Neuron-released oligomeric α-synuclein is an endogenous agonist of TLR2 for paracrine activation of microglia.

Abnormal aggregation of α-synuclein and sustained microglial activation are important contributors to the pathogenic processes of Parkinson's disease. However, the relationship between disease-associated protein aggregation and microglia-mediated neuroinflammation remains unknown. Here, using a combination of in silico, in vitro and in vivo approaches, we show that extracellular α-synuclein released from neuronal cells is an endogenous agonist for Toll-like receptor 2 (TLR2), which activates inflammatory responses in microglia. The TLR2 ligand activity of α-synuclein is conformation-sensitive; only specific types of oligomer can interact with and activate TLR2. This paracrine interaction between neuron-released oligomeric α-synuclein and TLR2 in microglia suggests that both of these proteins are novel therapeutic targets for modification of neuroinflammation in Parkinson's disease and related neurological diseases.

Enzyme-linked immunosorbent assays for α-synuclein with species and multimeric state specificities.

Abnormal intracellular deposition of aggregated α-synuclein is the characteristic feature of a number of neurological disorders, including Parkinson's disease (PD). Although α-synuclein is typically known as a cytosolic protein, a small amount is secreted by exocytosis in both monomeric and aggregated forms. The extracellular forms of α-synuclein in human body fluids, such as cerebrospinal fluid (CSF) and blood plasma, might be a diagnostic target for PD and related diseases. Here, we characterized a new set of monoclonal antibodies against α-synuclein, and using different combinations of antibodies, we established ELISA systems to specifically detect human α-synuclein, mouse and human α-synuclein together, and multimeric forms of α-synuclein in biological samples. By employing the Tyramide signal amplification method, the sensitivity of the assay was significantly improved to detect a concentration as low as ∼12.5 pg/ml. These assays might be useful tools for quantitative analysis of α-synuclein in various forms and with high sensitivity in diverse biological samples.

Dopamine promotes formation and secretion of non-fibrillar alpha-synuclein oligomers.

Parkinson's disease (PD) is characterized by selective and progressive degeneration of dopamine (DA)-producing neurons in the substantia nigra pars compacta (SNpc) and by abnormal aggregation of α-synuclein. Previous studies have suggested that DA can interact with α-synuclein, thus modulating the aggregation process of this protein; this interaction may account for the selective vulnerability of DA neurons in patients with PD. However, the relationship between DA and α-synuclein, and the role in progressive degeneration of DA neurons remains elusive. We have shown that in the presence of DA, recombinant human α-synuclein produces non-fibrillar, SDS-resistant oligomers, while ß-sheet-rich fibril formation is inhibited. Pharmacologic elevation of the cytoplasmic DA level increased the formation of SDS-resistant oligomers in DA-producing neuronal cells. DA promoted α-synuclein oligomerization in intracellular vesicles, but not in the cytosol. Furthermore, elevation of DA levels increased secretion of α-synuclein oligomers to the extracellular space, but the secretion of monomers was not changed. DA-induced secretion of α-synuclein oligomers may contribute to the progressive loss of the dopaminergic neuronal population and the pronounced neuroinflammation observed in the SNpc in patients with PD.

Transmission of Synucleinopathies in the Enteric Nervous System of A53T Alpha-Synuclein Transgenic Mice.

Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are characterized by abnormal deposition of α-synuclein aggregates in many regions of the central and peripheral nervous systems. Accumulating evidence suggests that the α-synuclein pathology initiates in a few discrete regions and spreads to larger areas in the nervous system. Recent pathological studies of PD patients have raised the possibility that the enteric nervous system is one of the initial sites of α-synuclein aggregation and propagation. Here, we evaluated the induction and propagation of α-synuclein aggregates in the enteric nervous system of the A53T α-synuclein transgenic mice after injection of human brain tissue extracts into the gastric walls of the mice. Western analysis of the brain extracts showed that the DLB extract contained detergent-stable α-synuclein aggregates, but the normal brain extract did not. Injection of the DLB extract resulted in an increased deposition of α-synuclein in the myenteric neurons, in which α-synuclein formed punctate aggregates over time up to 4 months. In these mice, inflammatory responses were increased transiently at early time points. None of these changes were observed in the A53T mice injected with saline or the normal brain extract, nor were these found in the wild type mice injected with the DLB extract. These results demonstrate that pathological α-synuclein aggregates present in the brain of DLB patient can induce the aggregation of endogenous α-synuclein in the myenteric neurons in A53T mice, suggesting the transmission of synucleinopathy lesions in the enteric nervous system.

Non-classical exocytosis of alpha-synuclein is sensitive to folding states and promoted under stress conditions.

Parkinson's disease is characterized by deposition of misfolded/aggregated alpha-synuclein proteins in multiple regions of the brain. Neurons can release alpha-synuclein; through this release, pathological forms of alpha-synuclein are propagated between neurons, and also cause neuroinflammation. In this study, we demonstrate that release of alpha-synuclein is consistently increased under various protein misfolding stress conditions in both neuroblastoma and primary neuron models. This release is mediated by a non-classical, endoplasmic reticulum (ER)/Golgi-independent exocytosis, and stress-induced release coincides with increased translocation of alpha-synuclein into vesicles. Both vesicle translocation and secretion were blocked by attachment of a highly stable, globular protein to alpha-synuclein, whereas forced protein misfolding resulted in an increase in both of these activities. Mass spectrometry analysis showed a higher degree of oxidative modification in secreted alpha-synuclein than in the cellular protein. Together, these results suggest that structurally abnormal, damaged alpha-synuclein proteins translocate preferentially into vesicles and are released from neuronal cells via exocytosis.

Direct transfer of alpha-synuclein from neuron to astroglia causes inflammatory responses in synucleinopathies.

Abnormal neuronal aggregation of alpha-synuclein is implicated in the development of many neurological disorders, including Parkinson disease and dementia with Lewy bodies. Glial cells also show extensive alpha-synuclein pathology and may contribute to disease progression. However, the mechanism that produces the glial alpha-synuclein pathology and the interaction between neurons and glia in the disease-inflicted microenvironment remain unknown. Here, we show that alpha-synuclein proteins released from neuronal cells are taken up by astrocytes through endocytosis and form inclusion bodies. The glial accumulation of alpha-synuclein through the transmission of the neuronal protein was also demonstrated in a transgenic mouse model expressing human alpha-synuclein. Furthermore, astrocytes that were exposed to neuronal alpha-synuclein underwent changes in the gene expression profile reflecting an inflammatory response. Induction of pro-inflammatory cytokines and chemokines correlated with the extent of glial accumulation of alpha-synuclein. Together, these results suggest that astroglial alpha-synuclein pathology is produced by direct transmission of neuronal alpha-synuclein aggregates, causing inflammatory responses. This transmission step is thus an important mediator of pathogenic glial responses and could qualify as a new therapeutic target.

Clearance and deposition of extracellular alpha-synuclein aggregates in microglia.

Abnormal deposition of alpha-synuclein in neurons and glia is implicated in many neurological diseases, such as Parkinson's disease and Dementia with Lewy bodies. Recently, evidence has emerged that this protein and its aggregates are secreted from neuronal cells, and this extracellular protein may contribute to the pathogenic process. Here, we show that all the major brain cell types (neurons, astrocytes, and microglia) are capable of clearing the extracellular alpha-synuclein aggregates by internalization and degradation. Among these cell types, microglia showed the highest rate of degradation. Upon activation by lipopolysaccharide, the degradation of the internalized alpha-synuclein aggregates was slowed, causing protein accumulation in the microglial cytoplasm. These results suggest that microglia may be the major scavenger cells for extracellular alpha-synuclein aggregates in brain parenchyma, and that clearance may be regulated by the activation state of these cells.

Assembly-dependent endocytosis and clearance of extracellular alpha-synuclein.

Abnormal folding and accumulation of alpha-synuclein is implicated in several neurological disorders including Parkinson's disease. Although alpha-synuclein is a typical cytoplasmic protein, a small amount of both monomeric and aggregated forms is secreted from cells and is present in human body fluids, such as cerebrospinal fluid. Extracellular alpha-synuclein aggregates have been shown to be neurotoxic, posing a challenge to any cell exposed to them. Here, we examine the internalization of various forms of extracellular alpha-synuclein, including fibrils, oligomers, and monomer, into neuronal cells and their subsequent degradation. Internalization of fibrillar alpha-synuclein could be inhibited by low temperature or the expression of a dominant-negative mutant dynamin-1 K44A, suggesting the endocytosis-mediated internalization. The internalized fibrils moved through the endosomal pathway and were degraded in the lysosome, which ultimately resulted in the clearance of the alpha-synuclein aggregates from the culture medium. Non-fibrillar oligomeric aggregates were also internalized via endocytosis and degraded by the lysosome. In contrast to aggregate uptake, the internalization of monomeric alpha-synuclein was unaffected by cold temperature and the expression of dynamin-1 K44A, consistent with direct translocation across the plasma membrane. Internalized monomers rapidly pass the plasma membrane, escaping the cells before being degraded by the cellular proteolytic systems. These results suggest that only aggregated forms of extracellular alpha-synuclein can be cleared by cell-mediated uptake and degradation, and this might represent a mechanism of preventing neurons from exposure to potentially toxic alpha-synuclein.