The current set of experiments demonstrates that treatment with the ADNP derived peptide NAP significantly affected the tubulin pool in rat neuronal, neuronal models (PC12) and glial cells in culture. In the neuronal differentiation model, the PC12 cell line, NAP-treatment significantly influenced α tubulin tyrosination cycle which is associated with MT dynamics in the living cell. This effect was coupled to increased MT network area which is directly related to neurite outgrowth. When tested in primary neurons, NAP also affected dynamic Tyr-MT invasion to growth cones. These was associated with neurotrophic effects in PC12 cells, where expression of the MT subunit, β3 tubulin, a marker for neuronal differentiation and neurite outgrowth, increased as a consequence of NAP treatment, similar to NGF treatment effect. The NAP effects on the MT pool were extended to PC12 cell protection against tubulin and tau loss from assembled MT in the cell in the face of toxic environment, high zinc concentrations.
We further showed here that NAP significantly affected the α-tubulin tyrosination cycle in rat primary astrocytes, increasing stable Glu-MT. NAP was previously shown to protect astrocytes from zinc intoxication 
. Importantly, reduction in stable Glu-MTs is a primary consequence of tau accumulation that initiates mechanisms underlying astrocyte dysfunction and death in human neurodegenerative glial tauopathies 
In the PC12 cell model of neuronal differentiation, NAP treatment showed a concentration-dependent effect on the α-tubulin tyrosination cycle, increasing the levels of long living MT, Glu-MT, and also of newly formed MT, Tyr-MT, although to a much lesser extent compared to Glu-MT. The NAP effects were compared to paclitaxel effects, a known MT stabilizing drug, corroborating previously described results of paclitaxel effects at concentrations ≥10−9
. Here, we extended the measurements to low concentrations of paclitaxel (below the nanomolar range) that have been shown before to confer neuroprotection 
. We further extended the measurements to the dynamic Tyr-MT that increased at sub-micromolar paclitaxel concentrations, in agreement with a potential protective effect. As expected, a more extensive concentration-dependent increase in the stable Glu-MT was observed. At very low concentrations, paclitaxel, unlike NAP, did not seem to affect the MT tyrosination cycle. Ratio analysis of Tyr-MT to Glu-MT indicated that NAP treatment resulted in a tendency toward “older”, stabilized MT network. In agreement, NAP treatment increased the levels of cellular polymerized tubulin in a time-dependent manner, as suggested before 
. When measured under control non-compromising conditions for 2 and 4 hrs. NAP treatment showed an apparent increase of up to 10% (2 hrs.) and a significant increase of ~15% (4 hrs.) in the tubulin content of the MT pool, as compared to the soluble tubulin pool in PC12 cells.
In contrast to NAP, the paclitaxel effect on the α-tubulin tyrosination cycle increased exponentially (when tested above nanomolar concentrations) and was coupled to a major shift in the MT pool resulting in a significantly higher level of polymerized tubulin (compared to sham controls), even after a 2 hr. incubation period. Thus, unlike NAP, paclitaxel promotes MT assembly and stabilizes MTs by shifting the dynamic equilibrium toward MT assembly, forming extremely stable and non-functional MTs and preventing depolymerisation 
. Paclitaxel also exhibited a significant effect on NIH 3T3 cells, while NAP had no effect on these cells. This result is in agreement with our previous work 
in which NAP had no protective effect against severe oxidative stress in NIH3T3 cells and no effect on cell division 
, while paclitaxel inhibits cell proliferation and arrests cell division 
Under compromised conditions of zinc intoxication (measured for 4 hrs.) when there is a loss of tubulin and tau from the MT pool, NAP treatment significantly increased the tubulin and tau association with the MT pool, protecting against cell death.
Previously, NAP was shown to provide protection against zinc intoxication in primary neuronal 
and in glial cells 
. While NAP protected against zinc intoxication and increased tau-MT association, paclitaxel treatment (5 µM) seemed to exacerbate the toxicity. This is in-line with published data showing that: 1] Paclitaxel reduces the affinity of tau to tubulin 
. 2] Paclitaxel displaces tau from MTs 
. 3] Pre-incubation of tubulin with tau resulted in decreased paclitaxel binding and reduced paclitaxel-induced MT polymerization 
. 4] High tau expression renders cancer cell resistant to paclitaxel treatment 
. Thus, the mechanism by which NAP affects the tubulin pool in the cell differs from paclitaxel at the level of tau-MT association.
Furthermore, regarding the association of zinc intoxication with tauopathy, high levels of zinc, up to 1000 µM, can be reached within amyloid plaques in Alzheimer’s disease 
, and there is accumulating evidence indicating that zinc enhances the development of amyloid pathology in Alzheimer’s disease 
, which in turn can induce tau pathology (e.g. 
). In this respect, a 4 hr. time lapse imaging of tau- Enhanced Green Fluorescent Protein (EGFP) proteins in cells treated with 250 µM zinc showed that the fluorescent MT network disappeared progressively. These previous results suggest that tau-EGFP proteins were detached from MTs and/or that MTs were depolymerized in presence of zinc 
, in agreement with the current data. Taken together, our current results support previous studies that indirectly suggested increased tau association with the MT pool and inhibition of tauopathy in the presence of NAP in vivo 
Regarding tau hyperphosphorylation, while previous data from other laboratories suggested a bimodal effect of zinc on tau hyperphosphorylation 
, the currently used conditions did not produce a significant zinc induction of tau hyperphosphrylation. Specifically, zinc intoxication did not result in a significant increase in tau phosphorylation when measured for example with the antibody recognizing p-Ser262 (
) or p-Tau202
), (data not shown).
The effect of NAP on the α-tubulin tyrosination cycle coupled with the results of polymerized vs. soluble tubulin at 2 hr. and at 4 hr. incubation periods and further coupled to zinc intoxication, led us to evaluate the effect of NAP on the MT network over an extended time period of 24 hrs. A concentration-dependent increase in mean MT network area/cell in response to NAP treatment was observed. These results suggest that the significant increase in the α-tubulin tyrosination cycle in polymerized MT eventually led to increased protective MT pool (4 hrs.) coupled with increased MT network area/cell (24 hrs.).
Previous observation indicated an effect of NAP on MT organization inside the cells 
, coupled with an ability to promote neurite outgrowth 
and axonal elongation, all of which requiring stable MT. Paclitaxel on the other hand causes polyneuropathy, mainly sensory, with characteristic features of distal axonal degeneration 
. In this respect, other studies showed that the protection against axonal degeneration associated with paclitaxel neuropathy is related to relatively lower levels of detyrosinated tubulin, compared to the unprotected state 
. Furthermore, MT stabilization alone is insufficient to generate cellular processes, since paclitaxel treatment did not alter the overall cell shape, despite the induction of MT bundling within the cell body of Sf9 cells from a moth ovary, while Sf9 tau expressing cells induced processes 
Sensory neurons treated with paclitaxel at concentrations above 7×10−8
M did not elongate extensions. When actively growing neurites are exposed to these levels of paclitaxel, neurite growth stops immediately and does not recommence. The broad processes of neurons cultured for 24 hr. with paclitaxel contain densely packed arrays of MTs that loop back at the ends of the process. In the presence of 7×10−9
M paclitaxel neurites do grow, although they are broader and less branched compared to control neurites 
. In contrast, here we show effects of NAP on microtubule invasion into the growth cone.
MT turnover in the growth cone is essential since application paclitaxel to neurons as above 
or locally to growth cones 
inhibits neurite growth. One important in vivo observation that was made in studies, with either paclitaxel 
or epothilone D (another MT stabilizing drug) 
, assessing their potential neuroprotection, is that the respective dose−response curves appeared to be U-shaped, with relatively low doses of the compounds (e.g. 100 times below the cumulative cancer chemotherapeutic dose, in the case of epothilone D) being required for efficacy. Similarly, while 10 nM paclitaxel prevents mutant human-tau-induced swelling of axonal segments, translocation of tau and MT to sub-membrane domains, reduction in the number of MTs along the axon, reversal of the MT polar orientation, impaired organelle transport, accumulation of macro-autophagosomes and lysosomes, compromised neurite morphology and degeneration, higher paclitaxel concentrations (100 nM) do not prevent these events from occurring and in fact facilitate them 
NAP short-term effects (tyrosination and MT area/cell and tubulin assembly into MT) showed a bimodal dose response curve in contrast to the sigmoidal dose response curve of MT binding drugs like paclitaxel and colchicine 
. The bimodal concentration-response curve was also observed in primary cortical neurons that were subjected to tetrodotoxin (TTX) electrical blockade 
. This may be partially explained by differential protection of neurons and glial cells, different status of cellular differentiation in the tissue culture 
and different protective epitopes on the NAP primary sequence (NAPVSIPQ) as indicated by systematic alanine amino acid replacement (Ala walk), 
. Furthermore, a bimodal concentration response curve was noted when measuring axonal length following NAP treatment in cerebellar granule neurons 
NAP was shown to promote neurite outgrowth in different primary neuronal cultures including hippocampal, cortical, cerebellar granule neurons and retinal ganglion neurons 
. β3-tubulin, an established marker for neurite outgrowth with expression primarily limited to neurons 
plays a role in early neuritogenesis, concomitantly or in coordination with other MT associated proteins (MAPs) 
, enhancing MT polymerization 
As introduced above, purified MTs enriched in β3-tubulin are considerably more dynamic than those composed of other β-tubulin isotypes 
. MTs with different isotype composition have different functions 
and display different dynamic properties 
. In terms of neuroprotection, the expression of β3-tubulin renders the MTs less sensitive to oxidative damage 
. It has been shown that β3-tubulin is conditionally expressed as an adaptive mechanism of resistance to a stressing microenvironment featuring oxygen-poor conditions and low nutrient supply 
unraveling a functional connection between β3-tubulin expression and cell survival. Importantly, mutations in the gene encoding β3-tubulin (TUBB3) cause an increase in Glu-MT with increased MT stability 
and see below. Modifications like detyrosination, are regulated by specific enzymes, and each can profoundly impact the capacity of the MT to interact with other proteins 
Here, we showed that NAP treatment increased the MT network area/cell, which precedes neurite outgrowth. We also showed that long-term treatment with NAP increased β3-tubulin expression, a marker for neurite outgrowth, neurodifferentiation and neuroprotection, providing a time course and an initial mechanistic background for the observed biological action.
When addressing the NAP effect of neurotrophism/neuroprotection, a previous study associated mutations in human β3-tubulin in perturbation of MT dynamics (increases in de-tyrosinated tubulin), kinesin interactions, and axon guidance 
, which are linked to a spectrum of human nervous system disorders that are now called the TUBB3 syndromes. Our original studies related NAP activity with β3-tubulin expression, which is not found in NIH3T3 
. In this respect, Sudo and Baas demonstrated that katanin severing of MT can lead to microtubule breakdown in axons. They have further shown that this process is accentuated by tau silencing and is inhibited to a large extent by NAP treatment. In contrast to axons, in minor processes, tau silencing was not required for microtubule severing by katanin, and NAP provided protection in the absence and in the presence of tau silencing. Furthermore, Sudo and Baas also showed that NAP protected against katanin – induced microtubule breakdown in RFL-6 fibroblasts only when β3-tubulin was ectopically expressed 
The basal level of β3-tubulin expression or other neuronal differentiation related proteins may explain the different dose response curves observed for NAP treatment in non-differentiated and differentiated PC12 cells and the dose response curves obtained after long treatment of NAP. In this manner, differences can also be found in the tubulin required protein components in cell protection vs. neurite outgrowth 
. Furthermore, specific MT associated proteins contributing to MT function may play a part in NAP action. Thus, Chen and Charness have shown that the NAP mechanism on axon growth requires Fyn kinase 
which interacts with tau 
, while in vivo, NAP protected against the microtubule associated protein MAP6 (STOP) deficiencies 
. Taken together, these studies suggest that NAP repairs the severed MT system, depending on the availability of MAPs and the primary composition of the MT backbone.
We have previously discovered a NAP-dependent reduction in activated glycogen synthase kinase-3-β (GSK3β) that is associated with the pathological hyperphosphorylation of tau, and the formation of neurofibrillary tangles 
. The NGF-related cascade of neurite outgrowth includes inhibition/inactivation of GSK3β 
, coupled to increases in β3-tubulin and MT polymerization/dynamics.
In contrast to the similar effects found on axonal/neurite outgrowth of NAP and NGF, our previous studies did not show similarities in neuroprotection against TTX. While NAP provided protection, NGF (as well as other neurotrophins) did not, at concentrations that are active in other systems 
. Furthermore, in PC12 cells expressing myotonic dystrophy type 1-associated CTG repeats, NGF treatment resulted in reduction in expression of the MT associated proteins MAP6/STOP, while NAP protects against STOP – associated deficiencies 
. The neurotrophic activity of either NAP or NGF were also associated with polyADP ribosylation in vitro 
and polyADP ribosylation was in turn associated with plasticity and memory formation 
The neuroprotective effect of NAP paralleled protection against apoptosis (cytochrome-c release), protection against caspase 3 activation 
and MT breakdown protection 
. Recent studies suggested that MT polymerization re-establishment by protective paclitaxel concentrations reduced amyloid β oligomers 
, which in turn have been associated with the formation of hyperphosphorylated tau 
. Thus, protection of MT polymerization by NAP has far reaching mechanistic consequences on protection in Alzheimer’s disease and related tauopathies. It is our working hypothesis that by subtle changes to the MT network paralleled by effects on key enzymes/pathways, NAP confers MT related neuroprotection.
The current studies provide additional weight to observed effects of NAP on tau-related pathology and MT dysfunction 
. Thus, NAP effects on the MT structures and associated impact on MT pool and tau recruitment to MT may provide an explanation for the resulting neuroprotection. NAP (davunetide) is in phase 2/3 clinical trial in progressive supranuclear palsy (PSP) a disease presenting MT deficiency, tau pathology with tangles sharing epitopes with tyrosinated and detyrosinated tubulin 
. Understanding of the NAP cellular mechanism of action as a first in class investigational compound paves the path to the discovery of novel ways to combat devastating dementias.