Absence of the Autophagy Adaptor SQSTM1/p62 Causes Childhood-Onset Neurodegeneration with Ataxia, Dystonia, and Gaze Palsy Tobias B. Haack, Erika Ignatius,

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Absence of the Autophagy Adaptor SQSTM1/p62 Causes Childhood-Onset Neurodegeneration with Ataxia, Dystonia, and Gaze Palsy Tobias B. Haack, Erika Ignatius, Javier Calvo-Garrido, Arcangela Iuso, Pirjo Isohanni, Camilla Maffezzini, Tuula Lönnqvist, Anu Suomalainen, Matteo Gorza, Laura S. Kremer, Elisabeth Graf, Monika Hartig, Riccardo Berutti, Martin Paucar, Per Svenningsson, Henrik Stranneheim, Göran Brandberg, Anna Wedell, Manju A. Kurian, Susan A. Hayflick, Paola Venco, Valeria Tiranti, Tim M. Strom, Martin Dichgans, Rita Horvath, Elke Holinski-Feder, Christoph Freyer, Thomas Meitinger, Holger Prokisch, Jan Senderek, Anna Wredenberg, Christopher J. Carroll, Thomas Klopstock The American Journal of Human Genetics Volume 99, Issue 3, Pages 735-743 (September 2016) DOI: 10.1016/j.ajhg.2016.06.026 Copyright © 2016 American Society of Human Genetics Terms and Conditions

Figure 1 Pedigrees of Investigated Families and Structure of SQSTM1 (A) Pedigrees of four families with mutations in SQSTM1. Mutation status of affected (closed symbols) and healthy (open symbols) family members. n.d., not determined. (B) Gene structure of SQSTM1 with known protein domains and motifs of the gene product and localization of the identified mutations. Intronic regions are not drawn to scale. Abbreviations are as follows: PB1, Phox 1 and Bem1p; ZZ, zinc finger; TRAF6, tumor necrosis factor receptor-associated factor 6; LIR, LC3-interaction region; KIR, Keap1-interacting; UBA, ubiquitin-associated. The American Journal of Human Genetics 2016 99, 735-743DOI: (10.1016/j.ajhg.2016.06.026) Copyright © 2016 American Society of Human Genetics Terms and Conditions

Figure 2 Neuroimaging Findings in SQSTM1/p62 Variant Individuals (A) Brain MRI (T2-weighted image, axial view) of individual F1:II.6 at the age of 33 years, demonstrating iron accumulation in the globus pallidus internus. (B) Brain MRI (T1-weighted image, sagittal view) of individual F2:II.2 at the age of 31 years, demonstrating mild cerebellar atrophy. (C) Brain MRI (T1-weighted image, sagittal view) of individual F3:II.1 at the age of 18 years showing mild to moderate upper vermian atrophy. The American Journal of Human Genetics 2016 99, 735-743DOI: (10.1016/j.ajhg.2016.06.026) Copyright © 2016 American Society of Human Genetics Terms and Conditions

Figure 3 Investigation of SQSTM1/p62 Protein and RNA Levels Western blot studies in SQSTM1/p62 variant fibroblast cell lines indicating that the homozygous variants c.2T>A (p.?) and c.286C>T (p.Arg96∗) result in a loss of SQSTM1/p62 protein. Immunoblotting was done with anti-SQSTM1/p62 antibody (F1:II.6 with Progen cat# GP62-C; F3:II.1 and F4:II.1,4 with Cell Signaling cat# 5114). In SQSTM1/p62 variant fibroblasts cell lines of F4:II.1,4, total RNA was isolated for qPCR analysis using Trizol. Reverse transcription for qRT-PCR analysis was performed using High Capacity cDNA Reverse Transcription Kit (Life Technologies). qRT-PCR was performed on a QuantStudio 6 (Life Technologies) with Platinum SYBR Green qPCR supermix-UDG (Life Technologies) and gene-specific primers. Error bars indicate ± SEM; ∗∗∗p ≤ 0.001; two-tailed unpaired t test. The first two columns refer to controls 1 and 2, and the last two columns refer to affected individuals F4:II.1 and F4:II.4. The American Journal of Human Genetics 2016 99, 735-743DOI: (10.1016/j.ajhg.2016.06.026) Copyright © 2016 American Society of Human Genetics Terms and Conditions

Figure 4 Investigation of Aggregation of Depolarized Mitochondria and Autophagosomal Formation (A) Naive (no exogenous PARKIN) control and SQSTM1/p62 variant fibroblasts were treated with 20 μM CCCP for 3 and 6 hr and fixed with 4% PFA. Mitochondria were immunostained with the mitochondrial SSBP and detected with a fluorescent-labeled secondary antibody (AF568, red), nuclei were stained with DAPI (blue), and images were acquired by confocal microscopy. Depolarization of mitochondria using the protonophore CCCP led to the collapse of the mitochondrial network in control and in SQSTM1/p62 variant cells. After 3 hr of CCCP treatment, perinuclear clustering of mitochondria can be observed in control cells, which is less evident in SQSTM1/p62 variant fibroblasts (arrows indicate distance from outer cell membrane to clustered mitochondria). No obvious differences between cell lines were observed after 6 hr of treatment. (B) Control and SQSTM1/p62 variant fibroblasts were transfected with YFP-Parkin (Addgene #23955),28 treated with CCCP (20 μM) for 2 hr, followed by 4% PFA fixation. Mitochondria were immunostained using antibodies against the mitochondrial protein TOM20 (Santa Cruz cat# sc-11414, RRID: AB_793274) and detected with a fluorescent-labeled secondary antibody (AF568, red). Images were acquired by confocal microscopy. Depolarization of mitochondria using the protonophore CCCP led to the collapse of the mitochondrial network in control and in SQSTM1/p62 variant cells. After 2 hr, mitochondrial clustering can be observed in control cells, with reduced clustering in SQSTM1/p62-deficient cells. Right panel: quantification of percent of cells showing a dispersed or aggregated mitochondrial distribution after 2 hr of CCCP treatment. n = 3, 25 cells per experiment. Morphology classification was done according to previous reports studying the function of p62/SQSTM1 in mitophagy. (C) SQSTM1/p62 variant and control cell lines were treated for 24 hr with the protonophore CCCP (20 μM) and fixed with 4% PFA. Autophagosomes were immunostained, using antibodies against the autophagosomal protein LC3II (MBL International cat# M152-3, RRID: AB_1279144) and detected with a fluorescent-labeled secondary antibody (AF488, green). Images were acquired by fluorescence microscopy and autophagosomes were counted manually. Right panel: quantification of the relative number of autophagosomes in the cell. Treatment with CCCP resulted in reduced autophagosome formation in SQSTM1-deficient cells. n = 3, 150 cells per experiment. (D) SQSTM1/p62 variant and control cell lines were treated for 24 and 48 hr with the protonophore CCCP (20 μM). Treatment with CCCP resulted in reduced LC3II levels formation in SQSTM1/p62-deficient cells. Right panel: quantification of the relative LC3II protein levels using ImageJ software. In (B)–(D), ∗p ≤ 0.05; ∗∗p ≤ 0.01; two-tailed unpaired t test. Error bars indicate ± SEM from the mean of three replicates. The American Journal of Human Genetics 2016 99, 735-743DOI: (10.1016/j.ajhg.2016.06.026) Copyright © 2016 American Society of Human Genetics Terms and Conditions