N-Docosahexaenoylethanolamide promotes development of hippocampal neurons

DHA (docosahexaenoic acid, C22:6,n-3) has been shown to promote neurite growth and synaptogenesis in embryonic hippocampal neurons, supporting the importance of DHA known for hippocampus-related learning and memory function. In the present study, we demonstrate that DHA metabolism to DEA (N-docosahexaenoylethanolamide) is a significant mechanism for hippocampal neuronal development, contributing to synaptic function. We found that a fatty acid amide hydrolase inhibitor URB597 potentiates DHA-induced neurite growth, synaptogenesis and synaptic protein expression. Active metabolism of DHA to DEA was observed in embryonic day 18 hippocampal neuronal cultures, which was increased further by URB597. Synthetic DEA promoted hippocampal neurite growth and synaptogenesis at substantially lower concentrations in comparison with DHA. DEA-treated neurons increased the expression of synapsins and glutamate receptor subunits and exhibited enhanced glutamatergic synaptic activity, as was the case for DHA. The DEA level in mouse fetal hippocampi was altered according to the maternal dietary supply of n-3 fatty acids, suggesting that DEA formation is a relevant in vivo process responding to the DHA status. In conclusion, DHA metabolism to DEA is a significant biochemical mechanism for neurite growth, synaptogenesis and synaptic protein expression, leading to enhanced glutamatergic synaptic function. The novel DEA-dependent mechanism offers a new molecular insight into hippocampal neurodevelopment and function.     

Docosahexaenoic acid promotes hippocampal neuronal development and synaptic function

Docosahexaenoic acid (DHA, 22:6 n -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.     

Synaptamide,endocannabinoid-like derivative of docosahexaenoic acid with cannabinoid-independent function

Docosahexaenoylethanolamide, the structural analog of the endogenous cannabinoid receptor ligand anandamide, is synthesized from docosahexaenoic acid (DHA) in the brain. Although docosahexaenoylethanolamide binds weakly to cannabinoid receptors, it stimulates neurite growth, synaptogenesis and glutamatergic synaptic activity in developing hippocampal neurons at concentrations of 10–100 nM. We have previously proposed the term synaptamide for docosahexaenoylethanolamide to emphasize its potent synaptogenic activity and structural similarity to anandamide. Synaptamide is subjected to hydrolysis by fatty acid amide hydrolase, and can be oxygenated to bioactive metabolites. The brain synaptamide content is dependent on the dietary DHA intake, suggesting an endogenous mechanism whereby diets containing adequate amounts of omega-3 fatty acids improve synaptogenesis in addition to well-recognized anti-inflammatory effects.     

Docosahexaenoic acid promotes neurite growth in hippocampal neurons

Docosahexanoic acid (22:6n-3; DHA) deficiency during development is associated with impairment in learning and memory, suggesting an important role of DHA in neuronal development. Here we provide evidence that DHA promotes neuronal differentiation in rat embryonic hippocampal primary cultures. DHA deficiency in vitro was spontaneously induced by culturing hippocampal cells in chemically defined medium. DHA supplementation improved DHA levels to values observed in freshly isolated hippocampus. We found that DHA supplementation in culture increased the population of neurons with longer neurite length per neuron and with higher number of branches. However, supplementation with arachidonic, oleic or docosapentaenoic acid did not have any effect, indicating specificity of the DHA action on neurite growth. Furthermore, hippocampal cultures obtained from n-3 fatty acid deficient animals contained a lower DHA level and a neuronal population with shorter neurite length per neuron in comparison to those obtained from animals with adequate n-3 fatty acids. DHA supplementation to the deficient group recovered the neurite length to the level similar to n-3 fatty acid adequate cultures. Our data demonstrates that DHA uniquely promotes neurite growth in hippocampal neurons. Inadequate neurite development due to DHA deficiency may contribute to the cognitive impairment associated with n-3 fatty acid deficiency.     

Mechanisms of n-3 fatty acid-mediated development and maintenance of learning memory performance

Docosahexaenoic acid (DHA, 22:6n-3) is specifically enriched in the brain and mainly anchored in the neuronal membrane, where it is involved in the maintenance of normal neurological function. Most DHA accumulation in the brain takes place during brain development in the perinatal period. However, hippocampal DHA levels decrease with age and in the brain disorder Alzheimer's disease (AD), and this decrease is associated with reduced hippocampal-dependent spatial learning memory ability. A potential mechanism is proposed by which the n-3 fatty acids DHA and eicosapentaenoic acid (20:5n-3) aid the development and maintenance of spatial learning memory performance. The developing brain or hippocampal neurons can synthesize and take up DHA and incorporate it into membrane phospholipids, especially phosphatidylethanolamine, resulting in enhanced neurite outgrowth, synaptogenesis and neurogenesis. Exposure to n-3 fatty acids enhances synaptic plasticity by increasing long-term potentiation and synaptic protein expression to increase the dendritic spine density, number of c-Fos-positive neurons and neurogenesis in the hippocampus for learning memory processing. In aged rats, n-3 fatty acid supplementation reverses age-related changes and maintains learning memory performance. n-3 fatty acids have anti-oxidative stress, anti-inflammation, and anti-apoptosis effects, leading to neuron protection in the aged, damaged, and AD brain. Retinoid signaling may be involved in the effects of DHA on learning memory performance. Estrogen has similar effects to n-3 fatty acids on hippocampal function. It would be interesting to know if there is any interaction between DHA and estrogen so as to provide a better strategy for the development and maintenance of learning memory.     

Role of RXR in neurite outgrowth induced by docosahexaenoic acid

We have previously demonstrated that docosahexaenoic acid (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 RXR-alpha at or above 10 microM but not at 1.5 microM where DHA induces neurite outgrowth. Arachidonic acid also activated RXR-alpha 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 RXR-alpha, although involvement of other isoforms or DHA metabolites cannot be excluded.     

Effects of docosahexaenoic acid on the survival and neurite outgrowth of rat cortical neurons in primary cultures

Effects of docosahexaenoic acid (DHA) on survival and neurite outgrowth were investigated in primary cultures of rat cortical neurons. Cell cultures were prepared from cortex on embryonic day 18 (E-18) for treatment with a series of DHA concentrations (12.5, 25, 50, 75, 100 and 200 microM). Docosahexaenoic acid (25-50 microM) significantly enhanced neuronal viability, but lower concentration of DHA (12.5 microM) did not show an obvious effect. In contrast, higher concentrations of DHA (100-200 microM) exerted the significant opposite effects by decreasing neuronal viability. Furthermore, treatment with 25 microM DHA significantly prevented the neurons from death after different culture days in vitro (DIV). Moreover, measurements from the cultures exposed to 25 microM DHA immediately after plating showed significant increases in the percentage of cells with neurites, the mean number of neurite branches, the total neuritic length per cell and the length of the longest neurite in each cell after 24 and 48 h in vitro (HIV). The DHA-treated neurons had greater growth-associated protein-43 (GAP-43) immunoactivity and higher phosphatidylserine (PS) and phosphatidylethanolamine (PE) contents, but lower phosphatidylcholine (PC) content than control neurons. The significant increased DHA contents were also observed in both PE and PS in the treated neurons. These findings suggest that optimal DHA (25 microM) may have positive effects on the survival and the neurite outgrowth of the cultured fetal rat cortical neurons, and the effects probably are related to DHA-stimulating neuron-specific protein synthesis and its enhancing the discrete phospholipid (PL) content through enrichment of DHA in the PL species.     

Neurite outgrowth from progeny of epidermal growth factor–responsive hippocampal stem cells is significantly less robust than from fetal hippocampal cells following grafting onto organotypic hippocampal slice cultures: Effect of brain‐derived neurotrophic factor

Epidermal growth factor (EGF)–responsive stem cells from both developing and adult central nervous system (CNS) can be expanded and induced to differentiate into neurons and glia in vitro. Because of their self‐renewal and multipotent properties, these cells can potentially provide an unlimited tissue source for neural grafting in neurodegenerative disorders. However, the capability of neurons derived from these stem cells to project axons to distant targets following grafting, thereby enabling the restoration of damaged CNS circuitry, remains unknown. We hypothesize that grafted EGF‐responsive stem cells and their progeny are not competent to project axons into distant target sites unless exposed to specific neurotrophic factors. We compared neurite outgrowth between gestation day 14 primary mouse hippocampal cells and EGF‐generated secondary neurospheres of postnatal mouse hippocampal stem cells, following grafting onto the CA3 region of organotypic hippocampal slice cultures prepared from postnatal rats. Neurite outgrowth from grafted cells was visualized using immunohistochemical staining for the mouse specific antigen M6. Fetal hippocampal cells showed extensive and specific neurite outgrowth into many regions of the slice, including the CA1 region and distant subiculum, by 7 days after grafting. In contrast, neurite outgrowth from neurosphere cells was nonspecific and restricted to the immediate surrounding region after either 7 or even 15 days following grafting. Application of brain‐derived neurotrophic factor (BDNF) (5 ng in 0.5 μL) to slices on day 1 after grafting significantly enhanced neurite outgrowth from neurosphere cells, but overall neurite outgrowth from neurosphere cells remained decreased compared to that from fetal hippocampal cells. These results underscore that EGF‐responsive stem cell‐derived neurons possess limited intrinsic capability for long‐distance neurite outgrowth compared to fetal neurons. However, neurite outgrowth from EGF‐responsive stem cell–derived neurons can be enhanced by treating with specific neurotrophic factors such as BDNF. © 1999 John Wiley & Sons, Inc. J Neurobiol 38: 391–413, 1999     

Effects of docosahexaenoic acid on mouse brain synaptic plasma membrane proteome analyzed by mass spectrometry and (16)O/(18)O labeling

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 (16)O/(18)O 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 O(18)/O(16) 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.     

Neurite outgrowth from progeny of epidermal growth factor-responsive hippocampal stem cells is significantly less robust than from fetal hippocampal cells following grafting onto organotypic hippocampal slice cultures: effect of brain-derived neurotrophic factor

Epidermal growth factor (EGF)-responsive stem cells from both developing and adult central nervous system (CNS) can be expanded and induced to differentiate into neurons and glia in vitro. Because of their self-renewal and multipotent properties, these cells can potentially provide an unlimited tissue source for neural grafting in neurodegenerative disorders. However, the capability of neurons derived from these stem cells to project axons to distant targets following grafting, thereby enabling the restoration of damaged CNS circuitry, remains unknown. We hypothesize that grafted EGF-responsive stem cells and their progeny are not competent to project axons into distant target sites unless exposed to specific neurotrophic factors. We compared neurite outgrowth between gestation day 14 primary mouse hippocampal cells and EGF-generated secondary neurospheres of postnatal mouse hippocampal stem cells, following grafting onto the CA3 region of organotypic hippocampal slice cultures prepared from postnatal rats. Neurite outgrowth from grafted cells was visualized using immunohistochemical staining for the mouse specific antigen M6. Fetal hippocampal cells showed extensive and specific neurite outgrowth into many regions of the slice, including the CA1 region and distant subiculum, by 7 days after grafting. In contrast, neurite outgrowth from neurosphere cells was nonspecific and restricted to the immediate surrounding region after either 7 or even 15 days following grafting. Application of brain-derived neurotrophic factor (BDNF) (5 ng in 0.5 microL) to slices on day 1 after grafting significantly enhanced neurite outgrowth from neurosphere cells, but overall neurite outgrowth from neurosphere cells remained decreased compared to that from fetal hippocampal cells. These results underscore that EGF-responsive stem cell-derived neurons possess limited intrinsic capability for long-distance neurite outgrowth compared to fetal neurons. However, neurite outgrowth from EGF-responsive stem cell-derived neurons can be enhanced by treating with specific neurotrophic factors such as BDNF.     

A role for caveolin-1 in post-injury reactive neuronal plasticity

Remodeling and plasticity in the adult brain require cholesterol redistribution and synthesis for the formation of new membrane components. Caveolin-1 is a cholesterol-binding membrane protein involved in cellular cholesterol transport and homeostasis. Evidence presented here demonstrates an up-regulation of caveolin-1 in the hippocampus, which was temporally correlated with an increase in synaptophysin during the reinnervation phase in a mouse model of hippocampal deafferentation. Using an in vitro model of neuronal reactive plasticity, we examined the effect of virally mediated overexpression of caveolin-1 on injured differentiated PC12 cells undergoing terminal remodeling. Three days post lesion, caveolin-1-overexpressing cells revealed increases in synaptophysin and GAP-43, two markers of neurite sprouting and synaptogenesis. Morphologically, caveolin-1-overexpressing cells showed a decrease in primary neurite outgrowth and branching as well as an increase in neurite density. Caveolin-1-overexpressing cells also revealed the presence of terminal swelling and beading along processes, consistent with a possible alteration of microtubules stability. Moreover, a focal enrichment of caveolin-1 immunofluorescence was observed at the bases of axonal and dendritic terminals of mouse primary hippocampal neurons. Altogether, these results indicate that caveolin-1 plays an active role in the regulation of injury-induced synaptic and terminal remodeling in the adult CNS.     

Membrane-type 5 matrix metalloproteinase is expressed in differentiated neurons and regulates axonal growth.

Expression of membrane-type (MT) 5 matrix metalloproteinase (MMP) in the mouse brain was examined. MT5-MMP was expressed in the cerebrum in embryos, but it declined after birth. In contrast, expression in the cerebellum started to increase postnatally and continued thereafter. The cells expressing MT5-MMP were postmitotic neurons that showed gelatinolytic activities. Specific expression of MT5-MMP was observed in the neurons but not in the glial cells when embryonal mouse carcinoma P19 cells were differentiated in vitro by retinoic acid treatment. Neurons isolated from dorsal root ganglia also expressed MT5-MMP, and it was localized at the edge of growth cone. Proteoglycans inhibit neurite extension and regulate synaptogenesis. The inhibitory effect of the proteoglycans on neurite extension of dorsal root ganglia neurons was effectively eliminated by recombinant MT5-MMP. Thus, MT5-MMP expressed in neurons may play a role in axonal growth that contributes to the regulation of neural network formation.     

OMgp LRR281～228：OMgp神经突起生长抑制功能的主要结构域

OMgp(oligodendrocyte-myelin glycoprotein)可以通过与MAG、nogo-66等神经再生抑制因子竞争结合同一受体NgR而诱使生长锥溃变和抑制神经突起的生长。以前的研究表明,在OMgp与NgR结合抑制神经突起生长的过程中,OMgp的亮氨酸富含重复序列(LRR)是必需的。为进一步了解OMgp LRR在神经突起生长中的作用及其结构与功能之间的关系,采用PCR-定点突变法对OMgp LRR结构域分段删除,表达了删除不同基因片段后的OMgp LRR蛋白,通过对表达有NgR的CHO细胞(NgR-CHO)的黏附实验和对原代培养神经细胞的抑制实验对其功能进行了研究。结果显示,分别删除了OMgp 25～56、57～133、134～180位氨基酸的OMgp LRR蛋白仍具有结合NgR-CHO和抑制原代培养的神经元突起生长的作用;而删除了第181～228位氨基酸的OMgp LRR蛋白则失去了对原代培养神经元的生长抑制作用,但仍然具有结合NgR的能力。表明OMgp181～228在OMgp的功能中具有重要的意义。删除了第181～228位氨基酸的OMgp LRR蛋白可望作为OMgp的竞争性抑制剂,用于中枢神经系统损伤后神经再生的治疗。     

Versican G3 domain regulates neurite growth and synaptic transmission of hippocampal neurons by activation of epidermal growth factor receptor

Versican is one of the major extracellular matrix (ECM) proteins in the brain. ECM molecules and their cleavage products critically regulate the growth and arborization of neurites, hence adjusting the formation of neural networks. Recent findings have revealed that peptide fragments containing the versican C terminus (G3 domain) are present in human brain astrocytoma. The present study demonstrated that a versican G3 domain enhanced cell attachment, neurite growth, and glutamate receptor-mediated currents in cultured embryonic hippocampal neurons. In addition, the G3 domain intensified dendritic spines, increased the clustering of both synaptophysin and the glutamate receptor subunit GluR2, and augmented excitatory synaptic activity. In contrast, a mutated G3 domain lacking the epidermal growth factor (EGF)-like repeats (G3deltaEGF) had little effect on neurite growth and glutamatergic function. Treating the neurons with the G3-conditioned medium rapidly increased the levels of phosphorylated EGF receptor (pEGFR) and phosphorylated extracellular signal-regulated kinase (pERK), indicating an activation of EGFR-mediated signaling pathways. Blockade of EGFR prevented the G3-induced ERK activation and suppressed the G3-provoked enhancement of neurite growth and glutamatergic function but failed to block the G3-mediated enhancement of cell attachment. These combined results indicate that the versican G3 domain regulates neuronal attachment, neurite outgrowth, and synaptic function of hippocampal neurons via EGFR-dependent and -independent signaling pathway(s). Our findings suggest a role for ECM proteolytic products in neural development and regeneration.     

Docosahexaenoic acid synthesis from n-3 fatty acid precursors in rat hippocampal neurons

Docosahexaenoic acid (DHA), the most abundant n-3 polyunsaturated fatty acid in the brain, has important functions in the hippocampus. To better understand essential fatty acid homeostasis in this region of the brain, we investigated the contributions of n-3 fatty acid precursors in supplying hippocampal neurons with DHA. Primary cultures of rat hippocampal neurons incorporated radiolabeled 18-, 20-, 22-, and 24-carbon n-3 fatty acid and converted some of the uptake to DHA, but the amounts produced from either [1-¹⁴C]α-linolenic or [1-¹⁴C]eicosapentaenoic acid were considerably less than the amounts incorporated when the cultures were incubated with [1-¹⁴C]22:6n-3. Most of the [1-¹⁴C]22:6n-3 uptake was incorporated into phospholipids, primarily ethanolamine phosphoglycerides. Additional studies demonstrated that the neurons converted [1-¹⁴C]linoleic acid to arachidonic acid, the main n-6 fatty acid in the brain. These findings differ from previous results indicating that cerebral and cerebellar neurons cannot convert polyunsaturated fatty acid precursors to DHA or arachidonic acid. Fatty acid compositional analysis demonstrated that the hippocampal neurons contained only 1.1–2.5 mol% DHA under the usual low-DHA culture conditions. The relatively low-DHA content suggests that some responses obtained with these cultures may not be representative of neuronal function in the brain.     

Effects of lipid-derivatized glycosaminoglycans (GAGs), a novel probe for functional analyses of GAGs, on cell-to-substratum adhesion and neurite elongation in primary cultures of fetal rat hippocampal neurons

The effects of glycosaminoglycans (GAG) on cell-to-substratum adhesion and neurite elongation were examined in primary cultures of fetal rat hippocampal neurons using tissue culture dishes coated with GAGs coupled to dipalmitoylphosphatidylethanolamine (PE), a novel probe for biological functions of GAGs. Both chondroitin sulfate conjugate to PE (CS-PE) and hyaluronic acid conjugate to PE (HA-PE) promoted neurite elongation from neurons in a dose-dependent manner when immobilized onto polylysine-coated dishes at various concentrations up to 1.0 microg/ml. The coating of CS-PE or HA-PE at a concentration higher than 1.0 microg/ml resulted in failure of neurite extension and adhesion of neurons to the substrata. In contrast, heparin conjugate to PE (HP-PE) did not exert any effects on neurite elongation or on cell attachment at these concentrations. These findings suggest that GAGs serve as a modulator for neurite elongation during neuronal network formation in the developing central nervous system.     

CRMP4 suppresses apical dendrite bifurcation of CA1 pyramidal neurons in the mouse hippocampus

Collapsin response mediator proteins (CRMPs) are a family of cytosolic phosphoproteins that consist of 5 members (CRMP 1–5). CRMP2 and CRMP4 regulate neurite outgrowth by binding to tubulin heterodimers, resulting in the assembly of microtubules. CRMP2 also mediates the growth cone collapse response to the repulsive guidance molecule semaphorin‐3A (Sema3A). However, the role of CRMP4 in Sema3A signaling and its function in the developing mouse brain remain unclear. We generated CRMP4?/? mice in order to study the in vivo function of CRMP4 and identified a phenotype of proximal bifurcation of apical dendrites in the CA1 pyramidal neurons of CRMP4?/? mice. We also observed increased dendritic branching in cultured CRMP4?/? hippocampal neurons as well as in cultured cortical neurons treated with CRMP4 shRNA. Sema3A induces extension and branching of the dendrites of hippocampal neurons; however, these inductions were compromised in the CRMP4?/? hippocampal neurons. These results suggest that CRMP4 suppresses apical dendrite bifurcation of CA1 pyramidal neurons in the mouse hippocampus and that this is partly dependent on Sema3A signaling. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2012