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Originally published In Press as doi:10.1074/jbc.M203159200 on October 2, 2002

J. Biol. Chem., Vol. 277, Issue 49, 47870-47877, December 6, 2002
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Parkin Accumulation in Aggresomes Due to Proteasome Impairment*

Eunsung Junn, Sang Seop Lee, Unsun T. Suhr, and M. Maral MouradianDagger

From the Genetic Pharmacology Unit, Experimental Therapeutics Branch, NINDS, National Institutes of Health, Bethesda, Maryland 20892-1406

Received for publication, April 2, 2002, and in revised form, September 24, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Parkinson's disease (PD) is characterized by loss of dopaminergic neurons in the substantia nigra and by the presence of ubiquitinated cytoplasmic inclusions known as Lewy bodies. alpha -Synuclein and Parkin are two of the proteins associated with inherited forms of PD and are found in Lewy bodies. Whereas numerous reports indicate the tendency of alpha -synuclein to aggregate both in vitro and in vivo, no information is available about similar physical properties for Parkin. Here we show that overexpression of Parkin in the presence of proteasome inhibitors leads to the formation of aggresome-like perinuclear inclusions. These eosinophilic inclusions share many characteristics with Lewy bodies, including a core and halo organization, immunoreactivity to ubiquitin, alpha -synuclein, synphilin-1, Parkin, molecular chaperones, and proteasome subunit as well as staining of some with thioflavin S. We propose that the process of Lewy body formation may be akin to that of aggresome-like structures. The tendency of wild-type Parkin to aggregate and form inclusions may have implications for the pathogenesis of sporadic PD.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The accumulation of insoluble intracellular protein aggregates is a hallmark of Parkinson's disease (PD)1 and several other neurodegenerative disorders. PD is characterized by the progressive degeneration of midbrain dopaminergic neurons (1) and by the accumulation of cytoplasmic inclusions known as Lewy bodies (2). Although the cause and effect relationship between these inclusions and cell death remains unclear, the molecular constituents of inclusions reveal clues about the events leading to their formation. For example, Lewy bodies are rich in ubiquitin and proteasome subunits (3, 4) consistent with the critical role of this protein degradation pathway in neuronal homeostasis and apoptosis in PD (5, 6). Other components of Lewy bodies include alpha -synuclein, Parkin, and ubiquitin C-terminal hydrolase L1 (UCH-L1) (7-10). Mutations in the cognate gene of each of these proteins are linked to inherited forms of PD (11-14). Additionally, overexpression of alpha -synuclein in transgenic models results in the formation of intracellular protein aggregates and locomotor dysfunction (15, 16).

Parkin, originally identified by positional cloning in families with autosomal recessive PD (13), is a ubiquitin-protein isopeptide ligase (E3) (17, 18). This 465-amino acid protein has mild homology to ubiquitin at its N terminus and contains two RING finger domains at its C terminus. Parkin exerts its ubiquitin ligase function through interactions between its RING finger domain and E2-conjugating enzymes. In addition to ubiquitinating a number of substrate proteins such as CDCrel-1, glycosylated alpha -synuclein, Pael-R, and synphilin-1 (9, 18-20), Parkin ubiquitinates itself as an early step in its proteasome-mediated degradation (18, 21).

To date, several studies (22, 23) have addressed the tendency of alpha -synuclein to aggregate as ubiquitinated inclusions, but no information is available about the ability of Parkin to aggregate. In this report, we show that overexpression of Parkin in the presence of a proteasome inhibitor leads to the accumulation of Parkin aggregates as single, large, eosinophilic peri-nuclear inclusions consisting of a core and a halo. These structures are similar to aggresomes (24), the formation of which results in the redistribution of several cellular factors, such as intermediate filaments, chaperones, and proteasome subunits to these inclusion bodies. Based on our observations, we suggest that the formation of Lewy bodies in the brains of PD patients is similar to the formation of aggresome-like structures when proteasome activity is impaired.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Antibodies-- The following antibodies were used in immunofluorescence studies. Mouse monoclonal anti-FLAG (M2)-fluorescein isothiocyanate conjugate (1:200), anti-FLAG (M2)-Cy3 conjugate (1:200), and mouse monoclonal gamma -tubulin (GTU-88) antibody (1:1000) were purchased from Sigma. Mouse monoclonal anti-HA-TRITC conjugate (1:100), rabbit polyclonal vimentin antibody (1:100), rabbit polyclonal ubiquitin antibody (1:100), goat polyclonal BIP/GRP78 antibody (1:200), rabbit polyclonal gamma -adaptin antibody (1:200), goat polyclonal LAMP-1 antibody (1:200), and secondary donkey anti-rabbit or goat IgG conjugated to rhodamine (1:200) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal Hsp70 antibody (1:200) was obtained from Upstate Biotechnology, Inc. (Waltham, MA). Rabbit polyclonal Hsp40 antibody (1:200) was from Stressgen (Victoria, British Columbia, Canada). Rabbit polyclonal alpha -subunit of 20 S proteasome antibody (1:250) was from Calbiochem. Goat polyclonal synphilin-1 antibody (1:200), rabbit polyclonal alpha -synuclein antibody (1:200), and rabbit polyclonal mannosidase II antibody (1:200) were from Chemicon (Temecula, CA).

cDNA Cloning-- Full-length Parkin cDNA was amplified by PCR using primers 5'-GCCGAATTCACCATGATAGTGTTTGTCAGGTTC-3' and 5'-GGCGGATCCCTACACGTCGAACCAGTGG-3'. These primers contained additional restriction enzyme cleavage sites to facilitate insertion of the product in pFLAG-CMV-2 (Sigma) to express FLAG- tagged Parkin, in pEGFP-C2 (Clontech, Palo Alto, CA) to express GFP-Parkin, and in pcDNA3.1 (Invitrogen). Ubiquitin cDNA was isolated by PCR from human adult brain cDNA library using primers 5'-GGAAGCTTAATGCAGATCTTCGTGAAGACTCTG-3' and 5'-GGCGAATTCTACCCACCCTGAGACGGAGTAC-3'. The amplified sequence was inserted into pHM6 (Roche Molecular Biochemicals) to express HA-tagged ubiquitin.

Cell Culture and Transfection-- COS-7 and human embryonic kidney HEK 293T cell lines were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. PC12 cells were cultured in Dulbecco's modified Eagle's medium containing 10% horse serum and 5% fetal bovine serum. Transfections were performed using FuGENE 6 reagent (Roche Molecular Biochemicals) according to the supplier's instructions. Cells were cultured for 24 h after transfection.

Immunoprecipitation and Western Blot-- Cells were lysed in a buffer containing PBS with 1% Triton X-100 and a mixture of protease inhibitors (Roche Molecular Biochemicals). After homogenizing with 20 strokes using a Dounce homogenizer, cells were centrifuged at 100,000 × g at 4 °C for 30 min. The soluble and insoluble fractions were used in Western blots for FLAG, alpha -synuclein, and synphilin-1. The Triton X-100-insoluble pellets were dissolved in a buffer (PBS plus 1% Triton X-100, 1% SDS) containing a mixture of protease inhibitors (Roche Molecular Biochemicals). After centrifugation, supernatant was diluted in 10× volume of the same buffer lacking SDS. Immunoprecipitations were performed with agarose-conjugated anti-FLAG (M2) (Sigma) followed by washing with lysis buffer four times. Immunoprecipitates or total cell lysates were analyzed by Western blots using mouse anti-ubiquitin antibody (P4D1) (Santa Cruz Biotechnology, Santa Cruz, CA) with ECL detection reagent (PerkinElmer Life Sciences).

Immunocytochemistry-- HEK 293T or COS-7 cells were cultured in 60-mm plates, transfected using FuGENE 6 transfection kit, and plated in collagen-coated Biocoat® slides (BD Biosciences) for 1 day. Before staining, 5 µM of the proteasome inhibitor MG-132 was added for 16 h. Cells were fixed in 4% formaldehyde in PBS for 20 min, washed with PBS three times, and permeabilized with 0.6% Triton X-100 in PBS for 5 min. After washing the cells again with PBS three times, they were blocked with 1% BSA in PBS for 20 min. Cells were incubated with primary antibody diluted in PBS and 1% BSA at 4 °C for 2 h. Cells were washed five times for 5 min each with PBS. Secondary antibodies were diluted in PBS with 1% BSA and incubated at 4 °C for 1 h. Cells were washed five times with PBS, mounted with ProLong® antifade mounting material (Molecular Probes, Eugene, OR) under a coverglass, and analyzed under a Zeiss (LSM 510) confocal microscope or epi-fluorescence microscope (Zeiss, Axiophot). For quantification of inclusions, 10 microscopic fields were randomly selected, and the percentage of inclusion-positive cells was counted among transfected cells expressing FLAG-Parkin. For nuclear staining, fixed cells were incubated with 10 µM 4',6-diamidino-2-phenylindole (DAPI) for 5 min after secondary antibody incubation. For thioflavin S staining, fixed cells were incubated with 0.1% thioflavin S (Sigma) for 5 min and washed with 70% ethanol three times before antibody staining.

Hematoxylin and Eosin Staining-- For H & E staining of MG-132-treated COS-7 cells transiently transfected with FLAG-Parkin, cells were washed with PBS twice and incubated with hematoxylin (Vector Laboratories, Burlingame, CA) at room temperature for 3 min. Cells were then rinsed with deionized water three times and destained with acidic alcohol for a few seconds. After rinsing the cells again with deionized water, bicarbonate solution (1 g/liter) was added, and cells were incubated for 3 min. After this bluing step, cells were washed again with deionized water and placed in 70% ethanol for 3 min, followed by staining with eosin (0.5 g of Eosin Y, 2.5 ml of acetic acid, 500 ml of 70% ethanol) for 1 min. Cells were then washed with three changes of 95% ethanol and dehydrated with absolute ethanol. Slides were dried, mounted, and analyzed under a light microscope.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To investigate the molecular properties of Parkin, cDNA constructs were used to transiently overexpress this protein in cell lines. HEK 293T cells transfected with N-terminally FLAG-tagged Parkin expressed a high molecular weight complex (HMW) of Parkin detected in the Triton X-100-insoluble fraction as well as monomeric Parkin detected in both Triton X-100-soluble and -insoluble fractions on Western blots (Fig. 1A). Expression of FLAG-Parkin in COS-7 cells reproduced this finding (data not shown). Similarly, the expression of non-tagged Parkin in PC12 cells also led to the formation of a HMW complex recognized by Parkin antibody (data not shown). Because Parkin contains 35 cysteine residues, which are vulnerable to oxidation and tend to form intra- and inter-molecular disulfide bridges, 20 mM dithiothreitol (DTT) was added onto the Triton X-100-insoluble fraction and boiled prior to Western blotting. DTT treatment failed to inhibit the formation of the HMW complex (Fig. 1B), suggesting that the HMW complex does not represent Parkin oxidation products.


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  		Fig. 1.   Parkin forms ubiquitin-positive insoluble HMW complexes. A, HEK 293T cells transiently transfected with FLAG-Parkin were divided into two dishes and were either untreated or treated with 5 µM MG-132 for 16 h. Cells were then lysed in a buffer containing 1% Triton X-100 and fractionated into soluble (S) and insoluble (I) fractions, followed by Western blotting using anti-FLAG (M2) antibody. Filled arrow points to monomeric Parkin. B, Triton X-100-insoluble fraction from HEK 293T cells transfected with FLAG-Parkin were either untreated (-) or treated (+) with 20 mM DTT prior to Western blot analysis. Filled arrow indicates monomeric Parkin. C, Triton X-100-insoluble fraction was subjected to immunoprecipitation with anti-FLAG (M2) antibody and followed by Western blot with anti-ubiquitin (P4D1) antibody. IP indicates the immunoprecipitated sample, and T indicates total protein prior to immunoprecipitation.

Incubation with the proteasome inhibitor MG-132 increased the amount of Parkin HMW complex in HEK 293T cells (Fig. 1A), suggesting that this complex contains ubiquitin-conjugated proteins. We then examined if Parkin could be covalently modified by ubiquitin as well. Western blot analysis of the FLAG-Parkin immunoprecipitate with anti-ubiquitin antibody showed that the HMW bands are ubiquitin-positive (Fig. 1C), consistent with previous reports showing Parkin ubiquitination (18, 21).

To study the intracellular formation and distribution of aggregated Parkin, fluorescence microscopy was employed to detect the expression of FLAG-Parkin in COS-7 cells. Following transient transfection, FLAG-Parkin was detected in the peri-nuclear and Golgi complex regions as demonstrated by co-localization with mannosidase II and the trans-Golgi network marker gamma -adaptin (Fig. 2, A and B), as reported previously (25, 26) in both human brain tissue and cell lines. We also found Parkin to be co-localized with the endoplasmic reticulum marker BIP/GRP78 (27) (Fig. 2C) and the lysosomal marker LAMP-1 (28) (Fig. 2D). Additionally, several small spherical inclusions were detected throughout the cytoplasm in some cells (Fig. 2E). The relative frequency of inclusion formation was 0.7% of transfected cells. Because Triton X-100-insoluble forms of FLAG-Parkin are ubiquitin-positive, we investigated whether these aggregates are ubiquitinated. Staining of the same cells with an anti-ubiquitin antibody revealed that most Parkin-positive inclusions also contain ubiquitin (Fig. 2E). These observations suggest that Parkin can aggregate when overexpressed and that this process is associated with its ubiquitination.


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  		Fig. 2.   Immunocytochemical detection of overexpressed Parkin. COS-7 cells were transfected with FLAG-Parkin and subjected to co-immunostaining with anti-FLAG antibody (green) along with the Golgi complex marker mannosidase II (A), trans-Golgi network marker gamma -adaptin (B), endoplasmic reticulum marker BIP/GRP78 (C), and lysosomal marker LAMP-1 (D), all with red fluorescence. E, COS-7 cells transfected with FLAG-Parkin were immunostained with anti-FLAG antibody (green) and ubiquitin (red). Intense green fluorescence appears as lime color. Arrows point to inclusions. Pre-adsorption with FLAG peptide at a concentration of 100 µg/ml was used as negative control and verified the specificity of Parkin staining (data not shown). Scale bars, 20 µm.

After treatment of FLAG-Parkin-transfected COS-7 cells with the proteasome inhibitor MG-132 for 15 h, about 20% of transfected cells manifested a single large, round, peri-nuclear inclusion (Fig. 3A) that appeared to impinge upon the nuclear envelope. Similar inclusions were observed in COS-7 cells expressing GFP-Parkin and in HEK 293T cells expressing FLAG-Parkin, indicating that this process is not unique to FLAG-tagged Parkin or to COS-7 cells (data not shown). Other inhibitors of the proteasome, such as lactacystin and PSI (23), induced the formation of similar inclusions in ~15-20% of transfected cells (Fig. 3B), indicating that the formation of these inclusions is due to proteasomal dysfunction rather than the unique effect of an individual drug. Double immunocytochemical studies revealed that these inclusions are localized at the Golgi complex as demonstrated by significant co-localization of Parkin immunoreactivity with the Golgi marker mannosidase II (29). Notably, the presence of these inclusions disrupts the normal morphology of the Golgi complex (Fig. 3C). We also investigated whether ubiquitin is present in these peri-nuclear, Parkin-containing inclusions. To this end, COS-7 cells were transiently co-transfected with FLAG-Parkin cDNA together with a plasmid encoding HA-tagged ubiquitin followed by treatment with MG-132 for 15 h. Double immunofluorescence microscopy revealed that the majority of FLAG-positive inclusions co-localize with HA immunoreactivity (Fig. 3D), indicating that Parkin-positive inclusions are ubiquitinated. Specificity of FLAG-Parkin immunoreactivity in these inclusions was verified by pre-adsorption with FLAG peptide resulting in absence of signal, whereas immunocytochemistry with ubiquitin antibody revealed positive staining of aggregates (Fig. 3E). We also analyzed non-Parkin-transfected, MG-132-treated COS-7 cells with ubiquitin immunostaining and found no peri-nuclear inclusions, implying that the formation of such inclusions is dependent on the overexpression of Parkin.


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  		Fig. 3.   Formation of a single, large peri-nuclear inclusion upon Parkin overexpression and proteasome inhibition. A, COS-7 cells transiently transfected with FLAG-Parkin were treated with 5 µM MG-132 for 16 h and processed for immunofluorescence using anti-FLAG antibody (green). Nuclei were counterstained blue with DAPI. B, cells transiently transfected with FLAG-Parkin were treated with either 5 µM MG-132, 5 µM lactacystin, or 5 µM PSI (Calbiochem) as well as with vehicle (Me2SO) for 16 h, and stained for FLAG. Inclusion-containing cells among transfected cells were counted in 10 randomly selected fields. Each microscopic field had 10-20 transfected cells. The data represent means ± S.E. Asterisks (analysis of variance, p < 0.001) indicate statistical significance compared with vehicle treatment. No significant differences were found among the three proteasome inhibitors. C, cells treated as in A were stained with FLAG (green) and with the Golgi complex marker mannosidase II (red). Arrowhead points to the disrupted Golgi morphology due to the presence of inclusion, and arrows indicate normal Golgi morphology. D, COS-7 cells transiently co-transfected with FLAG-Parkin and HA-ubiquitin were treated with 5 µM MG-132 for 16 h and subjected to immunostaining using anti-FLAG antibody (green) and anti-HA (red). Intense green fluorescence appears as lime color. E, Pre-adsorption with FLAG peptide eliminated FLAG-Parkin staining, whereas ubiquitin staining of the same cell marked the inclusion (red). Scale bars, 20 µm.

The large peri-nuclear inclusions containing Parkin had marked similarities to structures, termed aggresomes, formed by other proteins (27, 28, 30-32). Overexpression of certain proteins or inhibition of proteasome activity in cells expressing these proteins leads to the accumulation of stable, high molecular weight, detergent-insoluble, and multiubiquitinated aggregates in a large structure located peri-nuclearly on one side (24). Other characteristic features of the aggresome include its formation at the centrosome/microtubule organizing center (MTOC). To assess whether peri-nuclear Parkin aggregates are similar in structure to aggresomes, additional co-localization studies were carried out. Comparison of the fluorescence from Parkin aggregates with the staining pattern of gamma -tubulin, a marker for the centrosome/MTOC (33), revealed that both signals are co-localized, indicating a close physical relationship between the aggresome and centrosome (Fig. 4A). The presence of aggresome at the centrosome/MTOC suggests direct involvement of microtubules (MT) in their formation. To address this link, MT-disrupting drugs, such as nocodazole and vinblastine sulfate, were employed together with proteasome inhibition (Fig. 4B). Treatment with these drugs markedly abrogated the MG-132-induced accumulation of Parkin as a single large peri-nuclear aggresome. Rather, FLAG-Parkin immunofluorescence was observed throughout the cytoplasm with no obvious concentration near the centrosome/MTOC (Fig. 4C). These data suggest that an intact MT is required for the formation of Parkin-containing aggresomes through retrograde transport of misfolded proteins along microtubules as demonstrated previously (28, 30, 32) for the cystic fibrosis transmembrane conductance regulator, GFP-250, and superoxide dismutase.


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  		Fig. 4.   Inclusion bodies containing Parkin are aggresomes. A, FLAG-Parkin-transfected COS-7 cells were double-immunolabeled with FLAG (green) and gamma -tubulin (red). B, cells transiently transfected with FLAG-Parkin were co-treated with either 10 µg/ml nocodazole or 1 µM vinblastine sulfate together with 5 µM MG-132 for 16 h and stained for FLAG. Aggresome-containing cells among transfected cells were counted in 10 randomly selected fields. Each microscopic field had 10-20 transfected cells. The data represent means ± S.E. Asterisks (analysis of variance, p < 0.005) indicate statistical significance relative to MG-132 treatment alone. C, cells transiently transfected with FLAG-Parkin were co-treated with 10 µg/ml nocodazole and 5 µM MG-132 for 16 h and stained for FLAG. Note disruption of the aggresome structure. D, cells transiently transfected with FLAG-Parkin, treated with 5 µM MG-132 for 16 h, and double-stained for FLAG (green) and vimentin (red). Note the ring-like appearance of vimentin fluorescence (arrow). E, cells transiently transfected with FLAG-Parkin, treated with 5 µM MG-132 for 16 h, and then washed-out of MG-132 for 8 h. Staining was done as in D. Intense green fluorescence appears as lime color. Scale bars, 20 µm.

Among the components of intracellular inclusion bodies associated with some neurodegenerative diseases, including Parkinson and Alzheimer, are deposits of abnormal intermediate filament proteins such as neurofilaments (34, 35). Similarly, one of the characteristic features of aggresomes is the deposition of intermediate filaments such as vimentin, particularly redistributed to the aggresomal region forming a ring-like halo (28). FLAG-Parkin-transfected cells treated with a proteasome inhibitor elicited the same response, showing a ring of vimentin immunoreactivity around aggregated Parkin (Fig. 4D). Additionally, these inclusions are quite stable as they persist after an 8-h wash-out of MG-132 (Fig. 4E). Taken together, these observations indicate that the peri-nuclear inclusion containing Parkin has the structural characteristics of an aggresome.

The proteasome complex is involved in the degradation of incompletely folded or misfolded proteins. In the case of mutant huntingtin and GFP-250, proteasome subunits have been detected in their respective aggresomes (27, 30). Additionally, proteasome subunits are found in Lewy bodies of PD (4). To investigate whether Parkin-containing aggresomes also recruit the proteasome, we analyzed the distribution of the 20 S proteasome alpha -subunit. As expected, we found co-localization of the alpha -subunit with Parkin within aggresomes (Fig. 5A), supporting previous reports (8, 21) that the proteasome is involved in Parkin degradation.


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  		Fig. 5.   Co-localization of Parkin-containing aggresomes with proteasome and molecular chaperones. COS-7 cells were transfected and treated as in Fig. 3. A, cells were immunostained for FLAG (green) and alpha -subunit of 20 S proteasome (red). B and C, cells were immunostained for FLAG (green) and for either Hsp70 (red) (B) or Hsp40 (red) (C). Note the localization of Hsp70 and Hsp40 in the periphery of the aggresome (arrows). D, cells were double-labeled with FLAG (green) and BIP/GRP78 (red). Intense green fluorescence appears as lime color. Scale bars, 20 µm.

Molecular chaperones interact with aggregation-prone proteins and facilitate their re-folding or degradation (36). Hsp70, for example, has been shown to be recruited to the centrosomal locus upon inhibition of proteasomal function (37). Because aggresomes contain misfolded proteins destined for degradation, it is likely that such proteins could be complexed with chaperones. To investigate whether chaperones are associated with Parkin aggresomes, the distribution of Hsp70 and its co-chaperone, Hsp40, was examined. These chaperones were detected in the periphery of the aggresome forming a ring around Parkin immunoreactivity (Fig. 5, B and C), similar to the staining pattern of vimentin (Fig. 4D). Interestingly, Hsp70 and Hsp40 were recently found in Lewy bodies of PD brains and in neuronal inclusions of alpha -synuclein transgenic Drosophila (38). We also analyzed the localization of BIP/GRP78, an endoplasmic reticulum resident chaperone, within Parkin-containing aggresomes. Compared with its staining pattern in the absence of MG-132 (Fig. 2C), BIP/GRP78 redistributed to aggresomes in proteasome inhibitor-treated cells and co-localized with Parkin in the halo (Fig. 5D).

alpha -Synuclein is a major constituent of Lewy bodies (7), some of which also contain Parkin (9, 39). These findings prompted us to examine the presence of alpha -synuclein in Parkin aggresomes. alpha -Synuclein immunoreactivity was found localized primarily in the periphery of the aggresomes around Parkin aggregates (Fig. 6A). A halo-like staining for alpha -synuclein has also been observed in Lewy bodies (9, 40). Synphilin-1, originally identified as an alpha -synuclein interacting protein (41) and found in Lewy bodies (42), also interacts with and is ubiquitinated by Parkin (20). We detected synphilin-1 immunoreactivity in peri-nuclear aggresomes co-localizing with Parkin (Fig. 6B). However, the staining pattern for synphilin-1 is somewhat different from that of alpha -synuclein. Whereas alpha -synuclein is concentrated mainly in the periphery, synphilin-1 is located in the entire aggresome structure covering the core, consistent with a previous observation that synphilin-1 is concentrated primarily in the dense core of Lewy bodies (42). The presence of alpha -synuclein and synphilin-1 in Parkin-containing aggresomes was also confirmed in Western blot analyses (Fig. 6, C and D), demonstrating HMW smears of the respective proteins only in the insoluble fraction of MG-132-treated cells.


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  		Fig. 6.   Co-localization of Parkin-containing aggresome with alpha -synuclein and synphilin-1. COS-7 cells were transfected and treated as in Fig. 3. Cells were immunostained for FLAG (green) and alpha -synuclein (red) (A) or synphilin-1 (red) (B). Note that alpha -synuclein staining is localized primarily in the periphery of the aggresome, whereas synphilin-1 has a homogeneous staining pattern covering the core. Intense green fluorescence appears as lime color. Scale bars, 20 µm. The same samples as in Fig. 1A were probed with anti-alpha -synuclein (C) or anti-synphilin-1 (D) antibody to detect the HMW smears under aggresome-forming conditions. Note the presence of HMW smears in the insoluble pellet (I) with MG-132 treatment. S, soluble; WB, Western blot.

alpha -Synuclein forms aggregates having a structure similar to amyloid, characterized by beta -pleated sheet (43), which stains with thiazole dyes such as thioflavin S and T. Additionally, some Lewy bodies are thioflavin S-positive (2, 44). Because the Parkin-containing aggresome also includes alpha -synuclein and shows many features of a Lewy body, we double-stained Parkin-transfected, MG 132-treated cells with thioflavin S and Parkin. About 60% of the resultant aggresomes stained positively with thioflavin S, suggesting that these inclusions have amyloid-like features (Fig. 7A). Furthermore, similar to Lewy bodies, H & E staining revealed that Parkin-containing aggresomes are eosinophilic (Fig. 7B).


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  		Fig. 7.   Parkin-containing aggresome is thioflavin S-positive and is eosinophilic. COS-7 cells were transfected and treated as in Fig. 3. A, cells were stained with thioflavin S (green) followed by anti-FLAG (M2)-Cy3 antibody (red). Nuclei (blue) were stained with DAPI. Note that the Parkin-containing aggresome is thioflavin S-positive. B, cells were stained with hematoxylin and eosin (H&E). Whereas nuclei stain primarily with hematoxylin as deep purple color, aggresomes are eosinophilic staining pink. Scale bars, 20 µm.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The characterization of a newly recognized intracellular structure named aggresome has recently been described (24, 27, 28, 30-32). Aggresomes form by the accumulation and deposition of misfolded proteins in a single large structure surrounding the centrosome. Aggresomes have been detected in cells expressing cystic fibrosis transmembrane conductance regulator, presenilin-1, GFP-250, mutant huntingtin, superoxide dismutase, and prion protein when these molecules are overexpressed or when the cells are treated with drugs that inhibit proteasome activity. These observations, therefore, can provide clues about the pathogenesis of certain neurodegenerative disorders that are characterized by morphologically and biochemically distinct intracellular inclusions containing aggregated, ubiquitinated proteins often bundled with hyperphosphorylated and disordered intermediate filaments (45).

The accumulation of wild-type or mutant intracellular proteins due to misfolding or defects in the ubiquitin-proteasome degradation system is a common occurrence in PD (46). The major indication that altered protein handling is a critical factor in this disorder is the presence of proteinaceous, intracytoplasmic inclusions known as Lewy bodies in dopaminergic neurons of the substantia nigra and in other brain regions affected by the PD pathology (2). A wide range of proteins has been identified in Lewy bodies including alpha -synuclein, Parkin, ubiquitin, UCH-L1, synphilin-1, components of the ubiquitin-proteasome system, and protein adducts of 3-nitrotyrosine (3, 4, 7-10, 42, 47, 48). The presence of Parkin in Lewy bodies prompted us to investigate the aggregate-forming capability of Parkin in a cellular model. In the present report, we demonstrate that Parkin has a tendency to aggregate into inclusion bodies when overexpressed. These aggregates also contain ubiquitin, suggesting that the process of Parkin aggregation is coupled to its ubiquitination. But the relative frequency of microscopically visible aggregates is less than 1% although abundant amounts of Parkin exist as Triton X-100-insoluble high molecular complexes. Therefore, these HMW complexes could be viewed as small inclusions that cannot be detected by our conventional immunocytochemical methods or simply as oligomers that may act as seeds for the formation of larger inclusions when appropriate conditions prevail. Because Parkin is degraded by the proteasome, it is conceivable that the impaired proteasomal activity found in the parkinsonian nigra (49) could lead to slowed Parkin clearance, thus increasing the possibility for its aggregation. In fact, the frequency of inclusion formation in our experimental paradigm was markedly increased upon treatment with proteasome inhibitors jumping up to 20% of Parkin-transfected cells. The inclusions formed by MG-132 treatment are different from those seen in untreated control cells in several respects. Inclusions in cells with compromised proteasomal function appear as single large peri-nuclear structures resembling aggresomes, whereas those seen in untreated cells are small and scattered throughout the cytoplasm. Aggresomes are characterized by their content of gamma -tubulin, vimentin, chaperones, and proteasome subunits, all components present in Parkin-containing inclusions. Furthermore, alpha -synuclein and synphilin-1 are detected in Parkin-containing inclusions, consistent with the recent observation (20) that co-expression of these three proteins in cells results in the formation of ubiquitin-positive cytosolic inclusions. These three proteins are also known to be constituents of Lewy bodies in PD brains. These observations lead us to speculate that the formation of Lewy bodies containing Parkin is analogous to the formation of aggresome-like structures when proteasomal activity is compromised. Additionally, mitochondrial dysfunction has recently been reported (22) to result in alpha -synuclein aggregates structurally similar to aggresomes, pointing to another cellular insult that can cause protein clumping into single, large inclusions.

The parallels between the Lewy body and the aggresome are complex due to differences in the organization of microtubules and the centrosome in post-mitotic neurons versus dividing cells (50). Nevertheless, many similarities between aggresomes and Lewy bodies have been uncovered by our present findings. First, both types of inclusions share the same major constituent proteins including Parkin, alpha -synuclein, synphilin-1, ubiquitin, proteasome subunit, Hsp70, and Hsp40. Second, morphologically, the core and halo structure of Lewy bodies is preserved in the aggresome with alpha -synuclein in the periphery and synphlin-1 in the core (42). Third, some Lewy bodies have been reported to be localized in the juxtanuclear region (51) typical of the aggresome (24). Peri-nuclear alpha -synuclein-positive inclusions similar to Lewy bodies have also been seen in transgenic Drosophila overexpressing alpha -synuclein (38). Fourth, some Lewy bodies are thioflavin S-positive, which may result from the amyloid-like aggregation of alpha -synuclein because Lewy bodies do not contain amyloid beta -protein (52). Fifth, both Lewy bodies and Parkin-containing aggresomes are eosinophilic. And sixth, Lewy bodies contain neurofilament proteins (34), although the presence of gamma -tubulin and vimentin in these neuronal structures remains to be determined.

The stimuli that promote Parkin aggregation in the brain are likely to be diverse. Here we show that overexpressing this protein in a cellular model results in the formation of Parkin- and ubiquitin-positive inclusions. Thus, conditions that enhance Parkin expression (21) could promote its aggregation. On the other hand, proteasomal activity is reportedly down-regulated in PD brains (49), a condition that promotes the formation of Parkin-containing inclusions. Our findings with wild-type Parkin coupled with the fact that Parkin is present in Lewy bodies suggest that this protein is involved in the pathogenesis of not only inherited PD but of sporadic forms of the disease as well. In PD not associated with parkin mutations, sequestration of Parkin into inclusions, thereby inhibiting its E3 function, could be another mechanism leading to the accumulation of its toxic substrates, thus accelerating neuronal degeneration.

The foregoing observations demonstrate that Parkin is prone to aggregate into large peri-nuclear inclusion bodies when proteasomal activity is impaired. Whether Parkin-positive inclusions per se relate to cell toxicity remains to be determined. However, if the aggregation of Parkin in certain conformations is deleterious to neurons, its prevention or resolution would have therapeutic value in PD.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: NINDS, National Institutes of Health, 10 Center Dr., MSC 1406, Bethesda, MD 20892-1406. Tel.: 301-496-7872; Fax: 301-496-6609; E-mail: MouradianM@ ninds.nih.gov.

Published, JBC Papers in Press, October 2, 2002, DOI 10.1074/jbc.M203159200

    ABBREVIATIONS

The abbreviations used are: PD, Parkinson's disease; TRITC, tetramethylrhodamine isothiocyanate; HA, hemagglutinin; PBS, phosphate-buffered saline; BSA, bovine serum albumin; DAPI, 4',6-diamidino- 2-phenylindole; HMW, high molecular weight complex; DTT, dithiothreitol; GFP, green fluorescent protein; MTOC, microtubule organizing center; MT, microtubules; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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