Dyrk1A phosphorylates alpha-synuclein and enhances intracellular inclusion formation

Lewy bodies (LBs) are pathological hallmarks of Parkinson disease (PD) but also occur in Alzheimer disease (AD) and dementia of LBs. Alpha-synuclein, the major component of LBs, is observed in the brain of Down syndrome (DS) patients with AD. Dyrk1A, a dual specificity tyrosine-regulated kinase (Dyrk) family member, is the mammalian ortholog of the Drosophila minibrain (Mnb) gene, essential for normal postembryonic neurogenesis. The Dyrk1A gene resides in the human chromosome 21q22.2 region, which is associated with DS anomalies, including mental retardation. In this study, we examined whether Dyrk1A interacts with alpha-synuclein and subsequently affects intracellular alpha-synuclein inclusion formation in immortalized hippocampal neuronal (H19-7) cells. Dyrk1A selectively binds to alpha-synuclein in transformed and primary neuronal cells. Alpha-synuclein overexpression, followed by basic fibroblast growth factor-induced neuronal differentiation, resulted in cell death. We observed that accompanying cell death was increased alpha-synuclein phosphorylation and intracytoplasmic aggregation. In addition, the transfection of kinase-inactive Dyrk1A or Dyrk1A small interfering RNA blocked alpha-synuclein phosphorylation and aggregate formation. In vitro kinase assay of anti-Dyrk1A immunocomplexes demonstrated that Dyrk1A could phosphorylate alpha-synuclein at Ser-87. Furthermore, aggregates formed by phosphorylated alpha-synuclein have a distinct morphology and are more neurotoxic compared with aggregates composed of unmodified wild type alpha-synuclein. These findings suggest alpha-synuclein inclusion formation regulated by Dyrk1A, potentially affecting neuronal cell viability.     

Dorfin localizes to Lewy bodies and ubiquitylates synphilin-1

Parkinson's disease (PD) is a neurodegenerative disease characterized by loss of nigra dopaminergic neurons. Lewy bodies (LBs) are a characteristic neuronal inclusion in PD brains. In this study, we report that Dorfin, a RING finger-type ubiquityl ligase for mutant superoxide dismutase-1, was localized with ubiquitin in LBs. Recently, synphilin-1 was identified to associate with alpha-synuclein and to be a major component of LBs. We found that overexpression of synphilin-1 in cultured cells led to the formation of large juxtanuclear inclusions, but showed no cytotoxicity. Dorfin colocalized in these large inclusions with ubiquitin and proteasomal components. In contrast to full-length synphilin-1, overexpression of the central portion of synphilin-1, including ankyrin-like repeats, a coiled-coil domain, and an ATP/GTP-binding domain, predominantly led to the formation of small punctate aggregates scattered throughout the cytoplasm and showed cytotoxic effects. Dorfin and ubiquitin did not localize in these small aggregates. Overexpression of the N or C terminus of synphilin-1 did not lead to the formation of any aggregates. Dorfin physically bound and ubiquitylated synphilin-1 through its central portion, but did not ubiquitylate wild-type or mutant alpha-synuclein. These results suggest that the central domain of synphilin-1 has an important role in the formation of aggregates and cytotoxicity and that Dorfin may be involved in the pathogenic process of PD and LB formation by ubiquitylation of synphilin-1.     

Ubiquitination of alpha-synuclein and autophagy in Parkinson's disease

alpha-Synuclein is mutated in Parkinson's disease (PD) and is found in cytosolic inclusions, called Lewy bodies, in sporadic forms of the disease. A fraction of alpha-synuclein purified from Lewy bodies is monoubiquitinated, but the role of this monoubiquitination has been obscure. We now review recent data indicating a role of alpha-synuclein monoubiquitination in Lewy body formation and implicating the autophagic pathway in regulating these processes. The E3 ubiquitin-ligase SIAH is present in Lewy bodies and monoubiquitinates alpha-synuclein at the same lysines that are monoubiquitinated in Lewy bodies. Monoubiquitination by SIAH promotes the aggregation of alpha-synuclein into amorphous aggregates and increases the formation of inclusions within dopaminergic cells. Such effect is observed even at low monoubiquitination levels, suggesting that monoubiquitinated alpha-synuclein may work as a seed for aggregation. Accumulation of monoubiquitinated alpha-synuclein and formation of cytosolic inclusions is promoted by autophagy inhibition and to a lesser extent by proteasomal and lysosomal inhibition. Monoubiquitinated alpha-synuclein inclusions are toxic to cells and recruit PD-related proteins, such as synphilin-1 and UCH-L1. Altogether, the new data indicate that monoubiquitination might play an important role in Lewy body formation. Decreasing alpha- synuclein monoubiquitination, by preventing SIAH function or by stimulating autophagy, constitutes a new therapeutic strategy for Parkinson's disease.     

The co-chaperone carboxyl terminus of Hsp70-interacting protein (CHIP) mediates alpha-synuclein degradation decisions between proteasomal and lysosomal pathways

Alpha-synuclein is a major component of Lewy bodies, the pathological hallmark of Parkinson disease, dementia with Lewy bodies, and related disorders. Misfolding and aggregation of alpha-synuclein is thought to be a critical cofactor in the pathogenesis of certain neurodegenerative diseases. In the current study, we investigate the role of the carboxyl terminus of Hsp70-interacting protein (CHIP) in alpha-synuclein aggregation. We demonstrate that CHIP is a component of Lewy bodies in the human brain, where it colocalizes with alpha-synuclein and Hsp70. In a cell culture model, endogenous CHIP colocalizes with alpha-synuclein and Hsp70 in intracellular inclusions, and overexpression of CHIP inhibits alpha-synuclein inclusion formation and reduces alpha-synuclein protein levels. We demonstrate that CHIP can mediate alpha-synuclein degradation by two discrete mechanisms that can be dissected using deletion mutants; the tetratricopeptide repeat domain is critical for proteasomal degradation, whereas the U-box domain is sufficient to direct alpha-synuclein toward the lysosomal degradation pathway. Furthermore, alpha-synuclein, synphilin-1, and Hsp70 all coimmunoprecipitate with CHIP, raising the possibility of a direct alpha-synuclein-CHIP interaction. The fact that the tetratricopeptide repeat domain is required for the effects of CHIP on alpha-synuclein inclusion morphology, number of inclusions, and proteasomal degradation as well as the direct interaction of CHIP with Hsp70 implicates a cooperation of CHIP and Hsp70 in these processes. Taken together, these data suggest that CHIP acts a molecular switch between proteasomal and lysosomal degradation pathways.     

A light-inducible protein clustering system for in vivo analysis of α-synuclein aggregation in Parkinson disease

Neurodegenerative disorders refer to a group of diseases commonly associated with abnormal protein accumulation and aggregation in the central nervous system. However, the exact role of protein aggregation in the pathophysiology of these disorders remains unclear. This gap in knowledge is due to the lack of experimental models that allow for the spatiotemporal control of protein aggregation, and the investigation of early dynamic events associated with inclusion formation. Here, we report on the development of a light-inducible protein aggregation (LIPA) system that enables spatiotemporal control of α-synuclein (α-syn) aggregation into insoluble deposits called Lewy bodies (LBs), the pathological hallmark of Parkinson disease (PD) and other proteinopathies. We demonstrate that LIPA-α-syn inclusions mimic key biochemical, biophysical, and ultrastructural features of authentic LBs observed in PD-diseased brains. In vivo, LIPA-α-syn aggregates compromise nigrostriatal transmission, induce neurodegeneration and PD-like motor impairments. Collectively, our findings provide a new tool for the generation, visualization, and dissection of the role of α-syn aggregation in PD.

How do alpha-synuclein aggregates contribute to neuronal damage in Parkinson’s disease? To help address this question, this study presents a new optogenetic-based experimental model that allows for the induction and real-time monitoring of alpha-synuclein clustering in vivo.     

Monoubiquitylation of alpha-synuclein by seven in absentia homolog (SIAH) promotes its aggregation in dopaminergic cells

alpha-Synuclein plays a major role in Parkinson disease. Unraveling the mechanisms of alpha-synuclein aggregation is essential to understand the formation of Lewy bodies and their involvement in dopaminergic cell death. alpha-Synuclein is ubiquitylated in Lewy bodies, but the role of alpha-synuclein ubiquitylation has been mysterious. We now report that the ubiquitin-protein isopeptide ligase seven in absentia homolog (SIAH) directly interacts with and monoubiquitylates alpha-synuclein and promotes its aggregation in vitro and in vivo, which is toxic to cells. Mass spectrometry analysis demonstrates that SIAH monoubiquitylates alpha-synuclein at lysines 12, 21, and 23, which were previously shown to be ubiquitylated in Lewy bodies. SIAH ubiquitylates lysines 10, 34, 43, and 96 as well. Suppression of SIAH expression by short hairpin RNA to SIAH-1 and SIAH-2 abolished alpha-synuclein monoubiquitylation in dopaminergic cells, indicating that endogenous SIAH ubiquitylates alpha-synuclein. Moreover, SIAH co-immunoprecipitated with alpha-synuclein from brain extracts. Inhibition of proteasomal, lysosomal, and autophagic pathways, as well as overexpression of a ubiquitin mutant less prone to deubiquitylation, G76A, increased monoubiquitylation of alpha-synuclein by SIAH. Monoubiquitylation increased the aggregation of alpha-synuclein in vitro. At the electron microscopy level, monoubiquitylated alpha-synuclein promoted the formation of massive amounts of amorphous aggregates. Monoubiquitylation also increased alpha-synuclein aggregation in vivo as observed by increased formation of alpha-synuclein inclusion bodies within dopaminergic cells. These inclusions are toxic to cells, and their formation was prevented when endogenous SIAH expression was suppressed. Our data suggest that monoubiquitylation represents a possible trigger event for alpha-synuclein aggregation and Lewy body formation.     

Dityrosine cross-linking promotes formation of stable alpha -synuclein polymers. Implication of nitrative and oxidative stress in the pathogenesis of neurodegenerative synucleinopathies

Intracellular proteinaceous aggregates are hallmarks of many common neurodegenerative disorders, and recent studies have shown that alpha-synuclein is a major component of several pathological intracellular inclusions, including Lewy bodies in Parkinson's disease (PD) and glial cell inclusions in multiple system atrophy. However, the molecular mechanisms underlying alpha-synuclein aggregation into filamentous inclusions remain unknown. Since oxidative and nitrative stresses are potential pathogenic mediators of PD and other neurodegenerative diseases, we asked if oxidative and/or nitrative events alter alpha-synuclein and induce it to aggregate. Here we show that exposure of human recombinant alpha-synuclein to nitrating agents (peroxynitrite/CO(2) or myeloperoxidase/H(2)O(2)/nitrite) induces formation of nitrated alpha-synuclein oligomers that are highly stabilized due to covalent cross-linking via the oxidation of tyrosine to form o,o'-dityrosine. We also demonstrate that oxidation and nitration of pre-assembled alpha-synuclein filaments stabilize these filaments to withstand denaturing conditions and enhance formation of SDS-insoluble, heat-stable high molecular mass aggregates. Thus, these data suggest that oxidative and nitrative stresses are involved in mechanisms underlying the pathogenesis of Lewy bodies and glial cell inclusions in PD and multiple system atrophy, respectively, as well as alpha-synuclein pathologies in other synucleinopathies.     

Role of post-translational modifications on the alpha-synuclein aggregation-related pathogenesis of Parkinson’s disease

Together with neuronal loss, the existence of insoluble inclusions of alpha-synuclein (α-syn) in the brain is widely accepted as a hallmark of synucleinopathies including Parkinson’s disease (PD), multiple system atrophy, and dementia with Lewy body. Because the α-syn aggregates are deeply involved in the pathogenesis, there have been many attempts to demonstrate the mechanism of the aggregation and its potential causative factors including post-translational modifications (PTMs). Although no concrete conclusions have been made based on the previous study results, growing evidence suggests that modifications such as phosphorylation and ubiquitination can alter α-syn characteristics to have certain effects on the aggregation process in PD; either facilitating or inhibiting fibrillization. In the present work, we reviewed studies showing the significant impacts of PTMs on α-syn aggregation. Furthermore, the PTMs modulating α-syn aggregation-induced cell death have been discussed.      

Parkinson's disease transgenic mitochondrial cybrids generate Lewy inclusion bodies

Many models of Parkinson's disease (PD) have succeeded in replicating dopaminergic neuron loss or alpha-synuclein aggregation but not the formation of classical Lewy bodies, the pathological hallmark of PD. Our cybrid model of sporadic PD was created by introducing the mitochondrial genes from PD patients into neuroblastoma cells that lack mitochondrial DNA. Previous studies using cybrids have shown that information encoded by mitochondrial DNA in patients contributes to many pathogenic features of sporadic PD. In this paper, we report the generation of fibrillar and vesicular inclusions in a long-term cybrid cell culture model that replicates the essential antigenic and structural features of Lewy bodies in PD brain without the need for exogenous protein expression or inhibition of mitochondrial or proteasomal function. The inclusions generated by PD cybrid cells stained with eosin, thioflavin S, and antibodies to alpha-synuclein, ubiquitin, parkin, synphilin-1, neurofilament, beta-tubulin, the proteasome, nitrotyrosine, and cytochrome c. Future studies of these cybrids will enable us to better understand how Lewy bodies form and what role they play in the pathogenesis of PD.     

Parkin accumulation in aggresomes due to proteasome impairment

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.     

Alpha-synuclein and the pathogenesis of Parkinson's disease

Lesions known as Lewy bodies (LBs) and Lewy neurites (LNs) characterise brains of Parkinson's disease (PD) patients. Intracellular aggregation of alpha-synuclein (alpha-syn) appears to play a key role in the generation of LBs and LNs. Such aggregation in the presence of redox metals may initiate Fenton reaction-mediated generation of reactive oxygen species (ROS). ROS thus generated may result in cytotoxic mechanisms such as the induction of DNA single-strand breaks.     

Interaction between parkin and alpha-synuclein

A dramatic paradigm shift in understanding Parkinson's disease (PD) has emerged with implications for Alzheimer's disease (AD) because: (1) Mutations in the alpha-synuclein (AS) gene cause familial PD, (2) Antibodies to AS detect Lewy bodies (LBs) and dystrophic Lewy neurites in PD, dementia with LBs (DLB), sporadic AD and the LB variant of AD (LBVAD), (3) Insoluble AS filaments are recovered from DLB brains and purified LBs, (4) Recombinant AS assembles into LB-like filaments and residues 71–82 are essential for filament assembly, (5) AS transgenic mice and flies develop a PD-like phenotype, (6) Cortical LBs detected with antibodies to AS correlate with dementia in PD, DLB and LBVAD, (7) Antibodies to AS detect LBs in 50% of familial AD, sporadic AD and Down's syndrome brains, (8) AS forms glial cytoplasmic inclusions (GCIs) in multiple system atrophy, (9) Epitopes throughout AS in LBs and GCIs, (10) Filamentous AS aggregates in LBs, GCIs and related lesions contain nitrated tyrosines, (11) Cells transfected with AS and treated with nitric oxide generators develop LB-like AS inclusions, (12) Bigenic mice overexpressing mutant human APP and AS show an augmentation in AS inclusions. Thus, neurodegenerative diseases characterized by AS pathologies are synucleinopathies, and the filamentous AS lesions in these disorders may result in part from oxidative/nitrative damage to AS. Abnormal interactions of brain proteins may underlie synucleinopathies and other neurodegenerative disorders. Acknowledgements:
Supported by NIA/NIH and Alzheimer's Association.     

Transgenic mice overexpressing tyrosine-to-cysteine mutant human alpha-synuclein: a progressive neurodegenerative model of diffuse Lewy body disease

Abnormal aggregation of human alpha-synuclein in Lewy bodies and Lewy neurites is a pathological hallmark of Parkinson disease and dementia with Lewy bodies. Studies have shown that oxidation and nitration of alpha-synuclein lead to the formation of stable dimers and oligomers through dityrosine cross-linking. Previously we have reported that tyrosine-to-cysteine mutations, particularly at the tyrosine 39 residue (Y39C), significantly enhanced alpha-synuclein fibril formation and neurotoxicity. In the current study, we have generated transgenic mice expressing the Y39C mutant human alpha-synuclein gene controlled by the mouse Thy1 promoter. Mutant human alpha-synuclein was widely expressed in transgenic mouse brain, resulting in 150% overexpression relative to endogenous mouse alpha-synuclein. At age 9-12 months, transgenic mice began to display motor dysfunction in rotarod testing. Older animals aged 15-18 months showed progressive accumulation of human alpha-synuclein oligomers, associated with worse motor function and cognitive impairment in the Morris water maze. By age 21-24 months, alpha-synuclein aggregates were further increased, accompanied by severe behavioral deficits. At this age, transgenic mice developed neuropathology, such as Lewy body-like alpha-synuclein and ubiquitin-positive inclusions, phosphorylation at Ser(129) of human alpha-synuclein, and increased apoptotic cell death. In summary, Y39C human alpha-synuclein transgenic mice show age-dependent, progressive neuronal degeneration with motor and cognitive deficits similar to diffuse Lewy body disease. The time course of alpha-synuclein oligomer accumulation coincided with behavioral and pathological changes, indicating that these oligomers may initiate protein aggregation, disrupt cellular function, and eventually lead to neuronal death.     

Alpha-synuclein-induced aggregation of cytoplasmic vesicles in Saccharomyces cerevisiae

Aggregated alpha-synuclein (alpha-syn) fibrils form Lewy bodies (LBs), the signature lesions of Parkinson's disease (PD) and related synucleinopathies, but the pathogenesis and neurodegenerative effects of LBs remain enigmatic. Recent studies have shown that when overexpressed in Saccharomyces cerevisiae, alpha-syn localizes to plasma membranes and forms cytoplasmic accumulations similar to human alpha-syn inclusions. However, the exact nature, composition, temporal evolution, and underlying mechanisms of yeast alpha-syn accumulations and their relevance to human synucleinopathies are unknown. Here we provide ultrastructural evidence that alpha-syn accumulations are not comprised of LB-like fibrils, but are associated with clusters of vesicles. Live-cell imaging showed alpha-syn initially localized to the plasma membrane and subsequently formed accumulations in association with vesicles. Imaging of truncated and mutant forms of alpha-syn revealed the molecular determinants and vesicular trafficking pathways underlying this pathological process. Because vesicular clustering is also found in LB-containing neurons of PD brains, alpha-syn-mediated vesicular accumulation in yeast represents a model system to study specific aspects of neurodegeneration in PD and related synucleinopathies.     

Fatal attractions: abnormal protein aggregation and neuron death in Parkinson's disease and Lewy body dementia

The abnormal aggregation of proteins into fibrillar lesions is a neuropathological hallmark of several sporadic and hereditary neurodegenerative diseases. For example, Lewy bodies (LBs) are intracytoplasmic filamentous inclusions that accumulate primarily in subcortical neurons of patients with Parkinson's disease (PD), or predominantly in neocortical neurons in a subtype of Alzheimer's disease (AD) known as the LB variant of AD (LBVAD) and in dementia with LBs (DLB). Aggregated neurofilament subunits and alpha-synuclein are major protein components of LBs, and these inclusions may contribute mechanistically to the degeneration of neurons in PD, DLB and LBVAD. Here we review recent studies of the protein building blocks of LBs, as well as the role LBs play in the onset and progression of PD, DLB and LBVAD. Increased understanding of the protein composition and pathological significance of LBs may provide insight into mechanisms of neuron dysfunction and death in other neurodegenerative disorders characterized by brain lesions containing massive deposits of proteinacious fibrils.     

TorsinA and heat shock proteins act as molecular chaperones: suppression of alpha-synuclein aggregation

TorsinA, a protein with homology to yeast heat shock protein104, has previously been demonstrated to colocalize with alpha-synuclein in Lewy bodies, the pathological hallmark of Parkinson's disease. Heat shock proteins are a family of chaperones that are both constitutively expressed and induced by stressors, and that serve essential functions for protein refolding and/or degradation. Here, we demonstrate that, like torsinA, specific molecular chaperone heat shock proteins colocalize with alpha-synuclein in Lewy bodies. In addition, using a cellular model of alpha-synuclein aggregation, we demonstrate that torsinA and specific heat shock protein molecular chaperones colocalize with alpha-synuclein immunopositive inclusions. Further, overexpression of torsinA and specific heat shock proteins suppress alpha-synuclein aggregation in this cellular model, whereas mutant torsinA has no effect. These data suggest that torsinA has chaperone-like activity and that the disease-associated GAG deletion mutant has a loss-of-function phenotype. Moreover, these data support a role for chaperone proteins, including torsinA and heat shock proteins, in cellular responses to neurodegenerative inclusions.     

Molecular Chaperones, Alpha-Synuclein, and Neurodegeneration

Parkinson’s disease (PD) is a devastating neurological condition that affects about 1 % of people older than 65 years of age. In PD, dopaminergic neurons in the mid-brain slowly accumulate cytoplasmic inclusions (Lewy bodies, LBs) of the protein alpha-synuclein (α-syn) and then gradually lose function and die off. Cell death is thought to be causally linked to the aggregation/fibrillization of α-syn. This review focuses on new findings about the structure of α-syn, about how α-syn cooperates with Hsp70 and Hsp40 chaperones to promote neurotransmitter release, and about cell-to-cell transfer of pathogenic forms of α-syn and how Hsp70 might protect against this disease process.     

Exogenous α-synuclein fibrils induce Lewy body pathology leading to synaptic dysfunction and neuron death

Inclusions composed of α-synuclein (α-syn), i.e., Lewy bodies (LBs) and Lewy neurites (LNs), define synucleinopathies including Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Here, we demonstrate that preformed fibrils generated from full-length and truncated recombinant α-syn enter primary neurons, probably by adsorptive-mediated endocytosis, and promote recruitment of soluble endogenous α-syn into insoluble PD-like LBs and LNs. Remarkably, endogenous α-syn was sufficient for formation of these aggregates, and overexpression of wild-type or mutant α-syn was not required. LN-like pathology first developed in axons and propagated to form LB-like inclusions in perikarya. Accumulation of pathologic α-syn led to selective decreases in synaptic proteins, progressive impairments in neuronal excitability and connectivity, and, eventually, neuron death. Thus, our data contribute important insights into the etiology and pathogenesis of PD-like α-syn inclusions and their impact on neuronal functions, and they provide a model for discovering therapeutics targeting pathologic α-syn-mediated neurodegeneration.     

Rat alpha-synuclein interacts with Tat binding protein 1, a component of the 26S proteasomal complex

The alpha-synuclein gene, which encodes a brain presynaptic nerve terminal protein of unknown function, is linked to familial early-onset Parkinson's disease (PD). The finding that alpha-synuclein forms the major fibrillary component of Lewy bodies in brains of PD patients suggests that the two point mutations in alpha-synuclein (Ala(53)Thr, Ala(30)Pro) may promote the aggregation of alpha-synuclein into filaments. To address the role of alpha-synuclein in neurodegenerative diseases, we performed a yeast two-hybrid screen of a rat adult brain cDNA library using rat alpha-synuclein 2 (alphaSYN2). Here we report that alphaSYN2 interacts specifically with Tat binding protein 1, a subunit of the 700-kDa proteasome activator (PA700), the regulatory complex of the 26S proteasome and of the modulator complex, which enhances PA700 activation of the proteasome.     

Aggresomes formed by alpha-synuclein and synphilin-1 are cytoprotective

Lewy bodies (LBs), which are the hallmark pathologic features of Parkinson's disease and of dementia with LBs, have several morphologic and molecular similarities to aggresomes. Whether such cytoplasmic inclusions contribute to neuronal death or protect cells from the toxic effects of misfolded proteins remains controversial. In this report, the role of aggresomes in cell viability was addressed in the context of over-expressing alpha-synuclein and its interacting partner synphilin-1 using engineered 293T cells. Inhibition of proteasome activity elicited the formation of juxtanuclear aggregates with characteristics of aggresomes including immunoreactivity for vimentin, gamma-tubulin, ubiquitin, proteasome subunit, and hsp70. As expected from the properties of aggresomes, the microtubule disrupting agents, vinblastin and nocodazole, markedly prevented the formation of these inclusions. Similar to LBs, the phosphorylated form of alpha-synuclein co-localized in these synphilin-1-containing aggresomes. Although the caspase inhibitor z-VAD-fmk significantly reduced the number of apoptotic cells, it had no impact on the percentage of aggresome-positive cells. Finally, quantitative analysis revealed aggresomes in 60% of nonapoptotic cells but only in 10% of apoptotic cells. Additionally, alpha-synuclein-induced apoptosis was not coupled with increased prevalence of aggresome-bearing cells. Taken together, these observations indicate a disconnection between aggresome formation and apoptosis, and support a protective role for these inclusions from the toxicity associated with the combined over-expression of alpha-synuclein and synphilin-1.