Quantum-dot-labeled synuclein seed assay identifies drugs modulating the experimental prion-like transmission.

Synucleinopathies are neurodegenerative disorders including Parkinson disease (PD), dementia with Lewy body (DLB), and multiple system atrophy (MSA) that involve deposits of the protein alpha-synuclein (α-syn) in the brain. The inoculation of α-syn aggregates derived from synucleinopathy or preformed fibrils (PFF) formed in vitro induces misfolding and deposition of endogenous α-syn. This is referred to as prion-like transmission, and the mechanism is still unknown. In this study, we label α-syn PFF with quantum dots and visualize their movement directly in acute slices of brain tissue inoculated with α-syn PFF seeds. Using this system, we find that the trafficking of α-syn seeds is dependent on fast axonal transport and the seed spreading is dependent on endocytosis and neuronal activity. We also observe pharmacological effects on α-syn seed spreading; clinically available drugs including riluzole are effective in reducing the spread of α-syn seeds and this effect is also observed in vivo. Our quantum-dot-labeled α-syn seed assay system combined with in vivo transmission experiment reveals an early phase of transmission, in which uptake and spreading of seeds occur depending on neuronal activity, and a later phase, in which seeds induce the propagation of endogenous misfolded α-syn.

Preserved proteinase K-resistant core after amplification of alpha-synuclein aggregates: Implication to disease-related structural study.

Many pathological proteins related to neurodegenerative diseases are misfolded, aggregating to form amyloid fibrils during pathogenesis. One of the pathological proteins, alpha-synuclein (α-syn), accumulates in the brains of Parkinson disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA), which are designated as synucleinopathies. Recently, structural properties of abnormal accumulated proteins are suggested to determine the disease phenotype. However, the biochemical and structural characteristics of those accumulated proteins are still poorly understood. We previously reported the sequence and seed-structure-dependent polymorphic fibrils of α-syn and the polymorphism was identified by proteinase K-resistant cores determined by mass spectrometry (MS) analysis. In this study, we applied this method to analyze α-syn aggregates of MSA and DLB. To perform MS analysis on proteinase K-resistant cores, we first performed amplification of α-syn aggregates by seeding reaction and protein misfolding cyclic amplification (PMCA) to obtain a sufficient amount of aggregates. Using SDS insoluble fraction of the disease brain, we successfully amplified enough α-syn aggregates for MS analysis. We differentiated between mouse and human α-syn aggregates by MS analysis on proteinase K-resistant cores of the aggregates before and after amplification. The results suggest that structural properties of amplified α-syn fibrils are preserved after PMCA and these methods can be applicable in the study of pathological proteins of the neurodegenerative disorders.

Rapid dissemination of alpha-synuclein seeds through neural circuits in an in-vivo prion-like seeding experiment.

Accumulating evidence suggests that the lesions of Parkinson's disease (PD) expand due to transneuronal spreading of fibrils composed of misfolded alpha-synuclein (a-syn), over the course of 5-10 years. However, the precise mechanisms and the processes underlying the spread of these fibril seeds have not been clarified in vivo. Here, we investigated the speed of a-syn transmission, which has not been a focus of previous a-syn transmission experiments, and whether a-syn pathologies spread in a neural circuit-dependent manner in the mouse brain. We injected a-syn preformed fibrils (PFFs), which are seeds for the propagation of a-syn deposits, either before or after callosotomy, to disconnect bilateral hemispheric connections. In mice that underwent callosotomy before the injection, the propagation of a-syn pathology to the contralateral hemisphere was clearly reduced. In contrast, mice that underwent callosotomy 24 h after a-syn PFFs injection showed a-syn pathology similar to that seen in mice without callosotomy. These results suggest that a-syn seeds are rapidly disseminated through neuronal circuits immediately after seed injection, in a prion-like seeding experiment in vivo, although it is believed that clinical a-syn pathologies take years to spread throughout the brain. In addition, we found that botulinum toxin B blocked the transsynaptic transmission of a-syn seeds by specifically inactivating the synaptic vesicle fusion machinery. This study offers a novel concept regarding a-syn propagation, based on the Braak hypothesis, and also cautions that experimental transmission systems may be examining a unique type of transmission, which differs from the clinical disease state.