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

Abstract

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.

Sortilin acts as an endocytic receptor for α‐synuclein fibril

“…This hypothetical concept is supported by autopsy results in the brains of PD patients who received transplants of fetal mesencephalic tissues more than a decade ago; in these brains, αSYN‐positive LB‐like inclusions were confirmed within donor‐derived neurons 15,16 . Furthermore, the non‐cell‐autonomous spread of αSYN aggregates has been verified via neuronal co‐culture and animal models, which showed that misfolded αSYN released from neurons can transfer to neighboring cells and convert normal physiological αSYN to toxic misfolded species in a prion‐like manner 17–23 . Following its validation, this “prionoid” hypothesis is now widely accepted as a common phenomenon in the pathogenesis of neurodegenerative diseases involving aberrant protein aggregates 24 …”

“…The proposed mechanism underlying the mode of αSYN aggregate uptake by cells include endocytosis, macropinocytosis, extracellular vesicles, and direct cellular contact via nanotube tunnel 24 . Several studies have highlighted the importance of the endocytic process because low temperature and the genetic and chemical ablation of dynamin, a key determinant for vesicle scission, markedly decrease the internalization of αSYN in cell cultures and animal models 17,20,26 . Studies have sought to determine whether the endocytic uptake of αSYN is receptor‐mediated, because the existence of specific receptors for misfolded αSYN on the cell surface might explain the region‐ and cell‐type‐specific neurodegeneration in synucleinopathies 24,27 .…”

“…15,16 Furthermore, the noncell-autonomous spread of αSYN aggregates has been verified via neuronal co-culture and animal models, which showed that misfolded αSYN released from neurons can transfer to neighboring cells and convert normal physiological αSYN to toxic misfolded species in a prion-like manner. [17][18][19][20][21][22][23] Following its validation, this "prionoid" hypothesis is now widely accepted as a common phenomenon in the pathogenesis of neurodegenerative diseases involving aberrant protein aggregates. 24 Although the precise mechanisms are not understood, the uptake, release, synthesis, and degradation machineries are known to cooperatively regulate the cellular burden and intercellular transmission of αSYN.…”

“…endocytosis, fibril, glial cytoplasmic inclusion, Lewy body, multiple system atrophy, Parkinson's disease, receptor, sortilin, synucleinopathy, αsynuclein | 3 of 19 ISHIYAMA et al αSYN in cell cultures and animal models. 17,20,26 Studies have sought to determine whether the endocytic uptake of αSYN is receptor-mediated, because the existence of specific receptors for misfolded αSYN on the cell surface might explain the region-and cell-type-specific neurodegeneration in synucleinopathies. 24,27 Several molecules on the plasma membrane (PM) have been proposed as putative binding partners for αSYN fibrils 24,27 ; however, some of these membrane proteins are preferentially expressed in non-neuronal cells, and it is possible that unknown receptors for misfolded αSYN in neuronal and glial cells remain to be discovered.…”

The Pharmacorank Search Tool for the Retrieval of Prioritized Protein Drug Targets and Drug Repositioning Candidates According to Selected Diseases

“…A second repurposing candidate is riluzole, which has the ability to reduce the alpha-synuclein protein aggregation seeds [ 45 ]. The current indication for riluzole is amyotrophic lateral sclerosis [ 43 ].…”