Kazuyoshi Shinpo

Glycation—a sweet tempter for neuronal death

Glycation, one of the post-translational modifications of proteins, is a nonenzymatic reaction initiated by the primary addition of a sugar aldehyde or ketone to the amino groups of proteins. In the early stage of glycation, the synthesis of intermediates leading to the formation of Amadori compounds occurs. In the late stage, advanced glycation end products (AGE) are irreversibly formed after a complex cascade of reactions. Several AGEs have been characterized chemically, while other new compounds remain to be identified. To date, studies of the contribution of glycation to diseases have been primarily focused on its relationship to diabetes and diabetes-related complications. However, glucose-induced damage is not limited to diabetic patients. Although it does not cause rapid or remarkable cell damage, glycation advances slowly and accompanies every fundamental process of cellular metabolism. It has recently become clear that glycation also affects physiological aging and neurodegenerative diseases such as Alzheimers disease and amyotrophic lateral sclerosis. Glycation alters the biological activity of proteins and their degradation processes. Protein cross-linking by AGE results in the formation of detergent-insoluble and protease-resistant aggregates. Such aggregates may interfere with both axonal transport and intracellular protein traffic in neurons. In addition, glycation reactions lead to the production of reactive oxygen species. Conversely, glycation is promoted by oxidative stress. We speculate on the presence of synergism between glycation and oxidative stress. In this review, we provide an outline of glycation and propose some possible mechanisms of its cytotoxicity and defense systems against it.

Neurotoxicity of methylglyoxal and 3‐deoxyglucosone on cultured cortical neurons: Synergism between glycation and oxidative stress, possibly involved in neurodegenerative diseases

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 first 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. Neurosci. Res. 57:280–289, 1999.

Effect of 1,25-dihydroxyvitamin D3 on cultured mesencephalic dopaminergic neurons to the combined toxicity caused by L-buthionine sulfoximine and 1-methyl-4-phenylpyridine

A decrease in intracellular glutathione content may be related to the primary event in Parkinsons disease, so increasing the glutathione level may have a therapeutic benefit. The biologically active form of vitamin D, 1,25‐dihydroxyvitamin D3 [1,25‐(OH)2D3] has been recently reported to enhance the intracellular glutathione concentration in the central nervous system. Exposing rat cultured mesencephalic neurons for 24 hr to a mixture of L‐buthionine sulfoximine (BSO) and 1‐methyl‐4‐phenylpyridium ions (MPP+) resulted in a relatively selective damage to dopaminergic neurons. This damage has been accompanied by a reduction of intracellular glutathione levels. Low doses, i.e., 1–100 nM, of 1,25‐(OH)2D3 protect cultured dopaminergic neurons against this toxicity, although higher concentrations of this active form of vitamin D have been found to enhance the toxic effect. Generation of reactive oxygen species (ROS) by this toxicity has been attenuated in cultures being pretreated with low concentrations of 1,25‐(OH)2D3. Because the hormone increases the intracellular glutathione content in cultures, determining how this hormone suppresses ROS generation may involve the enhancement of the antioxidative system. These data suggest that low doses of 1,25‐(OH)2D3 are able to protect mesencephalic dopaminergic neurons against BSO/MPP+‐induced toxicity that causes a depletion in glutathione content. J. Neurosci. Res. 62:374–382, 2000.

Tumor necrosis factor enhancement of transient outward potassium currents in cultured rat cortical neurons

The effect of recombinant human tumor necrosis factor‐α (TNF) on voltage‐gated membrane currents of cultured neurons derived from embryonic rat cerebral cortex was studied using the whole‐cell patch‐clamp technique. Treatment of neurons with TNF resulted in an increase in outward potassium current density, dependent upon the concentration of TNF and the incubation time, without affecting other membrane currents such as barium and N‐methyl‐D‐aspartate (NMDA). Long exposures (12–48 hr) to TNF (10–100 ng/ml) increased transient outward potassium current (A‐current) density without affecting the parameters of activation and inactivation of the current. Prolonged exposures to TNF diminished its increasing effect on the A‐current. Since the increase of A‐current density induced by TNF is inhibited by both the anti‐TNF receptor antibody and cycloheximide treatment, the effect of TNF might be mediated through receptors and by de novo synthesis of the channel protein itself and/or modulating proteins associated with the channel activities. Results indicate that phosphatidylcholine‐specific phospholipase C and protein kinase C, but not ceramide, are involved in the signal transduction. In toxicological experiments, TNF had no neurotoxicity. Moreover, a 12 hr pretreatment of TNF protected neurons against NMDA‐induced neurotoxicity. This protective effect of TNF was canceled by 4‐aminopyridine, an A‐current blocker, suggesting that the increase of A‐current densities induced by TNF contributes to the neuroprotection. J. Neurosci. Res. 50:990–999, 1997. © 1997 Wiley‐Liss, Inc.

The effects of neuronal induction on gene expression profile in bone marrow stromal cells (BMSC)—a preliminary study using microarray analysis

Bone marrow stromal cells (BMSC) have been anticipated as a donor for cell type for transplantation therapy in various neurological disorders. However, their neurogenic capacity still remains undetermined. In this study, we aimed to clarify whether in vitro chemical treatment promotes their neuronal differentiation on the level of gene expression. Mice BMSC were cultured with medium supplemented with DMSO, retinoic acid, and basic fibroblast growth factor, and their morphology and expression of neuronal markers were evaluated. Subsequently, using microarray and RT-PCR techniques, the treatment-induced changes in the gene expression profile were analyzed. After exposure to the medium, the BMSC simulated a neuron-like appearance and increased their immunoreactivity for nestin and Tuj-1. Microarray analysis revealed that the BMSC per se express the multilineage cellular genes, including those associated with the neuron. Chemical treatment significantly decreased the expression of genes related to mesenchymal cells and increased the expression of 5 neuron-associated genes. Microarray and RT-PCR analyses also demonstrated that the BMSC express the genes for several growth factors including NGF-beta and BDNF, indicating their therapeutic role in protecting the injured central nervous system. The present results suggest that at least a certain subpopulation of the BMSC have the potential to alter their gene expression profile in response to the surrounding environment and may possibly protect the host tissue by secreting neuroprotective factors.

Selective vulnerability of spinal motor neurons to reactive dicarbonyl compounds, intermediate products of glycation, in vitro : implication of inefficient glutathione system in spinal motor neurons

We investigated the effects of two reactive dicarbonyl compounds, methylglyoxal (MG) and 3-deoxyglucosone (3-DG), on cultured spinal cord neurons. Incubation of cortical and spinal neurons with MG and 3-DG for 24 h induced neuronal death in a dose-dependent manner. Spinal motor neurons were more vulnerable than spinal non-motor neurons and cortical neurons. Treatments with glutathione (GSH)-augmenting agents showed protective effects against MG and 3-DG neurotoxicity. Motor neurons were better protected than non-motor neurons. Cotreatment, but not pretreatment, of aminoguanidine (AG), a known inhibitor of advanced glycation end-products (AGEs) from crosslinking, showed a protective effect on spinal neurons with no difference in protective rates between motor and non-motor spinal neurons. Treatments with GSH depleting agents enhanced the neurotoxicity of MG and 3-DG on spinal neurons. Motor neurons were more vulnerable than non-motor neurons with GSH-depleting treatments prior to MG and 3-DG exposures. These data demonstrate that spinal motor neurons are more vulnerable to dicarbonyl compounds, and this selectivity might be related to the relatively inefficient GSH system in spinal motor neurons.

Protective effects of the TNF-ceramide pathway against glutamate neurotoxicity on cultured mesencephalic neurons

Pretreatments with TNF-alpha and lower concentrations of C2-ceramide protected cultured mesencephalic neurons from excitotoxicity in a dose-dependent manner. These protective effects are reduced by cotreatment with N,N-dimethylsphingosine (DMS), an inhibitor of sphingosine kinase. Since the pretreatment with sphingosine-1-phosphate (SPP) showed a neuroprotective effect, our data suggest that protective effects of TNF and C2-ceramide could be attributable to their further metabolism to SPP.

Effect of proteasome inhibitor on cultured mesencephalic dopaminergic neurons.

Proteasomal dysfunction has been implicated in the pathogenesis of Parkinsons disease (PD). We examined the effect of a selective proteasomal inhibitor, epoxomicin, on primary cultured mesencephalic neurons. Exposing rat cultured mesencephalic neurons to epoxomicin for 24 h resulted in neurotoxicity in a dose-dependent manner. Epoxomicin caused mitochondrial dysfunction, reduction in reduced glutathione (GSH), and increased generation of free radicals. Neuronal damage was significantly blocked by antioxidative/GSH-augmenting agents. Epoxomicin also increased the expression of Bax and decreased that of Bcl-2, which may cause mitochondrial dysfunction and release of free radicals. Dopaminergic neurons were preferentially resistant to the toxicity of epoxomicin. Inhibiting the synthesis of tetrahydrobiopterin (BH(4)), which has been reported to have antioxidative function, increased the susceptibility of dopaminergic neurons, whereas increasing BH(4) levels protected non-dopaminergic neurons. These findings suggest that BH(4) is at least in part a contributing factor to grand the resistance to dopaminergic neurons against epoxomicin neurotoxicity. Our results suggest that proteasome inhibition causes the neurotoxicity in mesencephalic neurons, but that is not sufficient to reproduce the selective damage to dopaminergic neurons, such as that seen in PD.

Effect of geranylgeranylaceton on cellular damage induced by proteasome inhibition in cultured spinal neurons

We investigated the effect of two proteasome inhibitors, lactacystin and epoxomicin, on cultured spinal cord neurons. The incubation of spinal neurons with proteasome inhibitors for 24 hr induced neurotoxicity in a dose‐dependent manner. We found motor neurons to be more vulnerable to proteasome‐induced neurotoxicity than nonmotor neurons. The staining of cell bodies in treated motor neurons was markedly disrupted and showed characteristic granular patterns. Proteasome‐induced neurotoxicity is accompanied by apoptotic nuclear changes, posttranslational modification of the cellular proteins, generation of intracellular free radicals, reduction in the amount of reduced glutathione, and mitochondrial dysfunction. Neurotoxicity was reduced by the administration of low concentrations (1–100 nM) of geranylgeranylacetone (GGA), which is widely used as an antiulcer drug, although higher concentrations of this drug produced neurotoxicity in spinal cord neurons. GGA was found to induce the expression of heat shock protein 70 as well as thioredoxin, which may partly contribute to the protective effect of GGA. These data suggest that the inhibition of proteasome may play a role in the mechanism of neurodegenerative diseases of the spinal cord, such as amyotrophic lateral sclerosis, and that the use of GGA may be effective in the treatment of these conditions.

Detection of N epsilon-(carboxymethyl)lysine (CML) and non-CML advanced glycation end-products in the anterior horn of amyotrophic lateral sclerosis spinal cord.

INTRODUCTION The involvement of glycation in neurodegenerative diseases such as Alzheimers disease, Parkinsons disease and amyotrophic lateral sclerosis (ALS) was recently indicated. We previously reported the existence of an Amadori product, 1-hexitol-lysine (1-HL), which is formed in the early glycation reaction, in axonal spheroids of the anterior horn of the ALS spinal cord. OBJECTIVE The purpose of the present study was to confirm the occurrence of the later-stage glycation reaction that follows the early glycation reaction and leads to the formation of advanced glycation end products (AGEs). METHOD We examined whether N(epsilon)-(carboxymethyl)lysine (CML) and non-CML AGE are present in ALS spinal cords. RESULTS Immunohistochemical staining with anti-CML antibody revealed intense positivity in the cell bodies of the remaining atrophic motor neurons and in microglia. Microglia were also positive on staining with anti-non-CML antibody. Axonal spheroids were also positive on anti-non-CML-antibody staining. Vascular endothelial cells were slightly stained by both antibodies. CONCLUSIONS The presence of non-CML AGE in the anterior horn of the ALS spinal cord indicates that the later stage of the glycation reaction is involved in the pathology of ALS. The presence of CML in the anterior horn was also confirmed, and this may reflect augmented oxidative stress.