Late appearance of glutamate transporter defects in a murine model of ALS-parkinsonism dementia complex

Excitotoxicity has been widely hypothesized to play a major role in various neurodegenerative diseases. We have used a mouse model of ALS–parkinsonism dementia complex (ALS–PDC) of the Western Pacific to explore this hypothesis. Mice fed washed cycad flour, the major epidemiological link to ALS–PDC, showed significant and progressive motor, cognitive, and sensory behavioural deficits [Wilson, J.M., Khabazian, I., Wong, M.C., Seyedalikhani, A., Bains, J.S., Pasqualotto, B.A., Williams, D.E., Andersen, R.J., Simpson, R.J., Smith, R., Craig, U.K., Kurland, L.T., Shaw, C.A., 2002. Behavioral and neurological correlates of ALS-parkinsonism dementia complex in adult mice fed washed cycad flour. Neuromol. Med. 1 (3), 207–221]. In addition, glutamate transporter (GLT-1/EAAT2) levels measured by immunohistochemistry with antibodies specific for two glial glutamate transporter splice variants (GLT-1 and GLT-1B) were significantly down-regulated showing a ‘patchy’ loss of antibody label centered on blood vessels [Wilson, J.M., Khabazian, I., Pow, D.V., Craig, U.K., Shaw, C.A., 2003. Decrease in glial glutamate transporter variants and excitatory amino acid receptor down-regulation in a murine model of ALS-PDC. Neuromol. Med. 3 (2), 105–118]. Receptor binding assays showed decreased NMDA and AMPA receptor levels combined with increased GABA_A receptor levels in various CNS regions. The alterations in GLT-1 variants and the ionotropic receptors are consistent with an increased level of extracellular glutamate. The interaction between environmental toxicity and genetic susceptibility was also tested using mice expressing various Apolipoprotein E (ApoE) genotypes. Mice lacking the ApoE gene showed relative resistance to cycad-induced toxicity as measured by GLT-1B labeling, but all mice expressing the human ApoE isoforms showed a similar loss of GLT-1B. We have further shown that an isolated cycad toxin (β-sitosterol-β-d-glucoside, BSSG), previously shown to release glutamate in vitro [Wilson, J.M., Khabazian, I., Wong, M.C., Seyedalikhani, A., Bains, J.S., Pasqualotto, B.A., Williams, D.E., Andersen, R.J., Simpson, R.J., Smith, R., Craig, U.K., Kurland, L.T., Shaw, C.A., 2002. Behavioral and neurological correlates of ALS-parkinsonism dementia complex in adult mice fed washed cycad flour. Neuromol. Med. 1 (3), 207–221], can be directly toxic to motor neurons in vivo [Wilson, J.M., Petrik, M.S., Moghadasian, M.H., Shaw, C.A., 2005. Examining the interaction of apo E and neurotoxicity on a murine model of ALS-PDC. Can. J. Physiol. Pharmacol. 83 (2), 131–141]. However, BSSG-fed mice did not show altered GLT-1B labeling in the spinal cord suggesting that an initial excitotoxic mechanism may not be responsible for the final neuronal loss observed. While glutamate-mediated excitotoxicity is likely involved in the outcomes following cycad/BSSG exposure, the precise location in the cascade of events ultimately leading to neuronal death remains to be determined.     

Examining the interaction of apo E and neurotoxicity on a murine model of ALS-PDC

Epidemiological studies have shown a positive relationship between cycad flour consumption and the development of the neurodegenerative disorder, amyotrophic lateral sclerosis - parkinsonism - dementia complex (ALS-PDC). Apolipoprotein E (apo E) allele variations have been associated with genetic susceptibility in neurodegenerative diseases, including ALS-PDC. We have studied cycad toxicity in a mouse model of ALS-PDC with a particular interest in its impact on the central nervous system (CNS) in both apo E knock-out (KO) mice and their wild-type (WT) counterparts. Behavioral motor tests, motor neuron counts, and immunohistochemical staining in brain and spinal cord, as well as routine histological examinations on internal organs, were performed to evaluate cycad toxicity. Plasma cholesterol levels were also measured before and during the study. Cycad treatment was associated with higher levels of plasma cholesterol only in apo E KO mice; increased levels of plasma cholesterol did not result in increased athero genesis. Cycad-fed wild-type mice developed progressive behavioral deficits including ALS-PDC-like pathological outcomes, while cycad-fed apo E KO mice were not significantly affected. Cycad-fed wild-type mice had shorter gait length measurements along with higher active caspase-3 levels in the striatum, substantia nigra, primary motor cortex, and spinal cord as compared with corresponding controls. These changes were associated with decreased labeling for glutamate transporter 1B and tyrosine hydroxylase activity levels. No evidence of cycad toxicity was observed in internal organs of either wild-type or apo E KO mice. Our data demonstrate that apo E KO mice are less susceptible to cycad toxicity, suggesting a role for apo E as a possible genetic susceptibility factor for some forms of toxin-induced neurodegeneration.     

In Vitro Effects of Cholesterol β-d-Glucoside, Cholesterol and Cycad Phytosterol Glucosides on Respiration and Reactive Oxygen Species Generation in Brain Mitochondria

The cluster of neurodegenerative disorders in the western Pacific termed amyotrophic lateral sclerosis-parkinsonism dementia complex (ALS-PDC) has been repeatedly linked to the use of seeds of various species of cycad. Identification and chemical synthesis of the most toxic compounds in the washed cycad seeds, a variant phytosteryl glucosides, and even more toxic cholesterol β-D-glucoside (CG), which is produced by the human parasite Helicobacter pylori, provide a possibility to study in vitro the mechanisms of toxicity of these compounds. We studied in detail the effects of CG on the respiratory activities and generation of reactive oxygen species (ROS) by nonsynaptic brain and heart mitochondria oxidizing various substrates. The stimulatory effects of CG on respiration and ROS generation showed strong substrate dependence, suggesting involvement of succinate dehydrogenase (complex II). Maximal effects on ROS production were observed with 1 μmol CG/1 mg mitochondria. At this concentration the cycad toxins β-sitosterol-β-D-glucoside and stigmasterol-β-D-glucoside had effects on respiration and ROS production similar to CG. However, poor solubility precluded full concentration analysis of these toxins. Cholesterol, stigmasterol and β-sitosterol had no effect on mitochondrial functions studied at concentrations up to 100 μmol/mg protein. Our results suggest that CG may influence mitochondrial functions through changes in the packing of the bulk membrane lipids, as was shown earlier by Deliconstantinos et al. (Biochem Cell Biol 67:16-24, 1989). The neurotoxic effects of phytosteryl glucosides and CG may be associated with increased oxidative damage of neurons. Unlike heart mitochondria, in activated neurons mitochondria specifically increase ROS production associated with succinate oxidation (Panov et al., J Biol Chem 284:14448-14456, 2009).     

Synergistic versus antagonistic actions of glutamate and glutathione: the role of excitotoxicity and oxidative stress in neuronal disease.

The etiology of various age-related neurological diseases remains unknown. Sporadic forms ofAlzheimer's, Parkinson's and Lou Gehrig's disease have been linked to environmental factors that cause neuronal cell death either by excitotoxicity or by inducing oxidative stress. Our recent studies have demonstrated that various compounds not previously associated with these diseases, i.e. methionine sulfoximine (MSO), originally isolated from 'agenized' flour, and sitosterol glucoside (BSSG), isolated from the seed of the cycad, appear to be neurotoxins, likely acting by excitotoxic mechanisms. For these compounds, the primary excitotoxic effect appears to involve glutamate release followed by NMDA receptor activation. Lactate dehydrogenase assays demonstrate that both compounds cause rapid cell death in vitro. In addition, both compounds appear to alter antioxidant defense mechanisms, acting particularly on levels of reduced glutathione (GSH). In vivo application of MSO has historically been linked to behavioral abnormalities, including seizures, in various species. Our recent experiments have demonstrated that mice fed cycad flour containing sitosterol glucoside have severe behavioral abnormalities of motor and cognitive function, as well as significant levels of neurodegeneration in cortex, hippocampus, spinal cord and other CNS regions measured post mortem. The combined weight of excitotoxic action, in concert to a decline in antioxidant defenses, induced by molecules such as methionine sulfoximine and sitosterol glucoside is hypothesized to be causal to neuronal degeneration in various neurological diseases. Understanding the mechanisms of action of these and functionally related molecules may serve to focus attention on potential neurotoxins present in the human environment. Only once such molecules have been identified, can we begin to design appropriate pharmaceutical strategies to prevent or halt the progression of the age-related neurological diseases.     

Distribution of the neurotoxic nonprotein amino acid BMAA in Cycas micronesica

BMAA (β-methylamino-L-alanine), a nonprotein amino acid with neurotoxic properties occurs in the cycad Cycas micronesica Hill in Guam. BMAA may have originally played a role as an antiherbivory compound in the plant, but is now of great interest because of its possible link to ALS-PDC, a neurological disease among the Chamorro people of Guam. Biomagnified cycad neurotoxins may play a role because they accumulate in flying foxes of the genus Pteropus that are eaten during traditional feasts. Since flying foxes feed on the seed sarcotesta it is important to understand the distribution of BMAA in the various tissues of the cycad. Using HPLC techniques, we quantified both BMAA and glutamic acid (GLU) in all cycad tissues. Although GLU is distributed randomly throughout the plant, BMAA is concentrated in cycad reproductive organs, with the highest concentrations being found in the immature staminate sporangium and the outmost layer of the sarcotesta. This finding is consistent with the putative evolutionary role of BMAA as an antiherbivory compound, as well as the biomagnification of the compound in flying foxes that ingest the seed sarcotesta.  © 2003 The Linnean Society of London, Botanical Journal of the Linnean Society , 2003, 143 , 165–168.     

Methionine sulfoximine shows excitotoxic actions in rat cortical slices

Methionine sulfoximine (MSO) is a rare amino acid. It occurs in nature or as a by-product of some forms of food processing. A notable example of the latter was a former method for bleaching wheat flour, using nitrogen trichloride, the "agene process," in use for most of the first 50 years of this century. "Agenized" flour was found to be responsible for various neurological disorders in animals, and MSO was identified as the toxic factor. The agene process was subsequently discontinued in the United States and the United Kingdom circa 1950. MSO inhibits the synthesis of both glutathione and glutamine, and it is possible that its actions on the nervous system arise from alterations in the amount or distribution of these molecules. Structurally, MSO resembles glutamate, an observation that has also raised the possibility that it might have more direct glutamate-like actions on neurons. In the present investigation, we report excitatory and toxic actions of MSO in an in vitro preparation of adult rat cortex. Field potential recordings in this preparation show that MSO application evokes a sustained depolarization, which can be blocked by the N-methyl-D-aspartate (NMDA) antagonist L-(+)-2-amino-5-phosphonovalerate (AP5). However, competition assays using MSO on [3H]CGP-39653 (DL-(E)-2-amino-4-propyl-1-phosphono-3-pentenoate) binding in rat cortical homogenates show only 20% displacement of total binding, suggesting that MSO is acting indirectly, perhaps by releasing glutamate. To investigate this possibility, we measured glutamate release during MSO application. Time course and dose-response experiments with MSO showed significant [3H]glutamate release, which was partially attenuated by AP5. To assess cellular toxicity, we measured lactate dehydrogenase (LDH) release from cortical sections exposed to MSO. MSO treatment led to a rapid increase in LDH activity, which could be blocked by AP5. These data suggest that MSO acts by increasing glutamate release, which then activates NMDA receptors, leading to excitotoxic cell death. These data suggest the possibility that MSO in processed flour had excitotoxic actions that may have been contributing factors to some human neuronal disorders.     

Attenuation of Glutamate-Induced Excitotoxicity by Withanolide-A in Neuron-Like Cells: Role for PI3K/Akt/MAPK Signaling Pathway

Glutamate-induced excitotoxicity is one of the major underlying mechanisms for neurodegenerative diseases. Efforts are being made to treat such conditions with an array of natural compounds that can modulate the release of glutamate or the underlying mechanisms associated with it. Withania somnifera extract has potent pharmacologic activity similar to that of Korean Ginseng tea and is used to treat several neuronal disorders. However, to date, little efforts have been made to evaluate individual constituents of this plant for neurodegenerative disorders. Present study was carried out to investigate withanolide-A, one of the active constituents of Withania somnifera against glutamate-induced excitotoxicity in retinoic acid differentiated Neuro2a neuroblastoma cells. The results indicated that glutamate treatment for 2 h induced death in cells that was significantly attenuated by pre-treatment with MK-801 (specific NMDA receptor antagonist) and different concentrations of withanolide-A. Withanolide-A abated the glutamate-induced influx of intracellular calcium and excessive ROS production significantly. Further on, glutamate treatment resulted in increased levels of pro-apoptotic and decreased levels of anti-apoptotic proteins, and these protein levels were normalized by various doses of withanolide-A. All of these protective effects were partly due to inhibition of MAPK family proteins and activation of PI3K/Akt signaling. Thus, our results suggest that withanolide-A may serve as potential neuroprotective agent.     

Molecular mechanisms of glutamate receptor-mediated excitotoxic neuronal cell death

Excitotoxicity is one of the most extensively studied processes of neuronal cell death, and plays an important role in many central nervous system (CNS) diseases, including CNS ischemia, trauma, and neurodegenerative disorders. First described by Olney, excitotoxicity was later characterized as an excessive synaptic release of glutamate, which in turn activates postsynaptic glutamate receptors. While almost every glutamate receptor subtype has been implicated in mediating excitotoxic cell death, it is generally accepted that the N-methyl-D-aspartate (NMDA) subtypes play a major role, mainly owing to their high calcium (Ca²⁺) permeability. However, other glutamate receptor subtypes such as 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl) propionate (AMPA) or kainate receptors have also been attributed a critical role in mediating excitotoxic neuronal cell death. Although the molecular basis of glutamate toxicity is uncertain, there is general agreement that it is in large part Ca²⁺-dependent. The present review is aimed at summarizing the molecular mechanisms of NMDA receptor and AMPA/kainate receptor-mediated excitotoxic neuronal cell death.     

Glutamate release promotes growth of malignant gliomas

Glutamate neurotoxicity has been implicated in stroke, head trauma, multiple sclerosis and neurodegenerative diseases. Although recent data show that cultured glioma cells secrete glutamate, the growth potential of brain tumors has not yet been linked to an excitotoxic mechanism. Using bioluminescence detection of glutamate release from freshly prepared brain slices, we show that implanted glioma cells continue to secrete glutamate. Moreover, gliomas with high glutamate release have a distinct growth advantage in host brain that is not present in vitro. Treatment with the NMDA receptor antagonists MK801 or memantine slowed the growth of glutamate-secreting tumors in situ, suggesting that activation of NMDA receptors facilitates tumor expansion. These findings support a new approach for therapy of brain tumors, based upon antagonizing glutamate secretion or its target receptors.     

Enhanced NMDA Receptor-Mediated Modulation of Excitatory Neurotransmission in the Dorsal Vagal Complex of Streptozotocin-Treated,Chronically Hyperglycemic Mice

A variety of metabolic disorders, including complications experienced by diabetic patients, have been linked to altered neural activity in the dorsal vagal complex. This study tested the hypothesis that augmentation of N-Methyl-D-Aspartate (NMDA) receptor-mediated responses in the vagal complex contributes to increased glutamate release in the dorsal motor nucleus of the vagus nerve (DMV) in mice with streptozotocin-induced chronic hyperglycemia (i.e., hyperglycemic mice), a model of type 1 diabetes. Antagonism of NMDA receptors with AP-5 (100 μM) suppressed sEPSC frequency in vagal motor neurons recorded in vitro, confirming that constitutively active NMDA receptors regulate glutamate release in the DMV. There was a greater relative effect of NMDA receptor antagonism in hyperglycemic mice, suggesting that augmented NMDA effects occur in neurons presynaptic to the DMV. Effects of NMDA receptor blockade on mEPSC frequency were equivalent in control and diabetic mice, suggesting that differential effects on glutamate release were due to altered NMDA function in the soma-dendritic membrane of intact afferent neurons. Application of NMDA (300 μM) resulted in greater inward current and current density in NTS neurons recorded from hyperglycemic than control mice, particularly in glutamatergic NTS neurons identified by single-cell RT-PCR for VGLUT2. Overall expression of NR1 protein and message in the dorsal vagal complex were not different between the two groups. Enhanced postsynaptic NMDA responsiveness of glutamatergic NTS neurons is consistent with tonically-increased glutamate release in the DMV in mice with chronic hyperglycemia. Functional augmentation of NMDA-mediated responses may serve as a physiological counter-regulatory mechanism to control pathological disturbances of homeostatic autonomic function in type 1 diabetes.     

The Bacterial Endotoxin Lipopolysaccharide Causes Rapid Inappropriate Excitation in Rat Cortex

Abstract : There is mounting evidence that inflammation and associated excitotoxicity may play important roles in various neurodegenerative disorders, such as bacterial infections, Alzheimer's disease, AIDS dementia, and multiple sclerosis. The immunogen E. coli lipopolysaccharide (LPS, endotoxin) has been widely used to stimulate immune/inflammatory responses both systemically and in the CNS. Here, we show that exposure of parietal cortical slices from adult rats to LPS triggered very rapid (<2.5 min) and sustained releases of the neurotransmitters glutamate and noradrenaline, and of the neuromodulator adenosine. The responses to LPS declined rapidly following removal of the LPS and exhibited no tachyphylaxis to repeated exposures to LPS. The detoxified form of LPS had no effect. LPS-evoked release of [³H]noradrenaline, but not of glutamate or adenosine, appears to be partly due to the released glutamate acting at ionotropic receptors on the noradrenergic axons present in the cortical slices. LPS appears to release glutamate, which then acts at non-NMDA receptors to remove the voltage-sensitive Mg²⁺ block of NMDA receptors, thus permitting NMDA receptors to be activated and noradrenaline release to proceed. It seems possible that rapid, inappropriate excitation may occur in the immediate vicinity of gram-negative bacterial infections in the brain. If similar inappropriate excitations are also triggered by those immunogens specifically associated with Alzheimer's disease (β-amyloid), AIDS dementia (gp 120 and gp41), or multiple sclerosis (myelin basic protein), they might explain some of the acute, transient neurological and psychiatric symptoms associated with these disorders.     

Carbonyl stress and NMDA receptor activation contribute to methylglyoxal neurotoxicity

Methylglyoxal (MG) is a reactive alpha-ketoaldehyde physiologically generated as a by-product of glycolysis. MG that is able to form protein adducts resulting in advanced glycation end products accumulates under conditions associated with neurodegeneration such as impaired glucose metabolism or oxidative stress. In the present study, short-term exposure of human neuroblastoma SH-SY5Y cells to MG was associated with an early depolarization of the plasma membrane, glutamate release, and formation of reactive oxygen species. In addition, long-term exposure (24 h) of SH-SY5Y cells to MG caused a decrease in cell viability, intracellular ATP, and rhodamine 123 (Rh-123) fluorescence. ATP depletion and the decrease in Rh-123 fluorescence were prevented by carbonyl scavengers, the nitric oxide synthase inhibitor L-NAME, and N-methyl-d-aspartate (NMDA) receptor antagonists. Furthermore, the MG-induced glutamate release and the loss in cell viability were prevented by NMDA receptor antagonists. Therefore, MG renders cells more vulnerable to excitotoxicity. In conclusion, carbonyl scavengers as well as NMDA receptor antagonists may represent effective therapeutic tools to reduce the risk of pathophysiological changes associated with carbonyl stress in neurodegenerative diseases.     

Modeling the effect of glutamate diffusion and uptake on NMDA and non-NMDA receptor saturation.

One- and two-dimensional models of glutamate diffusion, uptake, and binding in the synaptic cleft were developed to determine if the release of single vesicles of glutamate would saturate NMDA and non-NMDA receptors. Ranges of parameter values were used in the simulations to determine the conditions when saturation could occur. Single vesicles of glutamate did not saturate NMDA receptors unless diffusion was very slow and the number of glutamate molecules in a vesicle was large. However, the release of eight vesicles at 400 Hz caused NMDA receptor saturation for all parameter values tested. Glutamate uptake was found to reduce NMDA receptor saturation, but the effect was smaller than that of changes in the diffusion coefficient or in the number of glutamate molecules in a vesicle. Non-NMDA receptors were not saturated unless diffusion was very slow and the number of glutamate molecules in a vesicle was large. The release of eight vesicles at 400 Hz caused significant non-NMDA receptor desensitization. The results suggest that NMDA and non-NMDA receptors are not saturated by single vesicles of glutamate under usual conditions, and that tetanic input, of the type typically used to induce long-term potentiation, will increase calcium influx by increasing receptor binding as well as by reducing voltage-dependent block of NMDA receptors.     

Identification of Gly-Pro-Glu (GPE), the aminoterminal tripeptide of insulin-like growth factor 1 which is truncated in brain, as a novel neuroactive peptide

A truncated form of IGF-1 which lacks the aminoterminal tripeptide Gly-Pro-Glu (GPE) is found in human brain. It was proposed that GPE may result from neural specific processing and also have a function within the CNS. GPE was synthesized and shown to inhibit glutamate binding to the N-methyl-D-aspartate (NMDA) receptor. Whilst the carboxyterminal glutamate was necessary for NMDA receptor binding, the aminoterminal glycine potentiated receptor crossreaction. Furthermore, GPE had a potent stimulatory effect on the potassium induced release of acetylcholine from rat cortical slices. A less potent stimulation of dopamine release from striatum was also observed. The specific competitive NMDA receptor antagonist, (+/-)2-amino-7-phosphonoheptanoate (AP7), inhibited the action of GPE on dopamine but not on acetylcholine release. These studies have identified GPE as a novel neuroactive peptide with a potent action on acetylcholine release and support the general concept that the proteolytic products of the IGF-1 precursor play a role in the regulation of brain function.     

突触前代谢型谷氨酸受体调节神经递质的释放

谷氨酸通过激活离子型受体（iGluR)介导快速兴奋性突触传递,参与脑内几乎所有生理过程。谷氨酸过量释放可导致与脑缺血,缺氧及变性疾病有关的兴奋毒作用,最终引起神经元的死亡。代谢型谷氨酸受体（mGluRs)是一个与G－蛋白偶联的受体家族,分三型共八个亚型。其中Ⅱ和Ⅲ型mGluRs主要位于突触前,发挥对谷氨酸释放的负反馈调节。Ⅲ型mGluRs中的mGluR7位于谷氨酸能末梢突触前膜的活性区,发挥自身受体的作用,对正常情况下突触传递过程的谷氨酸释放进行负反馈调节;而属于Ⅱ型的mGluR2及属于Ⅲ型的mGluR4和mGluR8,则位于远离突有膜活性区的外突触区,因而正常突触传递过程中释放的谷氨酸量不能激活它们。只有在突触传递增强的情况下才被激活,抑制递质的释放。国外,mGluRs还分布在GABA能纤维末梢,通过突触前机制抑制GABA的释放。对突触前膜受体尤其是位于外突触区的mGluRs受体的研究,将有可能开发出理想的工具药,从而预防和阻止谷氨酸过量释放引起的神经毒及神经元的死亡。     

Glutamate receptors on human melanocytes regulate the expression of MiTF

Glutamate is the major excitatory neurotransmitter in the central nervous system but has also important functions in the epidermis. It is involved in keratinocyte barrier function and in re-epithelialization processes after wounding. Recently, glutamate signalling has been suggested to be implicated in the development of melanoma. The present study examined the expression and functionality of metabotropic and ionotropic glutamate receptors on normal human melanocytes. We found that cultured melanocytes expressed the ionotropic glutamate receptors GluR2 and 4 [alpha-amino-3-hydroxy-5-methyl-4-isoxsazolepropionic acid (AMPA) receptors] and N-methyl-d-aspartate (NMDA) receptors 2A and 2C and possibly the metabotropic glutamate receptor 1. Melanocytes were also found to express specific glutamate transporters and decarboxylases, but appeared neither to produce nor to release l-glutamate. Stimulation with 10 or 100 microM AMPA or NMDA elevated intracellular calcium concentrations in melanocytes, and thus demonstrated the functionality of the glutamate receptors. Millimolar concentrations of l-glutamate did not induce melanocyte toxicity and had no stimulating effect on melanin production. However, blockage of AMPA and NMDA receptors with CFM-2, memantine or MK801 caused a rapid and reversible change in melanocyte morphology, which was associated with disorganisation of actin and tubulin microfilaments. After 24 h of treatment with the AMPA receptor inhibitor CFM-2, there was a sharp reduction in the expression of the crucial melanocyte differentiation and proliferation factor MiTF. The results of this study demonstrate a role for glutamate in melanocyte regulation that may have implications in melanocyte associated disorders.     

Expressed sequence tag analysis in <Emphasis Type="Italic">Cycas</Emphasis>, the most primitive living seed plant

Background

Cycads are ancient seed plants (living fossils) with origins in the Paleozoic. Cycads are sometimes considered a 'missing link' as they exhibit characteristics intermediate between vascular non-seed plants and the more derived seed plants. Cycads have also been implicated as the source of 'Guam's dementia', possibly due to the production of S(+)-beta-methyl-alpha, beta-diaminopropionic acid (BMAA), which is an agonist of animal glutamate receptors.

Results

A total of 4,200 expressed sequence tags (ESTs) were created from Cycas rumphii and clustered into 2,458 contigs, of which 1,764 had low-stringency BLAST similarity to other plant genes. Among those cycad contigs with similarity to plant genes, 1,718 cycad 'hits' are to angiosperms, 1,310 match genes in gymnosperms and 734 match lower (non-seed) plants. Forty-six contigs were found that matched only genes in lower plants and gymnosperms. Upon obtaining the complete sequence from the clones of 37/46 contigs, 14 still matched only gymnosperms. Among those cycad contigs common to higher plants, ESTs were discovered that correspond to those involved in development and signaling in present-day flowering plants. We purified a cycad EST for a glutamate receptor (GLR)-like gene, as well as ESTs potentially involved in the synthesis of the GLR agonist BMAA.

Conclusions

Analysis of cycad ESTs has uncovered conserved and potentially novel genes. Furthermore, the presence of a glutamate receptor agonist, as well as a glutamate receptor-like gene in cycads, supports the hypothesis that such neuroactive plant products are not merely herbivore deterrents but may also serve a role in plant signaling.

     

Genetic Program of Neuronal Differentiation and Growth Induced by Specific Activation of NMDA Receptors

Glutamate and its receptors are expressed very early during development and may play important roles in neurogenesis, synapse formation and brain wiring. The levels of glutamate and activity of its receptors can be influenced by exogenous factors, leading to neurodevelopmental disorders. To investigate the role of NMDA receptors on gene regulation in a neuronal model, we used primary neuronal cultures developed from embryonic rat cerebri in serum-free medium. Using Affymetrix Gene Arrays, we found that genes known to be involved in neuronal plasticity were differentially expressed 24 h after a brief activation of NMDA receptors. The upregulation of these genes was accompanied by a sustained induction of CREB phosphorylation, and an increase in synaptophysin immunoreactivity. We conclude that NMDA receptor activation elicits expression of genes whose downstream products are involved in the regulation of early phases of the process leading to synaptogenesis and its consolidation, at least in part through sustained CREB phosphorylation. Special issue dedicated to Dr. Anthony Campagnoni.     

N-Methyl-D-Aspartate Releases Excitatory Amino Acids in Rat Corpus Striatum In Vivo

There is a considerable amount of conflicting evidence from several studies as to the action of applied N-methyl-D-aspartate (NMDA) on the release of glutamate and aspartate in the brain. In the present study the effect of NMDA on extracellular levels of endogenous amino acids was investigated in conscious, unrestrained rats using intracerebral microdialysis. NMDA caused dose-related increases in extracellular levels of glutamate and aspartate; threonine and glutamine were unaffected. The NMDA-evoked release of glutamate and aspartate was significantly decreased by the specific NMDA receptor antagonist 3-[(+-)-2-carboxypiperazin-4-yl]-propyl-l-phosphonic acid. In addition, increasing the perfusate concentration (and therefore the extracellular concentration) of Ca2+ significantly enhanced the NMDA-evoked release of glutamate and aspartate, whereas removal of Ca2+ and addition of a high Mg2+ concentration to the perfusate caused a significant reduction in their NMDA-evoked release. Moreover, the NMDA-evoked release of glutamate and aspartate was reduced in decorticate animals. These results demonstrate that, in the striatum in vivo, NMDA causes selective release of endogenous glutamate and aspartate from neurone terminals and that this action occurs through an NMDA receptor-mediated mechanism. The ability of NMDA receptor activation to induce release of glutamate and aspartate, perhaps by a positive feedback mechanism, may be relevant to the pathologies underlying epilepsy and ischaemic and hypoglycaemic brain damage.