Illana Gozes

(1) Background: Recently, we showed aberrant nuclear/cytoplasmic boundaries/activity-dependent neuroprotective protein (ADNP) distribution in ADNP-mutated cells. This malformation was corrected upon neuronal differentiation by the ADNP-derived fragment drug candidate NAP (davunetide). Here, we investigated the mechanism of NAP nuclear protection. (2) Methods: CRISPR/Cas9 DNA-editing established N1E-115 neuroblastoma cell lines that express two different green fluorescent proteins (GFPs)—labeled mutated ADNP variants (p.Tyr718* and p.Ser403*). Cells were exposed to NAP conjugated to Cy5, followed by live imaging. Cells were further characterized using quantitative morphology/immunocytochemistry/RNA and protein quantifications. (3) Results: NAP rapidly distributed in the cytoplasm and was also seen in the nucleus. Furthermore, reduced microtubule content was observed in the ADNP-mutated cell lines. In parallel, disrupting microtubules by zinc or nocodazole intoxication mimicked ADNP mutation phenotypes and resulted in aberrant nuclear–cytoplasmic boundaries, which were rapidly corrected by NAP treatment. No NAP effects were noted on ADNP levels. Ketamine, used as a control, was ineffective, but both NAP and ketamine exhibited direct interactions with ADNP, as observed via in silico docking. (4) Conclusions: Through a microtubule-linked mechanism, NAP rapidly localized to the cytoplasmic and nuclear compartments, ameliorating mutated ADNP-related deficiencies. These novel findings explain previously published gene expression results and broaden NAP (davunetide) utilization in research and clinical development.

NAP (NAPVSIPQ, drug candidate name, davunetide) is the neuroprotective fragment of activity‐dependent neuroprotective protein (ADNP). Recent studies identified NAPVSIP as a Src homology 3 (SH3) domain‐ligand association site, responsible for controlling signalling pathways regulating the cytoskeleton. Furthermore, the SIP motif in NAP/ADNP was identified as crucial for direct microtubule end‐binding protein interaction facilitating microtubule dynamics and Tau microtubule interaction, at the microtubule end‐binding protein site EB1 and EB3. Most de novo ADNP mutations reveal heterozygous STOP or frameshift STOP aberrations, driving the autistic/intellectual disability‐related ADNP syndrome. Here, we report for the first time on a de novo missense mutation, resulting in ADNP containing NAPVISPQ E instead of NAPVSIPQ Q , in a child presenting developmental hypotonia, possibly associated with inflammation affecting food intake in early life coupled with fear of peer interactions and suggestive of a novel case of the ADNP syndrome. In silico modelling showed that the mutation Q (polar side chain) to E (negative side chain) affected the electrostatic characteristics of ADNP (reducing, while scattering the electrostatic positive patch). Comparison with the most prevalent pathogenic ADNP mutation, p.Tyr719*, indicated a further reduction in the electrostatic patch. Previously, exogenous NAP partially ameliorated deficits associated with ADNP p.Tyr719* mutations in transfected cells and in CRISPR/Cas9 genome edited cell and mouse models. These findings stress the importance of the NAP sequence in ADNP and as a future putative therapy for the ADNP syndrome.