Effects of sodium butyrate on oxidative stress and behavioral changes induced by administration of d-AMPH

Several evidences have demonstrated that oxidative stress has a central role in bipolar disorder (BD). Recently, studies have been suggested histone deacetylases (HDAC) as a possible target for new medications in treatment of mood disorders. In this study, we investigated the effects of sodium butyrate (SB, a histone deacetilase inhibitor) on oxidative stress in rats submitted to an animal model of mania induced by d-amphetamine (d-AMPH). Wistar rats were first given d-AMPH or saline (Sal) for 14 days, and then, between days 8 and 14, rats were treated with SB or Sal. Locomotor activity and risk-taking behavior were assessed by open-field test and oxidative stress was measured in prefrontal cortex, amygdala, hippocampus and striatum. The results showed that SB reversed and prevented d-AMPH-induced behavioral effects. The d-AMPH administration induced oxidative damage in all brain structures analyzed. Depending on the cerebral area and technique, SB was able to reverse this impairment. The present study reinforces the need for more studies of HDAC inhibitors as possible target for new medications in treatment for BD.     

Brain creatine kinase activity in an animal model of mania

There is evidence pointing to dysfunction at the mitochondrial level as an important target for the understanding of the pathophysiology of bipolar disorder (BD). We assessed creatine kinase (CK) activity in rats submitted to an animal model of mania which included the use of lithium and valproate. In the acute treatment, amphetamine (AMPH) or saline was administered to rats for 14 days, and between day 8 and 14, rats were treated with either lithium, valproate or saline. In the maintenance treatment, rats were pretreated with lithium, valproate or saline, and between day 8 and 14, AMPH or saline were administered. In both experiments, locomotor activity was assessed by open-field test and CK activity was evaluated in hippocampus, striatum, cerebellum, whole cortex and prefrontal cortex. Our results showed that mood stabilizers reversed AMPH-induced behavioral effects. Moreover, AMPH (acute treatment) inhibited CK activity in hippocampus, striatum and cortex, but not in cerebellum and prefrontal cortex, and administration of lithium or valproate did not reverse the enzyme inhibition. In the maintenance treatment, AMPH decreased CK activity in saline-pretreated rats in hippocampus, striatum and cortex, but not in cerebellum and prefrontal cortex. AMPH administration in lithium- or valproate-pretreated animals decreased CK activity in hippocampus, striatum and cortex. Our results showed that AMPH inhibited CK activity and that mood stabilizers were not able to reverse and/or prevent the enzyme inhibition. These findings reinforce the hypothesis that mitochondrial dysfunction plays an important role in the pathophysiology of BD.     

Histone hyperacetylation is associated with amelioration of experimental colitis in mice

Inhibitors of histone deacetylases (HDAC) are being studied for their antiproliferative effects in preclinical cancer trials. Recent studies suggest an anti-inflammatory role for this class of compounds. Because inflammatory bowel disease is associated with an increased risk of malignancies, agents with antiproliferative and anti-inflammatory properties would be of therapeutic interest. HDAC inhibitors from various classes were selected and evaluated for their in vitro capacity to suppress cytokine production and to induce apoptosis and histone acetylation. Valproic acid (VPA) and suberyolanilide hydroxamic acid (SAHA) were chosen for further studies in dextran sulfate sodium- and trinitrobenzene sulfonic acid-induced colitis in mice. In vitro, inhibition of HDAC resulted in a dose-dependent suppression of cytokine synthesis and apoptosis induction requiring higher concentrations of HDAC inhibitors for apoptosis induction compared with cytokine inhibition. Oral administration of either VPA or SAHA reduced disease severity in dextran sulfate sodium-induced colitis. The macroscopic and histologic reduction of disease severity was associated with a marked suppression of colonic proinflammatory cytokines. In parallel to the beneficial effect observed, a dose-dependent increase in histone 3 acetylation at the site of inflammation was shown under VPA treatment. Furthermore, SAHA as well as VPA treatment resulted in amelioration of trinitrobenzene sulfonic acid-induced colitis, which was associated with an increase of apoptosis of lamina propria lymphocytes. Inhibitors of HDAC reveal strong protective effects in different models of experimental colitis by inducing apoptosis and suppressing proinflammatory cytokines, thereby representing a promising class of compounds for clinical studies in human inflammatory bowel disease.     

Valproic acid induces functional heat-shock protein 70 via Class I histone deacetylase inhibition in cortical neurons: a potential role of Sp1 acetylation

Neuroprotective properties of the mood stabilizer valproic acid (VPA) are implicated in its therapeutic efficacy. Heat-shock protein 70 (HSP70) is a molecular chaperone, neuroprotective and anti-inflammatory agent. This study aimed to investigate underlying mechanisms and functional significance of HSP70 induction by VPA in rat cortical neurons. VPA treatment markedly up-regulated HSP70 protein levels, and this was accompanied by increased HSP70 mRNA levels and promoter hyperacetylation and activity. Other HDAC inhibitors – sodium butyrate, trichostatin A, and Class I HDAC-specific inhibitors MS-275 and apicidin, – all mimicked the ability of VPA to induce HSP70. Pre-treatment with phosphatidylinositol 3-kinase inhibitors or an Akt inhibitor attenuated HSP70 induction by VPA and other HDAC inhibitors. VPA treatment increased Sp1 acetylation, and a Sp1 inhibitor, mithramycin, abolished the induction of HSP70 by HDAC inhibitors. Moreover, VPA promoted the association of Sp1 with the histone acetyltransferases p300 and recruitment of p300 to the HSP70 promoter. Further, VPA-induced neuroprotection against glutamate excitotoxicity was prevented by blocking HSP70 induction. Taken together, the data suggest that the phosphatidylinositol 3-kinase/Akt pathway and Sp1 are likely involved in HSP70 induction by HDAC inhibitors, and induction of HSP70 by VPA in cortical neurons may contribute to its neuroprotective and therapeutic effects.     

Valproic acid reduces brain damage induced by transient focal cerebral ischemia in rats: potential roles of histone deacetylase inhibition and heat shock protein induction

Growing evidence from in vitro studies supports that valproic acid (VPA), an anti-convulsant and mood-stabilizing drug, has neuroprotective effects. The present study investigated whether VPA reduces brain damage and improves functional outcome in a transient focal cerebral ischemia model of rats. Subcutaneous injection of VPA (300 mg/kg) immediately after ischemia followed by repeated injections every 12 h, was found to markedly decrease infarct size and reduce ischemia-induced neurological deficit scores measured at 24 and 48 h after ischemic onset. VPA treatment also suppressed ischemia-induced neuronal caspase-3 activation in the cerebral cortex. VPA treatments resulted in a time-dependent increase in acetylated histone H3 levels in the cortex and striatum of both ipsilateral and contralateral brain hemispheres of middle cerebral artery occlusion (MCAO) rats, as well as in these brain areas of normal, non-surgical rats, supporting the in vitro finding that VPA is a histone deacetylase (HDAC) inhibitor. Similarly, heat shock protein 70 (HSP70) levels were time-dependently up-regulated by VPA in the cortex and striatum of both ipsilateral and contralateral sides of MCAO rats and in these brain areas of normal rats. Altogether, our results demonstrate that VPA is neuroprotective in the cerebral ischemia model and suggest that the protection mechanisms may involve HDAC inhibition and HSP induction.     

Inhibition of histone deacetylase on ventricular remodeling in infarcted rats

Histone deacetylase (HDAC) determines the acetylation status of histones and, thereby, controls the regulation of gene expression. HDAC inhibitors have been shown to inhibit cardiomyocyte growth in vitro and in vivo. We assessed whether HDAC inhibitors exert a beneficial effect on the remodeling heart in infarcted rats. At 24 h after ligation of the left anterior descending artery, male Wistar rats were randomized to vehicle, HDAC inhibitors [valproic acid (VPA) and tributyrin], an agonist of HDAC (theophylline), VPA + theophylline, or tributyrin + theophylline for 4 wk. Significant ventricular hypertrophy was detected as increased myocyte size at the border zone isolated by enzymatic dissociation after infarction. Cardiomyocyte hypertrophy and collagen formation at the remote region and border zone were significantly attenuated by VPA and tributyrin with a similar potency compared with that induced by the vehicle. Left ventricular shortening fraction was significantly higher in the VPA- and tributyrin-treated groups than in the vehicle-treated group. Increased synthesis of atrial natriuretic peptide mRNA after infarction was confirmed by RT-PCR, consistent with the results of immunohistochemistry and Western blot for acetyl histone H4. The beneficial effects of VPA and tributyrin were abolished by theophylline, implicating HDAC as the relevant target. Inhibition of HDAC by VPA or tributyrin can attenuate ventricular remodeling after infarction. This might provide a worthwhile therapeutic target.     

Induction of osteogenic differentiation of human mesenchymal stem cells by histone deacetylase inhibitors

Valproic acid (VPA) has been used as an anticonvulsant agent for the treatment of epilepsy, as well as a mood stabilizer for the treatment of bipolar disorder, for several decades. The mechanism of action for these effects remains to be elucidated and is most likely multifactorial. Recently, VPA has been reported to inhibit histone deacetylase (HDAC) and HDAC has been reported to play roles in differentiation of mammalian cells. In this study, the effects of HDAC inhibitors on differentiation and proliferation of human adipose tissue-derived stromal cells (hADSC) and bone marrow stromal cells (hBMSC) were determined. VPA increased osteogenic differentiation in a dose dependent manner. The pretreatment of VPA before induction of differentiation also showed stimulatory effects on osteogenic differentiation of hMSC. Trichostatin A (TSA), another HDAC inhibitor, also increased osteogenic differentiation, whereas valpromide (VPM), a structural analog of VPA which does not possess HDAC inhibitory effects, did not show any effect on osteogenic differentiation on hADSC. RT-PCR and Real-time PCR analysis revealed that VPA treatment increased osterix, osteopontin, BMP-2, and Runx2 expression. The addition of noggin inhibited VPA-induced potentiation of osteogenic differentiation. VPA inhibited proliferation of hADSC and hBMSC. Our results suggest that VPA enhance osteogenic differentiation, probably due to inhibition of HDAC, and could be useful for in vivo bone engineering using hMSC.     

Hdac6 knock-out increases tubulin acetylation but does not modify disease progression in the R6/2 mouse model of Huntington's disease

Huntington's disease (HD) is a progressive neurodegenerative disorder for which there is no effective disease modifying treatment. Following-on from studies in HD animal models, histone deacetylase (HDAC) inhibition has emerged as an attractive therapeutic option. In parallel, several reports have demonstrated a role for histone deacetylase 6 (HDAC6) in the modulation of the toxicity caused by the accumulation of misfolded proteins, including that of expanded polyglutamine in an N-terminal huntingtin fragment. An important role for HDAC6 in kinesin-1 dependent transport of brain-derived neurotrophic factor (BDNF) from the cortex to the striatum has also been demonstrated. To elucidate the role that HDAC6 plays in HD progression, we evaluated the effects of the genetic depletion of HDAC6 in the R6/2 mouse model of HD. Loss of HDAC6 resulted in a marked increase in tubulin acetylation throughout the brain. Despite this, there was no effect on the onset and progression of a wide range of behavioural, physiological, molecular and pathological HD-related phenotypes. We observed no change in the aggregate load or in the levels of soluble mutant exon 1 transprotein. HDAC6 genetic depletion did not affect the efficiency of BDNF transport from the cortex to the striatum. Therefore, we conclude that HDAC6 inhibition does not modify disease progression in R6/2 mice and HDAC6 should not be prioritized as a therapeutic target for HD.     

Prenatal exposure to valproate induces sex-, age-, and tissue-dependent alterations of cholesterol metabolism: Potential implications on autism

Here, we investigated the protein network regulating cholesterol metabolism in the liver and brain of adolescent and adult male and female rats prenatally exposed to valproate (VPA), a well validated experimental model of autism spectrum disorders (ASD). We were aimed at studying whether prenatal VPA exposure affected the proteins involved in cholesterol homeostasis in a sex-dependent manner. To this aim the protein network of cholesterol metabolism, in term of synthesis and plasma membrane trafficking, was analyzed by western blot in the liver and different brain areas (amygdala, cerebellum, cortex, hippocampus, nucleus accumbens, and dorsal striatum) of adolescent and adult male and female rats prenatally exposed to VPA. Our results show that physiological sex-dependent differences are present both in the liver and in brain of rats. Interestingly, VPA affects specifically the brain in an age- and region-specific manner; indeed, cerebellum, cortex, hippocampus and nucleus accumbens are affected in a sex-dependent way, while this does not occur in amygdala and dorsal striatum. Overall, we demonstrate that each brain area responds differently to the same external stimulus and males and females respond in a different way, suggesting that this could be related to the diverse incidences, between the sexes, of some neurodevelopmental pathologies such as autism, which displays a 3:1 male to female ratio.     

Cocaine

Cocaine HCl (0, 10, or 50 mg/kg) was injected into adult male ICR mice ip. Thirty minutes later, the brains were removed, and nine regions were isolated: olfactory bulbs, olfactory tubercles, prefrontal cortex, septum, striatum, amygdala, hypothalamus, hippocampus, and thalamus. Using high-performance liquid chromatography, concentrations of norepinephrine, dopamine, serotonin, and their major metabolites and the metabolite/neurotransmitter ratios were determined as an indicator of utilization. Serotonergic systems responded most dramatically. 5HIAA/5-HT decreases were seen in all the brain regions, except the septum, hippocampus, and olfactory bulbs. In most instances, the alterations were dose-dependent. The most profound changes were seen in the amygdala, prefrontal cortex, hypothalamus, and thalamus. For noradrenergic systems, significant responses were seen only in the amygdala, prefrontal cortex, and hypothalamus, but then only at the lower dose. The dopaminergic responses were more complex and not always dose-dependent. The DOPAC/DA ratio was decreased only in the amygdala and striatum at the lower dose, and the olfactory tubercles at the higher dose. It was increased in the septum. The HVA/DA ratios were decreased in the amygdala, prefrontal cortex, and hypothalamus, but only at the lower dose (like MHPG/NE). The 3MT/DA ratio was decreased in the thalamus at the lower dose and in the olfactory tubercles at the higher dose, whereas it was increased in the prefrontal cortex at the lower dose. The HVA and DOPAC routes of degradation were both utilized only by the amygdala. Thus, cocaine produced its most comprehensive effects in this nucleus, as well as the greatest absolute percentage changes for all three of the monoamine systems studied.     

VPA inhibits renal cancer cell migration by targeting HDAC2 and down-regulating HIF-1α

Cell migration plays major roles in human renal cancer-related death, but the molecular mechanisms remain unclear. Valproic acid (VPA) is a broad-spectrum inhibitor of class I and II histone deacetylases and shows great anticancer activity in a variety of human cancers. In this study, we found that VPA significantly inhibited cell migration but not proliferation of human renal cancer ACHN cells. Mechanistic studies found that VPA significantly inhibited the expression of HIF-1α. Knockdown of HIF-1α could obviously inhibited cell migration, while over-expression of HIF-1α markedly rescued the inhibition of VPA on cell migration. Further studies found that knockdown of HDAC2 completely mimicked the effects of VPA on HIF-1α and cell migration, and over-expression of HIF-1α could also rescue the effects of HDAC2 knockdown on cell migration. Collectively, these results indicated that the potential of specific inhibition of HDAC2 by small molecular chemicals may lead to future therapeutic agents in human renal cancer treatment.     

Inhibition of Mitochondrial Respiratory Chain in the Brain of Adult Rats After Acute and Chronic Administration of Methylphenidate

Methylphenidate (MPH) is frequently prescribed for the treatment of attention deficit/hyperactivity disorder. It was previously demonstrated that MPH altered brain metabolic activity. Most cell energy is obtained through oxidative phosphorylation, in the mitochondrial respiratory chain. However, there are still few studies about MPH effects on the brain of adult rats. Thus, in the present study we evaluated the effect of acute or chronic administration of MPH on the activities of mitochondrial respiratory chain complexes I–IV in the brain of adult rats. For acute administration, a single injection of MPH was given to 60-day-old rats. For chronic administration, MPH injections were given to 60-day-old rats once daily for 28 days. Our results showed that complexes I, II, III and IV were inhibited after acute or chronic MPH administration in the hippocampus, prefrontal cortex, striatum and cerebral cortex. On the other hand, cerebellum was not affected.     

Distinctions in metabolism of serotonin and dopamine in rat brain during latent inhibition

Latent inhibition (LI) is a behavioral phenomenon, in which repeated presenting of a non-reinforced stimulus retards conditioning to this stimulus when it is coupled with a reinforcer. In order to find specific serotonin (5-HT- and dopamine (DA) changes mediating the LI, the 5-HT and DA metabolism was investigated in certain brain regions. Oxidative deamination of 5-HT and DA by monoamine oxidase (MAO) was determined in the prefrontal cortex, striatim, amygdala, and hippocampus at preexposure and testing stages of the LI using the passive avoidance procedure in rats. Preexposed animals demonstrated high MAO activity for 5-HT deamination in the amygdala and striatum and lower MAO activity for DA deamination in the amygdala and hippocampus. After testing the LI, a high level of 5-HT deamination by MAO was revealed in the amygdala, white the lower level of 5-HT deamination by MAO was shown in the prefrontal cortex. At the same time, no changes in DA metabolism were found in all the brain regions studied. Thus, the role of dopaminergic system in the LI effect may be limited by the preexposure stage. The obtained evidence suggests that the enhanced 5-HT activity in the amygdala and striatum induced by the preexposed stimulus is a principal biochemical mechanism underlying the LI.     

Changes in brain serotonin metabolism and [3H]-serotonin receptor binding during recall of conditioned passive avoidance in rats

The content of serotonin and its metabolite 5-hydroxyindoleacetic acid, monoamine oxidase activity, and [3H]-serotonin radioligand receptor binding were examined in the prefrontal cortex, striatum, amygdala, hippocampus and periaqueductal gray matter at different time after one-trial passive avoidance training of rats. Changes in the serotonergic activity were observed only in rats, which showed retrieval of conditioned passive avoidance response. No serotonergic changes were found immediately and one day after training. Also, there were no changes in trained rats without retrieval of conditioned passive avoidance response or rats with experimental amnesia. The pattern of the involvement of brain structures in the retrieval process was also revealed. [3H]-serotonin binding was decreased in the amygdala, periaqueductal gray matter and striatum, whereas it did not change in the prefrontal cortex and hippocampus. At the same time, the serotonin content in these structures did not differ from that of intact rats. Deamination of serotonin by monoamine oxidase and active transport of 5-hydroxyindoleacetic acid from nerve terminals were increased in the amygdala and periaqueductal gray matter, whereas in the striatum serotonin catabolism was decreased. The obtained differences in serotonin catabo- lism suggest that the decrease in receptor binding of serotonin in these brain structures is provided by different synaptic processes: presynaptic changes in the striatum and postsynaptic receptor changes in the amygdala and periaqueductal gray matter. It is concluded that the decrease in the serotonergic activity in the amygdala and periaqueductal gray matter represents one of the mechanisms activating the emotiogenic system mediating the memory trace retrieval in inhibitory avoidance learning.     

A proapoptotic effect of valproic acid on progenitors of embryonic stem cell-derived glutamatergic neurons

Valproic acid (VPA) is a branched-chain saturated fatty acid with a long history of clinical use as an antiepileptic drug (AED). VPA is also known to inhibit histone deacetylases (HDACs) and to cause diverse effects on neural progenitor cells (NPCs) and neurons. Although the neuroprotective or neurodestructive effects of VPA have been investigated in heterogeneous cell populations, in this study, we used homogeneous populations of NPCs and glutamatergic cortical pyramidal neurons, which were differentiated from embryonic stem (ES) cells. At therapeutic concentrations, VPA had a proapoptotic effect on ES cell-derived NPCs of glutamatergic neurons, but not on their progeny. This effect of VPA most likely occurred through the inhibition of HDACs, because similar phenotypes were observed following treatment with other HDAC inhibitors (HDACis) such as trichostatin A and sodium butyrate. The proapoptotic phenotype was not observed when cells were exposed to a structural analog of VPA, valpromide (VPM), which has the same antiepileptic effect as VPA, but does not inhibit HDACs. Western blotting confirmed that treatment with HDACis, but not VPM, significantly increased the levels of histone H3 acetylation in NPCs. HDACi treatments did not affect the survival of neurons, although the acetylation levels were increased to a limited extent. These results, which are based on a homogeneous culture system, suggest that VPA inhibits HDAC activity and induces the apoptosis of NPCs that are fated to differentiate into glutamatergic neurons. The dose-dependent effects of VPA both on apoptosis and hyperacetylation of histone H3 in NPCs supported this notion. These cell type- and differentiation stage-specific effects of VPA imply that dysfunction of HDACs during pregnancy significantly increase the risk of congenital malformations associated with VPA administration.     

Increased Oxidative Parameters and Decreased Cytokine Levels in an Animal Model of Attention-Deficit/Hyperactivity Disorder

Attention-deficit/hyperactivity disorder (ADHD) is a highly heterogeneous disorder characterized by impairing levels of hyperactivity, impulsivity and inattention. Oxidative and inflammatory parameters have been recognized among its multiple predisposing pathways, and clinical studies indicate that ADHD patients have increased oxidative stress. In this study, we aimed to evaluate oxidative (DCFH oxidation, glutathione levels, glutathione peroxidase, catalase and superoxide dismutase activities) and inflammatory (TNF-α, IL-1β and IL-10) parameters in the most widely accepted animal model of ADHD, the spontaneously hypertensive rats (SHR). Prefrontal cortex, cortex (remaining regions), striatum and hippocampus of adult male SHR and Wistar Kyoto rats were studied. SHR presented increased reactive oxygen species (ROS) production in the cortex, striatum and hippocampus. In SHR, glutathione peroxidase activity was decreased in the prefrontal cortex and hippocampus. TNF-α levels were reduced in the prefrontal cortex, cortex (remaining regions), hippocampus and striatum of SHR. Besides, IL-1β and IL-10 levels were decreased in the cortex of the ADHD model. Results indicate that SHR presented an oxidative profile that is characterized by an increase in ROS production without an effective antioxidant counterbalance. In addition, this strain showed a decrease in cytokine levels, mainly TNF-α, indicating a basal deficit. These results may present a new approach to the cognitive disturbances seen in the SHR.