Suppression of dynamin GTPase decreases α-synuclein uptake by neuronal and oligodendroglial cells: a potent therapeutic target for synucleinopathy

Abstract

BackgroundThe intracellular deposition of misfolded proteins is a common neuropathological hallmark of most neurodegenerative disorders. Increasing evidence suggests that these pathogenic proteins may spread to neighboring cells and induce the propagation of neurodegeneration.ResultsIn this study, we have demonstrated that α-synuclein (αSYN), a major constituent of intracellular inclusions in synucleinopathies, was taken up by neuronal and oligodendroglial cells in both a time- and concentration-dependent manner. Once incorporated, the extracellular αSYN was immediately assembled into high-molecular-weight oligomers and subsequently formed cytoplasmic inclusion bodies. Furthermore, αSYN uptake by neurons and cells of the oligodendroglial lineage was markedly decreased by the genetic suppression and pharmacological inhibition of the dynamin GTPases, suggesting the involvement of the endocytic pathway in this process.ConclusionsOur findings shed light on the mode of αSYN uptake by neuronal and oligodendroglial cells and identify therapeutic strategies aimed at reducing the propagation of protein misfolding.

Effects of Serine 129 Phosphorylation on α-Synuclein Aggregation, Membrane Association, and Internalization

“…In the presence of the dynamin inhibitor dynasore, instead of the expected perinuclear accumulation, the ␣-syn immunoreactivity was excluded from the cell interior and retained at the periphery adjacent to the plasma membrane. Despite the qualitatively different intracellular localization, the overall signal for each ␣-syn isoform was not greatly reduced, indicating normal cell surface binding and sequestration, and consistent with a blockade of dynamindependent endocytosis (69,(72)(73)(74). Interestingly, the dynasore treatment accentuated the differential accumulation of the non-phosphorylated and phosphorylated WT ␣-syn, raising the possibility that the endocytosed ␣-syn may normally undergo trafficking to organelles with protein degradation machinery.…”

Lys-63-linked Ubiquitination by E3 Ubiquitin Ligase Nedd4-1 Facilitates Endosomal Sequestration of Internalized α-Synuclein

“…All recombinant proteins were expressed in the BL21(DE3)pLysS Escherichia coli strain and purified as described previously (3). The purity and identity of the recombinant proteins were verified by Coomassie Brilliant Blue (MP Biomedicals, OH) staining and Western blot analysis.…”

“…After the discovery of LB-like inclusions in the grafted neurons of PD patients who had previously received transplants of fetal mesencephalic neurons (1), increasing evidence has suggested that both monomeric and oligomeric aS can be secreted into the extracellular milieu (2), thereby affecting the physiological state of neighboring cells. Previous studies have revealed that the cellular uptake of fibrillar aS requires physiological temperatures and dynamin-1 (3,4), a master regulator of endocytic vesicle formation, suggesting the active participation of the endocytic machinery. However, aS incorporation into cells is not completely disabled by the inhibition of endocytosis (3), indicating that other pathways, such as direct penetration, macropinocytosis, pore formation, and diffusion, might be involved in aS internalization (5).…”

“…Previous studies have revealed that the cellular uptake of fibrillar aS requires physiological temperatures and dynamin-1 (3,4), a master regulator of endocytic vesicle formation, suggesting the active participation of the endocytic machinery. However, aS incorporation into cells is not completely disabled by the inhibition of endocytosis (3), indicating that other pathways, such as direct penetration, macropinocytosis, pore formation, and diffusion, might be involved in aS internalization (5). This notion is supported by previous studies showing that both monomers and oligomers of aS can freely passes though plasma membranes (4).…”

“…The intraneuronal aggregation of misfolded ␣-synuclein (aS), 3 known as a component of Lewy bodies (LB), is a pathological hallmark of Parkinson disease (PD). After the discovery of LB-like inclusions in the grafted neurons of PD patients who had previously received transplants of fetal mesencephalic neurons (1), increasing evidence has suggested that both monomeric and oligomeric aS can be secreted into the extracellular milieu (2), thereby affecting the physiological state of neighboring cells.…”