Role of phospholipase D1 in neurite outgrowth of neural stem cells

Employing neural stem cells from the brain cortex of E12 rat embryos, we investigated the possible role of phospholipase D (PLD) in the synaptogenesis and neurite formation of neural cells during differentiation. Expression level of PLD1 increased during neuronal differentiation of the neural stem cells, resulting in increased PLD activity. Expression level of synapsin I, a marker of synaptogenesis, also increased as the differentiation of neural stem cells progressed. To figure out the effect of PLD on synapsin I expression, we treated the neural stem cells with phorbol myristate acetate (PMA) to stimulate PLD activity. Increased PLD activity induced by PMA treatment resulted in elevated synapsin I expression and neurite outgrowth during neuronal differentiation. To further confirm the role of PLD in neurite outgrowth, we transfected the dominant-negative form of rat PLD1 cDNA (DN-rPLD1) into neural stem cells to downregulate PLD activity. Overexpression of DN-rPLD1 showed the complete inhibition of neurite outgrowth of neural stem cells under differentiation condition. While transfection of DN-rPLD1 did not affect the synapsin I expression, overexpression of rPLD1 resulted in increased synapsin I expression of the neural cells. These results suggest that PLD1 plays a critical role in neurite outgrowth during differentiation of the neural stem cells. In conclusion, this is the first evidence to show that PLD1 acts as an important regulator of neurite outgrowth in neural stem cell by promoting neuronal differentiation via increase of synapsin I expression.     

The ADNP Derived Peptide,NAP Modulates the Tubulin Pool: Implication for Neurotrophic and Neuroprotective Activities

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.     

Phosphatidylcholine biosynthesis via CTP:phosphocholine cytidylyltransferase 2 facilitates neurite outgrowth and branching

Hallmarks of neuronal differentiation are neurite sprouting, extension, and branching. We previously showed that increased expression of CTP:phosphocholine cytidylyltransferase beta2 (CTbeta2), an isoform of a key phosphatidylcholine (PC) biosynthetic enzyme, accompanies neurite outgrowth (Carter, J. M., Waite, K. A., Campenot, R. B., Vance, J. E., and Vance, D. E. (2003) J. Biol. Chem. 278, 44988-44994). CTbeta2 mRNA is highly expressed in the brain. We show that CTbeta2 is abundant in axons of rat sympathetic neurons and retinal ganglion cells. We used RNA silencing to decrease CTbeta2 expression in PC12 cells differentiated by nerve growth factor. In CTbeta2-silenced cells, numbers of primary and secondary neurites were markedly reduced, suggesting that CTbeta2 facilitates neurite outgrowth and branching. However, the length of individual neurites was significantly increased, and the total amount of neuronal membrane was unchanged. Neurite branching of PC12 cells is known to be inhibited by activation of Akt and promoted by the Akt inhibitor LY294002. Our experiments showed that LY294002 increases neurite sprouting and branching in control PC12 cells but not in CTbeta2-deficient cells. CTbeta2 was not phosphorylated in vitro by Akt. However, inhibition of Cdk5 by roscovitine blocked CTbeta2 phosphorylation and reduced neurite outgrowth and branching. These results highlight the importance of CTbeta2 in neurons for promoting neurite outgrowth and branching and represent the first identification of a lipid biosynthetic enzyme that facilitates these functions.     

Versican V1 isoform induces neuronal differentiation and promotes neurite outgrowth

The chondroitin sulfate proteoglycan versican is one of the major extracellular components in the developing and adult brain. Here, we show that isoforms of versican play different roles in neuronal differentiation and neurite outgrowth. Expression of versican V1 isoform in PC12 cells induced complete differentiation, whereas expression of V2 induced an aborted differentiation accompanied by apoptosis. V1 promoted neurite outgrowth of hippocampal neurons, but V2 failed to do so. V1 transfection enhanced expression of epidermal growth factor receptor and integrins, and facilitated sustained extracellular signal-regulated kinase/MAPK phosphorylation. Blockade of the epidermal growth factor receptor, beta1 integrin, or Src significantly inhibited neuronal differentiation. Finally, we demonstrated that versican V1 isoform also promoted differentiation of neural stem cells into neurons. Our results have implications for understanding how versican regulates neuronal development, function, and repair.     

Binding of Cdc42 to phospholipase D1 is important in neurite outgrowth of neural stem cells

We previously demonstrated that phospholipase D (PLD) expression and PLD activity are upregulated during neuronal differentiation. In the present study, employing neural stem cells from the brain cortex of E14 rat embryos, we investigated the role of Rho family GTPases in PLD activation and in neurite outgrowth of neural stem cells during differentiation. As neuronal differentiation progressed, the expression levels of Cdc42 and RhoA increased. Furthermore, Cdc42 and PLD1 were mainly localized in neurite, whereas RhoA was localized in cytosol. Co-immunoprecipitation revealed that Cdc42 was bound to PLD1 during differentiation, whereas RhoA was associated with PLD1 during both proliferation and differentiation. These results indicate that the association between Cdc42 and PLD1 is related to neuronal differentiation. To examine the effect of Cdc42 on PLD activation and neurite outgrowth, we transfected dominant negative Cdc42 (Cdc42N17) and constitutively active Cdc42 (Cdc42V12) into neural stem cells, respectively. Overexpression of Cdc42N17 decreased both PLD activity and neurite outgrowth, whereas co-transfection with Cdc42N17 and PLD1 restored them. On the other hand, Cdc42V12 increased both PLD activity and neurite outgrowth, suggesting that active state of Cdc42 is important in upregulation of PLD activity which is responsible for the increase of neurite outgrowth.     

KLHL1/MRP2 mediates neurite outgrowth in a glycogen synthase kinase 3beta-dependent manner

The actin-based cytoskeleton is essential for the generation and maintenance of cell polarity, cellular motility, and the formation of neural cell processes. MRP2 is an actin-binding protein of the kelch-related protein family. While MRP2 has been shown to be expressed specifically in brain, its function is still unknown. Here, we report that in neuronal growth factor (NGF)-induced PC12 cells, MRP2 was expressed along the neurite processes and colocalized with Talin at the growth cones. MRP2 mRNA and protein levels were up-regulated in PC12 cells following NGF stimulation. Moreover, treatment of PC12 cells with interfering RNAs for MRP2 and glycogen synthase kinase 3beta (GSK3beta) resulted in the inhibition of neurite outgrowth. A significant decrease in MRP2 expression levels was observed following GSK3beta inhibition, which was correlated with the inhibited neurite outgrowth, while GSK3beta overexpression was found to increase MRP2 expression levels. MRP2 interacted with GSK3beta through its NH2 terminus containing the BTB domain, and these molecules colocalized along neurite processes and growth cones in differentiated PC12 cells and rat primary hippocampal neurons. Additionally, increased associations of MRP2 with GSK3beta and MRP2 with actin were observed in the NGF-treated PC12 cells. Thus, this study provides, for the first time, insights into the involvement of MRP2 in neurite outgrowth, which occurs in a GSK3beta-dependent manner.     

Extremely Low-Frequency Electromagnetic Fields Promote In Vitro Neuronal Differentiation and Neurite Outgrowth of Embryonic Neural Stem Cells via Up-Regulating TRPC1

Exposure to extremely low-frequency electromagnetic fields (ELF-EMFs) can enhance hippocampal neurogenesis in adult mice. However, little is focused on the effects of ELF-EMFs on embryonic neurogenesis. Here, we studied the potential effects of ELF-EMFs on embryonic neural stem cells (eNSCs). We exposed eNSCs to ELF-EMF (50 Hz, 1 mT) for 1, 2, and 3 days with 4 hours per day. We found that eNSC proliferation and maintenance were significantly enhanced after ELF-EMF exposure in proliferation medium. ELF-EMF exposure increased the ratio of differentiated neurons and promoted the neurite outgrowth of eNSC-derived neurons without influencing astrocyes differentiation and the cell apoptosis. In addition, the expression of the proneural genes, NeuroD and Ngn1, which are crucial for neuronal differentiation and neurite outgrowth, was increased after ELF-EMF exposure. Moreover, the expression of transient receptor potential canonical 1 (TRPC1) was significantly up-regulated accompanied by increased the peak amplitude of intracellular calcium level induced by ELF-EMF. Furthermore, silencing TRPC1 expression eliminated the up-regulation of the proneural genes and the promotion of neuronal differentiation and neurite outgrowth induced by ELF-EMF. These results suggest that ELF-EMF exposure promotes the neuronal differentiation and neurite outgrowth of eNSCs via up-regulation the expression of TRPC1 and proneural genes (NeuroD and Ngn1). These findings also provide new insights in understanding the effects of ELF-EMF exposure on embryonic brain development.     

Neurite outgrowth in response to transfected N-CAM and N-cadherin reveals fundamental differences in neuronal responsiveness to CAMs

Different neuronal populations were used to compare the neurite outgrowth-promoting activities of N-CAM and N-cadherin expressed via gene transfer on the surface of nonneuronal cells. In contrast to a previously reported developmental loss of retinal ganglion cell responsiveness to N-CAM, these cells exhibited an increased and maintained responsiveness to N-cadherin over the same developmental period (E6-E11). N-CAM and N-cadherin responses could be specifically inhibited by their own antibodies, but not by antisera to the beta 1 integrin family or the L1/G4 glycoprotein. Cerebellar neurons showed qualitative differences in the nature of the dose-response curves for transfected N-CAM expression (highly cooperative) versus N-cadherin expression (linear). In addition "subthreshold" levels of N-CAM expression, which do not normally support neurite outgrowth, did so when coexpressed with functional levels of N-cadherin. These studies show fundamental differences in neuronal responsiveness to cell adhesion molecules and suggest a more dynamic regulation for N-CAM-dependent neurite outgrowth than for N-cadherin-dependent outgrowth.     

Involvement of corticotropin-releasing factor receptor 2 beta in differentiation of dopaminergic MN9D cells

Corticotropin releasing factor (CRF) mediates various responses to stress through CRF receptors 1 and 2. CRF receptor 2 has two forms, 2alpha and 2beta each of which appears to have distinct roles. Here we used dopaminergic neuron-derived MN9D cells to investigate the function of CRF receptor 2 in dopamine neurons. We found that n-butyrate, a histone deacetylase inhibitor, induced MN9D cell differentiation and increased gene expression of all CRF receptors. CRF receptor 2beta was minimally expressed in MN9D cells; however, its expression dramatically increased during differentiation. CRF receptor 2beta expression levels appeared to correlate with neurite outgrowth, suggesting CRF receptor 2beta involvement in neuronal differentiation. To validate this statement, we made a CRF receptor 2beta-overexpressing MN9D/CRFR2 beta stable cell line. This cell line showed robust neurite outgrowth and GAP43 overexpression, together with MEK and ERK activation, suggesting MN9D cell neuronal differentiation. From these results, we conclude that CRF receptor 2beta plays an important role in MN9D cell differentiation by activating the MEK/ERK signaling pathway.     

Evidence for Fibroblast Growth Factor-2 as a Mediator of Amphetamine-Enhanced Motor Improvement following Stroke

Previously we have shown that addition of amphetamine to physical therapy results in enhanced motor improvement following stroke in rats, which was associated with the formation of new motor pathways from cortical projection neurons of the contralesional cortex. It is unclear what mechanisms are involved, but amphetamine is known to induce the neuronal release of catecholamines as well as upregulate fibroblast growth factor-2 (FGF-2) expression in the brain. Since FGF-2 has been widely documented to stimulate neurite outgrowth, the present studies were undertaken to provide evidence for FGF-2 as a neurobiological mechanism underlying amphetamine-induced neuroplasticity. In the present study rats that received amphetamine plus physical therapy following permanent middle cerebral artery occlusion exhibited significantly greater motor improvement over animals receiving physical therapy alone. Amphetamine plus physical therapy also significantly increased the number of FGF-2 expressing pyramidal neurons of the contralesional cortex at 2 weeks post-stroke and resulted in significant axonal outgrowth from these neurons at 8 weeks post-stroke. Since amphetamine is a known releaser of norepinephrine, in vitro analyses focused on whether noradrenergic stimulation could lead to neurite outgrowth in a manner requiring FGF-2 activity. Primary cortical neurons did not respond to direct stimulation by norepinephrine or amphetamine with increased neurite outgrowth. However, conditioned media from astrocytes exposed to norepinephrine or isoproterenol (a beta adrenergic agonist) significantly increased neurite outgrowth when applied to neuronal cultures. Adrenergic agonists also upregulated FGF-2 expression in astrocytes. Pharmacological analysis indicated that beta receptors and alpha1, but not alpha2, receptors were involved in both effects. Antibody neutralization studies demonstrated that FGF-2 was a critical contributor to neurite outgrowth induced by astrocyte-conditioned media. Taken together the present results suggest that noradrenergic activation, when combined with physical therapy, can improve motor recovery following ischemic damage by stimulating the formation of new neural pathways in an FGF-2-dependent manner.     

N-cadherin and integrins: two receptor systems that mediate neuronal process outgrowth on astrocyte surfaces

Receptor-mediated interactions between neurons and astroglia are likely to play a crucial role in the growth and guidance of CNS axons. Using antibodies to neuronal cell surface proteins, we identified two receptor systems mediating neurite outgrowth on cultured astrocytes. N-cadherin, a Ca2(+)-dependent cell adhesion molecule, functions prominently in the outgrowth of neurites on astrocytes by E8 and E14 chick ciliary ganglion (CG) neurons. beta 1-class integrin ECM receptor heterodimers function less prominently in E8 and not at all in E14 neurite outgrowth on astrocytes. The lack of effect of integrin beta 1 antibodies on E14 neurite outgrowth reflects an apparent loss of integrin function, as assayed by E14 neuronal attachment and process outgrowth on laminin. N-CAM appeared not to be required for neurite outgrowth by either E8 or E14 neurons. Since N-cadherin and integrin beta 1 antibodies together virtually eliminated E8 CG neurite outgrowth on cultured astrocytes, these two neuronal receptors are probably important in regulating axon growth on astroglia in vivo.     

N-acetylglucosaminyltranferase VB expression enhances beta1 integrin- dependent PC12 neurite outgrowth on laminin and collagen

N-acetylglucosaminyltransferase VB (GnT-VB, -IX) is a newly discovered glycosyltransferase expressed exclusively in high levels in neuronal tissue during early development. Its homolog, GnT-V, is expressed in many tissues and modulates cell-cell and cell-matrix adhesion. The ability of GnT-VB to regulate cell-matrix interactions was initially investigated using the rat pheochromocytoma PC12 neurite outgrowth model. PC12 cells stably transfected with GnT-VB consistently showed an enhanced rate of nerve growth factor (NGF)-induced neurite outgrowth on collagen and laminin substrates. Levels of TrkA receptor phosphorylation and downstream ERK activation induced by NGF were not influenced by GnT-VB expression. No significant difference was observed in the rate of neurite outgrowth when cells were cultured on non-coated culture dishes, indicating that integrin-ECM interaction is required for the stimulatory effects. Neurite outgrowth induced by manganese-dependent activation of beta1 integrin on collagen and laminin substrates, however, showed a significant increase in neurite length for the PC12/GnT-VB cells, compared with control cells, suggesting that the enhancement is most likely mediated by alteration of beta1 integrin-ECM interaction by GnT-VB. These results demonstrate that GnT-VB expression can modulate the rate of neurite outgrowth by affecting beta1 integrin-ECM interaction.     

Calcium influx into neurons can solely account for cell contact- dependent neurite outgrowth stimulated by transfected L1

We have used monolayers of control 3T3 cells and 3T3 cells expressing transfected human L1 as a culture substrate for rat PC12 cells and rat cerebellar neurons. PC12 cells and cerebellar neurons extended longer neurites on human L1 expressing cells. Neurons isolated from the cerebellum at postnatal day 9 responded equally as well as those isolated at postnatal day 1-4, and this contrasts with the failure of these older neurons to respond to the transfected human neural cell adhesion molecule (NCAM). Human L1-dependent neurite outgrowth could be blocked by antibodies that bound to rat L1 and, additionally, the response could be fully inhibited by pertussis toxin and substantially inhibited by antagonists of L- and N-type calcium channels. Calcium influx into neurons induced by K+ depolarization fully mimics the L1 response. Furthermore, we show that L1- and K+(-)dependent neurite outgrowth can be specifically inhibited by a reduction in extracellular calcium to 0.25 microM, and by pretreatment of cerebellar neurons with the intracellular calcium chelator BAPTA/AM. In contrast, the response was not inhibited by heparin or by removal of polysialic acid from neuronal NCAM both of which substantially inhibit NCAM-dependent neurite outgrowth. These data demonstrate that whereas NCAM and L1 promote neurite outgrowth via activation of a common CAM-specific second messenger pathway in neurons, neuronal responsiveness to NCAM and L1 is not coordinately regulated via posttranslational processing of NCAM. The fact that NCAM- and L1-dependent neurite outgrowth, but not adhesion, are calcium dependent provides further evidence that adhesion per se does not directly contribute to neurite outgrowth.     

Modulators of cytoskeletal reorganization in CA1 hippocampal neurons show increased expression in patients at mid-stage Alzheimer's disease

During the progression of Alzheimer's disease (AD), hippocampal neurons undergo cytoskeletal reorganization, resulting in degenerative as well as regenerative changes. As neurofibrillary tangles form and dystrophic neurites appear, sprouting neuronal processes with growth cones emerge. Actin and tubulin are indispensable for normal neurite development and regenerative responses to injury and neurodegenerative stimuli. We have previously shown that actin capping protein beta2 subunit, Capzb2, binds tubulin and, in the presence of tau, affects microtubule polymerization necessary for neurite outgrowth and normal growth cone morphology. Accordingly, Capzb2 silencing in hippocampal neurons resulted in short, dystrophic neurites, seen in neurodegenerative diseases including AD. Here we demonstrate the statistically significant increase in the Capzb2 expression in the postmortem hippocampi in persons at mid-stage, Braak and Braak stage (BB) III-IV, non-familial AD in comparison to controls. The dynamics of Capzb2 expression in progressive AD stages cannot be attributed to reactive astrocytosis. Moreover, the increased expression of Capzb2 mRNA in CA1 pyramidal neurons in AD BB III-IV is accompanied by an increased mRNA expression of brain derived neurotrophic factor (BDNF) receptor tyrosine kinase B (TrkB), mediator of synaptic plasticity in hippocampal neurons. Thus, the up-regulation of Capzb2 and TrkB may reflect cytoskeletal reorganization and/or regenerative response occurring in hippocampal CA1 neurons at a specific stage of AD progression.     

Neurite formation by neurons derived from adult rat hippocampal progenitor cells is susceptible to myelin inhibition

Myelin-associated inhibitors expressed following injury to the adult central nervous system (CNS) induce growth cone collapse and retraction of the axonal cytoskeleton. Myelin-associated glycoprotein (MAG) is a bi-functional molecule that promotes neuritogenesis in some immature neurons during development then becomes inhibitory to neurite outgrowth as neurons mature. Progress is being made towards the elucidation of the downstream events that regulate myelin inhibition of regeneration in neuronal populations. However it is not known how adult-derived neural stem cells or progenitors respond to myelin during neuronal differentiation and neuritogenesis. Here we examine the effect of MAG on neurons derived from an adult rat hippocampal progenitor cell line (AHPCs). We show that, unlike their developmental counterparts, AHPC-derived neurons are susceptible to MAG inhibition of neuritogenesis during differentiation and display a 57% reduction in neurite outgrowth when compared with controls. We demonstrate that this effect can be overcome (by up to 69%) by activation of the neurotrophin, cyclic AMP and protein kinase A pathways or by Rho-kinase suppression. We also demonstrate that combination of these factors enhanced neurite outgrowth from differentiating neurons in the presence of MAG. This work provides important information for the successful generation of new neurons from adult neural stem cell populations within compromised adult circuitry and is thus directly relevant to endogenous repair and regeneration of the adult CNS.     

Neuroglobin Promotes Neurite Outgrowth via Differential Binding to PTEN and Akt

Neuroglobin, the third mammalian globin with a hexa-coordinated heme, exists predominantly in neurons of the brain. Neuroglobin plays an important role in neuronal death upon ischemia and oxidative stress. The physiological function of neuroglobin remains unclear. Here, we report a novel function of neuroglobin in neurite development. Knocking-down neuroglobin exhibited a prominent neurite-deficient phenotype in mouse neuroblastoma N2a cells. Silencing neuroglobin prevented neurite outgrowth, while ectopic expression of neuroglobin but not homologous cytoglobin promoted neurite outgrowth of N2a cells upon serum withdrawal. In primary cultured rat cerebral cortical neurons, neuroglobin was upregulated and preferentially distributed in neurites during neuronal development. Overexpression of neuroglobin but not cytoglobin in cultured cortical neurons promoted axonal outgrowth, while knocking-down of neuroglobin retarded axonal outgrowth. Neuroglobin overexpression suppressed phosphatase and tensin homolog (PTEN) but increased Akt phosphorylation during neurite induction. Bimolecular fluorescence complementation and glutathione S-transferase pull-down assays revealed that neuroglobin and various mutants (E53Q, E118Q, K119N, H64A, H64L, and Y44D) bound with Akt and PTEN differentially. Neuroglobin E53Q showed a prominent reduced PTEN binding but increased Akt binding, resulting in decreased p-PTEN, increased p-Akt, and increased neurite length. Taken together, we demonstrate a critical role of neuroglobin in neuritogenesis or development via interacting with PTEN and Akt differentially to activate phosphatidylinositol 3-kinase/Akt pathway, providing potential therapeutic applications of neuroglobin for axonopathy in neurological diseases.     

Rho‐associated kinase (ROCK) inhibitor,Y27632, promotes neurite outgrowth in PC12 cells in the absence of NGF

Neurite extension and retraction are very important processes in the formation of neuronal networks. A strategy for fostering axonal regrowth/regeneration of injured adult neurons is attractive therapeutically for various diseases such as traumatic brain injury, stroke and Alzheimer's disease. The Rho family of small GTPases, including Rac and Cdc42 have been shown to be involved in promoting neurite outgrowth. On the other hand, activation of RhoA induces collapse of growth cone and retraction of neurites. Rho‐associated kinase (ROCK) an effector molecule of RhoA, is downstream of a number of axonal outgrowth and growth cone collapse inhibition mechanisms. In the present study, we sought to identify the role of ROCK in neurite outgrowth in PC12 cells. Y27632, a specific inhibitor of ROCK, induced a robust increase in neurite outgrowth in these cells within 24–48 h as visualized by phase contrast microscopy. Staining with FITC‐tubulin or phalloidin show extended neurites in PC12 cells treated with Y27632, comparable to that with 100 ng/mL of NGF. Assessment of other biochemical markers of neurite outgrowth such as GAP43, neurofilament and tyrosine hydroxylase phosphorylation further indicates that inhibition of ROCK in PC12 cells causes differentiation of these cells to a neuronal phenotype.     

Peptide neuroprotection through specific interaction with brain tubulin

This study aimed to identify the neuronal target for the potent neuroprotective peptide NAP. When added to pheochromocytoma cells (neuronal model), NAP was found in the intracellular milieu and was co-localized with microtubules. NAP induced neurite outgrowth and protected primary neurons against microtubule-associated ZnCl2 toxicity. Rapid microtubule reorganization into distinct microtubules ensued after NAP addition to both pheochromocytoma cells and primary cerebral cortical neurons, but not to fibrobalsts. While binding neuronal tubulin and protecting pheochromocytoma cells against oxidative stress, NAP did not bind tubulin extracted from fibroblasts, nor did it protect those cells against oxidative stress. Affinity chromatography identified the brain-specific betaIII-tubulin as a major NAP binding protein. Paclitaxel (a microtubule aggregating agent that interacts with beta-tubulin) reduced NAP tubulin binding. Thus, the underlying mechanism for the neuroprotection offered by NAP is targeting neuronal microtubules that are essential for neuronal survival and function.