Pornima Phatak

Myelin staining of deep white matter in the dorsolateral prefrontal cortex in schizophrenia, bipolar disorder, and unipolar major depression

Neuroimaging and postmortem studies suggest the involvement of white matter disease in schizophrenia, bipolar disorder, and unipolar major depression. To date there is no published, collective study of myelin staining in these three psychiatric disorders. Deep white matter lesions, potentially affecting corticolimbic circuits, have been particularly implicated in late life depression and poor outcome bipolar disorder. We hypothesized that individuals with these disorders would manifest reduced deep white matter myelin staining compared to normal controls. Sixty transverse sections of fixed dorsolateral prefrontal cortex - 15 from individuals with each psychiatric disorder and 15 from normal controls - were stained according to the method of Kluver and Barrera. Myelin staining intensity was quantified by digital image analysis and expressed as a percent of grey matter staining for a given section. Mean deep (but not gyral) white matter myelin staining was less intense in all three psychiatric groups compared to control. This difference was statistically significant for the bipolar and unipolar groups, with a strong trend toward attenuated staining in the schizophrenic group. Our findings are consistent with postmortem and neuroimaging studies of affective disorders that indicate an increased prevalence of deep white matter lesions in unipolar and bipolar affective disorders.

Elevated Cerebrospinal Fluid Lactate Concentrations in Patients with Bipolar Disorder and Schizophrenia: Implications for the Mitochondrial Dysfunction Hypothesis

BACKGROUND Evidence is accumulating that mitochondrial dysfunction is involved in the pathophysiology of bipolar disorder and schizophrenia. Cerebrospinal fluid (CSF) concentration of lactate, a product of extra-mitochondrial glucose metabolism, is commonly elevated in individuals with mitochondrial disorders, especially those with neuropsychiatric symptoms. We tested the hypothesis that patients with bipolar disorder and schizophrenia would, on average, have elevated CSF lactate concentrations compared with healthy control subjects. METHODS The CSF lactate and CSF and plasma glucose concentrations were measured with a YSI (YSI, Yellow Springs, Ohio) 2300 STAT Plus Glucose & Lactate Analyzer in 15 samples from each of three groups of subjects: bipolar I disorder patients, schizophrenic patients, and healthy control subjects. RESULTS Mean CSF lactate concentrations were significantly higher in bipolar (1.76 +/- .38) and schizophrenic subjects (1.61 +/- .31) compared with control subjects (1.31 +/- .21 mmol/L). These differences persisted after adjusting means for CSF glucose concentration, which correlated positively with CSF lactate concentration. CONCLUSIONS This is the first report of increased CSF lactate concentrations in patients with bipolar disorder and schizophrenia. Elevated CSF lactate indicates increased extra-mitochondrial and anaerobic glucose metabolism and is consistent with impaired mitochondrial metabolism. Measuring CSF lactate concentration might help identify bipolar and schizophrenic patients with mitochondrial dysfunction who might benefit from research to elucidate and ultimately rectify possible mitochondrial pathology underlying these disorders.

Cerebrospinal fluid evidence of increased extra-mitochondrial glucose metabolism implicates mitochondrial dysfunction in multiple sclerosis disease progression

In contrast to relapse, the mechanisms of multiple sclerosis (MS) disease progression are less understood and appear not to be exclusively inflammatory in nature. In this pilot study we investigated the relationship between disturbed CNS energy metabolism and MS disease progression. We tested the hypothesis that cerebrospinal fluid (CSF) concentrations of sorbitol, fructose, and lactate, all metabolites of extra-mitochondrial glucose metabolism, would be elevated in secondary progressive (SP) MS patients and would be associated with worsening neurologic disability. We measured metabolite concentrations by gas chromatographic/mass spectrometric and enzymatic methods in archived CSF samples from 85 MS patients [31 relapsing-remitting (RR) and 54 SP patients] and 18 healthy controls. We found that concentrations of all three metabolites, but not concentrations of glucose or myoinositol, were significantly increased in CSF from SP and, to a lesser degree, RR patients, compared to controls. Furthermore, CSF concentrations of sorbitol and fructose (polyol pathway metabolites), but not lactate (anaerobic glycolysis metabolite), correlated positively and significantly with Expanded Disability Status Scale (EDSS) score, an index of neurologic disability in MS patients. We conclude that extra-mitochondrial glucose metabolism is increased in MS patients and is associated with disease progression evidenced by increasing EDSS score. As extra-mitochondrial glucose metabolism increases with impaired mitochondrial metabolism of glucose, these findings implicate mitochondrial dysfunction in the pathogenesis of MS disease progression. CSF metabolic profiling may be useful in clarifying the role of mitochondrial pathology in progression and in targeting and monitoring therapies for disease progression that aim to preserve or boost mitochondrial glucose metabolism.

Relationship of cerebrospinal fluid glucose metabolites to MRI deep white matter hyperintensities and treatment resistance in bipolar disorder patients

OBJECTIVES Both diabetes mellitus and magnetic resonance image (MRI) deep white matter hyperintensities (WMHs) are more common in bipolar disorder (BD) patients than in matched controls. Deep-as opposed to periventricular--WMHs and diabetes are associated with treatment resistance and poorer outcome. This study investigated whether brain glucose metabolism by the polyol pathway--a pathway linked to nervous tissue disease in diabetes--is related to deep WMH volume and treatment resistance in BD patients. METHODS Volumes of fluid-attenuated inversion recovery WMHs were quantified and correlated with cerebrospinal fluid (CSF) concentrations of glucose metabolites in 20 nondiabetic patients with BD and nondiabetic comparison subjects with schizophrenia (n = 15) or transient neurologic symptoms (neurologic controls, n = 15). RESULTS BD patients, but not schizophrenic patients, had significantly greater volumes of deep but not periventricular WMHs compared to neurologic controls. BD subjects also had significantly greater CSF concentrations of sorbitol and fructose (the polyol pathway metabolites of glucose) compared to controls. Significant positive correlations between CSF metabolites and WMH volumes were found only in the BD group and were between deep WMH volumes and CSF sorbitol (rho = 0.487, p = 0.029) and fructose (rho = 0.474, p = 0.035). An index of treatment resistance correlated significantly with deep WMH volume (rho = 0.578, p = 0.008), sorbitol (rho = 0.542, p = 0.013), and fructose (rho = 0.692, p = 0.001) in BD subjects but not in other subjects. CONCLUSIONS This is the first reported evidence of relationships between abnormal brain glucose metabolism and both deep WMHs and treatment resistance in a group of BD patients. Further studies are necessary to determine the significance of these findings to BD pathophysiology.

Overexpression of miR-214-3p in esophageal squamous cancer cells enhances sensitivity to cisplatin by targeting survivin directly and indirectly through CUG-BP1.

Based on its marked overexpression in multiple malignancies and its roles in promoting cell survival and proliferation, survivin is an attractive candidate for targeted therapy. Toward this end, a detailed understanding of the mechanisms regulating survivin expression in different cancer cells will be critical. We have previously shown that the RNA-binding protein (RBP) CUG-BP1 is overexpressed in esophageal cancer cells and post-transcriptionally regulates survivin in these cells. The objective of this study was to investigate the role of microRNAs (miRs) in regulating survivin expression in esophageal cancer cells. Using miR expression profiling analysis, we found that miR-214-3p is one of the most markedly downregulated miRs in two esophageal squamous cancer cell lines compared with esophageal epithelial cells. Interestingly, using miR target prediction programs, both survivin and CUG-BP1 mRNA were found to contain potential binding sites for miR-214-3p. Forced expression of miR-214-3p in esophageal cancer cells leads to a decrease in the mRNA and protein levels of both survivin and CUG-BP1. This effect is due to decreased mRNA stability of both targets. By contrast, silencing miR-214-3p in esophageal epithelial cells leads to an increase in both survivin and CUG-BP1 mRNA and protein. To determine whether the observed effect of miR-214-3p on survivin expression was direct, mediated through CUG-BP1, or both, binding studies utilizing biotin pull-down assays and heterologous luciferase reporter constructs were performed. These demonstrated that the mRNA of survivin and CUG-BP1 each contain two functional miR-214-3p-binding sites as confirmed by mutational analysis. Finally, forced expression of miR-214-3p enhances the sensitivity of esophageal cancer cells to cisplatin-induced apoptosis. This effect is abrogated with rescue expression of survivin or CUG-BP1. These findings suggest that miR-214-3p acts as a tumor suppressor and that its downregulation contributes to chemoresistance in esophageal cancer cells by targeting both survivin and CUG-BP1.

Overexpression of miR-199a-5p decreases esophageal cancer cell proliferation through repression of mitogen-activated protein kinase kinase kinase-11 (MAP3K11)

Studies examining the oncogenic or tumor suppressive functions of dysregulated microRNAs (miRs) in cancer cells may also identify novel miR targets, which can themselves serve as therapeutic targets. Using array analysis, we have previously determined that miR-199a-5p was the most downregulated miR in two esophageal cancer cell lines compared to esophageal epithelial cells. MiR-199a-5p is predicted to bind mitogen-activated protein kinase kinase kinase 11 (MAP3K11) mRNA with high affinity. In this study, we observed that MAP3K11 is markedly overexpressed in esophageal cancer cell lines. Forced expression of miR-199a-5p in these cells leads to a decrease in the mRNA and protein levels of MAP3K11, due to decreased MAP3K11 mRNA stability. A direct binding interaction between miR-199a-5p and MAP3K11 mRNA is demonstrated using biotin pull-down assays and heterologous luciferase reporter constructs and confirmed by mutational analysis. Finally, forced expression of miR-199a-5p decreases proliferation of esophageal cancer cells by inducing G2/M arrest. This effect is mediated, in part, by decreased transcription of cyclin D1, due to reduced MAP3K11-mediated phosphorylation of c-Jun. These findings suggest that miR-199a-5p acts as a tumor suppressor in esophageal cancer cells and that its downregulation contributes to enhanced cellular proliferation by targeting MAP3K11.

MiR-199a-3p decreases esophageal cancer cell proliferation by targeting p21 activated kinase 4

Although microRNA (miR) 199a-3p functions as a tumor suppressor in multiple malignancies, its expression and role in esophageal cancer have not been studied. Based on our previous observation that miR-199a-3p is markedly downregulated in esophageal cancer cell lines relative to esophageal epithelial cells, we examined the function of miR-199a-3p in these cells. MiR-199a-3p is predicted to bind with high affinity to the mRNA of p21 activated kinase 4 (PAK4). This kinase has been shown to be overexpressed in several malignancies and to modulate proliferation and motility. The current study is designed to determine whether miR-199a-3p regulates the expression of PAK4 in esophageal cancer cells and to understand the functional consequences of this interaction. Herein, we demonstrate reduced expression of miR-199a-3p in human esophageal cancer specimens and cell lines compared to esophageal epithelial cells, with associated increased expression of PAK4. Forced expression of miR-199a-3p decreases expression of PAK4 in esophageal cancer cell lines. Mechanistic studies reveal that miR-199a-3p binds to the 3’UTR of PAK4 mRNA. This interaction results in reduced levels of PAK4 mRNA due to decreased mRNA stability. Downregulation of PAK4 leads to decreased cyclin D1 (CD1) transcription and protein expression, resulting in markedly impaired cellular proliferation. When PAK4 expression is rescued, both CD1 transcription and protein return to baseline levels. Our results show that miR-199a-3p functions as a tumor suppressor in esophageal cancer cells through repression of PAK4. These findings suggest that both miR-199a-3p and PAK4 may be novel therapeutic targets in the treatment of esophageal cancer.

Abstract A37: Overexpression of microRNA (miR) 199a-3p reduces proliferation through the induction of G2/M arrest in esophageal cancer cells by targeting cyclin D1

Objectives: Downregulation of miR-199a-3p has been demonstrated in several malignancies. A potential role of miR-199a-3p as a tumor suppressor has been postulated based on its ability to regulate targets involved in proliferation, apoptosis, and migration. Using miR array analysis, we have previously shown that miR-199a-3p is one of the most markedly downregulated miRs in esophageal cancer cell lines compared to esophageal epithelial cells. To date, no information exists on the role of miR-199a-3p in esophageal cancer cells. In several miR-target sequence analysis programs, miR-199a-3p is predicted to bind cyclin D1 mRNA with high affinity. The objective of this study was to determine expression of cyclin D1 in esophageal cancer cells and to investigate the interaction between miR-199a-3p and cyclin D1 in these cells and to characterize the functional implications of this interaction. Methods: Studies were conducted in human esophageal epithelial (hESO) cells and in TE7 human squamous esophageal cancer cells. Levels of cyclin D1 protein expression were assessed by Western blot. Expression of miR-199a-3p and cyclin D1 mRNA in these cell lines was measured by real-time PCR. MiR-199a-3p function was tested through its overexpression and silencing. Binding of miR-199a-3p to cyclin D1 mRNA was examined using a biotinylated RNA pull-down assay. Cellular proliferation was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and cell cycle progression was determined by FACS analysis. Results: Levels of miR-199a-3p in TE7 esophageal cancer cells are reduced by more than 3 log-fold, as compared to hESO cells. Cyclin D1 protein expression is markedly elevated in TE7 cells compared to hESO cells. Cyclin D1 mRNA and protein expression levels decreased in a time-dependent manner following miR-199a-3p overexpression in TE7 cells. In reciprocal experiments, silencing miR-199a-3p in hESO cells resulted in increased cyclin D1 mRNA and protein levels. Binding of miR-199a-3p to cyclin D1 mRNA was confirmed by biotinylated RNA-pull down assay. Forced expression of miR-199a-3p in TE7 cells led to a marked decrease in cellular proliferation resulting from the induction of G2/M arrest. Conclusions: MiR-199a-3p expression is significantly reduced in TE7 esophageal cancer cells relative to hESO cells. MiR-199a-3p directly binds cyclin D1 mRNA, leading to its destabilization and a marked decreased in cyclin D1 protein expression. This interaction results in decreased cellular proliferation through the induction of G2/M arrest. These results suggest that the loss of miR-199a-3p in esophageal epithelial cells may be an important event in esophageal carcinogenesis. Citation Format: Pornima Phatak, Kimberly Byrnes, Jaladanki N. Rao, Douglas J. Turner, Jian Y. Wang, James M. Donahue. Overexpression of microRNA (miR) 199a-3p reduces proliferation through the induction of G2/M arrest in esophageal cancer cells by targeting cyclin D1. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr A37.

72 Regulation by miR-195 of Actin-Related Protein-2 Translation Modulates Gut Epithelial Restitution After Wounding

G A A b st ra ct s may interact with mucosal immune cells to modulate the gut immune system or alternatively, the organism may act directly on the epithelial cells to strengthen the tight junction. Using a reductionist approach, we employed mice enteroid, a 3D structure grown from mouse intestinal crypts to investigate what components of the Lactobacillus rhamnosus GG (LGG) is responsible for improving mucosal permeability in the absence of immune modulators. Mouse enteroids were maintained in culture for 3 wks. We employed IFNgamma to induce epithelial barrier damage by causing disruption of tight junction (Plos One 2011;6:e22967). Mouse enteroids were incubated for 24 hr with IFNgamma (20 mg/ml) with or without cell-free LGG supernatant (5 μl/well), LGG extracted DNA (10 μg/ml) or LGG cell wall (4 mg/ ml). Incubation with IFNgamma caused a 80% and 67% downregulation of gene expression of occludin and ZO1 (P<0.05). These changes were accompanied by disruption of barrier function. Addition of LGG supernatant prevented these changes and normalized occludin and ZO1 to control levels. In contrast, additions of LGG DNA extracts or cell wall were without effects. These observations indicate that metabolites secreted by Lactobacillus rhamnosus GG but not the bacterial DNA or cell wall are responsible to prevent IFNgamma-induced epithelial barrier damage. This effect occurs independent of immune modulating effects of Lactobacillus rhamnosus GG and is mediated by upregulation of the scaffold protein ZO1 and transmembrane protein occludin.

Abstract 4366: MicroRNA (miR) 199a-5p regulates mitogen-activated protein kinase 3-11 (MAP3K11) expression in esophageal cancer cells by modulating mRNA stability

Introduction: The MAP kinase signaling pathway regulates several targets involved in proliferation, migration, and apoptosis. MAP3K11 is a critical component of this pathway, whose overexpression has been demonstrated in multiple malignancies. To date, scant information is available on its expression or regulation in esophageal cancer cells. MiR 199a-5p has been shown to post-transcriptionally regulate targets involved with proliferation and apoptosis. Based on sequence analysis, miR 199a-5p is predicted to interact with MAP3K11 with high binding affinity. We hypothesize that miR199a-5p plays a clinically relevant role in regulating proliferative and metastatic properties of esophageal cancer cells through regulation of MAP3K11. The purpose of this study is to measure expression of MAP3K11 and miR-199a-5p in esophageal cancer cells and determine whether miR-199a-5p directly interacts with MAP3K11 mRNA. Methods: Studies were conducted in human esophageal epithelial (hESO) cells and in TE7 and TE10 human esophageal carcinoma cells. Global microRNA expression in these cell lines was determined by microarray analysis and confirmed by real-time PCR. Levels of protein expression were measured by Western blot. MiR-199a-5p function was tested through its overexpression and silencing. mRNA levels were determined using real-time PCR. MAP3K11 mRNA stability was determined by measuring half-life after exposure to Actinomycin D. Binding of miR-199a-5p to MAP3K11 mRNA was examined using a biotinylated RNA pull-down assay. Results: Levels of miR-199a-5p in TE7 and TE10 esophageal cancer cells are reduced by approximately 4,000 log-fold, as compared to hESO cells. MAP3K11 protein expression is markedly elevated in TE7 and TE10 cells compared to hESO cells. MAP3K11 mRNA and protein expression levels decreased in a time-dependent manner following miR-199a-5p overexpression in TE7 and TE10 cells. In reciprocal experiments, silencing miR-199a-5p in hESO resulted in increased MAP3K11 mRNA and protein levels. MAP3K11 mRNA stability is also reduced by ∼ 75% following overexpression of miR-199a-5p in TE7 cells. Binding of miR-199a-5p to MAP3K11 mRNA was confirmed by biotinylated RNA-pull down assay. Conclusions: MiR-199a-5p expression is significantly reduced in TE7 and TE10 esophageal cancer cells relative to hESO cells, while MAP3K11 is markedly over-expressed in TE7 and TE10 cells. MiR-199a-5p directly binds MAP3K11 mRNA and leads to its destabilization. Overexpression of miR-199a-5p in TE7 and TE10 cells results in decreased expression of MAP3K11. Taken together, these results support the supposition that the loss of miR-199a-5p in esophageal epithelial cells contributes to the esophageal oncogenesis. Citation Format: Kimberly A. Byrnes, Pornima Phatak, Daniel Mansour, Jaladanki N. Rao, Douglas Turner, Jian-Ying Wang, James M. Donahue. MicroRNA (miR) 199a-5p regulates mitogen-activated protein kinase 3-11 (MAP3K11) expression in esophageal cancer cells by modulating mRNA stability. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4366. doi:10.1158/1538-7445.AM2014-4366