Nucleolar stress controls mutant Huntington toxicity and monitors Huntington’s disease progression

www.nature.com/cddis ARTICLE OPEN Nucleolar stress controls mutant Huntington toxicity and monitors Huntington’s disease progression Aynur Sönmez1,2, Rasem Mustafa 1,3, Salome T. Ryll1,3, Francesca Tuorto 4, Ludivine Wacheul 2, Donatella Ponti3,5, Christian Litke 3, Tanja Hering6, Kerstin Kojer6, Jenniver Koch1, Claudia Pitzer 7, Joachim Kirsch3, Andreas Neueder ✉ Grzegorz Kreiner8, Denis L. J. Lafontaine 2, Michael Orth6,11, Birgit Liss 1,9,11 and Rosanna Parlato 1,3,10,11 6 , 1234567890();,: © The Author(s) 2021 Transcriptional and cellular-stress surveillance deficits are hallmarks of Huntington’s disease (HD), a fatal autosomal-dominant neurodegenerative disorder caused by a pathological expansion of CAG repeats in the Huntingtin (HTT) gene. The nucleolus, a dynamic nuclear biomolecular condensate and the site of ribosomal RNA (rRNA) transcription, is implicated in the cellular stress response and in protein quality control. While the exact pathomechanisms of HD are still unclear, the impact of nucleolar dysfunction on HD pathophysiology in vivo remains elusive. Here we identified aberrant maturation of rRNA and decreased translational rate in association with human mutant Huntingtin (mHTT) expression. The protein nucleophosmin 1 (NPM1), important for nucleolar integrity and rRNA maturation, loses its prominent nucleolar localization. Genetic disruption of nucleolar integrity in vulnerable striatal neurons of the R6/2 HD mouse model decreases the distribution of mHTT in a disperse state in the nucleus, exacerbating motor deficits. We confirmed NPM1 delocalization in the gradually progressing zQ175 knock-in HD mouse model: in the striatum at a presymptomatic stage and in the skeletal muscle at an early symptomatic stage. In Huntington’s patient skeletal muscle biopsies, we found a selective redistribution of NPM1, similar to that in the zQ175 model. Taken together, our study demonstrates that nucleolar integrity regulates the formation of mHTT inclusions in vivo, and identifies NPM1 as a novel, readily detectable peripheral histopathological marker of HD progression. Cell Death and Disease (2021)12:1139 ; https://doi.org/10.1038/s41419-021-04432-x INTRODUCTION Dysregulation of rRNA biogenesis represents an emerging mechanism in several progressive neurodegenerative diseases characterized by proteinopathy [1–6]. Ribosomal RNA synthesis in the nucleolus—the most prominent nuclear compartment and a multilayered bio-molecular condensate—is tightly linked to the cell wellbeing, and it is highly responsive to cellular stress [7, 8]. Nucleolar stress is a p53-dependent anti-tumoral surveillance pathway activated upon ribosome-biogenesis dysfunction [9]. The shape of the nucleolus, its size, and the number of nucleoli per nucleus may change upon stress and in disease, reflecting changes in its function [10]. These properties have started to be explored as disease biomarkers [11, 12]. Huntington’ disease (HD) is caused by the expansion of CAG repeats in exon 1 of the Huntingtin (HTT) gene [13]. This autosomal dominant mutation results in an abnormal polyglutamine expansion in the Huntingtin protein with toxic effects [14]. Typical clinical hallmarks include motor, cognitive, and psychiatric symptoms [15]. Dopaminoceptive medium spiny neurons (MSNs) of the striatum are particularly vulnerable to neurodegeneration, along with reduced connectivity in regional and whole-brain cortico-caudate networks that highly correlate with cognitive and motor deficits [16]. Other non-neuronal features include metabolic and immune problems, malfunction of skeletal muscle, and bodyweight loss [17]. The length of the expanded CAG tract in the mutant HTT gene partially accounts for the variability in the clinical HD onset [18]. Multiple pathophysiological mechanisms may contribute to HD [19]. Mutant HTT (mHTT) protein forms nuclear and cytoplasmic inclusions that interfere with almost all aspects of cell physiology, from nuclear transcription dysregulation to mitochondrial dysfunction, and compromised quality-control mechanisms, among many others [20, 21]. Previous studies showed that mHTT interferes with rDNA transcription and with the integrity of the nucleolus [3, 22–27]. In the striatum of the R6/2 transgenic mice, the de novo 1 Institute of Applied Physiology, Ulm University, Ulm, Germany. 2RNA Molecular Biology, Fonds de la Recherche Scientifique (F.R.S./FNRS), Université Libre de Bruxelles (ULB), Biopark campus, Gosselies, Belgium. 3Institute of Anatomy and Cell Biology, Heidelberg University, Heidelberg, Germany. 4Division of Biochemistry, Mannheim Institute for Innate Immunoscience (MI3), Medical Faculty Mannheim, Heidelberg University, Mannheim and Center for Molecular Biology of Heidelberg University (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany. 5Department of Medical-Surgical Sciences and Biotechnologies, University of Rome “Sapienza”, Rome, Italy. 6Department of Neurology, Ulm University, Ulm, Germany. 7Interdisciplinary Neurobehavioral Core (INBC), Heidelberg University, Heidelberg, Germany. 8Maj Institute of Pharmacology, Department of Brain Biochemistry, Polish Academy of Sciences, Krakow, Poland. 9Linacre & New College, University of Oxford, Oxford, UK. 10Division for Neurodegenerative Diseases, Department of Neurology, Mannheim Center for Translational Neuroscience, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany. 11These authors contributed equally: Michael Orth, Birgit Liss, Rosanna Parlato. ✉email: Edited by Professor Fabio Blandini Received: 12 July 2021 Revised: 12 November 2021 Accepted: 18 November 2021 Official journal of CDDpress A. Sönmez et al. 2 transcription of rRNA is impaired [26]. Several mechanisms have been proposed to explain how mHTT affects rRNA transcription [24, 25, 27]. mHTT protein acts on the acetyltransferase CBP (CREBbinding protein), required for the activity of the RNA polymerase I (RNA Pol I) [3, 24]. Moreover, mHTT mRNAs down-regulate rRNA transcription by interacting with the nucleolar protein nucleolin (NCL), that plays multiple roles in rRNA synthesis, ribosome biogenesis and nucleolar structure maintenance [25, 28, 29]. PGC1alpha (peroxisome proliferator-activated receptor gamma coactivator 1alpha), a master regulator of mitochondrial biogenesis, which is transcriptionally repressed by mHTT, also controls rDNA transcription in the nucleolus [27]. Importantly, brain derived neurotrophic factor (BDNF) known to sustain striatal neuron survival and downregulated in HD [30, 31], stimulates the activity of the transcription-initiation factor-IA (TIF-IA), essential for the recruitment of the RNA Pol I at the ribosomal promoters [32]. The nucleolus is also involved in protein quality control to prevent the irreversible aggregation of misfolded proteins, a mechanism often altered in several aggregate-forming neurodegenerative diseases [33, 34]. In particular, the nucleolar protein nucleophosmin-1 (NPM1) appears to have (...truncated)