Journal of Neuroscience Research 57:280289 (1999)

Neurotoxicity of Methylglyoxal and 3-Deoxyglucosone on Cultured Cortical Neurons: Synergism Between Glycation and Oxidative Stress, Possibly Involved in Neurodegenerative DiseasesSeiji Kikuchi,1* Kazuyoshi Shinpo,1 Fumio Moriwaka,1 Zenji Makita,2 Toshio Miyata,3 and Kunio Tashiro1of Neurology, Hokkaido University School of Medicine, Sapporo, Hokkaido, Japan of Internal Medicine, Hokkaido University School of Medicine, Sapporo, Hokkaido, Japan 3Institute of Medical Science and Department of Medicine, Tokai University School of Medicine, Isehara, Kanagawa, Japan2Department 1Department

In this study, we investigate the neurotoxicity of glycation, particularly early-stage glycation, and its mechanisms, which are possibly synergized with oxidative stress. Methylglyoxal (MG) and 3-deoxyglucosone (3DG), intermediate products of glycation, are known to further accelerate glycation and advanced glycation endproducts (AGEs) formation. Both compounds showed neurotoxicity on cultured cortical neurons and these effects were associated with reactive oxygen species production followed by neuronal apoptosis. Pretreatment with N-acetylcysteine induced neuroprotection against MG and 3DG. Cotreatment, but not pretreatment, with aminoguanidine protected neurons against the neurotoxicities of both compounds. The present study provides the rst evidence that MG and 3DG are neurotoxic to cortical neurons in culture. Interference with the process by which glycation and AGEs formation occur may provide new therapeutic opportunities to reduce the pathophysiological changes associated with neurodegeneration, if, as indicated here, the participation of glycoxidation in the pathogenesis of neurodegenerative diseases is essential. J. 1999 Wiley-Liss, Inc. Neurosci. Res. 57:280289, 1999. Key words: glycation; glycoxidation; neurotoxicity INTRODUCTION Glycation reactions are initiated by addition of sugar aldehyde or ketone groups to free amino groups mainly on lysine and arginine residues of proteins, or N-terminal amino groups of proteins by a nonenzymatic reaction known as the Maillard reaction (Monnier and Cerami, 1981; Sell and Monnier, 1989; Smith et al., 1995). A synthesis of intermediates up to Amadori1999 Wiley-Liss, Inc.

compound is caused in the early stage of glycation, and this reversible reaction depends on the concentration of sugars and incubation time. In the late stage of glycation, advance glycation endproducts (AGEs) are formed after a complex cascade of repeated dehydration, condensation, fragmentation, oxidation, and cyclization reactions, through intermediates such as 3-deoxyglucosone (3DG). These reactions are irreversible. The AGEs whose structures have been identied are as follows: N2-(carboxymethyl)lysine (CML) (Fu et al., 1996), pentosidine (Dyer et al., 1991; Grandhee and Monnier, 1991), pyrralin (Miyata and Monnier, 1992), imidazolon (Niwa et al., 1997), crosslin, and X1, while other new compounds remain to be identied. The change in the biological activity and abnormal degradation process of proteins such as superoxide dismutase-1 (SOD-1) (Arai et al., 1987) are brought about by glycation or AGEs formation. Moreover, there is a possibility that AGEs act as a protein crosslinker by forming aggregates that are detergentinsoluble and protease-resistant, and thereby inducing the change in intracellular localization (Smith et al., 1995). Such aggregates may interfere with the axonal transport. Both the formation of adducts and the evolution in AGEs modication of proteins are accelerated by oxygen in a process called glycoxidation (Baynes, 1991; Smith et al., 1995). On the other hand, the condensation of reducing sugars with protein amino groups and subseContract grant sponsor: Research Committee for CNS Degenerative Diseases, the Ministry of Health and Welfare of Japan. *Correspondence to: Dr. Seiji Kikuchi, Department of Neurology, Hokkaido University School of Medicine, Kita 14 Nishi 5 Kitaku, Sapporo, Hokkaido, Japan. E-mail: skikuti@med.hokudai.ac.jp Received 24 September 1998; Revised 11 March 1999; Accepted 5 April 1999

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Fig. 1. Methylglyoxal (MG) induces neuronal cell death in a concentration- and timedependent manner in cultured cortical neurons. A: Cells were exposed to 0 (a), 100 (b), and 500 (c) M MG for 24 hr. Cells were then xed and stained with anti-MAP2 antibody. Bar 10 m. B: Cells were exposed for 24, 48, or 72 hr at the indicated concentrations. Cell survivals were evaluated by counting the number of MAP2-positive neurons.

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quent Amadori rearrangement lead to redox-cycling action, resulting in site-specic damage of proteins (Hunt et al., 1988; Sakurai and Tsuchiya, 1988). These observations have led to the hypothesis that glycation-induced pathology results from a cycle of oxidative stress, increased chemical modication of proteins via the Maillard reaction, and further AGEs-dependent oxidative stress (Kaneto et al., 1996; Mullarkey et al., 1990; Sakurai and Tsuchiya, 1988; Yan et al., 1994). The contribution to disease made by glycation and AGEs has been studied thus far in relation to diabetes and diabetes-related complications (Myint et al., 1995; Ryle et al., 1997). However, it has become clear that glycation and AGEs also have an impact on physiological aging (senile cataract, arteriosclerosis, etc.; Vlassara et al., 1994), neurodegenerative diseases, such as Alzheimers and Parkinsons disease (Castellani et al., 1996), and amyotrophic lateral sclerosis (ALS) (Arai et al., 1987; Chou et al., 1998; Ookawara et al., 1992; Shibata et al., 1997). In Alzheimers disease (Horie et al., 1997; Takeda et al., 1996), senile plaques and neurobrillary tangles are sometimes stained positively by anti-AGEs antibodies (Smith et al., 1994). In the present study, we investigate the neurotoxicity of glycation, particularly early-stage glycation, and its mechanisms, which are possibly synergized with oxidative stress. For this purpose, we used a glycationaccelerated system, since cultured cells cannot be maintained over 2 weeks. If glucose is used as a reducing sugar, a much longer incubation may be needed to detect AGEs, even in test tubes (Grandhee and Monnier, 1991). 3DG and methylglyoxal (MG), intermediate products of glycation, are known to further accelerate glycation and AGEs formation (Che et al., 1997; Lo et al., 1994; Okado et al., 1996; Shinoda, 1994; Suzuki et al., 1998; Yamada et al., 1994). The pathomechanism of neurodegenerative diseases, in which many factors are thought to be involved, was examined from the viewpoint of the synergism between glycation and oxidation. The present study provides the rst evidence that the glycation intermediates, MG and 3DG, are neurotoxic on cortical neurons in culture. The possibility of a treatment is suggested (Edelstein and Brownlee, 1992) if the participation of glycoxidation in the pathogenesis of neurodegenerative diseases is essential and glycoxidation is the modulating factor of the neurotoxicity in these diseases. MATERIALS AND METHODS Materials 3-DG was kindly donated by Dr. F. Hayase (Department of Agricultural Chemistry, Meiji University, Ka-

nagawa, Japan) and T. Miyata (coauthor). MG was purchased from Sigma (St. Louis, MO). Neurobasal medium and B27 supplements were purchased from GibcoBRL Life Technologies (Gaithersburg, MD). Aminoguanidine (AMG) was from Wako Chemicals (Osaka, Japan). N-acetyl-L-cysteine (NAC) was from Katayama Chemical (Osaka, Japan). 2,7-Dichlorouorescin diacetate (DCF-DA) was from Molecular Probes (Eugene, OR). Polyclonal anti-AGEs antibody was prepared by one of the authors (Z. Makita). Cell Culture and Experimental Treatments Dissociated cell cultures were established from the cortices of 14-day Sprague-Dawley rat fetuses under deep anesthesia with diethylether. Cells were dissociated by incubation for 20 min in phosphate-buffered saline (PBS) containing 2 mg/ml trypsin and 0.2% DNase, followed by trituration. Cells were plated onto poly-L-lysine-coated 8-well chamber slides at a density of approximately 600/mm2 in Eagles minimum essential medium supplemented with 10% heat-inactivated fetal bovine serum. Twenty-four hr after plating, the culture medium was replaced with neurobasal medium containing B27 supplements. Experiments were performed on 10-day-old cultures. Under these culture conditions, more than 90% of the cells were found to be neurons. Cell cultures were exposed to MG or 3DG of various concentrations for 24 hr with or without AMG or NAC. Analyses of Neuronal Survival Neuronal survival was assessed by counting the number of morphologically undamaged neurons on immunocytochemical staining using monoclonal anti-microtubuleassociated protein 2 (anti-MAP2) antibody. After experimental treatments, cultures were xed in 4% paraformaldehyde for 30 min, and membranes were permeabilized with 0.2% Triton X-100. Cells were incubated with blocking solution (10% normal rabbit serum in PBS) for 30 min and then with monoclonal anti-MAP2 antibody in blocking solution at a dilution of 1:500 overnight at 4C. Biotinylated secondary antibody, ABC solution, and diaminobenzidine were used to visualize stained cells. Analysis of Neuronal Apoptosis and Detection of Reactive Oxygen Species (ROS) As a measure of apoptosis, cells were xed in 4% paraformaldehyde, membranes were permealized with 0.2% Triton X-100, and cells were stained with the uorescent DNA-binding dye Hoechst 33258 (1 mg/ml). Hoechst-stained cells were visualized and photographed under epiuorescence illumination (excitation, 340 mn; barrier lter, 510 nm).

Fig. 2. 3-Deoxyglucosone (3DG) induces neuronal cell death in a concentration- and time-dependent manner in cultured cortical neurons. A: Cells were exposed to 0 (a), 100 (b), and 500 (c) M 3DG for 24 hr. Cells were then xed and stained with anti-MAP2 antibody. Bar 10 m. B: Cells were exposed for 24, 48, or 72 hr at the indicated concentrations. Cell survivals were evaluated by counting the number of MAP2positive neurons.

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After incubation with the reagents described, ineternucleosomal DNA cleavage was detected by ladder formation as reported previously by Kaneto et al. (1996). ROS were detected with DCF-DA, which produces a green uorescence when oxidized. Cultures were incubated with 50 M DCF-DA for 30 min at 37C, rinsed three times with PBS, and visualized by epiuorescence microscopy (excitation, 488 nm; emission, 510 nm). Immunocytochemical Study for AGEs For the attempt to produce AGE on our cell culture system, cells were treated with 50 M 3DG for 3 days and then xed in 4% paraformaldehyde for 30 min, and membranes were permeabilized witn 0.2% Triton X-100. Cells were incubated with blocking solution (10% normal goat serum in PBS) for 30 min and then with polyclonal anti-AGE antibody in blocking solution at various dilutions (1:100, 1:500, 1:1,000, and 1:10,000) overnight at 4C. Biotinylated secondary antibody, ABC solution, and diaminobenzidine were used to visualize stained cells. RESULTS MG and 3DG Have Neurotoxic Effects on Cultured Cortical Neurons The effects of MG on neuronal survival were rst examined morphologically. Incubation of cortical neurons with 100 M MG for 24 hr resulted in decreases in cell numbers as well as in number and length of neurites (Fig. 1A). Treatment with MG resulted in a concentration- and time-dependent decrease in the number of viable neurons when cell survival was assessed 24 hr after treatment (Fig. 1B). LC50 for MG neurotoxicity was 130 M. Incubation of cortical neurons with 100 M 3DG for 24 hr also resulted in decreases in cell numbers as well as in number and length of neurites (Fig. 2A). Treatment with 3DG resulted in a concentration- and timedependent decrease in the number of viable neurons when assessed 24 hr after treatments (Fig. 2B). LC50 for 3DG neurotoxicity was 209 M. MG and 3DG Induce Neuronal Apoptosis Cells were treated with 100 M of MG or 100 M of 3DG, and apoptosis was examined after 30 min by assessment of chromatin condensation in cells stained with the Hoechst 33258 for 30 min. Treatment of neurons with both agents resulted in chromatin condensations (for MG; Fig. 3, for 3DG; data not shown). Nuclear fragmentation was demonstrared to start 30 min after the exposure to MG, and was increasing even after 3 hr. DNA ladder formation was induced after incubation with 100 M of MG for 3 and 24 hr (data not shown).

MG and 3DG Induce Production of Peroxide Cells treated with 50 M of MG for 30 min emitted higher levels of peroxide (Fig. 4b) than those treated with vehicle (Fig. 4a) as detected using the uorescence marker DCF-DA. Neurotoxicity of MG and 3DG Is Attenuated by NAC and AMG Cultures were pretreated for 24 hr with 1 mM NAC and then exposed to MG. Neuronal degenerations induced by MG were signicantly reduced in cultures pretreated with NAC (Fig. 5). Cultures were pretreated for 24 hr with 1 mM of AMG and then exposed to 3DG. Pretreatment with AMG showed no signicant protective effect against neurotoxicity induced by 3DG (data not shown). Cotreatment with AMG, however, resulted in signicant protection from the neurotoxicity of 3DG (Fig. 6). Exposure to 3DG Fails to Produce Positive AGEs Immunostaining For the attempt to produce AGEs in cultured cortical neurons, cultured cells were exposed to 50 M of 3DG for 3 days, which concentration caused an approximately 40% reduction in neuronal survival. There was no signicant staining for anti-AGEs antibody in cortical neurons using this method of exposure (data not shown). DISCUSSION In the present study, we investigated the neurotoxicity of glycation, particularly early-stage glycation, and its mechanisms, which are possibly synergized with oxidative stress. For this purpose, we used a glycationaccelerated system. MG and 3DG are known to accelerate glycation reactions and crosslinking of proteins. In cases of hyperglycemia, it has been shown that the concentrations of both compounds are elevated and that both compounds accelerate glycation reactions. MG (Che et al., 1997; Lo et al., 1994; Okado et al., 1996; Suzuki et al., 1998) is one of a series of dicarbonyl intermediates, which includes such intermediates as glucosone, deoxyglucosone, dehydroascorbate, and glyoxal, that have been identied in the Maillard reaction. MG is formed nonenzymatically by amine-catalysed sugar fragmentations, and by spontaneous decomposition of triose phosphate intermediates in glycolysis. It is also a product of the metabolism of acetol, an intermediate in the catabolism of both threonine and the ketone body acetone. MG reacts rapidly with amino, guanidino, and thiol functional groups in proteins leading to denaturation and crosslinking of proteins. The physiological signicance

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Fig. 3. MG induces apoptotic nuclear alterations in cultured cortical neurons. Cells were exposed to vehicle (a), or 100 M of MG for 30 min (b) and 3 hr (c), then were stained with Hoechst dye. MG induced nuclear condensation and fragmentation in neurons. Neurons were photographed using a uorescence microscope with a 20 objective.

Fig. 4. MG induces peroxide accumulation in cultured cortical neurons. Cells were exposed to 0 (a) or 50 M (b) of MG for 30 min. Cells were then washed and loaded with 50 M of 2,7-Dichlorouorescin diacetate (DCF-DA) for 30 min.

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of the modication of proteins by MG has been difficult to judge, since until recently, the physiological concentration of MG had not been determined. Lo et al. (1994) have shown that at physiological concentration, MG binds and irreversibly modies plasma proteins. 3DG (Okado et al., 1996; Shinoda, 1994; Suzuki et al., 1998; Yamada et al., 1994), another highly reactive carbonyl compound, is formed after the multiple dehydration and rearrangements of Amadori products. 3DG reacts again with free amino groups, leading to crosslinking via the formation of AGEs in the late stage of the Maillard reaction. Incubation of 3DG with proteins leads to the formation of pyrraline and pentosidine (Dyer et al., 1991), whose structures have identied them as AGEs occurring in human tissue proteins. Plasma concentrations of MG and 3DG reach 5 M and 1 M, respectively, under diabetic conditions (Phillips et al., 1993). Niwa et al. (1993) reported plasma concentration of 3DG in DM patients with nephropathy as 1,235 ng/ml (7.6 M), versus 314 ng/ml (1.9 M) in healthy subjects. These compounds are capable of inducing apoptotic cell death in the macrophage-derived cell line, U937, with 10300 M of MG and 101,000 M of 3DG (Okado et al., 1996). In addition, PC12 cells are susceptible to MG of 300 M and over, and to 3DG of 10 mM and over (Suzuki et al., 1998). Taken together, these results suggest that the concentrations of MG and 3DG used here were reasonable and were appropriate for investigating the acute phase of their neurotoxicity. Until now, the validity of the incubation time for glycation and AGEs formation has been investigated in test tubes using reducing sugars and proteins such as albumin, RNase, and collagen. Such investigations have shown that a period of several days to a month is needed, depending on the sugars used. However, the incubation time in our experiments (24 hr) is sufficient to examine the early phase of neurotoxicity of glycoxidation with MG and 3DG, although it may be insufficient to detect AGEs formation. Recently, Niwa et al. (1998) reported that 3DG and glyoxal accelerated the formation of CML, a chemically dened AGE, in the explant-cultured neurons of dorsal root ganglia. To our knowledge, there is no direct pathway through which CML is produced from 3DG. CML is, however, reported to be produced by lipid peroxidation under the condition of oxidant stress. Even though glyoxal is a known precursor of CML, glyoxal itself is also formed during lipid peroxidation. (Moreover, the signicance of glyoxal has not been elucidated even in diabetic complications.) It is the oxidant stress induced by 3DG and MG (a derivative of glyoxal) that was demonstrated in our study. Taken together, it is much more possible that, in their experiment (Niwa et al., 1998), CML was

Fig. 5. Effects of N-acetyl-L-cysteine (NAC) on MG neurotoxicity. Cells were preincubated for 24 hr in the presence or absence of 10 mM of NAC, then exposed for 24 hr to MG.

Fig. 6. Effects of AMG on 3DG neurotoxicity. Cells were exposed for 24 hr to 3DG in the presence or absence of 1 mM of AMG.

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synthesized by lipid peroxidation under the oxidant stress induced by 3DG and glyoxal, and that they did not fully elucidate the involvement of glycation in their system. Contrary to our study, neurotoxicity was not demonstrated in their study (Niwa et al., 1998), although they used 3DG up to 1 mM which was enough to bring about neurotoxicity in our study. Does it mean that CML is not toxic enough to cause neuronal death? There are several possibilities to explain this discrepancy. The most important point is the difference of culture system, i.e., our dissociated cultured neurons can be easily exposed to 3DG directly. On the other hand, their explant cultured neurons (Niwa et al., 1998) were surrounded by many non-neuronal cells which may have had protective effects on neurons. In the present study, we elucidated the neurotoxicity of early intermediates of glycation through oxidant stress. For the study of the neurotoxicity of AGE, which requires several cascade reactions after early intermediates, analyses with non-CML AGE are necessary because CML, itself, can not generate reactive oxygen species or act as a crosslinker. Long-term incubation (for example, with slice culture system) is also necessary to make non-CML AGE generate. LC50 of MG and 3DG were different, i.e., 130 M and 209 M, respectively. As mentioned above, in the macrophage-derived cell line, U937, the concentrations of MG and 3DG producing intracellular peroxide were essentially the same, while nuclear fragmentation (Hoechst stain) was detected by 200 M MG and 1,000 M 3DG (Shinoda, 1994). In PC12 cells, cell viability was more greatly diminished by MG treatment than by treatment with 3DG (Suzuki et al., 1998). These differences are possibly due to: (1) the cell permeability of MG (Che et al., 1997) and 3DG; (2) their reactivity with proteins; (3) the amount of reactive oxygen species produced during glycation; (4) the toxicity of glycation products as a protein crosslinker; or (5) the mechanisms of detoxication (Phillips et al., 1993; Suzuki et al., 1998). Previously, none of glycation products had been investigated from a toxicological point of view. Future studies will be needed to examine the difference between MG and 3DG toxicity using a DCF experiment to quantify the amount of ROS. With regard to the detoxication mechanism, MG is mainly inactivated enzymatically by the GSH-dependent glyoxalase pathway and NADPH-dependent aldose reductase pathway (Phillips et al., 1993). 3DG is mainly detoxied by NADPHdependent aldehyde reductase (Suzuki et al., 1998). In the aldehyde reductase (ALR) gene-transfected PC12 cells, the cytotoxicity of both MG and 3DG and apoptotic cell death were decreased (Suzuki et al., 1998). This suggests that intracellular ALR protects neural cells from cytotox-

icity of MG and 3DG, and that neural cells, which normally express a low level of ALR, might be susceptible to glycation. Future studies will be needed to investigate the detoxication mechanism in each cell type. The intracellular peroxide level was assessed using oxidant-sensitive uorescent DCF-DA. Fifty M of MG is sufficient to induce ROS in cultured cortical neurons in 30 min. As for U937 cells, peroxide production has been analyzed by uorescent activated cell sorter (FACS), with the result that MG over 10 M was shown to produce peroxide (Okado et al., 1996). NAC demonstrated neuroprotective action by treatment prior to the exposure to MG. NAC can raise intracellular GSH levels and thereby provide cells with the cosubstrate required to eliminate hydroperoxides, resulting in protection from ROS. In addition, NAC also reacts with MG directly, rapidly, and reversely to form the hemithioacetal adduct. To exclude the latter possibility, NAC was added simultaneously with the exposure to MG or added after it. We were unable to demonstrate a protective effect of NAC using this method of administration. The level of GSH induction and its time course are important subjects for future research. The concentration of NAC showing neuroprotective action was 1 mM, which is the same as that used previously (Kaneto et al., 1996). Although examination with other antioxidants will be necessary, the participation of oxidative stress was conrmed by the DCF experiment and by the protective action of NAC against the neurotoxicity of 3DG and MG. Aminoguanidine has been used already as an inhibitor of AGEs crosslinking in experimental diabetes. The primary mechanism of aminoguanidine is direct reaction with Amadori derivative fragments, such as 3DG, which prevents subsequent AGEs formation in susceptible proteins. It has been reported, however, that aminoguanidine has antioxidant properties in addition to the effects on glycation (Scaccini et al., 1994). Aminoguanidine also inhibits inducible nitric oxide synthase iNOS (Soulis et al., 1997). In our study, cotreatment of aminoguanidine protected cultured cortical neurons against 3DG neurotoxicity. This suggests that the protective action of aminoguanidine in our experiment was due to a direct reaction which did not require pretreatment to induce other proteins, such as iNOS. The uptake of aminoguanidine into the cell has not yet been conrmed. It is difficult to conclude the pathological signicance of glycation in neurodegenerative diseases. In the present study, we showed that glycation, even in the early stage, can be harmful to neurons. And if it is a modulating factor of the pathomechanism, interference with the process by which AGEs formation occurs may provide new therapeutic opportunities to reduce the pathophysiological changes associated with neurodegeneration. In

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