Transcriptomic Analysis of Metastatic Uveal Melanoma and Differences in Male and Female Patients.

BACKGROUND/AIM: Uveal melanoma is an ocular malignancy whose prognosis severely worsens following metastasis. In order to improve the understanding of molecular physiology of metastatic uveal melanoma, we identified genes and pathways implicated in metastatic vs non-metastatic uveal melanoma. PATIENTS AND METHODS: A previously published dataset from Gene Expression Omnibus (GEO) was used to identify differentially expressed genes between metastatic and non-metastatic samples as well as to conduct pathway and perturbagen analyses using Gene Set Enrichment Analysis (GSEA), EnrichR, and iLINCS. RESULTS: In male metastatic uveal melanoma samples, the gene LOC401052 is significantly down-regulated and FHDC1 is significantly up-regulated compared to non-metastatic male samples. In female samples, no significant differently expressed genes were found. Additionally, we identified many significant up-regulated immune response pathways in male metastatic uveal melanoma, including "T cell activation in immune response". In contrast, many top up-regulated female pathways involve iron metabolism, including "heme biosynthetic process". iLINCS perturbagen analysis identified that both male and female samples have similar discordant activity with growth factor receptors, but only female samples have discordant activity with progesterone receptor agonists. CONCLUSION: Our results from analyzing genes, pathways, and perturbagens demonstrate differences in metastatic processes between sexes.

In silico transcriptome screens identify epidermal growth factor receptor inhibitors as therapeutics for noise-induced hearing loss.

Noise-induced hearing loss (NIHL) is a common sensorineural hearing impairment that lacks U.S. Food and Drug Administration-approved drugs. To fill the gap in effective screening models, we used an in silico transcriptome-based drug screening approach, identifying 22 biological pathways and 64 potential small molecule treatments for NIHL. Two of these, afatinib and zorifertinib [epidermal growth factor receptor (EGFR) inhibitors], showed efficacy in zebrafish and mouse models. Further tests with EGFR knockout mice and EGF-morpholino zebrafish confirmed their protective role against NIHL. Molecular studies in mice highlighted EGFR's crucial involvement in NIHL and the protective effect of zorifertinib. When given orally, zorifertinib was found in the perilymph with favorable pharmacokinetics. In addition, zorifertinib combined with AZD5438 (a cyclin-dependent kinase 2 inhibitor) synergistically prevented NIHL in zebrafish. Our results underscore the potential for in silico transcriptome-based drug screening in diseases lacking efficient models and suggest EGFR inhibitors as potential treatments for NIHL, meriting clinical trials.

Adenosine Receptor mRNA Expression in Frontal Cortical Neurons in Schizophrenia.

Schizophrenia is a devastating neuropsychiatric disorder associated with the dysregulation of glutamate and dopamine neurotransmitter systems. The adenosine system is an important neuroregulatory system in the brain that modulates glutamate and dopamine signaling via the ubiquitously expressed adenosine receptors; however, adenosine A1 and A2A receptor (A1R and A2AR) mRNA expression is poorly understood in specific cell subtypes in the frontal cortical brain regions implicated in this disorder. In this study, we assayed A1R and A2AR mRNA expression via qPCR in enriched populations of pyramidal neurons, which were isolated from postmortem anterior cingulate cortex (ACC) tissue from schizophrenia (n = 20) and control (n = 20) subjects using laser microdissection (LMD). A1R expression was significantly increased in female schizophrenia subjects compared to female control subjects (t(13) = -4.008, p = 0.001). A1R expression was also significantly decreased in female control subjects compared to male control subjects, suggesting sex differences in basal A1R expression (t(17) = 2.137, p = 0.047). A significant, positive association was found between dementia severity (clinical dementia rating (CDR) scores) and A2AR mRNA expression (Spearman's r = 0.424, p = 0.009). A2AR mRNA expression was significantly increased in unmedicated schizophrenia subjects, suggesting that A2AR expression may be normalized by chronic antipsychotic treatment (F(1,14) = 9.259, p = 0.009). Together, these results provide novel insights into the neuronal expression of adenosine receptors in the ACC in schizophrenia and suggest that receptor expression changes may be sex-dependent and associated with cognitive decline in these subjects.

Altered purinergic receptor expression in the frontal cortex in schizophrenia.

ATP functions as a neurotransmitter, acting on the ubiquitously expressed family of purinergic P2 receptors. In schizophrenia (SCZ), the pathways that modulate extracellular ATP and its catabolism to adenosine are dysregulated. However, the effects of altered ATP availability on P2 receptor expression in the brain in SCZ have not been assessed. We assayed P2 receptor mRNA and protein expression in the DLPFC and ACC in subjects diagnosed with SCZ and matched, non-psychiatrically ill controls (n = 20-22/group). P2RX7, P2RX4 and male P2RX5 mRNA expression were significantly increased (p < 0.05) in the DLPFC in SCZ. Expression of P2RX7 protein isoform was also significantly increased (p < 0.05) in the DLPFC in SCZ. Significant increases in P2RX4 and male P2RX5 mRNA expression may be associated with antipsychotic medication effects. We found that P2RX4 and P2RX7 mRNA are significantly correlated with the inflammatory marker SERPINA3, and may suggest an association between upregulated P2XR and neuroinflammation in SCZ. These findings lend support for brain-region dependent dysregulation of the purinergic system in SCZ.

Antipsychotics impair regulation of glucose metabolism by central glucose.

Hypothalamic detection of elevated circulating glucose triggers suppression of endogenous glucose production (EGP) to maintain glucose homeostasis. Antipsychotics alleviate symptoms associated with schizophrenia but also increase the risk for impaired glucose metabolism. In the current study, we examined whether two acutely administered antipsychotics from different drug classes, haloperidol (first generation antipsychotic) and olanzapine (second generation antipsychotic), affect the ability of intracerebroventricular (ICV) glucose infusion approximating postprandial levels to suppress EGP. The experimental protocol consisted of a pancreatic euglycemic clamp, followed by kinomic and RNA-seq analyses of hypothalamic samples to determine changes in serine/threonine kinase activity and gene expression, respectively. Both antipsychotics inhibited ICV glucose-mediated increases in glucose infusion rate during the clamp, a measure of whole-body glucose metabolism. Similarly, olanzapine and haloperidol blocked central glucose-induced suppression of EGP. ICV glucose stimulated the vascular endothelial growth factor (VEGF) pathway, phosphatidylinositol 3-kinase (PI3K) pathway, and kinases capable of activating KATP channels in the hypothalamus. These effects were inhibited by both antipsychotics. In conclusion, olanzapine and haloperidol impair central glucose sensing. Although results of hypothalamic analyses in our study do not prove causality, they are novel and provide the basis for a multitude of future studies.

Adenosine, Schizophrenia and Cancer: Does the Purinergic System Offer a Pathway to Treatment?

For over a century, a complex relationship between schizophrenia diagnosis and development of many cancers has been observed. Findings from epidemiological studies are mixed, with reports of increased, reduced, or no difference in cancer incidence in schizophrenia patients. However, as risk factors for cancer, including elevated smoking rates and substance abuse, are commonly associated with this patient population, it is surprising that cancer incidence is not higher. Various factors may account for the proposed reduction in cancer incidence rates including pathophysiological changes associated with disease. Perturbations of the adenosine system are hypothesized to contribute to the neurobiology of schizophrenia. Conversely, hyperfunction of the adenosine system is found in the tumor microenvironment in cancer and targeting the adenosine system therapeutically is a promising area of research in this disease. We outline the current biochemical and pharmacological evidence for hypofunction of the adenosine system in schizophrenia, and the role of increased adenosine metabolism in the tumor microenvironment. In the context of the relatively limited literature on this patient population, we discuss whether hypofunction of this system in schizophrenia, may counteract the immunosuppressive role of adenosine in the tumor microenvironment. We also highlight the importance of studies examining the adenosine system in this subset of patients for the potential insight they may offer into these complex disorders.

Identification of activity-induced Egr3-dependent genes reveals genes associated with DNA damage response and schizophrenia.

Bioinformatics and network studies have identified the immediate early gene transcription factor early growth response 3 (EGR3) as a master regulator of genes differentially expressed in the brains of patients with neuropsychiatric illnesses ranging from schizophrenia and bipolar disorder to Alzheimer's disease. However, few studies have identified and validated Egr3-dependent genes in the mammalian brain. We have previously shown that Egr3 is required for stress-responsive behavior, memory, and hippocampal long-term depression in mice. To identify Egr3-dependent genes that may regulate these processes, we conducted an expression microarray on hippocampi from wildtype (WT) and Egr3-/- mice following electroconvulsive seizure (ECS), a stimulus that induces maximal expression of immediate early genes including Egr3. We identified 69 genes that were differentially expressed between WT and Egr3-/- mice one hour following ECS. Bioinformatic analyses showed that many of these are altered in, or associated with, schizophrenia, including Mef2c and Calb2. Enrichr pathway analysis revealed the GADD45 (growth arrest and DNA-damage-inducible) family (Gadd45b, Gadd45g) as a leading group of differentially expressed genes. Together with differentially expressed genes in the AP-1 transcription factor family genes (Fos, Fosb), and the centromere organization protein Cenpa, these results revealed that Egr3 is required for activity-dependent expression of genes involved in the DNA damage response. Our findings show that EGR3 is critical for the expression of genes that are mis-expressed in schizophrenia and reveal a novel requirement for EGR3 in the expression of genes involved in activity-induced DNA damage response.

mTOR kinase activity disrupts a phosphorylation signaling network in schizophrenia brain.

The AKT-mTOR signaling transduction pathway plays an important role in neurodevelopment and synaptic plasticity. mTOR is a serine/threonine kinase that modulates signals from multiple neurotransmitters and phosphorylates specific proteins to regulate protein synthesis and cytoskeletal organization. There is substantial evidence demonstrating abnormalities in AKT expression and activity in different schizophrenia (SZ) models. However, direct evidence for dysregulated mTOR kinase activity and its consequences on downstream effector proteins in SZ pathophysiology is lacking. Recently, we reported reduced phosphorylation of mTOR at an activating site and abnormal mTOR complex formation in the SZ dorsolateral prefrontal cortex (DLPFC). Here, we expand on our hypothesis of disrupted mTOR signaling in the SZ brain and studied the expression and activity of downstream effector proteins of mTOR complexes and the kinase activity profiles of SZ subjects. We found that S6RP phosphorylation, downstream of mTOR complex I, is reduced, whereas PKCα phosphorylation, downstream of mTOR complex II, is increased in SZ DLPFC. In rats chronically treated with haloperidol, we showed that S6RP phosphorylation is increased in the rat frontal cortex, suggesting a potential novel mechanism of action for antipsychotics. We also demonstrated key differences in kinase signaling networks between SZ and comparison subjects for both males and females using kinome peptide arrays. We further investigated the role of mTOR kinase activity by inhibiting it with rapamycin in postmortem tissue and compared the impact of mTOR inhibition in SZ and comparison subjects using kinome arrays. We found that SZ subjects are globally more sensitive to rapamycin treatment and AMP-activated protein kinase (AMPK) contributes to this differential kinase activity. Together, our findings provide new insights into the role of mTOR as a master regulator of kinase activity in SZ and suggest potential targets for therapeutic intervention.

Corticostriatal dysfunction and social interaction deficits in mice lacking the cystine/glutamate antiporter.

The astrocytic cystine/glutamate antiporter system xc- represents an important source of extracellular glutamate in the central nervous system, with potential impact on excitatory neurotransmission. Yet, its function and importance in brain physiology remain incompletely understood. Employing slice electrophysiology and mice with a genetic deletion of the specific subunit of system xc-, xCT (xCT-/- mice), we uncovered decreased neurotransmission at corticostriatal synapses. This effect was partly mitigated by replenishing extracellular glutamate levels, indicating a defect linked with decreased extracellular glutamate availability. We observed no changes in the morphology of striatal medium spiny neurons, the density of dendritic spines, or the density or ultrastructure of corticostriatal synapses, indicating that the observed functional defects are not due to morphological or structural abnormalities. By combining electron microscopy with glutamate immunogold labeling, we identified decreased intracellular glutamate density in presynaptic terminals, presynaptic mitochondria, and in dendritic spines of xCT-/- mice. A proteomic and kinomic screen of the striatum of xCT-/- mice revealed decreased expression of presynaptic proteins and abnormal kinase network signaling, that may contribute to the observed changes in postsynaptic responses. Finally, these corticostriatal deregulations resulted in a behavioral phenotype suggestive of autism spectrum disorder in the xCT-/- mice; in tests sensitive to corticostriatal functioning we recorded increased repetitive digging behavior and decreased sociability. To conclude, our findings show that system xc- plays a previously unrecognized role in regulating corticostriatal neurotransmission and influences social preference and repetitive behavior.

Kinome Array Profiling of Patient-Derived Pancreatic Ductal Adenocarcinoma Identifies Differentially Active Protein Tyrosine Kinases.

Pancreatic cancer remains one of the most difficult malignancies to treat. Minimal improvements in patient outcomes and persistently abysmal patient survival rates underscore the great need for new treatment strategies. Currently, there is intense interest in therapeutic strategies that target tyrosine protein kinases. Here, we employed kinome arrays and bioinformatic pipelines capable of identifying differentially active protein tyrosine kinases in different patient-derived pancreatic ductal adenocarcinoma (PDAC) cell lines and wild-type pancreatic tissue to investigate the unique kinomic networks of PDAC samples and posit novel target kinases for pancreatic cancer therapy. Consistent with previously described reports, the resultant peptide-based kinome array profiles identified increased protein tyrosine kinase activity in pancreatic cancer for the following kinases: epidermal growth factor receptor (EGFR), fms related receptor tyrosine kinase 4/vascular endothelial growth factor receptor 3 (FLT4/VEGFR-3), insulin receptor (INSR), ephrin receptor A2 (EPHA2), platelet derived growth factor receptor alpha (PDGFRA), SRC proto-oncogene kinase (SRC), and tyrosine kinase non receptor 2 (TNK2). Furthermore, this study identified increased activity for protein tyrosine kinases with limited prior evidence of differential activity in pancreatic cancer. These protein tyrosine kinases include B lymphoid kinase (BLK), Fyn-related kinase (FRK), Lck/Yes-related novel kinase (LYN), FYN proto-oncogene kinase (FYN), lymphocyte cell-specific kinase (LCK), tec protein kinase (TEC), hemopoietic cell kinase (HCK), ABL proto-oncogene 2 kinase (ABL2), discoidin domain receptor 1 kinase (DDR1), and ephrin receptor A8 kinase (EPHA8). Together, these results support the utility of peptide array kinomic analyses in the generation of potential candidate kinases for future pancreatic cancer therapeutic development.

Emerging Kinase Therapeutic Targets in Pancreatic Ductal Adenocarcinoma and Pancreatic Cancer Desmoplasia.

Kinase drug discovery represents an active area of therapeutic research, with previous pharmaceutical success improving patient outcomes across a wide variety of human diseases. In pancreatic ductal adenocarcinoma (PDAC), innovative pharmaceutical strategies such as kinase targeting have been unable to appreciably increase patient survival. This may be due, in part, to unchecked desmoplastic reactions to pancreatic tumors. Desmoplastic stroma enhances tumor development and progression while simultaneously restricting drug delivery to the tumor cells it protects. Emerging evidence indicates that many of the pathologic fibrotic processes directly or indirectly supporting desmoplasia may be driven by targetable protein tyrosine kinases such as Fyn-related kinase (FRK); B lymphoid kinase (BLK); hemopoietic cell kinase (HCK); ABL proto-oncogene 2 kinase (ABL2); discoidin domain receptor 1 kinase (DDR1); Lck/Yes-related novel kinase (LYN); ephrin receptor A8 kinase (EPHA8); FYN proto-oncogene kinase (FYN); lymphocyte cell-specific kinase (LCK); tec protein kinase (TEC). Herein, we review literature related to these kinases and posit signaling networks, mechanisms, and biochemical relationships by which this group may contribute to PDAC tumor growth and desmoplasia.

Adenosine Kinase Expression in the Frontal Cortex in Schizophrenia.

The adenosine hypothesis of schizophrenia posits that reduced availability of the neuromodulator adenosine contributes to dysregulation of dopamine and glutamate transmission and the symptoms associated with schizophrenia. It has been proposed that increased expression of the enzyme adenosine kinase (ADK) may drive hypofunction of the adenosine system. While animal models of ADK overexpression support such a role for altered ADK, the expression of ADK in schizophrenia has yet to be examined. In this study, we assayed ADK gene and protein expression in frontocortical tissue from schizophrenia subjects. In the dorsolateral prefrontal cortex (DLPFC), ADK-long and -short splice variant expression was not significantly altered in schizophrenia compared to controls. There was also no significant difference in ADK splice variant expression in the frontal cortex of rats treated chronically with haloperidol-decanoate, in a study to identify the effect of antipsychotics on ADK gene expression. ADK protein expression was not significantly altered in the DLPFC or anterior cingulate cortex (ACC). There was no significant effect of antipsychotic medication on ADK protein expression in the DLPFC or ACC. Overall, our results suggest that increased ADK expression does not contribute to hypofunction of the adenosine system in schizophrenia and that alternative mechanisms are involved in dysregulation of this system in schizophrenia.

Sex differences in DEK expression in the anterior cingulate cortex and its association with dementia severity in schizophrenia.

DEK is a chromatin-remodeling phosphoprotein found in most human tissues, but its expression and function in the human brain is largely unknown. DEK depletion in vitro induces cellular and molecular anomalies associated with cognitive impairment, including down-regulation of the canonical Wnt/ß-catenin signaling pathway. ToppGene analyses link DEK loss to genes associated with various dementias and age-related cognitive decline. To examine the role of DEK in cognitive impairment in severe mental illness, DEK protein expression was assayed by immunoblot in the anterior cingulate cortex (ACC) of subjects with schizophrenia. Cognitive impairment is a core feature of schizophrenia and cognitive function in subjects was assessed antemortem using the clinical dementia rating (CDR) scale. DEK protein expression was not significantly altered in schizophrenia (n = 20) compared to control subjects (n = 20). Further analysis revealed significant reduction in DEK protein expression in women with schizophrenia, and a significant increase in expression in men with schizophrenia, relative to their same-sex controls. DEK protein expression levels were inversely correlated with dementia severity in women. Conversely, in men, DEK protein expression and dementia severity were positively correlated. Notably, there was no sex difference in DEK protein expression in the control group, suggesting that this sex difference is specific to schizophrenia and not due to inherent differences in DEK expression between males and females. These results suggest a novel, sex-specific role for DEK in cognitive performance and highlight a putative sex-specific link between central nervous system DEK protein expression and a neuropsychiatric disease that is commonly associated with cognitive impairment.

Decreased protein S-palmitoylation in dorsolateral prefrontal cortex in schizophrenia.

Recent reports suggest abnormalities of neurotransmitter receptor trafficking, targeting, dendritic localization, recycling, and degradation in the brain in schizophrenia. We hypothesized that a potential explanation for these findings may be abnormal posttranslational modifications that influence intracellular targeting and trafficking of proteins between subcellular compartments. Dysregulation of protein palmitoylation is a strong candidate for such a process. S-palmitoylation is a reversible thioesterification of palmitoyl-groups to cysteine residues that can regulate trafficking and targeting of intracellular proteins. Using a biotin switch assay to study S-palmitoylation of proteins in human postmortem brain, we identified a pattern of palmitoylated proteins that cluster into 17 bands of discrete molecular masses, including numerous proteins associated with receptor signal transduction. Using mass spectrometry, we identified 219 palmitoylated proteins in human frontal cortex, and individually validated palmitoylation status of a subset of these proteins. Next, we assayed protein palmitoylation in dorsolateral prefrontal cortex from 16 schizophrenia patients and paired comparison subjects. S-palmitoylation was significantly reduced for proteins in most of the 17 schizophrenia bands. In rats chronically treated with haloperidol, the same pattern of palmitoylation was observed but the extent of palmitoylation was unchanged, suggesting that the diminution in protein palmitoylation in schizophrenia is not due to chronic antipsychotic treatment. These results indicate there are changes in the extent of S-palmitoylation of many proteins in the frontal cortex in schizophrenia. Given the roles of this posttranslational modification, these data suggest a potential mechanism reconciling previous observations of abnormal intracellular targeting and trafficking of neurotransmitter receptors in this illness.

Systematic evaluation of data-independent acquisition for sensitive and reproducible proteomics-a prototype design for a single injection assay.

Data-independent acquisition (DIA)-based proteomics has become increasingly complicated in recent years because of the vast number of workflows described, coupled with a lack of studies indicating a rational framework for selecting effective settings to use. To address this issue and provide a resource for the proteomics community, we compared 12 DIA methods that assay tryptic peptides using various mass-isolation windows. Our findings indicate that the most sensitive single injection LC-DIA method uses 6 m/z isolation windows to analyze the densely populated tryptic peptide range from 450 to 730 m/z, which allowed quantification of 4465 Escherichia coli peptides. In contrast, using the sequential windowed acquisition of all theoretical fragment-ions (SWATH) approach with 26 m/z isolation windows across the entire 400-1200 m/z range, allowed quantification of only 3309 peptides. This reduced sensitivity with 26 m/z windows is caused by an increase in co-eluting compounds with similar precursor values detected in the same tandem MS spectra, which lowers the signal-to-noise of peptide fragment-ion chromatograms and reduces the amount of low abundance peptides that can be quantified from 410 to 920 m/z. Above 920 m/z, more peptides were quantified with 26 m/z windows because of substantial peptide (13) C isotope distributions that parse peptide ions into separate isolation windows. Because reproducible quantification has been a long-standing aim of quantitative proteomics, and is a so-called trait of DIA, we sought to determine whether precursor-level chromatograms used in some methods rather than their fragment-level counterparts have similar precision. Our data show that extracted fragment-ion chromatograms are the reason DIA provides superior reproducibility. Copyright © 2015 John Wiley & Sons, Ltd.

Abnormal partitioning of hexokinase 1 suggests disruption of a glutamate transport protein complex in schizophrenia.

Excitatory amino acid transporter 2 (EAAT2) belongs to a family of Na(+) dependent glutamate transporters that maintain a low synaptic concentration of glutamate by removing glutamate from the synaptic cleft into astroglia and neurons. EAAT2 activity depends on Na(+) and K(+) gradients generated by Na(+)/K(+) ATPase and ATP. Hexokinase 1 (HK1), an initial enzyme of glycolysis, binds to mitochondrial outer membrane where it couples cytosolic glycolysis to mitochondrial oxidative phosphorylation, producing ATP utilized by the EAAT2/Na(+)/K(+) ATPase protein complex to facilitate glutamate reuptake. In this study, we hypothesized that the protein complex formed by EAAT2, Na(+)/K(+) ATPase and mitochondrial proteins in human postmortem prefrontal cortex may be disrupted, leading to abnormal glutamate transmission in schizophrenia. We first determined that EAAT2, Na(+)/K(+) ATPase, HK1 and aconitase were found in both EAAT2 and Na(+)/K(+) ATPase interactomes by immunoisolation and mass spectrometry in human postmortem prefrontal cortex. Next, we measured levels of glutamate transport complex proteins in subcellular fractions in the dorsolateral prefrontal cortex and found increases in the EAAT2B isoform of EAAT2 in a fraction containing extrasynaptic membranes and increased aconitase 1 in a mitochondrial fraction. Finally, an increased ratio of HK1 protein in the extrasynaptic membrane/mitochondrial fraction was found in subjects with schizophrenia, suggesting that HK1 protein is abnormally partitioned in this illness. Our findings indicate that the integrity of the glutamate transport protein complex may be disrupted, leading to decreased perisynaptic buffering and reuptake of glutamate, as well as impaired energy metabolism in schizophrenia.

Global signaling effects of a schizophrenia-associated missense mutation in neuregulin 1: an exploratory study using whole genome and novel kinome approaches.

Aberrant neuregulin 1-ErbB4 signaling has been implicated in schizophrenia. We previously identified a novel schizophrenia-associated missense mutation (valine to leucine) in the NRG1 transmembrane domain. This variant inhibits formation of the NRG1 intracellular domain (ICD) and causes decreases in dendrite formation. To assess the global effects of this mutation, we used lymphoblastoid cell lines from unaffected heterozygous carriers (Val/Leu) and non-carriers (Val/Val). Transcriptome data showed 367 genes differentially expressed between the two groups (Val/Val N = 6, Val/Leu N = 5, T test, FDR (1 %), α = 0.05, -log10 p value >1.5). Ingenuity pathway (IPA) analyses showed inflammation and NRG1 signaling as the top pathways altered. Within NRG1 signaling, protein kinase C (PKC)-eta (PRKCH) and non-receptor tyrosine kinase (SRC) were down-regulated in heterozygous carriers. Novel kinome profiling (serine/threonine) was performed after stimulating cells (V/V N = 6, V/L N = 6) with ErbB4, to induce release of the NRG1 ICD, and revealed significant effects of treatment on the phosphorylation of 35 peptides. IPA showed neurite outgrowth (six peptides) as the top annotated function. Phosphorylation of these peptides was significantly decreased in ErbB4-treated Val/Val but not in Val/Leu cells. These results show that perturbing NRG1 ICD formation has major effects on cell signaling, including inflammatory and neurite formation pathways, and may contribute significantly to schizophrenia pathophysiology.

Abnormal activity of the MAPK- and cAMP-associated signaling pathways in frontal cortical areas in postmortem brain in schizophrenia.

Recent evidence suggests that schizophrenia may result from alterations of integration of signaling mediated by multiple neurotransmitter systems. Abnormalities of associated intracellular signaling pathways may contribute to the pathophysiology of schizophrenia. Proteins and phospho-proteins comprising mitogen activated protein kinase (MAPK) and 3'-5'-cyclic adenosine monophosphate (cAMP)-associated signaling pathways may be abnormally expressed in the anterior cingulate (ACC) and dorsolateral prefrontal cortex (DLPFC) in schizophrenia. Using western blot analysis we examined proteins of the MAPK- and cAMP-associated pathways in these two brain regions. Postmortem samples were used from a well-characterized collection of elderly patients with schizophrenia (ACC=36, DLPFC=35) and a comparison (ACC=33, DLPFC=31) group. Near-infrared intensity of IR-dye labeled secondary antisera bound to targeted proteins of the MAPK- and cAMP-associated signaling pathways was measured using LiCor Odyssey imaging system. We found decreased expression of Rap2, JNK1, JNK2, PSD-95, and decreased phosphorylation of JNK1/2 at T183/Y185 and PSD-95 at S295 in the ACC in schizophrenia. In the DLPFC, we found increased expression of Rack1, Fyn, Cdk5, and increased phosphorylation of PSD-95 at S295 and NR2B at Y1336. MAPK- and cAMP-associated molecules constitute ubiquitous intracellular signaling pathways that integrate extracellular stimuli, modify receptor expression and function, and regulate cell survival and neuroplasticity. These data suggest abnormal activity of the MAPK- and cAMP-associated pathways in frontal cortical areas in schizophrenia. These alterations may underlie the hypothesized hypoglutamatergic function in this illness. Together with previous findings, these data suggest that abnormalities of intracellular signaling pathways may contribute to the pathophysiology of schizophrenia.

Expression of four housekeeping proteins in elderly patients with schizophrenia.

We compared protein expression by Western blot analysis in four areas of postmortem brain from patients with schizophrenia and control subjects for several proteins that are often used as controls for Western blot studies: beta-tubulin, actin, glyceraldehyde-3-phosphate dehydrogenase, and valosin-containing protein. We did not detect any differences in expression between subjects with schizophrenia and a comparison group. These results suggest that all four proteins are suitable loading controls for postmortem studies of schizophrenia.

Expression of transcripts for myelination-related genes in the anterior cingulate cortex in schizophrenia.

Several recent studies have found changes in the expression of genes functionally related to myelination and oligodendrocyte homeostasis in schizophrenia. These studies utilized microarrays and quantitative PCR (QPCR), methodologies which do not permit direct, unamplified examination of mRNA expression. In addition, these studies generally only examined transcript expression in homogenates of gray matter. In the present study, we examined the expression of myelination-related genes previously implicated in schizophrenia by microarray or QPCR. Using in situ hybridization, we measured transcript expression of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP), myelin-associated glycoprotein (MAG), transferrin (TF), quaking (QKI), gelsolin, myelin oligodendrocyte glycoprotein, v-erb-b2 erythroblastic leukemia viral oncogene homolog 3, erbb2 interacting protein, motility-related protein-1, SRY-box containing gene 10, oligodendrocyte transcription factor 2, peripheral myelin protein 22, and claudin-11 in both gray and white matter of the anterior cingulate cortex (ACC) in subjects with schizophrenia (n=41) and a comparison group (n=34). We found decreased expression of MAG, QKI, TF, and CNP transcripts in white matter. We did not find any differences in expression of these transcripts between medicated (n=31) and unmedicated (n=10) schizophrenics, suggesting that these changes are not secondary to treatment with antipsychotics. Finally, we found significant positive correlations between QKI and MAG or CNP mRNA expression, suggesting that the transcription factor QKI regulates MAG and CNP expression. Our results support the hypothesis that myelination and oligodendrocyte function are impaired in schizophrenia.