In the present study, we demonstrate that metabolism of DHA to DEA is a significant mechanism for hippocampal development. Neurite outgrowth, synaptogenesis and the expression of synapsins and glutamate receptors, which are known to be important for synaptic transmission and glutamatergic synaptic activity, were promoted by DEA treatment in developing hippocampal neurons. DEA effects on hippocampal development and glutamatergic synaptic activity are similar to those previously demonstrated with DHA [12
], suggesting that DEA is, at least in part, responsible for hippocampal development and function promoted by DHA.
The involvement of an FAAH-sensitive form of DHA was apparent in DHA-induced hippocampal development, at least for the neurite growth, synaptogenesis and expression of some synaptic proteins (). The metabolism of DHA to DEA was found to be active in developing hippocampi, and the biological activity of DEA was similar in nature to, but significantly higher than, that of DHA, strongly suggesting that DEA is a principal component for the observed DHA effects. Specific inhibitors of DEA biosynthesis may further confirm whether DEA mediates DHA effects on hippocampal development and function, when such inhibitors become available. The biochemical mechanisms for DEA synthesis are not clear at present. It is possible that similar biosynthetic pathways for producing AEA via NAPE (N
-arachidonyl phosphatidylethanolamine) [25
] may also be involved for DEA formation.
The hippocampal development in non-supplemented neurons was also sensitive to the FAAHI URB597 (). In addition to DEA, N
-oleoylethanolamide at a rather high concentration has been shown to promote neurite growth through PPARα in a fatty acid-depleted experimental condition [26
], suggesting that there are multiple mechanisms supporting the basic requirements for neurite growth, synaptogenesis and synaptic protein expression. The DEA- and DHA-dependent synaptic protein expression demonstrated in the present study may involve the activation of distinctive transcription factors that have yet to be identified. Nevertheless, involvement of PPARα is unlikely, as the expression of PPARα downstream target proteins, CPT1A or LPL, was not altered by DHA or DEA ().
Previous studies have indicated that NPD1 (neuroprotectin D1, also called 10,17S
-docosatriene), a DHA metabolite formed by lipoxygenation, can exert a neuroprotective function, particularly under pathophysiological conditions such as ischaemia [16
]. It has been also reported that 17-hydroxy-DHA, which is thought to be the immediate precursor of NPD1, is produced in ischaemic brains [27
]. For hippocampal neurite growth and synaptogenesis, we found that DEA, an N
-acylated ethanolamide, is a key active metabolite of DHA. The DEA content in the fetal hippocampi decreased with the reduction of DHA through maternal dietary depletion of n
−3 fatty acids (), which is consistent with the previous report that the DEA content in the pig brain can be increased by dietary inclusion of DHA [28
]. As the DEA level correlates with the DHA status, provision of DHA to the developing brain, as well as its metabolism to DEA, is important for neuronal development in offspring. In addition to ethanolamine, DHA has been shown to be N
-acylated to amino acids or neurotransmitters in the brain [29
], although their biological activity has yet to be determined.
The DEA-induced hippocampal synaptogenesis paralleled the enhancement of synaptic activity. The synaptic activity increase due to the DEA treatment was derived mostly from glutamatergic activity, as has been observed in DHA-treated neurons [12
]. Acute applications of DEA showed no effects on synaptic activity (), suggesting that availability of DHA-derived DEA during development for neurite growth, synaptogenesis and synaptic protein expression is an important aspect for enhanced synaptic activity. In this regard, DHA metabolism to DEA is a key mechanism leading to enhanced synaptic function through promoted hippocampal neurodevelopment.
It is well established that the endocannabinoid AEA exerts its biological effects principally through binding to G-protein-coupled CB (cannabinoid) receptors [30
]. It has been demonstrated that activation of CB1
receptors promotes hippocampal neurogenesis in both the embryonic and adult hippocampus [31
]. Although a possible role of DEA in CB1
-mediated signalling cannot be ruled out, involvement of CB1
in the DEA-induced hippocampal development is unlikely. It has been reported that DEA binding to CB1
is substantially weaker than AEA [32
]. Despite its higher binding capability as a natural ligand for CB1
, AEA showed minimal effects on hippocampal development in comparison with DEA (). Moreover, the AEA content observed in E18 hippocampi was significantly lower in comparison with DEA (), further supporting the unlikely involvement of a CB1
-mediated mechanism. Our present results appear to be in line with the previous finding that CB1
activation by AEA does not promote but inhibits neuronal progenitor cell differentiation [33
It has been reported that DHA is an endogenous ligand to RXR (retinoid X receptor) [34
]. It has been also shown that DHA can activate PPARγ, particularly as oxidized forms [35
]. At present, it is not clear whether DEA can also activate RXR or PPARγ. Nevertheless, DHA and DEA appear to target the same transcriptional activity, since DHA and DEA promote the expression of similar specific synaptic proteins. Since DEA is produced from DHA and DEA is significantly more effective than DHA for synaptic protein expression, the involvement of DEA in DHA-promoted hippocampal synaptic protein expression is strongly suggested. It appears possible that the effective DEA level can be reached in the hippocampal neuronal culture even from low micromolar concentrations of DHA, further supporting the role of DEA as an active component in DHA-mediated transcriptional activation for neuronal differentiation and specific synaptic protein expression.
In conclusion, the present study demonstrates that the metabolism of DHA to DEA is a novel and significant mechanism for hippocampal neuronal development and function. DEA produced from DHA promotes neurite development, synaptogenesis and expression of synapsins and glutamate receptors, which in turn leads to improved synaptic transmission. Since DEA is derived from DHA, compromised DEA-dependent hippocampal development may be an underlying mechanism for the learning disability and memory deficit associated with neural DHA depletion due to dietary deficiencies of n−3 fatty acids. More importantly, the DEA-dependent mechanism may offer new molecular targets for the regulation of hippocampal neuronal development and function.