Abstract
Spinocerebellar ataxias (SCAs) are devastating neurodegenerative disorders for which no curative or preventive therapies are available. Deregulation of brain cholesterol metabolism and impaired brain cholesterol turnover have been associated with several neurodegenerative diseases. SCA3 or Machado–Joseph disease (MJD) is the most prevalent ataxia worldwide. We show that cholesterol 24-hydroxylase (CYP46A1), the key enzyme allowing efflux of brain cholesterol and activating brain cholesterol turnover, is decreased in cerebellar extracts from SCA3 patients and SCA3 mice. We investigated whether reinstating CYP46A1 expression would improve the disease phenotype of SCA3 mouse models. We show that administration of adeno-associated viral vectors encoding CYP46A1 to a lentiviral-based SCA3 mouse model reduces mutant ataxin-3 accumulation, which is a hallmark of SCA3, and preserves neuronal markers. In a transgenic SCA3 model with a severe motor phenotype we confirm that cerebellar delivery of AAVrh10-CYP46A1 is strongly neuroprotective in adult mice with established pathology. CYP46A1 significantly decreases ataxin-3 protein aggregation, alleviates motor impairments and improves SCA3-associated neuropathology. In particular, improvement in Purkinje cell number and reduction of cerebellar atrophy are observed in AAVrh10-CYP46A1-treated mice. Conversely, we show that knocking-down CYP46A1 in normal mouse brain impairs cholesterol metabolism, induces motor deficits and produces strong neurodegeneration with impairment of the endosomal–lysosomal pathway, a phenotype closely resembling that of SCA3. Remarkably, we demonstrate for the first time both in vitro, in a SCA3 cellular model, and in vivo, in mouse brain, that CYP46A1 activates autophagy, which is impaired in SCA3, leading to decreased mutant ataxin-3 deposition. More broadly, we show that the beneficial effect of CYP46A1 is also observed with mutant ataxin-2 aggregates. Altogether, our results confirm a pivotal role for CYP46A1 and brain cholesterol metabolism in neuronal function, pointing to a key contribution of the neuronal cholesterol pathway in mechanisms mediating clearance of aggregate-prone proteins. This study identifies CYP46A1 as a relevant therapeutic target not only for SCA3 but also for other SCAs.
Similar content being viewed by others
References
Alves S, Cormier-Dequaire F, Marinello M, Marais T, Muriel MP, Beaumatin F et al (2014) The autophagy/lysosome pathway is impaired in SCA7 patients and SCA7 knock-in mice. Acta Neuropathol 128:705–722. https://doi.org/10.1007/s00401-014-1289-8
Alves S, Nascimento-Ferreira I, Auregan G, Hassig R, Dufour N, Brouillet E et al (2008) Allele-specific RNA silencing of mutant ataxin-3 mediates neuroprotection in a rat model of Machado–Joseph disease. PLoS One 3:e3341. https://doi.org/10.1371/journal.pone.0003341
Alves S, Nascimento-Ferreira I, Dufour N, Hassig R, Auregan G, Nobrega C et al (2010) Silencing ataxin-3 mitigates degeneration in a rat model of Machado–Joseph disease: no role for wild-type ataxin-3? Hum Mol Genet 19:2380–2394. https://doi.org/10.1093/hmg/ddq111
Alves S, Regulier E, Nascimento-Ferreira I, Hassig R, Dufour N, Koeppen A et al (2008) Striatal and nigral pathology in a lentiviral rat model of Machado–Joseph disease. Hum Mol Genet 17:2071–2083. https://doi.org/10.1093/hmg/ddn106
Aveleira CA, Botelho M, Carmo-Silva S, Pascoal JF (2015) Neuropeptide Y stimulates autophagy in hypothalamic neurons. Proc Natl Acad Sci 112:E1642–E1651. https://doi.org/10.1073/pnas.1416609112
Ayciriex S, Djelti F, Alves S, Regazzetti A, Gaudin M, Varin J et al (2017) Neuronal cholesterol accumulation induced by Cyp46a1 down-regulation in mouse hippocampus disrupts brain lipid homeostasis. Front Mol Neurosci 10:211. https://doi.org/10.3389/fnmol.2017.00211
Ayers JI, Fromholt S, Sinyavskaya O, Siemienski Z, Rosario AM, Li A et al (2015) Widespread and efficient transduction of spinal cord and brain following neonatal AAV injection and potential disease modifying effect in ALS mice. Mol Ther 23:53–62. https://doi.org/10.1038/mt.2014.180
Bjorkhem I (2006) Crossing the barrier: oxysterols as cholesterol transporters and metabolic modulators in the brain. J Intern Med 260:493–508. https://doi.org/10.1111/j.1365-2796.2006.01725.x
Bjorkhem I, Leoni V, Meaney S (2010) Genetic connections between neurological disorders and cholesterol metabolism. J Lipid Res 51:2489–2503. https://doi.org/10.1194/jlr.R006338
Bjorkhem I, Lutjohann D, Diczfalusy U, Stahle L, Ahlborg G, Wahren J (1998) Cholesterol homeostasis in human brain: turnover of 24S-hydroxycholesterol and evidence for a cerebral origin of most of this oxysterol in the circulation. J Lipid Res 39:1594–1600
Bjorkhem I, Meaney S (2004) Brain cholesterol: long secret life behind a barrier. Arterioscler Thromb Vasc Biol 24:806–815. https://doi.org/10.1161/01.ATV.0000120374.59826.1b
Blount JR, Tsou WL, Ristic G, Burr AA, Ouyang M, Galante H et al (2014) Ubiquitin-binding site 2 of ataxin-3 prevents its proteasomal degradation by interacting with Rad23. Nat Commun 5:4638. https://doi.org/10.1038/ncomms5638
Boussicault L, Alves S, Lamaziere A, Planques A, Heck N, Moumne L et al (2016) CYP46A1, the rate-limiting enzyme for cholesterol degradation, is neuroprotective in Huntington’s disease. Brain 139:953–970. https://doi.org/10.1093/brain/awv384
Branchu J, Boutry M, Sourd L, Depp M, Leone C, Corriger A et al (2017) Loss of spatacsin function alters lysosomal lipid clearance leading to upper and lower motor neuron degeneration. Neurobiol Dis 102:21–37. https://doi.org/10.1016/j.nbd.2017.02.007
Burk K, Fetter M, Abele M, Laccone F, Brice A, Dichgans J et al (1999) Autosomal dominant cerebellar ataxia type I: oculomotor abnormalities in families with SCA1, SCA2, and SCA3. J Neurol 246:789–797
Burlot MA, Braudeau J, Michaelsen-Preusse K, Potier B, Ayciriex S, Varin J et al (2015) Cholesterol 24-hydroxylase defect is implicated in memory impairments associated with Alzheimer-like Tau pathology. Hum Mol Genet 24:5965–5976. https://doi.org/10.1093/hmg/ddv268
Camargo N, Smit AB, Verheijen MH (2009) SREBPs: SREBP function in glia-neuron interactions. FEBS J 276:628–636. https://doi.org/10.1111/j.1742-4658.2008.06808.x
Chai Y, Shao J, Miller VM, Williams A, Paulson HL (2002) Live-cell imaging reveals divergent intracellular dynamics of polyglutamine disease proteins and supports a sequestration model of pathogenesis. Proc Natl Acad Sci USA 99:9310–9315. https://doi.org/10.1073/pnas.152101299
Chevy F, Humbert L, Wolf C (2005) Sterol profiling of amniotic fluid: a routine method for the detection of distal cholesterol synthesis deficit. Prenat Diagn 25:1000–1006. https://doi.org/10.1002/pd.1254
Cortes CJ, La Spada AR (2015) Autophagy in polyglutamine disease: imposing order on disorder or contributing to the chaos? Mol Cell Neurosci 66:53–61. https://doi.org/10.1016/j.mcn.2015.03.010
Cunha-Santos J, Duarte-Neves J, Carmona V, Guarente L, Pereira de Almeida L, Cavadas C (2016) Caloric restriction blocks neuropathology and motor deficits in Machado–Joseph disease mouse models through SIRT1 pathway. Nat Commun 7:11445. https://doi.org/10.1038/ncomms11445
Deverman BE, Pravdo PL, Simpson BP, Kumar SR, Chan KY, Banerjee A et al (2016) Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain 34:204–209. https://doi.org/10.1038/nbt.3440
Dietschy JM, Turley SD (2002) Control of cholesterol turnover in the mouse. J Biol Chem 277:3801–3804. https://doi.org/10.1074/jbc.R100057200
Djelti F, Braudeau J, Hudry E, Dhenain M, Varin J, Bieche I et al (2015) CYP46A1 inhibition, brain cholesterol accumulation and neurodegeneration pave the way for Alzheimer’s disease. Brain 138:2383–2398. https://doi.org/10.1093/brain/awv166
Durr A, Stevanin G, Cancel G, Duyckaerts C, Abbas N, Didierjean O et al (1996) Spinocerebellar ataxia 3 and Machado–Joseph disease: clinical, molecular, and neuropathological features. Ann Neurol 39:490–499. https://doi.org/10.1002/ana.410390411
Dzeletovic S, Babiker A, Lund E, Diczfalusy U (1995) Time course of oxysterol formation during in vitro oxidation of low density lipoprotein. Chem Phys Lipid 78:119–128
Hara T, Nakamura K, Matsui M, Yamamoto A, Nakahara Y, Suzuki-Migishima R et al (2006) Suppression of basal autophagy in neural cells causes neurodegenerative disease in mice. Nature 441:885–889. https://doi.org/10.1038/nature04724
Hudry E, Van Dam D, Kulik W, De Deyn PP, Stet FS, Ahouansou O et al (2010) Adeno-associated virus gene therapy with cholesterol 24-hydroxylase reduces the amyloid pathology before or after the onset of amyloid plaques in mouse models of Alzheimer’s disease. Mol Ther 18:44–53. https://doi.org/10.1038/mt.2009.175
Huynh DP, Yang HT, Vakharia H, Nguyen D, Pulst SM (2003) Expansion of the polyQ repeat in ataxin-2 alters its Golgi localization, disrupts the Golgi complex and causes cell death. Hum Mol Genet 12:1485–1496
Kawaguchi Y, Okamoto T, Taniwaki M, Aizawa M, Inoue M, Katayama S et al (1994) CAG expansions in a novel gene for Machado–Joseph disease at chromosome 14q32.1. Nat Genet 8:221–228. https://doi.org/10.1038/ng1194-221
Klockgether T, Skalej M, Wedekind D, Luft AR, Welte D, Schulz JB et al (1998) Autosomal dominant cerebellar ataxia type I. MRI-based volumetry of posterior fossa structures and basal ganglia in spinocerebellar ataxia types 1, 2 and 3. Brain 121(Pt 9):1687–1693
Korade Z, Kenworthy AK (2008) Lipid rafts, cholesterol, and the brain. Neuropharmacology 55:1265–1273. https://doi.org/10.1016/j.neuropharm.2008.02.019
Kotti TJ, Ramirez DM, Pfeiffer BE, Huber KM, Russell DW (2006) Brain cholesterol turnover required for geranylgeraniol production and learning in mice. Proc Natl Acad Sci USA 103:3869–3874. https://doi.org/10.1073/pnas.0600316103
Lukashchuk V, Lewis KE, Coldicott I, Grierson AJ, Azzouz M (2016) AAV9-mediated central nervous system-targeted gene delivery via cisterna magna route in mice. Mol Ther Methods Clin Dev 3:15055. https://doi.org/10.1038/mtm.2015.55
Lund EG, Guileyardo JM, Russell DW (1999) cDNA cloning of cholesterol 24-hydroxylase, a mediator of cholesterol homeostasis in the brain. Proc Natl Acad Sci USA 96:7238–7243
Lund EG, Xie C, Kotti T, Turley SD, Dietschy JM, Russell DW (2003) Knockout of the cholesterol 24-hydroxylase gene in mice reveals a brain-specific mechanism of cholesterol turnover. J Biol Chem. https://doi.org/10.1074/jbc.M303415200
Margolis RL, Ross CA (2001) Expansion explosion: new clues to the pathogenesis of repeat expansion neurodegenerative diseases. Trends Mol Med 7:479–482
Martin MG, Pfrieger F, Dotti CG (2014) Cholesterol in brain disease: sometimes determinant and frequently implicated. EMBO Rep 15:1036–1052. https://doi.org/10.15252/embr.201439225
Matilla-Duenas A, Ashizawa T, Brice A, Magri S, McFarland KN, Pandolfo M et al (2014) Consensus paper: pathological mechanisms underlying neurodegeneration in spinocerebellar ataxias. Cerebellum 13:269–302. https://doi.org/10.1007/s12311-013-0539-y
Mauch DH, Nagler K, Schumacher S, Goritz C, Muller EC, Otto A et al (2001) CNS synaptogenesis promoted by glia-derived cholesterol. Science 294:1354–1357. https://doi.org/10.1126/science.294.5545.1354
Meaney S, Hassan M, Sakinis A, Lutjohann D, von Bergmann K, Wennmalm A et al (2001) Evidence that the major oxysterols in human circulation originate from distinct pools of cholesterol: a stable isotope study. J Lipid Res 42:70–78
Mendonca LS, Nobrega C, Hirai H, Kaspar BK, Pereira de Almeida L (2015) Transplantation of cerebellar neural stem cells improves motor coordination and neuropathology in Machado–Joseph disease mice. Brain 138:320–335. https://doi.org/10.1093/brain/awu352
Menzies FM, Fleming A, Rubinsztein DC (2015) Compromised autophagy and neurodegenerative diseases. Nat Rev Neurosci 16:345–357. https://doi.org/10.1038/nrn3961
Moutinho M, Nunes MJ, Rodrigues E (2016) Cholesterol 24-hydroxylase: brain cholesterol metabolism and beyond. Biochem Biophys Acta 1861:1911–1920. https://doi.org/10.1016/j.bbalip.2016.09.011
Nascimento-Ferreira I, Nobrega C, Vasconcelos-Ferreira A, Onofre I, Albuquerque D, Aveleira C et al (2013) Beclin 1 mitigates motor and neuropathological deficits in genetic mouse models of Machado–Joseph disease. Brain 136:2173–2188. https://doi.org/10.1093/brain/awt144
Nascimento-Ferreira I, Santos-Ferreira T, Sousa-Ferreira L, Auregan G, Onofre I, Alves S et al (2011) Overexpression of the autophagic beclin-1 protein clears mutant ataxin-3 and alleviates Machado–Joseph disease. Brain 134:1400–1415. https://doi.org/10.1093/brain/awr047
Nobrega C, Carmo-Silva S, Albuquerque D, Vasconcelos-Ferreira A, Vijayakumar UG, Mendonca L et al (2015) Re-establishing ataxin-2 downregulates translation of mutant ataxin-3 and alleviates Machado–Joseph disease. Brain 138:3537–3554. https://doi.org/10.1093/brain/awv298
Nobrega C, Nascimento-Ferreira I, Onofre I, Albuquerque D, Conceicao M, Deglon N et al (2013) Overexpression of mutant ataxin-3 in mouse cerebellum induces ataxia and cerebellar neuropathology. Cerebellum 12:441–455. https://doi.org/10.1007/s12311-012-0432-0
Nobrega C, Nascimento-Ferreira I, Onofre I, Albuquerque D, Deglon N, de Almeida LP (2014) RNA interference mitigates motor and neuropathological deficits in a cerebellar mouse model of Machado–Joseph disease. PLoS One 9:e100086. https://doi.org/10.1371/journal.pone.0100086
Ohyama Y, Meaney S, Heverin M, Ekstrom L, Brafman A, Shafir M et al (2006) Studies on the transcriptional regulation of cholesterol 24-hydroxylase (CYP46A1): marked insensitivity toward different regulatory axes. J Biol Chem 281:3810–3820. https://doi.org/10.1074/jbc.M505179200
Onofre I, Mendonca N, Lopes S, Nobre R, de Melo JB, Carreira IM et al (2016) Fibroblasts of Machado–Joseph disease patients reveal autophagy impairment. Sci Rep 6:28220. https://doi.org/10.1038/srep28220
Orr HT, Zoghbi HY (2007) Trinucleotide repeat disorders. Annu Rev Neurosci 30:575–621. https://doi.org/10.1146/annurev.neuro.29.051605.113042
Oue M, Mitsumura K, Torashima T, Koyama C, Yamaguchi H, Furuya N et al (2009) Characterization of mutant mice that express polyglutamine in cerebellar Purkinje cells. Brain Res 1255:9–17. https://doi.org/10.1016/j.brainres.2008.12.014
Ramirez DM, Andersson S, Russell DW (2008) Neuronal expression and subcellular localization of cholesterol 24-hydroxylase in the mouse brain. J Comp Neurol 507:1676–1693. https://doi.org/10.1002/cne.21605
Reetz K, Costa AS, Mirzazade S, Lehmann A, Juzek A, Rakowicz M et al (2013) Genotype-specific patterns of atrophy progression are more sensitive than clinical decline in SCA1, SCA3 and SCA6. Brain 136:905–917. https://doi.org/10.1093/brain/aws369
Rub U, Brunt ER, Deller T (2008) New insights into the pathoanatomy of spinocerebellar ataxia type 3 (Machado–Joseph disease). Curr Opin Neurol 21:111–116. https://doi.org/10.1097/WCO.0b013e3282f7673d
Rub U, Brunt ER, Petrasch-Parwez E, Schols L, Theegarten D, Auburger G et al (2006) Degeneration of ingestion-related brainstem nuclei in spinocerebellar ataxia type 2, 3, 6 and 7. Neuropathol Appl Neurobiol 32:635–649. https://doi.org/10.1111/j.1365-2990.2006.00772.x
Russell DW, Halford RW, Ramirez DM, Shah R, Kotti T (2009) Cholesterol 24-hydroxylase: an enzyme of cholesterol turnover in the brain. Annu Rev Biochem 78:1017–1040. https://doi.org/10.1146/annurev.biochem.78.072407.103859
Samaranch L, Salegio EA, San Sebastian W, Kells AP, Foust KD, Bringas JR et al (2012) Adeno-associated virus serotype 9 transduction in the central nervous system of nonhuman primates. Hum Gene Ther 23:382–389. https://doi.org/10.1089/hum.2011.200
Scherzed W, Brunt ER, Heinsen H, de Vos RA, Seidel K, Burk K et al (2012) Pathoanatomy of cerebellar degeneration in spinocerebellar ataxia type 2 (SCA2) and type 3 (SCA3). Cerebellum 11:749–760. https://doi.org/10.1007/s12311-011-0340-8
Schols L, Amoiridis G, Buttner T, Przuntek H, Epplen JT, Riess O (1997) Autosomal dominant cerebellar ataxia: phenotypic differences in genetically defined subtypes? Ann Neurol 42:924–932. https://doi.org/10.1002/ana.410420615
Schols L, Bauer P, Schmidt T, Schulte T, Riess O (2004) Autosomal dominant cerebellar ataxias: clinical features, genetics, and pathogenesis. Lancet Neurol 3:291–304. https://doi.org/10.1016/S1474-4422(04)00737-9
Schuster DJ, Dykstra JA, Riedl MS, Kitto KF, Belur LR, McIvor RS et al (2014) Biodistribution of adeno-associated virus serotype 9 (AAV9) vector after intrathecal and intravenous delivery in mouse. Front Neuroanat 8:42. https://doi.org/10.3389/fnana.2014.00042
Sittler A, Muriel MP, Marinello M, Brice A, den Dunnen W, Alves S (2017) Deregulation of autophagy in postmortem brains of Machado–Joseph disease patients. Neuropathology. https://doi.org/10.1111/neup.12433
Sobo K, Le Blanc I, Luyet PP, Fivaz M, Ferguson C, Parton RG et al (2007) Late endosomal cholesterol accumulation leads to impaired intra-endosomal trafficking. PLoS One 2:e851. https://doi.org/10.1371/journal.pone.0000851
Sodero AO, Trovo L, Iannilli F, Van Veldhoven P, Dotti CG, Martin MG (2011) Regulation of tyrosine kinase B activity by the Cyp46/cholesterol loss pathway in mature hippocampal neurons: relevance for neuronal survival under stress and in aging. J Neurochem 116:747–755. https://doi.org/10.1111/j.1471-4159.2010.07079.x
Sodero AO, Vriens J, Ghosh D, Stegner D, Brachet A, Pallotto M et al (2012) Cholesterol loss during glutamate-mediated excitotoxicity. EMBO J 31:1764–1773. https://doi.org/10.1038/emboj.2012.31
Stefanescu MR, Dohnalek M, Maderwald S, Thurling M, Minnerop M, Beck A et al (2015) Structural and functional MRI abnormalities of cerebellar cortex and nuclei in SCA3, SCA6 and Friedreich’s ataxia. Brain 138:1182–1197. https://doi.org/10.1093/brain/awv064
Thelen KM, Falkai P, Bayer TA, Lutjohann D (2006) Cholesterol synthesis rate in human hippocampus declines with aging. Neurosci Lett 403:15–19. https://doi.org/10.1016/j.neulet.2006.04.034
Toonen LJA, Overzier M, Evers MM, Leon LG, van der Zeeuw SAJ, Mei H et al (2018) Transcriptional profiling and biomarker identification reveal tissue specific effects of expanded ataxin-3 in a spinocerebellar ataxia type 3 mouse model. Mol Neurodegener 13:31. https://doi.org/10.1186/s13024-018-0261-9
Torashima T, Koyama C, Iizuka A, Mitsumura K, Takayama K, Yanagi S et al (2008) Lentivector-mediated rescue from cerebellar ataxia in a mouse model of spinocerebellar ataxia. EMBO Rep 9:393–399. https://doi.org/10.1038/embor.2008.31
Vance JE (2012) Dysregulation of cholesterol balance in the brain: contribution to neurodegenerative diseases. Dis Models Mech 5:746–755. https://doi.org/10.1242/dmm.010124
Zerah M, Piguet F, Colle MA, Raoul S, Deschamps JY, Deniaud J et al (2015) Intracerebral gene therapy using AAVrh. 10-hARSA recombinant vector to treat patients with early-onset forms of metachromatic leukodystrophy: preclinical feasibility and safety assessments in nonhuman primates. Hum Gene Ther Clin Dev 26:113–124. https://doi.org/10.1089/humc.2014.139
Acknowledgements
This work was supported by NeurATRIS: A Translational Research Infrastructure for Biotherapies in Neurosciences, the Fondation pour la Recherche Médicale, Bioingénierie pour la Santé 2014 “Project DBS20140930765”, Paris Biotech Santé incubator, the SATT (Société d’Accélération de Transfert Technologique) Ile de France Innov, E.rare: E-Rare Joint Transnational Call for Proposals 2017 “Transnational Research Projects for Innovative Therapeutic Approaches for Rare Diseases”, Biotheralliance network from the Paris Saclay University and Brainvectis. This work was also financed by the European Regional Development Fund (ERDF), through the CENTRO 2020 Regional Operational Programme under project CENTRO-01-0145-FEDER-000008:BrainHealth 2020, through the COMPETE 2020—Operational Programme for Competitiveness and Internationalization and Portuguese national funds via FCT—Fundação para a Ciência e a Tecnologia, I.P., under projects POCI-01-0145-FEDER-016719 (PTDC/NEU-NMC/0084/2014), POCI-01-0145-FEDER-007440 (UID/NEU/04539/2013) and POCI-01-0145-FEDER-016390:CANCEL STEM, and through CENTRO 2020 and FCT under project CENTRO-01-0145-FEDER-022095:ViraVector; also by projects ESMI (JPCOFUND/0001/2015) and ModelPolyQ (JPCOFUND/0005/2015) under the EU Joint Program—Neurodegenerative Disease Research (JPND), the last two co-funded by the European Union H2020 program, GA No.643417 and national funds (FCT), and by the Richard Chin and Lily Lock Machado Joseph Disease Research Fund; and the National Ataxia Foundation. CN laboratory is supported by the French Muscular Dystrophy Association (AFM-Téléthon), the Ataxia UK, and the FCT. AM is supported by a Ph.D. fellowship from FCT (SFRH/BD/133192/2017).
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
Conflict of interest
The authors declare financial interest. CN, LM, NC, LPA and SA are inventors of patent applications claiming the use of AAV-CYP46A1 therapy in spinocerebellar ataxias.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Nóbrega, C., Mendonça, L., Marcelo, A. et al. Restoring brain cholesterol turnover improves autophagy and has therapeutic potential in mouse models of spinocerebellar ataxia. Acta Neuropathol 138, 837–858 (2019). https://doi.org/10.1007/s00401-019-02019-7
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00401-019-02019-7