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Research Articles: Neurobiology of Disease Human astrocytes transfer aggregated alpha-synuclein via tunneling nanotubes Jinar Rostami1, Staffan Holmqvist2,3,4, Veronica Lindström1, Jessica Sigvardson5, Gunilla T Westermark6, Martin Ingelsson1, Joakim Bergström1, Laurent Roybon2,3,4 and Anna Erlandsson1 1Molecular Geriatrics, Department of Public Health and Caring Sciences, Rudbeck Laboratory, Uppsala University, 751 85 Uppsala, Sweden 2Stem Cell laboratory for CNS Disease Modeling, Wallenberg Neuroscience Center, Department of Experimental Medical Science, BMC A10, Lund University, 22184 Lund, Sweden 3Strategic Research Area MultiPark, Lund University, 22184 Lund, Sweden 4Lund Stem Cell Center, Lund University, 22184 Lund, Sweden 5BioArctic AB, Warfvinges väg 35, 112 51 Stockholm, Sweden 6Department of Medical Cell Biology, BMC, Uppsala University, 751 85 Uppsala, Sweden DOI: 10.1523/JNEUROSCI.0983-17.2017 Received: 11 April 2017 Revised: 20 September 2017 Accepted: 11 October 2017 Published: 31 October 2017 Author contributions: J.R., s.H., V.L., G.T.W., M.I., J.B., L.R., and A.E. designed research; J.R. performed research; J.R. and A.E. analyzed data; J.R., s.H., V.L., G.T.W., M.I., J.B., L.R., and A.E. wrote the paper; J.S. contributed unpublished reagents/analytic tools. Conflict of Interest: The authors declare no competing financial interests. This study was supported by grants from the Swedish Research Council, the Parkinson Foundation, the Alzheimer Foundation, Åhlén Foundation, The Dementia Association Foundation, Crafoord Foundation, Kockska Foundation, Åke Wiberg Foundation, Lennart and Christina Kalén, Hedlunds Foundation and Brain Stem-Stem Cell Center of Excellence in Neurology, funded by Innovation Fund Denmark. The authors thank Linn Gallasch and Sofia Söllvander at Uppsala University for their support and Anders Ahlander at the SciLifeLab BioVis Facility, Uppsala University for technical assistance with TEM. Corresponding author: Anna Erlandsson, Department of Public Health and Caring Sciences/ Molecular Geriatrics, Rudbeck Laboratory, Uppsala University, SE-751 85 Uppsala, Sweden, E-mail: [email protected] Phone: +46-18-4715030 Cite as: J. Neurosci ; 10.1523/JNEUROSCI.0983-17.2017 Alerts: Sign up at www.jneurosci.org/cgi/alerts to receive customized email alerts when the fully formatted version of this article is published. Accepted manuscripts are peer-reviewed but have not been through the copyediting, formatting, or proofreading process. Copyright © 2017 Rostami et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. 1 Human astrocytes transfer aggregated alpha-synuclein via 2 tunneling nanotubes 3 4 Abbreviated title 5 Astrocytes transfer alpha-synuclein via TNTs 6 7 Authors 8 Jinar Rostami1, Staffan Holmqvist2,3,4, Veronica Lindström1, Jessica Sigvardson5, Gunilla T 9 Westermark6, Martin Ingelsson1, Joakim Bergström1, Laurent Roybon2,3,4 and Anna 10 Erlandsson1 11 12 Affiliations 13 1 Molecular Geriatrics, Department of Public Health and Caring Sciences, Rudbeck 14 Laboratory, Uppsala University, 751 85 Uppsala, Sweden 15 2 Stem Cell laboratory for CNS Disease Modeling, Wallenberg Neuroscience Center, 16 Department of Experimental Medical Science, BMC A10, Lund University, 22184 Lund, 17 Sweden 18 3 Strategic Research Area MultiPark, Lund University, 22184 Lund, Sweden 19 4 Lund Stem Cell Center, Lund University, 22184 Lund, Sweden 20 5 BioArctic AB, Warfvinges väg 35, 112 51 Stockholm, Sweden 21 6 Department of Medical Cell Biology, BMC, Uppsala University, 751 85 Uppsala, Sweden 22 23 1 24 Corresponding author 25 Anna Erlandsson 26 Department of Public Health and Caring Sciences / Molecular Geriatrics, 27 Rudbeck Laboratory, Uppsala University 28 SE-751 85 Uppsala, Sweden 29 E-mail: [email protected] Phone: +46-18-4715030 30 31 Number of pages: 33 32 Number of figures: 9 33 Number of multimedia: 3 34 Number of words for abstract: 153 35 Number of words for introduction: 645 36 Number of words for discussion: 1163 37 38 Acknowledgements 39 The authors declare that they have no competing interests. 40 This study was supported by grants from the Swedish Research Council, the Parkinson 41 Foundation, the Alzheimer Foundation, Åhlén Foundation, The Dementia Association 42 Foundation, Crafoord Foundation, Kockska Foundation, Åke Wiberg Foundation, Lennart and 43 Christina Kalén, Hedlunds Foundation and Brain Stem-Stem Cell Center of Excellence in 44 Neurology, funded by Innovation Fund Denmark. The authors thank Linn Gallasch and Sofia 45 Söllvander at Uppsala University for their support and Anders Ahlander at the SciLifeLab 46 BioVis Facility, Uppsala University for technical assistance with TEM. 47 2 48 Abstract 49 Many lines of evidence suggest that the Parkinson’s disease (PD) related protein alpha- 50 synuclein (α-SYN) can propagate from cell-to-cell in a prion-like manner. However, the 51 cellular mechanisms behind the spreading remain elusive. Here, we show that human 52 astrocytes, derived from embryonic stem cells, actively transfer aggregated α-SYN to nearby 53 astrocytes via direct contact and tunneling nanotubes (TNTs). Failure in the astrocytes’ 54 lysosomal digestion of excess α-SYN oligomers, results in α-SYN deposits in the trans-Golgi 55 network followed by endoplasmic reticulum swelling and mitochondrial disturbances. The 56 stressed astrocytes respond by conspicuously sending out TNTs, enabling intercellular 57 transfer of α-SYN to healthy astrocytes, which in return deliver mitochondria, indicating a 58 TNT-mediated rescue mechanism. Using a pharmacological approach to inhibit TNT 59 formation, we abolished the transfer of both α-SYN and mitochondria. Taken together, our 60 results highlight the role of astrocytes in α-SYN cell-to-cell transfer, identifying possible 61 pathophysiological events in the PD brain that could be of therapeutic relevance. 62 63 64 65 66 67 68 69 70 3 71 Significance statement 72 Astrocytes are the major cell type in the brain; yet their role in Parkinson’s disease 73 progression remains elusive. Here we show that human astrocytes actively transfer aggregated 74 alpha-synuclein (α-SYN) to healthy astrocytes, via direct contact and tunneling nanotubes 75 (TNTs), rather than degrade it. The astrocytes engulf large amounts of oligomeric α-SYN that 76 are subsequently stored in the trans-Golgi network region. The accumulation of α-SYN in the 77 astrocytes affects their lysosomal machinery and induces mitochondrial damage. The stressed 78 astrocytes respond by sending out TNTs, enabling intercellular transfer of α-SYN to healthy 79 astrocytes. Our findings highlight an unexpected role of astrocytes in the propagation of α- 80 SYN pathology via TNTs, revealing astrocytes as a potential target for therapeutic 81 intervention. 82 83 84 85 86 87 88 89 90 91 92 93 4 94 Introduction 95 Cellular inclusions in the brain, often referred to as Lewy bodies and Lewy neurites, are a 96 pathological hallmark for Parkinson’s disease (PD) and several other neurodegenerative 97 disorders, including dementia with Lewy bodies (DLB) and multiple system atrophy (MSA) 98 (Spillantini et al., 1998). The inclusions predominantly consist of insoluble fibrillary forms of 99 the alpha-synuclein (α-SYN) protein (Spillantini et al., 1997), but smaller soluble aggregates 100 are also present in the diseased brain. These aggregates, referred to as α-SYN oligomers, are 101 particularly neurotoxic (Danzer et al., 2007; Chinta et al., 2010; Winner et al., 2011; Luth et 102 al., 2014; Ogen-Shtern et al., 2016). Although, α-SYN deposits are primarily found in 103 neurons, they also appear frequently in glial cells at advanced disease stages (Tu et al., 1998; 104 Wakabayashi et al., 2000; Terada et al., 2003; Croisier and Graeber, 2006; Braak et al., 2007). 105 The consequences of astrocytic α-SYN inclusions for progression and spreading of PD 106 pathology however remain unknown. 107 108 Being the most abundant glial cell type in the nervous system, astrocytes play an important 109 role in maintaining brain homeostasis (Sofroniew and Vinters, 2010). The complex role of 110 astrocytes in the pathological brain is largely depending on their release and uptake of 111 substances from the microenvironment that they share with the neurons (Sofroniew and 112 Vinters, 2010). For example, astrocytes confer neuroprotection by removing excessive 113 extracellular glutamate, potassium and calcium, while they produce cytokines and 114 chemokines that could be harmful to neurons, if chronically released (Rappold and Tieu, 115 2010; Sofroniew and Vinters, 2010). 116 117 Reactive astrocytes effectively engulf dead cells, synapses and protein aggregates of amyloid 118 beta (Aβ) and α-SYN (Chang et al., 2000; Magnus et al., 2002; Sokolowski et al., 2011; Loov 5 119 et al., 2012; Chung et al., 2013; Fellner et al., 2013; Jones et al., 2013; Sollvander et al., 120 2016). Moreover, experimental evidence from in vitro and in vivo studies indicates that α- 121 SYN can transfer from cell-to-cell and thereby contribute to disease progression (Kordower et 122 al., 2008; Li et al., 2008; Desplats et