This Accepted Manuscript has not been copyedited and formatted. The final version may differ from this version. Research Articles: Neurobiology of Disease Impaired mitochondrial fatty acid synthesis leads to neurodegeneration in mice Remya R. Nair1, Henna Koivisto2, Kimmo Jokivarsi2, Ilkka J. Miinalainen3, Kaija J. Autio1, Aki Manninen1,4, Heikki Tanila2, J. Kalervo Hiltunen1 and Alexander J. Kastaniotis1 1Faculty of Biochemistry and Molecular Medicine, University of Oulu, FI-90014 Oulu, Finland 2A. I. Virtanen Institute, University of Eastern Finland, FI-70211 Kuopio, Finland 3Electron Microscopy Core Facility, Biocenter Oulu, University of Oulu, FI-90014 Oulu, Finland 4Virus Core Facility, Biocenter Oulu, University of Oulu, FI-90014 Oulu, Finland DOI: 10.1523/JNEUROSCI.3514-17.2018 Received: 13 December 2017 Revised: 31 August 2018 Accepted: 19 September 2018 Published: 28 September 2018 Author contributions: R.R.N., A.M., H.T., J.K.H., and A.J.K. designed research; R.R.N., H.K., K.J., I.M., K.J.A., H.T., and J.K.H. performed research; R.R.N., H.K., K.J., I.M., K.J.A., A.M., H.T., J.K.H., and A.J.K. analyzed data; R.R.N., H.K., K.J., I.M., K.J.A., A.M., H.T., J.K.H., and A.J.K. wrote the paper; A.M. contributed unpublished reagents/analytic tools. Conflict of Interest: The authors declare no competing financial interests. We are grateful to Dr Raija Soininen for the continuous support at various stages of this project and critical review of this manuscript. The Biocenter Oulu Transgenic Core facility, the Biocenter Oulu EM laboratory and the Biocenter Oulu Virus Core laboratory, which are all part of Biocenter Finland, are acknowledged for the support in generating mice, in the EM analysis and the lentivirus production, respectively. We thank Juha M. Kerätär for providing Mecrtm1c strain for the project. We thank Leena Ollitervo, Anna Sofia Kuusisto, Miriam Martens, Jessica Müller and Belen Sintes Garcia for technical help with histology samples and Anna von Schaewen and Ujjwal Suwal for their work on the quantitation of fluorescence image data. We are grateful to Dr. Randy Woltjer (Oregon Health and Science University) for the reviewing in H&E stained cerebellar sections. The project was funded by the Academy of Finland, the Sigrid Jusélius Foundation, Finnish Cultural Foundation and Biocenter Finland. Correspondence should be addressed to To whom correspondence should be addressed: Faculty of Biochemistry and Molecular Medicine, University of Oulu, P.O. Box 5400, FI-90014 Oulu, Finland; Tel. +358294481196; email address: [email protected] Cite as: J. Neurosci ; 10.1523/JNEUROSCI.3514-17.2018 Alerts: Sign up at www.jneurosci.org/cgi/alerts to receive customized email alerts when the fully formatted version of this article is published. Accepted manuscripts are peer-reviewed but have not been through the copyediting, formatting, or proofreading process. Copyright © 2018 the authors 1 Impaired mitochondrial fatty acid synthesis leads to neurodegeneration in mice 2 MECR and neurodegeneration 3 Remya R. Nair1#, Henna Koivisto2, Kimmo Jokivarsi2, Ilkka J. Miinalainen3, Kaija J. Autio1, Aki 4 Manninen1,4, Heikki Tanila2, J. Kalervo Hiltunen1 & Alexander J. Kastaniotis1* 5 1 Faculty of Biochemistry and Molecular Medicine, University of Oulu, FI-90014 Oulu, Finland; 6 2A. I. Virtanen Institute, University of Eastern Finland, FI-70211 Kuopio, Finland; 3Electron 7 Microscopy Core Facility, Biocenter Oulu, University of Oulu, FI-90014 Oulu, Finland; 4Virus 8 Core Facility, Biocenter Oulu, University of Oulu, FI-90014 Oulu, Finland 9 # Current address: MRC Harwell Institute, Harwell Science and Innovation campus, Didcot, 10 Oxfordshire OX11 0QG, United Kingdom. 11 12 *To whom correspondence should be addressed: Faculty of Biochemistry and Molecular Medicine, 13 University of Oulu, P.O. Box 5400, FI-90014 Oulu, Finland; Tel. +358294481196; email address: 14 [email protected] 15 16 Number of Pages: 45 17 Number of Figures: 15 18 Number of Tables: 3 19 Number of Words: 20 Abstract 229 21 Significance statement 112 22 Introduction 481 23 Discussion: 1497 24 The authors declare no competing financial interests. 1 25 Acknowledgements 26 We are grateful to Dr Raija Soininen for the continuous support at various stages of this project and 27 critical review of this manuscript. The Biocenter Oulu Transgenic Core facility, the Biocenter Oulu 28 EM laboratory and the Biocenter Oulu Virus Core laboratory, which are all part of Biocenter 29 Finland, are acknowledged for the support in generating mice, in the EM analysis and the lentivirus 30 production, respectively. We thank Juha M. Kerätär for providing Mecrtm1c strain for the project. 31 We thank Leena Ollitervo, Anna Sofia Kuusisto, Miriam Martens, Jessica Müller and Belen Sintes 32 Garcia for technical help with histology samples and Anna von Schaewen and Ujjwal Suwal for 33 their work on the quantitation of fluorescence image data. We are grateful to Dr. Randy Woltjer 34 (Oregon Health and Science University) for the reviewing in H&E stained cerebellar sections. The 35 project was funded by the Academy of Finland, the Sigrid Jusélius Foundation, Finnish Cultural 36 Foundation and Biocenter Finland. 37 38 2 39 Abstract 40 There has been a growing interest towards mitochondrial fatty acid synthesis (mtFAS) since the 41 recent discovery of a neurodegenerative human disorder termed MEPAN (mitochondrial enoyl 42 reductase protein associated neurodegeneration), caused by mutations in the mitochondrial enoyl- 43 CoA/ACP (acyl carrier protein) reductase (MECR) carrying out the last step of mtFAS. We show 44 here that MECR protein is highly expressed in mouse Purkinje cells (PCs). To elucidate mtFAS 45 function in neural tissue, we here generated a mouse line with a PC-specific knock-out (KO) of 46 Mecr, leading to inactivation of mtFAS confined to this cell type. Both sexes were studied. The 47 mitochondria in KO PCs displayed abnormal morphology, loss of protein lipoylation and reduced 48 respiratory chain enzymatic activities by the time these mice were 6 months of age, followed by 49 nearly complete loss of PCs by 9 months of age. These animals exhibited balancing difficulties 50 around 7 months of age and ataxic symptoms were evident from 8-9 months of age onwards. Our 51 data shows that impairment of mtFAS results in functional and ultrastructural changes in 52 mitochondria followed by death of PCs, mimicking aspects of the clinical phenotype. This KO 53 mouse represents a new model for impaired mitochondrial lipid metabolism and cerebellar ataxia 54 with a distinct and well-trackable cellular phenotype. This mouse model will allow the future 55 investigation of the feasibility of metabolite supplementation approaches towards the prevention of 56 neurodegeneration due to dysfunctional mtFAS. 57 58 3 59 Significance statement 60 We have recently reported a novel neurodegenerative disorder in humans termed MEPAN 61 (mitochondrial enoyl reductase protein associated neurodegeneration) (Heimer et al. 2016). The 62 cause of neuron degeneration in MEPAN patients is the dysfunction of the highly conserved 63 mitochondrial fatty acid synthesis (mtFAS) pathway due to mutations in MECR, encoding 64 mitochondrial 2-enoyl-ACP reductase. 65 The report presented here describes the analysis of the first mouse model suffering from mtFAS- 66 defect-induced neurodegenerative changes due to specific disruption of the Mecr gene in PCs. Our 67 work sheds a light on the mechanisms of neurodegeneration caused by mtFAS deficiency and 68 provides a test bed for future treatment approaches. 69 70 4 71 Introduction 72 Mitochondria are essential multipurpose organelles of intermediate and energy metabolism. 73 Especially the function of organs with high energy demands such as brain depends on flawless 74 mitochondrial operation for their optimal performance. Mitochondrial defects have been reported as 75 a cause for many neurodegenerative diseases (Johri and Beal, 2012), e.g. Alzheimer’s disease 76 (Moreira et al., 2010), Parkinson’s disease (Winklhofer and Haass, 2010), Huntington‘s disease 77 (Quintanilla and Johnson, 2009), Friedreich ataxia (Delatycki et al., 2000), as well as other 78 spinocerebellar ataxias (Zeviani et al., 2012). 79 As a reflection of the prokaryotic ancestry of the endosymbiotic organelle, mitochondria harbor a 80 fatty acid synthesis (mtFAS) pathway that closely follows the prokaryotic model, where each step 81 in the process is carried out by separate polypeptides. The enzymes in mammals involved with this 82 pathway have been identified (Hiltunen et al., 2010; Monteuuis et al., 2017). Octanoic acid is a 83 confirmed product of mtFAS and required as a precursor for endogenous synthesis of lipoic acid 84 (LA). In Saccharomyces cerevisiae, the inactivation of any of the enzymes of mtFAS leads to 85 respiratory defects, lack of cytochromes due to disturbed mitochondrial translation and respiratory 86 complex assembly, rudimentary mitochondria and loss of protein lipoylation (Kastaniotis et al., 87 2004; Hiltunen et al., 2009; Kursu et al., 2013). Evidence generated in yeast and mammalian 88 models indicates that, in addition to providing the LA precursor, mtFAS products play important 89 roles in mitochondrial biogenesis, affecting heme biosynthesis, regulating respiratory chain 90 assembly (Kursu et al. 2013, Zhu et al., 2017, Van Vranken et al. 2018) and participating in iron- 91 sulfur (Fe-S) cluster biosynthesis (Van Vranken et al., 2016, Cory et al., 2017). Two acylated acyl 92 carrier proteins (ACPs) of mtFAS, SDAP-α and SDAP-β, serve as structural components in the 93 mammalian complex I, (Caroll et al., 2013; Zhu et al., 2016). Acyl-ACPs have also been reported 94 to be required for the maturation of succinate dehydrogenase (Angerer et al., 2015). 5 95 MECR (mitochondrial enoyl-CoA reductase) catalyzes the NADPH-dependent reduction of enoyl 96 acyl carrier protein (ACP) to saturated acyl-ACP (Miinalainen et al., 2003). This is the last enzyme 97 of mtFAS pathway and essential for embryonic development in mouse (Nair et al., 2017).