Research Articles: Cellular/Molecular BRCA1–BARD1 regulates axon regeneration in concert with the Gqα–DAG signaling network https://doi.org/10.1523/JNEUROSCI.1806-20.2021 Cite as: J. Neurosci 2021; 10.1523/JNEUROSCI.1806-20.2021 Received: 13 July 2020 Revised: 20 January 2021 Accepted: 5 February 2021 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 © 2021 Sakai et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. Sakai et al., 1 1 BRCA1–BARD1 regulates axon regeneration in 2 concert with the GqD–DAG signaling network 3 4 Abbreviated Title: BRCA1–BARD1 regulates axon regeneration 5 6 Yoshiki Sakai, Hiroshi Hanafusa, Tatsuhiro Shimizu, Strahil Iv. 7 Pastuhov, Naoki Hisamoto, and Kunihiro Matsumoto 8 9 Division of Biological Science, Graduate School of Science, Nagoya University, 10 Chikusa-ku, Nagoya 464-8602, Japan 11 12 To whom correspondence should be addressed: 13 Kunihiro Matsumoto and Naoki Hisamoto 14 Division of Biological Science, Graduate School of Science, 15 Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan. 16 E-mail address: [email protected] (K. M.) 17 [email protected] (N. H.) 18 19 Y.S., H.H., N.H., and K.M. designed the experiments and analyzed data. Y.S., 20 N.H., and K.M. wrote the manuscript. Y.S., H.H., T.S., and S.P. performed 21 experiments. 22 23 Number of Pages: 37 pages, without figures and tables 24 Number of Figures and Tables: 11 figures and 2 tables 25 Number of words for Abstract, Introduction and Discussion: 26 198, 762, and 1022 words, respectively. 27 1 Sakai et al., 2 28 Acknowledgments 29 We thank Dr Ikue Mori, Caenorhabditis Genetic Center (CGC), National 30 Bio-Resource Project and C. elegans Knockout Consortium for materials. Some 31 strains were provided by the CGC, which is funded by NIH Office of Research 32 Infrastructure Programs (P40 OD10440). This work was supported by grants 33 from the Ministry of Education, Culture and Science of Japan (to S.P., N.H., and 34 K.M.) and the Project for Elucidating and Controlling Mechanisms of Aging and 35 Longevity from Japan Agency for Medical Research and Development, AMED, 36 under Grant Number JP20gm5010001 (to N.H.). T.S. was supported by a Japan 37 Society for the Promotion of Science Research Fellowship. 38 39 The authors declare no competing financial interests. 2 Sakai et al., 3 40 Abstract 41 42 The breast cancer susceptibility protein BRCA1 and its partner BARD1 43 form an E3 ubiquitin ligase complex that acts as a tumor suppressor in 44 mitotic cells. However, the roles of BRCA1–BARD1 in post-mitotic cells, 45 such as neurons, remain poorly defined. Here we report that BRC-1 and 46 BRD-1, the Caenorhabditis elegans orthologs of BRCA1 and BARD1, are 47 required for adult-specific axon regeneration, which is positively regulated 48 by the EGL-30 GqD–diacylglycerol (DAG) signaling pathway. This pathway 49 is down-regulated by DAG kinase (DGK), which converts DAG to 50 phosphatidic acid. We demonstrate that inactivation of DGK-3 suppresses 51 the brc-1 brd-1 defect in axon regeneration, suggesting that BRC-1–BRD-1 52 inhibits DGK-3 function. Indeed, we show that BRC-1–BRD-1 53 poly-ubiquitylates DGK-3 in a manner dependent on its E3 ligase activity, 54 causing DGK-3 degradation. Furthermore, we find that axon injury causes 55 the translocation of BRC-1 from the nucleus to the cytoplasm, where 56 DGK-3 is localized. These results suggest that the BRC-1–BRD-1 complex 57 regulates axon regeneration in concert with the GqD–DAG signaling 58 network. Thus, this study describes a new role for breast cancer proteins 59 in fully differentiated neurons and the molecular mechanism underlying 60 the regulation of axon regeneration in response to nerve injury. 3 Sakai et al., 4 61 Significance Statement 62 63 BRCA1–BARD1 is an E3 ubiquitin ligase complex acting as a tumor 64 suppressor in mitotic cells. The roles of BRCA1–BARD1 in post-mitotic 65 cells, such as neurons, remain poorly defined. We show here that C. 66 elegans BRC-1/BRCA1 and BRD-1/BARD1 are required for adult-specific 67 axon regeneration, a process that requires high diacylglycerol (DAG) 68 levels in injured neurons. The DAG kinase DGK-3 inhibits axon 69 regeneration by reducing DAG levels. We find that BRC-1–BRD-1 70 poly-ubiquitylates and degrades DGK-3, thereby keeping DAG levels 71 elevated and promoting axon regeneration. Furthermore, we demonstrate 72 that axon injury causes the translocation of BRC-1 from the nucleus to the 73 cytoplasm, where DGK-3 is localized. Thus, this study describes a new 74 role for BRCA1–BARD1 in fully-differentiated neurons. 4 Sakai et al., 5 75 Introduction 76 77 Genetic susceptibility to breast cancer is caused largely by mutations in the 78 BRCA1 and BRCA2 genes (Fackenthal and Olopade, 2007). These regulate a 79 wide range of biological processes, including DNA damage repair by 80 homologous recombination, gene silencing, cell cycle checkpoint, and 81 centrosome duplication, all of which are relevant to the regulation of cell 82 proliferation (Scully and Livingston, 2000; Moynahan and Jasin, 2010; Zhu et al., 83 2011; Li and Greenberg, 2012). BRCA1 exists primarily in a heterodimeric 84 complex with the BRCA1-associated RING domain protein 1 (BARD1) (Wu et al., 85 1996). It has been shown that this BRCA1–BARD1 complex possesses 86 E3-ubiquitin (Ub) ligase activity, and this activity can be disrupted by 87 cancer-derived mutations, underscoring the critical role of this enzymatic 88 function in suppressing tumorigenesis (Baer and Ludwig, 2002). To date, 89 intensive efforts have been devoted to understanding the tumor-suppressive 90 functions of BRCA1–BARD1 and BRCA2 in mitotic cells. However, their roles in 91 post-mitotic cells, such as neurons, remain poorly understood at the molecular 92 level. 93 Neurons are one type of post-mitotic cell, specialized for transmitting 94 information over long distances through axons. Although axons can be damaged 95 by various internal and external stresses, neurons have a conserved system of 96 regenerating axons post-injury, and failure of this system can cause sensory and 97 motor paralysis. This axon’s regenerative capacity is controlled by intrinsic 98 neuronal signaling pathways (He and Jin, 2016). Upon axon injury, Ca2+ and 99 cyclic adenosine monophosphate (cAMP) levels rise in severed neurons, which 100 drives various signaling pathways (Ghosh-Roy et al., 2010; Mar et al., 2014). For 101 instance, cAMP elevation activates cAMP-dependent protein kinase (PKA), 5 Sakai et al., 6 102 which promotes axonal regeneration through phosphorylation of various 103 downstream targets (Neumann et al., 2002; Bhatt et al., 2004; Gao et al., 2004). 104 However, the intrinsic signaling pathways that regulate regeneration in the adult 105 nervous system have yet to be fully elucidated. 106 The nematode Caenorhabditis elegans has recently emerged as an 107 attractive model to dissect the mechanisms of axon regeneration in the mature 108 nervous system (Yanik et al., 2004). Recent studies in C. elegans have identified 109 many signaling molecules that promote or inhibit axon regeneration (Chen et al., 110 2011; Nix et al., 2014; Kim et al., 2018). We have previously demonstrated that 111 the evolutionarily conserved JNK MAP kinase (MAPK) pathway, consisting of 112 MLK-1 MAPKKK–MEK-1 MAPKK–KGB-1 JNK, drives the initiation of axon 113 regeneration (Nix et al., 2011). Two different protein kinases act as MAP4Ks for 114 MLK-1 in a manner specific for different life stages. The Ste20-related kinase 115 MAX-2 phosphorylates and activates MLK-1 mainly at the L4 stage to promote 116 axon regeneration (Pastuhov et al., 2016). On the other hand, the protein kinase 117 C (PKC) ortholog TPA-1 can activate MLK-1 at the young adult stage, but not at 118 the L4 stage (Pastuhov et al., 2012). The GqD protein EGL-30 acts as a 119 component upstream of TPA-1. EGL-30 activates the phospholipase CE (PLCE) 120 EGL-8, which in turn generates diacylglycerol (DAG), an activator of TPA-1, from 121 phosphatidylinositol bisphosphate (Lackner et al., 1999). DAG kinases (DGKs) 122 antagonize the EGL-30 pathway by converting DAG to phosphatidic acid (PA) 123 (Miller et al., 1999). 124 We have recently found that BRC-2, the C. elegans ortholog of BRCA2, acts 125 as a regulator of axon regeneration (Shimizu et al., 2018). C. elegans also has 126 two genes, brc-1 and brd-1, which encode orthologs of mammalian BRCA1 and 127 BARD1, respectively (Figure 1A) (Boulton et al., 2004). BRC-1 and BRD-1 share 128 extensive sequence and domain conservation with their mammalian 6 Sakai et al., 7 129 counterparts, including RING and BRCT domains. Similar to mammalian 130 BRCA1–BARD1, BRC-1 heterodimerizes with BRD-1 to form a complex having 131 E3-Ub ligase activity (Polanowska et al., 2006). BRC-1–BRD-1 is involved in 132 DNA repair at sites damaged by ionizing radiation. Our finding that BRC-2 is 133 implicated in axon regeneration prompted us to explore the possibility that 134 BRC-1 and BRD-1 also participate in this process.