Introduction

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Neurotrophic factors such as nerve growth factor (NGF), glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 and -4/5 promote the differentiation, growth, and survival of neurons during development and adulthood (Henderson, 1996). Neurotrophic factors exert their action through activation of a number of signaling pathways, including activation of the mitogen-activated protein kinases (MAPK)/extracellular signal-regulated kinases (ERK) (Greene and Kaplan, 1995). The significant advances that have been achieved in understanding the mechanism of action and the biological role of these compounds support the use of neurotrophic factors in neurodegenerative diseases (Lindsay et al., 1994; Yuen and Mobley, 1995).

Animal models useful for the analysis of neurodegenerative pathogenic mechanisms have been developed, and several neurotrophic factors have been tested for their ability to prevent degeneration of lesioned neurons: in a rat model of transient forebrain ischemia, chronic infusions of BDNF significantly reduced the neuronal death occurring after the ischemic insult (Beck et al., 1994). A profound beneficial effect of GDNF has been demonstrated in models of Parkinson's disease (Gash et al., 1996; Winkler et al., 1996). NGF has shown a protective effect against excitatory amino acid-induced striatal brain lesions (Aloe, 1987; Frim et al., 1993a,b). These studies indicate that neurotrophic factors indeed have a therapeutic potential in neurodegenerative diseases. However, the endogenous neurotrophic factors are large peptides that do not pass the blood-brain barrier, and, therefore, treatments of disorders in the central nervous system with neurotrophic factors have necessitated intracranial injections or infusions. Potential therapeutic activation of neurotrophic systems is dependent on the identification of small molecules capable of crossing the blood-brain barrier, which directly or indirectly mimic or potentiate the activity of endogenous neurotrophic factors. In the present report, we describe such a molecule, NS521, a benzimidazolone that was identified in an in vitro screening for compounds with neurotrophic effects based on PC12 rat pheochromocytoma cells. PC12 cells have been used extensively as an in vitro model to study the neurotrophic effects of NGF (Greene and Tischler, 1982; Guroff, 1985). In addition to the effect on PC12 cells, NS521 also exhibits neuroprotective activity in two in vivo models of neuronal degeneration.

	Materials and Methods

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Cell Culture and Survival Experiments. PC12 cells obtained from Brian B. Rudkin (Ecole Normale Supérieure de Lyon, Lyon, France) were cultured in Dulbecco's modified Eagle's medium supplemented with 7.5% fetal bovine serum and 7.5% horse serum in a humidified incubator at 37°C and 5% CO₂. All tissue culture reagents were obtained from GIBCO/Life Technologies Ltd., Paisley, Scotland. For survival experiments, PC12 cells were seeded in 96-well plates (2.5 × 10³ cells/well) coated with calf skin collagen (Seromed, Biochrom VG, Berlin, Germany) in culture medium supplemented with 1000 pM mouse 7S NGF (Alomone Labs Ltd., Jerusalem, Israel). After 3 days, additional NGF was added (1000 pM NGF). After 6 days with NGF, where more than 95% of the cells appeared to be morphologically differentiated with neurites at least twice the length of the cell body diameter, the cells were incubated in medium without serum in the absence or presence of NS521 or NGF. Cell viability was evaluated by the ability to reduce the tetrazolium derivative, MTT (3-(4,5-dimethylethiazol-2-yl)-2,5-diphenyl tetrazolium bromide) into a blue formazan salt. Briefly, 10 µl of MTT solution (1.5 mg/ml in PBS) was added directly to each well containing cells and culture media. After a 4-h incubation, the formazan dye was solubilized by removing the media and adding 100 µl of 0.04 M HCl in 35% isopropanol. The dye development was quantified at 565 nm by using an enzyme-linked immunosorbent assay (ELISA) plate reader (ELX800; Biotek Instruments, Luton, UK). Statistical significant effects were tested by using Kruskal-Wallis test for many groups followed by Dunn's test or Dunnett's test as indicated for pairwise comparison of groups.

Detection of Fragmented DNA. DNA fragmentation was measured by quantitation of cytosolic oligonucleosome-bound DNA by using a cell death-detection ELISA kit (Boehringer Mannheim GmbH, Germany) following the manufacturer's instructions. Briefly, PC12 cells were seeded in 48-well plates (1 × 10⁴ cells/well) and differentiated, as described above for the survival experiments. After incubation in medium with serum and NGF or sodium azide or in medium without serum in the absence or presence of NS521, cells were collected from each well and the soluble DNA present in the cytoplasmic fraction was extracted. The extracted DNA then was bound by an antihistone antibody immobilized in an ELISA plate. After incubation and washing, anti-DNA peroxidase-conjugated antibody was added, and fragmented DNA present in the cytoplasmic fraction was determined photometrically as the amount of peroxidase retained in the immunocomplex using 2,2'-azino-di[3-ethylbenzthiazoline sulfonate] as a substrate.

MAPK Phosphorylation. PC12 cells were plated in a 48-well plate in culture medium (1 × 10⁵ cells/well) and incubated for 16 h. To reduce the basal level of phosphorylation of ERK1/2, the cells were starved for 1 h before performing the experiment in medium without serum supplemented with 1% BSA. Cells were stimulated in the presence or absence of NGF and NS521. After stimulation, the cells were lysed in sample buffer (400 mM Tris-HCl, pH 8.0/2% SDS/10 mM dithiothreitol) and cell lysates were applied on a precast, 8 to 18% gradient SDS-polyacrylamide gel (Amersham Pharmacia Biotech, Uppsala, Sweden). After electrophoresis, the proteins were transferred to a nitrocellulose membrane and immunoblotted by using a monoclonal antibody recognizing the 44- and 42-kDa MAPK isoforms, ERK1 and ERK2 (clone MK12, Transduction Laboratories, Lexington, KY), diluted 1:2500, followed by incubation with an alkaline phosphatase-conjugated anti-mouse IgG (Sigma, St. Louis, MO), diluted 1:5000. ERK1/ERK2 immunoreactivity was detected by using 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium as substrate.

Transient Forebrain Ischemia Model (2-VO Gerbils). Gerbils were anaesthetized with halothane, and right and left carotid arteries were located and occluded for 4 min. Animals were kept at normal body temperature before and after the operation by using heating lamps. During the operation, the gerbils were placed on heating pads, and the body temperature was controlled and maintained at 37 ± 0.5°C. Animals of the treated group received 30 mg/kg s.c. NS521 daily starting 15 min postocclusion. Four days later, the animals were sacrificed, and brains were removed and cooled to 70°C. Thereafter, the brains were sectioned in 20-µm-thick sections, of which five to seven with hippocampal tissue were selected and stained with H&E. Based on the degree of hippocampal damage, each hippocampus was categorized into one of four groups: group 1, no damage in the CA₁ layer; group 2, the CA₁ layer partly damaged; group 3, the CA₁ layer completely damaged; and group 4, damage in more than just the CA₁ layer. The total ischemia score was obtained as the sum of scores in the right and left hemispheres. Kendall's  test was used for statistic evaluation.

Nerve Regrowth in Rats. Male Wistar rats, 200 g (Moellegaard Breeding and Research Center L1. Skensred, Keoge, Denmark), were brought to the laboratory at least 5 days before the experiment. The rats were housed in Macrolon III cages (20 × 40 × 18 cm), with four rats in each cage, food (Altromin Biogaarden, Gentofte, Denmark) and tap water ad libitum, and a 12-h daily light-on/light-off schedule. On day 1 of the experiment, the animals were anesthetized by using halothane. The right sciatic nerve was dissected free, cut, sutured, and embedded in Tisseel (Immuno, Copenhagen, Denmark). NS521 was administered s.c. at 30 mg/kg (6 mg/ml in 0.9% NaCl, pH  7) once a day for 21 days, starting the day of the operation. Controls received NaCl at the same time intervals. On day 21, the animals were sacrificed and the sciatic nerve was removed, frozen, and sectioned in 20-µm sections at different distances from the lesion. The sections were stained with Luxol Fast Blue (Gurr; BDH, Poole, England), and the total number of myelinated fibers were counted unbiasedly by using the Olympus-DK CAST system (Olympus, New Hyde Park, NY). An ANOVA test combined with a Tukey test for pairwise multiple comparison (Jandel Sigma Stat, version 2.0) was used for the evaluation of data.

	Results

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Survival of Neuronal PC12 Cells after Serum Starvation. In vitro, NS521 (Fig. 1) was tested for neuroprotective effects in a model system of neuronal death based on PC12 pheochromocytoma cells. PC12 cells cultured for 6 days in the presence of NGF differentiate into postmitotic, neuron-like cells. To test for neuroprotective effects, NGF and serum were replaced by NS521 in various concentrations. After 1 to 4 days of incubation, the ability of the cell culture to reduce MTT was used to quantitate the viability of the culture. As a positive control, a parallel culture maintained without serum, but with NGF, was included. As seen in Fig. 2A, removal of NGF and serum caused a gradual decrease of viability of the culture. After 4 days of deprivation of trophic support, the viability was reduced to <25%. A significant effect on cell survival was seen by NGF and NS521 (P < .001, H = 34.801, with 3 df by Kruskall-Wallis test of "no addition" versus all other groups). NGF in a high concentration (1000 pM) was able rescue the majority of the cells. Approximately half of the PC12 cell population was rescued by 100 pM NGF, and the same amount of protection was achieved by 1 µM NS521. A statistically significant survival effect of NS521 could be detected in concentrations from 1 µM, and the effect was dose-dependent (Fig. 2B). In this experiment, 10 µM NS521 appeared to be as protective as 1000 pM NGF. The apparent variation in survival of the PC12 cells at equivalent doses seen in Fig. 2 may be due to sensitivity of the assay to small variations between the different experiments such as cell density or to a difference in potencies between the NGF batches used in different experiments. In contrast to NGF, NS521 did not preserve neurite outgrowth of the rescued cells (Fig. 3).

View larger version (8K): [in this window] [in a new window]   Fig. 1.   Chemical structure of NS521 (oxalic acid salt).

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Survival of PC12 cells determined by using MTT. PC12 cells were primed with NGF for 6 days before trophic factor deprivation. A, survival of primed PC12 cells cultured in serum-free medium supplemented with or without NGF or NS521 as indicated for 1, 2, 3, or 4 days maximum survival was determined in a parallel culture of primed PC12 cells incubated in the presence of serum and 1000 pM NGF. Data are means ± S.E.M. of 12 determinations originating from two individual experiments. B, dose-response curve for NS521 at day 4 after trophic factor withdrawal (P < .001, H = 78.088 with 9 df by Kruskall-Wallis test, no addition versus the NGF- or the NS521-treated groups). Data shown are the means ± S.E.M. of six determinations from a representative experiment. *P < .05 by pairwise comparison of all other groups with no addition (Dunn's test).

View larger version (147K): [in this window] [in a new window]   Fig. 3.   Morphologies of differentiated PC12 cells rescued by 1000 pM NGF (upper) and 1 µM NS521 (lower) from a representative experiment. Cells were differentiated as described in legend to Fig. 2, medium was changed to serum-free medium with or without NGF or NS521, and the cells were maintained for 4 days. Notice that, although the cells rescued with NS521 have lost their neurites, they look healthy and intact.

View larger version (44K): [in this window] [in a new window]   Fig. 4.   Survival of primed PC12 cells determined by MTT. MTT reduction (optical density measured at 565 nm) in cultures of primed PC12 cells incubated in serum-free medium supplemented with or without NGF or NS521 as indicated for either 4 days (open columns) or for 4 days with the indicated treatment followed by recovery for 4 days in 10% fetal calf serum and 1000 pM NGF (hatched columns). Data shown are the means ± S.E.M. of six determinations from a representative experiment (P < .001 by Kruskal-Wallis test, no addition versus all other groups, 8 df; H values are 49.388 and 43.953 for determinations before and after recovery, respectively). *, **Pairwise comparison of all other groups with no addition (P < .05, Dunnet's test).

View larger version (32K): [in this window] [in a new window]   Fig. 5.   DNA fragmentation in primed PC12 cells. PC12 cells were primed with NGF for 6 days followed by incubation in either serum-containing medium with 1000 pM NGF (NGF) or 50 mM sodium azide (azide). Alternatively primed PC12 cells were incubated in serum-free medium without any additives (control) or supplemented with 1 µM NS521 (NS521). DNA fragmentation was measured by quantification of cytosolic oligonucleosome-bound DNA by using a specific ELISA system after incubation for 0.5 h (open columns), 5 h (gray columns), 2 days (hatched columns), or 4 days (solid columns). DNA fragmentation data (absorbance at 405 nm) are means of two determinations from a representative experiment that has been repeated three times with similar results.

Effect on MAPK Phosphorylation in PC12 Cells. We investigated by immunoblot analysis whether NS521 had any effect on MAPK activation in PC12 cells by using an antibody recognizing the 44- and 42-kDa MAPK isoforms ERK1 and ERK2. Activation of ERK1 and ERK2 is associated with phosphorylation on specific threonine and tyrosine residues, which results in shifted electrophoretic mobility of the proteins in a polyacrylamide gel (Leevers and Marshall, 1992). As seen in Fig. 6, in lysates of control PC12 cells and cells treated for 5 to 40 min with 10 µM NS521, no phosphorylation of ERK1 and ERK2 could be detected. In contrast, NGF induced a substantial transient stimulation of phosphorylation of both isoforms after 5 and 10 min of stimulation. Furthermore, no effect of NS521 on ERK phosphorylation induced by a submaximal dose of NGF could be detected (data not shown).

View larger version (36K): [in this window] [in a new window]   Fig. 6.   ERK phosphorylation. Cells were prestarved in serum-free medium with 1% BSA for 1 h. After starvation, cells were left unstimulated or stimulated with 3000 pM NGF or 10 µM NS521 for 40 min, 20 min, 10 min, or 5 min as indicated. Cells were harvested and processed for SDS polyacrylamide gel electrophoresis. After electroblotting to nitrocellulose, the 44-kDa ERK1 and the 42-kDa ERK2 were immunodetected by using a monoclonal antibody recognizing both isoforms. A representative immunoblot is shown.

Transient Forebrain Ischemia Model. NS521 showed a significant (p < .002) protection against ischemic damage, when dosed 30 mg/kg s.c., 15 min postocclusion, and once a day for the following 2 days (Fig. 7); ischemic scores of 4 or less corresponding to no or only partial damage were seen in more than 30% of the animals treated with NS521. In contrast, approximately 95% of the untreated control animals exhibited ischemic score of 6 or more, corresponding to a significant hippocampal damage.

View larger version (15K): [in this window] [in a new window]   Fig. 7.   The protective effect of NS521 against hippocampal CA1 neuronal cell loss evoked by 4-min transient forebrain ischemia in gerbils. After staining, each hippocampus was categorized into one of four groups based on the degree of hippocampal damage: group 1, no damage in the CA1 layer; group 2, the CA1 layer partly damaged; group 3, the CA1 layer completely damaged; and group 4, damage in more than just the CA1 layer. The total ischemia score was obtained as the sum of the right and the left scores. Animals of the treated group received 30 mg/kg s.c. NS521 daily starting 15 min postocclusion. Kendall's  test was used for statistical evaluation.

Effect of NS521 on Nerve Regrowth in Rats. NS521 was tested in an animal model of peripheral nerve injury, the rat sciatic nerve-cut model. Significant protection of the initial retrograde degeneration (0.5 mm proximal to the nerve cut) was obtained by administration of NS521 (Fig. 8). An approximately 15% reduction of the total number of myelinated fibers was seen in the group treated with NS521 (30 mg/kg) as compared to an approximately 65% reduction in the nontreated control group (P < .001). Treatment with NS521 showed no effect on the total number of fibers distal to the lesion.

View larger version (15K): [in this window] [in a new window]   Fig. 8.   NS521 prevents retrograde degeneration of the sciatic nerve after transection. Rats with cut sciatic nerves were treated with NS521. On day 21 postcut, the lesions were evaluated by counting the total number of myelinated fibers in the sciatic nerve 0.5 mm proximal and 0.5 mm distal to the side of the cut as indicated on the figure. Means ± S.E.M. are shown for control, unoperated nerves (); operated, vehicle-treated nerves (); and operated nerves after daily treatment with 30 mg/kg s.c. NS521 (). An ANOVA test combined with Tukey test for pairwise multiple comparison was used for the evaluation of data.

	Discussion

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In the present study, we show evidence that a novel benzimidazolone, NS521, capable of crossing the blood-brain barrier, exhibits neuroprotective activity in vivo and in vitro. NS521 was identified in an in vitro screening for compounds with neuroprotective effects based on PC12 pheochromocytoma cells. In medium containing serum, PC12 cells divide and display many characteristics of adrenal chromaffin cells. Within several days of NGF exposure, PC12 cells stop dividing and acquire numerous properties of mature sympathetic neurons. NS521 inhibited the death of neuronal (NGF-pretreated) PC12 cells deprived of trophic support. PC12 cells deprived of serum and NGF have been used extensively as an in vitro model of neuronal apoptosis. Neuronal PC12 cells resemble primary cultures of sympathetic neurons, in that, death induced by withdrawal of trophic factors is apoptotic and inhibited by transcriptional and translational inhibitors (Martin et al., 1988; Mesner et al., 1992). Moreover, the known intracellular changes associated with apoptosis are indistinguishable in the two cell culture systems. The ability of NS521 to prevent or delay apoptosis may be limited to cells of neural origin, because the compound does not protect thymocytes from apoptosis induced by interleukin 2 deprivation or Fas, nor does it inhibit tumor necrosis factor -induced apoptosis in MCF-7 breast carcinoma cells and WEHI fibrosarcoma cells (data not shown). The loss of neurites after NS521 treatment may be PC12-specific because, unlike terminal differentiated neurons, this cell line is known to be able to dedifferentiate upon removal of NGF and resume proliferation provided by mitogenic stimuli.

DNA fragmentation is a hallmark of apoptosis in many cell types including PC12 (Batistatou and Greene, 1993). We were able to detect DNA fragmentation after incubation for only 0.5 h in serum-free medium by using an immunoassay for measuring DNA fragmentation. This very early DNA fragmentation could not be suppressed by NS521. It might be related to the inability of this compound to rescue PC12 cells that have not been preexposed to NGF (data not shown), because the very early DNA fragmentation might occur in a subpopulation of the PC12 culture that was not differentiated fully and, therefore, has a faster kinetics of apoptosis (Mesner et al., 1992). Although more than 95% of the culture appeared to be differentiated after 6 days with NGF, as judged from the morphology of the cells (data not shown), DNA fragmentation in a few percent of undifferentiated cells might give a substantial signal using the immunoassay. However, NS521 caused a transient inhibition of DNA fragmentation lasting from 5 h to at least 2 days after withdrawal of trophic support, thereby delaying the death process of the culture. The apparent discrepancy between the decreased cell death and DNA fragmentation at day 4 may be explained by a certain lag period from initiation of DNA fragmentation to actual cell death.

Neurotrophins, such as NGF, BDNF, and NT_3-5, bind to specific receptors present on the target cells. These receptors, which are structurally related and belong to the Trk family of receptor tyrosine kinases, mediate their biological responses to neurotrophins by activating multiple signaling pathways, including the activation of the MAPK isoforms ERK1 and ERK2 (Greene and Kaplan, 1995). Activation of the ERKs is essential for neuronal differentiation (Cowley et al., 1994) and also has been suggested to play a role in the NGF-mediated survival response in PC12 cells (Xia et al., 1995). The cellular mechanism of how NS521 mediates its effect on PC12 survival is unknown. However, we found no effect of the compound on ERK phosphorylation, which also makes it unlikely that the effect of NS521 on neuronal PC12 survival should be mediated through the physiological NGF receptor, TrkA.

In the present article, we also present evidence for significant neuroprotective effects of NS521 in animal models of ischemia and peripheral nerve injury. The CA₁ pyramidal neurons in the hippocampus have been shown to undergo selective and delayed cell death both in experimental animals and in humans after transient cerebral ischemia. In the gerbil model of transient global ischemia used in the present study, we found a significant neuroprotective effect of NS521. The mechanisms leading to the ischemic brain damage are complex, and the selective vulnerability of the CA₁ neurons are not fully understood. A decrease in blood flow and ATP levels, which is an inevitable consequence after ischemic insults, results in massive neuronal depolarization, leading to glutamate release and opening of voltage-gated Na⁺ and Ca²⁺ channels (reviewed in Koroshetz and Moskowitz, 1996). In particular, the release of glutamate leading to excessive activation of ionotrophic glutamate receptors is thought to play a major role in mediating neurotoxicity. The neuroprotective effect of NS521 is unlikely to be a result of the blockade of glutamate receptors, because the compound displays very low affinity to these receptors (IC₅₀ > 10 µM at [³H]a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, [³H]kainate, [³H]CGS-19755, and [³H]glycine-binding sites; data not shown). The delayed cell death of the CA₁ cells might occur by apoptosis (Nitatori et al., 1995). Thus, it is possible that NS521 protects the neurons by interfering with the apoptotic process.

Peripheral nerve regeneration after injury most often occurs spontaneously in humans. However, the functional recovery often is poor, because once the nerves finally reach the neuromuscular end plates, they have disappeared due to lack of activation. Just after injury, a retrograde degeneration of the myelinated nerve fibers is seen (i.e., "Wallerian die-back") (Chaudhry et al., 1992). In the present study, the neuroprotective effect of NS521 was studied after unilateral transection of the sciatic nerve in a rat model. NS521 showed a highly significant protection against the initial retrograde, nerve-fiber degeneration. Beneficial effects of NGF previously have been demonstrated in the same model, where the retrograde degeneration of the proximal nerve, reduction in axonal diameter, and decrease in neurofilament content in dorsal root ganglion neurons after transection of the sciatic nerve in rats can be attenuated by NGF (Gold et al., 1991).

We conclude that NS521 exerts robust neuroprotective activity in a cell culture system of neuronal death and in two in vivo models of neuronal degeneration. Although NS521 seems to delay the apoptotic death of neuronal PC12 cells deprived of growth factors rather than preventing it, this activity might have clinical relevance because the compound in the present study showed neuroprotective efficacy in models of global cerebral ischemia and nerve transection. It may be speculated that the neuroprotection seen in these in vivo models could be explained by the compound exerting an antiapoptotic effect during a critical period during which endogenous survival or protective mechanisms are restored. Further investigations on the cellular mechanisms of how NS521 exerts its effects are in progress.