Cellular and molecular dissection of neuromorphological deficits underlying ADNP syndrome and autism spectrum disorder

Kazuhito Toyooka

Activity‐dependent neuroprotective protein (ADNP) is shuttled to the cytoplasm to promote neuronal morphogenesis and functional cortical connectivity. Defective neuritogenesis is a contributing pathogenic mechanism behind a variety of neurodevelopmental disorders. Mutations in ADNP are among the most frequent underlying autism spectrum disorder. Adnp has a suggested role in neurite formation, but if defective neuritogenesis underlies the pathology of ADNP syndrome has yet to be explored. We found that Adnp knockdown using in utero electroporation of mouse layer 2/3 pyramidal neurons in the somatosensory cortex leads to neurite formation defects beginning at P0. We used ex vivo live imaging and found severe flaws in cellular dynamics in Adnp deficient neurons. These include failure of neurite retraction, slow growth speed, increased neurite stabilization, and intracellular swellings. These defects are sustained throughout development. At P15, we noted increased basal dendrite number, axon length, and interhemispheric axon innervation. Slight changes to neurite morphology can lead to significant scale changes in brain connectivity and function, which can have behavioral consequences. To assess potential changes to neuronal function, we performed ex vivo calcium imaging which revealed that Adnp deficient neurons were hyperexcitable. To further probe changes to neuronal activity, we utilized GRAPHIC, a novel synaptic tracing technology, to assess cortico‐cortical connectivity. We found increased interhemispheric connectivity between Adnp deficient layer 2/3 pyramidal neurons. To probe the molecular mechanism of changes to neuronal morphology, we performed a localization analysis of Adnp. We found that Adnp is shuttled from the nucleus to the cytoplasm upon neurite formation, and a 14‐3‐3 inhibitor, difopein, can block this shuttling. We also found that Adnp binds nuclear‐cytoplasmic shuttle 14‐3‐3ε. We conclude that Adnp is shuttled to the cytoplasm by 14-3-3ε, where it regulates neurite formation, maturation, and functional cortical connectivity upon neuritogenesis.


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