Mass Spectrometric Analysis of Lewy Body-Enriched α-Synuclein in Parkinson’s Disease

Abstract

Parkinson's disease (PD) is characterized by intraneuronal inclusions of aggregated α-synuclein protein (so-called Lewy bodies) in distinct brain regions. Multiple posttranslational modifications may affect the structure and function of α-synuclein. Mass spectrometry-based analysis may be useful for the characterization and quantitation of α-synuclein forms, but has proven challenging, mainly due to the insolubility of Lewy bodies in aqueous buffer. In the present study, we developed a novel method by combining differential solubilization with immunoprecipitation and targeted proteomics using liquid chromatography and tandem mass spectrometry. Brain tissue homogenization and sample preparation were modified to facilitate analysis of soluble, detergent-soluble, and detergent-insoluble protein fractions (Lewy body-enriched). The method was used to compare α-synuclein forms from cingulate cortex (affected) and occipital cortex (unaffected) in two study sets of PD patients and controls. We identified ∼20 modified α-synuclein variants, including species with N-terminal acetylation and C-terminal truncations at amino acids 103 and 119. The levels of α-synuclein forms Ac-α-syn 1−6 , α-syn 13−21 , α-syn 35−43 , α-syn 46−58 , α-syn 61−80 , and α-syn 81−96 except αsyn 103−119 were significantly increased in PD cingulate region compared to controls in the Lewy body-enriched α-synuclein fraction. In the soluble fraction, only Ac-α-syn 1−6 was significantly increased in PD compared to controls. None of the detected α-synuclein variants were Lewy body-specific, but acetylated forms should be examined further as potential biomarkers for abnormal α-synuclein accumulation.

Protein Quality Control Pathways at the Crossroad of Synucleinopathies

“…Deletion of only the last 10 amino acids is already sufficient to accelerate ␣-Syn aggregation in vitro, and this effect is stronger upon larger C-terminal truncations up to amino acids 102-120 [24][25][26]. ␣-Syn is subject to several post-translational modifications (PTMs), including N-terminal acetylation, ubiquitylation, SUMOylation, nitration, and phosphorylation [27][28][29][30][31][32], with diverse consequences for its function and propensity to aggregate (detailed below). Several roles have been ascribed to ␣-Syn, including facilitating the assembly of N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-complexes at presynaptic neuron terminals that mediate release of neurotransmitters [33,34], and induction of membrane curvatures [35], among many others [36].…”

SUMOylation in α-Synuclein Homeostasis and Pathology

“…Acetylation is also an outstanding α-synuclein PTM as it is found in Lewy bodies and brain regions affected in the disease ( Anderson et al, 2006 ; Ohrfelt et al, 2011 ; Kellie et al, 2014 ; Bhattacharjee et al, 2019 ). Obliteration of α-synuclein acetylation sites in vivo leads to its enhanced aggregation and toxicity in primary neurons and mice ( de Oliveira et al, 2017 ), implying that acetylation may prevent α-synuclein aggregation.…”

“…Later, it was identified by mass spectrometry analysis in both purified Lewy bodies ( Anderson et al, 2006 ; Bhattacharjee et al, 2019 ) and lysates of PD tissues ( Ohrfelt et al, 2011 ; Kellie et al, 2014 ), and shown to occur at different α-synuclein residues at the C- and N-termini (for more details see Table 1 ). Although not all reported α-synuclein truncations were found enriched in Lewy bodies or PD tissues ( Ohrfelt et al, 2011 ; Kellie et al, 2014 ; Bhattacharjee et al, 2019 ), the importance of truncations is implied by their increased tendency to aggregate in vitro , in cell lines and in vivo models ( Crowther et al, 1998 ; Liu et al, 2005 ; Tofaris et al, 2006 ; Periquet et al, 2007 ). Also, the C-terminally truncated α-synuclein species assemble into filaments that resemble those found in diseased brains ( Crowther et al, 1998 ) and leads to the formation of pathological inclusions and neurotoxicity in transgenic mice ( Tofaris et al, 2006 ).…”

Amyloid precipitation in biofluids using a structure-specific chemical antibody

“…Concentrations spiked were 1 pM, 100 pM, 1 nM, 10 nM and 100 nM and samples were prepared in triplicate. The immunoprecipitation method for CSF samples was performed according to Bhattacharjee et al 2019 with minor modifications 64,65 . Briefly, after overnight incubation at 4 °C the KingFisher magnetic particle processor (Thermo where P is the probability that the observed match is a random event.…”