Activity-dependent neuroprotective protein (ADNP), vital for brain formation and cognitive function, is mutated in autism and linked to neurodegenerative/psychiatric diseases. An eight-amino-acid peptide snippet of ADNP, NAP (NAPVSIPQ), identified as a smallest active fragment, includes the SxIP microtubule (MT) end-binding protein (EB) association motif, and enhances ADNP-EB3 interaction. Depletion of EB1 or EB3 abolishes NAP protection against zinc intoxication. Furthermore, NAP enhances Tau-MT interaction, and Tau regulates the localization and function of EB1 and EB3 in developing neuronal cells. Here, we asked how NAP (ADNP) enhances Tau-MT interactions and whether this is mediated by EBs. We showed, for we believe the first time, that NAP augmented endogenous EB1 comet density in the N1E-115 neuroblastoma neuronal model. This finding was substantiated by cell transfection with fluorescent EB1 and live cell imaging. NAP increased comet amounts, length and speed. At the molecular level, NAP enhanced EB3 homodimer formation, while decreasing EB1-EB3 heterodimer content and driving EB1- and EB3-Tau interactions (dramatic 20-fold increases), leading to recruitment of EB1/EB3 and Tau to MTs under zinc intoxication. Our previous results showed that while NAP protected neuronal-like cells against oxidative stress, it did not protect NIH3T3 fibroblasts. Here, NAP did not protect NIH3T3 cells against zinc intoxication, unless these cells were transfected with Tau. Interestingly, other MT associated proteins (MAPs) may replace Tau, thus, EB-Tau (MAPs) interaction is identified as a novel target for endogenous ADNP neuroprotection, and a future target for drug development, with NAP as a prototype.

Microtubules (MTs), key cytoskeletal elements in living cells, are critical for axonal transport, synaptic transmission, and maintenance of neuronal morphology. NAP (NAPVSIPQ) is a neuroprotective peptide derived from the essential activity-dependent neuroprotective protein (ADNP). In Alzheimer’s disease models, NAP protects against tauopathy and cognitive decline. Here, we show that NAP treatment significantly affected the alpha tubulin tyrosination cycle in the neuronal differentiation model, rat pheochromocytoma (PC12) and in rat cortical astrocytes. The effect on tubulin tyrosination/detyrosination was coupled to increased MT network area (measured in PC12 cells), which is directly related to neurite outgrowth. Tubulin beta3, a marker for neurite outgrowth/neuronal differentiation significantly increased after NAP treatment. In rat cortical neurons, NAP doubled the area of dynamic MT invasion (Tyr-tubulin) into the neuronal growth cone periphery. NAP was previously shown to protect against zinc-induced MT/neurite destruction and neuronal death, here, in PC12 cells, NAP treatment reversed zinc-decreased tau-tubulin-MT interaction and protected against death. NAP effects on the MT pool, coupled with increased tau engagement on compromised MTs imply an important role in neuronal plasticity, protecting against free tau accumulation leading to tauopathy. With tauopathy representing a major pathological hallmark in Alzheimer's disease and related disorders, the current findings provide a mechanistic basis for further development. NAP (davunetide) is in phase 2/3 clinical trial in progressive supranuclear palsy, a disease presenting MT deficiency and tau pathology.

With Alzheimer's disease (AD) exhibiting reduced ability of neural stem cell renewal, we hypothesized that de novo mutations controlling embryonic development, in the form of brain somatic mutations instigate the disease. A leading gene presenting heterozygous dominant de novo autism-intellectual disabilities (ID) causing mutations is activity-dependent neuroprotective protein (ADNP), with intact ADNP protecting against AD-tauopathy. We discovered a genomic autism ADNP mutation (c.2188C>T) in postmortem AD olfactory bulbs and hippocampi. RNA-Seq of olfactory bulbs also identified a novel ADNP hotspot mutation, c.2187_2188insA. Altogether, 665 mutations in 596 genes with 441 mutations in AD patients (389 genes, 38% AD-exclusive mutations) and 104 genes presenting disease-causing mutations (OMIM) were discovered. OMIM AD mutated genes converged on cytoskeletal mechanisms, autism and ID causing mutations (about 40% each). The number and average frequencies of AD-related mutations per subject were higher in AD subjects compared to controls. RNA-seq datamining (hippocampus, dorsolateral prefrontal cortex, fusiform gyrus and superior frontal gyrus-583 subjects) yielded similar results. Overlapping all tested brain areas identified unique and shared mutations, with ADNP singled out as a gene associated with autism/ID/AD and presenting several unique aging/AD mutations. The large fusiform gyrus library (117 subjects) with high sequencing coverage correlated the c.2187_2188insA ADNP mutation frequency to Braak stage (tauopathy) and showed more ADNP mutations in AD specimens. In cell cultures, the ADNP-derived snippet NAP inhibited mutated-ADNP-microtubule (MT) toxicity and enhanced Tau-MT association. We propose a paradigm-shifting concept in the perception of AD whereby accumulating mosaic somatic mutations promote brain pathology.

Given our recent discovery of somatic mutations in autism spectrum disorder (ASD)/intellectual disability (ID) genes in postmortem aged Alzheimer's disease brains correlating with increasing tauopathy, it is important to decipher if tauopathy is underlying brain imaging results of atrophy in ASD/ID children. We concentrated on activity-dependent neuroprotective protein (ADNP), a prevalent autism gene. The unique availability of multiple postmortem brain sections of a 7-year-old male, heterozygous for ADNP de novo mutation c.2244Adup/p.His559Glnfs*3 allowed exploration of tauopathy, reflecting on a general unexplored mechanism. The tested subject exhibited autism, fine motor delays, severe intellectual disability and seizures. The patient died after multiple organ failure following liver transplantation. To compare to other ADNP syndrome mutations, immortalized lymphoblastoid cell lines from three different patients (including ADNP p.Arg216*, p.Lys408Valfs*31, and p.Tyr719* heterozygous dominant mutations) and a control were subjected to RNA-seq. Immunohistochemistry, high-throughput gene expression profiles in numerous postmortem tissues followed. Comparisons to a control brain and to extensive datasets were used. Live cell imaging investigated Tau-microtubule interaction, protecting against tauopathy. Extensive child brain tauopathy paralleled by multiple gene expression changes was discovered. Tauopathy was explained by direct mutation effects on Tau-microtubule interaction and correction by the ADNP active snippet NAP. Significant pathway changes (empirical P value < 0.05) included over 100 genes encompassing neuroactive ligand-receptor and cytokine-cytokine receptor interaction, MAPK and calcium signaling, axon guidance and Wnt signaling pathways. Changes were also seen in steroid biosynthesis genes, suggesting sex differences. Selecting the most affected genes by the ADNP mutations for gene expression analysis, in multiple postmortem tissues, identified Tau (MAPT)-gene-related expression changes compared with extensive normal gene expression (RNA-seq) databases. ADNP showed relatively reduced expression in the ADNP syndrome cerebellum, which was also observed for 25 additional genes (representing >50% of the tested genes), including NLGN1, NLGN2, PAX6, SMARCA4, and SNAP25, converging on nervous system development and tauopathy. NAP provided protection against mutated ADNP disrupted Tau-microtubule association. In conclusion, tauopathy may explain brain-imaging findings in ADNP syndrome children and may provide a new direction for the development of tauopathy protecting drug candidates like NAP in ASD/ID.