Docosahexaenoic acid (DHA), the n-3 essential fatty acid that is highly enriched in the brain, increases neurite growth and synaptogenesis in cultured mouse fetal hippocampal neurons. These cellular effects may underlie the DHA-induced enhancement of hippocampus-dependent learning and memory functions. We found that N-docsahexaenoylethanolamide (DEA), an ethanolamide derivative of DHA, is a potent mediator for these actions. This is supported by the observation that DHA is converted to DEA by fetal mouse hippocampal neuron cultures and a hippocampal homogenate, and DEA is present endogenously in the mouse hippocampus. Furthermore, DEA stimulates neurite growth and synaptogenesis at substantially lower concentrations than DHA, and it enhances glutamatergic synaptic activities with concomitant increases in synapsin and glutamate receptor subunit expression in the hippocampal neurons. These findings suggest that DEA, an ethanolamide derivative of DHA, is a synaptogenic factor, and therefore we suggest utilizing the term ‘synaptamide’. This brief review summarizes the neuronal production and actions of synaptamide and describes other N-docosahexaenoyl amides that are present in the brain.
N-Docosahexaenoylethanolamide; Synaptamide; DHA; Hippocampus; Neuron; Anandamide; N-Docosahexaenoyl-amino acylamide
Docosahexaenoic acid (DHA, 22:6n-3), the major polyunsaturated fatty acid accumulated in the brain during development, has been implicated in learning and memory, but underlying cellular mechanisms are not clearly understood. Here, we demonstrate that DHA significantly affects hippocampal neuronal development and synaptic function in developing hippocampi. In embryonic neuronal cultures, DHA supplementation uniquely promoted neurite growth, synapsin puncta formation and synaptic protein expression, particularly synapsins and glutamate receptors. In DHA-supplemented neurons, spontaneous synaptic activity was significantly increased, mostly because of enhanced glutamatergic synaptic activity. Conversely, hippocampal neurons from DHA-depleted fetuses showed inhibited neurite growth and synaptogenesis. Furthermore, n-3 fatty acid deprivation during development resulted in marked decreases of synapsins and glutamate receptor subunits in the hippocampi of 18-day-old pups with concomitant impairment of long-term potentiation, a cellular mechanism underlying learning and memory. While levels of synapsins and NMDA receptor subunit NR2A were decreased in most hippocampal regions, NR2A expression was particularly reduced in CA3, suggesting possible role of DHA in CA3-NMDA receptor-dependent learning and memory processes. The DHA-induced neurite growth, synaptogenesis, synapsin, and glutamate receptor expression, and glutamatergic synaptic function may represent important cellular aspects supporting the hippocampus-related cognitive function improved by DHA.
docosahexaenoic acid; hippocampal development; long-term potentiation; neurite growth; synaptic function; synaptogenesis
Docosahexenoic acid (DHA, 22:6n-3) plays an important role in development of proper brain function in mammals. We have previously reported that DHA promotes synaptogenesis and synaptic function in hippocampal neurons while DHA-depletion in the brain due to n-3 fatty acid deficiency produces opposite effects. To gain insight into underlying molecular mechanisms, we investigated whether the brain DHA status affects the synaptic plasma membrane (SPM) proteome by using nanoLC/ESI-MS/MS and 16O/18O labeling. The DHA level in mouse brains was lowered by dietary depletion of n-3 fatty acids, and SPM was prepared by differential centrifugation followed by osmotic shock. SPM proteins from DHA-adequate and depleted brains were analyzed by nanoLC/ESI-MS/MS after SDS-PAGE, in-gel digestion and differential O18/O16 labeling. This strategy allowed comparative quantitation of more than 200 distinct membrane or membrane-associated proteins from DHA-adequate or depleted brains. We found that 18 pre- and postsynaptic proteins that are relevant to synaptic physiology were significantly down-regulated in DHA-depleted mouse brains. The protein network analysis suggests involvement of CREB and caspase-3 pathways in the DHA-dependent modulation of synaptic proteome. Reduction of specific synaptic proteins due to brain DHA-depletion may be an important mechanism for the suboptimal brain function associated with n-3 fatty acid deficiency.
Synaptic plasma membrane (SPM); synaptic proteins; docosahexaenoic acid (DHA); 18O labeling; nano-LC/ESI-MS/MS; brain
Docosahexaenoic acid (DHA), an omega-3 polyunsaturated fatty acid, is an essential component of membrane phosphatides and has been implicated in cognitive functions. Low levels of circulating or brain DHA are associated with various neurocognitive disorders including Alzheimer’s disease (AD), while laboratory animals, including animal models of AD, can exhibit improved cognitive ability with a diet enriched in DHA. Various cellular mechanisms have been proposed for DHA’s behavioral effects, including increases in cellular membrane fluidity, promotion of neurite extension, and inhibition of apoptosis. However, there is little direct evidence that DHA affects synaptic structure in living animals. Here we show that oral supplementation with DHA substantially increases the number of dendritic spines in adult gerbil hippocampus, particularly when animals are co-supplemented with a uridine source, uridine-5’-monophosphate (UMP), which increases brain levels of the rate-limiting phosphatide precursor CTP. The increase in dendritic spines (> 30%) is accompanied by parallel increases in membrane phosphatides, and in pre- and post-synaptic proteins within the hippocampus. Hence oral DHA may promote neuronal membrane synthesis to increase the number of synapses, particularly when co-administered with UMP. Our findings provide a possible explanation for the effects of DHA on behavior and also suggest a strategy to treat cognitive disorders resulting from synapse loss.
docosahexaenoic acid; uridine; membrane synthesis; spine formation; synaptogenesis; phosphatides
The pathology of traumatic brain injury (TBI) is characterized by the decreased capacity of neurons to metabolize energy and sustain synaptic function, likely resulting in cognitive and emotional disorders. Based on the broad nature of the pathology, we have assessed the potential of the omega-3 fatty acid docosahexaenoic acid (DHA) to counteract the effects of concussive injury on important aspects of neuronal function and cognition. Fluid percussion injury (FPI) or sham injury was performed, and rats were then maintained on a diet high in DHA (1.2% DHA) for 12 days. We found that DHA supplementation, which elevates brain DHA content, normalized levels of brain-derived neurotrophic factor (BDNF), synapsin I (Syn-1), cAMP-responsive element-binding protein (CREB), and calcium/calmodulin-dependent kinase II (CaMKII), and improved learning ability in FPI rats. It is known that BDNF facilitates synaptic transmission and learning ability by modulating Syn-I, CREB, and CaMKII signaling. The DHA diet also counteracted the FPI-reduced manganese superoxide dismutase (SOD) and Sir2 (a NAD+-dependent deacetylase). Given the involvement of SOD and Sir2 in promoting metabolic homeostasis, DHA may help the injured brain by providing resistance to oxidative stress. Furthermore, DHA normalized levels of calcium-independent phospholipase A2 (iPLA2) and syntaxin-3, which may help preserve membrane homeostasis and function after FPI. The overall results emphasize the potential of dietary DHA to counteract broad and fundamental aspects of TBI pathology that may translate into preserved cognitive capacity.
brain-derived neurotrophic factor; plasticity; Sir2; superoxide dismutase; traumatic brain injury
Omega-3 fatty acids (i.e., docosahexaenoic acid; DHA), similar to exercise, improve cognitive function, promote neuroplasticity, and protect against neurological lesion. In this study, we investigated a possible synergistic action between DHA dietary supplementation and voluntary exercise on modulating synaptic plasticity and cognition. Rats received DHA dietary supplementation (1.25% DHA) with or without voluntary exercise for 12 days. We found that the DHA-enriched diet significantly increased spatial learning ability, and these effects were enhanced by exercise. The DHA-enriched diet increased levels of pro-BDNF and mature BDNF, whereas the additional application of exercise boosted the levels of both. Furthermore, the levels of the activated forms of CREB and synapsin I were incremented by the DHA-enriched diet with greater elevation by the concurrent application of exercise. While the DHA diet reduced hippocampal oxidized protein levels, a combination of a DHA diet and exercise resulted in a greater reduction rate. The levels of activated forms of hippocampal Akt and CaMKII were increased by the DHA-enriched diet, and with even greater elevation by a combination of diet and exercise. Akt and CaMKII signaling are crucial step by which BDNF exerts its action on synaptic plasticity and learning and memory. These results indicate that the DHA diet enhance the effects of exercise on cognition and BDNF-related synaptic plasticity, a capacity that may be used to promote mental health and reduce risk of neurological disorders.
DHA; exercise; BDNF; omega-3 fatty acids; cognition
We have previously demonstrated that DHA at low micromolar concentrations has a remarkable effect on morphological differentiation of hippocampal neurons by increasing the population of neurons with more branches and longer neurites. In this study, possible involvement of the retinoid X receptor (RXR) in the DHA-induced hippocampal neurite outgrowth was evaluated as DHA is an endogenous ligand for RXR. Immunocytochemical examination revealed that all RXR isoforms, RXR-alpha, -beta 1, -beta 2 and -gamma, are expressed exclusively in neurons with distinctive intracellular distribution. The cell-based dual luciferase reporter assay indicated that DHA activates RXRα at or above 10 μM but not at 1.5 μM where DHA induces neurite outgrowth. Arachidonic acid also activated RXRα in a similar concentration range but with lower efficacy. Our results suggest that DHA-induced neurite outgrowth may not be mediated by direct activation of RXRα, although involvement of other isoforms or DHA metabolites can not be excluded.
RXR; docosahexaenoic acid; arachidonic acid; hippocampal; neuronal cells
Docosahexaenoic acid (DHA) is the major polyunsaturated fatty acid (PUFA) in the brain and a structural component of neuronal membranes. Changes in DHA content of neuronal membranes lead to functional changes in the activity of receptors and other proteins which might be associated with synaptic function. Accumulating evidence suggests the beneficial effects of dietary DHA supplementation on neurotransmission. This article reviews the beneficial effects of DHA on the brain; uptake, incorporation and release of DHA at synapses, effects of DHA on synapses, effects of DHA on neurotransmitters, DHA metabolites, and changes in DHA with age. Further studies to better understand the metabolome of DHA could result in more effective use of this molecule for treatment of neurodegenerative or neuropsychiatric diseases.
Docosahexaenoic acid (DHA); Polyunsaturated fatty acid (PUFA); Neurodegeneration; Depression; Anti-nociception
Enrichment of polyunsaturated fatty acids, particularly docosahexaenoic acid (DHA, 22:6n–3), in the brain is known to be critical for optimal brain development and function. Mechanisms for DHA’s beneficial effects in the nervous system are not clearly understood at present. DHA is incorporated into the phospholipids in neuronal membranes, which in turn can influence not only the membrane chemical and physical properties but also the cell signaling involved in neuronal survival, proliferation and differentiation. Our studies have indicated that DHA supplementation promotes phosphatidylserine (PS) accumulation and inhibits neuronal cell death under challenged conditions, supporting a notion that DHA is an important neuroprotective agent. This article summarizes our findings on the DHA-mediated membrane-related signaling mechanisms that might explain some of the beneficial effects of DHA, particularly on neuronal survival.
Dietary omega-3 fatty acid (i.e. docosohexaenoic acid (DHA)) and exercise are gaining recognition for supporting brain function under normal and challenging conditions. Here we evaluate the possibility that the interaction of DHA and exercise can involve specific elements of the synaptic plasma membrane. We found that voluntary exercise potentiated the effects of a 12-day DHA dietary supplementation regimen on increasing the levels of syntaxin 3 (STX-3) and the growth-associated protein (GAP-43) in the adult rat hippocampus region. STX-3 is a synaptic membrane-bound protein involved in the effects of DHA on membrane expansion. The DHA diet and exercise also elevated levels of the NMDA receptor subunit NR2B, which is important for synaptic function underlying learning and memory. The actions of exercise and DHA dietary supplementation reflected on enhanced learning performance in the Morris water maze as learning ability was associated with higher levels of STX-3 and NR2B. The overall findings reveal a mechanism by which exercise can interact with the function of DHA dietary enrichment to elevate the capacity of the adult brain for axonal growth, synaptic plasticity, and cognitive function.
Omega-3 fatty acid; Voluntary exercise; Syntaxin; Synaptic membrane; Hippocampus
The endocannabinoid anandamide (ANA) participates in the control of cell death inducing the formation of apoptotic bodies and DNA fragmentation. The aim of this study was to evaluate whether the ANA degrading enzyme, the fatty acid amide hydrolase (FAAH), would induce cellular death. Experiments were performed in cerebellar granule neurons cultured with the FAAH inhibitor, URB597 (25, 50 or 100 nM) as well as endogenous lipids such as oleoylethanolamide (OEA) or palmitoylethanolamide (PEA) and cellular viability was determined by MTT test. Neurons cultured with URB597 (25, 50 or 100 nM) displayed a decrease in cellular viability. In addition, if cultured with OEA (25 nM) or PEA (100 nM), cellular death was found. These results further suggest that URB597, OEA or PEA promote cellular death.
In rodents, many exogenous cannabinoid agonists including Δ9-THC and WIN 55,212-2 (WIN-2) have been shown to impair short-term memory (STM) by inhibition of hippocampal neuronal assemblies. However, the mechanisms by which endocannabinoids such as anandamide and 2-arachidonylglycerol (2-AG) modulate STM processes are not well understood. Here the effects of anandamide on performance of a Delayed Non-Match to Sample (DNMS) task (i.e., STM task) and concomitant hippocampal ensemble activity were assessed following administration of either URB597 (0.3, 3.0mg/kg), an inhibitor of the Fatty Acid Amide Hydrolase (FAAH), AM404 (1.5, 10.0mg/kg), a putative anandamide uptake/FAAH inhibitor, or R-methanandamide (3.0, 10.0mg/kg), a stable analogue of anandamide. Principal cells from hippocampal CA3/CA1 were recorded extracellularly by multi-electrode arrays in Long-Evans rats during DNMS task (1–30s delays) performance and tracked throughout drug administration and recovery. Both R-methanandamide and URB597 caused dose- and delay-dependent deficits in DNMS performance with suppression of hippocampal ensemble activity during the encoding (sample) phase. R-methanandamide induced effects were not reversed by capsaicine excluding a contribution of TRPV-1 receptors. AM404 produced subtle deficits at longer delay intervals but did not alter hippocampal neuronal activity during task-specific events. Collectively, these data indicate that endocannabinoid levels affect performance in a STM task and their pharmacological elevation beyond normal concentrations is detrimental also for the underlying physiological responses. They also highlight a specific window of memory processing, i.e. encoding, which is sensitive to cannabinoid modulation.
delayed non-match to sample; electrophysiology; endocannabinoid; hippocampus; short-term memory; single-unit recording; ensemble code
Deficiency in docosahexaenoic acid (DHA), a brain-essential omega-3 fatty acid, is associated with cognitive decline. Here we report that, in cytokine-stressed human neural cells, DHA attenuates amyloid-β (Aβ) secretion, an effect accompanied by the formation of NPD1, a novel, DHA-derived 10,17S-docosatriene. DHA and NPD1 were reduced in Alzheimer disease (AD) hippocampal cornu ammonis region 1, but not in the thalamus or occipital lobes from the same brains. The expression of key enzymes in NPD1 biosynthesis, cytosolic phospholipase A2 and 15-lipoxygenase, was altered in AD hippocampus. NPD1 repressed Aβ42-triggered activation of proinflammatory genes while upregulating the antiapoptotic genes encoding Bcl-2, Bcl-xl, and Bfl-1(A1). Soluble amyloid precursor protein-α stimulated NPD1 biosynthesis from DHA. These results indicate that NPD1 promotes brain cell survival via the induction of antiapoptotic and neuroprotective gene-expression programs that suppress Aβ42-induced neurotoxicity.
Bioactivities of Docosahexaenoic acid (DHA) and Eicosapentaenoic acid (EPA) depend on their chemical forms. The present study was to investigate short term effects of triglyceride (TG), ethyl ester (EE), free fatty acid (FFA) and phospholipid (PL) forms of omega-3 fatty acid (FA) on lipid metabolism in mice, fed high fat or low fat diet.
Male Balb/c mice were fed with 0.7% different Omega-3 fatty acid formulation: DHA bound free fatty acid (DHA-FFA), DHA bound triglyceride (DHA-TG), DHA bound ethyl ester (DHA-EE) and DHA bound phospholipid (DHA-PL) for 1 week, with dietary fat levels at 5% and 22.5%. Serum and hepatic lipid concentrations were analyzed, as well as the fatty acid composition of liver and brain.
At low fat level, serum total cholesterol (TC) level in mice fed diets with DHA-FFA, DHA-EE and DHA-PL were significantly lower than that in the control group (P < 0.05). Hepatic TG level decreased significantly in mice fed diets with DHA-TG (P < 0.05), DHA-EE (P < 0.05) and DHA-PL (P < 0.05), while TC level in liver was significantly lower in mice fed diets with TG and EE compared with the control group (P < 0.05). At high fat level, mice fed diets with DHA-EE and DHA-PL had significantly lower hepatic TC level compared with the control diet (P < 0.05). Hepatic PL concentration experienced a significant increase in mice fed the diet with PL at high fat level (P < 0.05). Furthermore, both at low and high fat levels, hepatic DHA level significantly increased and AA level significantly decreased in all forms of DHA groups (P < 0.05), compared to control groups at two different fat levels, respectively. Additionally, cerebral DHA level in mice fed diets with DHA-FFA, DHA-EE and DHA-PL significantly increased compared with the control at high fat level (P < 0.05), but no significant differences were observed among dietary treatments for mice fed diets with low fat level.
The present study suggested that not only total dietary fat content but also the molecular forms of omega-3 fatty acids contributed to lipid metabolism in mice. DHA-PL showed effective bioactivity in decreasing hepatic and serum TC, TG levels and increasing omega-3 concentration in liver and brain.
Omega-3 fatty acid; DHA; EPA; Lipid metabolism; Triglycerides; Ethyl ester; Phospholipids
The comparative effects of atropine and the indirect cannabinomimetics URB597 (a fatty acid amide hydrolase inhibitor) and URB602 (a monoacylglycerol lipase inhibitor) on functional and neurobehavioral endpoints following acute diisopropylfluorophosphate intoxication were studied. Male Sprague-Dawley rats were treated with vehicle or DFP (2.5 mg/kg, sc), immediately post-treated with either vehicle, atropine (16 mg/kg), URB597 (3 mg/kg), URB602 (10 mg/kg) or a combination of URB597 and URB602, and functional signs of toxicity as well as nocturnal motor activity were measured daily for seven consecutive days. Performance in the elevated plus maze (for anxiety-like behavior) and the forced swimming test (for depression-like behavior) was measured at days 6-8 and 27-29 after dosing. Twenty-four hours after dosing, DFP markedly reduced cholinesterase activity in selected brain regions and peripheral tissues (diaphragm and plasma). Substantial recovery of cholinesterase activity was noted at both 8 and 29 days after dosing but significant inhibition was still noted in some brain regions at the latest time-point. DFP elicited body weight reductions and typical signs of cholinergic toxicity, and reduced nocturnal ambulation and rearing. Atropine and the cannabinomimetics (alone and in combination) partially attenuated DFP-induced functional signs of toxicity. None of the post-treatments reversed the DFP-induced reduction in ambulation or rearing, however. No significant treatment-related effects on elevated plus maze performance were noted. DFP-treated rats exhibited decreased swimming and increased immobility in the forced swimming test at both time-points. None of the post-treatments had any effect on DFP-induced changes in immobility or swimming at day 8. At day 29, atropine and the combination of URB597/URB602 significantly blocked DFP-induced changes in immobility, while URB597 and the combination reversed DFP-induced changes in swimming. The results suggest that early blockade of muscarinic receptors and enhancement of eCB signaling can attenuate both acute and delayed effects elicited by DFP.
atropine; diisopropylfluorophosphate (DFP); elevated plus maze; fatty acid amide hydrolase (FAAH); forced swimming test; monoacylglycerol lipase (MAGL); motor activity; URB597; URB602
Fatty acid is amide hydrolase which reduce endogenous anandamide. Transient receptor potential vanilloid-1 (TRPV1) channels have been reported to have a role in the modulation of anxiety-like behaviors in rodents. In the present study, the effects of either endocannabinoid system or TRPV1 channels and their possible interaction on anxiety-like behaviors of the rats were explored.
Materials and Methods
Elevated plus-maze test of anxiety was used to induce anxiety. Capsaicin and AMG 9810 as TRPV1 agonist and antagonist respectively were injected into the dorsal hippocampus. URB 597 as selective FAAH inhibitor and AM 251 as CB1 receptor selective antagonist were also injected into the dorsal hippocampus. The effect of AMG 9810 on the response of URB 597 was also examined.
Intra-CA1 injection of URB 597 (0.001, 0.01 and 0.1 µg/rat) and AMG 9810 (0.003, 0.03 and 0.3 µg/rat) produced anxiolytic-like effects. Intra-CA1 infusion of capsaicin (0.003, 0.03 and 0.3 µg/rat) increased the anxiety-related behaviors and AM 251 (0.001, 0.01 and 0.1 µg/rat) did not significantly change the animals behavior. AMG 9810 at the dose of 0.003 µg/rat did not change the anxiolytic-like effect of URB 597.
The results of the present study demonstrated that both endocannabinoid system and TRPV1 receptors may affect anxiety-like behaviors. In addition, it seems that TRPV1 receptors are not involved in the effects of anandamide on anxiety-related behaviors in the CA1 region.
Anandamide; Anxiety; CA1 Region; Fatty-acid amide hydrolase; Rats; TRPV Cation Channels
Learning and memory depend on dendritic spine actin assembly and docosahexaenoic acid (DHA), an essential n-3 (omega-3) polyunsaturated fatty acid (PFA). High DHA consumption is associated with reduced Alzheimer’s disease (AD) risk, yet mechanisms and therapeutic potential remain elusive. Here, we report that reduction of dietary n-3 PFA in an AD mouse model resulted in 80%–90% losses of the p85α subunit of phosphatidylinositol 3-kinase and the postsynaptic actin-regulating protein drebrin, as in AD brain. The loss of postsynaptic proteins was associated with increased oxidation, without concomitant neuron or pre-synaptic protein loss. N-3 PFA depletion increased caspase-cleaved actin, which was localized in dendrites ultrastructurally. Treatment of n-3 PFA-restricted mice with DHA protected against these effects and behavioral deficits and increased antiapoptotic BAD phosphorylation. Since n-3 PFAs are essential for p85-mediated CNS insulin signaling and selective protection of postsynaptic proteins, these findings have implications for neurodegenerative diseases where synaptic loss is critical, especially AD.
Epidermal fatty acid-binding protein (E-FABP), a member of the family of FABPs, exhibits a robust expression in neurons during axonal growth in development and in nerve regeneration following nerve injury. This study examines the impact of E-FABP expression in normal neurite extension in differentiating pheochromocytoma cell (PC12) cultures supplemented with selected long chain free fatty acids (LCFFA). We found that E-FABP binds to a broad range of saturated and unsaturated LCFFAs, including those with potential interest for neuronal differentiation and axonal growth such as C22:6n-3 docosahexaenoic acid (DHA), C20:5n-3 eicosapentaenoic acid (EPA), and C20:4n-6 arachidonic acid (ARA). PC12 cells exposed to nerve growth factor (NGFDPC12) exhibit high E-FABP expression that is blocked by mitogen-activated protein kinase kinase (MEK) inhibitor U0126. Nerve growth factor-differentiated pheochromocytoma cells (NGFDPC12) antisense clones (NGFDPC12-AS) which exhibit low E-FABP expression have fewer/shorter neurites than cells transfected with vector only or NGFDPC12 sense cells (NGFDPC12-S). Replenishing NGFDPC12-AS cells with biotinylated recombinant E-FABP (biotin-E-FABP) protein restores normal neurite outgrowth. Cellular localization of biotin-E-FABP in NGFDPC12 was detected mostly in the cytoplasm and in the nuclear region. Treatment of NGFDPC12 with DHA, EPA, or ARA further enhances neurite length but it does not trigger further induction of TrkA or MEK phosphorylation or E-FABP mRNA observed in differentiating PC12 cells without LCFFA supplementation. Significantly, DHA and EPA neurite stimulating effects are higher in NGFDPC12-S than in NGFDPC12-AS cells. These findings are consistent with the scenario that neurite extension of differentiating PC12 cells, including further stimulation by DHA and EPA, requires sufficient cellular levels of E-FABP.
C20:5n-3 eicosapentaenoic acid; C22:6n-3 docosahexaenoic acid; epidermal fatty acid-binding protein; fatty acid binding; n-3/n-6 polyunsaturated fatty acids; neuronal differentiation
Preclinical and clinical evidence suggests that docosahexaenoic acid (DHA), an omega-3 fatty acid derived from diet or synthesized in the liver, decreases the risk of developing Alzheimer's disease (AD). DHA levels are reduced in the brain of subjects with AD, but it is still unclear whether human dementias are associated with dysregulations of DHA metabolism. A systems biological view of omega-3 fatty acid metabolism offered unexpected insights on the regulation of DHA homeostasis in AD1. Results of multi-organ lipidomic analyses were integrated with clinical and gene-expression data sets to develop testable hypotheses on the functional significance of lipid abnormalities observed and on their possible mechanistic bases. One surprising outcome of this integrative approach was the discovery that the liver of AD patients has a limited capacity to convert shorter chain omega-3 fatty acids into DHA due to a deficit in the peroxisomal D-bifunctional protein. This deficit may contribute to the decrease in brain DHA levels and contribute to cognitive impairment.
Docosahexaenoic acid (DHA) is required for normal brain function. The concentration of DHA in the brain depends on both diet and liver metabolism.
To determine rat brain DHA concentration and consumption in relation to dietary n-3 (omega-3) polyunsaturated fatty acid (PUFA) content and liver secretion of DHA derived from circulating α-linolenic acid (α-LNA).
Following weaning, male rats were fed for 15 weeks either: (1) a diet with a high DHA and α-LNA content, (2) an n-3 PUFA “adequate” diet containing 4.6% α-LNA but no DHA, or (3) an n-3 PUFA “deficient” diet containing 0.2% α-LNA and no DHA. Brain DHA consumption rates were measured following intravenous infusion in unanesthetized rats of [1-14C]DHA, whereas liver and brain DHA synthesis rates were measured by infusing [1-14C]α-LNA.
Brain DHA concentrations equaled 17.6 μm/g, 11.4 μm/g and 7.14 μm/g in rats on diets 1, 2 and 3, respectively. With each diet, the rate of brain DHA synthesis from α-LNA was much less than the brain DHA consumption rate, whereas the liver synthesis-secretion rate was 5-10 fold higher. Higher elongase 2 and 5 and desaturase Δ5 and Δ6 activities in liver than in brain accounted for the higher liver DHA synthesis rates; these enzymes were transcriptionally upregulated in liver but not in brain of rats fed the deficient diet.
While DHA is essential to normal brain function, this need might be covered by dietary α-LNA when liver metabolic conversion machinery is intact and the diet has a high α-LNA content.
docosahexaenoic acid; liver; brain; rat; n-3; omega-3; PUFA; imaging; metabolism; diet; synthesis; α-linolenic acid
Docosahexaenoic acid (DHA), the most abundant essential n-3 polyunsaturated fatty acid in the CNS, emerged recently together with eicosapentaenoic acid (EPA) and DHA/EPA metabolic derivatives as a major player in the resolution of inflammation. Protective antiinflammatory effects of DHA were reported in clinical studies and animal models of colitis, sepsis, and stroke. Here we report for the first time a beneficial effect of dietary n-3 fatty acids in experimental autoimmune encephalomyelitis (EAE), a model for human multiple sclerosis. In the present study we investigated the effects of DHA on the function of bone marrow-derived dendritic cells (DC) in CD4+ T cell stimulation and differentiation. Pretreatment of DC with DHA prevented LPS-induced DC maturation, maintaining an immature phenotype characterized by low expression of costimulatory molecules and lack of proinflammatory cytokine production (IL-12p70, IL-6 and IL-23). DHA-treated DC were poor stimulators of antigen-specific T cells in terms of proliferation and Th1/Th17 differentiation. This was associated with an increase in p27(kip1), a cell cycle arresting agent, and with decreases in Tbet, GATA-3 and RORγt, master transcription factors for Th1, Th2, and Th17. In contrast, T cells co-cultured with DC-DHA express higher levels of TGFβ and Foxp3, without exhibiting a functional Treg phenotype. Similar to the in vitro results, the beneficial effect of DHA in EAE was associated with reduced numbers of IFNγ- and IL-17-producing CD4+ T cells in both spleen and CNS.
Docosahexaenoic acid; Experimental autoimmune encephalomyelitis; IL-12; IL-23; Th1; Th17; Foxp3; Tbet; RORγt
AIM: To investigate the impact of arachidonic acid (AA) and docosahexaenoic acid (DHA) and their combination on colon cancer cell growth.
METHODS: The LS-174T colon cancer cell line was used to study the role of the prostaglandin precursor AA and the omega-3 polyunsaturated fatty acid DHA on cell growth. Cell viability was assessed in XTT assays. For analysis of cell cycle and cell death, flow cytometry and DAPI staining were applied. Expression of cyclooxygenase-2 (COX-2), p21 and bcl-2 in cells incubated with AA or DHA was examined by real-time RT-PCR. Prostaglandin E2 (PGE2) generation in the presence of AA and DHA was measured using a PGE2-ELISA.
RESULTS: AA increased cell growth, whereas DHA reduced viability of LS 174T cells in a time- and dose-dependent manner. Furthermore, DHA down- regulated mRNA of bcl-2 and up-regulated p21. Interestingly, DHA was able to suppress AA-induced cell proliferation and significantly lowered AA-derived PGE2 formation. DHA also down-regulated COX-2 expression. In addition to the effect on PGE2 formation, DHA directly reduced PGE2-induced cell proliferation in a dose-dependent manner.
CONCLUSION: These results suggest that DHA can inhibit the pro-proliferative effect of abundant AA or PGE2.
Colorectal carcinoma; Colon cancer; Omega-3; Omega-6; Polyunsaturated fatty acids; Arachidonic acid; Docosahexaenoic acid; Prostaglandin E2; Cyclooxygenase-2; Apoptosis
Oleoylethanolamide (OEA) and palmitoylethanolamide (PEA) are amides of fatty acids and ethanolamine named N-acylethanolamines or acylethanolamides. The hydrolysis of OEA and PEA is catalyzed by the fatty acid amide hydrolase (FAAH). A number of FAAH inhibitors that increase the levels of OEA and PEA in the brain have been developed, including URB597. In the present report, we examined whether URB597, OEA or PEA injected into wake-related brain areas, such as lateral hypothalamus (LH) or dorsal raphe nuclei (DRN) would promote wakefulness (W) in rats.
Methodology and Principal Findings
Male Wistar rats (250–300 g) were implanted for sleep studies with electrodes to record the electroencephalogram and electromyogram as well as a cannulae aimed either into LH or into DRN. Sleep stages were scored to determine W, slow wave sleep (SWS) and rapid eye movement sleep (REMS). Power spectra bands underly neurophysiological mechanisms of the sleep-wake cycle and provide information about quality rather than quantity of sleep, thus fast Fourier transformation analysis was collected after the pharmacological trials for alpha (for W; α = 8–12 Hz), delta (for SWS; δ = 0.5–4.0 Hz) and theta (for REMS; θ = 6.0–12.0 Hz). Finally, microdialysis samples were collected from a cannula placed into the nucleus accumbens (AcbC) and the levels of dopamine (DA) were determined by HPLC means after the injection of URB597, OEA or PEA. We found that microinjection of compounds (10, 20, 30 µg/1 µL; each) into LH or DRN during the lights-on period increased W and decreased SWS as well as REMS and enhanced DA extracellular levels.
URB597, OEA or PEA promoted waking and enhanced DA if injected into LH or DRN. The wake-promoting effects of these compounds could be linked with the enhancement in levels of DA and indirectly mediated by anandamide.
Primary dissociated neuronal cultures are widely used research tools to investigate of pathological mechanisms and to treat various central and peripheral nervous system problems including trauma and degenerative neuronal diseases. We introduced a protocol that utilizes hippocampal and cortical neurons from embryonic day 17 or 18 mice. We applied appropriate markers (GAP-43 and synaptophysin) to investigate whether neurite outgrowth and synaptogenesis can be distinguished at a particular period of time. GAP-43 was found along the neural processes in a typical granular pattern, and its expression increased proportionally as neurites lengthened during the early in vitro period. Unlike GAP-43, granular immunoreactive patterns of synaptophysin along the neurites were clearly found from day 2 in vitro with relatively high immunoreactive levels. Expression of synaptic markers from cortical neurons reached peak level earlier than that of hippocampal neurons, although neurite outgrowths of hippocampal neurons were faster than those of cortical neurons. The amount of peak synaptic markers expressed was also higher in cortical neurons than that in hippocampal neurons. These results strongly suggest the usefulness of primary cultured neurons from mice embryos for synaptic function and plasticity studies, because of their clear and typical patterns of morphology that establish synapses. Results from this study also suggest the proper amount of time in vitro according to neuronal types (cortical or hippocampal) when utilized in experiments related with synaptogenesis or synaptic activities.
primary neuronal culture; mouse embryo; neurite outgrowth; synaptogenesis; gap-43; synaptophysin
Neuropathic pain elevates spinal anandamide (AEA) levels in a way further increased when URB597, an inhibitor of AEA hydrolysis by fatty acid amide hydrolase (FAAH), is injected intrathecally. Spinal AEA reduces neuropathic pain by acting at both cannabinoid CB1 receptors and transient receptor potential vanilloid-1 (TRPV1) channels. Yet, intrathecal URB597 is only partially effective at counteracting neuropathic pain. We investigated the effect of high doses of intrathecal URB597 on allodynia and hyperalgesia in rats with chronic constriction injury (CCI) of the sciatic nerve. Among those tested, the 200 µg/rat dose of URB597 was the only one that elevated the levels of the FAAH non-endocannabinoid and anti-inflammatory substrates, oleoylethanolamide (OEA) and palmitoylethanolamide (PEA), and of the endocannabinoid FAAH substrate, 2-arachidonoylglycerol, and fully inhibited thermal and tactile nociception, although in a manner blocked almost uniquely by TRPV1 antagonism. Surprisingly, this dose of URB597 decreased spinal AEA levels. RT-qPCR and western blot analyses demonstrated altered spinal expression of lipoxygenases (LOX), and baicalein, an inhibitor of 12/15-LOX, significantly reduced URB597 analgesic effects, suggesting the occurrence of alternative pathways of AEA metabolism. Using immunofluorescence techniques, FAAH, 15-LOX and TRPV1 were found to co-localize in dorsal spinal horn neurons of CCI rats. Finally, 15-hydroxy-AEA, a 15-LOX derivative of AEA, potently and efficaciously activated the rat recombinant TRPV1 channel. We suggest that intrathecally injected URB597 at full analgesic efficacy unmasks a secondary route of AEA metabolism via 15-LOX with possible formation of 15-hydroxy-AEA, which, together with OEA and PEA, may contribute at producing TRPV1-mediated analgesia in CCI rats.