Infectious agents as disruptors of calcium homeostasis and possible implications for autism

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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




Infectious agents as disruptors of calcium homeostasis and possible implications for autism


One proposed etiology for autism is viral infection very early in development. Various viruses and other infectious pathogens have been implicated as etiological factors, including several members of herpes family, rubella, influenza, measles and mumps viruses. In addition, individual cases of prenatal syphilis and toxoplasmosis each have been reported in literature [15804954, 222139, 4947755].

The best association to date have been made between perinatal cytomegalovirus and rubella viruses and autism. Athough detailed large scale follow-up studies have yet to be carried out, numerous case reports describe perinatal cytomegalovirus infection in association with development of autism [12959425, 6315673, 6086566]. It is estimated that up to 15% of asymptomatic congenital infections by CMV in the neonatal period develop persistent problems, which often involve neurological disorders and which typically appear after a period of time, thus making diagnosis difficult or impossible [15973639]. With regards to rubella virus, one follow-up report on psychiatric and behavioral consequences of documented congenital infection recorded significantly high rates of mental retardation and behavioural pathology, including autism [702254].

Also worth noting is the finding that a large subset of subjects with autism shows evidence of bacterial and/or viral infections not present in age-matched controls [17265454], as well as a reduced response to vaccine antigens, especially Bortedella pertussis [link]. In additon, there appears to be a correlation between of virus serology and brain autoantibodies in autism (see Immunity/Inflammation).


Viruses

Animal models in which early viral infection results in behavioral changes later in life include the influenza virus model in pregnant mice and the Borna disease virus model in newborn Lewis rats. Neonatal Borna disease virus (BDV) infection of the rat brain is thought to be a useful model for studying the pathogenesis of neurodevelopmental abnormalities in humans, as it produces neurodevelopmental damage in rodents that is similar to some pathological and clinical features of autism and schizophrenia. Amongst other things neonatal BDV infection profoundly affects social behaviors in adult rats [16860408]. Another significant finding is that the same virus was found to produce differential neuroanatomical and behavioral abnormalities in genetically different inbred rat strains [12106670].

Of note is that neonatal rats infected with BDV do not mount an aggressive response to the virus like their adult counterparts, but instead develop a persistent infection with less obvious clinical symptoms. Postmortem examinations of the rats’ brains reveal significant loss of Purkinje and dentate gyrus granule cells, as well as degeneration of the parts of hippocampus [10321982]. Similarly, selective neuronal damage was observed in BDV replication in hippocampal slice cultures from rats, where damage and death of granular cells was accompanied by reduced density of mossy fiber axons [16140749].

BDV infection in rats is also associated with elevated expression of cytokine and chemokine mRNA, as well as abnormal levels of neurotransmitters, including increased tissue concentration of serotonin in parts of brain [15385249]. Significan abnormalities in brain levels of metallothioneins and zinc metabolism have also been observed, leading to suggestions that such disturbances may contribute to neurodevelopmental damage in perinatal viral infection [16612977] (see below).

Prenatal influenza viral infection in mice leads to development of abnormal emotional and cognitive functions and behaviours, including deficits in social interaction, in adult offspring [12064515, 12514227]. The infection causes differential expression of genes in brains of the infected mice [15906383] as well as atrophy of pyramidal neuorons and increased brain size in adulthood [12064515]. These findings were confirmed by another study, where it was noted that the alterations in gene expression became apparent only after a latency period, an observation which may be of significance in autism [15973158].

Upregulated expression of glial fibrillary acidic protein (GFAP), an important marker of gliosis, neuron migration, and reactive injury, has been recorded [12140787]. Of note is that significant elevations in levels of GFAP, as well as autoantibodies to the protein, have been observed in autism through plasma, cerebrospinal fluid and postmortem brain examinations [16147953, 9308986, 8353169]. In addition, reduced levels of Reelin have been observed in mouse offspring, implicating the involvement of this protein in neuronal migration abnormalities in mouse brains following viral infection [10208446] (see Brain re Reelin abnormalities in autism).

Developmental abnormalities in paediatric Human Immonodeficiency Virus (HIV) infections are of great relevance for establishing mechanisms that lead to autism. Children that are pre- or postnatally infected with the HIV subsequently developed neurological symptoms, including impairments or declines in cognitive, language, socio-emotional and motor development. In some cases regression/deterioration of previously acquired skills can happen several years after the original infection, the longest reported being in a child of 5 years of age, who previously exhibited age-appropriate behaviours and development of all areas of concern [7682171, 7770287, 8935240]. Most importantly, improvements in neurological distrurbances are often noted following antiviral or immune-modulation therapies. These improvements include diminishing of autistic symptoms and recovery, sometimes complete, of language and cognitive abilities, normalised play and social behaviour, and improvements in motor skills, muscle tone and visuo-spatial functions [2493377, 9493492] further reading: [link]

The cental mechanism behind the neurodevelopmental decline and behavioural problems in paediatric HIV infection is thought to be the neuronal injury caused by excessive increases of intracellular calcium induced by HIV viral proteins [16553776, 7847672]. In addition to HIV, numerous other viruses, including many that have been implicated in autism, code for proteins that are capable of inducing similar perturbances in calcium homeostasis in neurons and other types of cells. Calcium plays an important role in replication cycles and pathogenesis of many viral diseases and viruses have been proposed to influence calcium homeostasis of host cells through several different pathways.

An estimated third of the adults and half of the children with acquired immunodeficiency syndrome (AIDS) induced by HIV exibit neurological disturbances. This is usually refered to as AIDS-induced dementia, neuro-AIDS or HIV encephalitis, and the symptoms include language and attention problems, lethargy, motor and sensory dysfunction and abnormal reflexes. Affected children exibit many autistic symptoms, including language and socio-behavioral abnormalities (see above). Some of the pathologies reported in the brain of patients with AIDS are neuronal injury and loss, including myelin loss and axonal damage, and microglial activation.

Dysregulation of calcium homeostasis underlies neurological disturbances in neuro-AIDS. The virus seems to enter the brain in the early stages of infection and arising pathological processes in the brain and CNS are only in part affected by HIV-dependent processes in the periphery. Although the virus does not seem to directly infect the neurons, two of its identified viral proteins are neuroxic - the viral coat protein gp120 and the transcription regulator Tat. Both of these proteins can induce apoptosis of cultured neurons and can render neurons vulnerable to excitotoxicity and oxidative stress. Both gp120 and Tat disrupt neuronal calcium homeostasis by perturbing calcium-regulating systems in the plasma membrane and endoplasmic reticulum. By altering voltage-dependent calcium channels, glutamate receptor channels, and membrane transporters, these proteins promote excessive calcium entry, production of free radicals and mitochondrial dysfunction [12394783] (see Oxidative_Stress and Mitochondrial issues in autism). These effects are at least partly mediated via their effect on chemokine receptors, which are widely expressed on neurons and some of which are able to activate the calcium and cAMP-dependent transcription factor CREB (see Brain) [9826729]. In addition, glial cells were also found to contribute to chemokine upregulation in the CNS and so to contribute to neurological damage [15163738, 11744246]. (see chapter below re chemokine receptors on neurons and glial cells).

Of particular relevance to autism could be the observation of the ability of retroviruses to cause neurological damage in the absence of lympocyte infiltration, primarily through upregulation of chemokine MCP-1 [15163738].

Decreased plasma ratio of tryptophan observed in HIV infected subjects has also been sugested to have possible implications for development of neuro-AIDS. Low levels of tryptophan have been proposed to cause a decrease in serotonin synthesis in the brain, which is believed to impair functioning of some types of neurons [9026369]. Brain serotonin level is suspected to play an important role in developing brain and impairments of serotonin metabolism have been implicated in autism. Reduced brain levels of tryptophan and/or serotonin and its receptors have been recorded in other viral infections. Several in vitro studies have noted the inhibitory effects of serotonin and its receptors on the function of VGCC and neuronal migration during development [11976386] (see also Brain and Maternal_Factors).

Various calcium antagonists, acting on both voltage channels and glutamate operated NMDA receptors, are effective in vitro in reducing neuronal damage induced by HIV proteins. Reductions in glutamate levels also protected neurons from gp120-induced injury, suggesting that they act synergestically and that both are necessary for neuronal cell death [1656845]. Calcium channel blocker nimodipine has shown promise for HIV-induced neurological disorders in a Phase I/II clinical trial [9674806].

In addition to HIV, Maedi Visna virus, another lentil virus, often causes a variety of neurological symptoms in infected sheep. This effect once again is thought to be mediated via neurotoxic effects of its Tat protein and could be attenuated in vitro by application of calcium channel antagonists [8552302].

The effect of HIV proteins on chemokine receptors and modulation of calcium fluxes in immune cells is though to play an important role in HIV-induced immune dysfunction (see Immune/Inflammation). Similar mechanisms are thought to lie behind HIV disturbances of gastrointestinal function (see Gastrointestinal).

Several viruses from the herpes family have been implicated in the etiology of autism (see above). Some of the herpesviruses are neurotropic and are involved in the pathogenesis of neurological symptoms in infants. Cytomegalovirus (CMV) infection is the most frequent congenital infection in humans and can cause permanent damage, often following initial asymptomatic period. The most frequent symptoms are mental retardation, hearing loss, microcephaly as well as hydrocephalus, periventricular calcification, neuromuscular disorders and chorioretinitis or optic atrophy [6159568].

In vitro CMV was found to prevent induction of differentiation of human neural precursor cells into neurons. The virus arrested cell growth and induced apoptosis in infected cell cultures and this effect of CMV was proposed as a possible explanation for the abnormalities in brain development associated with congenital infection [16940505].
A study looking at intracellular calcium responses to CMV infection noted excessive influx of calcium shortly after the infection of cultured human fibroblasts, with substantial involvement of intracellular stores in addition to voltage gated plasma channels. Application of calcium channel blockers inhibited the rise in intracellular calcium levels. Authors of the study suggested that the observed effects of the virus on calcium metabolism may be related not only to the development of CMV pathology, but also to viral replication itself, since in other studies cyclic nucleotide modulators and calcium influx blockers were found to inhibit the replication of CMV [3029971].

CMV-induced rise of intracellular calcium is thought to be behind the ability of this virus to interfere in cellular differentiation pathways, including inhibition of macrophage differentiation, thus contributing to CMV immunosupressive effects [15470031].

Human herpesvirus 6 (HHV-6) encodes proteins that function as highly effective and adaptive chemokine receptor agonists, attracting cells that express those receptors. HHV-6 encoded U83A gene is able to induce significant calcium mobilization in cells expressing chemokine receptors CCR1, CCR4, CCR6, or CCR8. It has been proposed that this newly uncovered mechanism could have wider implications for neuroinflammatory diseases [16332987, 16365449]

(see also Gastrointestinal and Immunity/Inflammation re herpesvirus viral proteins, chemokine receptors and calcium homoestasis in immune cells, )

Significant abnormalities in calcium metabolism of influenza virus-infected cultured cells are brought on by enhancement of permeability of membrane calcium channels and also by mobilizing calcium from intercellular stores [3135328, 6626708]. Similar to CMV, influenza viral replication could be inhibited in vitro by a calcium channel blocker, as well as by a drug which binds to calmodulin and so inhibits calcium/calmodulin intracellular pathways that are necessary for virus assembly [6743023, 6808973].One of the proteins encoded by coxsackie virus, protein 2B, increases membrane permeability and release of calcium stored in endoplasmic reticulum, and in this way disrupts cellular calcium homeostasis, eventually causing membrane lesions that allow release of virus progeny [9218794, 12903773]. Similar effects have been noted following infection of cultured human fibroblasts by poliovirus, another enterovirus from Picornavirus family [7609085]. (see also Immune/Inflammation)

Coxsackievirus B3, a strain capable of inducing myocarditis, has been observed to increase calcium inflow through LTCC in cardiomyocytes, an affect which could be inhibited in vitro by aplication of calcium antagonist taurine [10375733]. Calcium blocker verapamil had similar effects in a murine model of virally-induced inflammation of heart muscle. Pretreatment with the drug before and during the infection, as well as administration of the drug several days after the infection significantly reduced the microvascular changes and myocardial necrosis, fibrosis, and calcification leading to cardiomyopathy [1331179].

Of particular relevance to autism could be the observation concerning the role of autoantibodies to beta adrenoceptors, since these g-protein linked receptors are expressed in the brain as well as the heart and their activation is linked to cAMP and membrane calcium channels. A study looking at the effects of autoantibodies against beta(1)-adrenoceptor in hepatitis virus myocarditis found that they were capable of inducing arrhythmias and/or impairment of heart mucle, an effect that is most likely mediated by LTCC [15069720].

In a similar manner cardiac abnormalities expressed in congenital heart block in newborns born to mothers with autoimmune disease are caused by maternal autoantibody-mediated disturbance of LTCC function [11257091] (see Maternal_Factors).

Mouse hepatitis virus, a murine coronavirus, induced rapid and transient increases in intercellelar calcium via LTCC in a small percentage of cells infected in vitro. It was concluded that the cells that responded with enhanced calcium signals were those that had been infected with multiple viruses and undergone rapid viral replication. Furthermore, several calcium channel blockers and the calcium chelator EGTA inhibited virus infection in this model [9417866]. Several viral proteins Human hepatitis C virus, appear to contribute to reactive oxygen species (ROS) generation by mechanisms that involve dysregulated calcium metabolism, including calcium uptake by mitochondria [16958669] (see also Mitochondria).

Mumps virus, which causes a persistent non-lytic infection, was shown to alter the function of membrane and intercellular calcium channels in cultured neuronal and glial cells in several studies. It has been suggested that this occurence may reflect a disturbed glia-nerve cell interactions [1647243, 1647243]. Although a reduction in the influx of calcium ions during early stages of infection by mumps virus was recorded, neuronal degradation was almost completely inhibited by nifedipine in one study and by dantrolene, a drug which inhibits release of calcium from intracellular stores, in another [1662994, 9372458].

Apart from its better-known immunosupressive role, it is believed that in rare cases measles virus may cause progressive neurodegenerative disease [14527283]. Inhibitory effect of the calcium antagonist, verapamil, on measles viral replication in cultured cells has been observed [8454440]. However another study concluded that this effect was probably not related to verapamil block of extracellular calcium entry. It is suggested that, similar to the abovementioned effects of calcium modulators on influenza virus, this is due to drug interferance in the calcium/calmodulin intracellular pathways that are necessary for virus assembly [16054245]. On the other hand the immunosupressive effect of measles virus likely caused by one of its protein binding to nucleoprotein receptor on cell surface, which triggers sustained calcium influx and inhibits spontaneous cell proliferation [14557619].

Rotavirus tends to effect gastrointestinal epithelial cells, causing acute gastroenteritis and occasionally lactose intolerance (see Gastrointestinal). Growing line of evidence in recent years is pointing to the ability of the virus to leave gastrointestinal tract and cause a broad range of systemic diseases in a number of different organs, including the CNS. Several case studies describe patients who developed seizures and other neurological problems following rotavirus infection. In one particula case diffuse inflammation of the brain was detected by MRI. Neurological symptoms in the patient included screaming fits and seizures, loss of language and social interaction, and reduction in muscle tone. These symptoms persisted in long after the disappearance of gastrointestinal problems [11731961, 12454200].

Animal studies detected replicating rotavirus in infected mice in, amongst other places, macrophages in the lungs and blood vessels, indicating a possible mechanism of its dissemination throughout the body. Interestingly, viral spreading and replication was noted even in the absence of diarrhea. Of note is that in the animals that were infected past a certain stage of development the virus seemed not to infect the brain, as was the case in younger animals. It was concluded that dissemination of rotavirus to the brain in mice is age restricted [16641274].

Rotavirus entry, activation of transcription, morphogenesis, cell lysis, particle release, and the distant action of viral proteins are calcium dependent processes. Viral protein synthesis modifies calcium homeostasis, and rotavirus infection induces increases in intracellular calcium concentration, most likely due to its protein NSP4-induced increases in membrane permeability and calcium influx. Application of LTCC antagonist was shown to reduce the amount of calcium entering the cells, thus suggesting that rotavirus infection of cultured cells activates and/or modulates a specific channel rather than forming a nonspecific ‘leak’ in the plasma membrane [11020376, 9971833].

The observation of the interaction of rotavirus protein NSP4 with membrane protein caveolin-1 during viral infection [16501093] could prove to be of some significance in autism and/or epilepsy, since caveolin-1 is expressed by neuronal cells and seems to exert significant effect on functioning on several ion channels, including VGCC, and on cellular excitability [16040758] (see Membrane_Metabolism). In additon, caveolin-1 seems to play an important role in some inflammatory pathways and in the maintainance of Blood Brain Barrier (BBB) – its loss is proposed to be a critical step in MCP-1-induced modulation of brain microvascular endothelial cells junctional protein expression and integrity [17023578]. Similarly, its downregulation in murine macrophages was shown to increase LPS-induced proinflammatory cytokine TNF-alpha and IL-6 production, but decrease anti-inflammatory cytokine IL-10 production [16357362].


Bacteria and other infectious agents

Bordetella pertussis is a gram-negative bacteria, the causative agent of pertussis (whooping cough). Its toxin interferes with intracellular communication by affecting the functioning of regulatory G-proteins and preventing them from interacting with cell membrane receptors. The close involvement of G-protein activation in regulation of opening times of voltage gated calcium channels has been observed in many types of cells including neurons [8382734, 2560167], insulin-secreting pancreatic betta cells and catecholamine-releasing adrenal chromaffin cells [15488596, 1714959] (see GI, Neurotransmitters and Hormones).

In animal studies the changes that were observed with regards to permeability of BBB and cellular calcium overload were thought to play a central role in the pathogenesis of infectious brain edema induced by injection of pertussis bacilli in rat brain. Treatment with nimodipine was neuroprotective – it dramatically reduced the damage of brain edema by inhibiting the excess of calcium influx and reducing the permeability of BBB (see BBB). Pretreatment of neurons by MK-801, a NMDA channel antagonist was observed to inhibit delayed calcium influx [12659709, 9812737]. Similarly to Borna Disease Virus infection of neonatal mice (see above), the reaction to pertussis toxin/vaccine and occurrence of toxin-induced encephalitis appears to be mouse-strain dependant [6933900]. In addition, it was found that mice that overexpress monocyte chemoattractant protein-1 (MCP-1) at high levels show greater vulnerability to pertuss toxin and that the disruption of CC chemokine receptor 2 (CCR2) abolished both CNS inflammation and encephalopathy in this mouse model, identifying the central role of CCR2 in pertussis-induced encephalopathy [12486156] (see below on role of chemokine receptors in neurological disorders).

A significant association was shown between neurological disorders and pertussis toxin in humans [6786580, 9755273]. Residual levels of active pertussis toxin and endotoxin are likely to be a major contributors to the reactogenicity of whole cell pertussis vaccines, and new guideliness and limits have been established by WHO for active pertussis toxin in acellular pertussis vaccine [link]. In addition, it has also been proposed that permeability changes in the cerebral vessels may be involved in the evolution of the encephalopathy attributed to the use of the vaccine [12780, 12503649].

Various other types of bacteria and their toxins interfere with calcium homeostasis of the host cell. Bacterial lipopolysaccharide (LPS) activation of mouse microglia in vitro leads to elevated basal calcium along with attenuated calcium signaling in response to stimulation, translating into reduced ability of activated microglia to respond to an external stimulus. In addition, the rise in calcium levels underlie characteristic features of microglial activation, such as release of nitric oxide (NO) and several cytokines and chemokines. In the absence of available calcium the LPS-stimulated secretion of cytokines and NO is greatly reduced.

One of the proposed ways of bacteria-induced rises in basal calcium is through formation of toxin-created calcium channels on cell membrane required for bacterial cell invasion, although more recent line of evidence suggests direct modulation of existing LTCC channels and their function [3917612, 6510944, 16732377, 12736823]. Addition of calcium channel blockers, calmodulin antagonists and/or chelation of extracellular calcium significantly reduce bacterial entry into host cells in vitro [12761148, 8382566, 16151220]. This same effect was also noted in invasion of host cells by Toxoplasma gondii [9309395, 15591836], a parasitic agent capable of passing on to humans and causing low grade encaphalitis followed by neurological and behavioural modifications [16000166].

Activation of various chemokine receptors expressed on cell surface by bacterial agents is proposed as another mechanism behind bacteria-induced rises in cell calcium levels (see below). Apart from the actions of their LPS, other bacterial membrane components are also known to act on various chemokine and toll-like receptors and influence their expression in different cell types, with CCR2, CCR5, TLR2 all being implicated [10559223, 14733721, 15885315]. TLR2 for example is essential for the recognition of peptidoglycan and lipoprotein/lipopeptides that are present in cell walls of a variety of micro-organisms, and rodent studies show that mice lacking this receptor do not exibit inflammatory response when exposed to these agents [12697090].

Encaphalitis caused by mycoplasma pneumoniae, a lung-infecting bacteria capable of causing neurological manifestation, is characterised by elevated levels of several cytokines and beta-chemokines in the lungs and the CNS [10441731, 16087054]. Changes in the intracellular calcium levels, most probably mediated through activation of toll-like receptors TLR2 and -6, are though to underlie the systemic inflammation and pathologenesis of mycoplasma genus of bacteria [16154916, 15312143]. In addition to the involvement of G-protein coupled receptors and plasma calcium channels, subsequent release of calcium ions from endoplasmic reticulum has also been observed [11953388].

Lipoprotein found on Treponema pallidum, causative agent of syphilis and implicated as one of the possible causative or contributing agent in autism (see above), induces CCR5 on human monocytes and enhances their susceptibility to infection by HIV [10608777]. Absence of the chemokine receptor CXCR2 results in reduced inflammation induced by Borrelia burgdorferi, another spirochete bacteria and the causative agent in Lyme disease and neuroborreliosis [15760769]. Similarly, infection of nul CXCR2 mice resulted in a significant decrease in susceptibility to development of experimental Lyme arthritis, suggesting that chemokine-mediated recruitment of neutrophils into the infected joint is a key requirement for the development of this condition [12847259].

Prion encelopathies are caracterised by neurotoxicity, neurodegeneration and microglial activation induced by mutated forms of the prion protein and the amyloid beta precursor protein (Abeta), and are thought to be mediated through their effects on calcium conductances through L-type calcium channels [9914452, 14622132, 10817932]. The so called channel hypothesis of Alzheimer's disease proposes that Abeta proteins accumulate in the brain and damage neurons by forming ion channels [12128087]. This hypothesis is further supported by neuroprotective effects shown by some calcium channel blockers in experimental Abeta neurotoxicity [17051460]. In addition, the involvement of presenilin enzymes in Azheimer’s disease is hypothesised to be unrelated to their role in formation of amyloid peptides, but more likely linked to ability of mutated forms of presenilins to directly disturb cellular calcium homeostasis, leading to excessive levels of intracellular calcium, formation of ROS and cellular death [9614221] (see Related_Conditions).



Chemokine receptors and calcium homeostasis in the brain

Chemokines belong to a family of chemotactic cytokines that direct the migration of immune cells towards sites of inflammation. They mediate their biological effects by binding to chemokine receptors on the cell surface. In adition to cytokines and chemokines, various other agents, like viral and bacterial proteins are capable of binding to these receptors and influencing cellular metabolism through this mechanism (see above). Since both chemokines and their receptors as well as various infectious agents have been implicated in the pathophysiology of a number of autoinflammatory diseases, including those involving inflammatory processes in the brain and CNS, chemokine receptor antagonists have often been suggested as potential treatment agents.

Chemokine receptors and their ligands are thought to have regulatory functions in the normal nervous system, where they participate in cell communication and may play a role in the control of cerebellar neuron survival and development. Since many of these receptors are widely expressed by various cells belonging to both the immune and the nervous systems, they have been suggested to serve as a ‘bridge’ between the two. This is further supported by the observed crosstalk between chemokine and neuropeptide receptors, such as opioid, vasoactive intestinal peptide, or adenosine receptors, whereas activation of one often has inhibitory effect on the other [16204635] (see also Related_Channels). In addition, activation and/or inhibition of these G-protein coupled receptors has a direct effect on functioning of membrane calcium channels and cellular calcium homeostasis.

In the CNS, activation of neuronal chemokine receptors by their ligands induces calcium transients and activates calcium cAMP-dependent gene transcription factor CREB (see Brain re its role in neuronal gene expression). Of note is that these effects were in some cases observed to be pertussis toxin-insensitive, indicating possible involvement of pathways that are not mediated by G-proteins [11958818]. Increases in calcium levels have also been observed in cultured embryonic and adult neural progenitor cells and embryonic neurons, with neurospheres grown from young mice exhibiting significantly larger increases in calcium following chemokine treatment than did neurospheres grown from older mice. It was suggested that chemokines may play an important role in control of progenitor cell migration in embryonic and adult brain [15048927, 11567789, 15465598] (see Brain re expression and function of calcium channels during brain development). In addition, the calcium/CREB-mediated upregulation of chemokine receptors mRNA has been observed in T lympocytes, whereby activation of for example CXCR4 by viral proteins leads to increased viral infectivity [11874984, 14563373] (see Immune/Inflammation re proposed involvement of cellular calcium loading in reduced T-cells responsiveness in autism).

Of special significance to developmental disorders could be the possible role played by chemokine receptor CCR2, as its involvement in neuronal communication and in particular cholinergic and dopaminergic neurotransmission has been observed. Its preferential ligand, chemokine MCP-1/CCL2 was shown to induce calcium transients in primary cultured neurons from various rat brain regions including the cortex, hippocampus, hypothalamus, and mesencephalon [16196033]. The near-universal induction of MCP-1 and CCL2 after neurological insult suggests that this chemokine plays a central role in the physiology of neuroinflammation – in animal studies chronic overexpression of MCP-1 in the CNS causes delayed encephalopathy and impaired microglial function in mice, and possible dysfunction of BBB has also been implicated. This experimental pertussis toxin-induced reversible mouse encephalopathy was completely dependant on MCP-1 overexpression, and mice lacking the MCP-1 gene showed absolute resistance [15857890]. Of note is that levels of MCP-1 were found in a postmortem examination to be significantly raised in brains and CSF (12-fold rise) of subject with autism compared to controls (see Immunity/Inflammation).

Similarly, overactivation of other chemokine receptors, as well as toll-like receptors, have been implicated in CNS inflammatory diseases [17151286, 15557205]. The presence of fractaline receptor CX3CR1 signalling has on the other hand recently been proposed as having protective effects against microglial neurotoxicity [16732273]

Apart from neurons, chemokine receptors are also widely expressed on brain glial cells, where their function modulates intracellular calcium signalling and calcium-regulated gene expression [15850656, 16547971, 12112372]. The abovementioned interactions between chemokine and opiod receptors and downstream effects on calcium-mediated pathways are thought to underlie the observed effects of opiod treatment and antagonism on viral infection and outcomes [15893700]. Cultured astrocytes simultaneously treated with opiates and viral protein Tat exibit exaggerated increases in chemokine release, including MCP-1, RANTES and IL-6, an effect that is mediated via the modulation of MAPK and CREB signaling pathways, and that can be prevented by applying a mu-opioid receptor antagonist [15630704, 15893700, 17162664] (see also Related_Receptors).



Genetic polymorphisms as predictors of inflammatory outcomes and chemokine receptors as treatment targets

Several chemokine receptor gene variants have been demonstrated to have detrimental effect on HIV infection and progression to AIDS, and various CCR5 genotypes in children determine both speed of disease progression as well as the degree of neurological impairment [14624371, 9696841].

Similar chemokine receptor-related genetic polymorphisms play a role in vulnerability of gastrointestinal tract to infectious agents, and chemokines and their receptors are thought to be viable therapeutic targets for the treatment of inflammatory bowel disease. A good example is that genetic deficiency in the chemokine receptor CCR1 was shown to protects against acute Clostridium difficile toxin A enteritis in mice - the toxin induced in all mice a significant increase in ileal fluid accumulation, epithelial damage, and neutrophil infiltration, but with all parameters being significantly lower in CCR1 and MIP-1alpha knockout mice [11875005].

Targeting CCR5 and its ligands has been suggested as a possible immune-modulating strategy since it appears to be involved in recruitment of immature MHC class II dendritic cells to the site of inflammation [15790880] (see also Immunity/Inflammation). In the case of HIV infection, various agents capable of modulating expression of multiple chemokine receptors are currently being developed as novel antiviral strategies [12936978, 12936978, 7000001].

The blockade of CCR2, CCR5 and CXCR3 by a novel chemokine antagonist TAK-779 prevents experimental colitis in murine studies by inhibiting the recruitment of inflammatory cells into the mucosa [16000328]. TAK-779 is currently in clinical trial for human use approval and is expected to be used as treatment agent in HIV infection and possibly Inflammatory Bowel Disease (see Gastrointestinal for inflammation and other gastrointestinal issues in autism).



Other issues for consideration

Observations that a virus can act both as an immunosupressor and as a stressor, capable of activating the hypothalamic-pituitary-adrenal axis and increasing brain concentrations of tryptophan and norepinephrine catabolite should be given some consideration in the context of brain development [2756050, 3509812] (see Hormones and Maternal_Serotonin).

A series of murine and studies pointing to links between viral infections and absorbtion and tissue/organ distribution of environmental toxins may be of great relevance. It has been observed for example that the intestinal absorption of cadmium increases during a common viral infection - in the infected animals the absorption of this metal was increased by 70% at low doses and was tripled at high doses compared to non-infected animals. The increased absorption also enhanced the accumulation of cadmium in all studied organs [9630849]. Redistribution of trace elements already present in the body has been noted, with brain levels of mercury increasing twofold during viral infection [16327074]. Mercury was shown to change virally-induced myocarditis in a direction compatible with the development of chronic disease and allow increased persistence of virus, indicating that heavy metals may interact and adversely affect viral replication and development of inflammatory disease. Of note is that in the inflammatory lesions of mice exposed to mercury, the myocardial contents of calcium, manganese, and iron were significantly increased compared to controls [11314973]. The observed changes in the patterns of accumulation, excretion and toxicity of the environmental pollutants during common virus infection has been proposed to be due to down-regulation of detoxifying processes in favour of acute-phase protein synthesis in the host in response to infection [11846173].

With regards to latent versus active viral infection, various factors are known to be capable of reactivating a latent virus, a process which appears to be calcium-dependent and involves elevations of cellular calcium levels [12692270]. An estimated three quarters of diagnosed autism cases seem to fall into category of regressive autism – a sudden loss of previously acquired language and sociobehavoural skills, usually occuring between 18-24 months of age, following a period of normal deveopment. The exact reason for this has been a subject of controversy, with parents and carers reports of regression following series of vaccination contrasted by epidemilogical studies reporting no causal link, leading to recent proposals to study incidence of autism in unvaccinated populations. Of note is that vaccinations have been proposed as capable of triggering reactivation of latent virus infections, via vaccine-induced immunomodulation [11103441]. This mechanism of an induced immune challenge having a trigger-like effect on latent viruses has been suggested to underlie development of CNS autoimmune diseases [11517396]. Pregnancy has been suggested as another immune-related event capable of triggering reactivation of latent viruses. Reactivation of HHV-6 seems common during pregnancy, and transfer of this herpesvirus to the fetus is estimated to occur in approximately 1% of pregnancies [10558965].

On the other hand, latent a viral infection, notably by herpesviruses, has also been shown to alter neuronal gene expression, including genes involed in neurotransmittion and cellular excitability, and latent viruses are therefore proposed to play a possible role in etiology of chronic disease [12915567].

Finally, following recent discoveries of human chromosomal integration of herpesviruses and their sites, the possible role in etiology of viral integration in chromosomal abnormalities and various human pathologies, including autism, should not be completely discounted [15255601, 10477678, 16597897, 16341055, 4285072] (see also Genetic_Factors).
















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