The Central Role of Excitotoxicity in Autism Spectrum Disorders Introduction In this discussion I shall define autism spectrum disorders as a group of disorders of higher cortical function ranging from attention deficit disorder to full blown autism itself. Despite divisions into numerous individually named disorders, Asperger’s, high autism, attention deficit hyperactivity disorder, etc, many feel that they represent a spectrum of related cognitive disorders. I do this recognizing that clinically, several may have characteristics that make them significantly different from the others. Recognizing these differences, I shall discuss their special physiology and biochemistry as the need arises. Recent evidence indicates that most neurological disorders, both acute and chronic, have a common set of pathological events despite their varying clinical presentation.1 At the center of this process is what has become known as excitotoxicity. Named in 1969 by Dr. John Olney, excitotoxicity is a phenomenon characterized by the triggering of neuronal excitation through over-stimulation of susceptible neurons by the excitatory amino acids, primarily glutamate and aspartate. 2 Using cloning techniques, scientists have characterized five sets of excitatory receptors that include NMDA, AMPA, kainate and two metabotrophic type receptors.3 We know the most about the NMDA receptor, which controls a voltage-gated calcium channel. Clustered around the calcium channel are various regulator receptors, including the zinc and magnesium sites that modulate the channel, so as to prevent over- activation, and a glycine receptor which enhances the signal during NMDA receptor activation. A phencyclidine receptor powerfully inhibits the opening of the calcium channel. Glutamate is the most abundant neurotransmitter in the central nervous system, yet it is also the most neurotoxic. It is for this reason that its concentration outside the neuron is so carefully controlled. This control is maintained by a family of glutamate transport proteins, which attach to the transmitter soon after its release. Soon after it is transported it to a nearby astrocyte, where it is deposited.4 Excess levels of glutamate, or other excitatory molecules, allow the calcium channel to remain open for a relatively long period of time. Calcium excess in the cytosol of the cell triggers the activation of inducible nitric oxide synthase and protein kinase C. The iNOS produces NO in excess, which begins to accumulate within the cell. When NO combines with the superoxide radical it forms the very destructive peroxynitrite radical. This radical is particularly injurious to the mitochondria, the chief source of energy for the neuron.5 At the same time, protein kinase C then activates phospholipase A2 within the neuron membrane, which brings about the release of arachidonic acid into the cytosol. Here the arachidonic acid is acted on by two enzymes, lipoxygenase and cyclooxygenase, which produce a series of potentially destructive eicosanoids. Of particular concern is the COX II enzyme, which brings about the accumulation of PGE2 and PGD2, both pro-inflammatory molecules. Interestingly, only glutamatergic neurons contain COX II enzymes, which are located on distal dendrites and are concentrated in dendritic spines.6 The accumulation of inflammatory eicosanoids leads to the production of free radicals, including the very destructive hydroxyl radical. As the process accelerates, the free radicals interact with the neuron’s numerous membrane structures, including nuclear, mitochondrial and cellular membranes. Once this process begins, a chain reaction within the membrane’s polyunsaturated fatty acids is initiated, a process we call lipid peroxidation. Numerous by-products are produced during lipid peroxidation, including the production of several aldehydic products. While the most abundant of these LPO products is malondialdhyde ( MDA), most destructive is a product called 4-hydroxynonenal.7 Recent research has shown that 4-HNE can produce extensive damage to the cell, including the prevention of dephosphorylation of excessively phosphorylated tau protein, significantly interfering with microtubule function.8 It has also been shown to inhibit glutathione reductase, which is needed to convert oxidized glutathione to its functional reduced form. 9 It has been demonstrated that children with active autoimmune diseases have significantly higher blood levels of 4-HNE than controls. 10 It is also known that 4-HNE can conjugate to synaptic proteins, where it impairs the transport of both glucose and glutamate. 11 This process is especially dangerous because several studies have shown that impaired energy supplies markedly enhances glutamate sensitivity. In fact, under such conditions, even normal levels of glutamate can produce neurotoxicity. 12 Peroxynitrite, by damaging mitochondrial membranes, DNA and electron transport enzymes, can also significantly reduce neuronal energy production. 13 It is known that numerous pathological events can trigger excitotoxicity, including ischemia, hypoxia, hypoglycemia, viral and bacteriological pathogens, toxic metals, trauma, autoimmune diseases, and free radical excess. It should also be recognized that there is an intimate relationship between excitotoxicity and free radical generation. Free radicals precipitate the release of glutamate in the brain and excitotoxins trigger the productions of large amounts of free radicals, both of the oxygen and nitrogen species. While this review of excitotoxicity is not complete it will provide the reader with a better understanding of the process. Seizures, Autism and Excitotoxicity It has been recognized that seizures are fairly common with several of the autism spectrum disorders. Approximately one third of autistic children have definable seizures or abnormal EEG seizure foci.14 Overt seizures are not necessary for regression. In many cases, abnormal EEG seizure foci have been found in the absence of clinical seizures.15 These abnormal seizure foci, with and without clinical seizures, are seen more commonly in autistic children who regress. Childs and Blair reported dramatic improvements after treatment with valproic acid in a pair of autistic twin boys who were found at age three to have absence seizures.16 The parents, on reflection, recalled symptoms consistent with seizures occurring at age two. These boys had symptoms characteristic of autism, including perseverative, non- purposeful and self-stimulatory behavior, a lack of symbolic play, poor eye contact, echolalic and non-communicative speech and a lack of response to discipline. In some autistic children one finds evidence of tuberous sclerosis, a condition associated with a high incidence of seizure disorders.17 Approximately 25% of children with tuberous sclerosis will be autistic. If you add pervasive developmental disorder the incidence increases to 40 to 45%. Among autistic children 1 to 4% will also have tuberous sclerosis. The incidence increases to 8 to 14% in autistic children with seizures. There is evidence that seizure foci in autistic children have been grossly underdiagnosed. In a recent study of children with Landeau Kleffner syndrome (LKS) as compared to autistic children with regression, researchers using a highly sensitive magnetoencephalographic technique ( MEG), found that out of 50 autistic children examined during stage II sleep, 82% demonstrated eipleptiform activity in the same region of the brain as seen in Landeau Kleffner syndrome.18 The difference in the two groups was that the LKS children demonstrated no epileptiform activity outside the left intra/perisylvian area whereas 75% of the regressive autistic children demonstrated seizure foci with independent activity, outside this area. The LKS children demonstrated propagation of the seizure to frontal and parietal regions on occasions, which could explain associated difficulties with socialization and behavior. During the examinations, standard EEG recordings were done simultaneously with the magnetoencephalographic recordings. While the MEG recordings demonstrated abnormal activity in 85% of cases combined, the standard EEG recordings demonstrated problems in only 68% of cases. This indicates that significant abnormalities are being overlooked during routine examinations. It is also possible that depth electrode recordings would detect even more abnormalities in subcortical areas, such as the amygdala and septal areas. That a persistent seizure focus discharge is pathologically damaging is graphically shown in the case of Landeau Kleffner syndrome. In this disorder, a persistent seizure focus results in a progressive loss of language function and social interaction, both higher cognitive functions. Of particular concern is that the seizures usually occur at nighttime and are very difficult to recognize by the parents or doctors, as we have seen. Recovery of language function depends on early seizure control. Another graphic demonstration of the connection between seizures, glutamate accumulation and cognitive deterioration is seen in the case of pyrodoxine-sensitive seizure in newborns. It has been shown that in the untreated child, CSF glutamate levels are 200X normal and seizures are uncontrollable.19 When given an intermediate dose of 5mg/kg/BW/day of pyrodoxine, the seizures cease, but mental deterioration continues. Glutamate levels at this dose were still 10X higher than normal. When using pyrodoxine at 10 mg/kg/BW/day there were no seizures, no cognitive deterioration, and glutamate levels are normal. It is interesting to note that some reported cases of pyridoxine-dependent seizures also had features of autism.20 While most cases of pyridoxine-dependent seizures are present at birth, cases have been reported that experienced their initial onset as late as 14 months after birth.21 It has been suggested that pyridoxine-dependent seizures are more common than is being reported, and that neurological deterioration can occur in the absence of seizures.22 A wide array of neurological symptoms can be seen on the basis of excitotoxic lesions produced with this syndrome, including, visual agnosia, squint, severe articulatory apraxia, and motor delay. We also know that the excitotoxic process associated with this syndrome can produce physical changes in the brain as seen on MRI and CT scans, usually with cortical and subcortical atrophy and progressive ventricular dilitation.23 Another demonstration of the importance of glutamate in seizure pathology comes from the study by Mathern and co-workers who demonstrated increased NMDA receptor content in cases of temporal lobe epilepsy associated with mesial hippocampal sclerosis, indicating dentate granule cell hyperexcitability.24 Others have shown degeneration of dendritic connections in epileptic hippocampal neurons characteristic of excitotoxicity. 25 Interestingly, a recent study found that the anatomic substrate of the limbic system, which included the subiculum/ CA1-CA3 area and the dentate gyrus/ CA4 area, was smaller in autistic subjects than matched controls.26 This. It has been observed that a percentage of autistic children improve when supplemented with zinc. It is known that the temporal lobes have the highest zinc content in the brain and that zinc plays a major role in reducing NMDA excitability.27 Zinc has also been found to reduce dentate granule cell hyperexcitability in epileptic humans.28 It is now known from experimental studies that seizures are intimately connected to the excitotoxic process.29 Not only can glutamate and aspartate precipitate seizures, especially when injected into the brain, but seizures themselves can stimulate the release of excitatory amino acids from the brain, most likely by stimulating free radical generation. Spontaneously discharging neurons, especially when the process is prolonged, are associated with energy loss, ischemia, and hypoxia, all of which precipitate excessive release of glutamate. There is considerable evidence that excitotoxicity is responsible for much of the pathological damage produced by prolonged seizures.30,31 This destructive process has been proposed as the mechanism for both the mirror focus seen with temporal lobe seizures and the cognitive deterioration associated with status epilepiticus. Cytopathological changes have been described in the hippocampus following prolonged seizures that closely resemble excitotoxic damage, with destruction of neurons in the CA1 and CA3 areas, and dendritic swelling in the hilus of the fascia dentata, as seen with cases of autism. Recent studies have shown that ketamine, a powerful NMDA receptor antagonist, can powerfully inhibit seizures, including status epilepticus.32 Of particular concern is the excitotoxic damage produced during limbic status epilepticus, a common form of epilepsy seen in autism spectrum disorders, and which may explain the above mentioned limbic atrophy in autism.33 Another excitotoxic substance associated with seizures is quinolinic acid.34 This excitotoxin is important for two reasons. First, it is a metabolic product of serotonin breakdown, and second it is released from both astrocytes and microglia when these cells are activated by various stimuli. Quinolinic acid acts at the NMDA receptor and, like glutamate, its activity can be blocked by MK-801. There is evidence that excessive accumulation of extraneuronal glutamate can inhibit oxidative phosphorylation. Studies using retinal cells have shown that high concentrations of glutamate can reduce complex I, II/III and IV, and that this inhibition can be completely blocked by MK-801.35 Several studies have shown that neuronal energy deficits dramatically increase excitotoxic sensitivity, even to the point where normal concentrations of glutamate can become excitotoxic. While glycine demonstrates inhibitory actions in the spinal cord, in the cerebrum it is excitatory. This is because it plays a major role in glutamate activation of the NMDA receptor. High concentrations of glycine have been shown to cause marked hyperexcitability and neurotoxicity in hippocampal brain slices.36 Kainate can induce kindling, when injected into the cortex or amygdala. The kindling response can occur without initiating seizures. Kindling without clinical seizures is something that has been observed in autism. Several studies have shown that kindling can produce excitotoxic lesions in the absence of clinical seizures, again, something important to consider in the autistic child.37 While neuronal degeneration can result from elevated levels of glutamate, a loss of dendritic connections can occur at much lower concentrations. There is also substantial evidence that elevated levels of glutamate during periods of critical brain formation can result in altered pathway development by over-stimulating growth cones.38 Glutamate levels are carefully regulated during early brain formation and disruptions in glutamate levels can result in alteration leading to either subtle or profound effects on brain function, depending on the timing and dose. Seizures, especially when prolonged, can result in such elevations of glutamate levels. It is also known that ischemia and hypoxia, not uncommon in prolonged seizures, can produce dramatic increases in glutamate levels for prolonged periods of time. These levels could have a profound effect on pathway formation as well as a loss of neurons, synaptic connections, and stem cells. It is known that after age two years, the developing brain contains more synaptic glutamate receptors than at birth, and that the number slowly declines over the next decade.39 This makes the infant brain especially vulnerable to excitotoxicity. The Role of Immune Stimulation It is recognized that activation of microglia, as well as astrocytes, during immune stimulation, can elicit excitotoxicity. 40 The mechanism involves a complex array of events primarily involving the release of numerous cytokines. It should be appreciated that microglial activation can occur during systemic immune challenge, as with vaccination.41,42 Microglial activation elicits the release of several cytokines including TNF-alpha, IL-1ß, IL-2, IL-6 and INF-gamma.43 In addition, cytokine activation of inflammatory eicosanoids occurs as well.44 Closely linked to this process is the generation of numerous species of reactive oxygen and nitrogen intermediates, including superoxide, hydrogen peroxide, hydroxyl radicals, peroxynitrite and 4-hydroxynonenal. These reactive intermediates not only damage synaptic connections, neurons, and cellular components, but also induce the release of glutamate from surrounding astrocytes.45 Of particular interest is the recent observation that microglial activation can also elicit the release of glutamate and quinolinic acid, two powerful excitotoxins, from the macrophage itself.46 Interaction with bacterial components, viruses and lipopolysacchrides can increase glutamate release two to three-fold above basal levels.47 Likewise, dexamethasone has been shown to reduce glutamate release following antigen exposure by 50%.48 It should also be appreciated that glutamate excess, as well as deficiency, interferes with long termed potentiation, which is critical for learning and memory.49 In addition, the growth and terminal distribution of developing brain pathways are also adversely affected by excess glutamate, especially when prolonged. Likewise, glutamate deficiency interferes with growth cone function, leading to “miswiring” of the brain’s circuitry. Anything that activates microglia, including viruses, ß-amyloid, mercury, aluminum, oxidized LDL and HDL, homocysteine and excitotoxins, can increase the accumulation of quinolinic acid.50 This raises concern about the use of L-tryptophan enhancing supplements and medications. Of particular concern is an imbalance between quinolinic acid and kynurenine formation, since the latter is a neuroprotectant. Another area of concern is the ability of immune microglial activation products to interfere with glutamate re-uptake. The glutamate transport family of proteins is particularly sensitive to inactivation by IL-1ß, TNF-alpha, mercury, peroxynitrite and 4-hydroxynonenal.51,52,53 Such interference with glutamate disposal has been associated with amyotrophic lateral sclerosis and possibly Alzheimer’s syndrome.54,55 All of these inhibitory factors can be seen in cases of over vaccination and autoimmunity. Mercury is a very powerful inhibitor of GLT-1, the glutamate transport protein, even in very small concentrations.56 Several studies have shown that children with autism frequently have significantly elevated mercury levels, with vaccines often being the only source of the mercury (as the preservative thimerosal). Mercury exposure from dental amalgam in rats produced significantly elevated levels of immune complexes in the renal glomeruli and vessel walls of numerous organs, including the brain.57 Based on what we know about overstimulation of the immune system, with concomitant prolonged microglial activation, removing the mercury from vaccines, while helpful, most likely will not eliminate the problem. Vijendra Singh and co-workers have found that 84% of autistic children examined demonstrated antibodies to myelin basic protein.58 This suggests that a state of autoimmunity to brain has occurred in the autistic child. It is known that autoimmune states are associated with high levels of cytokines and inflammatory mediators such as leukotrienes and prostaglandins.59 These inflammatory mediators increase brain oxidative stress and excitotoxicity. It is interesting to note that autoimmunity is also found in many of the adult neurodegenerative disorders, such as Alzheimer’s disease, ALS and Parkinson’s disease.60,61,62 Another possibility is the presence of a persistent virus or a stealth virus. When the immune system has been impaired, either genetically or by exhaustion, viruses can persist in tissues for long periods of time.63 Because the immune system is impaired, instead of killing the virus, the activated microglia continuously release neurotoxic mediators and a stream of free radicals. They also stimulate the release of glutamate and other excitotoxins, which further increases the production of destructive reactive intermediates. The first casualty is the synaptic connections, followed by the immature pathways forming during the brain growth spurt. That the measles virus enters the brain in cases of measles encephalomyelitis has been shown by protein sequencing.64 Viral entry into the brain can either induce a demyelinating syndrome (subacute sclerosing panencephalitis) or a non-demyelinating syndrome as characterized above. Giving children live measles viruses can possibly lead to invasion of the brain by these persistent viruses. In one study, using mice infected with hamster neurotropic measles virus, researchers found that after seven days post-inoculation, hippocampal brain slices produced 18X more quinolinic acid as compared to controls.65 Three-hydroxyanthranilic acid oxygenase, an astrocytic enzyme responsible for the production of quinolinic acid, increased its activity 3.3 fold on the seventh post-inoculation day. Quinolinic acid accumulation has been associated with HIV dementia as well, secondary to its release from activated microglia. The HIV viral envelope, gp 120 and tat proteins are neurotoxic by an excitotoxic mechanism. Blocking the NMDA receptor prevents quinolinic acid neurotoxicity. In mice, measles virus-induced encephalopathy associated neurotoxicity is also prevented by MK-801, an NMDA antagonist.66 Self-limited incidences of acute encephalopathy probably occur more often than are reported.67 This is because many pediatricians either do not recognized subtle neurological signs or dismiss them as the result of an overanxious mother. Chronic viral infections of the CNS, especially by stealth viruses, with waxing and waning symptoms, are frequently overlooked by those not trained in neurological care. Another study raises even more concern for atypical presentations of measles infections of the brain.68 In this study, it was found that hamster neurotropic virus could cause a non-inflammatory encephalopathy with degeneration of the hippocampal CA1 and CA3 regions. The excitotoxic reaction increased several days after the inoculation. In humans this could lead to varying degrees of memory loss and learning difficulties, since excitotoxin damage has been shown to interfere with long term potentiation (LTP). Within the last decade two cases of postvaccinal parkinsonism have been reported following inoculation for measles. One case occurred in a five- year-old boy who developed fever and a rigid-akinetic syndrome beginning 15 days after the vaccine. 69 A follow-up report at age seven, found that he was still suffering from parkinsonian symptoms. From these reports one must conclude that the virus localized within the striatum, eliciting an excitotoxic reaction of sufficient degree to produce parkinsonian symptoms.70,71 The fact that methamphetamine induces nigrostriatal dopaminergic toxicity by an excitotoxic mechanism questions the wisdom of placing children with autism spectrum disorders on such medications.72 By grouping vaccines together, especially live viral vaccines, one increases the stress on the immune system as well as increasing microglial activation within the brain. Not infrequently, very small children are given multiple vaccination during a single doctor’s visit. This can vary between three to nine vaccines at one sitting. This not only constitutes a heavy bacterial and/or viral antigen load, but contains powerful adjuvants to boost immunity so as to increase the likelihood of immunization. This has two effects. First, it overstimulates a dysfunctional immune system, leading to immune directed damage to the nervous system. Measles virus is known to induce autoimmune reactions to myelin basic protein.73 Second, it eventually exhaust the immune system leading to increased susceptibility to subsequent microbial infections or chronic viral infections. This scenario is more likely in the malnourished child, especially with vitamin A deficiencies. Experimentally, retinoids have been shown to significantly reduce the clinical severity of experimental allergic encephalomyelitis.74 Early nutrition has been shown to play a major role in immune function not only during the neonatal period, but also throughout life.75 Experimentally, using guinea pigs and rats, excitotoxic lesions within the hypothalamus have been shown to suppress both humoral and cell mediated immunity.76 Excitotoxin suppression of delayed type hypersensitivity may explain why subacute sclerosis panencephalitis is less often seen than excitotoxic lesions not directed at myelin. These excitotoxic-induced lesions in the hypothalamus have been shown to produce immune dysfunctions that persist throughout life. It has been observed that autistic children are frequently deficient in zinc, and zinc is known to play a role in neuroprotection.77,78 Part of the protection arises from the zinc portion of the NMDA receptor, which inhibits receptor activation by glutamate. Zinc is also involved in metallothionein, a protective molecule that increases with brain inflammation and intoxications with heavy metals, especially mercury.79 Under such conditions, zinc levels in the blood are seen to fall. Interestingly, prenatal exposure to caffeine from maternal consumption, induces decreased fetal levels of brain zinc.80 Magnesium levels have also been reported to be low in autistic children. Magnesium plays a major role in neuroprotection, primarily by inhibiting NMDA activation. Magnesium also acts as an antioxidant, with low levels being associated with doubling of free radical generation in both epithelial cells and neurons.81 Low magnesium also lowers cellular glutathione levels and increases excitotoxic neuronal death. Several studies have shown that low magnesium levels dramatically increase excitotoxicity.82 It has been shown that low magnesium plays a major role in encephalopathy associated with deficiency of thiamin and other “B” vitamins.83 In this study, rats made deficient only in thiamine or the other B-vitamins developed mild cytotoxic changes in their pontine tegmentum. Yet, when made hypomagnesmic the lesions were profoundly worsened. Hypomagnesmia has also been shown to inhibit GABA responses as well, which would increase cortical excitability.84 One of the principle cytokines released with microglial activation is tumor necrosis factor-alpha. While under normal conditions TNF-alpha acts as a neuroprotectant, it can also enhance excitotoxicity both by increasing reactive oxygen and nitrogen intermediates and by inhibiting glutamate re-uptake. TNF-alpha has been found to be elevated in several of the neurodegenerative disorders and with EAE.85 Cytokines have been shown to play a major role in neurodevelpment. For example, IL-1ß, IL-6 and TNF-alpha at physiological concentrations can effect the survival of both dopaminergic and sertonergic neurons in the embryo.86 At higher concentrations these cytokines significantly reduce the survival of the dopaminergic neurons, but not the sertonergic neurons. Recently Petitto and co-workers demonstrated, by using IL-2 knockout mice, that IL-2 was essential for the development and regulation of hippocampal neurons involved in spatial memory and learning.87 Likewise, IL-1 has been shown to have tropic functions within the brain.88,89 At higher concentrations, both IL-2 and IL-1ß have been shown to be cytodestructive, primarily by increasing free radical generation and blocking glutamate re-uptake.90 Besides increasing neuronal destruction through immune enhancement of excitotoxicity, viruses can also enhance excitotoxicity by inhibiting mitochondrial enzyme function. The polio virus, for example, has been shown to impair oxidative phosphorylation by inhibiting complex II of the electron transport chain.91 As stated, reductions in mitochondrial function significantly increase excitotoxity. Sytemic cytokines can also have effects on the nervous system, since they may enter by way of the circumventricular organs and through the impaired BBB.92 Cytokines can also interact with endothelial cells triggering the release of neuroactive substances within the brain and by altering the permeability of the blood-brain barrier. Innterleukin-2 has been shown to cause leaking of brain capillaries, leading to cerebral edema in cases of glioma patients treated with this cytokine. ` Cognitive impairments have been attributed to IL-2 and TNF infusions in humans. SPECT scans have demonstrated frontal lobe perfusion defects in these patients, which were suspected to be caused by changes in hypothalamic and/or frontal subcortical function.93 Treatment of patients with a variety of cytokines has demonstrated a two-phase effect, acute and chronic. The chronic phase, occurring after two weeks, is often characterized by psychomotor, cognitive and psychiatric abnormalities. Interferon-alpha infusions, even at low- doses, are also associated with numerous cognitive and psychological effects, including decreased attention span, an inability to concentrate, impaired short-tern memory, and hesitation of speech. Such patients often suddenly stop speaking and stare out into space. On rare occasions patients will progress to dementia. Many of these reactions are reminiscent of autism behavior. One set of symptoms associated with interferon-alpha use, that are also similar to that seen in autism, include uncontrollable overreaction to minor frustration, marked irritability, and a short temper.94 Even months later, such patients may become severely agitated, abusive, and withdrawn. Both interleukin - 1 and 2 infusions are associated with mental changes, including delusions, disorientation and seizures.95,96 There is evidence that IFN-alpha can enhance spontaneous activity in neurons in the cerebral, hippocampal and cerebellar cortices that can last several hours following a single exposure.97 It is not clear if this is a direct effect of interferon or if it is acting through enhanced glutamate release. Most of these clinical studies were on adult patients receiving therapeutic doses of cytokines to treat either viral illnesses or cancer. They demonstrate that peripherally administered cytokines can have a profound effect on CNS function. In the infant, with an immature brain undergoing rapid developmental changes, the neurotoxic effects of the cytokines would be expected to be more profound. Also, because most of the cytokines would be derived from activated microglia within the brain, smaller concentrations would be expected to have a greater effect than systemically administered cytokines. Finally, one problem frequently found in autistic children is an overgrowth of various fungal species, most often Candidia albicans, secondary to either the frequent use of broad-spectrum antibiotics or associated with immune depression. While concern with several of the organic acids released by the yeast organism is legitimate, and have been shown to have a profound effect on neurological function, of equal concern is immune activation of microglia in the brain secondary to systemic Candidia infection, or even infiltration of the brain itself. A recent study has shown that the Candidia organism can penetrate the BBB by budding and developing pseudohyphae inside human microvascular endothelial cells.98 Conclusion Epidemiological studies have shown that from 1960 until 1978, the incidence of autism was fairly stable nationwide, at about 100 to 200 new cases per year. Following the introduction of the MMR vaccine for the widespread inoculation of young children, the incidence of autism increased dramatically, and has continued to increase, with 1944 cases being reported in 1999 alone. In California there has been a 273% increase in severe autism cases over the past eleven years. While purely genetic disorders can explain a small subset of cases, most appear to involve children who are healthy until they receive their vaccination. Several of the vaccines are suspect, especially the MMR, DPT and HepB vaccines. Dr. Benard Rimland has pointed out that before the introduction of MMR vaccine, most autism cases occurred at birth. Yet, after MMR vaccine introduction most new cases were occurring around age 15 months, when the MMR vaccine was usually given. This does not exclude the possibility of pre-existing, genetic related immune defects that are triggered by the immunizations. Today, children are being given 33 doses of 10 types of vaccines before the age of five years. This represents a tremendous antigenic load for an immature immune system to deal with, especially when given so close together. Until recently, children were not only receiving a massive antigenic load but they were also exposed to very high concentrations of mercury. A child receiving all of their vaccinations often received as much as 62.5ug of mercury per visit, 100 times the exposure allowed by the EPA as safe for an infant. The oral polio vaccine and the measles vaccine were found to contain contaminant live viruses, which have been shown to disseminate to other organs, including the nervous system.99 The oral live polio vaccine has been shown to contain numerous pathogenic viruses, including HHV-6 SV-40 and possibly SIV. There is serious concern that stealth viruses may have infected millions of unsuspecting people due to contaminated vaccines. The mechanism by which vaccinations and/or other antigenic loads can precipitate the autistic syndrome is unknown. But we know that immune activation of the brain, especially when intense and prolonged, can precipitate the release of excitotoxins from astrocytes and microglia.100 Excitotoxicity is now known to be a major mechanism of neural destruction in cases of viral infections of the brain. Even without direct viral invasion, as seen in AIDS, immune activation can trigger the release of the excitotoxins quinolinic acid and glutamate. Chronic elevations of glutamate during critical brain growth periods can result in the development of faulty neural pathway circuitry, which can have profound effects on complex higher cortical functions as well as hypothalamic functions. Even transient interference during the period of rapid brain growth, can result in the apoptotic death of millions of developing neurons, and the loss of billions of synaptic connections.101 It should be appreciated that destruction of synaptic connection and dendrites can occur in the absence of neuron death itself, which means that it can occur at much lower levels of glutamate and aspartate, especially when antioxidant levels, cellular energy generation and/or magnesium levels are low.102 Intimately connected with excitotoxicity is free radical generation, including numerous oxygen and nitrogen intermediates. Peroxynitrite, a nitrogen intermediate derived from a union of nitric oxide and superoxide, is especially damaging to the mitochondria, leading to a loss of energy production. Low brain energy levels, no matter the cause, results in a dramatic increase in sensitivity to excitotoxicity. Both glutamate and reactive intermediates can induce microglial activation, leading to a release of inflammatory cytokines, lipid peroxidation products, inhibition of glutamate re-uptake, and eventual apoptosis and necrosis reactions. Glutamate excess has been shown to lead to glutathione depletion secondary to inhibition of cystine entry into the astrocyte (by way of its effects on the cystine transport xc system).103 A recent study indicates that glutathione may not only function as an antioxidant, but may act as a neuromodulator and neurotransmitter as well.104 As a neuromodulator, glutathione has been shown to down-regulate the excitotoxic NMDA receptor, thus blocking excitotoxicity.105 In addition, as stated, clinical seizures occur in approximately one third of autistic children. Excitotoxicity is intimately connected to seizures and explains the neural damage seen when they are prolonged or repeated. Less well appreciated is the fact that chronic seizure foci, even in the absence of clinical seizures, can produce significant neural damage by an excitotoxic mechanism. While the immature brain is less susceptible to neuron death than the mature brain, seizures in the developing brain result in irreversible changes in neuronal connectivity.106 A recent study found that repeated seizures during early life resulted in persistent changes in the CA1 pyramidal neurons in the hippocampus, which is related to observed behavioral changes.107 Mercury exposure is also intimately related to neonatal seizures. A recent study found that maternal exposure to mercury during pregnancy significantly increases epileptogenecity in the offspring.108 This is of special importance in women having dental amalgam, particularly if this amalgam is disturbed during the pregnancy. Of special concern as well is the recent discovery that glutamate, by activating the NMDA receptors on the BBB can disrupt the barrier, leading to free access of blood-born toxins to the CNS.109 In addition, free radicals themselves have been shown to open the BBB.110 Gupta and co-workers have shown that the developing BBB is highly vulnerable to single or repeated exposure of certain pesticides, and that the effect persist even after the offending agent is removed.111 It has been demonstrated that by blocking the NMDA receptor, one can significantly reduce neurovascular dysfunction seen with experimental allergic encephalomyelitis.112 It has been shown that humans develop the highest blood levels of glutamate of all known animals tested following MSG exposure.113 The immature brain is especially vulnerable to food-born excitotoxins, being 4X more sensitive than the adult brain.114 An explanation for this hypersensitivity of the immature brain lies in the observation that during brain development the NMDA receptor is more sensitive to glutamate and less responsive to magnesium protection.115 Food additive excitotoxins are found in virtually all process foods, with very high levels in many junk foods and diet foods.116 These are the types of foods often eaten in large quantities by children, but especially autistic children. With this knowledge of the central role played by excitotoxicity in the autistic syndrome, numerous options will be available for treatment. Many of the diets now being proposed for autistic children emphasize the elimination of foods that are known to be exceedingly high in excitotoxin additives, even though they are being eliminated for other reasons. They are also low in sugar. Autistic children have a high incidence of reactive hypoglycemia, which increases their risk of seizures and excitotoxicity. There is some evidence that Candidia infections may also increase the incidence and severity of hypoglycemia in autistic children.117 Many of the vitamins used to treat autism are antioxidants, which as we have seen, can significantly reduce excitotoxicity, as well as protect against the harmful effects of free radicals. Experimentally, vitamins E can completely abolish glutamate excitotoxicity in vitro. Metabolic stimulants also greatly reduce excitotoxicity. Thiamine, pyridoxine and nicotinamide have been shown to significantly reduce glutamate toxicity in vitro.118 Vitamin B6 can dramatically lower blood and tissue glutamate levels and raise seizure thresholds. In addition, along with folate and vitamin B12, it reduces homocysteine levels. While homocysteine is a marker for deficiencies of methionine metabolism, it is also metabolized into two very powerful excitotoxins, homocysteic acid and homocysteine sulfinic acid. Methylcobalamin is a glutamate receptor blocker as well.119 Pyridoxine’s ability to powerfully inhibit excitotoxity at least partially explains the often dramatic results reported by Bernard Rimland in treating autistic children with high dose pyridoxine/magnesium combinations.120 Magnesium and Zinc also powerfully inhibit excitotoxicity as well as act as co-factors in numerous enzymes systems, including energy generation. Low magnesium is associated with dramatic increases in free radical generation as well as glutathione depletion. High glutamate levels have also been shown to deplete cellular glutathione. Glutathione is vital since it is one of the few antioxidant molecules known to neutralize 4- hydroxynonenal and mercury. In addition, both malate and pyruvate protect against glutamate-mediated excitotoxicity.121 Of great interest is the use of selected flavonoids as antioxidants, anti-inflammatories and antimicrobals. The flavonoids are more powerful and versatile as antioxidants than are the vitamins.122 In addition, flavonoids have been shown to have effects on multiple enzyme systems, including protein kinase C, phospholipase A2, COX and LOX enzymes, iNOS, Na+/K+ ATPase, mitochondrial energy production, as well as cytokine production, all of which may be beneficial in protecting the brain. It should be pointed out that enrichment of the autistic child’s environment is also critical. Saari and co-workers have shown that enriched environments can override some of the problems produced by neonatal exposure to monosodium glutamate.123 Despite the central role played by excitotoxicity, it should be remembered that numerous other mechanism are at play as well, as detailed by William Shaw, Bernard Rimland and others. As a multifaceted disorder, autism requires a multifaceted approach, one that should include protection against excitotoxicity. References Lipton SA, Rosenberg PA. Excitatory amino acids as a final common pathway for neurological disorders. N Eng J Med 330: 613-622, 1994. Olney JW. Brain lesions, obesity, and other disturbances in mice treated with monosodium glutamate. Sci 165: 719-721, 1969. Gasic GP, Heinemann S. Receptors coupled to ionic channels: the glutamate receptor family. Curr Opinion Neurobiol 1: 20-26, 1991. Seal RP, Amara SG. Excitatory amino acid transporters: a family in flux. Ann rev Pharmacol Toxicol 39: 431-456, 1999. 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