2(3H)-Dihydrofranolactone metabolites from Pleosporales sp. NUH322 as anti-amyotrophic lateral sclerosis drugs.

Amyotrophic lateral sclerosis (ALS) is a devastating motor disease with limited treatment options. A domestic fungal extract library was screened using three assays related to the pathophysiology of ALS with the aim of developing a novel ALS drug. 2(3H)-dihydrofuranolactones 1 and 2, and five known compounds 3-7 were isolated from Pleosporales sp. NUH322 culture media, and their protective activity against the excitotoxicity of ß-N-oxalyl-L-α,ß-diaminopropionic acid (ODAP), an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamatergic agonist, was evaluated under low mitochondrial glutathione levels induced by ethacrynic acid (EA) and low sulfur amino acids using our developed ODAP-EA assay. Additional assays evaluated the recovery from cytotoxicity caused by transfected SOD1-G93A, an ALS-causal gene, and the inhibitory effect against reactive oxygen species (ROS) elevation. The structures of 1 and 2 were elucidated using various spectroscopic methods. We synthesized 1 from D-ribose, and confirmed the absolute structure. Isolated and synthesized 1 displayed higher ODAP-EA activities than the extract and represented its activity. Furthermore, 1 exhibited protective activity against SOD1-G93A-induced toxicity. An ALS mouse model, SOD1-G93A, of both sexes, was treated orally with 1 at pre- and post-symptomatic stages. The latter treatment significantly extended their lifespan (p = 0.03) and delayed motor deterioration (p = 0.001-0.01). Our result suggests that 1 is a promising lead compound for the development of ALS drugs with a new spectrum of action targeting both SOD1-G93A proteopathy and excitotoxicity through its action on the AMPA-type glutamatergic receptor.

Suppression of Superficial Microglial Activation by Spinal Cord Stimulation Attenuates Neuropathic Pain Following Sciatic Nerve Injury in Rats.

We evaluated the mechanisms underlying the spinal cord stimulation (SCS)-induced analgesic effect on neuropathic pain following spared nerve injury (SNI). On day 3 after SNI, SCS was performed for 6 h by using electrodes paraspinally placed on the L4-S1 spinal cord. The effects of SCS and intraperitoneal minocycline administration on plantar mechanical sensitivity, microglial activation, and neuronal excitability in the L4 dorsal horn were assessed on day 3 after SNI. The somatosensory cortical responses to electrical stimulation of the hind paw on day 3 following SNI were examined by using in vivo optical imaging with a voltage-sensitive dye. On day 3 after SNI, plantar mechanical hypersensitivity and enhanced microglial activation were suppressed by minocycline or SCS, and L4 dorsal horn nociceptive neuronal hyperexcitability was suppressed by SCS. In vivo optical imaging also revealed that electrical stimulation of the hind paw-activated areas in the somatosensory cortex was decreased by SCS. The present findings suggest that SCS could suppress plantar SNI-induced neuropathic pain via inhibition of microglial activation in the L4 dorsal horn, which is involved in spinal neuronal hyperexcitability. SCS is likely to be a potential alternative and complementary medicine therapy to alleviate neuropathic pain following nerve injury.

Stress-related over-enhancement of the hypothalamic-pituitary-adrenal axis causes experimental neurolathyrism in rats.

Neurolathyrism is a motor neuron disease that is caused by the overconsumption of grass peas (Lathyrus sativus L.) under stressful conditions. The neuro-excitatory ß-N-oxalyl-L-α,ß-diaminopropionic acid present in grass peas was proposed the causative agent of spastic paraparesis of the legs. Historical reports of neurolathyrism epidemics, studies of neurolathyrism animal models, and in vitro studies on the mechanism of ß-N-oxalyl-L-α,ß-diaminopropionic acid toxicity support the hypothesis that stress increases susceptibility to neurolathyrism. To elucidate the role of stress in neurolathyrism-induced motor dysfunction, we focused on the hypothalamic-pituitary-adrenal axis in a rodent model of neurolathyrism. Our results implicated increased glucocorticoid and neuroinflammation in the motor dysfunction (paraparesis) exhibited by the stress loaded rat models of neurolathyrism.

[Research in Motor Neuron Diseases Caused by Natural Substances: Focus on Pathological Mechanisms of Neurolathyrism].

Diseases of the motor-conducting system that cause moving disability affect socio-economic activity as well as human dignity. Neurolathyrism, konzo, and amyotrophic lateral sclerosis-parkinsonism-dementia complex (ALS-PDC) have attracted researchers to study the pathology of motor neuron (MN) diseases such as ALS. I have been studying neurolathyrism, which is caused by overconsumption of a legume grass pea (Lathyrys sativus L.). Among people who consume the legume as a food staple, many developed life-long paraparesis in their legs. ß-N-oxalyl-l-α,ß- diaminopropionic (l-ß-ODAP; BOAA), contained in this plant, is a neurotoxic analog of l-glutamic acid. We have clarified that in addition to the causal involvement of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) type glutamatergic receptor in MN death, a toxic role of group I metabotropic glutamate receptors as well as transient receptor potential channels were involved in the MN insult by l-ß-ODAP using primary MN culture. We have also established a neurolathyrism rat model by repeated, peripheral l-ß-ODAP treatment to newborn rats under mild stress. Rats showing hind-leg paraparesis with an incidence rate of around 25% were useful to study the in vivo pathology of MN disease. MNs of these rats were greatly decreased at their lumbo/sacral segments at various ages. Intra-parenchymal hemorrhage was consistently observed in paraparetic rats but not in cripple-free, treated rats. MN were depleted even at an acute period around bleeding spots, suggesting catastrophic neuro-vascular-glial interaction in this MN disease. Summaries of konzo and ALS-PDCs studies are also introduced.

New insights into the mechanism of neurolathyrism: L-ß-ODAP triggers [Ca2+]i accumulation and cell death in primary motor neurons through transient receptor potential channels and metabotropic glutamate receptors.

Neurolathyrism is a motor neuron (MN) disease caused by ß-N-oxalyl-L-α,ß-diaminopropionic acid (L-ß-ODAP), an AMPA receptor agonist. L-ß-ODAP caused a prolonged rise of intracellular Ca(2+) ([Ca(2+)]i) in rat spinal cord MNs, and the [Ca(2+)]i accumulation was inversely proportional to the MN's life span. The [Ca(2+)]i rise induced by L-ß-ODAP or (S)-AMPA was antagonized completely by NBQX, an AMPA-receptor blocker. However, blocking the L-type Ca(2+) channel with nifedipine significantly lowered [Ca(2+)]i induced by (S)-AMPA, but not that by L-ß-ODAP. Tetrodotoxin completely extinguished the [Ca(2+)]i rise induced by (S)-AMPA or kainic acid, whereas that induced by L-ß-ODAP was only attenuated by 65.6±6% indicating the prominent involvement of voltage-independent Ca(2+) entry. The tetrodotoxin-resistant [Ca(2+)]i induced by L-ß-ODAP was blocked by 2-APB, Gd(3+), La(3+), 1-(ß-[3-(4-methoxy-phenyl)propoxy]-4-methoxyphenethyl)-1H-imidazole hydrochloride (SKF-96365) and flufenamic acid, which all are blockers of the transient receptor potential (TRP) channels. Blockers of group I metabotropic glutamate receptors (mGluR I), 7-(hydroxyiminocyclopropan[b]chromen-1α-carboxylate ethyl ester (CPCCPEt) and 2-methyl-6-(phenylethynyl)-pyridine (MPEP) also lowered the [Ca(2+)]i rise by L-ß-ODAP. MN cell death induced by L-ß-ODAP was prolonged significantly with SKF-96365 as well as NBQX. The results show the involvement of TRPs and mGluR I in L-ß-ODAP-induced MN toxicity through prolonged [Ca(2+)]i mobilization, a unique characteristic of this neurotoxin.

Prostaglandin E2-induced cell death is mediated by activation of EP2 receptors in motor neuron-like NSC-34 cells.

Prostaglandin E2 (PGE2) was shown to induce neuronal death in the CNS. To characterize the neurotoxicity of PGE2 and E-prostanoid receptors (EP) in motor neurons, we investigated PGE2-induced cell death and the type(s) of EP responsible for mediating it in NSC-34, a motor neuron-like cell line. Immunoblotting studies showed that EP2 and EP3 were dominantly expressed in NSC-34 cells and motor neurons in mice. Exposure to PGE2 and butaprost, an EP2 agonist, but not sulprostone, an EP1/3 agonist, resulted in decreased viability of these cells. These results suggest that PGE2 induces cell death by activation of EP2 in NSC-34 cells.

Vascular insult accompanied by overexpressed heme oxygenase-1 as a pathophysiological mechanism in experimental neurolathyrism with hind-leg paraparesis.

Neurolathyrism (NL) is a motor neuron disease characterized by spastic paraparesis in the hind legs. ß-N-oxalyl-l-α,ß-diaminopropionic acid (l-ß-ODAP), a component amino acid of the grass pea (Lathyrus sativus L.), has been proposed as the cause of this disease. In our NL rat model, we previously reported that transient intra-parenchymal hemorrhage occurred in the lower spinal cord during the early treatment period. We show here a possible pathological role of the hemorrhage in motor neuron damage and paraparesis pathology. In the lumbo-sacral spinal cord, blood vessel integrity was lost with numerous TdT-mediated dUTP nick end-labeling-positive blood vessel-like structures occurring simultaneously with the hemorrhage. We observed a coincident >10-fold increase in heme oxygenase-1 (HO-1) only in the lower spinal cord. The early period of paraparesis in the lower leg was greatly suppressed by pretreatment with zinc protoporphyrin IX, a HO-1 inhibitor. In vitro, l-ß-ODAP was toxic to human umbilical vein endothelial cells compared to l-glutamate. The present data shed light on the role and the mechanism of vascular insult in causing dysfunction and moribund motor neurons in experimental NL.

Antagonism of NMDA receptors by butanesulfonyl-homospermine guanidine and neuroprotective effects in in vitro and in vivo.

The polyamine derivative BsHSPMG (butanesulfonyl-homospermine with guanidine group) was found to inhibit macroscopic currents strongly at heteromeric N-methyl-D-aspartate (NMDA) receptors (NR1/NR2A and NR1/NR2B) and Ca(2+)-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (homomeric glutamate receptor 1) receptors expressed in Xenopus laevis oocytes on voltage-clamp recording. The IC(50) values of BsHSPMG for NR1/NR2A, NR1/NR2B, NR1/NR2C, and NR1/NR2D receptors were 0.016, 0.021, 5.4, and 9.0 µM, respectively. BsHSPMG inhibited the activity of NR1/NR2A and NR1/NR2B receptors more strongly and did it for those of NR1/NR2C and NR1/NR2D receptors more weakly than a therapeutic drug of Alzheimer's disease, memantine. The inhibition by BsHSPMG was voltage-dependent, since it was prominent at -100 mV compared to that at -20 mV. Mutations including NR1 N616Q, E621Q, N650A, L655A, T807C, NR2B W559L, M562S, W607L, N616Q, and V620E, among others, reduced the inhibition by BsHSPMG, suggesting that BsHSPMG penetrates the channel pore of NMDA receptors deeply. The toxicity of BsHSPMG in neuroblastoma SH-SY5Y cells was much weaker than that of memantine. The effect of BsHSPMG was measured on the focal cerebral ischemia induced by occlusion (1 h) of the middle cerebral artery in mice. BsHSPMG applied before or after occlusion greatly reduced the volume of infarct in mice. These findings demonstrate that BsHSPMG penetrates the NMDA channel pore and exhibits neuroprotective effects against excitatory toxicity in mice.

Sulfur amino acids deficiency caused by grass pea diet plays an important role in the toxicity of L-ß-ODAP by increasing the oxidative stress: studies on a motor neuron cell line.

Neurolathyrism is a motor neuron disease caused by the overconsumption of grass pea (Lathyrus sativus L.) containing L-ß-ODAP. The precise mechanism to cause motor neuron degeneration has yet to be elucidated, but should agree with the epidemiological backgrounds. Considering the amino acid content of the legume, and the epidemiological link with prolonged unbalanced nutrition, the shortage of sulfur amino acids methionine and cysteine could affect the toxicity of L-ß-ODAP. We analyzed the effect of these amino acids in the media on the toxicity using primary motor neuron culture and a motor neuron cell line NSC-34. Deprivation of both methionine and cysteine exacerbated the toxicity of L-ß-ODAP by 66% compared to the complete medium. The glutathione content of these cells was greatly decreased in sulfur amino acid-deprived medium. L-ß-ODAP further lowered the content in the deprived media to be 32-44% of the controls compared to normal media being 62-74%. The increased motor neuron toxicity in this medium was neutralized by the addition of reduced glutathione ethyl ester or N-acetylcysteine suggesting the importance of the mitochondrial oxidative stress induced by L-ß-ODAP under sulfur amino acid-deficient conditions.

Hind-limb paraparesis in a rat model for neurolathyrism associated with apoptosis and an impaired vascular endothelial growth factor system in the spinal cord.

Neurolathyrism is a motor neuron disease characterized by lower limb paraparesis. It is associated with ingestion of a plant excitotoxin, beta-N-oxalyl-L-alphabeta-diaminopropionic acid (L-beta-ODAP), an agonist of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate-type glutamatergic receptors. Previously, a limited model of neurolathyrism was reported for the rat. To improve upon the model, we stressed rat pups by separation from their mothers, followed by the subcutaneous L-beta-ODAP treatment, resulting in a 4.6-fold higher incidence (14.0-15.6%) of the paraparesis compared with the prior study. The number and size of motor neurons in these rats were decreased only in the lumbar and sacral cord segments, at approximately 13-36 weeks after treatment. Only lumbar and sacral spinal cord tissue revealed pathological insults typical of physical and ischemic spinal cord injury in the surviving motor neurons. In addition, extensive but transient hemorrhage occurred in the ventral spinal cord parenchyma of the rat, and numerous TdT-mediated dUTP-biotin nick end-labeling (TUNEL)-positive cells were also observed. In parallel, vascular endothelial growth factor receptor (VEGFR)-2 (Flk-1) levels were significantly lowered in the lumbosacral spinal cord of the paraparetic rats compared with their controls, suggesting a failure of the VEGF system to protect neurons against L-beta-ODAP toxicity. We propose, based on these data, a novel pathological process of motor neuron death induced by peripheral L-beta-ODAP. For the first time, we present a model of the early molecular events that occur during chemically induced spinal cord injury, which can potentially be applied to other neurodegenerative disorders.

Differential effects of linear and cyclic polyamines on NMDA receptor activities.

The linear polyamine spermine enhances N-methyl-d-aspartate (NMDA) receptors activity at depolarized membrane potential and shows a voltage-dependent block. Spermine potentiates NMDA receptor currents in the presence of saturating concentrations of glutamate and glycine, but cyclic polyamines such as CP2323 do not. CP2323 inhibited the currents most potently amongst 10 kinds of cyclic polyamines tested. The inhibition was prominent at heteromeric NR1/NR2A and NR1/NR2B receptors but not at NR1/NR2C and NR1/NR2D receptors expressed in Xenopus oocytes. Inhibition by CP2323 was voltage-dependent, because the degree of inhibition was in the order -100mV>-70mV>-20mV. It was 10-100 times more prominent than inhibition by spermine. The inhibitory potency of both CP2323 and spermine was attenuated by the mutations around the vestibule of the channel pore at NR1 W563, N650, T807, and NR2B Y646. Inhibition by CP2323 was hardly affected by the mutations of NR1 N616 and E621, whereas inhibition by spermine was reduced by these mutations. The results suggest that CP2323 interacts with the vestibule region of the NMDA receptor and does not enter deep into the channel. Mutations of NR2B W607 greatly reduced the inhibition by CP2323 and spermine, suggesting that the mutation of this residue may cause the change of the channel structure. Neuroprotective effects of cyclic polyamines against cell damage caused by NMDA were compared with those of spermine in cultured rat hippocampal neurons. Addition of CP2323, but not spermine, into the medium attenuated the neurotoxicity induced by NMDA. These results indicate that CP2323 functions as a channel blocker of the NMDA receptor.

Cleft-type cyclophanes confer neuroprotection against excitatory neurotoxicity in vitro and in vivo through inhibition of NMDA receptors.

The cleft-type cyclophanes (ACCn, DNCn and TsDCn) were found to strongly inhibit macroscopic currents at heteromeric NMDA receptors (NR1/NR2) but not AMPA receptors expressed in Xenopus oocytes at voltage-clamp recording. The inhibition by cleft-type cyclophanes was voltage-dependent, because the inhibition was larger at -100 mV than at -20 mV. Mutations at NR1 N650, located in the vestibule of the channel pore, reduced the inhibition by DNCn and TsDCn, suggesting that the residue (N650) interacts with these cleft-type cyclophanes. Cell toxicity of TsDCn on SH-SY5Y cells was slightly weaker than that of memantine. The neuroprotective effects of cleft-type cyclophanes against cell damage caused by NMDA were investigated in cultured rat hippocampal neurons. Addition of 10 microM DNCn or TsDCn into the medium ablated the neurotoxicity induced by NMDA, and a similar effect was also observed with memantine. The neuroprotective effects of cleft-type cyclophanes were then assayed on NMDA-induced seizures in mice. Intracerebroventricular injection of TsDCn (5 mg/mouse) decreased the seizure induced by intraperitoneal injection of NMDA (115 mg/kg) in mice. The results demonstrate that these cleft-type cyclophanes interact directly with the extracellular mouth of the NMDA channel pore and exhibit neuroprotective effects on NMDA-induced excitatory toxicity in primary cultured neurons and mice.

Role of caspase-12 in amyloid beta-peptide-induced toxicity in organotypic hippocampal slices cultured for long periods.

Amyloid beta (Abeta) toxicity has been implicated in cell death in the hippocampus, but its specific mechanisms are poorly understood. In this study, Abeta-induced cell death was investigated in organotypic hippocampal slice cultures (OHCs) that were cultured for various periods in vitro. There were no obvious histological differences among slices cultured for 3 to 7 weeks in vitro. Although there was little neurotoxicity after treatment with Abeta25-35 in OHCs cultured for relatively shorter periods (3-5 weeks), age-dependent cell death was evident in OHCs cultured for relatively longer periods (6-7 weeks) after exposure to Abeta25-35. In OHCs cultured for 7 weeks, S-allyl-L-cysteine (SAC), a component of aged garlic extract, protected the cells in areas CA1 and CA3 and the dentate gyrus from Abeta25-35-induced toxicity. The immunoreactivity of cleaved caspase-12 was increased whereas that of glucose-regulated protein 78 was not altered after exposure to Abeta25-35. The increases in the cleaved caspase-12 were also reversed by simultaneously applied SAC. These results suggest that OHCs cultured for relatively longer periods are more susceptible to Abeta-induced toxicity and that the Abeta-induced cell death involves caspase-12-dependent pathways. It is also suggested that SAC is able to protect against the Abeta-induced neuronal cell death through the inhibition of the caspase-12-dependent pathway.

Monoamines directly inhibit N-methyl-D-aspartate receptors expressed in Xenopus oocytes in a voltage-dependent manner.

Dopamine has numerous functions in the brain and has been shown to modulate responses of N-methyl-D-aspartate (NMDA) receptors on thalamic and hippocampus neurons [N.G. Castro, M.C.F. de Mello, F.G. de Mello, Y. Aracava, Direct inhibition of the N-methyl-D-aspartate receptor channel by dopamine and (+)-SKF38393, Br. J. Pharmacol. 126 (1999) 1847-1855]. Thus, the effects of dopamine, serotonin, tyramine, epinephrine, norepinephrine, and octopamine on NMDA receptors were studied using voltage-clamp recording of recombinant NMDA receptors expressed in Xenopus oocytes. Serotonin and tyramine, in addition to dopamine, were found to inhibit macroscopic currents at heteromeric NMDA receptors, but not AMPA (GluR1/GluR2) receptors. Epinephrine, norepinephrine and octopamine also weakly inhibited macroscopic currents at NR1/NR2A and NR1/NR2B receptors. The inhibitory effects of these monoamines became prominent at -100 mV comparing those at -20 mV. Mutations at NR1 N616, NR2B N615, and NR2B N616, but not at NR1 W563 and NR1 N650, reduced the inhibitory effects by monoamines. These results indicate that these monoamines directly act on the narrowest region of channel pore.

Partial involvement of group I metabotropic glutamate receptors in the neurotoxicity of 3-N-oxalyl-L-2,3-diaminopropanoic acid (L-beta-ODAP).

Neurolathyrism is a human motoneuron disease caused by the overconsumption of grass pea (Lathyrus sativus) that contains a toxic non-protein amino acid, 3-N-oxalyl-L-2,3-diaminopropanoic acid (L-beta-ODAP). The preventive activities of various glutamatergic agents from acute neuronal death caused by L-beta-ODAP were studied using rat primary cortical neuron/glia culture. Nearly 80% of the rat primary cortical neurons were killed by 300 microM L-beta-ODAP within 24 h. Though antagonists acting on the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor prevented most of the toxicity, antagonists acting on group I metabotropic glutamatergic receptors (mGluRs), including (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA), (S)-alpha-methyl-4-carboxyphenylglycine (MCPG), and 2-methyl-6-(2-phenylethenyl)pyridine (SIB1893) partially and significantly prevented neuronal death due to L-beta-ODAP. These antagonists, within limited concentrations, did not have any inhibitory effects on the currents through AMPA receptors expressed in Xenopus oocytes. L-beta-ODAP itself did not induce the currents through group I mGluRs expressed in Xenopus oocytes. These results suggest that the neurotoxicity induced by L-beta-ODAP is partially mediated by the activation of group I mGluRs by an indirect mechanisms.

Polyamine transport, accumulation, and release in brain.

Cycling of polyamines (spermine and spermidine) in the brain was examined by measuring polyamine transport in synaptic vesicles, synaptosomes and glial cells, and the release of spermine from hippocampal slices. It was found that membrane potential-dependent polyamine transport systems exist in synaptosomes and glial cells, and a proton gradient-dependent polyamine transport system exists in synaptic vesicles. The glial cell transporter had high affinities for both spermine and spermidine, whereas the transporters in synaptosomes and synaptic vesicles had a much higher affinity for spermine than for spermidine. Polyamine transport by synaptosomes was inhibited by putrescine, agmatine, histidine, and histamine. Transport by glial cells was also inhibited by these four compounds and additionally by norepinephrine. On the other hand, polyamine transport by synaptic vesicles was inhibited only by putrescine and histamine. These results suggest that the polyamine transporters present in glial cells, neurons, and synaptic vesicles each have different properties and are, presumably, different molecular entities. Spermine was found to be accumulated in synaptic vesicles and was released from rat hippocampal slices by depolarization using a high concentration of KCl. Polyamines, in particular spermine, may function as neuromodulators in the brain.