Defective neuritogenesis is a contributing pathogenic mechanism underlying a variety of neurodevelopmental disorders. Single gene mutations in activity-dependent neuroprotective protein (ADNP) are the most frequent among autism spectrum disorders (ASDs) leading to the ADNP syndrome. Previous studies showed that during neuritogenesis, Adnp localizes to the cytoplasm/neurites, and Adnp knockdown inhibits neuritogenesis in culture. Here, we hypothesized that Adnp is localized in the cytoplasm during neurite formation and that this process is mediated by 14-3-3. Indeed, applying the 14-3-3 inhibitor, difopein, blocked Adnp cytoplasmic localization. Furthermore, co-immunoprecipitations showed that Adnp bound 14-3-3 proteins and proteomic analysis identified several potential phosphorylation-dependent Adnp/14-3-3 binding sites. We further discovered that knockdown of Adnp using in utero electroporation of mouse layer 2/3 pyramidal neurons in the somatosensory cortex led to previously unreported changes in neurite formation beginning at P0. Defects were sustained throughout development, the most notable included increased basal dendrite number and axon length. Paralleling the observed morphological aberrations, ex vivo calcium imaging revealed that Adnp deficient neurons had greater and more frequent spontaneous calcium influx in female mice. GRAPHIC, a novel synaptic tracing technology substantiated this finding, revealing increased interhemispheric connectivity between female Adnp deficient layer 2/3 pyramidal neurons. We conclude that Adnp is localized to the cytoplasm by 14-3-3 proteins, where it regulates neurite formation, maturation, and functional cortical connectivity significantly building on our current understanding of Adnp function and the etiology of ADNP syndrome.
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We would like to thank Dr. Peter Baas, Dr. Wen-Jun Gao, Dr. Elias Spiliotis, and Dr. Pat Levitt for their helpful comments, feedback, and fruitful discussions on this manuscript. We further thank Dr. Paschalis Theotokis for retrieval of results from ADNP mice and Dr. Tomomi Shimogori in Riken Center for Brain Science in Japan who provided the GRAPHIC vectors. This work has been supported by a research grant from the NINDS (R01NS096098-01A1 to KT and F31NS113404-01A1 to SMB), the NICHD (F31HD103405-01A1 to SAB), ERA-NET Neuron (ADNPinMED), Drs. Ronith and Armand Stemmer and Arthur Gerbi (French Friends of Tel Aviv University), Anne and Alex Cohen (Canadian Friends of Tel Aviv University) and Holly and Jonathan Strelzik (American Friends of Tel Aviv University) (IG).
NAP (CP201, davunetide) use is under patent protection (US patent nos. US7960334, US8618043, US10912819 and WO2017130190A1) (IG), PCT/IL2020/051010 (IG) and provisional patent applications have been submitted (IG inventor and contributing scientist SS). Davunetide is exclusively licensed to ATED Therapeutics LTD (IG, Co-Founder and Chief Scientific Officer).
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Bennison, S.A., Blazejewski, S.M., Liu, X. et al. The cytoplasmic localization of ADNP through 14-3-3 promotes sex-dependent neuronal morphogenesis, cortical connectivity, and calcium signaling. Mol Psychiatry 28, 1946–1959 (2023). https://doi.org/10.1038/s41380-022-01939-3
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