Extensive uptake of α-synuclein oligomers in astrocytes results in sustained intracellular deposits and mitochondrial damage
Introduction
Brains from patients with Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are characterized by Lewy bodies and Lewy neurites, intracellular inclusions predominantly consisting of insoluble α-synuclein fibrils (Spillantini et al., 1997). In healthy neurons, α-synuclein is highly abundant in the cytosol and presynaptic terminals, but its exact function remains unclear. During fibril formation, α-synuclein generates soluble intermediate aggregates, i.e. oligomers, which are particularly neurotoxic. For example, oligomeric α-synuclein has been shown to disrupt cellular membranes (Danzer et al., 2007, Winner et al., 2011) and induce mitochondrial dysfunction (Chinta et al., 2010, Luth et al., 2014).
Although α-synuclein deposits are primarily found in neurons, they also appear frequently in astrocytes at advanced disease stages (Braak et al., 2007, Croisier and Graeber, 2006, Terada et al., 2003, Tu et al., 1998, Wakabayashi et al., 2000). In contrast to neurons, astrocytes express very low levels of α-synuclein (Mori et al., 2002) and the glial inclusions therefore likely stem from adjacent neurons. Possibly, α-synuclein can spread from neurons to glial cells via the extracellular space or via direct cell-to-cell transfer (Angot et al., 2012, Hansen et al., 2011, Reyes et al., 2015). In line with this hypothesis, astrocytes have been demonstrated to readily take up extracellular α-synuclein in vitro (Fellner et al., 2013, Lee et al., 2010b, Rannikko et al., 2015), a process that also activates pro-inflammatory responses (Fellner et al., 2013, Lee et al., 2010b). Moreover, astroglial inclusions have been found in transgenic α-synuclein mice after intracerebral injections of fibrillar or soluble forms of α-synuclein (Sacino et al., 2014).
Astrocytes respond to pathological conditions through a process referred to as reactive astrogliosis. Thereby they upregulate their intermediate filaments, become hypertrophic, secrete various inflammatory mediators and transform to a phagocytic state (Buffo et al., 2010, Lööv et al., 2012; Lööv et al. 2015). We and others have shown that reactive astrogliosis is closely connected to α-synuclein pathology in mouse models of PD and DLB (Lindström et al., 2014, Neumann et al., 2002, Rockenstein et al., 2002). Moreover, reactive astrocytes have been demonstrated to be intimately associated with α-synuclein pathology in the human PD/DLB brain (Miklossy et al., 2006, Thannickal et al., 2007). Although there is growing evidence that astrocytes are highly involved in the pathology of PD/DLB, the functional consequence of α-synuclein deposition in astrocytes and their role in the disease progression remain unknown.
In the present study, we investigated the role of astrocytes in uptake, degradation and toxicity of α-synuclein oligomers. We found that these cells rapidly ingested large amounts of oligomers that were not efficiently degraded, resulting in the formation of ubiqutinylated, intracellular inclusions. The α-synuclein containing astrocytes remained viable, but displayed mitochondrial impairment.
Section snippets
Animals
All experiments were approved by the Uppsala County Animal Ethics Board, following the rules and regulations of the Swedish Animal Welfare Agency, and in compliance with the European Communities Council Directive (2010/63/EU). The mice were housed in a 12:12 dark:light cycle, kept in an enriched environment and given water and food ad libitum. Embryonic C57Bl6 mice were used for cell culture experiments and brains from adult (Thy-1)-h[A30P]α-synuclein mice (Kahle et al. 2000) were used for
Alpha-synuclein oligomers accumulate in astrocytes and oligodendrocytes
Co-cultures of astrocytes, neurons and oligodendrocytes were treated with 0.5 μM Cy3-labeled α-synuclein oligomers (Fig. 1A) for 24 h prior to fixation. Immunocytochemistry could not detect any uptake of α-synuclein oligomers in neurons (Fig. 1B). In contrast, immunocytochemical staining to GFAP and CNPase showed that α-synuclein oligomers co-localized with both astrocytes and oligodendrocytes (Fig. 1C–D). Confocal 3D images (Fig. 1E–F and Supplementary Fig. 1A–B) and Imaris 3D projection (Fig. 1
Discussion
Astrocytes, the most abundant glial cell type in the brain, have multiple functions that are tightly linked to pathological processes. Yet, their role in neurodegenerative diseases has been sparsely studied and the consequences of astrocytic α-synuclein inclusions remain unknown. The aim of the present study was to investigate the capacity of astrocytes to clear and degrade oligomers of the PD and DLB related protein α-synuclein. Moreover, we sought to clarify if astrocytic α-synuclein
Acknowledgements/conflict of interest disclosure
This study was supported by grants from the Swedish Research Council (2015-02671), the Swedish Parkinson Foundation (931/16), the Swedish Alzheimer Foundation (AF-646541), the U4 Ageing Brain network, the Åhlén Foundation (mC27h16, mA17/h14), the Dementia Association Foundation, Hedlunds Foundation (M-2016-0291), Magn Bergwalls stiftelse (2016-01714), Lennart and Christina Kalén, William N. & Bernice E. Bumpus Foundation Innovation Award, Marianne and Marcus Wallenberg Foundation, Swedish Brain
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