Extensive uptake of α-synuclein oligomers in astrocytes results in sustained intracellular deposits and mitochondrial damage

https://doi.org/10.1016/j.mcn.2017.04.009Get rights and content

Highlights

  • Astrocytes rapidly ingest large amounts of oligomeric α-synuclein.

  • Due to incomplete lysosomal degradation, the α-synuclein is intracellularly stored.

  • The accumulation of α-synuclein induces astrocytic mitochondrial impairments.

  • Our results emphasize an important role of astrocytes in α-synucleinopathies.

Abstract

The presence of Lewy bodies, mainly consisting of aggregated α-synuclein, is a pathological hallmark of Parkinson's disease (PD) and dementia with Lewy bodies (DLB). The α-synuclein inclusions are predominantly found in neurons, but also appear frequently in astrocytes. However, the pathological significance of α-synuclein inclusions in astrocytes and the capacity of glial cells to clear toxic α-synuclein species remain unknown. In the present study we investigated uptake, degradation and toxic effects of oligomeric α-synuclein in a co-culture system of primary neurons, astrocytes and oligodendrocytes. Alpha-synuclein oligomers were found to co-localize with the glial cells and the astrocytes were found to internalize particularly large amounts of the protein. Following ingestion, the astrocytes started to degrade the oligomers via the lysosomal pathway but, due to incomplete digestion, large intracellular deposits remained. Moreover, the astrocytes displayed mitochondrial abnormalities. Taken together, our data indicate that astrocytes play an important role in the clearance of toxic α-synuclein species from the extracellular space. However, when their degrading capacity is overburdened, α-synuclein deposits can persist and result in detrimental cellular processes.

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

References (51)

  • J. Miklossy et al.

    Role of ICAM-1 in persisting inflammation in Parkinson disease and MPTP monkeys

    Exp. Neurol.

    (2006)
  • F. Mori et al.

    Demonstration of alpha-synuclein immunoreactivity in neuronal and glial cytoplasm in normal human brain tissue using proteinase K and formic acid pretreatment

    Exp. Neurol.

    (2002)
  • K. Nakamura

    alpha-Synuclein and mitochondria: partners in crime?

    Neurotherapeutics

    (2013)
  • T. Nasstrom et al.

    The lipid peroxidation products 4-oxo-2-nonenal and 4-hydroxy-2-nonenal promote the formation of alpha-synuclein oligomers with distinct biochemical, morphological, and functional properties

    Free Radic. Biol. Med.

    (2011)
  • R. Ravin et al.

    Potency and fate specification in CNS stem cell populations in vitro

    Cell Stem Cell

    (2008)
  • J.F. Reyes et al.

    A cell culture model for monitoring alpha-synuclein cell-to-cell transfer

    Neurobiol. Dis.

    (2015)
  • L.H. Sanders et al.

    LRRK2 mutations cause mitochondrial DNA damage in iPSC-derived neural cells from Parkinson's disease patients: reversal by gene correction

    Neurobiol. Dis.

    (2014)
  • L.H. Sanders et al.

    Mitochondrial DNA damage: molecular marker of vulnerable nigral neurons in Parkinson's disease

    Neurobiol. Dis.

    (2014)
  • E. Angot et al.

    Alpha-synuclein cell-to-cell transfer and seeding in grafted dopaminergic neurons in vivo

    PLoS One

    (2012)
  • H. Appelqvist et al.

    The lysosome: from waste bag to potential therapeutic target

    J. Mol. Cell Biol.

    (2013)
  • H. Braak et al.

    Development of alpha-synuclein immunoreactive astrocytes in the forebrain parallels stages of intraneuronal pathology in sporadic Parkinson's disease

    Acta Neuropathol.

    (2007)
  • N. Braidy et al.

    Uptake and mitochondrial dysfunction of alpha-synuclein in human astrocytes, cortical neurons and fibroblasts

    Translational Neurodegeneration

    (2013)
  • Y. Chiba et al.

    Immunohistochemical localization of aggresomal proteins in glial cytoplasmic inclusions in multiple system atrophy

    Neuropathol. Appl. Neurobiol.

    (2012)
  • E. Croisier et al.

    Glial degeneration and reactive gliosis in alpha-synucleinopathies: the emerging concept of primary gliodegeneration

    Acta Neuropathol.

    (2006)
  • K.M. Danzer et al.

    Different species of alpha-synuclein oligomers induce calcium influx and seeding

    J. Neurosci.

    (2007)
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