Prion-Like Seeding of Misfolded \u03b1-Synuclein in the Brains of Dementia with Lewy Body Patients in RT-QUIC

Kazunori Sano,Ryuichiro Atarashi,Mari Yoshida,Noriyuki Nishida,Shigeo Murayama,Yasushi Iwasaki,Daisuke Ishibashi,Kenichi Mishima,Katsuya Satoh,Takehiro Nakagaki

doi:10.1007/s12035-017-0624-1

Abstract

The prion-like seeding of misfolded α-synuclein (αSyn) involved in the pathogenesis of Lewy body diseases (LBD) remains poorly understood at the molecular level. Using the real-time quaking-induced conversion (RT-QUIC) seeding assay, we investigated whether brain tissues from cases of dementia with Lewy bodies (DLB), which contain serine 129 (Ser129)-phosphorylated insoluble aggregates of αSyn, can convert Escherichia coli-derived recombinant αSyn (r-αSyn) to fibrils. Diffuse neocortical DLB yielded 50% seeding dose (SD50) values of 107~1010/g brain. Limbic DLB was estimated to have an SD50 value of ~105/g brain. Furthermore, RT-QUIC assay discriminated DLB from other neurological and neurodegenerative disorders. Unexpectedly, the prion-like seeding was reconstructed in reactions seeded with oligomer-like species, but not with insoluble aggregates of r-αSyn, regardless of Ser129 phosphorylation status. Our findings suggest that RT-QUIC using r-αSyn can be applied to detect seeding activity in LBD, and the culprit that causes prion-like seeding may be oligomeric forms of αSyn.

Highlights

The accumulation of abundant misfolded proteins in the brain is a defining feature of most neurodegenerative disorders
We examined whether serine 129 (Ser129) phosphorylation is crucial for αSyn fibril formation through a prion-like mechanism using recombinant αSyn (r-αSyn) phosphorylated at Ser129
The results of this study demonstrated that the formation of r-αSyn fibrils is induced by real-time quaking-induced conversion (RT-QUIC) using soluble r-αSyn only in the presence of brain homogenates (BH) from patients with dementia with Lewy bodies (DLB) (Fig. 3a, b)

Summary

Introduction

The accumulation of abundant misfolded proteins in the brain is a defining feature of most neurodegenerative disorders. Lewy body diseases (LBD), such as dementia with Lewy bodies (DLB) and Parkinson’s disease (PD), are characterized by the presence of Lewy bodies (LB), which are filamentous cytoplasmic inclusions composed mainly of aggregated αsynuclein (αSyn). The pathogenic mechanisms have not been fully elucidated, LBD is thought to occur through the accumulation of LB, i.e., aggregated αSyn, in neurons and neurites. The bulk of αSyn aggregates within LB are phosphorylated at serine 129 (Ser129), while αSyn in the normal brain undergoes very little phosphorylation. Phosphorylation at Ser129 is the dominant pathological characteristic and may be crucial in LB formation and the pathogenesis of LBD. Ser129 phosphorylation accelerates polymerization of recombinant αSyn (r-αSyn) [1], and overexpression of wild-type (WT) r-αSyn, but not nonphosphorylatable mutant with

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Conclusion