Bettina E. Kalisch

Differential contributions of de novo and maintenance DNA methyltransferases to object memory processing in the rat hippocampus and perirhinal cortex – a double dissociation

Epigenetic mechanisms are increasingly acknowledged as major players in memory formation. Specifically, DNA methylation is necessary for the formation of long‐term memory in various brain regions, including the hippocampus (HPC); however, its role in the perirhinal cortex (PRh), a structure critical for object memory, has not been characterized. Moreover, the mnemonic effects of selective DNA methyltransferase (DNMT) inhibition have not yet been investigated systematically, despite distinct roles for de novo (DNMT3a, 3b) and maintenance (DNMT1) methyltransferases. Consequently, we assessed the effects of various DNMT inhibitors within the HPC and PRh of rats using the object‐in‐place paradigm, which requires both brain regions. The non‐nucleoside DNA methyltransferase inhibitor RG‐108 impaired long‐term object‐in‐place memory in both regions. Furthermore, intracranial administration of Accell short‐interference RNA sequences to inhibit the expression of individual DNMTs implicated DNMT3a and DNMT1 in the HPC and PRh effects, respectively. mRNA expression analyses revealed a complementary pattern of results, as only de novo DNMT3a and DNMT3b mRNA was upregulated in the HPC (dentate gyrus) following object‐in‐place learning, whereas DNMT1 mRNA was selectively upregulated in the PRh. These results reinforce the established functional double dissociation between the HPC and PRh and imply the operation of different epigenetic mechanisms in brain regions dedicated to long‐term memory processing for different types of information.

Nitric oxide synthase inhibitors modulate nerve growth factor‐mediated regulation of amyloid precursor protein expression in PC12 cells

Nerve growth factor (NGF) can regulate nitric oxide synthase (NOS) expression and nitric oxide (NO) can modulate NGF‐mediated neurotrophic responses. In this study, the role of NO in NGF‐stimulated amyloid precursor protein (APP) levels was studied. PC12 cells were treated with either the non‐selective NOS inhibitor Nω‐nitro‐L‐arginine methylester (L‐NAME) or the inducible NOS selective inhibitor s‐methylisothiourea (S‐MIU), and the effect on NGF‐mediated increases in APP expression was determined. NGF significantly increased total APP protein levels following 96 h of treatment and this increase was prevented in cells pre‐treated with S‐MIU. Pre‐treatment of cells with actinomycin D also blocked this NGF‐mediated induction of APP, indicating de novo protein synthesis is necessary. Treatment with NGF increased APP promoter activity; however, this increase was only partially inhibited by pre‐treatment with S‐MIU and was increased in the presence of L‐NAME. This suggests that NO may be modulating other aspects of APP expression in addition to transcription. Inhibition of NGF signaling pathways was also investigated using inhibitors of mitogen‐activated protein (MAP) kinase (U0126), Akt (LY294002) and protein kinase C (PKC; U73122 and bisindolylmaleimide 1 (BIS‐1)) activation. Inhibition of each of these pathways prevented NGF‐mediated increases in APP protein expression; however, only BIS‐1 attenuated NGF‐mediated increases in promoter activation. This study indicates that NO is involved in the NGF‐mediated regulation of APP, in part at the level of APP transcription and could involve the modulation of NGF signal transduction pathways.

Effects of nerve growth factor and nitric oxide synthase inhibitors on amyloid precursor protein mRNA levels and protein stability.

We determined previously that nitric oxide (NO) modulates the nerve growth factor (NGF)-mediated increases in amyloid precursor protein (APP) levels in PC12 cells. To elucidate potential mechanisms, the effects of NGF and NO synthase (NOS) inhibitors on APP mRNA levels and protein stability were evaluated. Surprisingly, treatment of PC12 cells with NGF resulted in decreased levels of APP695 and APP751/770 mRNA. Therefore, the effect of NGF on APP protein stability was examined using the translation inhibitor, cycloheximide. Under these conditions, NGF did not alter the rate of APP degradation, suggesting that NGF may be enhancing the translation rate of APP. Since NOS inhibitors attenuate the NGF-mediated increase in APP levels, their effect on APP mRNA levels and protein stability was also assessed. S-methylisothiourea (S-MIU), selective for inducible NOS, decreased both APP695 and APP751/770 mRNA levels while the non-selective NOS inhibitor, Nω-nitro-L-arginine methylester (L-NAME) had no effect. In both control and NGF-treated PC12 cells, S-MIU increased the half-life of APP, with the greatest effect observed with the APP695 isoform. Based on these data we propose that in PC12 cells, NGF increases APP levels through enhanced translation rate and that NO, which modulates the NGF-induced increase in APP protein, also regulates APP mRNA levels and could play a role in APP processing.

Nitric oxide and histone deacetylases modulate cocaine-induced mu-opioid receptor levels in PC12 cells

BackgroundCocaine exposure has been reported to alter central μ-opioid receptor (MOR) expression in vivo. The present study employed an in vitro cellular model to explore possible mechanisms that may be involved in this action of cocaine.MethodsTo assess the effects of cocaine on MOR levels, two treatment regimens were tested in PC12 cells: single continuous or multiple intermittent. MOR protein levels were assessed by western blot analysis and quantitative PCR was used to determine relative MOR mRNA expression levels. To evaluate the role of nitric oxide (NO) and histone acetylation in cocaine-induced MOR expression, cells were pre-treated with the NO synthase inhibitor Nω-nitro-L-arginine methylester (L-NAME) or the non-selective histone acetyltransferase inhibitor curcumin.ResultsBoth cocaine treatment regimens significantly increased MOR protein levels and protein stability, but only multiple intermittent treatments increased MOR mRNA levels as well as c-fos mRNA levels and activator protein 1 binding activity. Both regimens increased NO production, and pre-treatment with L-NAME prevented cocaine-induced increases in MOR protein and mRNA levels. Single and multiple cocaine treatment regimens inhibited histone deacetylase activity, and pre-treatment with curcumin prevented cocaine-induced up-regulation of MOR protein expression.ConclusionsIn the PC12 cell model, both NO and histone deacetylase activity regulate cocaine-induced MOR expression at both the transcriptional and post-transcriptional levels. Based on these novel findings, it is hypothesized that epigenetic mechanisms are implicated in cocaine’s action on MOR expression in neurons.

Dissociable roles for histone acetyltransferases p300 and PCAF in hippocampus and perirhinal cortex-mediated object memory.

The importance of histone acetylation for certain types of memory is now well established. However, the specific contributions of the various histone acetyltransferases to distinct memory functions remain to be determined; therefore, we employed selective histone acetyltransferase protein inhibitors and short‐interference RNAs to evaluate the roles of CREB‐binding protein (CBP), E1A‐binding protein (p300) and p300/CBP‐associated factor (PCAF) in hippocampus and perirhinal cortex (PRh)‐mediated object memory. Rats were tested for short‐ (STM) and long‐term memory (LTM) in the object‐in‐place task, which relies on the hippocampus and PRh for spatial memory and object identity processing, respectively. Selective inhibition of these histone acetyltransferases by small‐interfering RNA and pharmacological inhibitors targeting the HAT domain produced dissociable effects. In the hippocampus, CBP or p300 inhibition impaired long‐term but not short‐term object memory, while inhibition of PCAF impaired memory at both delays. In PRh, HAT inhibition did not impair STM, and only CBP and PCAF inhibition disrupted LTM; p300 inhibition had no effects. Messenger RNA analyses revealed findings consistent with the pattern of behavioral effects, as all three enzymes were upregulated in the hippocampus (dentate gyrus) following learning, whereas only CBP and PCAF were upregulated in PRh. These results demonstrate, for the first time, the necessity of histone acetyltransferase activity for PRh‐mediated object memory and indicate that the specific mnemonic roles of distinctive histone acetyltransferases can be dissociated according to specific brain regions and memory timeframe.

Nitric Oxide Synthase Inhibitors Modulate Nerve-Growth-Factor Mediated Activation of Akt

Nitric oxide (NO) modulates nerve-growth-factor- (NGF-) mediated signaling and gene expression. In the present paper, the role of NO in NGF-mediated Akt activation in PC12 and IMR32 cells was investigated. Cells were treated with NGF (50 ng/mL) in the presence or absence of NO synthase (NOS) inhibitors and Akt phosphorylation assessed by western blot analysis. In both cell lines, Akt was phosphorylated within 15 min of NGF treatment. In PC12 cells, this level of phosphorylation was sustained for 60 min, while in IMR32 cells, the activation decreased after 30 min of NGF treatment. The nonselective NOS inhibitor Nω-nitro-L-arginine methylester (L-NAME; 20 mM) had no effect on NGF-mediated Akt phosphorylation in PC12 cells but in combination with NGF, the iNOS selective inhibitor s-methylisothiourea (S-MIU; 2.0 mM) maintained Akt phosphorylation up to 2 h. In IMR32 cells, both L-NAME and S-MIU prolonged the activation of Akt. Pretreatment with 50 μM U0126, a MAP kinase pathway inhibitor, also increased the activation of Akt in both cell lines. These data suggest that NO modulates the duration of phosphorylation of Akt in response to NGF and that this effect may, in part, be mediated by the effects of NO on the Ras-MAP kinase pathway.

Map kinase and PKC signaling pathways modulate NGF-mediated apoE transcription

The present study assessed the mechanisms by which nerve growth factor (NGF) increased the level of apolipoprotein E (apoE) in PC12 cells. NGF (50ng/mL) significantly increased apoE protein levels following 72h of treatment. Similarly NGF increased luciferase activity in cells transfected with a luciferase reporter construct containing a 500bp fragment of the apoE promoter, indicating NGF-induced apoE expression is regulated, at least in part, at the level of transcription. The non-selective nitric oxide synthase (NOS) inhibitor N(ɷ)-nitro-L-arginine methylester (L-NAME; 20mM) did not attenuate the NGF-mediated increase in luciferase activity, while the inducible NOS inhibitor s-methylisothiourea (S-MIU; 2mM) partially attenuated this action of NGF. Inhibition of MAP kinase activation with 50μM U0126 or pre-treatment with the PKC inhibitor bisindolylmaleimide 1 (BIS-1; 10μM) prevented the NGF-mediated activation of the apoE promoter. Pre-treatment with the phospholipase C (PLC) inhibitor U73122 (5μM) partially inhibited the NGF-induced increase in luciferase activity while the Akt inhibitor LY294002 (10μM) had no effect. These data suggest NGF-induced apoE transcription requires MAP kinase and PKC activation and that these TrkA signaling pathways may be modulated by NO.

The Relationship between Choline Acetyltransferase and Nitric Oxide Synthase Isoform Expression in Alzheimer's Disease

Nitric oxide (NO) plays an important role in cholinergic neurotransmission and has been implicated in the progression of Alzheimer’s disease (AD). Cholinergic neurotransmission has been associated with regulation of NO synthase (NOS) expression, and we determined previously that NO modulates choline acetyltransferase (ChAT) expression. In spite of the important links identified between NOS and ChAT, little is known about the relationship between these two enzymes in AD. Therefore in the present study, we compared the expression levels of ChAT and NOS in necropsy brain of individuals with AD and non-demented controls. ChAT and NOS levels were assessed in control and AD caudate, nucleus basalis of Meynert (nbM), cortex and hippocampus by radioenzymatic assay, immunoblot analysis and RT-PCR. We detected a significant decrease in ChAT activity in the cortex of AD cases, but no alterations in NOS activity were observed in any of the brain regions examined. At the mRNA level, no significant decrease in total ChAT mRNA was detected but the decrease in M-ChAT mRNA levels approached significance in the nbM. No difference in nNOS and iNOS mRNA and protein levels was observed between control and AD tissue in any of the four brain regions sampled, but a statistically significant decrease in eNOS mRNA levels was detected in both the cortex and hippocampus of AD brain. Finally, the levels of ChAT and NOS activity, protein or mRNA isoforms did not correlate in most of the brain regions examined, but a reduction in ChAT activity and eNOS mRNA in basal forebrain projection regions was observed. These data suggest that in general, there is no correlation between the levels of NOS and ChAT in control or AD subjects, but a reduction in eNOS levels in the hippocampus and cortex indicates there could be an interaction between eNOS containing cells and basal forebrain projections neurons in AD.

Dissociable roles of GADD45A/B in the rat perirhinal cortex and hippocampus for object memory: different forms of dna methylation?

This free journal suppl. entitled: Special Issue: 25th Biennial Meeting of the International Society for Neurochemistry jointly with the 13th Meeting of the Asian-Pacific Society for Neurochemistry in conjunction with the 35th Meeting of the Australasian Neuroscience Society 23–27 August 2015, Cairns, AustraliaThis free journal suppl. entitled: Special Issue: 25th Biennial Meeting of the International Society for Neurochemistry jointly with the 13th Meeting of the Asian-Pacific Society for Neurochemistry in conjunction with the 35th Meeting of the Australasian Neuroscience Society 23–27 August 2015, Cairns, Australia