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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

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Summary of abnormal biomedical findings in autism






Calcium homeostasis in the Central Nervous System – implications for brain development and autism



Mechanisms related to calcium homeostasis that influence neuronal growth, branching, differentiation, maturation, motiliy and structural organisation in developing brain are reviewed in this section, together with their possible role in the etiology of autism.


Various neuropathological and MRI studies have pointed to the following neurological abnormalities in autism:

* significantly elevated calcium levels in autistic brains compared to controls, followed by elevations of mitochondrial aspartate/glutamate carrier rates and mitochondrial metabolism rates (18607376, also see Mitochondrial dysfunction)

* abnormal neuronal migration in both brainstem and cerebellum and disorganised columns of the cerebral cortex.

* significant reduction in granular and Purkinje cell numbers, often accompanied by gliosis (proliferation of astrocytes in damaged areas of the brain).

* marked neuroglial activation and neuroinflammation (see Immunity/Inflammation).

abnormalities of cortical development has been observed in some cases, including areas of increased cortical thickness, high neuronal density, neuronal disorganization and poor differention of neurons.

* abnormalities in brain size and volume, recently linked to increased tissue water content in brain matter (see BBB).

reduced blood flow to parts of the brain (see BBB)

* abnormalities in the size and densitiy of neurons and less dendritic branching, with increased neuron density and shorter connecting fibers, pointing to delays in neuronal maturation.

* autoantibodies to brain proteins, notably to myelin basic protein, neuron-axon filament protein and glial fibrillary acidic protein (see Immunity/Inflammation).

[15546155, 20198484, 9619192, 18435417, 9758336, 15749244, 16819561, 16214373 ,14519452, 16924017]


Intracellular calcium homeostasis is essential for neuronal development and function and calcium influx through voltgate gated calcium channels (VGCC) regulates numerous processes in the central nervous system (CNS), including neuronal growth, differentiation, motility and excitability, secretion of neurotransmitters and hormones, synaptic plasticity, neurotoxicity and neuronal gene expression. Regulation of calcium entry through VGCC is also of major importance in sensory processing and motor function. Because of the critical role of calcium channels in signalling processes, disruption of their function can lead to profound disturbances in the structure and functioning of the nervous system. Elevated levels of intracellular calcium are involved in neurodegenerative mechanisms of the brain tissue and neurological disorders can be caused by mutations in genes encoding calcium channel subunits. There are currently several known human and mouse channelopathies of the CNS, including a recessive retinal disorder, X-linked congenital stationary night blindness, familial hemiplegic migraine, episodic ataxia type 2, and spinocerebellar ataxia. At present there are two known calcium channel genetic mutations directly linked to autism (see Genetic-Factors). Murine recessive neurological disorders as results of mutations in genes encoding calcium channels include the tottering, leaner, and rocker phenotypes with ataxia and absence epilepsy, and the rolling Nagoya phenotype with ataxia without seizures.

Ion entry into neurons occurs either through receptor-operated channels, for example GABA and NMDA channels, or through voltage-gated ion channels. Although it is recognised that calcium entry through both types of channels, as well as calcium relased from internal stores [15709700] may be important in the etiology of psychiatric disorders, this review will mainly focus on the role of voltage gated calcium channels, and L-type calcium channels (LTCC) in particular.

LTCC are localized on nerve terminals in the pre and postsynaptic parts, as well as on cell bodies. Although many cells throughout the body express LTCC, their densitity is higher in the brain, especially during both development and aging. Apart from neurons, calcium entry through LTCC is a major event in many processes in both microglia and astrocytes, the supporting cells of the CNS.

Calcium homeostasis in developing brain

Neuronal cell development is controlled by a tightly organised and regulated sequence of events that include cellular proliferation, differentiation, migration and maturation. Signalling by calcium ions plays a central role in these events.


Age-dependant changes in calcium signalling

VGCC are highly expressed during development and their function is critical for developing neurons. In postnatally developing brain, a transitional period for still developing neurons, there appears to be a critical window in development in which disturbances in calcium homeostasis may have significant consequences [16921238, 10493768].

In vitro neuronal cultures have exibited great differences in sensitivity to changes in levels of intracellular calcium, depending on the exact stage of development. These age-dependent changes in functioning of VGCC have so far been linked to the onset of several developmental events, including neuronal differentiation [7515527], neurite outgrowth and synaptogenesis [7790927, 7965045]. An age-dependent role of LTCC in developmental regulation of transmitter phenotype in neurons has also been demonstrated, whereas the expression of tyrosine hydroxylase (TH), a dopaminergic marker, in developing neurons was shown to be dependent on the activities of LTCC [9437025] (see Neurotransmitters).


Neuronal gene expression

A transcription factor is a protein that acts as a regulator of gene expression. CREB (cAMP response element-binding) proteins are transcription factors which bind to cAMP response elements in DNA and thereby increase or decrease the transcription of certain genes. CREB has been widely studied due to its role in diverse functions such as circadian rhythms, drug addiction and inflammatory pathways. Both CREB and several transcriptional regulators have been linked to epigenetic factors involved in cognitive and behavioural developmental disorders [15721740]. CREB deficient mice for example were shown to exibit less active and exploratory behaviours in novel environments, as well as memory deficits in spatial learning and fear conditioning [15233759, 15805310].

One of the ways in which calcium channels influence neuronal and many other activities is via signaling pathways that control gene expression. This involves regulation of various transcription factors, including CREB. Calcium entry specifically through LTCC is particularly important for transcriptional responses in neurons, muscle, pancreatic beta cells and osteoblasts. Through its stimulation of CREB nuclear calcium may modulate the expression of numerous genes including neurotransmitter receptors and transmembrane and scaffolding proteins, with the involvement of most having been implicated in autism (see Neurotransmitters and Genetic-Factors). LTCC in brain have been implicated in mediating many long term changes in neuronal activity, some having behavioural and cognitive modulation as their end results. This importance of LTCC function in gene expression has been observed in many diverse neurons, including those found in the hippocampus, cortex, striatum, retina, dorsal root ganglia and cerebellum. Early developing Purkinje neurons prior to the stage of dendritic development express a somatic calcium signaling pathway that communicates information from the cell membrane to the cytosol and nucleus [16035195] (see next).

Amongst other effects, opening of LTCC leads to CREB induced expression of brain derived neurotrophic factor (BDNF) and neuronal nitric oxide synthase (nNOS) [11572963, 14604759]. nNOS activity regulates the production of nitric oxide, excessive levels of which can be damaging to neurons, causing oxidative stress and cell death. Calcium channel mutant mice that display similarities with human neurological conditions, including autism, all exhibit varying degrees of cerebellar dysfunction and neuronal cell death, thought to be at least partly due to abnormal nNOS [12834873].

With regards to BDNF, its levels and levels of BDNF autoantibodies are known to be elevated in brains of individuals with autism, with one study observing them to be three times higher than controls, with on the other hand significantly reduced blood levels in adults with autism [16181614, 11431227, 16876305]. Excessive activation of LTCC causes granule cells to express BDNF, the release of which stimulates tyrosine kinase receptors (Trk)B to induce axonal branching, which may establish hyperexcitable dentate circuits implicated in epilepsy [15317847]. Exploring TrkB partial agonists as a possible treatment option for autism has been suggested [16023301]. The mechanism of calcium and BDNF signalling also plays a role in establishing granule cell synaptic transmission, including levels of expression of NMDA receptors, during cerebellar development [16221864].

Apart from BDNF, its neurotrophin family includes the growth factors Nerve Growth Factor (NGF) neurotrophin 3 (NT-3), and neurotrophin 4 (NT-4), some of which were also found to be elevated in autism in several studies [11357950, 16289943]. The same studies observed raised levels of neuropeptide vasoactive intestinal peptide (VIP) compared to controls. The expression level of VIP is influenced by calcium influx through LTCC, possibly through similar mechanisms [15197736]. On the other hand VIP is able to influence VGCC conductance through its known interaction with G-protein-coupled receptors [8772132, 15109935].

In addition, significant elevations of neuropeptide vasopressin (AVP), with concurent reductions in levels of apenin, a neuropetide that could counteract AVP action, have been observed in autism. Again the involvement of raised calcium levels and CREB activities has been suggested in the expression of vasopressin gene [3607454, 9389510].

Possible involvement of Homer and Shank protein complexes in the LTCC activation of CREB has been suggested [15689539, 12716953], as localized calcium responses, regulated by interactions with PDZ domain proteins, are deemed necessary for this activation. It should be mentioned that loss of the SHANK3/PROSAP2 gene has been proposed to be responsible for the main neurological developmental deficits observed in 22q13 deletion syndrome, characterised by delays in speech and motor deveopment [16284256] (see Genetic-Factors). Chromosomal deletions of SHANK3 have recently been identified in a small number of individuals with autism.

With reference to CREB-related activities possibly being relevant in the etiology of autism, it should be added that sex hormone estradiol has been noted to regulate CREB activity via its direct and/or indirect effect on LTCC, and that considerable overlap between behaviors and processes reliant on CREB and those that are influenced by estradiol has been noted [15901789] (see Gender Differences).

A reduced MET gene expression has been implicated in autism susceptibility. A study analysis of the gene encoding the pleiotropic MET receptor tyrosine kinase observed a decrease in the promoter activity of the gene and altered binding of specific transcription factor complexes in autism sample [17053076]. MET signaling plays a role in neuronal growth and maturation as well as in the immune function and gastrointestinal repair, two areas with frequently reported medical complications in autism. The expression of tyrosine kinase receptors is linked to calcium signalling pathways, and sudden changes in the levels of intercellular calcium from both external and internal sources results in changes in levels of MET tyrosine phosphorylation. This regulatory effect of calcium is mediated through calcium-linked proteins and enzymes [1651934, 2111905, 2005882].

It has been suggested that an important subset of developing hippocampal interneurons expressing inhibitory GABA and GAD enzymes express LTCC and that these channels likely regulate the development of these interneurons [16154277, 11085875], as well as the expression levels of GAD and GABA (see Neurotransmitters).

Increasing evidence suggests that the observed down-regulation of Reelin mRNA in neurological disorders may be caused by the dysfunction of epigenetic regulatory pathways in these interneurons [17065238]. Reelin is a protein that is found mainly in the brain and that acts on migrating neuronal precursors and controls correct cell positioning in several areas of the brain. It is secreted by Cajal-Retzius cells and by the external granule cell layer in the cerebellum and its release rate depends solely on its synthesis rate. Abnormalities in the expression levels of Reelin protein and mRNA, as well as those ofr Reln receptor VLDLR in frontal and cerebellar areas of autistic brains versus control subjects have been observed, implicating impaired Reelin signalling in autism [15820235]. Several linkage studies have so far failed to establish a firm genetic basis of this abnormality [15048647, 15048648].

It merits a mention in this context that the level of Reelin can be affected significantly following exposure to x-radiation [10744063]. Whether the effect of x-radiation on calcium channel conductance could be one likely mechanism behind this effect remains to be established [9096258]. Of equal interest is the observation that in rodents prenatal viral infection leads to significant reduction in production of Reelin [10208446] (see Viruses).

In addition to CREB, LTCC activate a number of other transcription factors such as NFAT, MEF-2, and SRF. The nuclear factor of activated T-cells (NFATc) was originally characterized in the immune system, but is now known to play an important role in brain function as well [link].

Another suggested mechanism for this privileged role of calcium channels in epigenetic pathways is the role of the calcium channel-associated transcription regulator (CCAT). CCAT binds to a nuclear protein and in this way regulates the expression of a wide variety of genes involved in neuronal signaling and excitability. The nuclear localization of CCAT is regulated both developmentally and by changes in intracellular calcium. If confirmed, this mechanism would provide a more direct way in which VGCC activate gene transcription in excitable cells [17081980].


Purkinje neurons


Purkinje cells are a class of GABAergic neurons located in the cerebellar cortex. These cells exhibit a highly intricate dendritic arbour, with a large number of dendritic spines. These cells are of central importance for bodily functions of balance and coordination. Cerebellar abiotrophy is a condition affecting some animals in which Purkinje cells begin to atrophy shortly after birth, and this often results in symptoms such as ataxia, intention tremors, hyperreactivity, stiff or high-stepping gait, apparent lack of awareness of where the feet are, and a general inability to determine space and distance. A similar condition known as cerebellar hypoplasia occurs when Purkinje cells either fail to develop or die prenatally.

The proliferation and survival of GABAeric neurons, including Purkinje neurons, in developing brain seems to be dependent on highly regulated calcium influx through VGCC. Increasing or decreasing calcium currents through these channels was observed to have profound effects on survival of cell cultures. This rate of dependence and survival seems to be linked to the exact stage of development (see above) [10366697]. As an illustration, the age-dependent effect of ethanol, a toxic environmental factor, on developing Purkinje neurons is well known, with ethanol being able induce to mitochondrial damage and ultimately cell death [12204202]. These effects are likely due to ethanol-induced changes in VGCC function and altered calcium signalling [16555300](see Mitochondria).

Young and undeveloped Purkinje neurons without dendrites in culture express only the high-threshold calcium current, which increases approximately by half in amplitude during development, thus indicating the importance of calcium conductances in development and maturation of early Purkinje neurons [1377238]. One prominent function of calcium entry through LTCC in these neurons is that this signalling pathway appears to convey developmental cues directly to the nucleus, thus influencing activation of gene transcription factors, CREB in particular (see above), and expression of cellular proteins. This effect can be additionally amplifed by release of calcium from intercellular stores [16035195, 16555300, 11007898].


Neuronal differentiation, growth, branching, migration and structural organisation

Calcium signaling regulates both axonal and dendritic branching in most types of developing neurons, including Purkinje neurons [11248350]. Some of the mechanism of this effect is via abovementioned calcium-dependent activation of CREB, its effects on cytoskeleton and its regulation of the expression levels of neurotransmitters and neurothrophins and activation of protein tyrosine kinases [15581694, 15882639, 8845164]. Calcium regulation of neurite growth and growth cone motility is a process that is dependent on activation G-proteins and is sensitive to pertussis toxin treatment.

Optimum levels of calcium influx promote normal dendritic and axonal elongation and growth cone movements, which are involved in neuronal directional pathfinding and target recognition. These activities are essential for assembly of functional circuits within the developing nervous system and for regeneration following damage. Changes in levels of intercellular calcium and its way of entry into the cells thus can have profound effects on the structure and function of these neuronal networks [3121806]. Calcium transients regulate growth cone advance by direct effects on the growth cone. These transients are mediated primarily by LTCC and silencing them with channel blockers can in some circumstances promote axon outgrowth [12574421]. Several factors that are though to influence neurite outgrowth, for example serotonin and acetylcholinesterase, are suggested to exert that influence through activities of LTCC [120313512376732, 10437116]. Brain serotonin levels play an important role in developing brain and impairments of serotonin metabolism have been implicated in autism. In addition, a recent study has described maternal serotonin levels as being of central importance for developing fetal brain (see Maternal_Factors). In addition, significant preturbations in brain levels of tryptophan and/or serotonin and its receptors have been recorded in some viral infections [8158981, 3509812] (see also Viruses). 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, 11494406, 12401168]. On the other hand, calcium signals and functioning of VGCC play an important role in serotonin metabolism and secretion, and in the regulation of serotonin receptors expression and function (see Neurotransmitters).

It has been suggested that calcium signals through LTCC influence differentiation of neural stem/progenitor cells (NSC). A study looking at NSC derived from the brain cortex of postnatal mice observed that their differentiation is strongly correlated with the expression of LTCC, and that influx of calcium ions through these channels plays a key role in promoting neuronal differentiation [1094458, 16519658].

In addition, calcium transients also play a central role in controlling migration and organisation of both neuronal and non-neuronal cells in the developing CNS [15820385, 16720042, 16029198, 15712206]. In vitro, neurons migrate in association with nonneuronal cells to form cellular aggregates. Changes in those cell complexes in cultured embryonic chick ciliary ganglion were observed in response to treatments that increased or decreased intracellular calcium concentration. Application of thimerosal, a compound that stimulates calcium mobilization from internal stores, increased the amplitude of spontaneous nonneuronal oscillations and the area of migrating nonneuronal cells as well as the velocity of the neuronal-nonneuronal cell complex [16720042].

In mouse ‘weaver’ phenotype the genetic mutation impairs migration of the cerebellar granular neurons and induces neuronal death during the first two weeks of postnatal life. Upregulation of calcium channels was found to contribute to the migration deficiency of these neurons. Loss of these neurons could be attenuated by application of LTCC blockers [8707831].

Increases in intracellular calcium levels via upregulation of VGCC activates calcium-calmodulin-dependent protein kinase (CaMK) and calcineurin phophatase (CaN), which play an important role in development and synaptic organization of granule cells during early postnatal period [16793900].


Neuronal apoptosis

LTCC involvement in neuronal apoptosis (cell death) is probably at least partly due to mitochondrial injury induced by excessive calcium influx and ROS (see Mitochondria and Oxidative Stress). Several LTCC antagonists are able to attenuate cell injury and death in culture, as being induced either by some pathogens, including Amyloid beta protein implicated in etiology of Alzheimer’s disease [15303126, 16321794, 15006551, 10964602] (see Related Disorders).


Synapse formation and synaptic plasticity

It has been hypothesised that autism and related symptoms could in part be a result of disruption of synaptic plasticity in developing brain [15362161]. Synaptic plasticity is the ability of the synapses between two neurons to change in strength. One of the mechanism underlying synaptic plasticity involves regulation of gene transcription and changes in the levels of key proteins at synapses [9437025]. Several recent studies have established a role of LTCC in long-term potentiation (LTP), a long lasting enhancement in efficacy of the synapses between the neurons, thought to be the cellular basis of learning and memory [16251435]. One good example is cpg15, a gene that encodes a membrane-bound ligand that regulates neurite growth and synaptic maturation, and whose expression level is thought to be at least partly influnced through LTCC activation of CREB [14664806]. LTCC are crucially involved in regulation of synapses of auditory inner hair cells, which includes regulation of the expression of potassium channels on those synapses [16828974] (see Motor/Sensory).


Calcium homoestasis in neuroglia

Glial cells are cells that provide support and nutrition to neurons. Astrocytes, the largest and most abundantly expressed glial cells, form connective tissue of the brain and carry out various functions, including induction of neuronal growth and differentiation, participation in maintenance of blood brain barrier and cerebral blood flow, regulation of ion concentration in the extracellular space and modulation of synaptic transmission. Astrocytes accummulate in areas where neurons have been damaged (gliosis). Microglia are macrophages that have immunoprotective role in the brain and play an important role in inflammatory responses.

Changes in intracellular calcium levels are an important signal for communication between glial cells and neurons, and recent evidence points to voltage gated calcium channels as playing an important regulatory role in these processes. For example transient increases in calcium levels in astrocytes either from external sources or from internal stores can result in release of glutamate and modulatation of synaptic transmission in surrounding neurons [1496686712555202]. Furthermore, reactive gliosis as well as glial cell injury and death is thought to be mediated by upregulation of VGCC [9502793, 9736645]. Astroglial release of proteins which enhance neuronal survival and induce neuronal growth and differentiation can be blocked by calcium antagonists and mimicked by Bay K 8644, a calcium channel agonist, indicating the importance of calcium homoestasis in these events [1397177].

For importance of calcium homeostasis in regulation of cerebral blood flow as well as maintenance of blood brain barrier by astrocytes see BBB.

Microglia play an important role in CNS inflammatory responses, and its migratory and secretory responses can be modulated by increases of calcium via LTCC. Several proteins and lipopolysaccharides are know to be able to exert such influence, either directly or through activation of chemokine receptors (see Immune/Inflammation) [10858625, 9914452, 12805281]. Similar mechanism of rises in calcium levels following activation of chemokine receptors has been observed in oligodendrocytes, whose main function is to myelinate axons [16095689]. Stimulation of CXCR4 receptors and subsequent elevation of calcium is in these neuroglial cells is a G protein-linked event [16837851]. It may be of interest in this context that an experimental mouse model of multiple sclerosis was succesfully treated with calcium antagonists bepridil and nitrendipine [15296830]. Furthermore, It has been proposed that oligodendrocytes, alongside astrocytes, play an important role in regulating potassium levels (see Epilepsy/Seizures).






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