Neuropathological Mechanisms of Seizures in Autism Spectrum Disorder.

This manuscript reviews biological abnormalities shared by autism spectrum disorder (ASD) and epilepsy. Two neuropathological findings are shared by ASD and epilepsy: abnormalities in minicolumn architecture and γ-aminobutyric acid (GABA) neurotransmission. The peripheral neuropil, which is the region that contains the inhibition circuits of the minicolumns, has been found to be decreased in the post-mortem ASD brain. ASD and epilepsy are associated with inhibitory GABA neurotransmission abnormalities including reduced GABAA and GABAB subunit expression. These abnormalities can elevate the excitation-to-inhibition balance, resulting in hyperexcitablity of the cortex and, in turn, increase the risk of seizures. Medical abnormalities associated with both epilepsy and ASD are discussed. These include specific genetic syndromes, specific metabolic disorders including disorders of energy metabolism and GABA and glutamate neurotransmission, mineral and vitamin deficiencies, heavy metal exposures and immune dysfunction. Many of these medical abnormalities can result in an elevation of the excitatory-to-inhibitory balance. Fragile X is linked to dysfunction of the mGluR5 receptor and Fragile X, Angelman and Rett syndromes are linked to a reduction in GABAA receptor expression. Defects in energy metabolism can reduce GABA interneuron function. Both pyridoxine dependent seizures and succinic semialdehyde dehydrogenase deficiency cause GABA deficiencies while urea cycle defects and phenylketonuria cause abnormalities in glutamate neurotransmission. Mineral deficiencies can cause glutamate and GABA neurotransmission abnormalities and heavy metals can cause mitochondrial dysfunction which disrupts GABA metabolism. Thus, both ASD and epilepsy are associated with similar abnormalities that may alter the excitatory-to-inhibitory balance of the cortex. These parallels may explain the high prevalence of epilepsy in ASD and the elevated prevalence of ASD features in individuals with epilepsy.

Downregulation of GABAA receptor protein subunits α6, ß2, δ, ε, γ2, θ, and ρ2 in superior frontal cortex of subjects with autism.

We measured protein and mRNA levels for nine gamma-aminobutyric acid A (GABAA) receptor subunits in three brain regions (cerebellum, superior frontal cortex, and parietal cortex) in subjects with autism versus matched controls. We observed changes in mRNA for a number of GABAA and GABAB subunits and overall reduced protein expression for GABAA receptor alpha 6 (GABRα6), GABAA receptor beta 2 (GABRß2), GABAA receptor delta (GABRδ), GABAA receptor epsilon (GABRε), GABAA receptor gamma 2 (GABRγ2), GABAA receptor theta (GABRθ), and GABAA receptor rho 2 (GABRρ2) in superior frontal cortex from subjects with autism. Our data demonstrate systematic changes in GABAA&B subunit expression in brains of subjects with autism, which may help explain the presence of cognitive abnormalities in subjects with autism.

The viral theory of schizophrenia revisited: abnormal placental gene expression and structural changes with lack of evidence for H1N1 viral presence in placentae of infected mice or brains of exposed offspring.

Researchers have long noted an excess of patients with schizophrenia were born during the months of January and March. This winter birth effect has been hypothesized to result either from various causes such as vitamin D deficiency (McGrath, 1999; McGrath et al., 2010), or from maternal infection during pregnancy. Infection with a number of viruses during pregnancy including influenza, and rubella are known to increase the risk of schizophrenia in the offspring (Brown, 2006). Animal models using influenza virus or Poly I:C, a viral mimic, have been able to replicate many of the brain morphological, genetic, and behavioral deficits of schizophrenia (Meyer et al., 2006, 2008a, 2009; Bitanihirwe et al., 2010; Meyer and Feldon, 2010; Short et al., 2010). Using a murine model of prenatal viral infection, our laboratory has shown that viral infection on embryonic days 9, 16, and 18 leads to abnormal expression of brain genes and brain structural abnormalities in the exposed offspring (Fatemi et al., 2005, 2008a,b, 2009a,b). The purpose of the current study was to examine gene expression and morphological changes in the placenta, hippocampus, and prefrontal cortex as a result of viral infection on embryonic day 7 of pregnancy. Pregnant mice were either infected with influenza virus [A/WSN/33 strain (H1N1)] or sham-infected with vehicle solution. At E16, placentas were harvested and prepared for either microarray analysis or for light microscopy. We observed significant, upregulation of 77 genes and significant downregulation of 93 genes in placentas. In brains of exposed offspring following E7 infection, there were changes in gene expression in prefrontal cortex (6 upregulated and 24 downregulated at P0; 5 upregulated and 14 downregulated at P56) and hippocampus (4 upregulated and 6 downregulated at P0; 6 upregulated and 13 downregulated at P56). QRT-PCR verified the direction and magnitude of change for a number of genes associated with hypoxia, inflammation, schizophrenia, and autism. Placentas from infected mice showed a number of morphological abnormalities including presence of thrombi and increased presence of immune cells. Additionally, we searched for presence of H1N1 viral-specific genes for M1/M2, NA, and NS1 in placentas of infected mice and brains of exposed offspring and found none. Our results demonstrate that prenatal viral infection disrupts structure and gene expression of the placenta, hippocampus, and prefrontal cortex potentially explaining deleterious effects in the exposed offspring without evidence for presence of viral RNAs in the target tissues.

Comparative gene expression study of the chronic exposure to clozapine and haloperidol in rat frontal cortex.

Antipsychotic drugs (APDs) are effective in treating some of the positive and negative symptoms of schizophrenia. APDs take time to achieve a therapeutic effect which suggests that changes in gene expression are involved in their efficacy. We hypothesized that there would be altered expression of specific genes associated with the etiology or treatment of schizophrenia in frontal cortex of rats that received chronic treatment with a typical APD (haloperidol) vs. an atypical APD (clozapine). Rats were administered clozapine, haloperidol, or sterile saline intraperitoneally daily for 21days. Frontal cortices from clozapine-, haloperidol-, and saline-treated rats were dissected and subjected to microarray analysis. We observed a significant (1.5 fold, p<0.05) downregulation of 278 genes and upregulation of 73 genes in the clozapine-treated brains vs. controls and downregulation of 451 genes and upregulation of 115 genes in the haloperidol-treated brains vs. control. A total of 146 genes (130 downregulated and 16 upregulated) were significantly altered by both clozapine and haloperidol. These genes were classified by functional groups. qRT-PCR (quantitative real-time polymerase chain reaction) analysis verified the direction and magnitude of change for a group of nine genes significantly altered by clozapine and 11 genes significantly altered by haloperidol. Three genes verified by qRT-PCR were altered by both drugs: Bcl2-like 1 (Bcl2l1), catechol-O-methyltransferase (Comt), and opioid-binding protein/cell adhesion molecule-like (Opcml). Our results show that clozapine and haloperidol cause changes in levels of many important genes that may be involved in etiology and treatment of schizophrenia.

Levels of phosphodiesterase 4A and 4B are altered by chronic treatment with psychotropic medications in rat frontal cortex.

Our laboratory has recently demonstrated altered expression of phosphodiesterase (PDE) 4A and 4B in subjects with autism, bipolar disorder, and schizophrenia, suggesting disrupted cAMP signaling in these diagnostic groups. In the current study, we measured expression of PDEs in rat frontal cortex (FC) following chronic treatment with clozapine, fluoxetine, haloperidol, lithium, olanzapine, valproic acid (VPA), or sterile saline for 21 days. Western blotting experiments showed decreased expression of PDE4A subtypes in FC following treatment with clozapine, haloperidol, lithium, and VPA. PDE4B subtypes were similarly reduced in FC following treatment with clozapine, fluoxetine, and lithium. We also measured levels of nine PDE subtypes via qRT-PCR in FC and found significant upregulation of PDE1A and PDE8B following treatment with olanzapine, while treatment with lithium reduced expression of mRNA for PDE8B. Our results demonstrate altered expression of PDE4A and PDE4B in response to a variety of psychotropic medications suggesting potentially new therapeutic avenues for treatment of neuropsychiatric diseases.

mRNA and protein levels for GABAAalpha4, alpha5, beta1 and GABABR1 receptors are altered in brains from subjects with autism.

We have shown altered expression of gamma-aminobutyric acid A (GABA(A)) and gamma-aminobutyric acid B (GABA(B)) receptors in the brains of subjects with autism. In the current study, we sought to verify our western blotting data for GABBR1 via qRT-PCR and to expand our previous work to measure mRNA and protein levels of 3 GABA(A) subunits previously associated with autism (GABRalpha4; GABRalpha5; GABRbeta1). Three GABA receptor subunits demonstrated mRNA and protein level concordance in superior frontal cortex (GABRalpha4, GABRalpha5, GABRbeta1) and one demonstrated concordance in cerebellum (GABBetaR1). These results provide further evidence of impairment of GABAergic signaling in autism.

The role of lithium in modulation of brain genes: relevance for aetiology and treatment of bipolar disorder.

Bipolar disorder is a debilitating disorder of the brain with a lifetime prevalence of 1.0% for bipolar I, 1.1% for bipolar II disorder and 2.4-4.7% for subthreshold bipolar disorder. Medications, including lithium, have demonstrated efficacy in the treatment of bipolar disorder, but their molecular targets and mode of action are largely unknown. A few studies have begun to shed light on potential targets of lithium treatment that may be involved in lithium's therapeutic effect. We have recently conducted a microarray study of rat frontal cortex following chronic treatment (21 days) with lithium. Chronic treatment with lithium led to a significant (at least 1.5-fold) down-regulation of 151 genes and up-regulation of 57 genes. We discuss our results in the context of previous microarray studies involving lithium and gene-association studies to identify key genes associated with chronic lithium treatment. A number of genes associated with bipolar disorder, including Comt (catechol-O-methyltransferase), Vapa (vesicle-associated membrane protein-associated protein A), Dtnb (dystrobrevin beta) and Pkd1 (polycystic kidney disease 1), were significantly altered in our microarray dataset along with genes associated with synaptic transmission, apoptosis and transport among other functions.

Abnormal expression of myelination genes and alterations in white matter fractional anisotropy following prenatal viral influenza infection at E16 in mice.

Prenatal viral infection has been associated with the development of schizophrenia and autism. Our laboratory has previously shown that viral infection causes deleterious effects on brain structure and function in mouse offspring following late first trimester (E9) and late second trimester (E18) administration of influenza virus. We hypothesized that middle second trimester infection (E16) in mice may lead to a different pattern of brain gene expression and structural defects in the developing offspring. C57BL6 mice were infected on E16 with a sublethal dose of human influenza virus or sham-infected using vehicle solution. Male offspring of the infected mice were collected at P0, P14, P35, and P56, their brains removed and cerebella dissected and flash frozen. Microarray, DTI and MRI scanning, as well as qRT-PCR and SDS-PAGE and western blotting analyses were performed to detect differences in gene expression and brain atrophy. Expression of several genes associated with myelination, including Mbp, Mag, and Plp1 were found to be altered, as were protein levels of Mbp, Mag, and DM20. Brain imaging revealed significant atrophy in cerebellum at P14, reduced fractional anisotropy in white matter of the right internal capsule at P0, and increased fractional anisotropy in white matter in corpus callosum at P14 and right middle cerebellar peduncle at P56. We propose that maternal infection in mouse impacts myelination genes.

Prenatal viral infection of mice at E16 causes changes in gene expression in hippocampi of the offspring.

The hippocampus governs memory formation and emotional regulation, and there is widespread evidence of hippocampal dysfunction in psychiatric disorders, including schizophrenia and autism. There is abundant evidence that prenatal viral infection may play a role in the development of these two disorders. In the current study, we have examined gene expression and structural changes of the hippocampi of exposed neonates following maternal infection at embryonic day (E) 16 (middle second trimester). We observed significant changes in gene expression in the offspring at postnatal day (P) 0 (birth), P14 (childhood), and P56 (adulthood), including a number of candidate genes for autism and schizophrenia. qRT-PCR verified the direction and magnitude of change for 5 of the genes from the microarray data set and revealed mRNA changes for additional genes associated with schizophrenia and autism. MRI revealed a decrease in hippocampal volume at P35 (adolescence). Our results demonstrate altered gene expression and reduced hippocampal volume in the offspring following prenatal viral infection at E16.

Chronic psychotropic drug treatment causes differential expression of Reelin signaling system in frontal cortex of rats.

Disruption of the Reelin and GABAergic signaling systems have been observed in psychiatric disorders including autism, schizophrenia, bipolar disorder, and major depression. Less is known of therapeutic interventions that may help ameliorate the effects of these disruptions. The current study investigated whether chronic administration of psychotropic medications (clozapine, fluoxetine, haloperidol, lithium, olanzapine, and valproic acid) used in the treatment of psychiatric disorders alters levels of Reelin, its receptor Vldlr, downstream molecules Gsk3 beta, Dab-1, and Gad65/67 in rat prefrontal cortex as measured by qRT-PCR and SDS-PAGE and western blotting. qRT-PCR revealed that mRNAs for Reelin, Vldlr, Dab-1, Gsk3 beta, and Gad65 were each significantly altered by at least one of the drugs tested, and in the case of Reelin, Dab-1, and Gsk3 beta, by multiple drugs. To verify our results, we also performed SDS-PAGE and western blotting experiments. Again, several of the protein products for Reelin, Vldlr, Dab-1, Gsk3 beta, Gad65, and Gad67 were also significantly altered by multiple drugs. The present results suggest that the Reelin signaling and GABAergic systems are affected by commonly used psychotropic medications. These changes may help explain the efficacy of these drugs and provide further support for the investigation of the Reelin and GABAergic signaling systems as therapeutic targets for the treatment of neuropsychiatric diseases.

Expression of GABA(B) receptors is altered in brains of subjects with autism.

Autism is a neurodevelopmental disorder that is often comorbid with seizures. Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in brain. GABA(B) receptors play an important role in maintaining excitatory-inhibitory balance in brain and alterations may lead to seizures. We compared levels of GABA(B) receptor subunits GABA(B) receptor 1 (GABBR1) and GABA(B) receptor 2 (GABBR2) in cerebellum, Brodmann's area 9 (BA9), and BA40 of subjects with autism and matched controls. Levels of GABBR1 were significantly decreased in BA9, BA40, and cerebellum, while GABBR2 was significantly reduced in the cerebellum. The presence of seizure disorder did not have a significant impact on the observed reductions in GABA(B) receptor subunit expression. Decreases in GABA(B) receptor subunits may help explain the presence of seizures that are often comorbid with autism, as well as cognitive difficulties prevalent in autism.

GABA(A) receptor downregulation in brains of subjects with autism.

Gamma-aminobutyric acid A (GABA(A)) receptors are ligand-gated ion channels responsible for mediation of fast inhibitory action of GABA in the brain. Preliminary reports have demonstrated altered expression of GABA receptors in the brains of subjects with autism suggesting GABA/glutamate system dysregulation. We investigated the expression of four GABA(A) receptor subunits and observed significant reductions in GABRA1, GABRA2, GABRA3, and GABRB3 in parietal cortex (Brodmann's Area 40 (BA40)), while GABRA1 and GABRB3 were significantly altered in cerebellum, and GABRA1 was significantly altered in superior frontal cortex (BA9). The presence of seizure disorder did not have a significant impact on GABA(A) receptor subunit expression in the three brain areas. Our results demonstrate that GABA(A) receptors are reduced in three brain regions that have previously been implicated in the pathogenesis of autism, suggesting widespread GABAergic dysfunction in the brains of subjects with autism.

Phosphodiesterase-4A expression is reduced in cerebella of patients with bipolar disorder.

OBJECTIVE: The cAMP-specific phosphodiesterase-4 (PDE4) gene family has four members (PDE4 A, B, C, and D) and is the target of several potential therapeutic inhibitors. Recently, PDE4A5 has been shown to bind with disrupted in schizophrenia 1 (DISC1), which has been identified as a risk factor for schizophrenia, bipolar disorder, and major depression. We sought to examine whether PDE4A5 expression was altered in cerebella of patients with schizophrenia, bipolar disorder, and major depression. METHODS: We measured protein levels of PDE4A isoforms in cerebella of patients with schizophrenia, bipolar disorder, and major depression versus matched controls using sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blotting. RESULTS: We observed that specific isoforms of PDE4A were reduced in cerebella of patients with bipolar disorder, whereas there was no change in patients with schizophrenia or major depression. CONCLUSION: Our results are the first to show that PDE4A expression is altered in patients with bipolar disorder and provide potential new therapeutic avenues for treatment of this disorder.

Dopamine and serotonin levels following prenatal viral infection in mouse--implications for psychiatric disorders such as schizophrenia and autism.

Prenatal viral infection has been associated with neurodevelopmental disorders such as schizophrenia and autism. It has previously been demonstrated that viral infection causes deleterious effects on brain structure and function in mouse offspring following late first trimester (E9) and middle-late second trimester (E18) administration of influenza virus. Neurochemical analysis following infection on E18 using this model has revealed significantly altered levels of serotonin, 5-hydroxyindoleacetic acid, and taurine, but not dopamine. In order to monitor these different patterns of monoamine expression in exposed offspring in more detail and to see if there are changes in the dopamine system at another time point, pregnant C57BL6J mice were infected with a sublethal dose of human influenza virus or sham-infected using vehicle solution on E16. Male offspring of the infected mice were collected at P0, P14, and P56, their brains removed and cerebellum dissected and flash frozen. Dopamine and serotonin levels were then measured using HPLC-ED technique. When compared to controls, there was a significant decrease in serotonin levels in the cerebella of offspring of virally exposed mice at P14. No differences in levels of dopamine were observed in exposed and control mice, although there was a significant decrease in dopamine at P14 and P56 when compared to P0. The present study shows that the serotonergic system is disrupted following prenatal viral infection, potentially modelling disruptions that occur in patients with schizophrenia and autism.

Chronic psychotropic drug treatment causes differential expression of connexin 43 and GFAP in frontal cortex of rats.

Astrocytic markers glial fibrillary acidic protein (GFAP) and connexin 43 (CX43) are known to have altered expression in brains of subjects with psychiatric disorders including autism and major depression. The current study investigated whether GFAP and CX43 expressions are affected by several commonly used psychotropic medications (clozapine, fluoxetine, haloperidol, lithium, olanzapine, and valproic acid). Using SDS-PAGE and western blotting technique, we observed that CX43 protein expression in prefrontal cortex was significantly increased following chronic treatment with fluoxetine and clozapine, while it was significantly decreased by haloperidol and lithium. GFAP protein expression was significantly decreased following chronic treatment with clozapine and valproic acid. These results suggest that astroglial markers GFAP and CX43 could be potential targets for therapeutic intervention.

Expression of astrocytic markers aquaporin 4 and connexin 43 is altered in brains of subjects with autism.

Neuroanatomical studies have revealed extensive structural brain abnormalities in subjects with autism. Recently, studies have provided evidence of neuroglial responses and neuroinflammation in autism. The current study investigated whether two astrocytic markers, aquaporin 4 and connexin 43, are altered in brains from subjects with autism. Postmortem brain tissues from Brodmann's Area 40 (BA40, parietal cortex), Brodmann's Area 9 (BA9, superior frontal cortex), and cerebella of subjects with autism and matched controls were subject to SDS-PAGE and western blotting. Connexin 43 expression was increased significantly in BA9. Aquaporin 4 expression was decreased significantly in cerebellum. These data suggest that changes are apparent in markers for abnormal glial-neuronal communication (connexin 43 and aquaporin 4) in brains of subjects with autism.

The role of cerebellar genes in pathology of autism and schizophrenia.

Schizophrenia and autism are neurodevelopmental diseases that have genetic as well as environmental etiologies. Both disorders have been associated with prenatal viral infection. Brain imaging and postmortem studies have found alterations in the structure of the cerebellum as well as changes in gene expression. Our laboratory has developed an animal model using prenatal infection of mice with human influenza virus that has demonstrated changes in behavior, pharmacology, structure, and gene expression in the brains of exposed offspring. In the current communication we describe altered expression of cerebellar genes associated with development of brain disorder in a mouse model for schizophrenia and autism and correlate these changes with those involved in the pathology of these two disorders.

PDE4B polymorphisms and decreased PDE4B expression are associated with schizophrenia.

Schizophrenia has a complex genetic underpinning and variations in a number of candidate genes have been identified that confer risk of developing the disorder. We report in the present studies that several single nucleotide polymorphisms (SNPs) and a two-SNP haplotype in PDE4B are associated with an increased incidence of schizophrenia in two large populations of Caucasian and African American patients. The SNPs in PDE4B associated with schizophrenia occur in intronic sequences in the vicinity of a critical splice junction that gives rise to the expression of PDE4B isoforms with distinct regulation and function. We also observed specific decreases in phosphodiesterase 4B (PDE4B) isoforms in brain tissue obtained postmortem from patients diagnosed with schizophrenia and bipolar disorder. PDE4B metabolically inactivates the second messenger cAMP to regulate intracellular signaling in neurons throughout the brain. Thus, the present observations suggest that dysregulation of intracellular signaling mediated by PDE4B is a significant factor in the cause and expression, respectively, of schizophrenia and bipolar disorder and that targeting PDE4B-regulated signaling pathways may yield new therapies to treat the totality of these disorders.

Maternal infection leads to abnormal gene regulation and brain atrophy in mouse offspring: implications for genesis of neurodevelopmental disorders.

Prenatal viral infection has been associated with development of schizophrenia and autism. Our laboratory has previously shown that viral infection causes deleterious effects on brain structure and function in mouse offspring following late first trimester (E9) administration of influenza virus. We hypothesized that late second trimester infection (E18) in mice may lead to a different pattern of brain gene expression and structural defects in the developing offspring. C57BL6J mice were infected on E18 with a sublethal dose of human influenza virus or sham-infected using vehicle solution. Male offsping of the infected mice were collected at P0, P14, P35 and P56, their brains removed and prefrontal cortex, hippocampus and cerebellum dissected and flash frozen. Microarray, qRT-PCR, DTI and MRI scanning, western blotting and neurochemical analysis were performed to detect differences in gene expression and brain atrophy. Expression of several genes associated with schizophrenia or autism including Sema3a, Trfr2 and Vldlr were found to be altered as were protein levels of Foxp2. E18 infection of C57BL6J mice with a sublethal dose of human influenza virus led to significant gene alterations in frontal, hippocampal and cerebellar cortices of developing mouse progeny. Brain imaging revealed significant atrophy in several brain areas and white matter thinning in corpus callosum. Finally, neurochemical analysis revealed significantly altered levels of serotonin (P14, P35), 5-Hydroxyindoleacetic acid (P14) and taurine (P35). We propose that maternal infection in mouse provides an heuristic animal model for studying the environmental contributions to genesis of schizophrenia and autism, two important examples of neurodevelopmental disorders.