Alpha-Synuclein Induces Lysosomal Rupture and Cathepsin Dependent Reactive Oxygen Species Following Endocytosis

Abstract

α-synuclein dysregulation is a critical aspect of Parkinson's disease pathology. Recent studies have observed that α-synuclein aggregates are cytotoxic to cells in culture and that this toxicity can be spread between cells. However, the molecular mechanisms governing this cytotoxicity and spread are poorly characterized. Recent studies of viruses and bacteria, which achieve their cytoplasmic entry by rupturing intracellular vesicles, have utilized the redistribution of galectin proteins as a tool to measure vesicle rupture by these organisms. Using this approach, we demonstrate that α-synuclein aggregates can induce the rupture of lysosomes following their endocytosis in neuronal cell lines. This rupture can be induced by the addition of α-synuclein aggregates directly into cells as well as by cell-to-cell transfer of α-synuclein. We also observe that lysosomal rupture by α-synuclein induces a cathepsin B dependent increase in reactive oxygen species (ROS) in target cells. Finally, we observe that α-synuclein aggregates can induce inflammasome activation in THP-1 cells. Lysosomal rupture is known to induce mitochondrial dysfunction and inflammation, both of which are well established aspects of Parkinson's disease, thus connecting these aspects of Parkinson's disease to the propagation of α-synuclein pathology in cells.

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

“…The cellular mechanisms by which extracellular or lumenal ␣-syn can encounter endogenous ␣-syn, which is predominantly cytosolic, are unclear, although one pathway that could mediate this encounter is via the rupture of endocytic vesicle membranes by internalized ␣-syn (20). Damage to transport vesicles could therefore permit misfolded lumenal ␣-syn to gain access to the cytosolic compartment where it may have an opportunity to propagate ␤-sheet structure to endogenous normal ␣-syn.…”

“…Cells were maintained in a 37°C incubator with 5% CO 2 . SH-SY5YchGal3 cells were made according to the protocol as described previously (20).…”

“…First, protein and solute concentrations, as well as total volume, were standardized across all four samples, according to previously published aggregation conditions (20). Specifically, 200 l of purified ␣-syn (1 mg/ml) was prepared in a pH 7.4 buffer containing the following solute concentrations: 323.3 mM NaCl, 20 mM Tris-HCl, 9 mM Na 2 HPO 4 , 2.43 mM KCl, and 1.62 mM KH 2 PO 4 .…”

“…The loss of vesicle membrane integrity permits galectin-3 access to lumenally oriented ␤-galactosides that are bound by the carbohydrate recognition domain present in galectin-3, and therefore it has been utilized to monitor vesicle rupture induced by bacterial and viral pathogens during infection (30,31). We have previously reported that ␣-syn aggregates induce galectin-3 relocalization to intracellular vesicles containing ␣-syn following endocytosis (20). We therefore used this assay to investigate the ability of endocytosed non-phosphorylated and phosphorylated ␣-syn to rupture vesicular membranes and to test the hypothesis that diseaseassociated familial missense mutations or post-translational modifications can differentially affect the potency of ␣-syn entry via this disruptive mechanism.…”

The neural chaperone proSAAS blocks α-synuclein fibrillation and neurotoxicity

“…Oligomers are thought to represent the cytotoxic form (61). Overexpression and/or oligomerization of α-synuclein causes changes in membrane permeability (62), mitochondrial dysfunction (63), disruptions in endoplasmic reticulum/Golgi trafficking (64), lysosomal leakage (65), and impairment and inhibition of proteasomes (66,67). Our finding of substoichiometric blockade of α-synuclein fibrillation, coupled with the demonstration of immunoreactive proSAAS within Lewy bodies, indicates that proSAAS could function upstream of cytotoxic processes by interacting with intracellular α-synuclein monomers in a transition state (68) that would otherwise propagate toward toxic fibril formation, or by interacting with oligomers to prevent further oligomerization, fibrillation, and cytotoxicity.…”