Quantitative proteomics of dorsolateral prefrontal cortex reveals an early pattern of synaptic dysmaturation in children with idiopathic autism.

Autism spectrum disorder (ASD) is a developmental disorder with a rising prevalence and unknown etiology presenting with deficits in cognition and abnormal behavior. We hypothesized that the investigation of the synaptic component of prefrontal cortex may provide proteomic signatures that may identify the biological underpinnings of cognitive deficits in childhood ASD. Subcellular fractions of synaptosomes from prefrontal cortices of age-, brain area-, and postmortem-interval-matched samples from children and adults with idiopathic ASD vs. controls were subjected to HPLC-tandem mass spectrometry. Analysis of data revealed the enrichment of ASD risk genes that participate in slow maturation of the postsynaptic density (PSD) structure and function during early brain development. Proteomic analysis revealed down regulation of PSD-related proteins including AMPA and NMDA receptors, GRM3, DLG4, olfactomedins, Shank1-3, Homer1, CaMK2α, NRXN1, NLGN2, Drebrin1, ARHGAP32, and Dock9 in children with autism (FDR-adjusted P < 0.05). In contrast, PSD-related alterations were less severe or unchanged in adult individuals with ASD. Network analyses revealed glutamate receptor abnormalities. Overall, the proteomic data support the concept that idiopathic autism is a synaptopathy involving PSD-related ASD risk genes. Interruption in evolutionarily conserved slow maturation of the PSD complex in prefrontal cortex may lead to the development of ASD in a susceptible individual.

Quantitative proteomics of forebrain subcellular fractions in fragile X mental retardation 1 knockout mice following acute treatment with 2-Methyl-6-(phenylethynyl)pyridine: Relevance to developmental study of schizophrenia.

The fragile X mental retardation 1 knockout (Fmr1 KO) mouse replicates behavioral deficits associated with autism, fragile X syndrome, and schizophrenia. Less is known whether protein expression changes are consistent with findings in subjects with schizophrenia. In the current study, we used liquid chromatography tandem mass spectrometry (LC-MS/MS) proteomics to determine the protein expression of four subcellular fractions in the forebrains of Fmr1 KO mice vs. C57BL/6 J mice and the effect of a negative allosteric modulator of mGluR5-2-Methyl-6-(phenylethynyl)pyridine (MPEP)-on protein expression. Strain- and treatment-specific differential expression of proteins was observed, many of which have previously been observed in the brains of subjects with schizophrenia. Western blotting verified the direction and magnitude of change for several proteins in different subcellular fractions as follows: neurofilament light protein (NEFL) and 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNP) in the total homogenate; heterogeneous nuclear ribonucleoproteins C1/C2 (HNRNPC) and heterogeneous nuclear ribonucleoprotein D0 (HNRNPD) in the nuclear fraction; excitatory amino acid transporter 2 (EAAT2) and ras-related protein rab 3a (RAB3A) in the synaptic fraction; and ras-related protein rab 35 (RAB35) and neuromodulin (GAP43) in the rough endoplasmic reticulum fraction. Individuals with FXS do not display symptoms of schizophrenia. However, the biomarkers that have been identified suggest that the Fmr1 KO model could potentially be useful in the study of schizophrenia.

The effects of prenatal H1N1 infection at E16 on FMRP, glutamate, GABA, and reelin signaling systems in developing murine cerebellum.

Prenatal viral infection has been identified as a potential risk factor for the development of neurodevelopmental disorders such as schizophrenia and autism. Additionally, dysfunction in gamma-aminobutyric acid, Reelin, and fragile X mental retardation protein (FMRP)-metabotropic glutamate receptor 5 signaling systems has also been demonstrated in these two disorders. In the current report, we have characterized the developmental profiles of selected markers for these systems in cerebella of mice born to pregnant mice infected with human influenza (H1N1) virus on embryonic day 16 or sham-infected controls using SDS-PAGE and Western blotting techniques and evaluated the presence of abnormalities in the above-mentioned markers during brain development. The cerebellum was selected in light of emerging evidence that it plays roles in learning, memory, and emotional processing-all of which are disrupted in autism and schizophrenia. We identified unique patterns of gene and protein expression at birth (postnatal day 0 [P0]), childhood (P14), adolescence (P35), and young adulthood (P56) in both exposed and control mouse progeny. We also identified significant differences in protein expression for FMRP, very-low-density lipoprotein receptor, and glutamic acid decarboxylase 65 and 67 kDa proteins at specific postnatal time points in cerebella of the offspring of exposed mice. Our results provide evidence of disrupted FMRP, glutamatergic, and Reelin signaling in the exposed mouse offspring that explains the multiple brain abnormalities observed in this animal model. © 2016 Wiley Periodicals, Inc.

GABAA and GABAB receptor dysregulation in superior frontal cortex of subjects with schizophrenia and bipolar disorder.

Schizophrenia and bipolar disorder are complex psychiatric disorders that affect millions of people worldwide. Evidence from gene association and postmortem studies has identified abnormalities of the gamma-aminobutyric acid (GABA) signaling system in both disorders. Abnormal GABAergic signaling and transmission could contribute to the symptomatology of these disorders, potentially through impaired gamma oscillations which normally occur during cognitive processing. In the current study, we examined the protein expression of 14 GABAA and two GABAB receptor subunits in the superior frontal cortex of subjects with schizophrenia, bipolar disorder, and healthy controls. Analyses of Variance (ANOVAs) identified significant group effects for protein levels for the α1, α6, ß1, ß3, δ, ɛ, and π GABAA receptor subunits and R1 and R2 GABAB receptor subunits. Follow-up t tests confirmed changes for these subunits in subjects with schizophrenia, subjects with bipolar disorder, or both groups. Alterations in stoichiometry of GABA receptor subunits could result in altered ligand binding, transmission, and pharmacology of GABA receptors in superior frontal cortex. Thus, impaired GABAergic transmission may negatively contribute to symptoms such as anxiety or panic as well as impaired learning and information processing, all of which are disrupted in schizophrenia and bipolar disorder. Taken together, these results provide additional evidence of GABAergic receptor abnormalities in these disorders.

Early Chronic Fluoxetine Treatment of Ts65Dn Mice Rescues Synaptic Vesicular Deficits and Prevents Aberrant Proteomic Alterations.

Down syndrome (DS) is the most common form of inherited intellectual disability caused by trisomy of chromosome 21, presenting with intellectual impairment, craniofacial abnormalities, cardiac defects, and gastrointestinal disorders. The Ts65Dn mouse model replicates many abnormalities of DS. We hypothesized that investigation of the cerebral cortex of fluoxetine-treated trisomic mice may provide proteomic signatures that identify therapeutic targets for DS. Subcellular fractionation of synaptosomes from cerebral cortices of age- and brain-area-matched samples from fluoxetine-treated vs. water-treated trisomic and euploid male mice were subjected to HPLC-tandem mass spectrometry. Analysis of the data revealed enrichment of trisomic risk genes that participate in regulation of synaptic vesicular traffic, pre-synaptic and post-synaptic development, and mitochondrial energy pathways during early brain development. Proteomic analysis of trisomic synaptic fractions revealed significant downregulation of proteins involved in synaptic vesicular traffic, including vesicular endocytosis (CLTA, CLTB, CLTC), synaptic assembly and maturation (EXOC1, EXOC3, EXOC8), anterograde axonal transport (EXOC1), neurotransmitter transport to PSD (SACM1L), endosomal-lysosomal acidification (ROGDI, DMXL2), and synaptic signaling (NRXN1, HIP1, ITSN1, YWHAG). Additionally, trisomic proteomes revealed upregulation of several trafficking proteins, involved in vesicular exocytosis (Rab5B), synapse elimination (UBE3A), scission of endocytosis (DBN1), transport of ER in dendritic spines (MYO5A), presynaptic activity-dependent bulk endocytosis (FMR1), and NMDA receptor activity (GRIN2A). Chronic fluoxetine treatment of Ts65Dn mice rescued synaptic vesicular abnormalities and prevented abnormal proteomic changes in adult Ts65Dn mice, pointing to therapeutic targets for potential treatment of DS.

Consensus paper: pathological role of the cerebellum in autism.

There has been significant advancement in various aspects of scientific knowledge concerning the role of cerebellum in the etiopathogenesis of autism. In the current consensus paper, we will observe the diversity of opinions regarding the involvement of this important site in the pathology of autism. Recent emergent findings in literature related to cerebellar involvement in autism are discussed, including: cerebellar pathology, cerebellar imaging and symptom expression in autism, cerebellar genetics, cerebellar immune function, oxidative stress and mitochondrial dysfunction, GABAergic and glutamatergic systems, cholinergic, dopaminergic, serotonergic, and oxytocin-related changes in autism, motor control and cognitive deficits, cerebellar coordination of movements and cognition, gene-environment interactions, therapeutics in autism, and relevant animal models of autism. Points of consensus include presence of abnormal cerebellar anatomy, abnormal neurotransmitter systems, oxidative stress, cerebellar motor and cognitive deficits, and neuroinflammation in subjects with autism. Undefined areas or areas requiring further investigation include lack of treatment options for core symptoms of autism, vermal hypoplasia, and other vermal abnormalities as a consistent feature of autism, mechanisms underlying cerebellar contributions to cognition, and unknown mechanisms underlying neuroinflammation.

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.

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.

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.

The neurodevelopmental hypothesis of schizophrenia, revisited.

While multiple theories have been put forth regarding the origin of schizophrenia, by far the vast majority of evidence points to the neurodevelopmental model in which developmental insults as early as late first or early second trimester lead to the activation of pathologic neural circuits during adolescence or young adulthood leading to the emergence of positive or negative symptoms. In this report, we examine the evidence from brain pathology (enlargement of the cerebroventricular system, changes in gray and white matters, and abnormal laminar organization), genetics (changes in the normal expression of proteins that are involved in early migration of neurons and glia, cell proliferation, axonal outgrowth, synaptogenesis, and apoptosis), environmental factors (increased frequency of obstetric complications and increased rates of schizophrenic births due to prenatal viral or bacterial infections), and gene-environmental interactions (a disproportionate number of schizophrenia candidate genes are regulated by hypoxia, microdeletions and microduplications, the overrepresentation of pathogen-related genes among schizophrenia candidate genes) in support of the neurodevelopmental model. We relate the neurodevelopmental model to a number of findings about schizophrenia. Finally, we also examine alternate explanations of the origin of schizophrenia including the neurodegenerative model.

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.

Viral regulation of aquaporin 4, connexin 43, microcephalin and nucleolin.

The current study investigated whether human influenza viral infection in midpregnancy leads to alterations in proteins involved in brain development. Human influenza viral infection was administered to E9 pregnant Balb/c mice. Brains of control and virally-exposed littermates were subjected to microarray analysis, SDS-PAGE and western blotting at three postnatal stages. Microarray analysis of virally-exposed mouse brains showed significant, two-fold change in expression of multiple genes in both neocortex and cerebellum when compared to sham-infected controls. Levels of mRNA and protein levels of four selected genes were examined in brains of exposed mice. Nucleolin mRNA was significantly decreased in day 0 and day 35 neocortex and significantly increased in day 35 cerebellum. Protein levels were significantly upregulated at days 35 and 56 in neocortex and at day 56 in cerebellum. Connexin 43 protein levels were significantly decreased at day 56 in neocortex. Aquaporin 4 mRNA was significantly decreased in day 0 neocortex. Aquaporin 4 protein levels decreased in neocortex significantly at day 35. Finally, microcephalin mRNA was significantly decreased in day 56 neocortex and protein levels were significantly decreased at 56 cerebellum. These data suggest that influenza viral infection in midpregnancy in mice leads to long-term changes in brain markers for enhanced ribosome genesis (nucleolin), increased production of immature neurons (microcephalin), and abnormal glial-neuronal communication and neuron migration (connexin 43 and aquaporin 4).

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.

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.

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.

Metabotropic glutamate receptor 5 tracer [18F]-FPEB displays increased binding potential in postcentral gyrus and cerebellum of male individuals with autism: a pilot PET study.

BACKGROUND: Autism is a neurodevelopmental disorder that is first manifested during early childhood. Postmortem experiments have identified significantly elevated expression of metabotropic glutamate receptor 5 (mGluR5) in cerebellar vermis and prefrontal cortex of individuals with autism. METHODS: In the current study we employed the mGluR5 tracer [18F]-3-fluoro-5-[(pyridin-3-yl)ethynyl]benzonitrile ([18F]-FPEB) to quantify mGluR5 binding in vivo in adults with autism vs. healthy controls using positron emission tomography (PET). RESULTS: We identified significantly higher [18F]-FPEB binding potential in the postcentral gyrus and cerebellum of individuals with autism. There was a significant negative correlation between age and [18F]-FPEB binding potential in the cerebellum but not in the postcentral gyrus. In the precuneus, [18F]-FPEB binding potential correlated positively with the lethargy subscale score for the Aberrant Behavioral Checklist (ABC). In cerebellum, there were significant negative correlations between [18F]-FPEB binding potential and ABC total score, ABC hyperactivity subscale score, and the ABC inappropriate speech subscale score. CONCLUSIONS: These novel findings demonstrate for the first time that mGluR5 binding is altered in critical brain areas of subjects with autism, suggesting abnormal glutamate signaling in these regions. Finally, the correlations between altered [18F]-FPEB binding potential in the cerebellum and precuneus suggest that some autistic symptoms may be influenced by abnormal glutamate signaling.

Expression of phosphodiesterase 4 is altered in the brains of subjects with autism.

The cyclic adenosine monophosphate-specific phosphodiesterase-4 (PDE4) gene family is the target of several potential therapeutic inhibitors and the PDE4B gene has been associated with schizophrenia and depression. Little, however, is known of any connection between this gene family and autism, with limited effective treatment being available for autism. We measured the expression of PDE4A and PDE4B by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting in Brodmann's area 40 (BA40, parietal cortex), BA9 (superior frontal cortex), and cerebellum from subjects with autism and matched controls. We observed a lower expression of PDE4A5, PDE4B1, PDE4B3, PDE4B4, and PDE4B2 in the cerebella of subjects with autism when compared with matched controls. In BA9, we observed the opposite: a higher expression of PDE4AX, PDE4A1, and PDE4B2 in subjects with autism. No changes were observed in BA40. Our results demonstrate altered expressions of the PDE4A and PDE4B proteins in the brains of subjects with autism and might provide new therapeutic avenues for the treatment of this debilitating disorder.

Altered subcellular localization of fragile X mental retardation signaling partners and targets in superior frontal cortex of individuals with schizophrenia.

Schizophrenia is a severe, debilitating, neurodevelopmental disorder that affects 1% of the world's population. Recent findings from our laboratory have identified reduced levels of fragile X mental retardation protein (FMRP) and several downstream FMRP targets in superior frontal cortex of individuals with schizophrenia. We hypothesized that altered subcellular expression of FMRP and its signaling partners may explain these changes. In the current study we employed subcellular fractionation and western blotting to determine levels of FMRP, phosphorylated-FMRP as well as selected signaling partners [protein phosphatase 2A catalytic subunit (PP2AC), p70 S6 kinase (p70 S6K), and amyloid-ß A4 precursor protein (APP)] in the total homogenate, nuclear, and rough endoplasmic reticulum fractions in superior frontal cortex of individuals with schizophrenia versus controls (N=12/group). In total homogenate of individuals with schizophrenia, we identified significantly lower levels of FMRP, phosphorylated-FMRP, and PP2AC. In the nuclear fraction of individuals with schizophrenia we found significantly higher levels of PP2AC, p70 S6K, APP 120 kDa, and APP 88 kDa proteins. Finally, in rough endoplasmic reticulum of individuals with schizophrenia, we identified significantly lower protein levels of p70 S6K and APP 120 kDa. These results provide evidence for a potential mechanism to explain altered FMRP expression in schizophrenia.