ORIGINAL ARTICLE The NAP motif of activity-dependent neuroprotective protein (ADNP) regulates dendritic spines through microtubule end binding proteins S Oz 1 , O Kapitansky 1 , Y Ivashco-Pachima 1 , A Malishkevich 1 , E Giladi 1 , N Skalka 2 , R Rosin-Arbesfeld 2 , L Mittelman 3 , O Segev 4,5 , JA Hirsch 4 and I Gozes 1,5 The NAP motif of activity-dependent neuroprotective protein (ADNP) enhanced memory scores in patients suffering from mild cognitive impairment and protected activities of daily living in schizophrenia patients, while fortifying microtubule (MT)-dependent axonal transport, in mice and flies. The question is how does NAP fortify MTs? Our sequence analysis identified the MT end-binding protein (EB1)-interacting motif SxIP (SIP, Ser-Ile-Pro) in ADNP/NAP and showed specific SxIP binding sites in all members of the EB protein family (EB1–3). Others found that EB1 enhancement of neurite outgrowth is attenuated by EB2, while EB3 interacts with postsynaptic density protein 95 (PSD-95) to modulate dendritic plasticity. Here, NAP increased PSD-95 expression in dendritic spines, which was inhibited by EB3 silencing. EB1 or EB3, but not EB2 silencing inhibited NAP-mediated cell protection, which reflected NAP binding specificity. NAPVSKIPQ (SxIP = SKIP), but not NAPVAAAAQ mimicked NAP activity. ADNP, essential for neuronal differentiation and brain formation in mouse, a member of the SWI/SNF chromatin remodeling complex and a major protein mutated in autism and deregulated in schizophrenia in men, showed similar EB interactions, which were enhanced by NAP treatment. The newly identified shared MT target of NAP/ADNP is directly implicated in synaptic plasticity, explaining the breadth and efficiency of neuroprotective/neurotrophic capacities. Molecular Psychiatry (2014) 19, 1115–1124; doi:10.1038/mp.2014.97; published online 2 September 2014 INTRODUCTION Activity-dependent neuroprotective protein (ADNP), 1,2 mutated in ≥0.17% of autism spectrum disorder cases (one of the most frequent autism spectrum disorder-associated genes known to- date), 3 deregulated in schizophrenia, 4,5 predicting the onset of frontotemporal dementia 6 and identified by complete proteomics as the only protein significantly decreasing in Alzheimer's disease (AD) patient serum (compared with matched controls), 7 takes part in chromatin remodeling as a component of the essential SWItch/ sucrose nonfermentable (SWI/SNF) complex. 8,9 While regulating 4400 genes during embryonic development, 10–12 ADNP is not confined to the cell nucleus, 13–15 and interacts with key regulators of the autophagy process, linked to cytoplasmic microtubules (MTs). 5 MTs, key cytoskeletal filaments found in all eukaryotic cells, provide structural support, segregation of chromosomes and intracellular trafficking. Neuronal MTs are essential for axonal transport and synaptic transmission. 16,17 MT +end tracking proteins (+TIPs), including end-binding proteins (EBs), have a role in axonal outgrowth and interact with MT-associated proteins. 18 Of the three mammalian EB family members (EB1, EB2 and EB3), 19 EB3 is preferentially expressed in the brain and is used to track MT dynamics. 20,21 Although present in axons and growth cones, EB3 is predominantly localized within the dendritic compartment and has been characterized as a major regulator of dendritic spine plasticity by influencing actin dynamics within the spine. 22–24 EB1, sharing 54% identity with EB3, has been implicated in axonal transport. 23 EB1/EB3, but not EB2 promote persistent MT growth by suppressing catastrophes, 25 increase neurite number in pheochromocytoma (PC12) cells, while EB2 blocks neurite elongation, 26 attenuating EB1 activity. 27 EB2 does not form dimers with either EB1 or EB3, while EB1/3 dimerization (but not necessarily heterodimerization) is a prerequisite for +TIP partner binding. 28,29 The short peptide motif, Ser-x-Ile-Pro (SxIP) is used by numerous +TIPs for localization to MT tips in an EB1-dependent manner, 30 and EB3 has also been suggested to interact with this motif, for example in plexin. 31 EB3 binds directly to p140Cap (Src kinase signaling inhibitor1, SRCN1, containing an SIP motif). Localized at the post- synaptic density, 24 p140Cap interacts with synaptosomal-associated protein of 25 kDa (SNAP-25) to regulate spine formation. 32 Structure activity mapping of ADNP fragments identified NAP (NAPVSIPQ), containing a SIP motif, as a neuroprotective peptide. 1 In AD models, NAP protected against MT-associated tau pathology and cognitive decline. 33–35 NAP treatment regulated the MT cellular pool, affecting MT dynamics/stability in neuronal and glial cells, which was associated with dynamic MT invasion into the growth cone and enlisting of tau to the MT shaft under stress conditions 36 as well as protecting against MT severing in the face 1 Department of Human Molecular Genetics and Biochemistry, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; 2 Department of Anatomy, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; 3 Department of Interdepartmental Services, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; 4 Department of Biochemistry and Molecular Biology, George Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel and 5 Sagol School of Neuroscience and Adams Super Center for Brain Studies, Tel Aviv University, Tel Aviv, Israel. Correspondence: Professor I Gozes, Adams Super Center for Brain Studies, The Edersheim Levie-Gitter fMRI Institute, The Lily and Avraham Gildor Chair for the Investigation of Growth Factors, The Dr. Diana and Zelman Elton (Elbaum) Laboratory for Molecular Neuroendocrinology, Sagol School of Neuroscience, Department of Human Molecular Genetics and Biochemistry, Sackler Faculty of Medicine, Tel Aviv University, Einstein Street, Tel Aviv 69978, Israel. E-mail: igozes@post.tau.ac.il Received 5 March 2014; revised 14 June 2014; accepted 8 July 2014; published online 2 September 2014 Molecular Psychiatry (2014) 19, 1115 – 1124 © 2014 Macmillan Publishers Limited All rights reserved 1359-4184/14 www.nature.com/mp