Research Articles: Cellular/Molecular Golgi-Dependent Copper Homeostasis Sustains Synaptic Development and Mitochondrial Content https://doi.org/10.1523/JNEUROSCI.1284-20.2020 Cite as: J. Neurosci 2020; 10.1523/JNEUROSCI.1284-20.2020 Received: 22 May 2020 Revised: 2 October 2020 Accepted: 9 November 2020 This Early Release article has been peer-reviewed and accepted, but has not been through the composition and copyediting processes. The final version may differ slightly in style or formatting and will contain links to any extended data. Alerts: Sign up at www.jneurosci.org/alerts to receive customized email alerts when the fully formatted version of this article is published. Copyright © 2020 the authors 1 2 Golgi-Dependent Copper Homeostasis Sustains Synaptic 3 Development and Mitochondrial Content. 4 5 Cortnie Hartwig*1, Gretchen Macías Méndez*2, Shatabdi Bhattacharjee*3, Alysia D. Vrailas- 6 Mortimer*4, Stephanie A. Zlatic1, Amanda A. H. Freeman5, Avanti Gokhale1, Mafalda Concilli6, 7 Erica Werner1, Christie Sapp Savas1, Samantha Rudin-Rush1, Laura Palmer1, Nicole Shearing1, 8 Lindsey Margewich4, Jacob McArthy4, Savanah Taylor4, Blaine Roberts7, Vladimir Lupashin8, 9 Roman S. Polishchuk6, Daniel N. Cox3, Ramon A. Jorquera2,9#, Victor Faundez1# 10 11 Abbreviated title: Copper Homeostasis Sustains Synaptic Mitochondria 12 13 14 Departments of Cell Biology1 and Biochemistry7, and the Center for the Study of Human Health5 USA, 15 30322, Emory University, Atlanta, Georgia, USA, 30322. Neuroscience Department, Universidad Central 16 del Caribe, Bayamon, Puerto Rico2. Neuroscience Institute, Center for Behavioral Neuroscience, Georgia 17 State University, Atlanta, Georgia 303023. School of Biological Sciences Illinois State University, Normal, 18 IL 6179014. Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli 80078, Italy6. Department of 19 Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, AK, 722058. Institute 20 of Biomedical Sciences, Santiago, Chile9. 21 22 #Address Correspondence to RJ ([email protected]) and VF ([email protected]) 23 * These authors contributed equally 24 25 Number of pages: 36 26 Number of figures: 10 plus 2 extended data 27 Number of words for abstract, introduction, and discussion: 153, 666, and 1490 28 29 Acknowledgements 30 This work was supported by grants from the National Institutes of Health 1RF1AG060285 to VF, 31 R01NS108778 to RJ, R15AR070505 to AVM, Telethon TIGEM-CBDM9 to RP, R01NS086082 to 32 DNC, R01GM083144 to VL, and 5K12GM000680-19 to CH. We are indebted to the Faundez lab 33 members for their comments. Stocks obtained from the Bloomington Drosophila Stock Center 34 (NIH P40OD018537) were used in this study. 35 36 Conflict of Interests 37 There are no interests to declare by all authors 2 38 Abstract 39 40 Rare genetic diseases preponderantly affect the nervous system causing 41 neurodegeneration to neurodevelopmental disorders. This is the case for both Menkes and 42 Wilson disease, arising from mutations in ATP7A and ATP7B, respectively. The ATP7A and 43 ATP7B proteins localize to the Golgi and regulate copper homeostasis. We demonstrate genetic 44 and biochemical interactions between ATP7 paralogs with the Conserved Oligomeric Golgi 45 complex, or COG complex, a Golgi apparatus vesicular tether. Disruption of Drosophila copper 46 homeostasis by ATP7 tissue-specific transgenic expression caused alterations in epidermis, 47 aminergic, sensory, and motor neurons. Prominent among neuronal phenotypes was a 48 decreased mitochondrial content at synapses, a phenotype that paralleled with alterations of 49 synaptic morphology, transmission, and plasticity. These neuronal and synaptic phenotypes 50 caused by transgenic expression of ATP7 were rescued by downregulation of COG complex 51 subunits. We conclude that the integrity of Golgi-dependent copper homeostasis mechanisms, 52 requiring ATP7 and COG, are necessary to maintain mitochondria functional integrity and 53 localization to synapses. 54 55 56 57 Significance Statement 58 59 Menkes and Wilson disease affect copper homeostasis and characteristically afflict the 60 nervous system. However, their molecular neuropathology mechanisms remain mostly 61 unexplored. We demonstrate that copper homeostasis in neurons is maintained by two factors 62 that localize to the Golgi apparatus, ATP7 and the COG complex. Disruption of these 63 mechanisms affect mitochondrial function and localization to synapses as well as 64 neurotransmission and synaptic plasticity. These findings suggest communication between the 65 Golgi apparatus and mitochondria through homeostatically controlled cellular copper levels and 66 copper-dependent enzymatic activities in both organelles. 67 68 69 70 71 72 73 74 75 2 3 76 77 Introduction 78 Rare diseases include genetic maladies that disproportionately affect the nervous 79 system with phenotypes ranging from neurodegeneration to behavioral impairments (Sanders 80 et al., 2019; Lee et al., 2020). The vast collection of mutated human genes and neurological 81 symptomatology offers a wide field for the discovery of novel cellular mechanisms necessary 82 for neuronal function. Here we focus on rare neurological diseases affecting ATP7A, ATP7B, and 83 the COG complex subunit genes, whose products localize to the Golgi complex at steady state 84 (Kaler, 2011; Polishchuk and Lutsenko, 2013; Climer et al., 2015). We report that these ATP7 85 paralogs and the COG complex converge to maintain copper homeostasis and, unexpectedly, 86 mitochondrial distribution in neurons. 87 Mutations in the ATP7A cause Menkes disease, a systemic copper-depletion affliction 88 caused by intestinal copper malabsorption. Menkes is characterized by childhood systemic and 89 neurological phenotypes. Menkes’ brain phenotypes result from this systemic copper depletion 90 and span from intellectual disability to widespread gray matter neurodegeneration. (Menkes, 91 1988, 1999; Kaler, 2011; Zlatic et al., 2015; Hartwig et al., 2019; Guthrie et al., 2020). These 92 Menkes disease phenotype are caused in part by defects in the activity of copper-dependent 93 enzymes that either traverse the Golgi complex or localize to mitochondria (Lutsenko et al., 94 2007; Zlatic et al., 2015). In contrast, with the systemic depletion of copper observed in 95 Menkes patients, cell-autonomous ATP7A gene defects cause a cellular overload of copper due 96 to impaired copper efflux from cells (Camakaris et al., 1980; Morgan et al., 2019). This ATP7A 97 cell-autonomous copper phenotype can be recapitulated systemically in Wilson disease. Wilson 98 disease is caused by mutations in the ATP7A paralogue, ATP7B, a copper transporter gene 99 expressed preferentially in the liver. Wilson disease leads to organismal copper overload due to 100 defective copper excretion by the liver. This systemic copper overload causes liver damage, 101 psychiatric symptoms, and lenticular neurodegeneration at late stages of disease (Lutsenko et 102 al., 2007; Kaler, 2011). 103 ATP7A and ATP7B are P-type ATPases residing in the Golgi complex at steady state 104 where they contribute to cellular copper homeostasis. These ATPases sequester copper into the 105 Golgi lumen away from the cytoplasm by a mechanism that requires ATP hydrolysis to drive 106 copper transport. Furthermore, after a copper challenge, these ATPases are translocated to the 107 plasma membrane where they pump copper outside of the cell (Petris et al., 1996; Kaler, 2011; 108 Polishchuk and Lutsenko, 2013; Polishchuk et al., 2014). In turn, extracellular copper is taken up 109 by the activity of a plasma membrane copper transporter CTR1 (Kuo et al., 2001; Lutsenko et 110 al., 2007; Kaler, 2011). The expression of ATP7A and CTR1 is controlled by the activity of the 111 COG complex, which tethers vesicles to the Golgi complex (Comstra et al., 2017). The COG 112 complex is an octamer localized to the Golgi apparatus and required for incoming vesicle fusion 113 with the Golgi. Elimination of any one of the eight COG subunits leads to Golgi fragmentation 3 4 114 due to destabilization and degradation of the whole octamer (Ungar et al., 2002; Zolov and 115 Lupashin, 2005; Climer et al., 2015; Bailey Blackburn et al., 2016). Human mutations in seven of 116 the eight COG subunits cause a group of diseases collectively known as congenital disorder of 117 glycosylation type II (Wu et al., 2004; Kranz et al., 2007; Foulquier, 2009; Climer et al., 2015). 118 These rare disorders are characterized, in part, by neurodevelopmental pathology and 119 behavioral phenotypes which are loosely similar to neurological phenotypes in Menkes disease. 120 However, whether congenital disorder of glycosylation type II neurological phenotypes may be 121 linked to copper metabolism remains unanswered (Climer et al., 2018). 122 The convergence of neurological, neurodevelopmental, and psychiatric phenotypes in 123 these three diseases with proteins localizing to the Golgi complex, point to fundamental, 124 neuronal mechanisms necessary to maintain cellular copper homeostasis. These phenotypes 125 raise the question: how are the activities of copper transporters and the COG complex 126 coordinated to maintain copper homeostasis in cells, tissues, and organisms? Here we 127 demonstrate that ATP7 paralogues and the COG complex, both Golgi localized machineries, 128 control synapse development, neurotransmission, and the subcellular localization of 129 mitochondria at synapses. 130 4 5 131 MATERIAL AND METHODS 132 133