Glutamate Synthesis

Secondary excitotoxicity is thought to be more relevant to chronic neurodegenerative disorders than the classical acute form. In this pathway increased glutamate levels are not required as disturbances with the neuronal resting membrane potential cause glutamate receptors to be over-activated by physiological levels of glutamate. In a neuron which is affected by another pathological process and shows reduced ATP production, Na+ dysfunction or impaired glucose metabolism and as a result has an abnormal membrane potential, the voltage-dependent Mg2+ block within NMDA receptors may be eliminated. This will cause pathological over-activation of the receptor. This shows a mechanism where in excitotoxicity is possible without increased levels of glutamate or alteration to glutamate receptors.
Glutamate can have an indirect toxic effect by depleting intracellular glutathione. The cystine-glutamate antiporter transports cystine into the cell and is driven by the movement of glutamate out of the cell. Cystine and glutamate compete to bind to the antiporter so increased levels of glutamate reduce the amount of cystine entering the cell. Cystine is a precursor for intracellular glutathione which has multiple functions within the cell, notably protection to the cell from oxidative damage and a direct effect on K+ conductance which
alters neuronal excitability. 1 Activation of glutamate receptors is also a main pathway for reactive oxygen species (ROS) production by calcium dependent activation of the arachidonic acid cascade and calpain. ROS cause widespread cellular injury and necrosis by damaging proteins, lipids and DNA within the cell causing dysfunction to many of the cell’s organelles. This can be resisted with molecules that absorb ROS such as glutathione however as glutamate influx also reduces the amount of intracellular glutathione, glutamate excitotoxicity can cause oxidative damage fatal to the neurone.
The final pathway for glutamate excitotoxicity is via chronic blockade of the glutamate transporter reuptake system. Excitotoxic events have the potential to cause chronic neurone pathology. After exposure of organotypic spinal slice cultures (OSCs) to low concentrations of the excitotoxin quinolinic acid, for an extended period of time excitotoxic effects were observed. Quinolinic acid acts as an EAA agonist and decreases the rate of removal of glutamate causing glutamate over-activation. It has been found that pharmaceutical blockade of EAATs in rat spinal cord explants resulted in rapid motor neuron degeneration, over a course of two weeks. 9
Rat OSCs treated with threo-hydroxyaspartate (THA), an inhibitor of glutamate uptake, have provided a model of ALS that can be used to screen potential therapeutic drugs, such as glial cell line-derived neurotrophic factor (GDNF). THA treatment causes slow, selective loss of motor neurons in the ventral horn due to glutamate transport inhibition. This in vitro model is based on the role of glutamate toxicity in ALS however the development/progression of this disease shows that other factors are involved including disorganization of neurofilaments and protein misfolding. In the study by Kosuge et al an OSC mouse model of FALS, G93A, was established to test GDNF which has been shown to promote survival of motor neurons in vivo and vitro. In the presence of GDNF, the rate of neuron survival increased in both G93A and control mice but was much more significant in G93A. Additionally, concentrations of the cleaved form of caspase-12 were increased after THA in G93A but not in control mice and the activation of caspase-12 was lowered by OSCs cultured with GDNF. Caspase 12 regulates the ER stress pathway which leads to apoptosis. Kosuge suggests that the pathway responsible for motoneuronal death in G93A mice involves not only excitotoxicity but also other mechanisms including the caspase-12-dependent ER stress response. Although the results from this study support the caspase-12 pathway’s role, the mechanisms at work in the THA-induced model are not fully understood and may behave differently to a genuine FALS case. 5
Motor neurons possess three types of ionotropic glutamate receptors, AMPA, NMDA and KA receptors. Each type is expressed in the motor cortex, brainstem and spinal cord however in each region the relative proportions of their subunit expression changes. AMPA receptors mediate fast excitatory transmission by making the neurone more permeable to Na+ and K+ ions, removing the Mg2+ blockade of NMDA receptors and leading to calcium influx, the late component of excitatory transmission. AMPA receptors are usually impermeable to Ca2+ due to the presence of GluR2 subunits which undergo RNA editing to encode an arginine molecule which is positively charged. The role of KA receptors is still not understood. Motor-neuronal non-NMDA receptors have low expression of GluR2 and have a higher calcium permeability contributing to excitotoxicity. However it was found that transgenic mice lacking the GluR2 subunit did not suffer from a motor neuron disease [72], suggesting that the absence of GluR2 subunit does not cause ALS but modifies motor-neuronal excitation in ALS. Disruption of GluR2 in a rodent mSOD1 model accelerated motor-neuronal degeneration [73].
It appears that certain receptors are targeted more frequently by glutamate.
Thus motor neurone groups that tend to be spared in ALS, oculomotor (III) nucleus, express a lower density of NMDA receptor binding sites and an increased density of AMPA binding sites compared with vulnerable motor neuron groups. This indicates differences in normal glutamate neurotransmission in spared and vulnerable motor neurone groups. Recent studies have found a specific, non-competitive NMDA receptor blocker, memantin, was able to delay the disease progression and prolong life span in mSOD1 mouse model through both subcutaneous and oral administrations [81][82].
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Savaskan et al showed that selenium deficiency increases susceptibility of neurones to glutamate excitotoxicity and that this may play a role in the development and onset of excitotoxic brain lesions, such as stroke and epilepsy. First, excitotoxic conditions were induced in neurons by treatment with an excess of glutamate which reduced cell survival by >80%. It was found that glutamate-induced cell death could be prevented with synchronous administering of selenite in a concentration-dependent manner. The median effective concentration of selenite was 37 nM with a maximal effect at 100 nM within the physiological range as shown in human studies in vivo. Glutamate-induced cell death could be prevented in rescue experiments where selenite was added hours after glutamate damage. Peroxide levels were taken and it was found that glutamate excess caused high concentrations of peroxides but this was prevented with selenite treatment. Glutamate treatment led to increased intranuclear NF-kB levels, which could be inhibited by increasing selenite concentrations. Selenium had no direct effect on NF-kB binding activity, suggesting that selenium in the form of a protein is involved in anti-apoptotic mechanisms.
Finally a comparative study of rats with either a selenium adequate or insufficient diet was made. In the kainate model of excitotoxicity the reduced selenium rats presented remarkably higher seizure rates compared with subjects on a selenium-adequate diet. There was also an increased number of caspase-3 positive cells in selenium deficient rats, and neuronal cell loss in the hippocampus was significantly higher. This shows that selenium deficiency increases susceptibility to kainate-induced excitotoxicity resulting in seizure activity and cell damage in vivo. 1
In motor neurons, astrocytic EAAT2 has the greatest contribution to the removal of synaptic glutamate. Motor cortex and spinal cord extract analysis from deceased patients with SALS and FALS showed a loss of EAAT2 in the majority of patients [39], and a significant reduction in expression and activity of EAAT2 was also observed in rodent models with mSOD1 [40][41]. This suggests that affected motor neurons had higher glutamatergic input and would be more vulnerable to elevated glutamate. Clear alterations are found in patients with ALS, but this is unlikely to be a hereditary condition. Overexpression of EAAT2 in rodent SOD1 models caused delayed onset of motor loss [46], and stimulation of EAAT2 with a b-lactam antibiotic significantly increased the life span of the rodent SOD1 subjects with the delayed onset [47], which supports the theory that EAAT2 loss contributes to ALS pathogenesis. 7
Brain tumours induce pathogenic changes by proliferating, invading surrounding tissues and promoting neuronal cell death through glutamate excitotoxicity. Studies have shown that astrocyte elevated gene-1 (AEG-1) has a critical role in malignant glioma progression and that its expression correlates with the clinical stages of glioma. In Lee et al’s study AEG-1 expression was also found to significantly correlate with reduction in the expression of EAAT2 in glioma patient samples. It was found that AEG-1 interacts directly with the repressor protein YY1 and the coactivator protein CBP which helps activate expression of EAAT2. This causes AEG-1 mediated EAAT2 repression which impairs glutamate uptake and contributes to glutamate excitotoxicity and neurodegeneration. 3