Oguz Gozen

A unique IBMPFD-related P97/VCP mutation with differential binding pattern and subcellular localization.

p97/VCP is a hexameric AAA type ATPase that functions in a variety of cellular processes such as endoplasmic reticulum associated degradation (ERAD), organelle biogenesis, autophagy and cell-cycle regulation. Inclusion body myopathy associated with Paget disease of the bone and frontotemporal dementia (IBMPFD) is an autosomal dominant disorder which has been attributed to mutations in p97/VCP. Several missense mutations affecting twelve different amino acids have been identified in IBMPFD patients and some of them were suggested to be involved in the observed pathology. Here, we analyzed the effect of all twelve p97/VCP variants on ERAD substrates and their cofactor binding abilities. While all mutants cause ERAD substrate accumulation, P137L mutant p97/VCP differs from other IBMPFD mutants by having a unique solubility profile and subcellular localization. Intriguingly, although almost all mutants exhibit enhanced p47 and Ufd1-Npl4 binding, the P137L mutation completely abolishes p97/VCP interactions with Ufd1, Npl4 and p47, while retaining its gp78 binding. While recombinant R155C mutant protein consistently interacts with both Ufd1 and VIM of gp78, P137L mutant protein lost binding ability to Ufd1 but not to VIM in vitro. The differential impairments in p97/VCP interactions with its functional partners and function should help our understanding of the molecular pathogenesis of IBMPFD.

CART expression in limbic regions of rat brain following forced swim stress : Sex differences

Our previous studies showed the modulation of cocaine and amphetamine regulated transcript (CART) positive neurons and CART mRNA by adrenalectomy and corticosterone replacement in hypothalamic nuclei of male rat brain. More recently, we have shown by CART immunohistochemistry that restraint and forced swim (FS) stress have sexually dimorphic and regionally specific effects on CART expression in the hypothalamic nuclei of male and female Sprague-Dawley rats. This study aimed to evaluate the effects of FS stress on CART peptide expression in hypothalamus, amygdala and hippocampus of male and female (in or near estrus) Sprague-Dawley rats. Initially basal CART levels in regions of interest were determined in male and female rats; no sex differences were observed. In FS test, rats were forced to swim on two consecutive days, in a Plexiglas cylinder for 15 and 6 min, respectively. Rats were decapitated on the second day, 10 min after the stress procedure. Hypothalami, amygdalae and hippocampi were dissected and homogenized. CART peptide expression in these regions was measured by Western blotting. In males, FS increased CART expression in hypothalamus and amygdala. On the other hand, in females, FS lowered CART expression in amygdala. CART expression in hippocampus was not affected by the stress procedure in either sex. Our results suggest sexually dimorphic modulation of CART expression in hypothalamus and amygdala by FS procedure. Although modulation of the CART peptide by glucocorticoids and gonadal hormones appears likely, future studies are needed to elucidate the underlying mechanisms in the involvement of CART peptide in stress response.

Forced swim stress elicits region-specific changes in CART expression in the stress axis and stress regulatory brain areas

CART mRNA and peptides are highly expressed in the anatomical structures composing the hypothalamo-pituitary-adrenal (HPA) axis and sympatho-adrenal system. Anatomical and functional studies suggest that CART peptides may have a role in the regulation of the neuroendocrine and autonomic responses during stress. Our previous study showed that CART peptides increased significantly in the male hypothalamus and amygdala 10min after the forced swim stress. The present study aimed to examine the effect of forced swim stress on CART peptide expression in selected brain regions, including those where CART peptide expression has not been reported before (frontal cortex, pons, medulla oblongata), as well as in endocrine glands related to stress in male Sprague Dawley rats. A total of 16 (n=8) animals were used, including control groups. Rats were subjected to forced swim on two consecutive days, and sacrificed on the second day, 2h after the termination of the stress procedure. Frontal cortex, pons, medulla oblongata, hypothalamus, pituitary and adrenal glands were dissected and homogenized. CART peptide expression in these tissues was measured by Western Blotting and six different CART peptide fragments were identified. Our results showed that forced swim stress elicited region-specific changes in CART peptide expression. CART was upregulated in the frontal cortex, hypothalamus, medulla oblongata and adrenal gland while there was no change in the pons and pituitary. Enhanced CART peptide fragments in these brain regions and adrenal glands may have a role in the regulation of the HPA and sympatho-adrenal axis activity during stress response.

The epigenetic effect of nicotine on dopamine D1 receptor expression in rat prefrontal cortex.

Nicotine is a highly addictive drug and exerts its effect partially through causing dopamine release, thereby increasing intrasynaptic dopamine levels in the brain reward systems. Dopaine D1 receptor (DRD1) mRNAs and receptors are localized in reward‐related brain regions, which receive cholinergic input. The aim of this study is to evaluate whether nicotine administration affects the expression of DRD1s, and if so, whether epigenetic mechanisms, such as histone acetylation, are involved. Twenty Male Sprague Dawley rats received nicotine (0.4 mg/kg/day, s.c.) or saline injections for 15 days. After nicotine/saline treatment, rats were perfused with saline; prefrontal cortex (PFC), corpus striatum (STR), and ventral tegmental area (VTA) were dissected. Homogenates were divided into two parts for total RNA isolation and histone H4 acetylation studies. DRD1 mRNA expression was significantly higher in the PFC of the nicotine‐treated group compared with controls; similar trends were observed in the VTA and STR. To study epigenetic regulation, the 2kb upstream region of the DRD1 gene promoter was investigated for histone H4 acetylation in PFC samples. After chromatin immunoprecipitation with anti‐acetyl histone H4 antibody, we found an increase in histone acetylation by two different primer pairs which amplified the −1365 to −1202 (P < 0.005) and −170 to +12 (P < 0.05) upstream regions of the DRD1 promoter. Our results suggest that intermittent subcutaneous nicotine administration increases the expression of DRD1 mRNA in the PFC of rats, and this increase may be due to changes in histone H4 acetylation of the 2kb promoter of the DRD1 gene. Synapse 67:545–552, 2013.

Hippocampal neuronal nitric oxide synthase (nNOS) is regulated by nicotine and stress in female but not in male rats

NO (nitric oxide) produced in limbic brain regions has important roles in the regulation of autonomic nervous system and HPA axis activity, anxiety, fear learning, long-term memory formation, and depression. NO is synthesized from l-arginine in a reaction catalyzed by nitric oxide synthase (NOS). Neuronal nitric oxide synthase (nNOS), one of the three isoforms of NOS, is synthesized constitutively in nerve cells. Increasing evidence indicates that nNOS expression in the nervous system may be regulated by stress and nicotinic receptors. Furthermore, data obtained from several studies suggest that signaling pathways induced by stress and nicotinic receptors may converge on various signal transduction molecules to regulate nNOS expression in brain. In the present study, we used Western Blot analysis to test the effect of forced swim stress, chronic nicotine administration, and the combined effect of both procedures on nNOS expression in the hippocampus, amygdala and frontal cortex of the male and female rat brain. Basal nNOS levels of the three brain regions examined did not show sex differences. However, forced swim stress and chronic nicotine administration increased nNOS expression in the hippocampus of female rats. When stress and nicotine were applied together, no additional increment was observed. Stress and nicotine did not regulate nNOS expression in the amygdala and the frontal cortex of either sex. Data obtained from the present study indicate that the regulation of stress and nicotine induced-nNOS expression in rat hippocampus shows sexual dimorphism and nNOS expression in the female rat hippocampus increases by nicotine and stress.

Region- and sex-specific changes in CART mRNA in rat hypothalamic nuclei induced by forced swim stress

Cocaine and amphetamine regulated transcript (CART) mRNA and peptides are highly expressed in the paraventricular (PVN), dorsomedial (DMH) and arcuate (ARC) nuclei of the hypothalamus. It has been suggested that these nuclei regulate the hypothalamic-pituitary-adrenal (HPA) axis, autonomic nervous system activity, and feeding behavior. Our previous studies showed that forced swim stress augmented CART peptide expression significantly in whole hypothalamus of male rats. In another study, forced swim stress increased the number of CART-immunoreactive cells in female PVN, whereas no effect was observed in male PVN or in the ARC nucleus of either sex. In the present study, we evaluated the effect of forced swim stress on CART mRNA expression in PVN, DMH and ARC nuclei in both male and female rats. Twelve male (stressed and controls, n=6 each) and 12 female (stressed and controls, n=6 each) Sprague-Dawley rats were used. Control animals were only handled, whereas forced swim stress procedure was applied to the stressed groups. Brains were dissected and brain sections containing PVN, DMH and ARC nuclei were prepared. CART mRNA levels were determined by in situ hybridization. In male rats, forced swim stress upregulated CART mRNA expression in DMH and downregulated it in the ARC. In female rats, forced swim stress increased CART mRNA expression in PVN and DMH, whereas a decrease was observed in the ARC nucleus. Our results show that forced swim stress elicits region- and sex-specific changes in CART mRNA expression in rat hypothalamus that may help in explaining some of the effects of stress.

Gene expression of pro-opiomelanocortin and melanocortin receptors is regulated in the hypothalamus and mesocorticolimbic system following nicotine administration

Pro-opiomelanocortin (POMC)-derived peptides and their receptors have been shown to play important roles in natural and drug-induced reward and reinforcement. Reward process may involve the regulation of POMC gene expression and the gene expression of POMC-derived peptide receptors. The present study investigated the alterations observed in the transcript levels of POMC, melanocortin 3 (MC3R), melanocortin 4 (MC4R) and mu-opioid receptors (MOR) in the hypothalamus and mesocorticolimbic system during nicotine exposure. Rats were injected subcutaneously for 5days with one of the three doses (0.2, 0.4 or 0.6mg/kg/day, free base) of nicotine and were decapitated one hour after a challenge dose on the sixth day. mRNA levels of POMC in the hypothalamus, MC3R in the ventral tegmental area (VTA), MC4R and MOR in the medial prefrontal cortex (mPFC), nucleus accumbens, dorsal striatum, amygdala, lateral hypothalamic area and VTA were measured by quantitative real-time PCR. Our results showed that treatment with 0.6mg/kg/day nicotine upregulated POMC mRNA in the hypothalamus and MC4R mRNA in the mPFC. Additionally, all three nicotine doses increased MC3R mRNA expression in the VTA. On the other hand, none of the nicotine doses altered MOR mRNA levels in the mesocorticolimbic system and associated limbic structures. These results suggest that nicotine may enhance melanocortin signaling in the mesocorticolimbic system and this alteration may be an important mechanism mediating nicotine reward.

Nicotine regulates cocaine-amphetamine-Regulated Transcript (Cart) in the mesocorticolimbic system

Cocaine‐and‐Amphetamine Regulated Transcript (CART) mRNA and peptides are intensely expressed in the brain regions comprising mesocorticolimbic system. Studies suggest that CART peptides may have a role in the regulation of reward circuitry. The present study aimed to examine the effect of nicotine on CART expression in the mesocorticolimbic system. Three different doses of nicotine (0.2, 0.4, 0.6 mg/kg free base) were injected subcutaneously for 5 days, and on day 6, rats were decapitated following a challenge dose. CART mRNA and peptide levels in medial prefrontal cortex (mPFC), nucleus accumbens (NAc), dorsal striatum (DST), amygdala (AMG), lateral hypothalamic area (LHA), and ventral tegmental area (VTA) were measured by quantitative real‐time PCR (qPCR) and Western Blot analysis, respectively. In the mPFC, 0.4 and 0.6 mg/kg nicotine, decreased CART peptide levels whereas there was no effect on CART mRNA levels. In the VTA, a down‐regulation of CART peptide expression was observed with 0.2 and 0.6 mg/kg nicotine. Conversely, 0.4 and 0.6 mg/kg nicotine increased CART mRNA levels in the AMG without affecting the CART peptide expression. Nicotine did not regulate CART mRNA or CART peptide expression in the NAc, DST, and LHA. We conclude that nicotine regulates CART expression in the mesocorticolimbic system and this regulation may play an important role in nicotine reward. Synapse 70:283–292, 2016.

Chronic nicotine-induced changes in gene expression of delta and kappa-opioid receptors and their endogenous ligands in the mesocorticolimbic system of the rat.

Delta and kappa opioid receptors (DOR and KOR, respectively) and their endogenous ligands, proenkephalin (PENK) and prodynorphin (PDYN)‐derived opioid peptides are proposed as important mediators of nicotine reward. This study investigated the regulatory effect of chronic nicotine treatment on the gene expression of DOR, KOR, PENK and PDYN in the mesocorticolimbic system. Three groups of rats were injected subcutaneously with nicotine at doses of 0.2, 0.4, or 0.6 mg/kg/day for 6 days. Rats were decapitated 1 hr after the last dose on day six, as this timing coincides with increased dopamine release in the mesocorticolimbic system. mRNA levels in the ventral tegmental area (VTA), lateral hypothalamic area (LHA), amygdala (AMG), dorsal striatum (DST), nucleus accumbens, and medial prefrontal cortex were measured by quantitative real‐time PCR. Our results showed that nicotine upregulated DOR mRNA in the VTA at all of the doses employed, in the AMG at the 0.4 and 0.6 mg/kg doses, and in the DST at the 0.4 mg/kg dose. Conversely, PDYN mRNA was reduced in the LHA with 0.6 mg/kg nicotine and in the AMG with 0.4 mg/kg nicotine. KOR mRNA was also decreased in the DST with 0.6 mg/kg nicotine. Nicotine did not regulate PENK mRNA in any brain region studied.

Nicotinic cholinergic and dopaminergic receptor mRNA expression in male and female rats with high or low preference for nicotine

ABSTRACT Background: Nicotine exerts its central actions through nicotinic acetylcholine receptors (nAChRs), which in turn regulate major neurotransmitter systems including dopamine. Nicotinic and dopaminergic systems play significant roles in physiological functions, neuropsychiatric disorders, and addiction. Objectives: To evaluate possible differences in the expression of nAChR subunit and dopamine receptor (DR) mRNAs following voluntary nicotine intake. Methods: Male and female rats (n = 67) were exposed to long-term free-choice oral nicotine (24 hours/day, 6 weeks); rats with maximum and minimum nicotine preference/intake were selected. The mRNA levels of genes encoding α4,β2,α5, and α7 nAChR subunits and DR Drd1and Drd2 subtypes were evaluated in the striatum (STR), prefrontal cortex (PFC), and hippocampus using quantitative real-time polymerase chain reaction in selected rats (n = 30) and their control groups (n = 15). Results: In addition to baseline differences, expression changes were observed in the mRNA levels of evaluated genes in rats exposed to voluntary oral nicotine in a brain region-, sex-, and preference-related manner. Nicotine intake is correlated negatively with Chrnb2, Chrna7 and positively with Drd1 expression. In the cholinergic system, regional differences in Chnrb2 and Chrna5, sex differences in Chrna4 and Chrna5, and nicotine preference effects in the expression of all subunits except α4 were observed. Chrna5 was lower in maximum than in minimum preferring, and in male than female rats, supporting the inhibitory role of the α5 subunit in nicotine dependence. Nicotine increased Drd2 mRNA expression only in minimum preferring female rats in STR and PFC. Conclusion: Modulation of nAChR and DR gene expression by nicotine may have clinical implications and aid drug development. Pharmaceuticals targeting the nicotinic cholinergic and dopaminergic systems might be expected to have differential efficacy that varies with the patient’s sex or smoking status.