The specificity of the toxic effect of SOD1 mutations on motor neurons arises from the convergence of several risk factors
a) Initially, a lower motor neuron receives signals to fire by the release of glutamate (Glu) from an upstream neuron, either an upper motor neuron or an interneuron. This signal is converted within the motor neuron into action potentials that stimulate the release of acetylcholine (orange) at its axon terminus, triggering muscle contraction.
b) In neurons and astrocytes, superoxide dismutase 1 (SOD1) accumulates during aging, forming mutant SOD1 aggregates, either by an inherently unstable conformation or by self-induced oxidative damage. This triggers a loss of overall protein-folding chaperone activity and inhibits the removal of other damaged proteins by choking the 20S proteasome. Neurofilaments, especially in axons, become disorganized, inhibiting transport of components along the axon. Caspase 1 is chronically activated.
c) Inhibition of chaperone and proteasome activity, loss of axonal transport capacity and an accelerated SOD1-mutant burden force chronic deficits in motor neurons. Similar damage in astrocytes suppresses the accumulation and activity of glutamate transporters (EAAT2) that are necessary for recovering synaptic glutamate and for preventing repetitive motor neuron firing. Such disproportionate firing produces excessive calcium entry through calcium-permeable glutamate receptors, activating caspase 3, which serves as the executioner for motor neuron death through the degradation of key cellular components.
Normal Glutamate Regulation:
When nerve cells signal to one another, glutamate is released from one nerve cell ending into the synaptic cleft. Receptors on the second nerve cell ending bind to the glutamate and “excite” the second nerve cell. Extra glutamate is soaked up by astrocytes through glutamate transporters.
Glutamate Regulation in SOD1-Linked ALS:
In SOD1-linked ALS, researchers suspect that the mutant SOD1 protein generates free radicals that, in turn, inactivate the glutamate transporters on astrocytes. Glutamate collects in the synaptic cleft and the second nerve cell becomes fatally overexcited. This phenomenon is known as glutamate excitotoxicity.
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Researches report mutant SOD1 protein inactivates glutamate transporters
New research from MDA grantees Matthias Hediger of Brigham and Women’s Hospital and Robert Brown of Massachusetts General Hospital, both in Boston, has established the first direct link between oxidative stress and problems with glutamate transport in the SOD1-linked familial form of ALS. The researchers’ work appears in the May issue of Nature Neuroscience.
The finding is important because it helps explain the relationship between two different cellular problems — abnormal glutamate transport and excess free radical production — that are known to occur in both sporadic ALS and SOD1-linked familial ALS.
(The rare SOD1-linked form of the disease is caused by defects in the gene that codes for a protein called superoxide dismutase 1, and is hereditary. Some 75 to 80 percent of people with ALS have the sporadic form, of which the cause is unknown. It occurs without a family history of the disease.)
Researchers have long suspected that nerve cell death in sporadic ALS may be caused by a problem known as glutamate excitotoxicity. Glutamate is a small chemical found in the central nervous system that serves as a messenger to bridge the physical gap, called the synaptic cleft, between two nerve cells, or neurons.
When neurons signal to one another, glutamate is released from one neuron into the synaptic cleft, where it can bind to receptors on the next neuron. Normally, after a signal passes between neurons, cells called astrocytes “vacuum up” the leftover glutamate with special proteins on their surfaces known as glutamate transporters.
Glutamate excitotoxicity occurs when the neurons are exposed to glutamate for too long or in too large quantities and become overstimulated. This overstimulation can lead to a chain reaction of events that are destructive to nerve cells, resulting in the paralysis that occurs in ALS.
Early studies suggested that extra glutamate could be a problem in sporadic ALS. This is the logic behind the drug riluzole, taken by people with ALS, which works to reduce the amount of glutamate released during cell signaling.
In 1998, MDA grantee Jeffrey D. Rothstein of Johns Hopkins University in Baltimore added credence to this theory with his report that some people with sporadic ALS may not be making the glutamate transporter properly, and thus clearing glutamate too slowly from the spaces between their nerve cells.
It’s less clear what role abnormal glutamate transport may play in the SOD1-linked form of familial ALS. A large body of evidence suggests that SOD1-linked ALS is caused by free radical damage due to abnormal activities of the mutant SOD1 protein (free radicals are highly reactive charged ions that can damage components of cells). Hediger wondered if the free radical damage, known as oxidative stress, was affecting glutamate transport in SOD1-linked ALS.
“There was some evidence suggesting that glutamate transport is impaired in the SOD1-linked form of the disease,” Hediger says, “but it wasn’t known if oxidative stress is causally linked to impaired transport.”
Now Hediger and Brown have found that the mutant SOD1 can directly inactivate a specific type of glutamate transporter.
“Our results establish a link between exposure to oxidative damage and the ability of the body to mop up glutamate,” Hediger says.
The discovery was made when Hediger laboratory member Davide Trotti examined the activity of human glutamate transporters and mutant SOD1 proteins in frog egg cells, which are large and can be injected easily with foreign DNA. Trotti found that egg cells making the glutamate transporter were able to take up glutamate, but egg cells making both the glutamate transporter and the mutant SOD1 had problems with glutamate uptake. The researchers also found that the effect of the mutant SOD1 on the glutamate transporter could be nullified if strong antioxidant chemicals were delivered to the cells.
These results suggest that problems with glutamate transport in some people with sporadic ALS may be caused by oxidative damage, even though they have normal SOD1. The findings also reinforce the logic behind the use of antioxidants and riluzole in slowing the course of sporadic ALS. Further research is needed to uncover the causes of oxidative stress in people with sporadic ALS.
Mutant SOD1 caused intrinsic damage to motor neurons. The reduction of mutant SOD1 in motor neurons delays the onset of disease and extends the lifespan of transgenic ALS mice. Reduction of mutant SOD1 in microglia affects disease duration after onset, suggesting a role for microglia in the propagation of the disease. Although there is cell-autonomous damage caused by mutant SOD1 in the motor neurons, this may not be sufficient to trigger and propagate ALS pathogenesis. The in vitro experiments of Nagai et al. and Di Georgio et al. suggest that activated astrocytes might be important mediators of mutant SOD1 toxicity that results in motor neuron death. EAAT, glutamate transporter; ER, endoplasmic reticulum; Glu, glutamate.
Compartmentalization of brain glutamate
The blood–brain barrier has a very low permeability to glutamate (Glu). Essentially all glutamate in the brain is synthesized by transamination of alpha-ketoglutarate in both neurons and glia. In glial cells, glutamate is converted to glutamine (Gln) before it is released into the extracellular space. Glutamine is taken up by neurons and converted to glutamate before being packaged into synaptic vesicles. Glutamate is then recycled into the transmitter pool after uptake and conversion into glutamine by astrocytes. Glutamine also functions as a carrier of excess ammonium, and is transported across the blood–brain barrier to be disposed of by the circulation. In young animals, the blood–brain barrier has a higher permeability to glutamate, and excessive dietary uptake of glutamate can be excitotoxic. The blood–brain barrier becomes impermeable to glutamate at seven days after birth, and circulating glutamate no longer represents a threat to neuronal survival after this period. The concentration of glutamate in the cerebrospinal fluid is about 1 uM, but can increase to 20 uM under pathological conditions that are characterized by defects in the blood–brain barrier and/or by cellular damage, such as stroke, trauma, multiple sclerosis and meningitis.
Glutamate plays an essential role in many physiological functions and is the major excitatory transmitter in the mammalian central nervous system (CNS) (Collingridge and Singer, 1990; Danysz et al., 1995; Collingridge and Bliss, 1995). However, under various conditions neurons can become so sensitive to glutamate that it actually kills them through receptor-mediated depolarization and calcium influx (Obrenovitch and Urenjak, 1997; Parsons et al., 1998). This phenomenon was described for the first time by John Olney and called “excitotoxicity” (Rothman and Olney, 1987). It has been implied that excitotoxicity is involved in many types of acute and chronic insults to the CNS including neurodegenerative disorders (Choi, 1995). Disturbance of glutamate homeostasis probably plays a pivotal role in the execution of pathological changes in many disease states and may be triggered by a wide variety of factors that facilitate the neurotoxic potential of endogenous glutamate such as; increase in glutamate release, malfunctioning of neuronal and glial uptake, energy deficits, neuronal depolarization, changes in glutamate receptor properties or expression patterns, free radical formation, the presence of toxic proteins such a ß-amyloid and tau in Alzheimer’s disease (AD) etc (Danysz et al., 1995; Beal, 1995; Obrenovitch and Urenjak, 1997; Parsons et al., 1998). Such excitotoxic effects can be pronounced during acute events such as ischaemic stroke and trauma or milder but prolonged in chronic neurodegenerative diseases such as Alzheimer’s disease, Parkinson´s disease, Huntington´s disease and amyotrophic lateral sclerosis (ALS) (Starr, 1995; Beal, 1995; Plaitakis et al., 1996; Shaw and Ince, 1997). Glutamatergic dysfunction is also involved in the symptomatology of disorders such schizophrenia, anxiety, depression (Danysz et al., 1995; Parsons et al., 1998) as well as in the development of disorders associated with long term plastic changes in the CNS such as chronic pain, drug tolerance, dependence, addiction, partial complex seizures and tardive dyskinesia (Danysz et al., 1995; Trujillo and Akil, 1995; Dickenson, 1997; Parsons et al., 1998).
Glutamate receptors are divided into metabotropic (coupled to intracellular second messengers modifying IP3 and cAMP concentrations) and ionotropic receptors (directly coupled to an ion channel).
Ionotropic glutamate receptors
Ionotropic glutamate receptors were originally classified on the basis of three selective, synthetic agonists, quisqualate, kainate and N-methyl-D-aspartate (NMDA). After the discovery of metabotropic receptors it became clear that quisqualate also interacts with them. Since that time quisqualate-sensitive ionotropic receptors are classified by the more selective agonist a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). All ionotropic glutamate receptors can form heteromeric subunit assemblies (composed of different subunits) which have different physiological and pharmacological properties and are differentially distributed throughout the CNS (Mcbain and Mayer, 1994; Hollmann and Heinemann, 1994; Danysz et al., 1995; Parsons et al., 1998; Danysz and Parsons, 1998; Cancela et al., 1999). Both AMPA (Rosenmund et al., 1998) and NMDA receptors (Laube et al., 1998) are probably largely formed from tetrameric, heteromeric assemblies of different subunits (Mansour et al., 2001).
Metabotropic glutamate receptors
Metabotropic glutamate receptors are divided into three major groups I-III. Group I metabotropic receptors consists of two receptor subtypes mGluR1 which has four splice variants and mGluR5 which has two splice variants. Both are largely postsynaptic and positively coupled to phospholipase C (PLC). PLC promotes the conversion of PIP2 to diacylglycerol (DAG) and IP3. DAG activates membrane-bound PKC which in turn can phosphorylate ionotropic glutamate receptors. IP3 has numerous intracellular effects including stimulation of Ca2+release from intracellular stores. Group II (mGluR2/3) and group III ( (mGluR4/6/7/8) receptors differ in their sequence homology, but are both coupled to a different effector system i.e. they decrease the activity of adenylate cyclase. They are both largely located on presynaptic neurones and glia.
These receptors are not the subject of this review as it is only very recently that agents with therapeutic potential have become available. However, it seems likely that allosteric modulation of these receptors may provide a valid strategy for the development of new pharmaceuticals in the near future (Gasparini et al., 2002).
Glutamate uptake
Glutamate clearance and, as a consequence, glutamate concentration and diffusion in the extracellular space, is associated with the degree of astrocytic coverage of its neurons (Oliet et al., 2001). Glutamate is eliminated from the synaptic cleft by specific transporters. The genes encoding glutamate transporter proteins have been cloned both from rats and humans (Arriza et al., 1994; Malandro and Kilberg, 1996). The human transporters EAAT1 and EAAT2 (rat equivalents GLAST and GLT1) are found in astroglia and microglia and are widely distributed in the CNS (highest in the cortex). Human EAAT3 (rat EAAC1) is restricted to neurons but is also found outside of the CNS. Human EAAT4 is expressed by cerebellar neurons.
N-Acetyl-aspartyl-glutamate (NAAG)
N-Acetyl-aspartyl-glutamate (NAAG) is abundant in the mammalian CNS which has led to the hypothesis that this dipeptide is the storage form of glutamate. The membrane bound metallopeptidase NAALADase (N-acetyl-a-linked-acidic dipeptidase, or glutamate carboxypeptidase II, E.C. 3.4.17.21) is co-localized with NAAG in the CNS and converts NAAG to NAA and glutamate (Blakely et al., 1988). It seems likely that NAAG is also a weak partial agonist at NMDA receptors with low intrinsic activity and an agonist at mGluR3 receptors (Neale et al., 2000). The former effect is however, seen at concentrations beyond therepeutically relevant levels. Inhibition of NAALADase could be useful in numerous CNS disorders associated with disturbances in glutamatergic transmission by decreasing the concentration of glutamate and increasing the concentration of NAAG. Guilford pharmaceuticals have followed this approach with their NAALADase inhibitors such as 2- (phosphonomethyl)-pentanedioic acid (PMPA, GPI 5000) and second generation compounds like GPI 5693 and GPI 16072 (Jackson and Slusher, 2001).
Synaptic Plasticity
In many regions of the CNS, Ca2+ influx through NMDA receptors can trigger two forms of synaptic plasticity: long-term depression (LTD) and long-term potentiation (LTP) which are believed to resemble some elementary features of memory formation at the neuronal level (Malenka, 1994; Bear and Malenka, 1994; Edwards, 1995; Collingridge and Bliss, 1995; Rison and Stanton, 1995; Baudry, 1996; Jeffery, 1997). The voltage-dependent blockade of NMDA receptors by Mg2+ and their high Ca2+ permeability renders them inherently suited for their role in mediating synaptic plasticity (Herron et al., 1986). NMDA receptor channels are only activated in the presence of a local strong depolarization induced by strong AMPA receptors activation and concurrent GABAergic dis-inhibition via feedback effects of GABA on GABAB autoreceptors. As a result, the Mg2+ blockade of NMDA receptors is transiently fully relieved allowing Ca2+ to flow into the postsynaptic neurone. This Ca2+ influx triggers a cascade of secondary messengers which ultimately activate a number of enzymes such as protein kinase C (PKC), phospholipase A2 (PLA2), phospholipase C (PLC), Ca2+/calmodulin-dependent protein kinase II (CaM kinase II), etc. (Abraham and Tate, 1997) (Grant and Silva, 1994; Lisman, 1994; Pasinelli et al., 1995; Benowitz and Routtenberg, 1997; Lan et al., 2001; Bayer et al., 2001). Consequently, these processes lead to fixation of changes in postsynaptic AMPA receptors such as an increase in their affinity and/or number (Maren et al., 1993; Ambros-Ingerson and Lynch, 1993; Ambros-Ingerson et al., 1993; Benke et al., 1998) but see (Kessler et al., 1991) and, possibly through retrograde signals (arachidonic acid, nitric oxide), modulate presynaptic glutamatergic terminals influencing transmitter release (Lynch, 1989; Odell et al., 1991; Kato et al., 1991; Schaechter and Benowitz, 1993; Kato et al., 1994; Luo and Vallano, 1995).
There is accumulating evidence that LTP and LTD share some common mechanisms, although LTD occurs with increases in postsynaptic Ca2+, that are insufficient to induce LTP (Artola and Singer, 1993; Christie et al., 1994; Cummings et al., 1996; Derrick and Martinez Jr, 1996; Hansel et al., 1996; Kirkwood et al., 1996; Tsumoto et al., 1996; Tsumoto and Yasuda, 1996; Christie et al., 1996; Artola et al., 1996). LTP and LTD have been extensively studied as cellular models of learning and memory. Although hippocampal long-term potentiation and spatial learning are impaired by NMDA receptor blockade see (Jeffery, 1997) learning deficits can be almost completely prevented if rats are pretrained in a different water maze (Bannerman et al., 1995; Saucier and Cain, 1995). NMDA receptors may therefore not be required for encoding the spatial representation of a specific environment but rather in other forms of memory important for learning this task (Morris, 1996). Recent evidence indicates that LTP is not only important for synaptic plasticity in the mature CNS but also in the formation of conducting glutamatergic synapses in the developing mammalian brain (Durand et al., 1996).
One form of hippocampal LTP involves the activation of the NMDA receptors and a rise in postsynaptic Ca2+ in the CA1 region but there is still considerable debate as to the site at which the increase in synaptic strength is expressed e.g. (Stricker et al., 1996; Stricker et al., 1996; Isaac et al., 1996; Isaac et al., 1996). Presynaptic mechanisms should be reflected in a change in release probability. This can be measured at excitatory synapses on cultured hippocampal neurones by analysis of the progressive block of NMDA receptor-mediated synaptic currents by the essentially irreversible open channel blocker dizocilpine ((+)MK-801) (Rosenmund et al., 1993). This technique was used to demonstrate that release probability was not affected after the induction of LTP making a presynaptic mechanism unlikely (Manabe and Nicoll, 1994). Moreover, recent reports indicate that a high proportion of synapses in hippocampal area CA1 transmit with NMDA receptors but not AMPA receptors, making these synapses effectively non-functional at normal resting potentials due to Mg2+blockade (Liao et al., 1995; Nicoll and Malenka, 1995; Montgomery et al., 2001; Montgomery and Madison, 2002). These silent synapses acquire AMPA-type responses following LTP induction. Furthermore, this form of LTP is accompanied by an increase in the conductance of postsynaptic AMPA receptors (Bibb et al., 2001; Bibb et al., 2001). Taken together, these findings challenge the view that LTP in CA1 involves a presynaptic modification, and suggest instead a simple postsynaptic mechanism for both induction and expression of LTP.
Nerve cells pass signals to each other and to their target organs by releasing messenger molecules, called transmitters. Many are simple amino acids such as the one called glutamate.
The message is intended to tell the recipient neuron whether to fire off its own neurotransmitters. As with all neurotransmitters, glutamate docks at specific recognition molecules on the receiving neuron. Glutamate is then swiftly cleared from the nerve cell junctions to keep the message brief. Prolonged excitation is toxic to nerve cells, and neurobiologists recognize that glutamate can cause harm when the messages are overwhelming, as in stroke or epilepsy.
Glutamate’s toxicity is apparently due to calcium flooding the cell. Calcium is supposed to briefly enter the neuron with each signal and triggers the cell to fire off its own signals and adjust its own activities accordingly. But prolonged calcium inside the cell evidently can do damage, and will even activate programmed cell death.
Research in the early 1990s determined that ALS patients have raised levels of glutamate in the fluid bathing the brain and spinal cord. In fact, 40 percent of sporadic cases of ALS are characterized by this elevated glutamate in cerebrospinal fluid (CSF). Abundant evidence points to glutamate as a destructive factor in ALS. The first and so far only approved specific treatment for ALS is riluzole, a drug that modulates glutamate.
The transporter that clears glutamate is called EAAT2 (an “excitatory amino acid transporter;” as glutamate is one of the amino acids that serve as transmitters). In ALS, transport of glutamate is slowed into the glial cells that surround the junctions of motor neurons. Indeed, a mutation has been identified in an ALS patient that prevented the transporter from working properly.
Researchers are working towards gene therapy approaches to deliver the glutamate transporter molecule to cells affected by ALS. Other avenues towards control of glutamate in ALS are also under active investigation.
OVERVIEW
Prolonged excitation is toxic to nerve cells. Neurobiologists recognize that the nerve cell messenger, glutamate, can cause harm when its messages are overwhelming. Normally glutamate is swiftly cleared from the nerve cell junctions to keep the messages brief. Molecules called transporters aid in keeping glutamate in proper concentrations around nerve cells. Abundant evidence points to glutamate as a destructive factor in ALS and investigators are working to find out how this can be changed. Gene therapy approaches are under investigation to deliver glutamate transporters to cells affected by ALS. Other avenues towards control of glutamate in ALS are also under active investigation.
Outside the community of biomedical scientists, glutamate is probably best known as “monosodium glutamate” or “MSG” which is used as a flavor or taste enhancer in food. It is usually available together with other food additives and spices in most large food stores. Some people may also have heard the term “Chinese restaurant syndrome” which is a sudden fall in blood pressure with subsequent fainting after ingestion of very spicy food. Excessive use of MSG has been suggested to be the cause, but this is controversial. The use of glutamate as a food additive, however, is not the reason for the enormous scientific interest in glutamate.
- Glutamate is the major excitatory transmitter in the brain
The main motivation for the ongoing World Wide research on glutamate is due to the role of glutamate in the signal transduction in the nervous systems of apparently all complex living organisms, including man. Glutamate is considered to be the major mediator of excitatory signals in the mammalian central nervous system and is involved in most aspects of normal brain function including cognition, memory and learning.
- Glutamate is toxic, not in spite of its importance, but because of it
Glutamate does not only mediate a lot of information, but also information which regulates brain development and information which determines cellular survival, differentiation and elimination as well as formation and elimination of nerve contacts (synapses). From this it follows that glutamate has to be present in the right concentrations in the right places for the right time. Both too much and too little glutamate is harmful. This implies that glutamate is both essential and highly toxic at the same time.
- It took a long time to realize that glutamate is a neurotransmitter
It may sound astonishing, but it took the scientific community a long time to realize that glutamate is a neurotransmitter although it was noted already 70 years ago that glutamate is abundant in the brain and that it plays a central role in brain metabolism. Ironically, the reason for the delay seems to have been its overwhelming importance. Brain tissue contains as much as 5 - 15 mmol glutamate pr kg, depending on the region, more than of any other amino acid. Glutamate is one of the ordinary 20 amino acids which are used to make proteins and takes parts in typical metabolic functions like energy production and ammonia detoxification in addition to protein synthesis. It was hard to believe that a compound with so many functions and which is present virtually everywhere in high concentrations could play an additional role as transmitter.
- How glutamate works as a transmitter
Like other signaling substances (neurotransmitters and hormones) the signaling effect of glutamate is not dependent on the chemical nature of glutamate, but on how cells are programmed to respond when exposed to glutamate. Only cells with glutamate receptor proteins (“glutamate receivers”) on their surfaces are sensitive to glutamate. Glutamate exerts its signaling function by binding to and thereby activating these receptor proteins. Several subtypes of glutamate receptors have been identified: NMDA, AMPA/kainate and metabotropic receptors (mGluR).
Although the individual receptor subtypes show specific (restricted) localizations, glutamate receptors of one type or another are found virtually everywhere. Most of the nerve cells, and even glial cells, have glutamate receptors.
- Glutamate must be kept inside the cells (intracellularly)
At first glance this looks like an impossible system. A closer look, however, reveals that glutamate is not present everywhere. It is almost exclusively located inside the cells. The intracellular location of some 99.99 % of brain glutamate is the reason why this system can work. This is essential because glutamate receptors can only be activated by glutamate binding to them from the outside. Hence, glutamate is relatively inactive as long as it is intracellular.
The volume of brain cells and of the meshwork formed by their intermingled extensions, constitute about 80 % of brain tissue volume. This network is submerged in a fluid, the extracellular fluid which represents the remaining 20 % of brain tissue volume. The normal (resting) concentration of glutamate in this fluid is low, in the order of a few micromolar. In contrast, the glutamate concentration inside the cells is several thousand times higher, at around 1 - 10 millimolar. The highest glutamate concentrations are found in nerve terminals and the concentration inside synaptic vesicles may be as high as 100 millimolar.
- The glutamate transporters remove glutamate from the extracellular fluid
It follows from the description above that the mechanisms which can maintain low extracellular concentrations of glutamate are essential for brain function. The only (significant) mechanism for removal of glutamate from the extracellular fluid is cellular uptake of glutamate; the so called “glutamate uptake”. This uptake is mediated by a family of special transporter proteins which act as pumps. These proteins bind glutamate, one molecule at the time, and transfer them into the cells. In agreement with the abundance of glutamate and the ubiquity of glutamate receptors, brain tissue displays a very high glutamate uptake activity. This was noted already in 1949, although its true importance was not recognized until after the excitatory action of glutamate was discovered in the 1950s and 1960s.
Glutamate is taken up into both glial cells and nerve terminals. The former is believed to be the more important from a quantitative point of view. Glutamate taken up by astroglial cells is converted to glutamine. Glutamine is inactive in the sense that it cannot activate glutamate receptors, and is released from the glial cells into to extracellular fluid. Nerve terminals take up glutamine and convert glutamine back to glutamate. This process is referred to as the glutamate-glutamine, and is important because it allows glutamate to be inactivated by glial cells and transported back to neurons in an inactive (non-toxic) form.
Bensimon, et al. (1994) hypothesized that riluzole may have beneficial effects on people with diseases such as amyotrophic lateral sclerosis (ALS) which involve overactivation of glutamate receptors. ALS is a progressive and fatal disorder affecting nerve cells. The cause of the disease is unknown, and no treatment is available that influences survival.
Many hypotheses about the cause of the disease are currently being studied. One of these hypotheses involves glutamate. Studies have reported that increased glutamate concentrations in the brain result in nerve cell death. Given this possible role of glutamate in ALS progression, the researchers sought to assess the effects of riluzole in people with ALS.
The researchers conducted a trial in 155 participants with ALS in France for one year. The participants were given either 50-mg of riluzole twice a day or a placebo. Survival and changes in ability to function were used as tests for the drug’s effectiveness. A secondary test used to examine the drug’s effectiveness was change in muscle strength.
After 12 months, 58 percent in the placebo group were still alive, compared with 74 percent in the riluzole group. The deterioration of muscle strength and functional ability was significantly slower in the riluzole group than in the placebo group.
Side effects of riluzole included stiffness, mild increase in blood pressure, and increase in the levels of the enzyme aminotransferase, which sometimes result in elevations of toxic ammonia. High levels of ammonia have been associated with brain damage, although the reason for ammonia toxicity is still unknown. While aminotransferase elevations were more frequent with riluzole treatment, the elevations were well tolerated and did not cause severe adverse effects in most of the participants in this study. More studies need to be conducted to understand this side effect of riluzole.
On the whole, it appears that these reported side effects may worsen the quality of life, but such consequences may be outweighed by the effect of the drug in improving muscle function and survival rates. The mechanism by which riluzole improves muscle function and survival rates is still unknown. However, the results of this study indicate that riluzole may have a beneficial effect in people with diseases that involve glutamate toxicity such as ALS and HD.
Rosas, et al. (1999) hypothesized that riluzole treatment may have beneficial effects in people with HD. The researchers conducted a 6-week trial of riluzole in eight participants with HD. The participants were treated with 50 mg of riluzole twice a day and were observed for changes in chorea (involuntary dance-like movements), dystonia (prolonged muscle contractions), and total functional capacity(TFC) scores. TFC is a standardized scale used to assess the capacity to work, handle finances, perform domestic chores and self-care tasks, and live independently. The brain lactate levels of the participants were also studied. Lactate is a by-product of anaerobic metabolism that is often used as a measure of energy metabolism efficiency in cells. Low lactate levels would indicated high aerobic respiration and high energy yields. High lactate levels on the other hand, would indicate that cells are unable to perform aerobic respiration and had to resort to the less-efficient anaerobic respiration instead. Changes in lactate levels were then used by the researchers to test the effects of riluzole on energy metabolism.
The researchers found that the chorea rating score of the participants who took riluzole improved by 35% compared to their scores before treatment. Discontinuation of treatment resulted in worsened chorea, indicating that riluzole was indeed associated with the improved chorea. No significant changes were seen on the dystonia or TFC scores.
Lactate levels were lower in the riluzole-treated participants compared to their levels before treatment. However, the researchers reported concerns about inaccuracies in lactate measurements due to limitations in their instruments and measuring methods. Whether or not the decreased lactate levels associated with riluzole indicate improved energy metabolism remains to be determined.
In this study, no significant adverse effects were observed after 6 weeks of treatment. The most frequent side effect was diarrhea; other symptoms quickly resolved without the need for medical intervention.
The results of this study also suggest a possible role for riluzole in the treatment of chorea in people with HD. However, the mechanism by which riluzole might alter or prevent disease progression is still ambiguous. More studies need to be conducted to determine whether and how riluzole can slow the progression of HD and protect nerve cells.
It is thought that riluzole inhibits the release of glutamate by interfering with sodium (Na+) channelsthat are required for normal glutamate release. Figure 1 shows how riluzole inhibits glutamate release.
* Na+ channels = gated ion channels that are necessary for glutamate release. Riluzole and lamotrigine interfere with these channels.
The mechanism by which riluzole disrupts the effects of glutamate on target cells is slightly more complicated. Let us first go over what happens in a normal glutamate-receiving cell in order to understand the effects of riluzole on these cells in a patient with HD. Various types of glutamate receptors are found in nerve cells. One type of glutamate receptor allows the entry of ions into the cell upon glutamate binding, resulting in various changes inside the cell. Among these receptors are NMDA receptors, discussed in the section HD and Glutamate. A second type of glutamate receptors causes cellular changes by initiating a messenger cascade, which involves the activation and deactivation of various molecules and pathways that can cause changes inside the nerve cell.
In a messenger cascade, the binding of glutamate is a “message” that is being sent to the nerve cell. This message is passed on from one molecule to another, until it reaches its final destination. Scientists have discovered that glutamate binding “tells” the cell to release calcium from its stores. In HD cells, the overactivation of the glutamate receptors results in overactivation of the messenger cascades and consequently, increased calcium release. High amounts of calcium in the nerve cells are known to cause cell death, which is one possible explanation of how HD nerve cells die. Figure 2 shows a diagram depicting the molecules involved in the messenger cascade as well as the final effects of the cascade.
Riluzole may disrupt glutamate activity by interfering with the activity of certain proteins involved in the messenger cascade. Once the cascade is inhibited, changes induced by glutamate such as calcium release and the associated cell death might eventually be delayed.
