Abstract
The Lewy bodies (LBs) are the pathological hallmark of Parkinson's disease (PD). More than 90% of α-synuclein (α-syn) within LBs is phosphorylated at the serine-129 residue [pSer129 α-syn (p-α-syn)]. Although various studies have revealed that this abnormally elevated p-α-syn acts as a pathological biomarker and is involved in the pathogenic process of PD, the exact pathophysiological mechanisms of p-α-syn are still not fully understood. Therefore, the development of specific and reliable tools for p-α-syn detection is important. In this study, we generated a novel p-α-syn mouse monoclonal antibody (C140S) using hybridoma technology. To further identify the characteristics of C140S, we performed several in vitro assays using recombinant proteins, along with ex vivo assays utilizing the brains of Thy1-SNCA transgenic (Tg) mice, the preformed fibril (PFF)-treated neurons, and the brain sections of patients with PD. Our C140S specifically recognized human and mouse p-α-syn proteins both in vitro and ex vivo, and similar to commercial p-α-syn antibodies, the C140S detected higher levels of p-α-syn in the midbrain of the Tg mice. Using immunogold electron microscopy, these p-α-syn particles were partly deposited in the cytoplasm and colocalized with the outer mitochondrial membrane. In addition, the C140S recognized p-α-syn pathologies in the PFF-treated neurons and the amygdala of patients with PD. Overall, the C140S antibody was a specific and potential research tool in the detection and mechanistic studies of pathogenic p-α-syn in PD and related synucleinopathies.
Highlights
IntroductionCharacteristic pathological changes of Parkinson’s disease (PD) include the formation of intracellular inclusions termed, “Lewy bodies” (LBs) and “Lewy neurites” (LNs) [1, 2]
Parkinson’s disease (PD) is one of the most common movement disorders in the world
The results showed that C54S and C140S exclusively bound to α-syn phosphorylated at the serine-129 residue (P1, peptides 2 (P2), and p-α-syn) without cross-reactivity with αsyn and β-synuclein, and the results showed no difference from the commercial p-α-syn#1 antibody
Summary
Characteristic pathological changes of PD include the formation of intracellular inclusions termed, “Lewy bodies” (LBs) and “Lewy neurites” (LNs) [1, 2]. Little is known about how α-syn abnormally aggregates and becomes the main component of LBs and LNs in the relevant brain regions. Pathological forms of α-syn can transfer from cell-to-cell in a prion-like manner, which might further contribute to the spread of α-syn pathology and promote the neurodegenerative process [5–7]. While it is not clear what mechanisms regulate α-syn transmission, we still know little about the pathophysiological roles of the secreted α-syn
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