NAP (davunetide) preferential interaction with dynamic 3-repeat Tau explains differential protection in selected tauopathies.

The microtubule (MT) associated protein Tau is instrumental for the regulation of MT assembly and dynamic instability, orchestrating MT-dependent cellular processes. Aberration in Tau post-translational modifications ratio deviation of spliced Tau isoforms 3 or 4 MT binding repeats (3R/4R) have been implicated in neurodegenerative tauopathies. Activity-dependent neuroprotective protein (ADNP) is vital for brain formation and cognitive function. ADNP deficiency in mice causes pathological Tau hyperphosphorylation and aggregation, correlated with impaired cognitive functions. It has been previously shown that the ADNP-derived peptide NAP protects against ADNP deficiency, exhibiting neuroprotection, MT interaction and memory protection. NAP prevents MT degradation by recruitment of Tau and end-binding proteins to MTs and expression of these proteins is required for NAP activity. Clinically, NAP (davunetide, CP201) exhibited efficacy in prodromal Alzheimer's disease patients (Tau3R/4R tauopathy) but not in progressive supranuclear palsy (increased Tau4R tauopathy). Here, we examined the potential preferential interaction of NAP with 3R vs. 4R Tau, toward personalized treatment of tauopathies. Affinity-chromatography showed that NAP preferentially interacted with Tau3R protein from rat brain extracts and fluorescence recovery after photobleaching assay indicated that NAP induced increased recruitment of human Tau3R to MTs under zinc intoxication, in comparison to Tau4R. Furthermore, we showed that NAP interaction with tubulin (MTs) was inhibited by obstruction of Tau-binding sites on MTs, confirming the requirement of Tau-MT interaction for NAP activity. The preferential interaction of NAP with Tau3R may explain clinical efficacy in mixed vs. Tau4R pathologies, and suggest effectiveness in Tau3R neurodevelopmental disorders

Distinct Impairments Characterizing Different ADNP Mutants Reveal Aberrant Cytoplasmic-Nuclear Crosstalk

(1) Background: Activity-dependent neuroprotective protein (ADNP) is essential for neuronal structure and function. Multiple de novo pathological mutations in ADNP cause the autistic ADNP syndrome, and they have been further suggested to affect Alzheimer’s disease progression in a somatic form. Here, we asked if different ADNP mutations produce specific neuronal-like phenotypes toward better understanding and personalized medicine. (2) Methods: We employed CRISPR/Cas9 genome editing in N1E-115 neuroblastoma cells to form neuron-like cell lines expressing ADNP mutant proteins conjugated to GFP. These new cell lines were characterized by quantitative morphology, immunocytochemistry and live cell imaging. (3) Results: Our novel cell lines, constitutively expressing GFP-ADNP p.Pro403 (p.Ser404* human orthologue) and GFP-ADNP p.Tyr718* (p.Tyr719* human orthologue), revealed new and distinct phenotypes. Increased neurite numbers (day 1, in culture) and increased neurite lengths upon differentiation (day 7, in culture) were linked with p.Pro403*. In contrast, p.Tyr718* decreased cell numbers (day 1). These discrete phenotypes were associated with an increased expression of both mutant proteins in the cytoplasm. Reduced nuclear/cytoplasmic boundaries were observed in the p.Tyr718* ADNP-mutant line, with this malformation being corrected by the ADNP-derived fragment drug candidate NAP. (4) Conclusions: Distinct impairments characterize different ADNP mutants and reveal aberrant cytoplasmic-nuclear crosstalk

NAP enhances tau-microtubule interactions and microtubule polymerization under zinc intoxication.

<p>(<b>a</b>) Zinc exposure (400 µM) resulted in PC12 cell death which was protected against by NAP treatment. Results of mitochondrial activity (MTS cell viability) are shown –100– is 100% survival – control w/o zinc treatment. (ANOVA, ***p<0.0001, n = 18/group, post hoc comparison were made in reference to vehicle+Zn treatment), upper panel. The lower panel shows a similar experiment with 5 µM paclitaxel (ANOVA, ***p<0.0001, n = 10/group, post hoc comparisons made in reference to vehicle, or to vehicle+Zn treatment). (<b>b</b>) Cell lysates were separated into polymerized (<i>P</i>) or soluble (<i>S</i>) protein fractions as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051458#pone-0051458-g002" target="_blank">Figure 2</a>. Aliquots of equal volumes for each pair were separated on adjacent lanes by SDS polyacrylamide gel electrophoresis, the blot was probed with antibodies recognizing α-tubulin, or total tau or actin and the percentage of the polymerized fraction was calculated for each ‘<i>P</i>’ and ‘<i>S</i>’ pair. (<b>c</b>) The intensity of each band was quantified by densitometry, and the percentage of the polymerized fraction was determined. (Actin, ANOVA, p = 0.4774, n = 6; Tubulin, ANOVA, p = 0.007, n = 6; TAU, ANOVA, p = 0.0022, n = 5, post hoc determinations were against the zinc-treated group for each of the variables).</p

NAP effect on Tyr-MT invasion to growth cones.

<p>(a) Glu-MTs (green) do not invade the peripheral domain of the growth cone. NAP clearly affected Tyr-MT (red) invasion to the peripheral domain of the growth cone in terms of number and length of this invasions. Actin is labeled in blue (Coumarin Phalloidin) and cell membranes are stained in cyan (Dil). Bars: 7.5 µm. (b) Analysis of MT invasion - the total area in the image covered by actin and membrane dyed using DiI determined the growth cone area (T). From this determined area we subtracted the area occupied by Glu-MT (proximal growth cone area, P) which gave us our region of interest (ROI), as illustrated, with a representative picture and termed A_tyr (area including Tyr-MT). (c). The percentage of the ROI penetrated by Tyr-MT was obtained, calculated and graphed, right hand side, (t-test, p<0.0001, n = 22/treatment group).</p

Treatment with NAP increases total polymerized α-tubulin network area in PC12 cells.

<p>(<b>a</b>) Concentration response analysis of MT network area/cell as determined by the outer boundaries of fluorescently probed MT (monoclonal anti α-tubulin) in PC12 or NIH3T3 cells, evaluated by image analysis of confocal images. Cultured cells were exposed for 24 hr. to different concentrations of NAP (10⁻¹⁸M - 10⁻⁶M) and compared to vehicle-treated cells and paclitaxel, 5 µM treated cells. (PC12 - ANOVA, p = 0.0081, n = 87 cells per treatment; NIH3T3 - ANOVA, p = 0.3892, n = 90). Left side, Representative field-images of PC12 cells treated with paclitaxel with fluorescently probed MT. Bar: 10 µm (<b>b</b>) Representative field-images of PC12 cells with fluorescently probed MT (control and NAP-treated as indicated). Bars: 10 µm.</p

NAP affects the α-tubulin tyrosination cycle.

<p>Glu-MT and Tyr-MT fluorescence levels in cultured cells after exposure for 2 hrs to different drug concentrations. Cells were permeabilized during fixation to remove non-polymerized tubulin subunits, therefore only tubulin incorporated into MT was assessed. (<b>a</b>) Concentration response analysis of fluorescence intensity units (FAU) normalized to Hoechst signal in paclitaxel-treated PC12 cells evaluated by “In cell western”, microplate reader method (Glu-MT, ANOVA, p<0.0001, n = 9 per treatment), (Tyr-MT, ANOVA, p<0.0001, n = 9 per treatment). The dotted line indicates the paclitaxel concentration, 5 µM, used in the following experiments as a control. (<b>b</b>) Concentration-response analysis of fluorescence intensity units (FAU) normalized to Hoechst signal in NAP- (10⁻¹⁸M - 10⁻⁶M) or vehicle-treated cells. PC12 cells were evaluated by “In cell western”, microplate reader method. (Glu-MT, ANOVA, p<0.0001, n = 9 per treatment), (Tyr-MT, ANOVA, p = 0.0139, n = 9 per treatment) (<b>c</b>) Concentration response analysis of fluorescence intensity ratio levels (Tyr-MT/Glu-MT) in PC12 cells treated with NAP (10⁻¹⁸M - 10⁻⁶M) or vehicle, evaluated by image analysis of confocal images. The control compound was paclitaxel, 5 µM, (ANOVA, p = 0.0001, n>34 cells per treatment). The fluorescence intensities of both Tyr-MT and Glu-MT in the colchicine-treated cells were below the threshold level of measurements (see d). (<b>d</b>) Comparison of the effect on Glu-MT and Tyr-MT and integrity of the MT network. Control compounds were paclitaxel, 5 µM and colchicine, 2 µM (bottom row). PC12 stained for Tyr-MT (red), Glu-MT (green), Hoechst (blue). Bars: 10 µm. (<b>e</b>) Concentration response analysis of fluorescence intensity ratio levels (Tyr-MT/Glu-MT) in rat cerebral cortical astrocytes evaluated by image analysis of confocal images. (ANOVA, p<0.0001, n = 27). The fluorescence intensities of both Tyr-MT and Glu-MT in the colchicine-treated cells were below the threshold level of measurements (see f). (<b>f</b>) Comparison of the effect on Glu-MT and Tyr-MT and integrity of the microtubular network. Control compounds were paclitaxel, 5 µM and colchicine, 2 µM (bottom row). Rat cerebral cortical astrocytes stained for Tyr-MT (red), Glu-MT (green), Hoechst (blue). Bars: 10 µm. (<b>g</b>) Concentration response analysis of fluorescence intensity ratio levels (Tyr-MT/Glu-MT) in NIH3T3 cells evaluated by image analysis of confocal images. (ANOVA, p = 0.9966, n = 18).</p

NAP affects β3-tubulin expression levels in PC12 cells.

<p>Protein expression levels of β3-tubulin were measured in PC12 cells maintained 8 days in culture. Comparisons were made after exposure to vehicle or different concentrations of NAP (10⁻¹⁸M - 10⁻⁶M). Control cultures were treated with medium only or with NGF (a) Concentration response analysis of protein expression levels were evaluated by western blotting of whole cell extracts with β3-tubulin immunoreactivity (top), and actin, control immunoreactivity (bottom). (b) Densitometric quantification of protein expression performed with ImageJ software. (ANOVA, p = 0.0009, n = 5, post hoc comparison are made in reference to vehicle treatment).</p

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

(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