Detection of elevated levels of soluble  -synuclein oligomers in post-mortem brain extracts from patients with dementia with Lewy bodies

Abstract

A number of neurodegenerative diseases including Parkinson's disease, dementia with Lewy bodies (DLB) and multiple system atrophy are characterized by the formation and intraneuronal accumulation of fibrillar aggregates of alpha-synuclein (alpha-syn) protein in affected brain regions. These and other findings suggest that the accumulation of alpha-syn in the brain plays an important role in the pathogenesis of these diseases. However, more recently it has been reported that early amyloid aggregates or 'soluble oligomers' are the pathogenic species that lead to neurodegeneration and neuronal cell death rather than the later 'mature fibrils'. In this study, we investigated the presence of alpha-syn oligomers in brain lysates prepared from frozen post-mortem brains of normal, Alzheimer's disease and DLB patients. The brain extracts were subjected to high speed centrifugation, to remove insoluble alpha-syn aggregates, followed by specific detection of soluble oligomers in the supernatants by employing FILA-1, an antibody that specifically binds to alpha-syn aggregates, but not to alpha-syn monomers, or to tau or beta-amyloid aggregates. Using this novel enzyme-linked immunosorbent assay (ELISA) method to quantify the amounts of alpha-syn oligomers in the brain extracts, our data clearly show an increase in the levels of soluble oligomers of alpha-syn in the DLB brains compared to those with Alzheimer's disease and the controls (P < 0.0001). Our findings provide strong evidence to support the contention that elevated soluble oligomers of alpha-syn are involved in the pathogenesis of DLB. Furthermore, these findings establish FILA-1 as a very sensitive tool for the detection of oligomeric forms of alpha-syn in human brain lysates.

Sequential Extraction of Soluble and Insoluble Alpha-Synuclein from Parkinsonian Brains

“…This methodology can not only be extended with appropriate modifications to the study of α-syn biology in α-syn transgenic animals but also to other neurodegenerative diseases featuring abnormal deposits of aggregated proteins such as AD, MSA, DLB, HD and frontotemporaldementias. However the western blot data described here could also be validated using enzyme-linked immunosorbent assays using antibodies for specific forms of α-syn 31 .…”

“…The SDS is an anionic detergent and solubilises α-syn oligomers that can include membrane bound forms of α-syn, whereas urea, a chaotropic reagent denatures the insoluble aggregated and fibrillar or amyloidogenic forms of α-syn 20 . In this regard a systematic study by Paleologou et al 31 examined the stability of α-syn oligomers and fibrils with varying concentrations of urea (6.5 -8 M) or SDS (0.25-2%) and reported that oligomers stable in SDS concentrations but not with high concentrations of urea. Their FILA-1 antibody, specific for α-syn oligomers and fibrils detected higher concentrations of fibrils at 6.5 M urea concentrations under non-denaturing conditions.…”

Novel One-step Immunoassays to Quantify α-Synuclein

“…The production of a ␣-synuclein oligomer-specific antibody would present the opportunity for the development of a more conformation-dependent immunoassay. Of note, such an antibody has been recently described for fibrillar ␣-synuclein (42,43). However, it indis-FIGURE 7.…”

CFS Biomarkers in Parkinson’s Disease

“…This achievement is likely to be fundamental to the systematic use of -synuclein as a biomarker in a clinical setting (Parnetti, 2011). Another perspective, which highlights the direction toward a combination of biomarkers rather than toward a single marker, is the combination of CSF total -syn and -syn oligomers, with the latter found increased in PD in two recent papers (Paleologou et al, 2009;Tokuda et al, 2010). Whether -syn is a marker of trait or a marker of progression is under investigation.…”