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Introduction

Clinical findings in autism and relevance of dysfunctional calcium signalling in
:

    Brain Development
     Neurotransmitters
     Hormones
     Motor/Sensory Disturbances
     Blood Brain Barrier
     Epilepsy/Seizures
     Immunity and Inflammation
     Gastrointestinal Issues
     Membrane Metabolism
     Oxidative Stress
     Mitochondrial Dysfunction
     Gender Differences

Dysregulating Factors:
     Genetic Factors
     Hypoxia/Ischemia
     Toxins
     Infectious Agents
     Other

Conclusion

Links

Contact

 

Summary of abnormal biomedical findings in autism






Oxidative stress in autism



Oxidative stress is defined as an imbalance between pro-oxidants and anti-oxidants, resulting in damage to cell by reactive oxygen species (ROS). Reactive oxygen species include oxygen ions, free radicals and peroxides. They form as a natural byproduct of the normal metabolism of oxygen and have important roles in a number of biological processes, such as the killing of bacteria. During times of environmental stress ROS levels can increase dramatically which can result in significant damage to cell structures, especially in absence of anti-oxidant defences, such as the enzymes superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase or antioxidant vitamins A, C and E and polyphenol antioxidants. Vitamin E plays an important role in cellular defence against lipid peroxidation – a degradation of cell membrane by free radicals.

There is mounting evidence that abnormalities of ROS and nitric oxide (NO) may underlie a wide range of neuropsychiatric disorders. Abnormal methionine metabolism, high levels of homocysteine and oxidative stress are also generally associated with neuropsychiatric disorders. NO signalling has been implicated in a number of physiological functions such as noradrenaline and dopamine releases. It is thought to have neuroprotective effects at low to moderate concentrations, but excessive NO production can cause oxidative stress to neurons thus impairing their function.

Studies comparing the level of homocysteine and other biomarkers in children with autism to controls showed that in children with autism there were highter levels of homocysteine, which was negatively correlated with glutathione peroxidase activity, low human paraoxonase 1 arylesterase activity, suboptimal levels of vitamin B 12 [16297937, 12445495] and increased levels of NO [12691871, 14960298].

Lipid peroxidation was found to be elevated in autism indicating increased oxidative stress. Moderate to dramatic increases in isoprostane levels [16081262, 16908745], decreased levels of phosphatidylethanolamine and increased levels phosphatidylserine [16766163] were observed in children with autism as compared to controls. Levels of major antioxidant proteins transferrin (iron-binding protein) and ceruloplasmin (copper-binding protein) were found to be significantly reduced in sera of autistic children. A strong correlation was observed between reduced levels of these proteins and loss of previously acquired language skills [15363659].

Another study measured levels of metabolites in methionine pathways in autistic children and found that plasma methionine and the ratio of S-adenosylmethionine (SAM) to S-adenosyl-homocysteine (SAH), an indicator of methylation capacity, were significantly decreased in the autistic children relative to controls. In addition, plasma levels of cysteine, glutathione, and the ratio of reduced to oxidized glutathione, indicative of antioxidant capacity and redox homeostasis, were significantly decreased in autistic group. The same study evaluated common polymorphic variants known to modulate these metabolic pathways in 360 autistic children and 205 controls. Differences in allele frequency and/or significant gene-gene interactions were found for relevant genes encoding the reduced folate carrier (RFC 80G), transcobalamin II (TCN2 776G), catechol- O-methyltransferase (COMT 472G), methylenetetrahydrofolate reductase (MTHFR 677C and 1298A), and glutathione-S-transferase (GST M1). The authors propose that an increased vulnerability to oxidative stress may be a contributive factor to the development and clinical manifestations of autism [16917939].

Oxidative damage in autism is also associated with altered expression of brain neurotrophins critical for normal brain growth and differentiation. An increase in 3-nitrotyrosine (3-NT), a marker of oxidative stress damage to proteins in autistic cerebella has been reported. Altered levels of brain NT-3 are likely to contribute to autistic pathology not only by affecting brain axonal targeting and synapse formation but also by further exacerbating oxidative stress and possibly contributing to Purkinje cell abnormalities (19357934).

A study looking into cellular and mitochondrial glutathione redox imbalance in lymphoblastoid cells derived from children with autism found that, compared to controls, autism LCLs exhibit a reduced glutathione reserve capacity in both cytosol and mitochondria that may compromise antioxidant defense and detoxification capacity under prooxidant conditions (19307255).

Several murine studies showed that defective homocysteine remethylation can be caused by deficiency of either methionine synthase enzyme that catalyzes the folate-dependent remethylation of homocysteine to methionine or deficiency of folic acid that produces oxidative stress and endothelial dysfunction in the cerebral microcirculation. [16043641].

In addition to genetic defects such as cystathionine beta-synthase (CBS) or MTHFR, additional factor that can contribute to increasing plasma homocysteine levels is the nutritional status of vitamin B12, vitamin B6, or folate deficiencies.

Folate deficiency induces neurotoxicity by multiple routes – apart from increasing oxidative stress by increasing the levels of homocysteine, it can also contribute to increases in cytosolic calcium and to subsequent mitochondrial and DNA damage (see Mitochondria). Folate deprivation was shown to induce calcium influx initially through the LTCC, and subsequently through NMDA channels and from internal stores [15038821].


Oxidative stress and calcium signalling

Recent data supports the speculation that calcium and reactive oxygen species are two cross-talking messengers in various cellular processe. The results of various studies have shown that calcium is essential for production of ROS. Elevation of intracellular calcium level is responsible for activation of ROS-generating enzymes and formation of free radicals by the mitochondria respiratory chain. It has been shown that the effective macrophage redox defense against Chlamydia pneumoniae depends on LTCC channel activation [12736823]. Hydrogen peroxide, a membrane-permeable form of reactive oxygen species, was shown to enhance invard calcium current in cultured dentate granule cells. This enhancement was cancelled by glutathione, an antioxidant, and nifedipine, an LTCC channel blocker, suggesting that oxidative stress induced by hydrogen peroxide selectively regulates the activity of LTCC [14746893, 9152045]. Nimodipine, another calcium channel blocker, was also shown to suppress ROS formation [10601165, 15820440, 9489715], and verapamil was both protective against oxidative stress and ameliorated morphological changes and dysfunction of mitochondria (see Mitochondria) [16644187].

On the other hand, an increase in intracellular calcium concentration may be stimulated by ROS. Hydrogen peroxide has been recently shown to accelerate the overall channel opening process in voltage-dependent calcium channels in plant and animal cells. In addition to outer membrane calcium channels, IP3 receptors as well as the ryanodine receptors of sarcoplasmic reticulum have also been demonstrated to be redox-regulated [14616077].

The rise in intracellular calcium activates, amongst other things, nitric oxide synthetases, a group of enzymes responsible for the synthesis of nitric oxide. A study looking into mechanisms of NO synthase in the developing rat cortex found that, quote: “… depolarization following GABA-A receptor activation leads to opening of L-type voltage-gated calcium channels, resulting in an increased calcium influx, which in turn leads to phosphorylation and, thus, activation, of the transcription factor CREB; the phosphorylated CREB can then induce BDNF, as well as nNOS " [14604759, 9153595] (see also Brain_Development).

In another study, application of dihydropyridine calcium channel blockers had protective effects against endothelial cell oxidative injury due to combined nitric oxide and superoxide. Nisoldipine, nicardipine and nifedipine all attenuated oxidative-insult induced by loss of reduced glutathione, with nisoldipine demonstrating greatest protection [11820858].

It has been shown that in red blood cells increases in intracellular calcium contentrations lead to a decrease of membrane protein methyl esterification and a subsequent impairment of S-adenosylmethionine synthesis (SAM). After the removal of extra calcium from the cells the levels of methyl esterification returned to normal [3081340].

Excessive lipid peroxidation is implicated in the pathogenesis of neurodegenerative disorders and is brought apon by free radicals action on cell membrane in the absence of inadequate antioxidant defence. Lipid peroxidation has been shown to modulate the activity of VGCC. In one study a prolonged exposure to a lipid-peroxidation enhancer resulted in neuronal death, which was prevented by treatment with glutathione and attenuated by the LTCC blocker nimodipine. It was concluded that the modulation of calcium channel activity in response to lipid peroxidation may play important roles in the responses of neurons to oxidative stress in both physiological and pathological settings [12006588].

Homocysteine is found to overstimulate NMDA receptors, leading to excessive calcium influx and possible neuronal damage [10797837].

Similar to the abovementioned cross talk between ROS and calcium, glutathione, as well as being influenced by calcium, also seems to have a critical role in gating the VGCC. Inhibition of glutathione reductase by carmustine in vitro resulted in depletion of glutathione and oxidative stress, and an influx of extracellular calcium through LTCC. This increase in intracellular calcium was dependent on the presence of extracellular calcium and could be inhibited by calcium blockers nimodipine or nitrendipine. In addition, this effect was also suppressed in cells that were treated with an antioxidant deferoxamine, and enhanced in cells that were pretreated with an inhibitor of glutathione synthesis, buthionine sulfoximine [15321730].

It may be of relevance to note that calcium channels in pancratic islets are very sensitive to levels of glutathione. Membrane thiols are thought to play an important role in insulin sectretion due to their effect on calcium influx via those channels [2424631]. (see Pancreatic function/GI)


Causes of oxidative stress

Apart from poor nutritional status and/or genetic factors, various enviromental agents have also been implicated as causative agents in disturbances in methylation pathways and increased oxidative stress. Oxidative damage due to increased generation of reactive oxygen species and reactive nitrogen species is a feature of many viral infections. The increasing prevalence of HIV-associated cognitive impairment has been the subject of many recent studies, the result of which provide overwhelming evidence for oxidative stress in mediating neuronal injury in patients with HIV induced dementia. These studies also suggest that patients with apolipoprotein E4 allele are more susceptible to neuronal oxidative damage [17034352].

Raised homocysteine levels alongside folate deficiency has been observed in HIV infected children [11737242] and in other viral and mycoplasmal infections, including influenza A and B, human parvovirus, rubella, infectious mononucleosis and Mycoplasma pneumoniae [3033086] [12214730].

One study looking at absorbtion of folate in HIV infected patients has found that absorption of folic acid appears to be significantly impaired in HIV disease, irrespective of the stage of the disease or gastro-intestinal complaints. The authors presented data to support their hypothesis that the virus can cause an enteropathy in the absence of opportunist infection [1680150].

Hepatitis C induced oxidative stress is also widely studied, with numerous studies showing Hepatitis infection causing a state of chronic oxidative stress. These viruses have been associated with changes in mitochondrial structure and function, including increased calcim uptake [16958669]. (see Mitochondria)

Furthermore there seems to be a direct correlation between levels Hepatitis A, B and C viral infections and levels of gluthatione, whereby increased viral activity precedes decreased glutathione levels [11366543, 17036398].

Oxidative injury is a also a component of acute encephalitis caused by herpes simplex virus type 1, reovirus, murine leukemia virus, and subacute sclerosing panencephalitis caused by measles virus [15944946].

Various enviromental toxins have been shown to cause oxidative damage to the cell. Metal-mediated formation of free radicals causes various modifications to DNA bases, enhanced lipid peroxidation, and altered calcium and sulfhydryl homeostasis. For a group of metals that include mercury, cadmium and nickel, one route for their toxicity is depletion of glutathione and bonding to sulfhydryl groups of proteins [15892631] [8512585].

A study looking at the effects of both methylmercury and inorganic mercury on cell oxidative stress and intracellular calcium concentration in rat cerebellar granule neuron cultures tested neuroprotective effects of several agents that selectively interfere with these mechanisms. The results suggested that disruption of redox equilibrium and calcium homeostasis contribute equally to inorganic mercury cell damage, whereas oxidative stress is the main cause of methylmercury neurotoxicity [11599010].

A study looking at functional activities of insulin-like growth factor-1, dopamine-stimulated methionine synthase, and folate-dependent methylation of phospholipids in normal development found that these pathways were interrupted by neurodevelopmental toxins, such as ethanol and heavy metals. Study results raised the possibility that these toxins might exert adverse effects on methylation pathways [14745455]. Reversible alterations of oxidative stress biomarkers resulting from in utero and neonatal exposures of airborne manganese have also been documented [16943606]. (see also Toxic_Agents)

Investigation of cellular mechanisms affected by lowered membrane cholesterol in Smith-Lemli-Opitz syndrome found decreased folate uptake parallel to enhanced calcium permeability (see Membrane).


Additonal issues for consideration:
implications of calcium signalling in oxalate formation and deficiencies of magnesium and B vitamins

Deficiencies of several forms of vitamins B have been shown in vitro and in rodent studies to enhance calcium influx through VGCC. For example it has been observed that depolarization and activation of LTCC occur during experimental thiamine deficiency, and it was proposed that this mechanim may play a role in histological brain lesions/damage induced by thiamine deficiency [9669323].

In the context of reports on beneficial effects of supplementiation of vitamin B12 in autism, as well as simultaneous supplementation of magnesium and vitamin B6 [links], it is of interest that several studies observed that simultaneous deficiencies of magnesium and pyridoxine may in fact act synergistically on impairing the function of LTCC. Deficient rats were found to exibit excessive influx of calcium into the intracellular compartments. Lowering magnesium in resulted in elevation of calcium in cultured canine celebral vascular cells, and this calcium entry could be blocked by exposing the cells to vitamins B6, B12, or folic acid, simultaneously or individually [10553943, 16459799823019].

Of some relevance could be the observation that simulaneous application of magnesium and pyridoxine significantly decreased formation of oxalate in a small-scale human study [7992461]. It is proposed that regulation of calcium fluxes through LTCC by these agents may at least in part underlie their effects in mammals, as it does in plants [link], as application of various calcium channel blockers has been shown to decrease oxalate formation in human and in animal studies. [8322624, 1845698, 10354288]


Conclusion:
Following the above findings it is suggested that folate and cobalamin deficencies observed in neurological disorders may be caused by a combination of genetic polymorphisms as well as impaired absorbtion and disturbances in metabolic pathways due to viral and other infections and enviromental toxic load, mediated in great part through enhanced calcium signalling. The involvement of viral infections and enviromental toxins as causes of disturbed folate metabolism and oxidative stress and their relation to calcium homeostasis may be of relevance to autism and neurological disorders and requires further investigation.






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HIV and Autism