Toyo-oka, K. | Search bioRxiv

bioRxiv2020

High-Throughput Kinase Inhibitor Screening Reveals Roles for Aurora and Nuak Kinases in Neurite Initiation and Dendritic Branching

Kinases are essential regulators of a variety of cellular signaling processes, including neurite formation - a foundational step in neurodevelopment. Aberrant axonal sprouting and failed regeneration of injured axons are associated with conditions like traumatic injury, neurodegenerative disease, and seizures. Investigating the mechanisms underlying neurite formation will allow for identification of potential therapeutics. We used a kinase inhibitor library to screen 493 kinase inhibitors and observed that 45% impacted neuritogenesis in Neuro2a (N-2a) cells. Based on the screening, we further investigated the roles of Aurora kinases A, B, and C and Nuak kinases 1 and 2. The roles of Aurora and Nuak kinases have not been thoroughly studied in the nervous system. Inhibition or overexpression of Aurora and Nuak kinases in primary cortical neurons resulted in various neuromorphological defects, with Aurora A regulating neurite initiation, Aurora B and C regulating neurite initiation and elongation, all Aurora kinases regulating arborization, and all Nuak kinases regulating neurite initiation and elongation and arborization. Our high-throughput screening and analysis of Aurora and Nuak kinases revealed their functions and may contribute to the identification of therapeutics.

neuroscience↗

bioRxiv2020

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

Neurite formation is the earliest stage of neuronal morphogenesis, where primitive dendrites and the primitive axon emerge from a spherical neuron and begin to elongate. Defective neuritogenesis is a contributing pathogenic mechanism behind a variety of neurodevelopmental disorders. Activity-dependent neuroprotective protein (Adnp) is essential to embryonic and postnatal brain development, and mutations in ADNP are among the most frequent underlying autism spectrum disorder (ASD). We found that knockdown of Adnp in vitro and in vivo in mouse layer 2/3 pyramidal neurons leads to increased neurite initiation and defective neurite elongation, suggesting that Adnp has distinct roles in each. In vivo analysis revealed that deficits begin at P0 and are sustained throughout development, the most notable of which include increased neurite stabilization, disrupted angle of the apical dendrite, increased basal dendrite number, and increased axon length. Because small changes in neuronal morphology can have large-scale effects on neuronal function and connectivity, we performed ex vivo calcium imaging to assess spontaneous function of layer 2/3 pyramidal neurons deficient in Adnp. This revealed that Adnp deficient neurons had a greater spontaneous calcium influx and a higher proportion of cells firing action potentials. Next, we utilized GRAPHIC, a novel synaptic tracing technology, to assess interhemispheric cortical connectivity. We found increased interhemispheric excitatory connectivity between Adnp deficient layer 2/3 pyramidal neurons. Because Adnp is a multifunctional protein with both transcription factor and cytoskeletal activity, we performed localization analysis of Adnp as neurons underwent neurite formation to probe the mechanism of our morphological defects. We found that Adnp is shuttled from the nucleus to the cytoplasm upon differentiation and this shuttling can be blocked via application of a global 14-3-3 inhibitor, difopein. Furthermore, we found that Adnp binds nuclear-cytoplasmic shuttle 14-3-3{varepsilon}. We conclude that Adnp is shuttled from the nucleus to the cytoplasm by 14-3-3{varepsilon}, where it regulates neuronal morphology, maturation, cortical connectivity, and calcium signaling.

neuroscience↗