α-Synuclein is central in Parkinson's disease pathogenesis. Although initially α-synuclein was considered a purely intracellular protein, recent data suggest that it can be detected in the plasma and CSF of humans and in the culture media of neuronal cells. To address a role of secreted α-synuclein in neuronal homeostasis, we have generated wild-type α-synuclein and β-galactosidase inducible SH-SY5Y cells. Soluble oligomeric and monomeric species of α-synuclein are readily detected in the conditioned media (CM) of these cells at concentrations similar to those observed in human CSF. We have found that, in this model, α-synuclein is secreted by externalized vesicles in a calcium-dependent manner. Electron microscopy and liquid chromatography–mass spectrometry proteomic analysis demonstrate that these vesicles have the characteristic hallmarks of exosomes, secreted intraluminar vesicles of multivesicular bodies. Application of CM containing secreted α-synuclein causes cell death of recipient neuronal cells, which can be reversed after α-synuclein immunodepletion from the CM. High- and low-molecular-weight α-synuclein species, isolated from this CM, significantly decrease cell viability. Importantly, treatment of the CM with oligomer-interfering compounds before application rescues the recipient neuronal cells from the observed toxicity. Our results show for the first time that cell-produced α-synuclein is secreted via an exosomal, calcium-dependent mechanism and suggest that α-synuclein secretion serves to amplify and propagate Parkinson's disease-related pathology.

Parkinson’s disease and related synucleinopathies are characterized by the abnormal accumulation of alpha-synuclein aggregates, loss of dopaminergic neurons, and gliosis of the substantia nigra. Although clinical evidence and in vitro studies indicate disruption of the Blood-Brain Barrier in Parkinson’s disease, the mechanisms mediating the endothelial dysfunction is not well understood. Here we leveraged the Organs-on-Chips technology to develop a human Brain-Chip representative of the substantia nigra area of the brain containing dopaminergic neurons, astrocytes, microglia, pericytes, and microvascular brain endothelial cells, cultured under fluid flow. Our αSyn fibril-induced model was capable of reproducing several key aspects of Parkinson’s disease, including accumulation of phosphorylated αSyn (pSer129-αSyn), mitochondrial impairment, neuroinflammation, and compromised barrier function. This model may enable research into the dynamics of cell-cell interactions in human synucleinopathies and serve as a testing platform for target identification and validation of novel therapeutics.

Approximately 90% of alpha-synuclein (α-Synuclein) deposited in Lewy bodies is phosphorylated at serine 129 suggesting that the accumulation of phosphorylated α-Synuclein is critical in the pathogenesis of Parkinson’s disease. However, in vivo experiments addressing the role of phosphorylated α-Synuclein in the progression of Parkinson’s disease have produced equivocal data. To clarify a role of Ser129 phosphorylation of α-Synuclein in pathology progression we performed stereotaxic injections targeting the mouse striatum with three fibrilar α-Synuclein types: wt-fibrils, phosphorylated S129 fibrils and, phosphorylation incompetent, S129A fibrils. Brain inoculation of all three fibrilar types caused seeding of the endogenous α-Synuclein. However, phosphorylated fibrils triggered the formation of more α-Synuclein inclusions in the Substantia Nigra pars compacta (SNpc), exacerbated pathology in the cortex and caused dopaminergic neuronal loss and fine motor impairment as early as 60 days post injection. Phosphorylated fibril injections also induced early changes in the innate immune response including alterations in macrophage recruitment and IL-10 release. Our study further shows that S129 phosphorylation facilitated α-Synuclein fibril uptake by neurons. Our results highlight the role of phosphorylated fibrilar α-Synuclein in pathology progression in vivo and suggest that targeting phosphorylated α-Synuclein assemblies might be important for delaying inclusion formation.

Genetic, biochemical, and animal model studies strongly suggest a central role for α-synuclein in the pathogenesis of Parkinson's disease. α-synuclein lacks a signal peptide sequence and has thus been considered a cytosolic protein. Recent data has suggested that the protein may be released from cells via a non-classical secretory pathway and may therefore exert paracrine effects in the extracellular environment. However, proof that α-synuclein is actually secreted into the brain extracellular space in vivo has not been obtained. We developed a novel highly sensitive ELISA in conjugation with an in vivo microdialysis technique to measure α-synuclein in brain interstitial fluid. We show for the first time that α-synuclein is readily detected in the interstitial fluid of both α-synuclein transgenic mice and human patients with traumatic brain injury. Our data suggest that α-synuclein is physiologically secreted by neurons in vivo. This interstitial fluid pool of the protein may have a role in the propagation of synuclein pathology and progression of Parkinson's disease.

Misfolding and aggregation of alpha‐synuclein (α‐synuclein) with concomitant cytotoxicity is a hallmark of Lewy body related disorders such as Parkinson’s disease, dementia with Lewy bodies, and multiple system atrophy. Although it plays a pivotal role in pathogenesis and disease progression, the function of α‐synuclein and the molecular mechanisms underlying α‐synuclein‐induced neurotoxicity in these diseases are still elusive. Many in vitro and in vivo experimental models mimicking α‐synuclein pathology such as oligomerization, toxicity and more recently neuronal propagation have been generated over the years. In particular, cellular models have been crucial for our comprehension of the pathogenic process of the disease and are beneficial for screening of molecules capable of modulating α‐synuclein toxicity. Here, we review α‐synuclein based cell culture models that reproduce some features of the neuronal populations affected in patients, from basic unicellular organisms to mammalian cell lines and primary neurons, to the cutting edge models of patient‐specific cell lines. These reprogrammed cells known as induced pluripotent stem cells (iPSCs) have garnered attention because they closely reproduce the characteristics of neurons found in patients and provide a valuable tool for mechanistic studies. We also discuss how different cell models may constitute powerful tools for high‐throughput screening of molecules capable of modulating α‐synuclein toxicity and prevention of its propagation. image This article is part of the Special Issue “Synuclein”.

Alpha‐synuclein (ASYN) is central in Parkinson’s disease pathogenesis. Converging pieces of evidence suggest that the levels of ASYN expression play a critical role in both familial and sporadic Parkinson’s disease. To elucidate the mechanism underlying wild type (WT) ASYN‐mediated neurotoxicity, we have generated a novel Tet‐Off SHSY‐5Y cell line, conditionally expressing WT ASYN. Induction of human WT ASYN in retinoic acid‐differentiated SHSY‐5Y cells leads to accumulation of soluble ASYN oligomers, in the absence of inclusions, and to gradual cellular degeneration. Morphologically, the death observed is non‐apoptotic. Caspases other than caspase 3, including caspase 9, are activated and caspase inhibition diminishes death by acting at a point upstream of cytochrome c release. Application of Scyllo‐inositol, an oligomer‐stabilizing compound, prevents neuronal death in this model. These findings are consistent with a model in which oligomeric ASYN triggers the initial activation of the apoptotic pathway, which is however blocked downstream of the mitochondrial checkpoint, thus leading to a death combining in a unique fashion both apoptotic and non‐apoptotic features. This novel inducible cell model system may prove valuable in the deciphering of WT ASYN‐induced pathogenic effects and in the assessment and screening of potential therapeutic strategies.