Assembly-dependent endocytosis and clearance of extracellular a-synuclein
Introduction
a-Synuclein (a-syn) is an abundant neuronal protein found in presynaptic terminals (Cookson, 2005). There is a large body of evidence suggesting that abnormal aggregation of a-syn is an important component of Parkinson's disease (PD) pathogenesis. Genetic linkage analyses have identified three missense mutations, as well as duplication or triplication of the locus containing the a-syn gene, in the inherited forms of parkinsonism (Farrer, 2006). Importantly, fibrillar aggregates of a-syn seem to be the main constituents of Lewy bodies and Lewy neurites, protein inclusion bodies that characterize PD-afflicted tissues (Spillantini, Crowther, Jakes, Hasegawa, & Goedert, 1998). Thus, aggregation of a-syn is not just a feature of familial PD, it is also associated with sporadic PD. Animal models that overexpress a-syn show neuronal loss and Lewy body-like inclusion formation, further supporting the importance of a-syn in the neurodegenerative processes (Maries, Dass, Collier, Kordower, & Steece-Collier, 2003). Synuclein pathology is also present in other neurological disorders, such as dementia with Lewy bodies, the Lewy body variant of Alzheimer's disease, and in multiple system atrophy (Jellinger, 2003).
a-syn is a classical cytosolic protein and it was generally assumed that the pathogenic effects of the protein were limited to the cytoplasm of single cells. However, recent studies of extracellular a-syn suggest the protein exerts extracellular pathogenic actions as well. A portion of a-syn has been identified in vesicles and it can be secreted from cells via exocytosis (Lee, Patel, & Lee, 2005). In humans, a-syn is present in blood plasma and cerebrospinal fluid in both monomeric and oligomeric forms (Borghi et al., 2000, El-Agnaf et al., 2006, Lee et al., 2006, Tokuda et al., 2006). Quantification by ELISA, specific to the oligomeric forms, has shown that the body fluid levels of a-syn oligomers are elevated in PD patients (El-Agnaf et al., 2006), implicating at least some forms of extracellular a-syn in the disease. Extracellular a-syn is cytotoxic when added to the culture medium (Albani et al., 2004; Bodles, Guthrie, Harriott, Campbell, & Irvine, 2000; Du et al., 2003; El-Agnaf et al., 1998; Forloni, Bertani, Calella, Thaler, & Invernizzi, 2000; Lee, Cho, et al. 2004; Seo et al., 2002; Sung et al., 2001). Moreover, in neuron/microglia mixed cell cultures, the aggregated forms of extracellular a-syn can induce microglial activation, producing dopaminergic neurotoxicity (Zhang et al., 2005). It has also been shown that exposure to a-syn stimulates production of pro-inflammatory factors from human astrocytes and astrocytoma cells (Klegeris et al., 2006). Therefore, removal of a-syn from the extracellular space has emerged as a critical problem with direct implication for neuronal function and survival.
Previous studies showed that extracellular a-syn can be internalized into cells, but the mechanism of internalization is controversial (Ahn, Paik, Chung, & Kim, 2006; Sung et al., 2001). Here, we show that the mechanism of internalization of extracellular a-syn is dependent on the assembly state of the protein; aggregated forms, both fibrils and oligomers, enter into cells via receptor-mediated endocytosis, while monomeric a-syn passively diffuse across the plasma membrane. Internalized a-syn aggregates move through the endosomal pathway and are degraded by the lysosomes. Neuron-mediated uptake and degradation leads to the clearance of extracellular a-syn aggregates. Based on these results, assembly-dependent endocytosis and degradation might represent a novel mechanism for the removal of extracellular a-syn aggregates.
Section snippets
Materials
All-trans retinoic acid, z-Leu-Leu-al (MG132), proteinase K (PK), and protease inhibitor cocktail were purchased from Sigma (St. Louis, MO). Bafilomycin A1 was purchased from Calbiochem (San Diego, CA). OPTI-PREP reagent was purchased from Accurate Chemicals and Scientific Corp. (Westbury, NY). Dynamin-1 K44A/pCB1 vector was a kind gift from Dr. Marc Caron. Recombinant human wild type a-syn was purchased from ATGen (Sungnam, Korea). The following primary antibodies are used: a-syn polyclonal
Receptor-mediated endocytosis of a-syn aggregates and trafficking through the endosomal organelles
Aggregates were prepared from recombinant human a-syn, and their structural and sedimentational properties as well as migration on the denaturing gel electrophoresis were examined. Both AFM and EM images of the aggregate samples show homogeneous fragmented (presumably by sonication) fibrils with the width of approximately 15 nm (Fig. 1A and B). CD spectrometry confirmed the enrichment of the ß-pleated sheet structure in fibrils (Fig. 1C). Most of the fibrils were dissolved into monomers under
Discussion
There is an increasing body of evidence that a-syn might exert pathogenic functions in the extracellular space, in addition to the much studied intracellular cytotoxic effects. In tissue culture systems, extracellular a-syn is toxic to cells and in most cases, the toxicity is associated with aggregated a-syn forms (Albani et al., 2004; Bodles et al., 2000; Du et al., 2003; El-Agnaf et al., 1998; Forloni et al., 2000; Lee, Cho, et al., 2004; Seo et al., 2002; Sung et al., 2001). Extracellular
Acknowledgements
We thank Lei Cho for technical assistance and Marc Caron for plasmid. This work was supported by a grant from Brain Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology (M103KV010021-06K2201-02110), by bio core-technology program of the Ministry of Science and Technology (2007-04303), and by a grant of the Health & Medical Technology R&D Project, Ministry of Health & Welfare (A060395), Republic of Korea.
References (31)
- et al.
Full length alpha-synuclein is present in cerebrospinal fluid from Parkinson's disease and normal subjects
Neuroscience Letters
(2000) - et al.
Aggregates from mutant and wild-type alpha-synuclein proteins and NAC peptide induce apoptotic cell death in human neuroblastoma cells by formation of beta-sheet and amyloid-like filaments
FEBS Letters
(1998) - et al.
Multiple ligand interaction of alpha-synuclein produced various forms of protein aggregates in the presence of Abeta25-35, copper, and eosin
Brain Research
(2001) - et al.
Formation and removal of alpha-synuclein aggregates in cells exposed to mitochondrial inhibitors
Journal of Biological Chemistry
(2002) - et al.
Effects of alpha-synuclein immunization in a mouse model of Parkinson's disease
Neuron
(2005) - et al.
Epitope mapping and specificity of the anti-alpha-synuclein monoclonal antibody Syn-1 in mouse brain and cultured cell lines
Neuroscience Letters
(2003) Dynamin and endocytosis
Current Opinions in Cell Biology
(2002)- et al.
Induction of neuronal cell death by Rab5A-dependent endocytosis of alpha-synuclein
Journal of Biological Chemistry
(2001) - et al.
Proteolytic cleavage of extracellular secreted {alpha}-synuclein via matrix metalloproteinases
Journal of Biological Chemistry
(2005) - et al.
Decreased alpha-synuclein in cerebrospinal fluid of aged individuals and subjects with Parkinson's disease
Biochemical Biophysical Research Communications
(2006)
Dynamin and beta-arrestin reveal distinct mechanisms for G protein-coupled receptor internalization
Journal of Biological Chemistry
Analysis of alpha-synuclein-associated proteins by quantitative proteomics
Journal of Biological Chemistry
Amino acid sequence motifs and mechanistic features of the membrane translocation of alpha-synuclein
Journal of Neurochemistry
Protective effect of TAT-delivered alpha-synuclein: relevance of the C-terminal domain and involvement of HSP70
FASEB Journal
Toxicity of non-abeta component of Alzheimer's disease amyloid, and N-terminal fragments thereof, correlates to formation of beta-sheet structure and fibrils
European Journal of Biochemistry
Cited by (394)
Understanding the potential causes of gastrointestinal dysfunctions in multiple system atrophy
2023, Neurobiology of DiseaseThe Hidden Cell-to-Cell Trail of α-Synuclein Aggregates
2023, Journal of Molecular BiologyThe unique neuropathological vulnerability of the human brain to aging
2023, Ageing Research ReviewsSpreading of alpha-synuclein between different cell types
2023, Behavioural Brain ResearchThe theoretical problems of “prodrome” and “phenoconversion” in neurodegeneration
2023, Handbook of Clinical Neurology