NAP (Davunetide): The Neuroprotective ADNP Drug Candidate Penetrates Cell Nuclei Explaining Pleiotropic Mechanisms

Maram Ganaiem; Nina D Gildor; Shula Shazman; Gidon Karmon; Yanina Ivashko-Pachima; Illana Gozes

doi:10.3390/cells12182251

NAP (Davunetide): The Neuroprotective ADNP Drug Candidate Penetrates Cell Nuclei Explaining Pleiotropic Mechanisms

Cells. 2023 Sep 11;12(18):2251. doi: 10.3390/cells12182251.

Authors

Maram Ganaiem¹, Nina D Gildor¹, Shula Shazman^{2

3}, Gidon Karmon¹, Yanina Ivashko-Pachima¹, Illana Gozes¹

Affiliations

¹ The Elton Laboratory for Molecular Neuroendocrinology, Department of Human Molecular Genetics and Biochemistry, Faculty of Medicine, Sagol School of Neuroscience and Adams Super Center for Brain Studies, Tel Aviv University, Tel Aviv 6997801, Israel.
² Department of Mathematics and Computer Science, The Open University of Israel, Raanana 4353107, Israel.
³ Department of Information Systems, The Max Stern Yezreel Valley College, Yezreel Valley, Afula 1930600, Israel.

Abstract

(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.

Keywords: ADNP syndrome; CRISPR/Cas9; NAP (davunetide); activity-dependent neuroprotective protein (ADNP); green fluorescent protein (GFP); immunocytochemistry; ketamine; live cell imaging; reverse transcription polymerase chain reaction (RT-PCR).

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Cell Nucleus / metabolism
Ketamine*
Neuroprotective Agents* / pharmacology
Neuroprotective Agents* / therapeutic use
tau Proteins / metabolism

Substances

davunetide
Neuroprotective Agents
Ketamine
tau Proteins

Grants and funding

This study was partially supported by grants and donations for research conducted at the laboratory of I.G. from the European Research Area Network (ERA-NET) Neuron ADNPinMED, Ronith and Armand Stemmer as well as Arthur Gerbi (French Friends of Tel Aviv University), Holly and Jonathan Strelzik (American Friends of Tel Aviv University), and Anne and Alex Cohen (Canadian Friends of Tel Aviv University). I.G. is Director of the Elton Laboratory for Molecular Neuroendocrinology and the former first incumbent of the Lily and Avraham Gildor Chair for the Investigation of Growth Factors. This work partially fulfils the Ph.D. thesis requirements of M.G. at the Miriam and Sheldon G. Adelson Graduate School, Faculty of Medicine, Tel Aviv University, who was also generously supported by a Neubauer Family Foundation Student Scholarship.