Aging is associated with altered inflammatory, arachidonic acid cascade, and synaptic markers, influenced by epigenetic modifications, in the human frontal cortex.

Aging is a risk factor for Alzheimer's disease (AD) and is associated with cognitive decline. However, underlying molecular mechanisms of brain aging are not clear. Recent studies suggest epigenetic influences on gene expression in AD, as DNA methylation levels influence protein and mRNA expression in postmortem AD brain. We hypothesized that some of these changes occur with normal aging. To test this hypothesis, we measured markers of the arachidonic acid (AA) cascade, neuroinflammation, pro- and anti-apoptosis factors, and gene specific epigenetic modifications in postmortem frontal cortex from nine middle-aged [41 ± 1 (SEM) years] and 10 aged subjects (70 ± 3 years). The aged compared with middle-aged brain showed elevated levels of neuroinflammatory and AA cascade markers, altered pro and anti-apoptosis factors and loss of synaptophysin. Some of these changes correlated with promoter hypermethylation of brain derived neurotrophic factor (BDNF), cyclic AMP responsive element binding protein (CREB), and synaptophysin and hypomethylation of BCL-2 associated X protein (BAX). These molecular alterations in aging are different from or more subtle than changes associated with AD pathology. The degree to which they are related to changes in cognition or behavior during normal aging remains to be evaluated.

Dietary n-6 PUFA deprivation downregulates arachidonate but upregulates docosahexaenoate metabolizing enzymes in rat brain.

BACKGROUND: Dietary n-3 polyunsaturated fatty acid (PUFA) deprivation increases expression of arachidonic acid (AA 20:4n-6)-selective cytosolic phospholipase A(2) (cPLA(2)) IVA and cyclooxygenase (COX)-2 in rat brain, while decreasing expression of docosahexaenoic acid (DHA 22:6n-3)-selective calcium-independent iPLA(2) VIA. Assuming that these enzyme changes represent brain homeostatic responses to deprivation, we hypothesized that dietary n-6 PUFA deprivation would produce changes in the opposite directions. METHODS: Brain expression of PUFA-metabolizing enzymes and their transcription factors was quantified in male rats fed an n-6 PUFA adequate or deficient diet for 15weeks post-weaning. RESULTS: The deficient compared with adequate diet increased brain mRNA, protein and activity of iPLA(2) VIA and 15-lipoxygenase (LOX), but decreased cPLA(2) IVA and COX-2 expression. The brain protein level of the iPLA(2) transcription factor SREBP-1 was elevated, while protein levels were decreased for AP-2α and NF-κB p65, cPLA(2) and COX-2 transcription factors, respectively. CONCLUSIONS: With dietary n-6 PUFA deprivation, rat brain PUFA metabolizing enzymes and some of their transcription factors change in a way that would homeostatically dampen reductions in brain n-6 PUFA concentrations and metabolism, while n-3 PUFA metabolizing enzyme expression is increased. The changes correspond to reported in vitro enzyme selectivities for AA compared with DHA.

Dose-dependent changes in neuroinflammatory and arachidonic acid cascade markers with synaptic marker loss in rat lipopolysaccharide infusion model of neuroinflammation.

BACKGROUND: Neuroinflammation, caused by six days of intracerebroventricular infusion of bacterial lipopolysaccharide (LPS), stimulates rat brain arachidonic acid (AA) metabolism. The molecular changes associated with increased AA metabolism are not clear. We examined effects of a six-day infusion of a low-dose (0.5 ng/h) and a high-dose (250 ng/h) of LPS on neuroinflammatory, AA cascade, and pre- and post-synaptic markers in rat brain. We used artificial cerebrospinal fluid-infused brains as controls. RESULTS: Infusion of low- or high-dose LPS increased brain protein levels of TNFα, and iNOS, without significantly changing GFAP. High-dose LPS infusion upregulated brain protein and mRNA levels of AA cascade markers (cytosolic cPLA2-IVA, secretory sPLA2-V, cyclooxygenase-2 and 5-lipoxygenase), and of transcription factor NF-κB p50 DNA binding activity. Both LPS doses increased cPLA2 and p38 mitogen-activated protein kinase levels, while reducing protein levels of the pre-synaptic marker, synaptophysin. Post-synaptic markers drebrin and PSD95 protein levels were decreased with high- but not low-dose LPS. CONCLUSIONS: Chronic LPS infusion has differential effects, depending on dose, on inflammatory, AA and synaptic markers in rat brain. Neuroinflammation associated with upregulated brain AA metabolism can lead to synaptic dysfunction.

Lamotrigine blocks NMDA receptor-initiated arachidonic acid signalling in rat brain: implications for its efficacy in bipolar disorder.

An up-regulated brain arachidonic acid (AA) cascade and a hyperglutamatergic state characterize bipolar disorder (BD). Lamotrigine (LTG), a mood stabilizer approved for treating BD, is reported to interfere with glutamatergic neurotransmission involving N-methyl-d-aspartate receptors (NMDARs). NMDARs allow extracellular calcium into the cell, thereby stimulating calcium-dependent cytosolic phospholipase A2 (cPLA2) to release AA from membrane phospholipid. We hypothesized that LTG, like other approved mood stabilizers, would reduce NMDAR-mediated AA signalling in rat brain. An acute subconvulsant dose of NMDA (25 mg/kg) or saline was administered intraperitoneally to unanaesthetized rats that had been treated p.o. daily for 42 d with vehicle or a therapeutically relevant dose of LTG (10 mg/kg.d). Regional brain AA incorporation coefficients k* and rates J in, and AA signals, were measured using quantitative autoradiography after intravenous [1-14C]AA infusion, as were other AA cascade markers. In chronic vehicle-treated rats, acute NMDA compared to saline increased k* and J in in widespread regions of the brain, as well as prostaglandin (PG)E2 and thromboxane B2 concentrations. Chronic LTG treatment compared to vehicle reduced brain cyclooxygenase (COX) activity, PGE2 concentration, and DNA-binding activity of the COX-2 transcription factor, NF-κB. Pretreatment with chronic LTG blocked the acute NMDA effects on AA cascade markers. In summary, chronic LTG like other mood stabilizers blocks NMDA-mediated signalling involving the AA metabolic cascade. Since markers of the AA cascade and of NMDAR signalling are up-regulated in the post-mortem BD brain, mood stabilizers generally may be effective in BD by dampening NMDAR signalling and the AA cascade.

Neuroinflammation and synaptic loss.

Neuroinflammation plays a critical role in the progression of many neurodegenerative, neuropsychiatric and viral diseases. In neuroinflammation, activated microglia and astrocytes release cytokines and chemokines as well as nitric oxide, which in turn activate many signal transduction pathways. The cytokines, interleukin-1 beta and tumor necrosis factor alpha, regulate transcription of a number of genes within the brain, which can lead to the formation of pro-inflammatory products of the arachidonic acid cascade. Formation of pro-inflammatory agents and associated cytotoxic products during neuroinflammation can be detrimental to neurons by altering synaptic proteins. Neuroinflammation as well as excitotoxic insults reduce synaptic markers such as synaptophysin and drebrin. Neurodegenerative, neuropsychiatric illnesses and viral infections are accompanied by loss of both pre- and post-synaptic proteins. These synaptic changes may contribute to the progressive cognitive decline and behavioral changes associated with these illnesses.

Chronic olanzapine treatment decreases arachidonic acid turnover and prostaglandin E2 concentration in rat brain.

The atypical antipsychotic, olanzapine (OLZ), is used to treat bipolar disorder, but its therapeutic mechanism of action is not clear. Arachidonic acid (AA, 20:4n-6) plays a critical role in brain signaling and an up-regulated AA metabolic cascade was reported in postmortem brains from bipolar disorder patients. In this study, we tested whether, similar to the action of the mood stabilizers lithium, carbamazepine and valproate, chronic OLZ treatment would reduce AA turnover in rat brain. We administered OLZ (6 mg/kg/day) or vehicle i.p. to male rats once daily for 21 days. A washout group received 21 days of OLZ followed by vehicle on day 22. Two hours after the last injection, [1-¹4C]AA was infused intravenously for 5 min, and timed arterial blood samples were taken. After the rat was killed at 5 min, its brain was microwaved, removed and analyzed. Chronic OLZ decreased plasma unesterified AA concentration, AA incorporation rates and AA turnover in brain phospholipids. These effects were absent after washout. Consistent with reduced AA turnover, OLZ decreased brain cyclooxygenase activity and the brain concentration of the proinflammatory AA-derived metabolite, prostaglandin E2, In view of up-regulated brain AA metabolic markers in bipolar disorder, the abilities of OLZ and the mood stabilizers to commonly decrease prostaglandin E2, and AA turnover in rat brain phospholipids, albeit by different mechanisms, may be related to their efficacy against the disease.

Altered expression of apoptotic factors and synaptic markers in postmortem brain from bipolar disorder patients.

Bipolar disorder (BD) is a progressive psychiatric disorder characterized by recurrent changes of mood and is associated with cognitive decline. There is evidence of excitotoxicity, neuroinflammation, upregulated arachidonic acid (AA) cascade signaling and brain atrophy in BD patients. These observations suggest that BD pathology may be associated with apoptosis as well as with disturbed synaptic function. To test this hypothesis, we measured mRNA and protein levels of the pro-apoptotic (Bax, BAD, caspase-9 and caspase-3) and anti-apoptotic factors (BDNF and Bcl-2) and of pre- and post-synaptic markers (synaptophysin and drebrin), in postmortem prefrontal cortex (Brodmann area 9) from 10 BD patients and 10 age-matched controls. Consistent with the hypothesis, BD brains showed significant increases in protein and mRNA levels of the pro-apoptotic factors and significant decreases of levels of the anti-apoptotic factors and the synaptic markers, synaptophysin and drebrin. These differences may contribute to brain atrophy and progressive cognitive changes in BD.

Chronic NMDA administration to rats increases brain pro-apoptotic factors while decreasing anti-Apoptotic factors and causes cell death.

BACKGROUND: Chronic N-Methyl-d-aspartate (NMDA) administration to rats is reported to increase arachidonic acid signaling and upregulate neuroinflammatory markers in rat brain. These changes may damage brain cells. In this study, we determined if chronic NMDA administration (25 mg/kg i.p., 21 days) to rats would alter expression of pro- and anti-apoptotic factors in frontal cortex, compared with vehicle control. RESULTS: Using real time RT-PCR and Western blotting, chronic NMDA administration was shown to decrease mRNA and protein levels of anti-apoptotic markers Bcl-2 and BDNF, and of their transcription factor phospho-CREB in the cortex. Expression of pro-apoptotic Bax, Bad, and 14-3-3zeta was increased, as well as Fluoro-Jade B (FJB) staining, a marker of neuronal loss. CONCLUSION: This alteration in the balance between pro- and anti-apoptotic factors by chronic NMDA receptor activation in this animal model may contribute to neuronal loss, and further suggests that the model can be used to examine multiple processes involved in excitotoxicity.

Decreased GRK3 but not GRK2 expression in frontal cortex from bipolar disorder patients.

Overactivation of G-protein-mediated functions and altered G-protein regulation have been reported in bipolar disorder (BD) brain. Further, drugs effective in treating BD are reported to up-regulate expression of G-protein receptor kinase (GRK) 3 in rat frontal cortex. We therefore hypothesized that some G-protein subunits and GRK levels would be reduced in the brain of BD patients. We determined protein and mRNA levels of G-protein beta and gamma subunits, GRK2, and GRK3 in post-mortem frontal cortex from 10 BD patients and 10 age-matched controls by using immunoblots and real-time RT-PCR. There were statistically significant decreases in protein and mRNA levels of G-protein subunits beta and gamma and of GRK3 in BD brain but not a significant difference in the GRK2 level. Decreased expression of G-protein subunits and of GRK3 may alter neurotransmission, leading to disturbed cognition and behaviour in BD.

Chronic administration of mood stabilizers upregulates BDNF and bcl-2 expression levels in rat frontal cortex.

Brain-derived neurotrophic factor (BDNF) and B-cell lymphoma-2 (Bcl-2) proteins are neuroprotective factors involved in neuronal signaling, survival and plasticity. Both can be regulated by cyclic AMP response element binding (CREB) protein. Decreased levels of BDNF and Bcl-2 are implicated in the pathogenesis of bipolar disorder. The present study investigated whether chronically administered mood stabilizers would increase BDNF and/or Bcl-2 levels in rat brain. Real time RT-PCR, sandwich ELISA and Western blotting were used to measure BDNF and Bcl-2 mRNA and protein levels in the frontal cortex of rats chronically administered carbamazepine (CBZ) or lamotrigine (LTG) to produce plasma concentrations therapeutically relevant to bipolar disorder. Chronic CBZ and LTG significantly increased BDNF and Bcl-2 mRNA and protein levels in the frontal cortex. A common mechanism of action of mood stabilizers in the treatment of bipolar disorder may involve neuroprotection mediated by upregulation of brain BDNF and Bcl-2 expression.

Chronic NMDA administration increases neuroinflammatory markers in rat frontal cortex: cross-talk between excitotoxicity and neuroinflammation.

Chronic N-Methyl-D: -aspartate (NMDA) administration, a model of excitotoxicity, and chronic intracerebroventricular lipopolysaccharide infusion, a model of neuroinflammation, are reported to upregulate arachidonic acid incorporation and turnover in rat brain phospholipids as well as enzymes involved in arachidonic acid metabolism. This suggests cross-talk between signaling pathways of excitotoxicity and of neuroinflammation, involving arachidonic acid. To test whether chronic NMDA administrations to rats can upregulate brain markers of neuroinflammation, NMDA (25 mg/kg i.p.) or vehicle (1 ml saline/kg i.p.) was administered daily to adult male rats for 21 days. Protein and mRNA levels of cytokines and other inflammatory markers were measured in the frontal cortex using immunoblot and real-time PCR. Compared with chronic vehicle, chronic NMDA significantly increased protein and mRNA levels of interleukin-1beta, tumor necrosis factor alpha, glial fibrillary acidic protein and inducible nitric oxide synthase. Chronic NMDA receptor overactivation results in increased levels of neuroinflammatory markers in the rat frontal cortex, consistent with cross-talk between excitotoxicity and neuroinflammation. As both processes have been reported in a number of human brain diseases, NMDA receptor inhibitors might be of use in treating neuroinflammation in these diseases.

Decrease in the AP-2 DNA-Binding Activity and in the Protein Expression of AP-2 a and AP-2 b in Frontal Cortex of Rats Treated with Lithium for 6 Weeks.

This corrects the article DOI: 10.1038/ajg.2016.125.

RETRACTED: Chronic administration of carbamazepine down-regulates AP-2 DNA-binding activity and AP-2alpha protein expression in rat frontal cortex.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of author Stanley Rapoport, with approval from Biological Psychiatry Editor, John H. Krystal, MD. The National Institutes of Health has found that Dr. Jagadeesh S. Rao engaged in research misconduct by falsifying data in Figures 1, 3, and 5 of the aforementioned manuscript. No other authors were implicated in the data falsification

Chronic treatment with mood stabilizers increases membrane GRK3 in rat frontal cortex.

BACKGROUND: G-protein receptor kinases (GRKs) are a family of serine/threonine kinases involved in the homologous desensitization of agonist activated G-protein coupled receptors (GPCRs). G-protein coupled receptor supersensitivity, possibly as a result of decreased GRK, has been suggested in affective disorders. METHODS: We used immunobloting to determine if chronic, therapeutically relevant doses of lithium (Li+), carbamazepine (CBZ), and valproate (VPA), would increase GRK2/3 protein levels in rat frontal cortex. RESULTS: Chronic Li+ (24%) and CBZ (44%) significantly increased GRK3 in the membrane but not cytosol fractions. Chronic VPA had no effect on GRK3. G-protein receptor kinase 2 protein levels were unchanged by all treatments. The GRK3 membrane to cytosol ratio was increased significantly in Li+ and CBZ treated rats. CONCLUSIONS: These results show that chronically administered Li+ and CBZ, but not VPA, increase the translocation of GRK3 from cytosol to membrane, possibly correcting supersensitivity of GPCRs in bipolar disorder.

Chronic fluoxetine increases cytosolic phospholipase A(2) activity and arachidonic acid turnover in brain phospholipids of the unanesthetized rat.

RATIONALE: Fluoxetine is used to treat unipolar depression and is thought to act by increasing the concentration of serotonin (5-HT) in the synaptic cleft, leading to increased serotonin signaling. The 5-HT(2A/2C) receptor subtypes are coupled to a phospholipase A(2) (PLA(2)). We hypothesized that chronic fluoxetine would increase the brain activity of PLA(2) and the turnover rate of arachidonic acid (AA) in phospholipids of the unanesthetized rat. MATERIALS AND METHODS: To test this hypothesis, rats were administered fluoxetine (10 mg/kg) or vehicle intraperitoneally daily for 21 days. In the unanesthetized rat, [1-(14)C]AA was infused intravenously and arterial blood plasma was sampled until the animal was killed at 5 min and its brain was subjected to chemical, radiotracer, or enzyme analysis. RESULTS: Using equations from our fatty acid model, we found that chronic fluoxetine compared with vehicle increased the turnover rate of AA within several brain phospholipids by 75-86%. The activity and protein levels of brain cytosolic PLA(2) (cPLA(2)) but not of secretory or calcium-independent PLA(2) were increased in rats administered fluoxetine. In a separate group of animals that received chronic fluoxetine followed by a 3-day saline washout, the turnover of AA and activity and protein levels of cPLA(2) were not significantly different from controls. The protein levels of cyclooxygenases 1 and 2 as well as the concentration of prostaglandin E(2) in rats chronically administered fluoxetine did not differ significantly from controls. CONCLUSION: The results support the hypothesis that fluoxetine increases the cPLA(2)-mediated turnover of AA within brain phospholipids.

Chronic lamotrigine does not alter the turnover of arachidonic acid within brain phospholipids of the unanesthetized rat: implications for the treatment of bipolar disorder.

RATIONALE: Drugs that are effective in treating the manic phase of bipolar disorder (lithium, carbamazepine, and valproate) upon chronic administration to rats decrease the turnover of arachidonic acid in their brain phospholipids. Lamotrigine may not be effective in the manic phase, but is effective in delaying the depressive phase and for treating rapid cycling bipolar disorder. Thus, lamotrigine provides a pharmacological tool to differentiate if downregulation of arachidonic acid turnover is specific to drugs effective in the manic phase of bipolar disorder. MATERIALS AND METHODS: To test this hypothesis, rats were administered lamotrigine (10 mg kg(-1) day(-1)) or vehicle intragastrically once daily for 42 days. In the unanesthetized rat, [1-(14)C]arachidonic acid was infused intravenously and arterial blood plasma was sampled until the animal was killed at 5 min, and its microwaved brain was subjected to chemical and radiotracer analysis. RESULTS: Using equations from our fatty acid model, we found that chronic lamotrigine compared with vehicle did not alter the net incorporation rate of plasma arachidonic acid into brain phospholipids, nor did it alter the turnover of arachidonic acid within brain phospholipids. CONCLUSION: Chronic lamotrigine, which is effective in the depressive phase or rapid cycling bipolar disorder does not alter brain arachidonic acid turnover in the unanesthetized rat. These results are consistent with the hypothesis that drugs effective in treating the manic phase of bipolar disorder decrease brain arachidonic acid turnover.

Brain metabolism of nutritionally essential polyunsaturated fatty acids depends on both the diet and the liver.

Plasma alpha-linolenic acid (alpha-LNA, 18:3n-3) and linoleic acid (LA, 18:2n-6) do not contribute significantly to the brain content of docosahexaenoic acid (DHA, 22:6n-3) or arachidonic acid (AA, 20:4n-6), respectively, and neither DHA nor AA can be synthesized de novo in vertebrate tissue. Therefore, measured rates of incorporation of circulating DHA and AA into brain exactly represent their rates of consumption by brain. Positron emission tomography (PET) has been used to show, based on this information, that the adult human brain consumes AA and DHA at rates of 17.8 and 4.6 mg/day, respectively, and that AA consumption does not change significantly with age. In unanesthetized adult rats fed an n-3 PUFA "adequate" diet containing 4.6% alpha-LNA (of total fatty acids) as its only n-3 PUFA, the rate of liver synthesis of DHA was more than sufficient to maintain brain DHA, whereas the brain's rate of DHA synthesis is very low and unable to do so. Reducing dietary alpha-LNA in the DHA-free diet led to upregulation of liver but not brain coefficients of alpha-LNA conversion to DHA and of liver expression of elongases and desaturases that catalyze this conversion. Concurrently, brain DHA loss slowed due to downregulation of several of its DHA-metabolizing enzymes. Dietary alpha-LNA deficiency also promoted accumulation of brain docosapentaenoic acid (22:5n-6), and upregulated expression of AA-metabolizing enzymes, including cytosolic and secretory phospholipases A(2) and cyclooxygenase-2. These changes, plus reduced levels of brain derived neurotrophic factor (BDNF) and cAMP response element-binding protein (CREB) in n-3 PUFA diet deficient rats, likely render their brain more vulnerable to neuropathological insults.

Antimanic therapies target brain arachidonic acid signaling: lessons learned about the regulation of brain fatty acid metabolism.

Bipolar disorder is a major medical, social and economic burden worldwide. However, the biochemical basis of the disorder and the mechanisms of action of effective antibipolar disorder drugs remain elusive. In this paper, we review how combining a kinetic approach to studying the turnover of fatty acids within brain phospholipids of unanesthetized rats along with chronic administration of antimanic drugs (lithium, valproate and carbamazepine) at therapeutically relevant doses, shows that the brain arachidonic acid cascade is a common target of these drugs. The overlapping effects of the three drugs are decreased turnover of arachidonic acid but not of docosahexaenoic acid in rat brain phospholipids, and decreased brain cyclooxygenase-2 and prostaglandin E(2). Whereas lithium and carbamazepine target the transcription of the arachidonic acid-selective calcium-dependent cytosolic phospholipase A(2), valproate is a non-competitive inhibitor of an arachidonic acid-selective acyl-CoA synthetase. Two potential models of bipolar disorder, chronic N-methyl-d-aspartate and n-3 polyunsaturated fatty acid deprivation, opposite to the antimanic drugs, increase the turnover and markers of the arachidonic acid cascade in rat brain. These observations support the hypothesis proposed by Rapoport and colleagues that the arachidonic acid cascade is a common target of mood stabilizers and that by targeting substrate-specific enzymes the turnover of individual fatty acids can be regulated within the brain.