Regular articleIntercellular transfer of pathogenic α-synuclein by extracellular vesicles is induced by the lipid peroxidation product 4-hydroxynonenal
Introduction
Parkinson's disease (PD), dementia with Lewy bodies, and multiple system atrophy are age-related neurodegenerative disorders characterized by accumulations of α-synuclein in the soma (Lewy bodies) and neurites (Baba et al., 1998, Jellinger, 2011, Spillantini et al., 1997, Spillantini et al., 1998a, Spillantini et al., 1998b, Wakabayashi et al., 1998). α-synuclein is a highly conserved 140-amino acid presynaptic protein, which associates with membranes and is believed to play a role in the regulation of neurotransmitter release (Abeliovich et al., 2000, Burre et al., 2010, George et al., 1995, Iwai et al., 1995, Vargas et al., 2014, Withers et al., 1997). Mutations in the gene encoding α-synuclein cause dominantly inherited PD (Kruger et al., 1998, Lesage et al., 2013, Polymeropoulos et al., 1997, Zarranz et al., 2004), and duplication or triplication of the α-synuclein gene (Chartier-Harlin et al., 2004, Farrer et al., 2004, Kara et al., 2014, Singleton et al., 2003) indicating that overproduction of α-synuclein is sufficient to trigger PD during aging. When mutated or overexpressed, α-synuclein monomers tend to assemble into helically folded tetramers that resist aggregation in the cytosol (Bartels et al., 2011).
Normally, α-synuclein is degraded in proteasomes and by autophagy in lysosomes (Cuervo et al., 2004, Mak et al., 2010, Snyder et al., 2003, Webb et al., 2003, Xilouri et al., 2013). Knockdown of the autophagy pathway proteins Atg5 or Atg7 results in the accumulation of α-synuclein aggregates and neurological deficits similar to PD (Hara et al., 2006, Komatsu et al., 2006), while overexpression of wild-type α-synuclein or its mutants inhibits autophagy (Chen et al., 2015, Chew et al., 2011, Choubey et al., 2011, Huang et al., 2012; Winslow et al., 2010). In addition to ubiquitination (Lee et al., 2008b, Nonaka et al., 2005), α-synuclein can be posttranslationally modified by phosphorylation (Anderson et al., 2006, Hasegawa et al., 2002, Okochi et al., 2000) and acetylation (Trexler and Rhoades, 2012), which may influence its aggregation and degradation. One age-related factor that may contribute to the aggregation and cytotoxicity of α-synuclein is oxidative stress. In this regard, the lipid peroxidation product 4-hydroxynonenal (HNE) is implicated in PD because of its accumulation in Lewy bodies (Castellani et al., 2002, Yoritaka et al., 1996) and because it can covalently modify α-synuclein (Näsström et al., 2011, Qin et al., 2007) and can impair lysosome function in neurons (Zhang et al., 2017). However, if and how HNE might cause intracellular accumulation and transcellular propagation of neurotoxic α-synuclein species is unknown.
Studies of PD patients at various stages of the disease process suggested that α-synuclein pathology spreads transcellularly and may occur in peripheral neurons before spreading rostrally to the brainstem, and thence to the midbrain and cerebral cortex (Braak et al., 2003, Braak et al., 2006). Recent studies have shown that when preformed α-synuclein fibrils are introduced into the olfactory bulb (Rey et al., 2016) or striatum (Luk et al., 2012), the α-synuclein pathology spreads in a retrograde manner. In addition, when Lewy body extracts from PD patient brains were injected into the brains of monkeys, they induced a spreading α-synuclein pathology and neurodegeneration (Recasens et al., 2014). The mechanism by which α-synuclein pathology is transmitted between neurons is unclear but may involve a prion-like seeding process (Goedert, 2015). It has also been reported that neurons can secrete pathogenic forms of amyloid β-peptide or α-synuclein in extracellular vesicles (EVs; also known as exosomes) in experimental models relevant to Alzheimer's disease and PD, respectively (Danzer et al., 2012, Eitan et al., 2016). However, the possible involvement of membrane lipid peroxidation in the extrusion of α-synuclein in EVs and their transcellular pathogenicity is unknown. Here, we report that HNE modifies α-synuclein, which triggers intracellular accumulation and subsequent extrusion of toxic α-synuclein species in and on the surface of EVs. The EVs with HNE-modified α-synuclein are then internalized by and transported within adjacent neurons, thus propagating α-synuclein pathology and causing neuronal degeneration.
Section snippets
Reagents
HNE was from Cayman Chemical Company (Ann Arbor, MI, USA). Anti-α-synuclein antibody (C20) was from Santa Cruz (Dallas, TX, USA; catalog # sc-7011), anti-human α-synuclein antibody (Syn211) was from Thermo Fisher Scientific (Waltham, MA, USA; catalog # MS-1572). Anti-S129 phosphorylated α-synuclein antibody was from Wako (Richmond, VA, USA; catalog # 015-25191). Anti-actin antibody was from Sigma (St. Louis, MO, USA; catalog #A2066). Anti-HNE antibody was from Alpha Diagnostic (San Antonio, TX,
Overexpression of human α-synuclein results in the accumulation of α-synuclein aggregates and HNE-modified proteins
To establish a cellular system for the production and release in EVs of potentially pathogenic forms of α-synuclein, we overexpressed wild-type human α-synuclein in HEK cells (HEK-wt-SNCA cells) and then performed immunoblot analyses of cell lysates prepared in radioimmunoprecipitation assay buffer. Lysates with the same amount of protein were loaded and separated in NuPAGE Bis-Tris gels. The analysis demonstrated that levels of monomeric α-synuclein (approximately 17 kDa) were more than
Discussion
Our findings demonstrate that: (1) EVs containing α-synuclein oligomers can be released from cultured primary neurons and a cell line overexpressing human α-synuclein; (2) EVs containing α-synuclein can be internalized by axons of healthy primary neurons, wherein they are transported retrogradely to the cell body and dendrites; (3) α-synuclein is present within EVs and on their surface, and the surface-associated α-synuclein plays a role in the internalization of the EVs by neurons; (4)
Disclosure statement
The authors have no actual or potential conflicts of interest.
Acknowledgements
The authors thank Dr Ruiqian Wan for technical assistance with stereotaxic injections. This work was supported by Intramural Research Program of National Institute on Aging.
References (87)
- et al.
Mice lacking alpha-synuclein display functional deficits in the nigrostriatal dopamine system
Neuron
(2000) - et al.
Lysosomal dysfunction increases exosome-mediated alpha-synuclein release and transmission
Neurobiol. Dis.
(2011) - et al.
Phosphorylation of Ser-129 is the dominant pathological modification of alpha-synuclein in familial and sporadic Lewy body disease
J. Biol. Chem.
(2006) - et al.
Gastric alpha-synuclein immunoreactive inclusions in Meissner's and Auerbach's plexuses in cases staged for Parkinson's disease-related brain pathology
Neurosci. Lett.
(2006) - et al.
Staging of brain pathology related to sporadic Parkinson's disease
Neurobiol. Aging
(2003) - et al.
Hydroxynonenal adducts indicate a role for lipid peroxidation in neocortical and brainstem Lewy bodies in humans
Neurosci. Lett.
(2002) - et al.
Alpha-synuclein cooperates with CSPalpha in preventing neurodegeneration
Cell
(2005) - et al.
Alpha-synuclein locus duplication as a cause of familial Parkinson's disease
Lancet
(2004) - et al.
Enhanced autophagy from chronic toxicity of iron and mutant A53T alpha-synuclein: implications for neuronal cell death in Parkinson disease
J. Biol. Chem.
(2011) - et al.
Mutant A53T alpha-synuclein induces neuronal death by increasing mitochondrial autophagy
J. Biol. Chem.
(2011)
Impact of lysosome status on extracellular vesicle content and release
Ageing Res. Rev.
Catalytic site-specific inhibition of the 20S proteasome by 4-hydroxynonenal
FEBS Lett.
Characterization of a novel protein regulated during the critical period for song learning in the zebra finch
Neuron
Distinct alpha-synuclein strains differentially promote tau inclusions in neurons
Cell
Phosphorylated alpha-synuclein is ubiquitinated in alpha-synucleinopathy lesions
J. Biol. Chem.
The precursor protein of non-A beta component of Alzheimer's disease amyloid is a presynaptic protein of the central nervous system
Neuron
Synuclein deposition and non-motor symptoms in Parkinson disease
J. Neurol. Sci.
Possible involvement of proteasome inhibition in aging: implications for oxidative stress
Mech. Ageing Dev.
4-Hydroxynonenal, an aldehydic product of membrane lipid peroxidation, impairs glutamate transport and mitochondrial function in synaptosomes
Neuroscience
Lipid peroxidation products reduce lysosomal protease activities in human retinal pigment epithelial cells via two different mechanisms of action
Exp. Eye Res.
Effects of lipid peroxidation products on lipofuscinogenesis and autophagy in human retinal pigment epithelial cells
Exp. Eye Res.
Lysosomal degradation of alpha-synuclein in vivo
J. Biol. Chem.
Roles of the lipid peroxidation product 4-hydroxynonenal in obesity, the metabolic syndrome, and associated vascular and neurodegenerative disorders
Exp. Gerontol.
The lipid peroxidation products 4-oxo-2-nonenal and 4-hydroxy-2-nonenal promote the formation of α-synuclein oligomers with distinct biochemical, morphological, and functional properties
Free Radic. Biol. Med.
Constitutive phosphorylation of the Parkinson's disease associated alpha-synuclein
J. Biol. Chem.
Intrastriatal injection of pre-formed mouse alpha-synuclein fibrils into rats triggers alpha-synuclein pathology and bilateral nigrostriatal degeneration
Neurobiol. Dis.
The roles of PINK1, parkin, and mitochondrial fidelity in Parkinson's disease
Neuron
Lysines, Achilles' heel in alpha-synuclein conversion to a deadly neuronal endotoxin
Ageing Res. Rev.
Effect of 4-hydroxy-2-nonenal modification on alpha-synuclein aggregation
J. Biol. Chem.
Mitochondrial dysfunction and mitophagy in Parkinson's: from familial to sporadic disease
Trends Biochem. Sci.
Aggregated and monomeric alpha-synuclein bind to the S6' proteasomal protein and inhibit proteasomal function
J. Biol. Chem.
Calcium and Parkinson's disease
Biochem. Biophys. Res. Commun.
Exosomes: current knowledge of their composition, biological functions, and diagnostic and therapeutic potentials
Biochim. Biophys. Acta
Exogenous alpha-synuclein fibrils induce Lewy body pathology leading to synaptic dysfunction and neuron death
Neuron
Alpha-Synuclein is degraded by both autophagy and the proteasome
J. Biol. Chem.
Delayed localization of synelfin (synuclein, NACP) to presynaptic terminals in cultured rat hippocampal neurons
Brain Res. Dev. Brain Res.
Aggregation of alpha-synuclein in Lewy bodies of sporadic Parkinson's disease and dementia with Lewy bodies
Am. J. Pathol.
alpha-Synuclein occurs physiologically as a helically folded tetramer that resists aggregation
Nature
Animal models of Parkinson's disease
Bioessays
Mitochondrial dysfunction in Parkinson's disease
J. Neurochem.
Alpha-synuclein promotes SNARE-complex assembly in vivo and in vitro
Science
A53T human alpha-synuclein overexpression in transgenic mice induces pervasive mitochondria macroautophagy defects preceding dopamine neuron degeneration
J. Neurosci.
Impaired degradation of mutant alpha-synuclein by chaperone-mediated autophagy
Science
Cited by (51)
Roles of extracellular vesicles in ageing-related chronic kidney disease: Demon or angel
2023, Pharmacological ResearchThe Hidden Cell-to-Cell Trail of α-Synuclein Aggregates
2023, Journal of Molecular BiologyExtracellular vesicles released after cranial radiation: An insight into an early mechanism of brain injury
2022, Brain ResearchCitation Excerpt :One potential mechanism leading to this observed difference between the increase in GFAP measured in the EVs and GFAP in the brain tissue could be a compensatory mechanism to dispose of oxidatively modified proteins due to the cranial radiation. It has been shown that EVs are being used to remove oxidatively modified proteins from the brain (Zhang, 2018). Thus, it is possible that the observed increase in GFAP in EVs but not in the brain tissue between the two groups is due, in part, to increased removal of oxidatively modified proteins by EVs.
T cells, α-synuclein and Parkinson disease
2022, Handbook of Clinical NeurologyCitation Excerpt :While the mechanism by which α-Syn is released from cells remains to be elucidated, its release may be caused by cell death in the CNS or enteric nervous system (ENS), turnover of red blood cells and platelets that express high levels of α-Syn in the periphery, or secretion via exosomes from multivesicular bodies due to lysosomal dysfunction. Various uptake pathways of α-Syn fibrils have been reported over the last several years, including via heparan sulfate proteoglycans (HSPGs) (Holmes et al., 2013), the immune receptor Lag3 (Mao et al., 2016), and endocytic uptake of extracellular vesicles (Minakaki et al., 2018; Zhang et al., 2018a). Internalized α-Syn “seeds” could be trafficked through the endocytic pathway (Apetri et al., 2016; Masaracchia et al., 2018).
The potential roles of excitatory-inhibitory imbalances and the repressor element-1 silencing transcription factor in aging and aging-associated diseases
2021, Molecular and Cellular NeuroscienceCitation Excerpt :The disrupted calcium homeostasis worsens stress resistance resulting in further Aβ accumulation (Mattson, 2009). Neurons attempt to combat this by expelling Aβ in extracellular vesicles which can propagate Aβ pathology across networks, progressing the disease (Eitan et al., 2016; Zhang et al., 2018). Thus, ApoE4, HNE, oxidative stress, and Aβ pathology can render neurons more vulnerable to hyperexcitation and cell death.
Clinical Studies and Therapies in Parkinson’s Disease: Translations from Preclinical Models
2021, Clinical Studies and Therapies in Parkinson's Disease: Translations from Preclinical Models