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Brain Deletion of Serf2 Shifts Amyloid Conformation in a Mouse Model bioRxiv preprint doi: https://doi.org/10.1101/2021.01.05.423442; this version posted February 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Brain deletion of Serf2 shifts amyloid conformation in a mouse model 2 E. Stroo1*, L. Janssen1*#, O. Sin1, W. Hogewerf1, M. Koster2, L. Harkema2, S.A. Youssef2,3, N. 3 Beschorner4, A.H.G. Wolters5, B. Bakker1, A. Thathiah6,7, F. Foijer1, B. van de Sluis2, J. van Deursen8, 4 M. Jucker4, A. de Bruin2,3, E.A.A. Nollen1# 5 1European Research Institute for the Biology of Ageing, University of Groningen, University Medical 6 Centre Groningen, The Netherlands 7 2Department of Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University, 8 The Netherlands 9 3Department of Pediatrics, Molecular Genetics Section, University of Groningen, University Medical 10 Centre Groningen, The Netherlands 11 4Department of Cellular Neurology, Hertie-Institute for Clinical Brain Research, University of 12 Tübingen, Tübingen, Germany. 13 5Department of Biomedical Sciences of Cells and Systems, University Medical Centre Groningen, The 14 Netherlands 15 6VIB Center for the Biology of Disease, KU Leuven Center for Human Genetics, University 16 of Leuven, Leuven, Belgium 17 7 Department of Neurobiology, University of Pittsburgh Brain Institute, University of 18 Pittsburgh School of Medicine, Pittsburgh, PA USA 19 8Mayo Clinic, United States of America 20 *These authors contributed equally 21 #Corresponding authors: [email protected] or [email protected] 22 Keywords: Small EDRK-rich factor 2, SERF, protein aggregation, amyloid-beta, amyloid 23 polymorphisms bioRxiv preprint doi: https://doi.org/10.1101/2021.01.05.423442; this version posted February 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 24 Highlights 25 • Loss of SERF2 slows embryonic development and causes perinatal lethality 26 • SERF2 affects proliferation in a cell-autonomous fashion 27 • Brain-specific Serf2 knockout does not affect viability or Aβ production 28 • Brain deletion of Serf2 shifts the amyloid conformation of Aβ bioRxiv preprint doi: https://doi.org/10.1101/2021.01.05.423442; this version posted February 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 29 Abstract 30 Neurodegenerative diseases like Alzheimer, Parkinson and Huntington disease are characterized by 31 aggregation-prone proteins that form amyloid fibrils through a nucleation process. Despite the shared 32 β-sheet structure, recent research has shown that structurally different polymorphs exist within fibrils 33 of the same protein. These polymorphs are associated with varying levels of toxicity and different 34 disease phenotypes. MOAG-4 and its human orthologs SERF1 and SERF2 have previously been 35 shown to modify the nucleation and drive amyloid formation and protein toxicity in vitro and in C. 36 elegans. To further explore these findings, we generated a Serf2 knockout (KO) mouse model and 37 crossed it with the APPPS1 mouse model for Aβ amyloid pathology. Full-body KO of Serf2 resulted 38 in a developmental delay and perinatal lethality due to insufficient lung maturation. Therefore, we 39 proceeded with a brain-specific Serf2 KO, which was found to be viable. We examined the Aβ 40 pathology at 1 and 3 months of age, which is before and after the start of amyloid deposition. We 41 show that SERF2 deficiency does not affect the production and overall Aβ levels. Serf2 KO-APPPS1 42 mice displayed an increased intracellular Aβ accumulation at 1 month and a higher number of Aβ 43 deposits compared to APPPS1 mice with similar Aβ levels. Moreover, conformation-specific dyes and 44 electron microscopy revealed a difference in the structure and amyloid content of these Aβ deposits. 45 Together, our results reveal that SERF2 causes a structural shift in Aβ aggregation in a mammalian 46 brain. These findings indicate that a single endogenous factor may contribute to amyloid 47 polymorphisms, allowing for new insights into this phenomenon’s contribution to disease 48 manifestation. bioRxiv preprint doi: https://doi.org/10.1101/2021.01.05.423442; this version posted February 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 49 Introduction 50 Protein aggregation is a pathological hallmark shared by several age-related neurodegenerative 51 diseases, such as Alzheimer (AD), Parkinson and Huntington disease. The amyloid-like aggregates 52 that accumulate in each of these diseases are composed of disease-specific proteins, i.e., amyloid-beta 53 (Aβ) and tau in AD(Glenner & Wong, 1984; Masters et al, 1985; Grundke-Iqbal et al, 1986a, 1986b), 54 alpha-synuclein (α-Syn) in Parkinson disease(Langley & Charlesworth, 1997) and Huntingtin (HTT) 55 in Huntington disease(Scherzinger et al, 1999). While the exact molecular mechanisms underlying the 56 disease pathology remain to be elucidated, genetic evidence indicates that these aggregation-prone 57 proteins play a key role in the disease processes. In AD multiple causative mutations have been found 58 in three genes: the amyloid precursor protein (APP)(Goate et al, 1991; Chartier-Harlin et al, 1991; 59 Citron et al, 1992; Eckman et al, 1997; Murrell et al, 2000; Kumar-Singh et al, 2000; Cruts et al, 60 2003; Kumar-Singh et al, 2006; Tomiyama et al, 2008), presenilin-1 (PSEN1)(Schellenberg et al, 61 1992; Sherrington et al, 1995; Borchelt et al, 1996; Lemere et al, 1996; Guerreiro et al, 2010) and 62 presenilin-2 (PSEN2)(Rogaev et al, 1995; Citron et al, 1997; Müller et al, 2014; Guerreiro et al, 2010) 63 (for a full list of known AD-linked mutations see https://www.alzforum.org/mutations). The APP 64 gene encodes the precursor protein from which Aβ is produced through sequential cleavage by a β- 65 and γ-secretase. The PSEN genes encode the main catalytic subunit of the γ-secretase complex. The 66 fact that all the causative mutations affect the production, length and/or structure of the Aβ peptides 67 and are sufficient to cause the disease(Chartier-Harlin et al, 1991; Citron et al, 1997; Cruts et al, 2003; 68 Eckman et al, 1997; Guerreiro et al, 2010; Goate et al, 1991; Kumar-Singh et al, 2000, 2006; Lemere 69 et al, 1996; Müller et al, 2014; Murrell et al, 2000; Rogaev et al, 1995; Schellenberg et al, 1992; 70 Sherrington et al, 1995; Tomiyama et al, 2008; Borchelt et al, 1996; Citron et al, 1992), is arguably 71 the strongest piece of evidence for the involvement of Aβ in the disease process. Causative mutations 72 generally affect the Aβ peptide in one of three ways: (i) an overall increase in Aβ production(Haass et 73 al, 1995; Citron et al, 1992), (ii) a shift in the production of shorter variants, like Aβ40, to more 74 aggregation-prone, longer variants, like Aβ42 (or Aβ43)(Suzuki et al, 1994; Duff et al, 1996; Murayama 75 et al, 1999; Murrell et al, 2000; Borchelt et al, 1996), (iii) Aβ peptides with a modified core region, bioRxiv preprint doi: https://doi.org/10.1101/2021.01.05.423442; this version posted February 12, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 76 resulting in an increased propensity for aggregation(Nilsberth et al, 2001; Miravalle et al, 2000; Ni et 77 al, 2011; Tomiyama et al, 2008). Taken together, these mutations do not necessarily increase the 78 amount of Aβ, but rather increase the likelihood of Aβ aggregation. Similarly, several mutations 79 in the SNCA gene that have been linked to Parkinson disease have been shown to alter α-Syn 80 aggregation(Conway et al, 1998; Narhi et al, 1999; Conway et al, 2000; Ghosh et al, 2013, 2014; Choi 81 et al, 2004). In Huntington disease, any expansion of a polyglutamine stretch in the HTT gene that 82 exceeds 36 repeats has been shown to facilitate HTT aggregation and cause the disease(Cooper et al, 83 1998). Hence, changes in the folding and aggregation of these disease proteins are believed to be at the 84 basis of the observed pathology. In addition, there is increasing evidence that distinct structural 85 variants exist between aggregates of the same disease protein and that these polymorphs are associated 86 with varying degrees of toxicity(Nekooki-Machida et al, 2009; Sun et al, 2015; Cohen et al, 2015; 87 Qiang et al, 2017; Bousset et al, 2013; Heise et al, 2008; Li et al, 2018; Guerrero-Ferreira et al, 2019; 88 Gath et al, 2014; Cohen et al, 2009) . For instance, recent studies with brain samples from human AD 89 patients have shown that variations in the disease type and disease progression correlate with the 90 presence of structurally different aggregates and fibrils(Lu et al, 2013; Rasmussen et al, 2017; Cohen 91 et al, 2015; Qiang et al, 2017). The knowledge that small changes in the structural conformation of 92 these aggregates can have far-reaching effects on the disease manifestation could provide a new 93 avenue for the development of therapeutics. Until now, most research into potential treatments of AD 94 has focused on either reducing the overall amount of aggregating proteins or preventing aggregation, 95 not modifying the aggregation process. 96 In a previous study, our group performed a genetic screen in Caenorhabditis elegans (C.
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