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NEUROPHARMACOLOGY

A Novel Neurotrophic Agent, T-817MA [1-{3-[2-(1-Benzothiophen-5-yl) Ethoxy] Propyl}-3-azetidinol Maleate], Attenuates Amyloid--Induced Neurotoxicity and Promotes Neurite Outgrowth in Rat Cultured Central Nervous System Neurons

Research Laboratories, Toyama Chemical Co., Ltd., Toyama, Japan (K.H., H.Y., Y.T., A.T., T.F., N.I., A.S., M.N.); and Fifth Department of Internal Medicine, School of Medicine, Fukuoka University, Fukuoka, Japan (T.Y.)

Received January 11, 2005; accepted March 25, 2005.

	Abstract

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Progressive neuronal loss in Alzheimer's disease (AD) is considered to be a consequence of the neurotoxic properties of amyloid- peptides (A). T-817MA (1-{3-[2-(1-benzothiophen-5-yl) ethoxy] propyl}-3-azetidinol maleate) was screened as a candidate therapeutic agent for the treatment of AD based on its neuroprotective potency against A-induced neurotoxicity and its effect of enhancing axonal regeneration in the sciatic nerve axotomy model. The neuroprotective effect of T-817MA against A(1-42) or oxidative stress-induced neurotoxicity was assessed using a coculture of rat cortical neurons with glia. T-817MA (0.1 and 1 µM) was strongly protective against A(1-42)-induced (10 µM for 48 h) or H₂O₂-induced (100 µM for 24 h) neuronal death. T-817MA suppressed the decrease of GSH levels induced by H₂O₂ exposure (30 µM for 4 h) in cortical neuron culture; therefore, T-817MA was likely to alleviate oxidative stress. Besides the neuroprotective effect, T-817MA (0.1 and 1 µM) promoted neurite outgrowth in hippocampal slice cultures and reaggregation culture of rat cortical neurons. T-817MA also increased the growth-associated protein 43 content in the reaggregation culture of cortical neurons. These findings suggest that T-817MA exerts neuroprotective effect and promotes neurite outgrowth in rat primary cultured neurons. Based on these neurotrophic features, T-817MA may have a potential for disease modification and be useful for patients with neurodegenerative diseases, such as AD.

Neurotrophic factors have been studied as one of the potential future therapies for AD (Tariot and Federoff, 2003). Neurotrophic factors can support the remaining neurons and protect them against disease progression in animal and cell culture models of neurodegenerative diseases (Mattson et al., 2001). In the adult nervous system, neurotrophic factors can also regulate neuronal plasticity by promoting nerve growth following injury (Gillespie, 2003) and thereby promote functional restoration (Lim et al., 2003). These features suggest that activation of neurotrophic pathways can contribute to the modification and prevention of disease progression in patients with AD.

T-817MA (1-{3-[2-(1-benzothiophen-5-yl) ethoxy] propyl}-3-azetidinol maleate) is a newly synthesized agent that was screened as a candidate therapeutic agent for the treatment of AD. Screening was carried out based on the neuroprotective potency against A-induced neurotoxicity and enhancing effect on axonal regeneration in the rat sciatic nerve axotomy model (daily treatment with T-817MA for 14 days enhanced the maximal regeneration distance of sciatic nerve axons measured using an electrophysiological analysis; Y. Nakada, unpublished observations) in the expectation of obtaining a neurotrophic agent. In the present study, to determine whether T-817MA exerts neurotrophic potency on the central nervous system (CNS), we evaluated its neuroprotective effect and neurite outgrowth promoting effect. Neuroprotective effect of T-817MA was assessed in amyloid-(1-42)-[A(1-42)] or H₂O₂-induced neuronal damages by using a coculture of rat cortical neurons with glia. The neurite outgrowth promoting effect was assessed using a hippocampal slice culture and cultured reaggregates of rat cortical neurons. The therapeutic potential of T-817MA in AD also is discussed.

	Materials and Methods

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Animals
Pregnant female Wistar/ST rats were purchased from Japan SLC, Inc. (Shizuoka, Japan) and kept in individual aluminum cages with laboratory bedding in an air-conditioned room on a 12-h light/dark cycle. The animals were given free access to a commercial diet (MF; Oriental Yeast Co., Ltd. Tokyo, Japan) and water. Neonatal rats were housed with their mother rat. All the experiments were performed in accordance with the Guide for Care and Use of Laboratory Animals at Toyama Chemical Co., Ltd. (Tokyo, Japan) and the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Materials
T-817MA was synthesized at Toyama Chemical Co., Ltd. The chemical structure of T-817MA is shown in Fig. 1. T-817MA was dissolved in distilled water at a concentration of 10 mM and diluted to 1, 0.1, and 0.01 µM with Dulbecco's modified Eagle's medium (DMEM; Nissui, Tokyo, Japan) on the day of use. A(1-42): -amyloid(1-42) · HCl was purchased from AnaSpec Inc. (San Jose, CA). A(1-42) was sonicated in distilled water at a concentration of 250 µM and then incubated at 37°C for 24 h. Fetal bovine serum (FBS) was purchased from JRH Biosciences (Lenexa, KS). FBS was heat-inactivated at 56°C for 30 min. Monoclonal anti-microtubule-associated protein 2 (MAP2) antibody (clone HM-2, mouse ascites fluid) and anti-glial fibrillary acidic protein were purchased from Sigma-Aldrich (St. Louis, MO). Goat polyclonal anti-growth-associated protein 43 (GAP-43) antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). VECTASTAIN ABC-PO Goat IgG Kit and VECTASTAIN ABC-PO Mouse IgG Kit were purchased from Vector Laboratories (Burlingame, CA). Recombinant human insulin-like growth factor-1 (IGF-1) was purchased from PeproTech (Rocky Hill, NJ).

View larger version (8K): [in this window] [in a new window]   Fig. 1. Chemical structure of T-817MA.

Neuroprotective Effect against A(1-42)-Induced Toxicity
Neuron/Glia Coculture. We prepared primary cortical neurons plated onto glial monolayer cultures as described previously (Pike et al., 1993). Primary cultures of cortical glial cells were prepared from 1- or 2-day-old neonatal rats. Neonatal rats were decapitated, and their whole brains were isolated. Cortices were dissected under a microscope and incubated at 37°C for 20 min with phosphate-buffered saline (PBS) devoid of Ca²⁺ and Mg²⁺ (Nissui) containing 0.25% trypsin (Invitrogen, Carlsbad, CA) and 40 units/ml DNase I (Sigma-Aldrich). Trypsinization was terminated by addition of 25% FBS. The dissociated cells were suspended in Eagle's minimum essential medium (Nissui) containing 10% FBS, 2 mM glutamine, and 25 µg/ml gentamicin sulfate. The cells were cultured in 75-cm² flasks in a humidified CO₂ incubator with 5% CO₂/95% air at 37°C for 3 weeks to form a monolayer. The glial cells were then harvested and replated on 24-well plates at a density of 400 cells/mm² and then maintained for another 1 week. Cortical neurons were prepared from rat embryos (day 18 gestation). Cortices were dissected under the microscope and incubated with PBS containing 0.25% trypsin and 40 units/ml DNase I at 37°C for 20 min. Trypsinization was terminated by the addition of 25% FBS. Dissociated cortical cells were suspended in DMEM containing 10% FBS, 2 mM glutamine, and 25 µg/ml gentamicin sulfate. They were subsequently seeded onto the cortical glial monolayer culture. Three or four days after plating cortical cells, the entire medium was replaced with DMEM containing 1% (v/v) N-2 supplement (Invitrogen), and 10 µM cytosine arabinoside (Nacalai Tesque, Kyoto, Japan) was added to halt glial proliferation. The cells were then incubated for 7 days in this medium. A(1-42) was added to the coculture at a concentration of 10 µM. T-817MA was added simultaneously with A(1-42) application at concentrations of 0 (control), 0.01, 0.1, and 1 µM, and the cells were further incubated for 48 h. For the normal group, the preparations were maintained in the medium with neither T-817MA nor A(1-42).

Assay for the Viability of Neurons. Neuronal cell viability was quantified by measuring MAP2 immunoreactivity. This method is favorable for measuring neuronal survival in the presence of glial cells without resorting to the counting of neurons (Brooke et al., 1999). Measurement was performed using monoclonal anti-MAP2 antibody and VECTASTAIN ABC-PO mouse IgG kit in accordance with the manufacturer's manual. In brief, the cultured cells were fixed with ice-cold methanol for more than 30 min and washed with PBS three times. Following the blocking procedure with horse serum for 20 min, the cells were incubated for 30 min with anti-MAP2 antibody (1:3000 dilution). The cells were then incubated with biotinylated secondary antibody (included in the kit) for 30 min, followed by incubation with VECTASTAIN ABC Reagent for 30 min. Peroxidase activity was estimated using o-phenylenediamine and H₂O₂ (Maus et al., 2002). The cells were incubated with o-phenylenediamine solution (10 mg/ml o-phenylenediamine, 0.03% hydrogen peroxide, 0.05 mM citric acid, and 0.1 mM disodium hydrogen phosphate; Wako Pure Chemicals, Osaka, Japan) for 3 min. Thereafter, the reaction was terminated using 0.05 M sulfuric acid (Wako Pure Chemicals). Each solution was diluted with 1 ml of distilled water. The absorbance of each reaction solution was measured at 490 nm. The procedures were all performed at room temperature.

Neuroprotective Effect against H₂O₂-Induced Toxicity
Assessment in Neuron/Glia Coculture. A cortical neuron/glia coculture was prepared as described above. T-817MA was added to the cocultures at concentrations of 0 (control), 0.01, 0.1, and 1 µM, and the cells were subsequently incubated for 5 min or 24 h. H₂O₂ was then added to the coculture at a concentration of 100 µM, and the cells were incubated for another 24 h. For the normal group, the preparations were maintained in the medium with neither T-817MA nor H₂O₂. Neuronal cell viability was quantified by measuring the MAP2 immunoreactivity as described above.

Assessment in Cortical Neuron Culture. Primary cultures of cortical neurons were prepared from rat embryos (gestational day 18). Dissociated cortical cells were suspended in DMEM containing 10% FBS, 2 mM glutamine, and 25 µg/ml gentamicin sulfate. The cells were seeded onto 24-well plates precoated with poly-L-lysine (molecular weight, 30,000-70,000; Sigma-Aldrich) at a density of 1000 cells/mm². Forty-eight hours after cell seeding, cytosine arabinoside was added to the cultures (10 µM) and removed by medium exchange after 24 h. In this condition, as shown by immunocytochemical studies using anti-MAP2 monoclonal antibody, the cultures were highly enriched in neurons, and less than 10% of the cells exhibited immunoreactivity with a rabbit antibody raised against glial fibrillary acid protein (data not shown). T-817MA was added to the cultures at concentrations of 0 (control), 0.01, 0.1, and 1 µM. Twenty-four hours following T-817MA application, H₂O₂ was added at a concentration of 30 µM, and the cells were incubated for another 24 h in the continuous presence of T-817MA. For the normal group, the preparations were maintained in the medium with neither T-817MA nor H₂O₂. Neuronal cell viability was quantified by measuring the MAP2 immunoreactivity as described above.

Measurement of the Intracellular GSH Content in Primary Cortical Neuron Culture. T-817MA was added to the primary culture of purified neuron at concentrations of 0.01, 0.1, and 1 µM. Twenty-four hours following T-817MA application, H₂O₂ was added at a concentration of 30 µM. The cells were then incubated for another 4 h in the continuous presence of T-817MA. The intracellular GSH content in the cultured neurons was measured in accordance with a previously reported method (Hiraku et al., 2002) with some modifications. In brief, the cells were washed once with ice-cold PBS, followed by the addition of 100 mM perchloric acid, and then harvested. The harvested cells were homogenized for 5 s with a microhomogenizer (Seiko Instruments, Chiba, Japan) and then centrifuged at 10,000g for 10 min at 4°C. The GSH content in the supernatant was measured using a high-pressure liquid chromatography system consisting of an L-7100 pump (Hitachi Ltd., Tokyo, Japan) and ECD-300 electrochemical detector (Eicom, Kyoto, Japan) equipped with a Symmetry C18 column (i.d., 4.6 mm x 25 cm; Waters, Milford, MA), a WE-AU gold electrode (Eicom), and a 50-mm GS-50 gasket (Eicom). The mobile phase consisted of 4.4 mM phosphate buffer, pH 2.5, 88 mg/l 1-octanesulfonic acid, 4.4 mg/l EDTA · 2Na, and 12% methanol. The measurement was carried out at room temperature at a flow rate of 0.8 ml/min. The voltage of the gold electrode was set at +600 mV against the Ag/AgCl reference electrode (RE-100; Eicom). Authentic GSH (Sigma-Aldrich) was simultaneously measured as an external standard under these conditions. Neuronal cell viability was also quantified by measuring the MAP2 immunoreactivity as described above.

Evaluation of Neurite Outgrowth Promoting Effect
Hippocampal Slice Culture. Organotypic hippocampal slice culture was prepared in accordance with the previously reported method (Stoppini et al., 1991) with some modifications. Hippocampal slices were prepared from 7-day-old rat pups. The dorsal hippocampus was isolated and cut into transverse slices of 350-µm thickness with a tissue chopper (Mickle Laboratory Engineering, Guilford, UK). The slices were placed onto dishes precoated with poly-L-lysine (three slices in each dish) and cultured in interface configuration with DMEM containing 12.5 mM HEPES, 1% (v/v) B-27 supplement (Invitrogen), 2 mM glutamine, and 25 µg/ml gentamicin sulfate. T-817MA was added at concentrations of 0 (control), 0.01, 0.1, and 1 µM at the initiation of the slice culture. The culture was then incubated at 37°C in 5% CO₂/95% air for 8 days. During the culture period, half the volume of the medium in each dish was changed every 2 or 3 days.

Reaggregation Culture of Cortical Neurons. Reaggregation culture of the cortical neurons was carried out referring to a previously reported method (Gao et al., 1992). The cortical neurons were harvested from rat embryos (embryonic day 18). Dissociated cortical cells were suspended in DMEM containing 10% FBS, 2 mM glutamine, and 25 mg/ml gentamicin sulfate and seeded onto 100-mm noncoated dishes at a density of 50,000 cells/mm² and then cultured for 4 days. As a result of this procedure, cortical neurons formed reaggregates and floated in the medium. The suspended reaggregates of neurons were collected and seeded onto six-well plates (for evaluating neurite length) or 24-well plates (for measuring contents of MAP2 and GAP-43) precoated with poly-L-lysine. The reaggregates of cells were cultured for 3 days. T-817MA was then added at concentrations of 0 (control), 0.01, 0.1, and 1 µM, and the reaggregates of cells were cultured further for 4 days. All the cultures were incubated in the atmosphere of humidified 5% CO₂/95% air at 37°C.

Measurement of Neurite Outgrowth. Following treatment of T-817MA, cultured slices or cortical reaggregation culture was fixed with methanol. The neurites generated from slices or cell reaggregates were subsequently immunostained (Schreyer et al., 1997) with a goat polyclonal anti-GAP-43 antibody using VECTASTAIN ABC-PO Goat IgG Kit. Neurite length was defined as the distance from the edge of a slice or a reaggregate to the neurite tip. Neurite outgrowth was evaluated by measuring the length of the longest neurite in each slice or reaggregate under a microscope using a micrometer. The outgrowth was measured in three slices or three reaggregates in each dish, and their mean value was regarded as the representative value of the dish. All measurements were performed in a blinded manner. GAP-43 content was quantified by enzyme immunoassay (Schreyer et al., 1997) using VECTASTAIN ABC-PO kit with goat polyclonal anti-GAP-43 antibody.

Statistical Analysis
The results are represented as mean with S.E.M. Statistical significance of the differences between the two groups was analyzed by an analysis of variance (F-test), followed by Student's t test. Statistical significance of the T-817MA treatment groups from the control group was evaluated by Dunnett's test. These analyses were performed using SAS release 8.2 (SAS Institute Japan Ltd., Tokyo, Japan). P < 0.05 (two-tailed) was considered to be significant.

	Results

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Neuroprotective Effect of T-817MA
A(1-42)-Induced Neuronal Death. The protective effect of T-817MA on cortical neurons against A(1-42)-induced neuronal death was investigated in the neuron/glia coculture. Typical microscope images of MAP2 immunocytochemistry are shown in Fig. 2, A-C. Many neurons that formed clusters were stained on the glial monolayer in the absence of A(1-42) (normal). On the other hand, few neurons were detected with 10 µMA(1-42) treatment (control). T-817MA treatment preserved the cortical neurons in the presence of A(1-42). To evaluate the number of surviving neurons, MAP2 immunoreactivity, which is known to correlate the number of neurons (Brooke et al., 1999), was measured. The exposure of A(1-42) significantly reduced MAP2 immunoreactivity. T-817MA significantly prevented this reduction at 0.1 and 1 µM (Fig. 2D). A peptide A(42-1) did not reduce MAP2 immunoreactivity on the neuron/glia coculture (data not shown), which suggested that A(1-42) specifically induced the neuronal damage.

View larger version (43K): [in this window] [in a new window]   Fig. 2. Effect of T-817MA on A(1-42)-induced neuronal death. A(1-42)-induced neuronal death and the neuroprotective effect of T-817MA were assessed using rat neuron/glia coculture. Images of MAP2 immunocytochemistry showed typical responses: neurons with vehicle treatment (normal; A), 10 µM A(1-42) treatment for 48 h (control; B), and cotreatment of 1 µM T-817MA with 10 µM A(1-42) treatment for 48 h (C). Calibration bar indicates 200 µm. D, neuronal survival was estimated as MAP2 immunoreactivity using enzyme immunoassay. Results are expressed as absorbance at 490 nm. Columns and bars indicate the mean with S.E.M. (n = 6). **, P < 0.01 versus control (Dunnett's test) and ##, P < 0.01 versus normal (Student's t test).

H₂O₂-Induced Neuronal Death. Previous studies indicated that oxidative stress is supposed to contribute to A-induced neuronal damages (Markesbery et al., 2001; Butterfield et al., 2002). On this basis, the effect of T-817MA on H₂O₂-induced neuronal damage was investigated in the neuron/glia coculture. The 100 µM H₂O₂ treatment greatly reduced the number of surviving neurons in the culture (Fig. 3, control). When T-817MA was pretreated for 24 h, T-817MA significantly prevented this neuronal damage at 0.1 and 1 µM (Fig. 3A). Conversely, a brief (5 min) pretreatment with T-817MA, followed by its continuous presence with H₂O₂, did not rescue the H₂O₂-treated neurons from death (Fig. 3B).

View larger version (18K): [in this window] [in a new window]   Fig. 3. Effect of T-817MA on H2O2-induced neuronal death. H2O2-induced (100 µM for 24 h) neuronal death and the neuroprotective effect of T-817MA were assessed using rat neuron/glia coculture. T-817MA was pretreated for 24 h (A) or for 5 min (B) and was continuously existed with H2O2. Neuronal survival was estimated as MAP2 immunoreactivity using enzyme immunoassay. Results are expressed as absorbance at 490 nm. Columns and bars indicate the mean with S.E.M. (n = 6). *, P < 0.05, **, P < 0.01 versus control (Dunnett's test) and #, P < 0.05; ##, P < 0.01 versus normal (Student's t test).

T-817MA Attenuated H₂O₂-Induced Reduction of Intracellular GSH Contents. A similar protective effect of T-817MA was observed in the primary cortical neuron culture. H₂O₂ exposure at 30 µM for 24 h induced neuronal death (Fig. 4A, control). Twenty-four hours of pretreatment, followed by the continuous presence of T-817MA, prevented this oxidative stress-induced neuronal death at 0.1 and 1 µM (Fig. 4A). To investigate the effect of T-817MA on this oxidative stress, intracellular GSH content was measured as an index of the intracellular oxidative condition in rat cortical neurons under H₂O₂ exposure. In this experiment, primary cultures of cortical neurons were exposed to 30 µM H₂O₂ for 4 h. Viability of neurons was not altered at 4 h following 30 µM H₂O₂ exposure (Fig. 4B). On the other hand, GSH content was significantly reduced by this stress (Fig. 4C). The effect of T-817MA was examined with pretreatment for 24 h, then continuous presence for 4 h with 30 µM H₂O₂ exposure. T-817MA almost completely prevented such GSH reduction at 0.1 and 1 µM (Fig. 4C). T-817MA alone did not exert significant effect on the intracellular GSH content in the normal medium (data not shown).

View larger version (21K): [in this window] [in a new window]   Fig. 4. Effect of T-817MA on H2O2-induced neuronal death and GSH reduction. Primary cultures of cortical neurons were pretreated with several concentrations of T-817MA for 24 h and then exposed to 30 µM H2O2 for 24 h (A) or 4 h (B) in the continuous presence of T-817MA. Neuronal survival was estimated as MAP2 immunoreactivity using enzyme immunoassay. C shows GSH contents in the culture assessed with 4-h H2O2 treatment, the same condition as B. Results are expressed as absorbance at 490 nm (A and B) or GSH contents (nanograms per well). Columns and bars indicate the mean with S.E.M. (n = 6). *, P < 0.05; **, P < 0.01 versus control (Dunnett's test) and #, P < 0.05; ##, P < 0.01 versus normal (Student's t test).

T-817MA Promoted Neurite Outgrowth
We further investigated the neurite outgrowth promoting action of T-817MA by using two types of cultured neurons: the hippocampal slice culture and the cortical reaggregation culture. Hippocampal slices with 1 µM T-817MA treatment generated more and much longer neurites than control slices (Fig. 5, A and B). T-817MA significantly increased the neurite length at 0.1 and 1 µM (Fig. 5C).

View larger version (49K): [in this window] [in a new window]   Fig. 5. Effect of T-817MA on neurite outgrowth in rat hippocampal slice cultures. Typical images of neurite response with GAP-43-immunostained neurites are shown in control (A) and 1 µM T-817MA for 8 days (B). Calibration bar, 500 µm. C, the neurite outgrowth was quantified by measuring the distance from the edge of the slice to the tip of the longest neurite (micrometers). Data are shown as mean with S.E.M. (n = 10). **, P < 0.01 versus control (Dunnett's test).

Likewise, neurite outgrowth promotion of T-817MA was observed in the cortical reaggregation culture. Typical photomicrographs revealed that T-817MA treatment induced reaggregates to generate longer neurites than control slices (Fig. 6, A and B). T-817MA significantly promoted neurite outgrowth at 0.1 and 1 µM (Fig. 6C). In addition to the neurite length, we also quantified the immunoreactivity of neurites for GAP-43, which is specifically located in axons. GAP-43 immunoreactivity was significantly increased by T-817MA (Fig. 6D). The effect of IGF-1, which has potential to promote neurite outgrowth in vitro (Kim et al., 1997), was evaluated in this cortical reaggregation culture as a reference experiment. IGF-1 promoted neurite outgrowth; GAP-43 immunoreactivity (A_{490 nm}) was 0.236 with 100 ng/ml IGF-1, which was higher than the control value (A_{490 nm}, 0.158). This result indicated that the cortical reaggregation culture was useful for assessing neurite outgrowth promotion.

View larger version (60K): [in this window] [in a new window]   Fig. 6. Effect of T-817MA on neurite outgrowth in rat cortical reaggregation cultures. Typical images of neurite response with GAP-43-immunostained neurites are shown in control (A) and 1 µM T-817MA for 4 days (B). Calibration bar, 500 µm. C, the neurite outgrowth was quantified by measuring the distance from the edge of the aggregate to the tip of the longest neurite (micrometers). Quantitative analysis was performed by GAP-43 immunoreactivity (D) using enzyme immunoassay, and its results are expressed as absorbance at 490 nm. Data were shown as mean with S.E.M. (C: n = 8; D: n = 4). **, P < 0.01 versus control (Dunnett's test).

	Discussion

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AD is a neurodegenerative condition characterized by progressive neuronal loss, which may be a consequence of the neurotoxic properties of the A (Selkoe, 1991; Naslund et al., 2000). The current treatment with acetylcholinesterase inhibitors focuses on the activation of the remaining functional capacities (Tariot and Federoff, 2003). Although drugs focusing on neuroprotection have been actively developed recently, causal therapy for such neurodegenerative diseases is unavailable.

T-817MA was screened as a candidate therapeutic agent for AD. The present data indicate that T-817MA exerts a neuroprotective effect and promotes neurite outgrowth in rat primary cultured neurons. Considering these neurotrophic properties, T-817MA would modify or prevent pathological deterioration in AD.

In the present study, we demonstrated that T-817MA exerted a neuroprotective effect against A(1-42)-induced and H₂O₂-induced neurotoxicity in the cortical neuron/glia coculture. These results indicate that T-817MA exerts a protective effect on an in vitro model of neuropathology in AD. A neurotoxicity is supposed to be associated with oxidative stress (Markesbery et al., 2001; Butterfield et al., 2002) and the reduction of endogenous antioxidant processes (Olivieri et al., 2001). Therefore, to understand the neuroprotective effect of T-817MA, we focused our interest on oxidative stress-induced cell death.

T-817MA exerted its neuroprotective effect when the cells were pretreated with T-817MA for 24 h before the H₂O₂ exposure. Unlike antioxidants (Fuson et al., 1999), T-817MA was unable to protect neurons when it was applied just before oxidative stress exposure. Based on this result, it is supposed that T-817MA may exert its neuroprotective effect through the modulation of endogenous antioxidative mechanisms, rather than scavenging the reactive oxygen species. In the current study, we used a neuron/glia coculture because A application failed to induce clear neurotoxicity in the primary culture of the enriched cortical neurons. Previous studies indicated that coexistence of glial cells enhanced A-induced neurotoxicity by modifying the redox status in the neuron/glia coculture (Qin et al., 2002; Abramov et al., 2003, 2004). To demonstrate that T-817MA interacts with neurons themselves, we also investigated H₂O₂-induced neuronal damage in a primary cortical neuron culture. In this culture set, H₂O₂ (30 µM for 24 h) induced neuronal damage similar to that observed in the neuron/glia coculture. The neuroprotective effect of T-817MA against H₂O₂-induced damage was also observed in the cortical neuron culture, thereby indicating that T-817MA might act on neuronal cells themselves. In this cortical neuron culture, brief exposure (4 h) of H₂O₂ did not significantly affect neuronal viability; meanwhile, this treatment reduced the GSH content in the neurons. GSH is an important intracellular antioxidant that protects the neurons against a variety of reactive oxygen species (Schulz et al., 2000). Decrease in GSH was supposed to contribute to signaling events occurring during apoptotic neuronal death (Kane et al., 1993). Disturbance of GSH homeostasis may either lead to or result from oxidative stress in neurodegenerative disorders, and the treatments that inhibit GSH degradation may result in slowing the disease progression (Schulz et al., 2000). In our current experiment, the reduction of endogenous antioxidant processes might have preceded the neuronal damage. In this brief oxidative stress condition, T-817MA attenuated the preceding reduction of the intracellular GSH content, although T-817MA had no effect in the normal condition (data not shown). Based on these results, we assumed that T-817MA maintained the intracellular GSH content and resulted in preventing cell death under the oxidative stress condition. In addition, pretreatment of T-817MA was necessary for neuroprotection in H₂O₂-induced neuronal death in the neuron/glia coculture. These results indicate that T-817MA promotes endogenous antioxidant processes to protect the neurons from H₂O₂ stress. According to the hypothesis that A(1-42)-induced neuronal damage in the neuron/glia coculture was mediated by reactive oxygen species generated from astrocytes (Abramov et al., 2004), promotion of the antioxidant processes in neurons might also contribute to the neuroprotective effect of T-817MA against A(1-42)-induced neuronal damage in the neuron/glia coculture, although effects of T-817MA on glia cells cannot be excluded. Much evidence suggests that oxidative stress may be responsible for dysfunction or death of neuronal cells in AD (Markesbery et al., 2001; Mattson et al., 2001; Butterfield et al., 2002). Oxidative stress in particular has been shown to be one of the earliest changes in disease pathogenesis in AD (Nunomura et al., 2001). On these presuppositions, the antioxidative effect of T-817MA, which might be able to slow the disease progression in the early stage of AD, would be beneficial for AD treatment.

In the present study, T-817MA significantly increased neurite outgrowth. To evaluate neurite outgrowth, we used an anti-GAP-43 antibody. GAP-43 is expressed in an axon and a growth cone at high levels during periods of axon elongation (Meiri et al., 1986; Schreyer et al., 1997; Goslin et al., 1998) and was reported to be a useful tool for visualizing the sprouting of neuronal axons (McKinney et al., 1997). In the quantitative analysis with the cortical reaggregation culture, T-817MA also significantly increased GAP-43 immunoreactivity, reflecting an increase in the neurite length. Based on these results, T-817MA is believed to promote the effect of axonal outgrowth in CNS neurons.

In AD, A deposition is considered to cause disruption of the neural network including progressive synaptic degeneration and neuronal loss, which consequently results in cognitive dysfunction and behavioral abnormalities (Stepanichev et al., 2004). Hence, reconstructing the damaged neural network is a possible therapeutic target of the disease. The neurite projection has the potential to form a target-oriented and active synapse and subsequently could reproduce functional connections (Stoppini et al., 1993; Li et al., 1994; McKinney et al., 1999). Therefore, promoting neurite outgrowth is supposed to be essential for reconstructing the damaged neural network in AD and various other neurodegenerative diseases. It is reported that some substances that possess neurite outgrowth promoting effect in vitro are useful in the treatment of AD (Gillespie, 2003; O'Neill et al., 2004; Tohda et al., 2004).

In the present in vitro investigations, T-817MA was demonstrated to have a neuroprotective effect and a neurite outgrowth promoting effect at the same concentration range. Although the subcellular mechanisms underlying these pharmacological effects are not known, this set of features is similar to that of neurotrophins. These features are supposed to be important for the maintenance of the nervous system and for regulating certain aspects of neuronal survival. Activation of neurotrophic signaling pathways can protect neurons in animal and cell culture models of neurodegenerative diseases such as AD. Neurotrophic factors may protect neurons against age-related degeneration by modulating neurodegenerative cascades and stimulating survival-promoting mechanisms (Mattson et al., 2001). For example, brain-derived neurotrophic factor stimulates the production of various factors, such as antioxidant enzymes and antiapoptotic protein for protection against oxidative insult relevant to the pathogenesis of AD and other neurodegenerative diseases (Mattson et al., 2001). IGF-1 has a well-described neuroprotective effect against excitotoxic, metabolic, and oxidative insults in various experimental models for AD, and it promotes neurogenesis and synaptic formation throughout the brain, and IGF-1 is actively transported across the blood-brain barrier (Heck et al., 1999; Mattson et al., 2001; Wei et al., 2002; Gasparini and Xu, 2003). Therefore, IGF-1 has been indicated as a potential therapeutic target of AD (Gasparini and Xu, 2003). Despite these benefits, in general, the therapeutic application of neurotrophic factors themselves to neurodegenerative diseases is strictly limited because of their poor stability and poor CNS penetration of many of the neurotrophic factors. Considering these limitations, a neurotrophic factor-like small chemical molecule, such as T-817MA, having good CNS penetration (brain level of T-817MA was approximately 10 times higher than blood level after oral administration; A. Takagi, unpublished inhouse data) may be more favorable for therapeutic use from the viewpoint of drug delivery.

In conclusion, T-817MA exerts a neuroprotective effect and promotes neurite outgrowth in rat primary cultured neurons, indicating that this compound may have a potential for disease modification and be useful for patients with neurodegenerative diseases, such as AD.

	Footnotes

 
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.105.083543.

ABBREVIATIONS: AD, Alzheimer's disease; A, amyloid- peptides; T-817MA, 1-{3-[2-(1-benzothiophen-5-yl) ethoxy] propyl}-3-azetidinol maleate; CNS, central nervous system; A(1-42), amyloid-(1-42); DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; MAP2, microtubule-associated protein 2; GAP-43, growth-associated protein 43; IGF-1, insulin-like growth factor-1; PBS, phosphate-buffered saline with neither Ca²⁺ nor Mg²⁺.

Address correspondence to: Kazunari Hirata, Research Laboratories, Toyama Chemical Co., Ltd, 2-4-1 Shimookui, Toyama, 930-8508, Japan. E-mail: kazunari_hirata{at}toyama-chemical.co.jp

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