14-3-3 shuttles Activity-dependent neuroprotective protein to the cytoplasm to promote appropriate neuronal morphogenesis, cortical connectivity and calcium signaling

Abstract

30Neurite formation is the earliest stage of neuronal morphogenesis, where primitive 31 dendrites and the primitive axon emerge from a spherical neuron and begin to elongate. Defective 32 neuritogenesis is a contributing pathogenic mechanism behind a variety of neurodevelopmental 33 disorders. Activity-dependent neuroprotective protein (Adnp) is essential to embryonic and 34 postnatal brain development, and mutations in ADNP are among the most frequent underlying 35 autism spectrum disorder (ASD). We found that knockdown of Adnp in vitro and in vivo in mouse 36 layer 2/3 pyramidal neurons leads to increased neurite initiation and defective neurite elongation, 37suggesting that Adnp has distinct roles in each. In vivo analysis revealed that deficits begin at P0 38 and are sustained throughout development, the most notable of which include increased neurite 39 stabilization, disrupted angle of the apical dendrite, increased basal dendrite number, and increased 40 axon length. Because small changes in neuronal morphology can have large-scale effects on 41 neuronal function and connectivity, we performed ex vivo calcium imaging to assess spontaneous 42 function of layer 2/3 pyramidal neurons deficient in Adnp. This revealed that Adnp deficient 43 neurons had a greater spontaneous calcium influx and a higher proportion of cells firing action 44 potentials. Next, we utilized GRAPHIC, a novel synaptic tracing technology, to assess 45 interhemispheric cortical connectivity. We found increased interhemispheric excitatory 46 connectivity between Adnp deficient layer 2/3 pyramidal neurons. Because Adnp is a 47 57Neuritogenesis is a fundamental step of cortical development essential for establishing 58 correct neuronal morphology, connectivity, and function (1, 2). Immature neurons have a spherical 59 morphology that upon maturation develops to extend a single axon and multiple dendrites (3)(4)(5)(6). 60This complex feat is driven by neuronal polarization which causes an initial break in symmetry 61 within an immature neuron, seamlessly followed by the two stages of neuritogenesis: neurite 62 initiation followed by neurite elongation (4, 7, 8). Following neuronal polarization, actin 63 aggregates form the sites of primitive neurites, precursors to the axon and dendrites (4, 9, 10). At 64 these sites, actin rich filopodia and lamellipodia rapidly extend and retract before being stabilized 65 by microtubule invasion. Microtubules then drive neurite elongation as primitive neurites are lead 66 to their appropriate destinations where they differentiate into a single axon and multiple dendrites 67 (8,(11)(12)(13)(14). It is of current interest which proteins are key players in this early developmental 68 process, particularly those which are causatively mutated in neurodevelopmental disorders. 69Following neurite formation, dendrites mature and develop intricately branched, complex 70 structures that form synaptic contacts with neighboring neurons that, when formed correctly, lead 71 to functional connectivity (1, 2, 15). The sit...