A Femtomolar Acting Octapeptide Interacts with Tubulin and Protects Astrocytes against Zinc Intoxication*

An octapeptide was previously described that protects neurons against a wide variety of insults directly and indirectly as a result of interactions (at femtomolar concentrations) with supporting glial cells. The current study set out to identify the octapeptide binding molecules so as to understand the high affinity mechanisms of cellular protection. Studies utilizing affinity chromatography of brain extracts identified tubulin, the brain major protein, as the octapeptide-binding ligand. Dot blot analysis with pure tubulin and the biotinylated octapeptide verified this finding. When added to cerebral cortical astrocytes, the octapeptide (10-15–10-10 m) induced a rapid microtubule reorganization into distinct microtubular structures that were stained by monoclonal tubulin antibodies and visualized by confocal microscopy. Fluorescein-labeled octapeptide induced a similar change and was detected in the intracellular milieu, even when cells were incubated at 4 °C or at low pH. In a cell-free system, the octapeptide stimulated tubulin assembly into microtubules. Furthermore, treatment of astrocytes with zinc chloride resulted in microtubule disassembly and cell death that was protected by the octapeptide. In conclusion, the results suggest that the octapeptide crosses the plasma membrane and interacts directly with tubulin, the microtubule subunit, to induce microtubule reorganization and improved survival. Because microtubules are the key component of the neuronal and glial cytoskeleton that regulates cell division, differentiation, and protection, this finding may explain the breadth and efficiency of the cellular protective capacities of the octapeptide.

The octapeptide NAP, 1 Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln (single-letter code: NAPVSIPQ), is a femtomolar-acting neuroprotective peptide derived from activity-dependent neuroprotective protein (ADNP) (1,2). The discovery of NAP resulted from studies on the major neuropeptide, vasoactive intestinal peptide (VIP). VIP, released from nerve cells, provides neuronal protection by inducing the synthesis and secretion of neuroprotective proteins from astrocytes (3,4). ADNP was identified as a VIP-responsive glial protein (1,5), and VIP neuroprotection was suggested to be mediated in part through increased synthesis of ADNP (5). Decreased ADNP synthesis by antisense oligodeoxynucleotides resulted in increased p53 expression and cell death (2).
NAP, an eight-amino acid peptide derived from ADNP that was identified by peptide activity scanning, was shown to be neuroprotective in a wide variety of systems and against multiple neurotoxins (6). NAP provided neuroprotection at remarkably low concentrations against the Alzheimer's disease toxin (␤-amyloid peptide) (1,7), the toxic envelope protein of the human immunodeficiency virus (HIV; gp120) (1), glucose deprivation (7), electrical blockade (tetrodotoxin) (1), oxidative stress (hydrogen peroxide) (8), dopamine toxicity, and decreased glutathione (9), in vitro. Further studies indicated an exceptionally broad range of neuroprotective concentrations against excitotoxicity (N-methyl-D-aspartate; 10 Ϫ16 -10 Ϫ8 M) (1). Treatment of mixed neuroglial populations identified two peaks of activity at femtomolar and at nanomolar concentrations; in contrast, treatment of enriched neuronal populations indicated only one peak of activity, at nanomolar concentrations of NAP. These results suggest a direct and an indirect mode of action for NAP, with the latter involving glial cells (1,7,10).
In vivo, NAP was identified as a potent neuroprotective peptide in a mouse model of apolipoprotein E deficiency (knockout) (1) and in a rat model of cholinotoxicity (11). The lipid carrier, apolipoprotein E, has been implicated as a risk factor in Alzheimer's disease, and because the knock-out mice exhibit short-term memory deficits that are ameliorated by chronic peptide treatment, NAP holds promise for future treatment against disease-related short-term memory deficits. Furthermore, Alzheimer's disease is associated with death of cholinergic neurons. Rats treated with the cholinotoxin ethylcholine, aziridium, provide a model of cholinotoxicity. A week following a single intracerebroventricular injection of the cholinotoxin, NAP administration ensued using daily intranasal application. Significant improvements in short-term spatial memory in NAP-treated animals were observed (11). Cognition enhancement was also found in the Morris water maze paradigm in middle-aged rats treated daily (by intranasal administration) with NAP (12).
In acute in vivo models of neuronal damage, a single NAP subcutaneous injection after closed head injury dramatically reduced mortality and facilitated clinical recovery (motor ability, balancing, and alertness) in mice (13). Water accumulation (edema) was reduced by 70% in the NAP-treated mice. Magnetic resonance imaging demonstrated significant brain tissue * This work was supported in part by the Institute for the Study of Aging and by the Israel Science Foundation, U.S.-Israel Binational Science Foundation, Institute for the Study of Aging, and Allon Therapeutics, Inc. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ This work is submitted in partial fulfillment of the requirements for a Ph.D. at Tel Aviv University.
ʈ Holds the Lily and Avraham Gildor Chair for the Investigation of Growth Factors. To whom correspondence should be addressed. Tel.: 972-3-6407240; Fax: 972-3-6408541; E-mail: igozes@post.tau.ac.il. 1 The abbreviations and trivial terms used are: NAP, octapeptide containing the amino acid sequence of NAPVSIPQ; ADNP, activitydependent neuroprotective protein; VIP, vasoactive intestinal peptide; PIPES, 1,4-piperazinediethanesulfonic acid; recovery in NAP-treated animals. Pretreatment with NAP by daily subcutaneous injections for the first 3 months of life followed by head trauma at the age of 4 months resulted in faster recovery and enhanced performance in a learning and memory paradigm (14). Middle cerebral artery occlusion in rats is manifested in stroke-like symptoms, including infarct formation, neuronal cell death, motor disabilities, and sensory impairments. A single intravenous NAP injection up to 4 h following artery occlusion resulted in a significant reduction in infarct size, marked protection against neuronal death (apoptosis), and defense against motor and sensory disabilities (15). Both closed head injury and middle cerebral artery occlusion can be considered as acute brain damage with severe secondary outcome that could be prevented, in part, by neuroprotection. In both cases, oxidative stress that is a result of the initial insult may exacerbate the damage. In this respect, fetal demise and growth restriction caused by overexposure of pregnant mice to alcohol are thought to be because of severe oxidative damage. In pregnant mice exposed to one intraperitoneal injection of alcohol, fetal demise was inhibited by a single injection of NAP (16).
Given the breadth of the protective activities of NAP, its mechanism of action is of great interest. Potential signal transduction pathways include cGMP production (17) and interference with inflammatory mechanisms, tumor necrosis factor ␣, and MAC1-related changes (13,14,18). NAP-associated protection against oxidative stress (8,9), glucose deprivation (7), and apoptotic mechanisms (15) suggests interference with fundamental processes. The current study identifies the tubulin subunit of microtubules as the primary target of NAP, disclosing that indeed NAP interacts with a key component of the cell and facilitates survival.

EXPERIMENTAL PROCEDURES
Affi-Gel 10 NAP Affinity Chromatography-A protein lysate was prepared from 1-day-old rat brains in a buffer containing the following ingredients: 150 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl, pH 4.5, 0.1% Triton X-100, 1% Nonidet P-40, and a protease inhibitor mixture (Roche Diagnostics). DNA was fragmented by sonication. Cell debris was discarded following 30 min of centrifugation at 30,000 ϫ g. An affinity column containing NAP was prepared using elongated NAP (KKKG-GNAPVSIPQC, synthesized as before) (1,11) and Affi-Gel 10 in 0.2 M NaHCO 3 , 0.5 M NaCl, pH 7.5. Further column preparation was according to the manufacturer's instructions (Amersham Biosciences). The brain extract prepared as above was loaded (2 mg/ml) on the column at 20°C and incubated for an hour; the column was then washed with phosphate-buffered saline until all unbound protein eluted (as confirmed by protein assay (Bradford; BioRad). NAP-binding proteins were eluted in 0.1 M glycine (pH 3.0), and the eluted protein fractions were adjusted to pH 7.5 with Tris-HCl buffer. Electrophoresis on a 12% polyacrylamide SDS-containing gel was then performed (2).
Sulfolink Coupling Gel NAP Affinity Chromatography-The second isolation efforts utilized a different affinity column, sulfolink coupling gel (Pierce). Binding of CKKKGGNAPVSIPQ was performed according FIG. 1. Affinity chromatography: isolation of NAP-binding proteins. NAP was bound to either Affi-Gel 10 or sulfolink coupling gel, and protein extracts from 1-day-old rat brains were loaded on the resulting affinity columns (see "Experimental Procedures"). Proteins from the different stages of the purification procedure were separated on a 12% polyacrylamide SDS-containing gel followed by GelCode Blue protein stain (Pierce). The stained gel lanes depicted on the figure contain the following protein samples: lanes 1 and 4, protein molecular mass markers (BioRad); lane 2, initial brain extract; lane 3, NAPbinding proteins that were eluted with 0.1 M glycine, pH 3.0, from an Affi-Gel 10-NAP affinity column; lane 5, NAP-sulfolink wash through proteins; lane 6, NAP-binding proteins displaced (eluted) from the NAPsulfolink affinity column by competition with excess soluble NAP. (MW, the molecular mass in kDa).

FIG. 2. Identification of tubulin as a NAP-binding protein.
A, sequence analysis. The major NAP-binding protein band observed in Fig. 1, lane 3 (ϳ50,000 daltons) was excised, and subjected to ingel proteolysis with trypsin and mass spectrometry. Results identified the sequence depicted on the figure. B, dot blot analysis of biotin-labeled NAP and tubulin. Purified tubulin (Sigma) was bound to a nitrocellulose membrane. The membrane was then incubated in a blocking solution, followed by a second incubation with biotin-labeled NAP and detection by avidin-horseradish peroxidase conjugate and ECLϩ. For further details, see "Experimental Procedures." to the manufacturer's instructions. Brain extract was prepared as above. Binding was performed at 4°C for 20 h, washing was as above, and elution was performed with NAP (NAPVSIPQ) in 2 mg/ml phosphate-buffered saline (2 ml/2-ml column) at 4°C for 20 h.
Sequence Analysis-To further identify the NAP-binding protein, the polyacrylamide gel portion containing the major purified protein band was subjected to in-gel proteolysis with trypsin and mass spectrometry analysis by the Smoler Protein Center, Department of Biology, The Technion, Israel Institute of Technology.
Cell Cultures-Rat cerebral cortical cells from newborn pups were prepared as before (1). In short, cerebral cortical tissue was incubated for 20 min at 37°C in Hanks' balanced salt solution containing 15 mM HEPES, pH 7.3 (Biological Industries, Beit Haemek, Israel), and trypsin. Dissociated cerebral cortical cells were added to the culture dish with 5% horse serum in Dulbecco's modified Eagle's medium. Cells were plated in a ratio of 1 cortex/two 75-cm 2 cell culture flasks (polystyrene; Corning Glass). The medium was changed 1 day after plating. Cells were split after 10 days of incubation, plated in 24-well plates (each flask into 60 wells containing 250 l of medium), and incubated for an additional 2 weeks. In some experiments, ZnCl 2 (150 M; Sigma) (20) was added for an additional incubation of 16 h. NAP (10 Ϫ15 -10 Ϫ10 M) was added either together with ZnCl 2 or 3 h after the addition of the ZnCl 2 solution. incubated for 15 min to 24 h. After incubation, cells were extensively washed and fixed in 4% paraformaldehyde. After fixation, Triton X-100 (0.2%) was added to allow antibody cellular penetration using mouse monoclonal tubulin antibodies (TUB2.5) (21) and rhodamine-labeled secondary goat antimouse IgG (Jackson ImmunoResearch, West Grove, PA). Fluorescently stained cells were analyzed using a Zeiss confocal laser scanning microscope. The Zeiss LSM 410 inverted (Oberkochen, Germany) is equipped with a 25-milliwatt krypton-argon laser (488 and 568 maximum lines) and 10-milliwatt helium-neon laser (633 maximum lines). A ϫ40 numeric aperture/1.2 C Apochromat water-immersion lens (Axiovert 135 M; Zeiss) was used for all imaging.
Statistical Analysis-All values are given as means Ϯ S.E. from a number of independent experiments as indicated in the figure legends. Results were analyzed for statistical significance by Student's t test and analysis of variance followed by Dunnet's multiple comparisons test.

Isolation of NAP-binding Proteins by Affinity
Chromatography-Brain homogenates were chosen as a putative enriched source for NAP-interacting molecules. Extracts were subjected to affinity chromatography comprising NAP bound to two different solid supports, Affi-Gel 10 and sulfolink coupling gel. Elution of the NAP-interacting molecules was obtained by either reducing the pH or by competing the binding to the insoluble NAP with excess free soluble NAP. Electrophoresis on a 12% polyacrylamide SDS-containing gel revealed a major purified protein band at about 50,000 daltons (Fig. 1); this protein was subjected to further characterization.
Tubulin Is a NAP-binding Protein-When the gel portion containing the purified ϳ50,000-dalton protein band was submitted to in-gel proteolysis with trypsin and mass spectrometry analysis, this NAP-binding protein was identified as rat-␣ tubulin (molecular mass 50,242), gi223556 (Fig. 2A). The identification of tubulin included the characterization of 6 different tryptic peptides ( Fig. 2A, bold). A dot blot assay performed with spotted tubulin indicated NAP binding to tubulin (Fig. 2B).
NAP Interaction with Tubulin/Microtubules: Confocal Microscopy-To establish an association between tubulin (22,23) and NAP in the living cell, we initiated confocal microscopy analysis of fluorescent NAP and immunodetection of tubulin (21). To visualize the microtubule structure, monoclonal ␤-tubulin antibodies (TUB2.5) (21) were used to stain primary astrocyte cultures. Either fluorescein-labeled NAP or native NAP was added to 2-week-old astrocyte cultures. Astrocytes were used as a model because previous results have indicated that, although nanomolar concentrations of NAP protected neuronal enriched cultures against ␤-amyloid toxicity (7), a more potent protection at femtomolar concentrations of NAP was observed when neurons were plated on a bed of astrocytes (1). A time course experiment suggested that the microtubule reorganization was apparent in astrocytes 2 h after NAP application, with an additional condensation 4 h after application, and returned to the original morphology 24 h after NAP application (Fig. 3A). Mitotic spindles were not noticeable. Similar microtubule reorganizations were observed with NAP at concentrations ranging from 10 Ϫ15 to 10 Ϫ10 M with fluoresceinlabeled and with native NAP. Evaluation of the number of cells undergoing microtubule reorganization following NAP treatment showed maximal organization at 2-4 h with a decline at 24 h (Fig. 3B). A control peptide, C2 (VLGGGSALL), that does not protect cells in vitro (24) did not induce a microtubuleassociated morphological change (Fig. 3A).
Detection of Fluorescein-labeled NAP-After a 2-h incubation at 37°C, fluorescein-labeled NAP was detected inside the cell (Fig. 4A). A critical question is whether NAP induces microtubule reorganization through interaction with a surface receptor or whether it is a pore-forming peptide that interacts with the FIG. 6. NAP protects astrocytes against zinc intoxication. Two-weekold astrocyte cultures were incubated with zinc chloride (ZnCl 2 ; 150 M) alone or with NAP at various concentrations (10 Ϫ15 -10 Ϫ10 M, designated NAP 10 Ϫ15 , NAP 10 Ϫ12 , NAP 10 Ϫ10 M). After a 16-h incubation period, cell viability was analyzed by metabolic activity assays (MTS) as described under "Experimental Procedures." The data shown in the bar graph represent the means Ϯ S.E. derived from three independent experiments with each experimental point performed in five repeats; ***, p Ͻ 0.001 versus ZnCl 2 treatment. Results are represented as fraction (-fold increase) from control untreated cultures that were given a value of 1.
FIG. 7. NAP protects astrocytes against zinc-associated microtubule rearrangement. Two-week-old astrocyte cultures were cotreated with ZnCl 2 (150 ⌴) and NAP (10 Ϫ15 M) for 4 h. Cells were then subjected to microtubule staining as in Fig. 3. In short, tubulin was detected with mouse monoclonal antibodies (TUB2.5) (21) and secondary rhodamine-labeled goat anti-mouse IgG. Bars, 20 m. lipid bilayer and is then internalized into cells. To evaluate potential surface labeling, the initial incubation was carried out at 4°C and in a parallel experiment at pH 3.0. When NAP (10 Ϫ15 M) was incubated with astrocytes at pH 3.0 for 15 min (Fig. 4B), microtubule reorganization was apparent and fluorescein-labeled NAP was visualized inside the cells. At 4°C, although microtubule reorganization did not take place because microtubules undergo disassembly at 4°C (25), a dosedependent intracellular accumulation of NAP was apparent (Fig. 4B).
NAP Promotes Tubulin Assembly-Using a high through-put analysis kit containing bovine tubulin (Cytoskeleton), tubulin assembly was determined in the presence of increasing NAP concentrations. Measurements included absorbance determinations at 350 nm. Although 10 Ϫ18 M NAP did not influence microtubule assembly in the test tube (data not shown), 10 Ϫ15 M NAP stimulated microtubule assembly in a similar way to paclitaxel (Fig. 5). Paclitaxel, a known tubulin stabilizing agent also suggested as a neuroprotective agent (26), was used as a positive control. NAP at 10 Ϫ10 M promoted tubulin assembly to the same degree as at a concentration of 10 Ϫ15 M (data not shown). C2, a peptide that was utilized as a negative control in the cellular assay (Fig. 3A), did not affect microtubule assembly as well.
NAP Protects against Zinc Intoxication-When 2-week-old astrocyte cultures were exposed to ZnCl 2 (16 h of incubation), there was an 88 Ϯ 0.008% reduction in cellular metabolic activity (Fig. 6). This marked reduction in metabolic activity (associated with cellular viability) was coupled to changes in microtubule organization observed 4 h after the addition of ZnCl 2 (Fig. 7). NAP added together with ZnCl 2 at concentrations ranging from 10 Ϫ15 to 10 Ϫ10 M protected completely against ZnCl 2 intoxication (Fig. 6, p Ͻ 0.01). Furthermore, the cellular microtubule network assumed the same organization when NAP was added in the presence or in the absence of ZnCl 2 (Fig. 7). Cell counts 4 h after the addition of NAP or ZnCl 2 , or both, showed that of 100 astrocytes treated with ZnCl 2 , 73 showed condensed tubulin structures as depicted in Fig. 7. In contrast, all the NAP-treated cells showed organized microtubular structures, and of 101 cells treated with ZnCl 2 and NAP, 72 cells showed organized microtubular structures as depicted in Fig. 7.

DISCUSSION
The present report outlines a mechanism of action for the short protective peptide, NAP. Affinity chromatography coupled to mass spectrometry identified tubulin, the subunit protein of microtubules, as a major NAP-binding protein. NAP internalization into astrocytes was observed at low pH and low temperatures, suggesting cellular penetration without a classical peptide receptor. Incubation of astrocytes with NAP resulted in time-dependent microtubule reorganization. Furthermore, NAP accelerated microtubule assembly in vitro. Finally, NAP protected astrocytes against ZnCl 2 toxicity, a protection that was paralleled with microtubule reorganization.
Generally speaking, cellular membranes are composed of lipid-protein bilayers that are freely permeable to small, nonionic lipophilic compounds and are inherently impermeable to polar compounds, macromolecules, and therapeutic or diagnos- tic agents. However, protein and peptide sequences have been described that have the ability to translocate other polypeptides that are fused to them across a cell membrane. Examples include the VP22 translocation domain from herpes simplex virus and signal peptides such as the Kaposi fibroblast growth factor h region. Furthermore, toxin molecules also have the ability to transport polypeptides across cell membranes. Often, such polypeptides are composed of at least two parts (called "binary toxins"): a translocation or binding domain and a separate toxin domain. Typically, the translocation domain binds to a cellular receptor, and then the toxin is transported into the cell. Several bacterial toxins, including Clostridium perfringens toxin, diphtheria toxin, Pseudomonas exotoxin A, pertussis toxin, Bacillus anthracis toxin, and pertussis adenylate cyclase, have been used in attempts to deliver peptides to the cell cytosol as internal or amino-terminal fusions (27)(28)(29)(30)(31)(32). Sequence comparisons (Table I) suggest that NAP shares a remarkable structural similarity with the above mentioned variety of translocation proteins, although not necessarily with the precise translocation domain (Table I). Typically, these toxins, like NAP, act at very low concentrations (33). In both cases the mechanism of action may involve a nucleation process that does not require equimolar concentrations (38).
NAP was also shown to be active in an all D-amino acid conformation, suggesting a non-chiral mechanism of activity that may not require a surface receptor (34). A number of the previous studies of non-chiral effects of peptides implied membrane interaction through the formation of pores, as in the case of the antibiotics, magainin and cecropin A (35). Another example of peptides that produced biological actions in the D-or Lform is the amyloidogenic peptide A␤-(1-42) that is associated with Alzheimer's disease neurodegeneration (36). A␤-  forms cation channels in model membranes (37). NAP was recently shown to bind to and inhibit toxic A␤ aggregation, suggesting structural interaction between these two peptides and a chaperone action for NAP that will support the microtubule association described above (38). Furthermore, a direct interaction between the A␤ peptide and tubulin has been suggested (39). Other studies have identified binding of antimitotic peptides to tubulin (40). Furthermore, VP22, which can transverse membranes and which exhibits structural similarity with NAP (Table I), was shown to enhance microtubule bundling. However, VP22 is a 38,000-dalton protein, and the microtubule rearrangement domain in VP22 seems to require a big portion of this protein (41).
Our findings are of importance because it has been known since the earliest electron microscopic studies of Alzheimer's disease that microtubules are decreased in number in the diseased brain as compared with the healthy brain (42). In the present study, we have seen an interaction with ␣ tubulin (Fig.  2A), and acetylated ␣ tubulin immunoreactivity has been inferred as a marker of stable microtubules (43).
Astrocytes constitute a major cell population in the brain and have been associated with neuronal survival; they function mainly as supporting cells (e.g. Refs. 1, 3, 4, 10, 24, 45). Furthermore, specific astrocyte populations serve as neuronal stem cells (46,47). Thus, astrocyte maintenance may constitute an importance aspect when designing therapeutic interventions against neuronal trauma and neurodegenerative diseases (48,49).
A recent report (50) suggests that Alzheimer's disease is associated with down-regulation of cytoskeletal protein synthesis and up-regulation of inflammatory response proteins. These findings are in agreement with our results suggesting a downregulation of the macrophage/microglial marker MAC1 (18) after NAP treatment.
In conclusion, NAP, which provides protection at femtomolar concentrations, seems to interact with the glial tubulin cytoskeleton after cellular internalization, a process which does not seem to require a conventional peptide-receptor interaction. The NAP doses required for tubulin polymerization concurred with the doses required for cellular protection against ZnCl 2 intoxication that was associated with microtubule reorganization. NAP mimicked the effects observed with paclitaxel, which is also suggested as a cytoprotective agent (26,51). Furthermore, neurosteroids (pregnenolone) are suggested to offer cytoprotection through interaction with the neural microtubule-associated protein type 2 and to increase both the rate and extent of tubulin polymerization (44). Because the tubulin cytoskeleton is a key component of the maintenance and stability of the nervous tissue, the results presented above explain in part the breadth and potency of the protective effect of NAP.