Introduction

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Vasoactive intestinal peptide (VIP) provides neuroprotection in vitro for neurons in the central and peripheral nervous system (Brenneman and Eiden, 1986; Kaiser and Lipton, 1990; Pincus et al., 1990; Gozes et al., 1991, 1995; Klimaschewski, 1997). In vivo neuroprotection by VIP and VIP derivatives (Gressens et al., 1994; Said, 1996; Said et al., 1996) has been demonstrated in animal models capturing facets of Alzheimer's disease (Gozes et al., 1996, 1997a, 1999). Previous studies have indicated that the neuroprotective actions of VIP were contingent in part on the presence of glial cells (Brenneman et al., 1990) expressing high-affinity VIP-binding sites (Gozes et al., 1991). VIP has been shown to be a secretagogue for several astroglial-derived substances that can increase the survival of central nervous system neurons, including interleukin-1 and other cytokines, chemokines, protease inhibitors (Brenneman et al., 1997; 1999), and activity-dependent neurotrophic factor (ADNF; Brenneman and Gozes, 1996). ADNF derives its name from the protection it offers neurons from apoptosis associated with electrical blockade by tetrodotoxin (Gozes et al., 1997b).

ADNF (14,000 Da and pI = 8.3 ± 0.25) has the following characteristics (Brenneman and Gozes, 1996; Gozes and Brenneman, 1996). First, it provides in vitro neuroprotection at femtomolar concentrations (Brenneman and Gozes, 1996). Second, peptide fragments of 14 amino acids (ADNF-14; Val-Leu-Gly-Gly-Gly-Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala; Brenneman and Gozes, 1996), and of nine amino acids (ADNF-9; Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala; Brenneman et al., 1998) mimic and surpass the activity of the entire protein. And third, ADNF exhibits a high degree of homology to a chaperonin, a protein associated with proper protein folding, heat shock protein 60 (hsp60; containing the sequence Val-Leu-Gly-Gly-Gly-Cys-Ala-Leu-Leu-Arg-Cys-Ile-Pro-Ala; differences from ADNF are in bold; Brenneman and Gozes, 1996; Gozes and Brenneman, 1996). Hsp60-like protein was identified in the extracellular milieu of both astrocytes and neuronal cell lines and VIP has been shown to augment the secretion of an hsp60-like protein (Bassan et al., 1998). Antiserum to hsp60 produced neuronal cell death that was prevented by ADNF (Brenneman and Gozes, 1996) and antibodies prepared against ADNF specifically recognized the active peptides (ADNF-14/9) and induced apoptosis in cerebral cortical cultures (Gozes et al., 1997b). Such antibodies were recently used to screen an expression cDNA library of neuroglial origin. A novel ADNF-14/9-like active peptide (NAP; Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln) with a greater in vivo neuroprotective efficacy compared with ADNF-9, in apolipoprotein E-deficient mice, was disclosed. NAP constitutes a portion of a new VIP-responsive activity-dependent neuroprotective protein [(ADNP) cDNA, including 2484 base pairs of open reading frame, encoding 828 amino acids with a calculated molecular mass of 92,062 kDa (Bassan et al., 1999)]. Femtomolar concentrations of both ADNF-9 (Brenneman et al., 1998) and NAP (Bassan et al., 1999) provided neuroprotection in vitro against a variety of toxins, including the -amyloid peptide, the neurotoxin associated with Alzheimer's disease.

The focus of this report was the investigation of the neuroprotective properties of the ADNF-9 and NAP in animals exposed to the cholinotoxin, ethylcholine aziridium (AF64A), a blocker of choline uptake (Fisher et al., 1989). An intact cholinergic system is required for normal brain function, whereas Alzheimer's disease is associated with the death of cholinergic cells. Thus, rats treated with AF64A provide an accepted model for testing in vivo efficacy of drugs that protect against cognitive impairments that may result from cholinotoxicity (Fisher et al., 1989; Gozes et al., 1996, 1999). We now show that intranasal administration of ADNF-9 and of NAP provided neuroprotection against short-term memory loss associated with AF64A cholinotoxicity. NAP was more efficacious in protecting against loss of cholinergic functions. Furthermore, when administered intranasally, radioactively labeled NAP was found intact in the brain, 30 min after application, and dissipated an hour after the application, suggesting an interesting candidate for future drug design.

	Materials and Methods

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Animals. Male Wistar rats (300-350 g; Harlan Laboratories, Jerusalem, Israel) were used for the cholinotoxicity assays.

Peptide Syntheses. Peptides were synthesized with solid-phase technology and purified to homogeneity by HPLC (Gozes et al., 1999). Purity and identity was ascertained with amino acid analysis and electrospray ionization mass spectrometry (Micromass, Manchester, UK). Additional peptides were purchased from Peptide Technologies (Bethesda, MD).

Cholinotoxicity in Rats and Assessment of Short-Term Spatial Memory in a Water Maze. Rats were subjected to two daily tests in a water maze, including a hidden platform (Morris, 1984; Gordon et al., 1995; Gozes et al., 1997a). Every day for the first test, both the platform and the animal were situated in a new location with regards to the pool (with the pool being immobile). The experiment was performed as follows: the animal was positioned on the platform for 0.5 min then placed in the water. The time required to reach the platform (indicative of learning and intact reference memory) was measured (first test). After 0.5 min on the platform, the animal was placed back in the water (in the previous position) for a second test and search for the hidden platform (retained in the previous position). The time required to reach the platform in the second trial was recorded, indicative of short-term (working) memory. Animals were tested for 4 days to eliminate random memory-defective animals. The best performers were injected i.c.v. at a rate of 0.21 µl/min with AF64A (Sigma Chemical Co., St. Louis, MO; 3 nmol/2 µl/side); control animals received an injection of saline (Gozes et al., 1996). Animals were allowed to recover for 1 week, followed by daily exposure to intranasal administration of 40 µl of 5% Sefsol (Sigma Chemical Co., Rehovot, Israel) and 20% isopropanol (control) or containing 0.5 µg of peptide (experimental; Gozes et al., 1996). After a week of peptide treatment, the animals were subjected to two daily tests in the water maze (as described above). During the test period, animals also were given an intranasal administration of peptide or vehicle (carrier) an hour before the daily tests. To avoid bias related to changes in motor activity in the various treatment groups, a probe trial test that assessed spatial memory also was used as follows. After 4 days of training and testing, the platform was removed and on day 5, the animals were subjected to swimming in the pool (120 s) without the platform; in these experiments, the time spent in the quadrant of the pool where the platform used to be was recorded. Measurements were performed with the HVS video tracking system (HVS Image Ltd., Hampton, UK).

Biodistribution after Intranasal Administration. NAP (mol. wt. = 824.9) was synthesized to include hydroprolines and those were exchanged to produce ³H-labeled peptide (NAP, propyl 3-3,4-[³H]; American Radiolabeled Chemicals, St. Louis, MO). The specific activity was 50 Ci/mmol. The purity and identity of NAP was ascertained with HPLC Zorbax SB-C18 (250 × 4.6 mm) 5 µm, and elution with a 5 to 25% methanol gradient in 0.1% trifluoroacetic acid over 20 min and detection by UV at 220 nm and ³H detector -Ram. Two and a half microliters of a solution containing 1 mCi/ml was applied to each nostril of a (200-300 g) male Wistar rat. At designated time points, rats were sacrificed and tissues were solubilized (100 mg in 1 ml of Luma Solve; Lumac bv., Landgraaf, the Netherlands) at 55°C for 12 h. Radioactivity was determined after the addition of Optiflour (10 ml/100 mg; Packard, Groningen, the Netherlands) in a beta scintillation counter.

Measurements of Cholinergic Activity. Choline acetyl transferase (ChAT) activity was measured according to Fonnum (1975) as before (Gozes et al., 1997a). At the termination of the behavioral experiment, animals were sacrificed and brains (cerebral cortex) dissected and processed as described in Gozes et al. (1997a). Comparisons were made among controls, AF64A-treated-, and AF64A + peptide-treated animals.

Statistical Analyses. Statistical tests used one-way ANOVA with pairwise multiple comparison procedures (Student-Newman-Keuls method).

	Results

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Intranasal Administration of ADNF-9 Protects against Short-Term Memory Loss Associated with AF64A Treatment In Vivo. Because ADNF-9 is a short hydrophobic peptide, we tested the possibility that it may affect brain functions through intranasal administration. Assessments of spatial learning and memory were performed in a water maze, by measurements of the time required to find a hidden platform. Two daily tests were performed. The platform location and the animal's starting point were held constant within each pair of daily trials, but the location of the platform and the animal's starting point were changed every day. In the first daily test, indicative of reference memory, the AF64A-treated animals were retarded compared with control animals as was obvious on the second test day (P < .016). Treatment with ADNF-9 resulted in an apparent insignificant improvement (Fig. 1A). In contrast, in the second daily test [indicative of intact short-term memory (Gordon et al., 1995)], AF64A-treated animals were markedly retarded (P < .001 on all experimental days) and ADNF-9-AF64A-treated animals exhibited significant improvements and reduced latencies throughout the experiment (Fig. 1B; P < .001). ADNF-9 treatment of control (sham-lesion) animals did not change their performance. Figure 1C depicts the results of the probe trial that assessed spatial memory. After 4 days of training and testing, the platform was removed and on day 5, the animals were subjected to swimming in a pool without the platform. It was apparent from the probe trial that the time spent in the quadrant of the pool where the platform was previously positioned was significantly increased (P < .001) in the AF64A-treated animals that were given ADNF-9. The peptide-treated animals were not significantly different from the control (sham-lesion) animals.

View larger version (10K): [in this window] [in a new window]   Fig. 1.   AF64A-treated rats exhibit an impairment in learning and memory that is ameliorated by intranasal administration of ADNF-9. Two daily water maze tests (A and B, respectively) were performed on adult rats. Groups tested were control animals treated with vehicle (20 animals; ); AF64A-treated animals intranasally administered with vehicle (27 animals; ); control animals treated by intranasal administration of ADNF-9 (12 animals; ); AF64A-treated animals intranasally administered with ADNF-9 (19 animals; ). A, latency measured in seconds (mean ± S.E.) to reach the hidden platform in its new daily location (indicative of intact reference memory; Gordon et al., 1995) is depicted. Tests were performed over four consecutive days. *P < .016 compared with control. B, latency measured in seconds to reach the hidden platform 0.5 min after being on it (indicative of intact working memory processes; Gordon et al., 1995; Gozes et al., 1997a) tested over four consecutive days. There were no differences between animals treated with vehicle and untreated animals (data not shown). *P < .001 compared with all experimental groups. C, on day 5 of testing, the platform was removed, and a spatial probe test was performed. The animals were allowed to swim for 120 s, and the time spent by the animal at the platform quadrant was recorded. *P < .001 compared with all experimental groups.

Intranasal Administration of NAP Protects against Short-Term Memory Loss Associated with AF64A Treatment In Vivo. An experiment similar to the one described for ADNF-9 was performed with NAP in control animals and AF64A-treated animals. Herein, the peptide also was administered by intranasal application. On day 1, in the first daily test, immediately after placement on the hidden platform (testing reference memory), NAP-treated animals were significantly improved compared with vehicle-treated controls (Fig. 2A; P < .001). ADNF-9-treated animals did not exhibit this behavior (Fig. 1A). As was indicated above, AF64A treatment resulted in reduced performance in the water maze paradigm and NAP-treated AF64A-impaired animals were significantly different from vehicle-treated AF64A-impaired animals on the 4th day of testing (Fig. 2A; P < .041). In the second daily test, indicative of short-term memory, NAP-treated AF64A-impaired animals were improved throughout the experiment and reached control levels already on test day 2 (Fig. 2B; P < .001). After 4 days of training and testing, the platform was removed and on day 5, the animals were subjected to swimming in a pool without the platform (as described above). Results showed that the time spent in the quadrant of the pool where the platform was previously positioned was significantly increased (Fig. 2C; P < .001) in the AF64A-treated animals that were given NAP compared with AF64A-vehicle-treated animals. Furthermore, peptide-treated groups (control-sham-lesion, or AF64A-lesion) were not significantly different from control (sham-lesion) animals and an apparent insignificant improvement was noted in the NAP-treated groups compared with control (sham-lesion) animals (Fig. 2C).

View larger version (10K): [in this window] [in a new window]   Fig. 2.   AF64A-treated rats exhibit impairments in learning and memory that are ameliorated by intranasal administration of NAP. The same experiment reported in Fig. 1 (A, B, C, respectively) was repeated, except that the peptide used was NAP and the number of animals per each experimental group was 10 to 20, and 27 for the AF64A-treated group. , control; , control + NAP; , AF-64A; , AF-64A + NAP. A, *P < .001 NAP-treated are significantly different than respective vehicle-treated control or AF64 animals; **P < .001 NAP-treated are significantly different than the respective vehicle-treated AF64 animals; B, *P < .001 compared with all experimental groups.

Bioavailability and Stability of NAP. In the above-mentioned water maze tests, NAP administration resulted in an apparently enhanced behavioral improvement (Fig. 1) compared with ADNF-9 application (Fig. 2). Previously, ADNF-9 was less effective than NAP in ameliorating memory deficits in the apolipoprotein E-deficient mice (Bassan et al., 1999) and PBS solutions of ADNF-9 lost biological activity upon storage at temperatures 4°C (Brenneman et al., 1998). We thus decided to evaluate the bioavailability and stability of NAP as a future therapeutic. A time course of distribution of [³H]NAP that was applied intranasally was measured in the various organs of the body. Results (Fig. 3A) demonstrated high levels of total radioactivity (calculated as femptomoles NAP per gram tissue) in the intestine and liver, with highest levels in the intestine, 30 min after administration. The total radioactivity in the brain (cortex) was highest 60 min after administration (Fig. 3B). Each animal received 5 µl of [³H]NAP containing 2.5 million dpm (22.75 pmol). If distributed homogeneously in the 250-g rat, then 91 fmol/g of tissue is assumed (with 300-g rats having 75.5 fmol/g of tissue). Our results indicated 45 fmol/g of tissue. Reversed phase-HPLC suggested that the peptide was intact in the brain 30 min after application (Fig. 3C). Of the 807.8 fmol/g of tissue eluted from the column, 98 fmol/g of tissue comigrated with intact NAP, suggesting that at least 12% of the material was intact in the brain 30 min after application. Sixty minutes after application, of the 1198.9 fmol/g of tissue eluted from the column, only 2% coeluted with intact radioactive NAP (Fig. 3D). These results suggested that the half-life of NAP in the cortex is ~15 min. Close examination of Fig. 3, A and B, showed higher levels of radioactive NAP in the blood than in the cortex, especially 3 h after administration, a time when the peptide is probably completely broken down (Fig. 3D). Thus, the increased level of radioactivity in the blood, at later times after peptide application may reflect peptide breakdown and dissipation. To examine whether the peptide is present in brain tissue, rather than within cerebral blood vessels, an additional experiment was performed. Herein, 200-g male rats were treated as described above and 30 min after peptide application (a time when the peptide is still intact; Fig. 3C) brains were dissected and small, visible blood vessels were carefully removed. Results demonstrated that although some of the radioactivity was due to small, visible blood vessels, most of it was found in the apparent brain tissue, with visible blood vessel contribution being insignificant (Fig. 3E). Furthermore, the cerebellum (free of small, visible blood vessels), which is further away from the olfactory bulb than the cortex, had apparently less radioactive peptide accumulation. However, the difference between the cerebral cortex and the cerebellum was not significant, suggesting rapid peptide distribution (Fig. 3, B and E).

View larger version (34K): [in this window] [in a new window]   Fig. 3.   Intranasally applied [3H]NAP reaches the body and the brain. A, animals were sacrificed at indicated times after administration and tissue samples were weighed and assayed (in duplicate) for radioactivity in a beta counter; a mean of four animals is depicted. B, brains were dissected at indicated time points and radioactivity monitored. C and D, intact [3H]NAP reached the brain after intranasal administration. Radioactive tissue samples (cerebral cortex) were homogenized and subjected to low-speed centrifugation. Supernatants [30 min after application;  (C) and 60 min after application;  (D)] were analyzed by HPLC fractionation against [3H]NAP stock (). Samples were monitored for radioactivity (disintegrations per minute) in a beta counter. All results were calculated to depict radioactivity as femtomoles of NAP per gram of tissue. E, experiment was repeated with three additional animals that weighted 200 g each instead of 250 to 300 g in A to D and small, visible blood vessels were removed with watchmaker's forceps (no. 5).

AF64A-Treated Animals Exhibit a Reduction in Choline Acetyl Transferase Activity: Protection by NAP. Enzymatic assays on brain extracts derived from AF64A-treated animals and sham-treated controls (three animals per group, each in triplicates) revealed a very minor reduction (11 + 2.6%) in choline acetyl transferase activity at the termination of the experiment (Fig. 4A). However, AF64A-animals that were treated with ADNF-9 showed a 36 + 5% reduction in cholinergic activity (P < .001), suggesting that ADNF-9 did not improve cholinergic functions, but the combination of AF64A treatment and ADNF-9 may have had an additive deleterious effect. In contrast, NAP treatment of AF64A-animals resulted in increased cholinergic activity indistinguishable from control (sham-operated) values (Fig. 4A; 100% choline acetyl transferase activity indicated 130 pmol/mg of protein/min). Because the AF64A-animals treated with ADNF-9 showed a reduction in cholinergic activity, another test was used to separate the immediate and the long-term effects of the peptides. Thus, in four groups of animals, three were treated with AF64A, allowed a week for recovery, and then two groups were treated (intranasally) with either ADNF-9 or NAP. After 5 treatment days, the animals were allowed to recover for 2 days and then subjected to daily water-maze tests (Figs. 1 and 2). The difference between this experiment and the experiments described above (Figs. 1 and 2) is that the animals did not receive a daily intranasal application of peptides before the behavioral test. Under these conditions, ADNF-9 treatment did not improve cognitive functions as depicted in Fig. 4B (second daily test in the water maze). In contrast, NAP-treated AF64A animals were not significantly different from control rats and were significantly faster in finding the hidden platform in the water maze compared with ADNF-9-treated AF64A-rats (P < .022).

View larger version (29K): [in this window] [in a new window]   Fig. 4.   A, intranasal application of NAP prevents reduction in choline acetyl transferase activity in AF64A-treated rats. Incorporation of radiolabeled choline into acetylcholine is shown. Results were calibrated against control (100%). Experiments with three animals per group (each in triplicate) were conducted and analyzed as described in the text. B, AF64A-treated rats exhibit impairments in learning and memory, long-lasting effects of NAP, but not of ADNF-9 treatment. Ten male rats (as described in Materials and Methods) were used per experimental group. Four groups were used, three were treated with AF64A and one group was treated with saline (control). The rats were allowed a week for recovery, and then two AF64A groups were treated (intranasally) with either ADNF-9 or NAP. After 5 treatment days, the animals were allowed to recover for 2 days and then subjected to daily water-maze tests (Figs. 1 and 2). The difference between this experiment and the experiments in Figs. 1 and 2 is that the animals did not receive a daily intranasal application of peptides before the behavioral test. The figure depicts the second daily test indicative of short-term memory. , control; , AF64A; , AF64A + NAP; , AF64A + ADNF-9. *P < .022 the AF64A + ADNF-9 group compared with AF6YA + NAP; **P < .013 AF64A compared with control, AF64A + ADNF-9 compared with control.

	Discussion

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This study has demonstrated in vivo efficacy for ADNF-like peptide neuroprotection. Intranasal administration of ADNF-9 or NAP protected against loss of short-term memory associated with AF64A treatment. NAP administration also improved reference memory in control animals. Furthermore, NAP protected against reductions in choline acetyl transferase activity, as was demonstrated for apolipoprotein E-deficient mice by Bassan et al. (1999). NAP distribution in the brain and the body was rapid. The calculated half-life of NAP in the brain after intranasal administration was ~15 min. HPLC analysis clearly indicated that NAP is metabolized in vivo to multiple fragments, suggesting the possibility of active metabolites.

Previous studies with the ADNF secretagogue VIP have shown that treatment with a specific VIP hybrid antagonist (Gozes et al., 1991) of newborn rat pups or developing embryos resulted in neurodegeneration as follows: 1) severe microcephally (Gressens et al., 1994); 2) delays in the acquisition of developmental milestones of behavior (Wu et al., 1997); 3) disturbances in the diurnal rhythm of motor behavior (Gozes et al., 1995); and 4) distortions in neuronal structure (Hill et al., 1994). Treatment of adult animals with the same VIP antagonist resulted in impairments in spatial memory, when the antagonist was applied i.c.v. (Glowa et al., 1992). Partial blockade of VIP expression in transgenic animals also has demonstrated the same impairments in the adult animal (Gozes et al., 1993). These results, coupled with the developmental regulation of brain VIP gene expression and the marked decline with aging (Gozes et al., 1988; Zhou et al., 1995; Krajnak et al., 1998), suggest an involvement of VIP in brain development and complex functions.

Lipophilic VIP analogs were designed that surpassed the neuroprotective activity attributed to the intact peptide. Stearyl-Nle¹⁷-VIP (SNV; Gozes et al., 1995) offered protection against cholinotoxicity (Gozes et al., 1996) and against developmental retardation and memory deficits in apolipoprotein E-deficient mice (Gozes et al., 1997a). Recent studies have suggested that the VIP/SNV neuroprotective action against excitotoxicity (Gressens et al., 1997, 1999) and the VIP growth factor effects on developing embryos were mimicked by ADNF and prevented, at least in part, by ADNF antibodies (Glazner et al., 1999). Furthermore, incubation of astrocytes with VIP resulted in 2- to 3-fold increases in the mRNA encoding activity-dependent neuroprotective protein (ADNP), the protein containing the NAP sequence, that is recognized by ADNF antibodies (Bassan et al., 1999).

The mechanism by which ADNF/ADNP and ADNF/ADNP-derived peptides provide neuroprotection remains an enigma. Studies of ADNF-9 action in mixed (glia plus neurons) versus glia-depleted neuronal cultures indicated that ADNF-9 can act directly on neurons, although the potency of the peptide was 10,000-fold greater in mixed cultures, suggesting additional active molecules (Brenneman et al., 1998). Kinetic studies showed that exposure to ADNF-9 for only 2 h was sufficient to produce a 4-day protection against the cell-killing action of tetrodotoxin (Brenneman et al., 1998). Treatment with bafilomycin A1 (an inhibitor of receptor-mediated endocytosis) for 2 h prevented this ADNF-9-associated neuroprotection. Part of the neuroprotection offered by ADNF-9 may involve regulated increases in chaperonins, proteins that maintain proper folding of other intracellular proteins. An example is hsp60 that was recently shown to be reduced in neurons upon incubation with a toxic fragment of the -amyloid peptide. Incubation with ADNF-9 resulted in rapid increases in hsp60 mRNA and protein and protected neurons against death associated with -amyloid toxicity (Zamostiano et al., 1999).

Mattson and colleagues (Guo et al., 1999) studied ADNF-9-mediated protection in mouse hippocampal neurons derived from control and mutant presinilin-1 knock-in mice (associated with overexpression of the toxic -amyloid peptide and early-onset Alzheimer's disease). Their results showed that a pretreatment with ADNF-9 or with basic fibroblast growth factor (bFGF) before exposure to glutamate excitotoxicity resulted in reduced oxiradical production and increased mitochondrial function, providing significant protection. Furthermore, the Ca²⁺ influx response to glutamate was suppressed in neurons pretreated with ADNF-9 and bFGF. This study places ADNF-9 on a par with bFGF, both factors interrupting excitotoxic neurodegenerative cascades promoted by the presenilin-1 mutation.

NAP, like ADNF-9, albeit with a much broader range of effective concentrations, was neuroprotective in vitro against N-methyl-D-aspartate (Brenneman et al., 1998; Bassan et al., 1999). In the current study, NAP, compared with ADNF-9, was more efficacious in maintaining choline acetyl transferase activity in vivo (Fig. 4; Bassan et al., 1999). The insignificant reduction in cortical choline acetyl transferase in the cerebral cortex after AF64A injection has been described in Lamberty et al. (1992). AF64A + ADNF-9 treatment resulted in ~40% reduction in choline acetyl transferase activity (Fig. 4) yet performance improved in the maze (Fig. 1). These results suggest that AF64A may cause changes in the maze test by a mechanism other than destruction of cholinergic neurons. ADNF-9 treatment might be detrimental to cholinergic neurons in vivo as also may be suggested from the behavioral tests (Fig. 4B); furthermore, in spinal cord cultures, ADNF antiserum produced a 20% decrease in choline acetyl transferase activity compared with controls (Gozes et al., 1997b). The different effects of ADNF-9 and NAP on cholinergic activity (Fig. 4A) and in the behavioral tests (Fig. 4B) suggest that the two peptides act through different mechanisms to improve cognitive functions, with ADNF-9 having an immediate short-term effect. Furthermore, animals treated with NAP by intranasal application exhibited increased learning and memory abilities in the water maze test compared with ADNF-9-treated animals (Figs. 1 and 2). Similarly, injection of NAP (and not of ADNF-9) to newborn apolipoprotein E-deficient mice prevented short-term memory deficits in the 3-week-old pups (Bassan et al., 1999). The longer peptide ADNF-14 was even less efficacious than ADNF-9 in vivo (Bassan et al., 1999). These studies suggest a wide range of neuroprotective activities for NAP. Indeed, NAP (over a wide range of concentrations) provided protection against buthionine sulfoximine-induced decreases (70-90%) in neuroblastoma cell viability (Offen et al., 2000). Buthionine sulfoximine, a selective inhibitor of glutathione synthesis, causes a marked decline in reduced glutathione in neuronal cell models leading to decreased viability (Offen et al., 2000). Thus, the mechanism of neuroprotection by NAP may be mediated through raising cellular resistance against oxidative stress, a general mechanism affecting cell survival. Furthermore, preliminary toxicology studies have shown no toxic effects for this peptide (supported by National Institute on Aging, Bethesda, MD, through a contract with MPI Research Inc., Mattawan, MI).

In conclusion, the demonstrated in vivo efficacy of NAP coupled with its bioavailability and apparent stability identify it as an attractive lead compound for the development of therapeutic agents against neurodegenerative diseases. Currently, drugs for symptomatic treatment of Alzheimer's disease target directly the function of the cholinergic system. An example is tacrine, an inhibitor of acetylcholine esterase (van Reekum et al., 1997). However, growth factors treatment may afford a broader range of neuroprotection, hence studies on in vivo effects of neurotrophic factors provide important basic information and open new horizons for drug design. Future experiments regarding the mechanism of action of ADNF-9 and NAP should expand our understanding of neuronal fate, survival, and death.