Miklós Sántha

The gene encoding proline dehydrogenase modulates sensorimotor gating in mice

Hemizygous cryptic deletions of the q11 band of human chromosome 22 have been associated with a number of psychiatric and behavioural phenotypes, including schizophrenia. Here we report the isolation and characterization of PRODH, a human homologue of Drosophila melanogaster sluggish-A (slgA), which encodes proline dehydrogenase responsible for the behavioural phenotype of the slgA mutant. PRODH is localized at chromosome 22q11 in a region deleted in some psychiatric patients. We also isolated the mouse homologue of slgA (Prodh), identified a mutation in this gene in the Pro/Re hyperprolinaemic mouse strain and found that these mice have a deficit in sensorimotor gating accompanied by regional neurochemical alterations in the brain. Sensorimotor gating is a neural filtering process that allows attention to be focused on a given stimulus, and is affected in patients with neuropsychiatric disorders. Furthermore, several lines of evidence suggest that proline may serve as a modulator of synaptic transmission in the mammalian brain. Our observations, in conjunction with the chromosomal location of PRODH, suggest a potential involvement of this gene in the 22q11-associated psychiatric and behavioural phenotypes.

Effect of Dopamine Uptake Inhibition on Brain Catecholamine Levels and Locomotion in Catechol-O-methyltransferase-Disrupted Mice

Two different uptake processes terminate the synaptic action of released catecholamines in brain: the high-affinity uptake to presynaptic nerve terminals (uptake1, followed by oxidation by monoamine oxidase, MAO) or glial cells uptake (uptake2, followed by O-methylation by catechol-O-methyltransferase, COMT, and/or oxidation by MAO). For dopaminergic neurons, uptake by the high-affinity dopamine transporter (DAT) is the most effective mechanism, and the contribution of glial COMT remains secondary under normal conditions. In the present study we have characterized the role of COMT using COMT-deficient mice in conditions where DAT is inhibited by 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)-piperazine (GBR 12909) or cocaine. In mice lacking COMT, GBR 12909 results in total brain tissue dopamine levels generally higher than in wild-type mice but no such potentiation was ever seen in striatal extracellular fluid. Dopamine accumulation in nerve endings is more evident in striatum and hypothalamus than in cortex. Both GBR 12909 and cocaine induced hyperlocomotion in mice lacking COMT. Unexpectedly, hyperactivity induced by 20 mg/kg GBR 12909 was attenuated only in male COMT knockout mice, i.e., they had an inability to sustain the hyperactivity induced by DAT inhibition. Furthermore, attenuation of hyperlocomotion was observed also after cocaine treatment in both C57BL/6 (at 5 and 15 mg/kg) and 129/Sv (at 30 mg/kg) genetic background COMT-deficient male mice. Despite the possible interaction between DAT and extraneuronal uptake (and subsequently COMT), the role of COMT in dopamine elimination is still minimal in conditions when DAT is inhibited.

Apolipoprotein B100 acts as a molecular link between lipid‐induced endoplasmic reticulum stress and hepatic insulin resistance

Accumulation of unfolded and misfolded proteins in the endoplasmic reticulum (ER) results in ER stress and lipid overload‐induced ER stress has been implicated in the development of insulin resistance. Here, evidence is provided for a molecular link between hepatic apolipoprotein B100 (apoB100), induction of ER stress, and attenuated insulin signaling. First, in vivo upregulation of hepatic apoB100 by a lipogenic diet was found to be closely associated with ER stress and attenuated insulin signaling in the liver. Direct in vivo overexpression of human apoB100 in a mouse transgenic model further supported the link between excessive apoB100 expression and hepatic ER stress. Human apoB100 transgenic mice exhibited hypertriglyceridemia and hyperglycemia. In vitro, accumulation of cellular apoB100 by free fatty acid (oleate) stimulation or constant expression of wild‐type or N‐glycosylation mutant apoB50 in hepatic cells induced ER stress. This led to perturbed activation of glycogen synthase kinase 3 and glycogen synthase by way of the activation of c‐Jun N‐terminal kinase and suppression of insulin signaling cascade, suggesting that dysregulation of apoB was sufficient to disturb ER homeostasis and induce hepatic insulin resistance. Small interfering (si)RNA‐mediated attenuation of elevated apoB level in the apoB50‐expressing cells rescued cells from lipid‐induced ER stress and reversed insulin insensitivity. Conclusion: These findings implicate apoB100 as a molecular link between lipid‐induced ER stress and hepatic insulin resistance. (HEPATOLOGY 2009.)

Over-expression of dopamine D2 receptor and inwardly rectifying potassium channel genes in drug-naive schizophrenic peripheral blood lymphocytes as potential diagnostic markers

Schizophrenia is one of the most common neuropsychiatric disorders affecting nearly 1% of the human population. Current diagnosis of schizophrenia is based on complex clinical symptoms. The use of easily detectable peripheral molecular markers could substantially help the diagnosis of psychiatric disorders. Recent studies showed that peripheral blood lymphocytes (PBL) express subtypes of D1 and D2 subclasses of dopamine receptors. Recently, dopamine receptor D3 (DRD3) was found to be over-expressed in schizophrenic PBL and proposed to be a diagnostic and follow-up marker for schizophrenia. In this study we screened PBL of 13 drug-naive/drug-free schizophrenic patients to identify additional markers of schizophrenia. One of the benefits of our study is the use of blood samples of non-medicated, drug-naive patients. This excludes the possibility that changes detected in gene expression levels might be attributed to the medication rather than to the disorder itself. Among others, genes for dopamine receptor D2 (DRD2) and the inwardly rectifying potassium channel (Kir2.3) were found to be over-expressed in microarray analysis. Increased mRNA levels were confirmed by quantitative real-time PCR (QRT-PCR) using the SybrGreen method and dual labeled TaqMan probes. The use of both molecular markers allows a more rapid and precise prediction of schizophrenia and might help find the optimal medication for schizophrenic patients.

Survival motor neuron SMN1 and SMN2 gene promoters: identical sequences and differential expression in neurons and non-neuronal cells

Spinal muscular atrophy (SMA) is a recessive disorder involving the loss of motor neurons from the spinal cord. Homozygous absence of the survival of motor neuron 1 gene (SMN1) is the main cause of SMA, but disease severity depends primarily on the number of SMN2 gene copies. SMN protein levels are high in normal spinal cord and much lower in the spinal cord of SMA patients, suggesting neuron-specific regulation for this ubiquitously expressed gene. We isolated genomic DNA from individuals with SMN1 or SMN2 deletions and sequenced 4.6 kb of the 5′ upstream regions of the these. We found that these upstream regions, one of which is telomeric and the other centromeric, were identical. We investigated the early regulation of SMN expression by transiently transfecting mouse embryonic spinal cord and fibroblast primary cultures with three transgenes containing 1.8, 3.2 and 4.6, respectively, of the SMN promoter driving β-galactosidase gene expression. The 4.6 kb construct gave reporter gene expression levels five times higher in neurons than in fibroblasts, due to the combined effects of a general enhancer and a non-neuronal cell silencer. The differential expression observed in neurons and fibroblasts suggests that the SMN genes play a neuron-specific role during development. An understanding of the mechanisms regulating SMN promoter activity may provide new avenues for the treatment of SMA.

Overexpression of Hsp27 ameliorates symptoms of Alzheimer's disease in APP/PS1 mice

Hsp27 belongs to the small heat shock protein family, which are ATP-independent chaperones. The most important function of Hsp27 is based on its ability to bind non-native proteins and inhibit the aggregation of incorrectly folded proteins maintaining them in a refolding-competent state. Additionally, it has anti-apoptotic and antioxidant activities. To study the effect of Hsp27 on memory and synaptic functions, amyloid-β (Aβ) accumulation, and neurodegeneration, we generated transgenic mice overexpressing human Hsp27 protein and crossed with APPswe/PS1dE9 mouse strain, a mouse model of Alzheimers disease (AD). Using different behavioral tests, we found that spatial learning was impaired in AD model mice and was rescued by Hsp27 overexpression. Electrophysiological recordings have revealed that excitability of neurons was significantly increased, and long-term potentiation (LTP) was impaired in AD model mice, whereas they were normalized in Hsp27 overexpressing AD model mice. Using anti-amyloid antibody, we counted significantly less amyloid plaques in the brain of APPswe/PS1dE9/Hsp27 animals compared to AD model mice. These results suggest that overexpression of Hsp27 protein might ameliorate certain symptoms of AD.

Noladin ether, a putative endocannabinoid, inhibits μ-opioid receptor activation via CB2 cannabinoid receptors

We examined the occurrence of possible changes in mRNA expression and the functional activity of opioid receptors after acute in vivo and in vitro treatment with the putative endogenous cannabinoid noladin ether. While noladin ether (NE) demonstrates agonist activity at CB1 cannabinoid receptors, recent data indicate that NE acts as a full agonist at CB2 cannabinoid receptors too. Considering the functional interactions between opioids and cannabinoids, it is of interest to examine whether NE affects the opioid system. To that end, we studied the influence of NE on mu-opioid receptor (MOR) mRNA expression and MOR mediated G-protein signaling. We used real-time PCR and [35S]GTPgammaS binding assays to examine the changes of MOR mRNA levels and the capability of the mu-opioid agonist peptide ([D-Ala2,(NMe)Phe4,Gly5-ol]enkephalin (DAMGO) in activating regulatory G-proteins via MORs in forebrain membrane fractions of wild-type (w.t., CB1+/+) and CB1 receptor deficient transgenic mice (knockout, CB1-/-). We found, that the expression of MOR mRNAs significantly decreased both in CB1+/+ and CB1-/- forebrain after a single injection of NE at 1 mg/kg when compared to control. Consequently, MOR-mediated signaling is attenuated after acute in vivo treatment with NE in both CB1+/+ and CB1-/- mice. Inhibition on MOR mediated activation is observed after in vitro NE administration as well. Radioligand binding competition studies showed that the noticed effect of NE on MOR signaling is not mediated through MORs. Both in vivo and in vitro attenuations of NE can be antagonized by the CB2 selective antagonist SR144528. Taken together, our data suggest that the NE caused pronounced decrease in the activity of MOR is mediated via CB2 cannabinoid receptors.

CB2 cannabinoid receptor antagonist SR144528 decreases mu-opioid receptor expression and activation in mouse brainstem: Role of CB2 receptor in pain

Formerly considered as an exclusively peripheral receptor, it is now accepted that CB(2) cannabinoid receptor is also present in limited amounts and distinct locations in the brain of several animal species, including mice. However, the possible roles of CB(2) receptors in the brain need to be clarified. The aim of our work was to study the mu-opioid receptor (MOR) mRNA expression level and functional activity after acute in vivo and in vitro treatments with the endocannabinoid noladin ether (NE) and with the CB(2) receptor antagonist SR144528 in brainstem of mice deficient in either CB(1) or CB(2) receptors. This study is based on our previous observations that noladin ether (NE) produces decrease in the activity of MOR in forebrain and this attenuation can be antagonized by the CB(2) cannabinoid antagonist SR144528, suggesting a CB(2) receptor mediated effect. We used quantitative real-time PCR to examine the changes of MOR mRNA levels, [(35)S]GTPgammaS binding assay to analyze the capability of mu-opioid agonist DAMGO to activate G-proteins and competition binding assays to directly measure the ligand binding to MOR in mice brainstem. After acute NE administration no significant changes were observed on MOR signaling. Nevertheless pretreatment of mice with SR144528 prior to the administration of NE significantly decreased MOR signaling suggesting the involvement of SR144528 in mediating the effect of MOR. mRNA expression of MORs significantly decreased both in CB(1) wild-type and CB(1) knockout mice after a single injection of SR144528 at 0.1mg/kg when compared to the vehicle treated controls. Consequently, MOR-mediated signaling was attenuated after acute in vivo treatment with SR144528 in both CB(1) wild-type and CB(1) knockout mice. In vitro addition of 1microM SR144528 caused a decrease in the maximal stimulation of DAMGO in [(35)S]GTPgammaS binding assays in CB(2) wild-type brainstem membranes whereas no significant changes were observed in CB(2) receptor knockouts. Radioligand binding competition studies showed that the noticed effect of SR144528 on MOR signaling is not mediated through MORs. Our data demonstrate that the SR144528 caused pronounced decrease in the activity of MOR is mediated via CB(2) cannabinoid receptors.

Hepatic mitochondrial and ER stress induced by defective PPARα signaling in the pathogenesis of hepatic steatosis

Emerging evidence demonstrates a close interplay between disturbances in mitochondrial function and ER homeostasis in the development of the metabolic syndrome. The present investigation sought to advance our understanding of the communication between mitochondrial dysfunction and ER stress in the onset of hepatic steatosis in male rodents with defective peroxisome proliferator-activated receptor-α (PPARα) signaling. Genetic depletion of PPARα or perturbation of PPARα signaling by high-fructose diet compromised the functional activity of metabolic enzymes involved in mitochondrial fatty acid β-oxidation and induced hepatic mitochondrial stress in rats and mice. Inhibition of PPARα activity further enhanced the expression of apolipoprotein B (apoB) mRNA and protein, which was associated with reduced mRNA expression of the sarco/endoplasmic reticulum calcium ATPase (SERCA), the induction of hepatic ER stress, and hepatic steatosis. Restoration of PPARα activity recovered the metabolic function of the mitochondria and ER, alleviated systemic hypertriglyceridemia, and improved hepatic steatosis. These findings unveil novel roles for PPARα in mediating stress signals between hepatic subcellular stress-responding machinery and in the onset of hepatic steatosis under conditions of metabolic stress.

Biglycan protects cardiomyocytes against hypoxia/reoxygenation injury: role of nitric oxide.

Biglycan, a proteoglycan component of extracellular matrix, has been suspected to contribute to the development of atherosclerosis, but overexpression of biglycan in transgenic mice has been shown to induce cardioprotective genes including nitric oxide (NO) synthases in the heart. Therefore, here we hypothesized if exogenous administration of biglycan exerts cytoprotection. Primary cardiomyocytes from neonatal rats were subjected to 150 min hypoxia and 2 h reoxygenation. Mortality of cardiomyocytes was dose-dependently attenuated by pretreatment with 1-100 nM biglycan. Biglycan enhanced eNOS mRNA and protein, and significantly increased NO content of cardiomyocytes. The NO synthase inhibitor l-nitro-arginine-methyl-ester significantly attenuated the cytoprotective effect of biglycan. This is the first demonstration that biglycan leads to cytoprotection against hypoxia/reoxygenation injury, and that this phenomenon is partially mediated by an NO-dependent mechanism.