibrillar condition with the empty vector However, of α-synuclein did not result in in the cell surface of DAT m.o.i. and Reduction in cell surface DAT was by the molecular of DAT from kDa to kDa The to the endoglycosidase and the is the biosynthetic intermediate J. Neurosci. 1999; Scholar). the is obtained in these that the proteins the in an In of the that the protein and the not be in this These results suggest that prefibrillar α-synuclein aggregates the impaired protein trafficking and maturation through the biosynthetic by Golgi Consistent with the that the of of DAT is not by α-synuclein the and of endoplasmic to remain in the presence of α-synuclein aggregates. the a the cytoplasm. This was of the α-synuclein aggregation or structural of in the cytoplasm suggesting that the α-synuclein aggregates have effect on the In to the prefibrillar no was found between the fibrillar inclusions and the Golgi fragmentation. an in Golgi fragmentation before the of fibrillar inclusions the of the cell that not during the day a in the number of fibrillar inclusions and was observed in the cells with fibrillar of were with fibrillar in Golgi were and the fibrillar inclusions and cells the slightly of that the juxtanuclear fibrillar inclusions The compact Golgi that is to inclusion has also been previously in a study using a of protein R. J. Cell Biol. 1999; Scholar). These of Golgi might be by the in the of of prefibrillar intermediates to fibrillar of the prefibrillar aggregates into the inclusions before the might to the compact Golgi to the inclusion inclusions be formed from prefibrillar species, of which is of Golgi fragmentation. In the that Golgi fragmentation before the formation of inclusions and that cells the compact Golgi the presence of inclusions suggest that the inclusions not be the of the of the of the fibrillar inclusions with α-synuclein and Golgi that the inclusions might be a consequence of an attempt by the cell to remove abnormal protein aggregates and impaired from the cytoplasm. Consistent with this lysosomes and mitochondria in the juxtanuclear at to be an early stage of inclusion formation and and are also in the compact inclusions and and which to be in the showed a in the juxtanuclear in the presence of the prefibrillar α-synuclein aggregates and These showed no of fragmentation and to be the used in this study in cellular with or with the study also showed the of Golgi and mitochondria in the juxtanuclear In and lysosomes can be found in this area at the early stage of inclusion formation and chaperone were also the in the cells with α-synuclein aggregates H.-J. Lee S.-J. J. Biol. Chem. 2002; 277: Scholar). has been suggested that the juxtanuclear as the in which the system and system their to remove abnormal proteins and damaged R. 2002; Scholar, Cell Biol. 2000; Scholar, Lee S. J. Cell Biol. 1999; Scholar). the of lysosomes and mitochondria in the juxtanuclear be the of of the cell against the protein aggregates. these results suggest that the formation of α-synuclein aggregates the of the cellular which is by the of these aggregates and impaired in the juxtanuclear with the and analysis of the cells were infected with adeno/α-syn at m.o.i. incubated for and for as described under and inclusions have compact with we to be an early stage of inclusion formation. In this mitochondria lysosomes and are in the juxtanuclear and this the stage the compact inclusion be found in the cells that were infected with empty viral for the and for A cytotoxic effect of α-synuclein has been in cell M. Choi J. B. J. Neurosci. 1999; Scholar, W. J. 2002; Scholar, A. M. J. Biol. Chem. 2001; Scholar, J. Neurosci. 2000; Scholar). However, the nature of species has not been To the cytotoxic effect of α-synuclein we cell viability as a of the of the aggregates. of did not the viability and Fig. day In cell viability was aggregates formed the and day correlation between in cell viability and α-synuclein aggregation was also found in the were formed at at m.o.i. and and on the the viability was A and the Golgi in cell viability before the of the fibrillar inclusions day 3 at the m.o.i. of and also Fig. and that the cytotoxic effect might be by the prefibrillar aggregates. These results suggest that the cytotoxic effect of α-synuclein on to the prefibrillar In this we biological effects of α-synuclein fragmentation of the and cell The that these effects with the formation of and not with the that of these effects on the formation of The prefibrillar intermediates to be for these of the effects in the presence of prefibrillar and before the formation of fibrillar We were to specific effects of prefibrillar intermediates from of monomers and fibrillar inclusions by of the features of experimental the a aggregates in to an amount of α-synuclein the This an to the effects of aggregates from of the to naturally prefibrillar aggregates and fibrillar inclusions analysis of This study showed that the aggregation process of α-synuclein in to the in test tube, prefibrillar intermediates in the course of fibrillar inclusions H.-J. Lee S.-J. J. Biol. Chem. 2002; 277: Scholar). such a of the inclusion formation to the effects of prefibrillar intermediates and fibrillar of human and have shown that the Golgi fragmentation was with the neurodegenerative of the has been found in human neurodegenerative including AD, ALS, CJD, and A. Cell Biol. 1998; Scholar, A. K. K. 2000; Scholar, A. K. M. 2002; Scholar). transgenic 1 showed Golgi fragmentation in motor neurons in an the role of Golgi fragmentation in the early stage of neurodegeneration A. Proc. Natl. Acad. Sci. U. S. A. Scholar). study this pathological of neurodegenerative the Golgi to the prefibrillar aggregate of results the correlation between Golgi fragmentation and cell are not to suggest the between these the of the in maturation and trafficking of proteins and we that might be to Golgi that transgenic non-fibrillar α-synuclein aggregates in neurons from presynaptic in system M. M. A. A. Science. 2000; Scholar). inclusion bodies are or or a of a has been of the in study that (i) Golgi fragmentation and cell are observed before the formation of inclusion bodies in mixed cell and that (ii) α-synuclein aggregates are into the area in the juxtanuclear with cellular such as and These results are with the that inclusion bodies not be to are consequences of the to remove abnormal protein aggregates and damaged from the cytoplasm. In a study using model in which of human α-synuclein Lewy inclusions and of a molecular chaperone hsp70 alleviated the neurodegenerative phenotype the number of inclusion bodies (8Auluck P.K. Chan H.Y. Trojanowski J.Q. Lee V.M.-Y. Bonini N.M. Science. 2002; 295: 865-868Google Scholar). This study also against the role of α-synuclein-positive inclusion in the process. The of inclusion bodies and cell has also been in other protein aggregation A phenotype and the formation of inclusion bodies have been in cell and that proteins S.M. H.Y. 1998; Scholar, S. 1998; Scholar, B. A. H.Y. Neuron. 1999; Scholar, Proc. Natl. Acad. Sci. U. S. A. 1999; Scholar, S. Science. 2000; Scholar). In study using a transgenic mouse model that protein before the of disease the inclusion bodies can be the is Proc. Natl. Acad. Sci. U. S. A. 2000; 97: Scholar). of the the presence of inclusion bodies not to be for the cell However, these results not the possibility that the of inclusion formation might the we refer to the process as the cellular to the inclusion formation the aggregation. For and of proteins and such as and from the as in the early stage of α-synuclein inclusion formation, make the cells to secondary The the cellular of the system has been to be a consequence of protein aggregation that can to cell R.M. Science. 2001; Scholar). with the also to the Golgi fragmentation. For such as a of Golgi proteins to J. S. Cell 1999; Scholar). the Golgi fragmentation observed in study be a secondary consequence of the system with the aggregates or of the trafficking at the of to the of α-synuclein than the effect of the α-synuclein aggregates. We not have to support of these but the of the Golgi that the α-synuclein Golgi fragmentation than the of In of the Golgi of Golgi to in the Golgi with the This with was observed during the Golgi in which proteins were than suggesting the of Golgi from other to the W. B. Biol. 1998; Scholar). the juxtanuclear inclusion bodies α-synuclein has been obtained for the presence of fibrillar α-synuclein aggregates the juxtanuclear These the is not at the structural of prefibrillar intermediates into fibrils are and in the H.-J. Lee S.-J. J. Biol. Chem. 2002; 277: Scholar). A study showed that α-synuclein fibrils were in than Lee S.-J. Rochet J.C. Ding T.T. J.C. Lansbury Jr., P.T. Biochemistry. 2001; suggesting an nature of In other studies using proteins or that are not in disease, fibrils were to the with non-fibrillar which precede formation of fibrils, in cell M. J. M. 2002; Scholar). formation of fibrillar inclusion bodies might the cells from the cytotoxicity of not by the prefibrillar aggregates from the cytoplasm but also by the for to In the results in this suggest that the pathogenic of α-synuclein not from but from to the prefibrillar aggregates. fibrillar inclusions not be the of the the cell abnormal protein aggregates that are formed the cytoplasm. the formation of aggregates and the of prefibrillar intermediates to fibrillar inclusions might be for the pathogenic of LB diseases. We and A. for on the We also T. Lansbury for antibody, for DAT and for the for the

Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.