Studies on the Cytoplasmic Inclusions Detected in Parkinson's disease
α-Synuclein-positive cytoplasmic inclusions are a pathological hallmark of several neurodegenerative disorders, including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). Synphilin-1, interaction partner of α-Synuclein is a major component of inclusion bodies, but it is unknown how synphiln-1 contributes to the cellular and biochemical mechanisms of PD, and its normal functions and biochemical properties are poorly understood. To determine the protein interaction partners of synphilin-1, we performed a yeast two-hybrid screen. We identified a new interacting protein LIM domain only 7 protein, LMO7. This protein localized in the nucleus, cytoplasm and cell surface, particularly adhesion junctions and contains a PDZ and LIM domain, both of which mediate protein-protein interactions. In this study, LMO7 interacts with α-Synuclein interacting protein, synphilin-1 and revealed that the co-expression with synphilin-1 results in the formation of cytoplasmic inclusions in cultured HEK293 and SY5Y cells. Synphilin-1 interacts preferentially with the C-terminal LIM domain of LMO7 and LMO7 interacts with the ankyrin domain of synphilin-1. These findings have important implications for understanding the molecular mechanism by which Lewy-body-associated proteins interact through synphilin-1. We immunostained sections of brains from patients with Parkinson's disease and demonstrated that LMO7, as well as synphilin-1, accumulates in the inclusion bodies. To define the role of LMO7 in the formation of these inclusion bodies, we performed a co-transfection with synphilin-1 and LMO7 using cultured HEK293 cells. This assay showed that LMO7 in the formation of these inclusion bodies promotes the formation of cytoplasmic inclusions. α-Synuclein, synphilin-1 and its interacting partner LMO7 are among constituent proteins in these aggregates. The presence of ubiquitin and proteasome subunits in these inclusions supports a role for this protein degradation pathway in the processing of proteins involved in this disease. Treatment with proteasome inhibitors resulted in attenuation of degradation and the accumulation of high molecular weight ubiquitinated LMO7 in immunoprecipitation /immunoblot experiments. Additionally, proteasome inhibitors stimulated the formation of peri-nuclear inclusions which were immunoreactive for LMO7, ubiquitin and synphilin-1. These observations indicate that LMO7 is ubiquitinated and degraded by the proteasome. Accumulation of ubiquitinated LMO7 due to impaired clearance results in its aggregation as peri-nuclear inclusions. These results suggest that LMO7 could serve as a neuropathological marker in patients with α-Synucleinopathies because it is strongly accumulated with synphilin-1 in the inclusions of their brain cells. They also suggest that LMO7 could be a potential therapeutic target for α-Synucleinopathies.
Parkinson's disease, Lewy body, Cytoplasmic Inclusions, PDZ, LIM
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