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Mouse Models for Hereditary Spastic Paraplegia Uncover a Role Of University of Dundee Mouse models for hereditary spastic paraplegia uncover a role of PI4K2A in autophagic lysosome reformation Khundadze, Mukhran; Ribaudo, Federico; Hussain, Adeela; Stahlberg, Henry; Brocke- Ahmadinejad, Nahal; Franzka, Patricia Published in: Autophagy DOI: 10.1080/15548627.2021.1891848 Publication date: 2021 Licence: CC BY Document Version Publisher's PDF, also known as Version of record Link to publication in Discovery Research Portal Citation for published version (APA): Khundadze, M., Ribaudo, F., Hussain, A., Stahlberg, H., Brocke-Ahmadinejad, N., Franzka, P., Varga, R-E., Zarkovic, M., Pungsrinont, T., Kokal, M., Ganley, I. G., Beetz, C., Sylvester, M., & Hübner, C. A. (2021). Mouse models for hereditary spastic paraplegia uncover a role of PI4K2A in autophagic lysosome reformation. Autophagy. https://doi.org/10.1080/15548627.2021.1891848 General rights Copyright and moral rights for the publications made accessible in Discovery Research Portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from Discovery Research Portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain. • You may freely distribute the URL identifying the publication in the public portal. Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 23. Sep. 2021 Autophagy ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/kaup20 Mouse models for hereditary spastic paraplegia uncover a role of PI4K2A in autophagic lysosome reformation Mukhran Khundadze, Federico Ribaudo, Adeela Hussain, Henry Stahlberg, Nahal Brocke-Ahmadinejad, Patricia Franzka, Rita-Eva Varga, Milena Zarkovic, Thanakorn Pungsrinont, Miriam Kokal, Ian G. Ganley, Christian Beetz, Marc Sylvester & Christian A. Hübner To cite this article: Mukhran Khundadze, Federico Ribaudo, Adeela Hussain, Henry Stahlberg, Nahal Brocke-Ahmadinejad, Patricia Franzka, Rita-Eva Varga, Milena Zarkovic, Thanakorn Pungsrinont, Miriam Kokal, Ian G. Ganley, Christian Beetz, Marc Sylvester & Christian A. Hübner (2021): Mouse models for hereditary spastic paraplegia uncover a role of PI4K2A in autophagic lysosome reformation, Autophagy, DOI: 10.1080/15548627.2021.1891848 To link to this article: https://doi.org/10.1080/15548627.2021.1891848 © 2021 The Author(s). Published by Informa View supplementary material UK Limited, trading as Taylor & Francis Group. Published online: 09 Mar 2021. Submit your article to this journal Article views: 498 View related articles View Crossmark data Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=kaup20 AUTOPHAGY https://doi.org/10.1080/15548627.2021.1891848 RESEARCH PAPER Mouse models for hereditary spastic paraplegia uncover a role of PI4K2A in autophagic lysosome reformation Mukhran Khundadzea,‡, Federico Ribaudoa,‡, Adeela Hussaina, Henry Stahlberga, Nahal Brocke-Ahmadinejadb, Patricia Franzkaa, Rita-Eva Vargaa, Milena Zarkovica, Thanakorn Pungsrinonta, Miriam Kokala, Ian G. Ganleyc, Christian Beetzd, Marc Sylvesterb, and Christian A. Hübnera aInstitute of Human Genetics, University Hospital Jena, Friedrich-Schiller-University Jena, Jena, Germany; bCore Facility Mass Spectrometry, Institute of Biochemistry and Molecular Biology, Medical Faculty, University of Bonn, Bonn, Germany; cMRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, Scotland; dInstitute of Clinical Chemistry, University Hospital Jena, Friedrich-Schiller- University Jena, Germany; Current Affiliation: Centogene GmbH, Rostock, Germany ABSTRACT ARTICLE HISTORY Hereditary spastic paraplegia (HSP) denotes genetically heterogeneous disorders characterized by leg Received 29 May 2020 spasticity due to degeneration of corticospinal axons. SPG11 and SPG15 have a similar clinical course Revised 1 February 2021 and together are the most prevalent autosomal recessive HSP entity. The respective proteins play a role Accepted 15 February 2021 for macroautophagy/autophagy and autophagic lysosome reformation (ALR). Here, we report that spg11 KEYWORDS and zfyve26 KO mice developed motor impairments within the same course of time. This correlated with Autophagy; lysosome; enhanced accumulation of autofluorescent material in neurons and progressive neuron loss. In agree­ neurodegeneration; PI4K2A; ment with defective ALR, tubulation events were diminished in starved KO mouse embryonic fibroblasts spg11; spg15 (MEFs) and lysosomes decreased in neurons of KO brain sections. Confirming that both proteins act in the same molecular pathway, the pathologies were not aggravated upon simultaneous disruption of both. We further show that PI4K2A (phosphatidylinositol 4-kinase type 2 alpha), which phosphorylates phosphatidylinositol to phosphatidylinositol-4-phosphate (PtdIns4P), accumulated in autofluorescent deposits isolated from KO but not WT brains. Elevated PI4K2A abundance was already found at autolysosomes of neurons of presymptomatic KO mice. Immunolabelings further suggested higher levels of PtdIns4P at LAMP1-positive structures in starved KO MEFs. An increased association with LAMP1-positive structures was also observed for clathrin and DNM2/dynamin 2, which are important effectors of ALR recruited by phospholipids. Because PI4K2A overexpression impaired ALR, while its knockdown increased tubulation, we conclude that PI4K2A modulates phosphoinositide levels at auto­ lysosomes and thus the recruitment of downstream effectors of ALR. Therefore, PI4K2A may play an important role in the pathogenesis of SPG11 and SPG15. Abbreviations: ALR: autophagic lysosome reformation; AP-5: adaptor protein complex 5; BFP: blue fluorescent protein; dKO: double knockout; EBSS: Earle’s balanced salt solution; FBA: foot base angle; GFP: green fluorescent protein; HSP: hereditary spastic paraplegia; KO: knockout; LAMP1: lysosomal- associated membrane protein 1; MAP1LC3B/LC3: microtubule-associated protein 1 light chain 3 beta; MEF: mouse embryonic fibroblast; SQSTM1/p62: sequestosome 1; PI4K2A: phosphatidylinositol 4-kinase type 2 alpha; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns4P: phosphatidylinositol-4-phosphate; RFP: red fluorescent protein; SPG: spastic paraplegia gene; TGN: trans-Golgi network; WT: wild type Introduction Hereditary spastic paraplegia (HSP) refers to a group of identification. Mutations in SPG11 (SPG11 vesicle traffick­ neurodegenerative disorders, which are characterized by the ing associated, spatacsin) are the most common cause of degeneration of upper motoneuron axons thus resulting in autosomal recessively inherited HSP [2]. Affected patients lower limb spasticity and weakness [1]. Additional symp­ often suffer from mental retardation, ataxia, Parkinsonism, toms such as cognitive deficits, a thinning of the corpus retinopathy or polyneuropathy and show a thinning of the callosum, optic atrophy, cerebellar atrophy, amyotrophy, corpus callosum and cortical atrophy. Clinically SPG11 peripheral nerve involvement, and seizures can occur and patients cannot be distinguished from SPG15 patients, characterize complicated HSPs. Up to date more than 80 another autosomal recessive form of HSP [3], which is different genetic loci have been identified, which are termed caused by mutations in ZFYVE26/Spastizin (zinc finger as SPGs with a running number reflecting the order of its FYVE-type containing 26). Therefore, it was concluded CONTACT Mukhran Khundadze [email protected]; Christian A. Hübner [email protected] Institute of Human Genetics, University Hospital Jena, Friedrich-Schiller-University Jena, Am Klinikum 1, 07747 Jena, Germany. ‡These authors share first authorship on this work. Supplemental data for this article can be accessed here. © 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 2 M. KHUNDADZE ET AL. that SPG11 and ZFYVE26 may act in the same cellular unbiased mass spectrometry approach identified PI4K2A pathway. Indeed, both SPG11 and ZFYVE26 co-precipitate (phosphatidylinositol 4-kinase type 2 alpha) in autofluor­ with other proteins [4], which were later identified as escent material isolated from brains of spg11 KO and members of the newly identified adaptor protein (AP) com­ zfyve26 KO but not WT mice. Immunostainings of brain plex AP-5 [5]. sections further revealed that PI4K2A abundance is AP complexes mediate intracellular membrane traffick­ already increased at autolysosomes of KO mice before ing along endocytic and secretory transport pathways. It is onset of overt neurodegeneration. PI4K2A overexpression assumed that AP-5 serves as a backup system for the in U2-OS cells inhibited autolysosomal tubulation and retrieval of proteins from late endosomes to the trans increased the association of clathrin and DNM2/dynamin Golgi, because knockdown [6] or disruption
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