Enhanced Lysosomal Pathology Caused by β-Synuclein Mutants Linked to Dementia with Lewy Bodies

Abstract

Two missense mutations (P123H and V70M) of ␤-synuclein (␤-syn), the homologue of ␣-syn, have been recently identified in dementia with Lewy bodies. However, the mechanism through which these mutations influence the pathogenesis of dementia with Lewy bodies is unclear. To investigate the role of the ␤-syn mutations in neurodegeneration, each mutant was stably transfected into B103 neuroblastoma cells. Cells overexpressing mutated ␤-syn had eosinophilic cytoplasmic inclusion bodies immunopositive for mutant ␤-syn, and electron microscopy revealed that these cells were abundant in various cytoplasmic membranous inclusions resembling the histopathology of lysosomal storage disease. Consistent with these findings, the inclusion bodies were immunopositive for lysosomal markers, including cathepsin B, LAMP-2, GM2 ganglioside, and ATP13A2, which has recently been linked to PARK9. Notably, formation of these lysosomal inclusions was greatly stimulated by co-expression of ␣-syn, was dependent on the phosphorylation of ␣-syn at Ser-129, and was more efficient with the A53T familial mutant of ␣-syn compared with wild type. Furthermore, the inclusion formation in cells overexpressing mutant ␤-syn and transfected with ␣-syn was significantly suppressed by treatment with autophagy-lysosomal inhibitors, which were associated with impaired clearance of syn proteins and enhanced apoptosis, indicating that formation of lysosomal inclusions might be protective. Collectively, the results demonstrated unambiguously that overexpression of ␤-syn mutants (P123H and V70M) in neuroblastoma cells results in an enhanced lysosomal pathology. We suggest that these missense mutations of ␤-syn might play a causative role in stimulating neurodegeneration.

Protective Role of Endogenous Gangliosides for Lysosomal Pathology in a Cellular Model of Synucleinopathies

“…The following antibodies were used: monoclonal anti-␣-syn (syn-1) and anti-␤-syn antibodies were from BD Biosciences (Franklin Lakes, NJ); rabbit polyclonal antiAtg5 and anti-beclin-1 antibodies and monoclonal anti-␤-actin (AC-15) antibody were from Sigma; monoclonal anti-ubiquitin antibody was from Chemicon (Temecula, CA); rabbit polyclonal anti-cathepsin B was from EMD Biosciences (San Diego, CA); rabbit polyclonal anti-cathepsin D (H-75) antibody and goat polyclonal anti-lysosome-associated membrane protein-2 (LAMP-2) antibody (C-20) were from Santa Cruz Biotechnology (Santa Cruz, CA); anti-V5 was from Bethyl Laboratories (Montgomery, TX); rabbit polyclonal anti-LAMP-2 (Igp96) antibody was from Invitrogen (Carlsbad, CA); rabbit polyclonal anti-ATP13A2 antibody was from ABR (Golden, CO); rabbit polyclonal anti-LC3B serum was from Novus (Littleton, CO); monoclonal anti-␣-syn LB509 antibody was from Wako Pure Chemical Industries (Saitama, Japan); rabbit polyclonal anti-phospho-mammalian target of RAP (mTOR)(Ser2448), anti-mTOR, anti-phospho-p70 S6 kinase (Ser371), anti-p70 S6 kinase, anti-phospho-4E-BP1 (Thr37/46), anti-4E-BP1, anti-HSP70, and anti-cleaved caspase-3 antibodies (Asp175) were from Cell Signaling (Beverly, MA); guinea pig anti-P62 was from Progen (Heidelberg, Germany); rabbit anti-GM2 antibody was previously described 24 ; cholera toxin subunit B conjugated with Alexa Fluor 488, Alexa Fluor 488-conjugated antigoat and anti-rabbit antibodies, Alexa Fluor 555-conjugated anti-mouse antibody, and Alexa Fluor 647-conjugated anti-guinea pig antibody were from Molecular Probes (Eugene, OR).…”

“…24 These cells were cultured in Dulbecco's modified Eagle's medium (high glucose) containing 10% fetal calf serum (BioWest, Nuaille, France) and 1% v/v penicillin/streptomycin (Invitrogen) in a 5% CO 2 /95% air atmosphere. For the transfection of P123H ␤-syn-overexpressing B103 neuroblastoma cells, cells were transfected with control vectors, wild-type ␣-syn, or familial PD-linked A53T ␣-syn.…”

“…Further, the number of inclusions were increased when these cells were further transfected with ␣-syn, thus providing a cellular model to investigate the relationship between gangliosides and synuclein pathology. 24 Using this cellular model of synucleinopathies, here we show that treatment of these cells with D-Threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) resulted in various features of lysosomal pathology. PDMP is a ceramide analogue that efficiently inhibits the cell's glucosylceramide synthase activities and has been widely used as a tool for studying various effects of endogenous glycosphingolipids, including gangliosides biosynthesized from glucosylceramide.…”

The Activation of P2X7 Receptor Impairs Lysosomal Functions and Stimulates the Release of Autophagolysosomes in Microglial Cells

“…Light and electron microscopic analyses were performed as described previously (27,28). Briefly, MG6 cells were inoculated at a density of 2.5 ϫ 10 6 cells into 60-mm petri dishes (BD Falcon).…”

Gangliosides as a Double-Edged Swordin Neurodegenerative Disease

“…Results in cell-free systems indicate that gangliosides may protect against synucleinopathies, and we have shown that changes in endogenous ganglioside levels affect lysosomal pathology in a cellular model of synucleinopathy (Wei et al, 2009a;Wei et al, 2007). In this work, DLB-linked P123H -synuclein neuroblastoma cells transfected with -synuclein were used as a model system because these cells have extensive formation of lysosomal inclusion bodies.…”