ABSTRACT
Autophagy is a pivotal intracellular process by which cellular macromolecules are degraded upon various stimuli. A failure in the degradation of autophagic substrates such as impaired organelles and protein aggregates leads to their accumulations, which are characteristics of many neurodegenerative diseases. Pharmacological activation of autophagy has thus been considered a prospective therapeutic approach for treating neurodegenerative diseases. Among a number of autophagy-inducing agents, trehalose has received attention for its beneficial effects in different disease models of neurodegeneration. However, how trehalose promotes autophagy has not been fully revealed. We investigated the influence of trehalose and other disaccharides upon autophagic flux and aggregation of α-synuclein, a protein linked to Parkinson's disease. In differentiated human neuroblastoma and primary rat cortical neuron culture models, treatment with trehalose and other disaccharides resulted in accumulation of lipidated LC3 (LC3-II), p62, and autophagosomes, whereas it decreased autolysosomes. On the other hand, addition of Bafilomycin A1 to trehalose treatments had relatively marginal effect, an indicative of autophagic flux blockage. In concordance with these results, the cells treated with trehalose exhibited an incremental tendency in α-synuclein aggregation. Secretion of α-synuclein was also elevated in the culture medium upon trehalose treatment, thereby significantly increasing intercellular transmission of this protein. Despite the substantial increase in α-synuclein aggregation, which normally leads to cell death, cell viability was not affected upon treatment with trehalose, suggesting an autophagy-independent protective function of trehalose against protein aggregates. This study demonstrates that, although trehalose has been widely considered an autophagic inducer, it may be actually a potent blocker of the autophagic flux.
Subject(s)
Parkinson Disease/drug therapy , Protein Aggregation, Pathological/drug therapy , Trehalose/administration & dosage , alpha-Synuclein/genetics , Animals , Autophagosomes/drug effects , Autophagy/drug effects , Cell Survival/drug effects , Disaccharides/administration & dosage , Humans , Lysosomes , Microtubule-Associated Proteins/chemistry , Microtubule-Associated Proteins/genetics , Neurons/drug effects , Parkinson Disease/genetics , Parkinson Disease/pathology , Primary Cell Culture , Protein Aggregation, Pathological/genetics , Protein Aggregation, Pathological/pathology , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , RatsABSTRACT
The accumulation of abnormal protein aggregates is a major characteristic of many neurodegenerative disorders, including Parkinson's disease (PD). The intracytoplasmic deposition of α-synuclein aggregates and Lewy bodies, often found in PD and other α-synucleinopathies, is thought to be linked to inefficient cellular clearance mechanisms, such as the proteasome and autophagy/lysosome pathways. The accumulation of α-synuclein aggregates in neuronal cytoplasm causes numerous autonomous changes in neurons. However, it can also affect the neighboring cells through transcellular transmission of the aggregates. Indeed, a progressive spreading of Lewy pathology among brain regions has been hypothesized from autopsy studies. We tested whether inhibition of the autophagy/lysosome pathway in α-synuclein-expressing cells would increase the secretion of α-synuclein, subsequently affecting the α-synuclein deposition in and viability of neighboring cells. Our results demonstrated that autophagic inhibition, via both pharmacological and genetic methods, led to increased exocytosis of α-synuclein. In a mixed culture of α-synuclein-expressing donor cells with recipient cells, autophagic inhibition resulted in elevated transcellular α-synuclein transmission. This increase in protein transmission coincided with elevated apoptotic cell death in the recipient cells. These results suggest that the inefficient clearance of α-synuclein aggregates, which can be caused by reduced autophagic activity, leads to elevated α-synuclein exocytosis, thereby promoting α-synuclein deposition and cell death in neighboring neurons. This finding provides a potential link between autophagic dysfunction and the progressive spread of Lewy pathology.
Subject(s)
Autophagy , Exocytosis , Extracellular Space/metabolism , alpha-Synuclein/metabolism , Adenine/analogs & derivatives , Adenine/pharmacology , Animals , Autophagy/drug effects , Autophagy-Related Protein 7 , Cell Line , Exocytosis/drug effects , Humans , Mice , Mice, Knockout , Microtubule-Associated Proteins/deficiency , Microtubule-Associated Proteins/metabolism , Phagosomes/drug effects , Phagosomes/metabolism , Protein Structure, Quaternary , Protein Transport/drug effects , alpha-Synuclein/chemistry , alpha-Synuclein/toxicityABSTRACT
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 â¼12.5 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.
Subject(s)
Enzyme-Linked Immunosorbent Assay/methods , Epitopes/immunology , Protein Multimerization , alpha-Synuclein/isolation & purification , Adult , Animals , Antibodies, Monoclonal/isolation & purification , Antibody Specificity , COS Cells , Chlorocebus aethiops , Female , Humans , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Parkinson Disease/blood , Parkinson Disease/cerebrospinal fluid , Parkinson Disease/immunology , Protein Multimerization/immunology , Young Adult , alpha-Synuclein/immunology , alpha-Synuclein/metabolismABSTRACT
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.
