Proteomic identification of novel proteins in cortical lewy bodies

Abstract

Lewy body (LB) inclusions are one of the pathological hallmarks of Parkinson's disease (PD) and dementia with Lewy bodies (DLB). One way to better understand the process leading to LB formation and associated pathogenesis responsible for neurodegeneration in PD and DLB is to examine the content of LB inclusions. Here, we performed a proteomic investigation of cortical LBs, obtained by laser capture microdissection from neurons in the temporal cortex of dementia patients with cortical LB disease. Analysis of over 2500 cortical LBs discovered 296 proteins; of those, 17 had been associated previously with brainstem and/or cortical LBs. We validated several proteins with immunohistochemical staining followed by confocal microscopy. The results demonstrated that heat shock cognate 71 kDa protein (also known as HSC70, HSP73, or HSPA10) was indeed not only colocalized with the majority of LBs in the temporal cortex but also colocalized to LBs in the frontal cortex of patients with diffuse LB disease. Our investigation represents the first extensive proteomic investigation of cortical LBs, and it is expected that characterization of the proteins in the cortical LBs may reveal novel mechanisms by which LB forms and pathways leading to neurodegeneration in DLB and/or advanced PD. Further investigation of these novel candidates is also necessary to ensure that the potential proteins in cortical LBs are not identified incorrectly because of incomplete current human protein database.

Figure 1

Classification of proteins identified in cortical Lewy bodies. Pie chart depicting the 296 proteins characterized by LC‐MS/MS. Functional classification of a given protein was based on the one that is best known, although typically, multiple functions may have been associated with that particular protein. Notably, a significant portion of the proteome is novel without known functions.

Figure 2

Confocal analysis of the relationship between SNCA and HSC70. Upper panel shows the triple staining results of SNCA, HSC70 and DAPI in the cortical brain sections of dementia with Lewy body (DLB) patients. Sections were stained with antibodies against SNCA and HSC70 simultaneously, along with nuclei staining (DAPI), after microwave antigen retrieval. The localization of each antibody was revealed by secondary antibodies conjugated with fluorescence probe for SNCA (green) and HSC70 (red), respectively. Yellow color in the merge pane indicates colocalization of the two antibodies. An enlarged view of a LB is shown in the inset of merged column. Lower panel shows the triple staining results of SNCA, HSC70 and DAPI in the substantia nigra of DLB patients. The experimental conditions were identical to those described for cortical sections.