Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy are caused by α-synuclein aggregates. At present, there is no good biochemical method defining α-synuclein aggregates formed in vivo versus oligomers as a means to investigate α-synuclein aggregation and its mechanisms of neurodegeneration. A simple method, therefore, for the selective and sensitive detection of α-synuclein aggregates suited for screening purposes would be useful. Since in contrast to prions a proper detection of α-synuclein aggregates by Western blot analysis is difficult, we developed a protein aggregate filtration (PAF) assay. It takes advantage of the inherent insolubility of aggregated α-synuclein using microfiltration to separate it from soluble isoforms. For the first time, this assay even makes quantitative comparisons possible. We describe how the PAF assay can be applied to human brain tissue and animal and cell culture models, as well as used as a screening method for the subcellular location of α-synuclein aggregates. Since it detects the pathological isoform instead of surrogate markers, the PAF assay may have also potential in diagnosis of PD and DLB.
Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple systemic atrophy (MSA) are α-synucleinopathies characterized by depositions of aggregated α-synuclein (1,2,3). For a long time, Lewy bodies, which are large juxtanuclear inclusions of aggregated α-synuclein (3), were considered to be the pathophysiological determinant in PD and DLB. However, the small number of Lewy bodies relative to the total neuron count did not correlate with the severity of clinical symptoms in DLB (4). Recently, we reported that there are enormous amounts of small α-synuclein aggregates localized at presynaptic terminals, which, rather than Lewy bodies, may better explain cognitive impairment in DLB (5). This finding suggests that Lewy bodies are the result of aggresome formation acting as a neuronal protection mechanism through the axonal retrograde transport of small presynaptic α-synuclein aggregates (6,7). The massive accumulation of α-synuclein aggregates at presynaptic terminals was linked to an almost complete loss of dendritic spines. This strongly suggests a novel mechanism of neurodegeneration for DLB and possibly also for PD in which synaptic dysfunction is caused by presynaptic accumulation of α-synuclein aggregates (5).
To investigate the process of α-synuclein aggregation in vivo as well as the molecular mechanisms of neurodegeneration in DLB, PD, and MSA in more detail, we need to analyze corresponding cell culture and animal models. A simple biochemical screening method for the sensitive and selective detection of α-synuclein aggregates in liquid samples such as homogenates would therefore be very useful.
Unlike with prions, the detection of α-synclein aggregates by Western blot analysis has proven difficult due to their inherent insolubility (8). The resulting smearing of α-synuclein reactivity at higher molecular weights categorized them not only as aggregates (9) but also as oligomers (10). Thus a simple biochemical method properly defining α-synuclein aggregates versus oligomers is lacking. For both the specific detection of aggregates by their selective insolubility in detergents followed by extraction with urea (9) and the detection of oligomers (10), ultracentrifugation steps are essential, rendering the process relatively tedious. Additionally, the insolubility of α-synuclein aggregates has hampered so far any quantitative approach to the α-synuclein aggregate burden.Materials and Methods Brain Homogenate Preparation
Samples for brain homogenates were taken from the frontal cortex (gyrus frontalis medialis) in DLB cases and from the substantia nigra in PD. They were homogenized in 9 vol (w/v) MSE buffer (10 mM MOPS/KOH pH 7.4, 0.3 M sucrose, and 1 mM EDTA) including protease inhibitors (1 mM PMSF, 0.2 mM TPCK, and 0.2 mM TLCK) with a glass/teflon homogenizer by 12 strokes.Cell Culture
Wild-type human α-synuclein was cloned into the SalI/XbaI site of pCIneo (Promega, Mannheim, Germany). SH-SY5Y cells were transfected with αSYN-pCIneo using Lipofectamin (Invitrogen, Karlsruhe, Germany) according to the manufacturer's instructions. Forty-eight hours after transfection, cells were grown in DMEM (Biochrom, Berlin, Germany) and supplemented with 10% FCS, penicillin/streptomycin, L-glutamine, 10 µM retinoic acid (Sigma, Taufkirchen, Germany), and 200 µg/mL G418 (Gibco, Karlsruhe, Germany). Overexpression of α-synuclein was checked by Western blot analysis. After 5 days, the cells were treated with freshly prepared FeCl2 (100 µM). Cells were harvested 24 h later and analyzed by the protein aggregate filtration (PAF) assay we developed.Western Blot Analysis
Brain homogenates were centrifuged at 16,000 g for 5 min and the supernatant was aspirated. The pellet fraction containing α-synuclein aggregates was dissolved in sample buffer containing 5% SDS and 8 M urea and applied to a SDS gel with a 12.5% (w/v) acrylamide separating gel, including 8 M urea. After transfer to nitrocellulose membrane, the blot was incubated with the antibody LB509 (1:10,000; Zymed, Berlin, Germany) and immunoreactivity was visualized by chemiluminescence using HRP-coupled goat-anti-mouse antibody. Western blot analysis for syntaxin and synaptophysin was performed as described previously (5).Protein Aggregate Filtration Assay for α-synuclein Aggregates
After centrifuging brain homogenates at 16,000 g for 5 min, supernatants were aspirated and saved for later analysis. The pellet fractions were subjected to digestion with DNase I (Fluka, Buchs, Switzerland) in 4% N-tetradecyl-N,N-dimethyl-3-amonio-1-propane sulfonate (SB14; Sigma) for 15 min at 37°C. Finally, pellet and supernatant fractions were adjusted to a final concentration of 5% SLS (Fluka).