Docosahexaenoic acid (DHA, 22:6n-3) is an omega-3 fatty acid essential for proper brain development. N-docosahexaenoylethanolamine (synaptamide), an endogenous metabolite of DHA, potently promotes neurogenesis, neuritogenesis and synaptogenesis; however, the underlying molecular mechanism is not known. Here, we demonstrate orphan G-protein coupled receptor 110 (GPR110, ADGRF1) as the synaptamide receptor, mediating synaptamide-induced bioactivity in a cAMP-dependent manner. Mass spectrometry-based proteomic characterization and cellular fluorescence tracing with chemical analogues of synaptamide reveal specific binding of GPR110 to synaptamide, which triggers cAMP production with low nM potency. Disruption of this binding or GPR110 gene knockout abolishes while GPR110 overexpression enhances synaptamide-induced bioactivity. GPR110 is highly expressed in fetal brains but rapidly decreases after birth. GPR110 knockout mice show significant deficits in object recognition and spatial memory. GPR110 deorphanized as a functional synaptamide receptor provides a novel target for neurodevelopmental control and new insight into mechanisms by which DHA promotes brain development and function.

N-methyl-D-aspartate receptors (NMDAR) have a recognized role in neuronal plasticity while their excessive activation results in excitotoxic death. Therefore, NMDAR antagonists are considered for neuroprotective interventions. However, there is also an emerging role of NMDAR in supporting neuronal survival. Thus, during CNS development, basal NMDAR activity suppresses neuronal apoptosis while moderate NMDAR activation may, at least under some conditions, protect against excitotoxic/ischemic insults. These suggest that while protecting from excitotoxicity, NMDAR antagonists would also reduce pro-survival activity of NMDAR. Hence, the identification of the switches controlling pro-survival vs. pro-excitotoxic outcome of NMDAR stimulation may lead to development of NMDAR antagonists that selectively block the excitotoxicity while enhancing the protective NMDAR signaling. On the other hand, the existence of anti-apoptotic/pro-proliferative NMDAR signaling in transformed cells may result in new strategies to attack cancer. This review focuses on the emerging field of neuroprotective signaling mediators that are implicated in pro-survival activity of NMDAR. We discuss the evidence implicating either NR2B or nonNR2B NMDAR in mediating the protection. We also present the reports linking NMDAR-mediated protection to the activation of survival signaling kinases including ERK and Akt, or suppression of a pro-apoptotic kinase, GSK-3beta. The protective role of transcription factors is also discussed. Finally, we review the existing evidence suggesting that NMDAR support survival by regulating the pro-survival trophic factor signaling and/or the cell death machinery. Although NMDAR provide a major survival input to CNS neurons, the NMDAR-activated protective signaling is poorly understood and, therefore, deserves further research effort.

Serum response factor (SRF)-mediated transcription contributes to developmental and adult brain plasticity. Therefore, we investigated the role of a newly identified SRF coactivator, MKL1, in the regulation of SRF-driven transcription in rat forebrain neurons. MKL1 expression was found in newborn rat cortical or hippocampal neurons in culture as well as in adult rat forebrain. Immunostaining demonstrated constitutive nuclear localization of MKL1 in the CA1 region of the hippocampus, in the deep layers of the neocortex, and in cultured neurons. Overexpression of MKL1 in primary cortical neurons elevated SRF-driven transcription and enhanced its stimulation by BDNF. In addition, inhibition of endogenous MKL1 by overexpression of a dominant-negative MKL1 mutant or by small interfering RNA reduced BDNF activation of SRF-driven transcription. In neurons, endogenous MKL1 was associated with SRF-regulated chromatin regions of several endogenous genes including c-fos, JunB, Srf, and Cyr61. BDNF activation of MKL1/SRF-driven transcription was dependent on the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway, which also led to MKL1 phosphorylation. Finally, synaptic activity stimulation of SRF-driven transcription was reduced by inhibition of endogenous MKL1. Conversely, synaptic activity enhanced transcription by overexpressed MKL1. MKL1 regulation by synaptic activity was mediated through the NMDA receptor-activated ERK1/2. These results suggest that neuronal MKL1 contributes to SRF-regulated gene expression induced by BDNF or synaptic activity. In addition, MKL1 appears as a novel mediator of the signaling between ERK1/2 and SRF. Moreover, MKL1 is a likely regulator of SRF-driven transcription programs that underlie neuronal plasticity.

Docosahexaenoic acid (DHA) has been shown to promote neuronal differentiation of neural stem cells (NSCs) in vivo and in vitro. Previously, we found that N-docosahexenoyethanolamine (synaptamide), an endogenous DHA metabolite with endocannabinoid-like structure, promotes neurite growth, synaptogenesis and synaptic function. In this study, we demonstrate that synaptamide potently induces neuronal differentiation of NSCs. Differentiating NSCs were capable of synthesizing synaptamide from DHA. Treatment of NSCs with synaptamide at low nanomolar concentrations significantly increased the number of MAP2 and Tuj-1 positive neurons with concomitant induction of PKA/CREB phosphorylation. Conversely, PKA inhibitors or PKA knockdown abolished the synaptamide-induced neuronal differentiation of NSCs. URB597, a fatty acid amide hydrolase inhibitor, elevated the level of DHA-derived synaptamide and further potentiated the DHA- or synaptamide-induced neuronal differentiation of NSCs. Similarly, NSCs obtained from fatty acid amide hydrolase (FAAH) KO mice exhibited greater capacity to induce neuronal differentiation in response to DHA or synaptamide compared to the wild type NSCs. Neither synaptamide nor DHA affected NSC differentiation into GFAP-positive glia cells. These results suggest that endogenously produced synaptamide is a potent mediator for neurogenic differentiation of NSCs acting through PKA/CREB activation.

Traumatic brain injury (TBI) induces secondary injury mechanisms, including cell cycle activation (CCA), that leads to neuronal death and neurological dysfunction. We recently reported that delayed administration of roscovitine, a relatively selective cyclin-dependent kinase (CDK) inhibitor, inhibits CCA and attenuates neurodegeneration and functional deficits following controlled cortical impact (CCI) injury in mice. Here we evaluated the neuro-protective potential of CR8, a more potent second-generation roscovitine analog, using the mouse CCI model. Key CCA markers (cyclin A and B1) were significantly up-regulated in the injured cortex following TBI, and phosphorylation of CDK substrates was increased. Central administration of CR8 after TBI, at a dose 20 times less than previously required for roscovitine, attenuated CCA pathways and reduced post-traumatic apoptotic cell death at 24 h post-TBI. Central administration of CR8, at 3 h after TBI, significantly attenuated sensorimotor and cognitive deficits, decreased lesion volume, and improved neuronal survival in the cortex and dentate gyrus. Moreover, unlike roscovitine treatment in the same model, CR8 also attenuated post-traumatic neurodegeneration in the CA3 region of the hippocampus and thalamus at 21 days. Furthermore, delayed systemic administration of CR8, at a dose 10 times less than previously required for roscovitine, significantly improved cognitive performance after CCI. These findings further demonstrate the neuroprotective potential of cell cycle inhibitors following experimental TBI. Given the increased potency and efficacy of CR8 as compared to earlier purine analog types of CDK inhibitors, this drug should be considered as a candidate for future clinical trials of TBI.

To identify the intracellular signaling pathways that mediate the pro-survival activity of NMDA receptors (NMDARs), we studied effects of exogenous NMDA on cultured rat cortical and hippocampal neurons that were treated with a phosphatidylinositol-3-kinase (PI3K) inhibitor, LY294002. NMDA at 5 or 10 lM protected against LY294002-induced apoptosis, suggesting NMDAR-mediated activation of a survival signaling pathway that is PI3K-independent. NR2B-specific NMDAR blockers antagonized anti-apoptotic effects of NMDA, indicating a critical role of NR2B NMDARs in the neuroprotection. NMDA at 10 lM suppressed LY294002-induced activation of a pro-apoptotic kinase, glycogen synthase kinase 3b (GSK3b). GSK3b activation by LY294002 was associated with decreased levels of inhibitory GSK3b phosphorylation at the Ser9 residue. However, NMDA did not prevent the LY294002-mediated decline of phospho-Ser9 levels. In addition, NMDA inhibited cortical neuron apoptosis induced by the overexpression of either wild type (wt) or Ser9Ala mutant form of GSK3b, suggesting that NMDA suppressed GSK3b in a Ser9-independent manner. Finally, inhibition of NR2B NMDARs reduced the NMDA protection against overexpression of GSK3bwt. These data indicate that moderate stimulation of NR2B NMDAR protects against inhibition of PI3K by a Ser9-independent inhibition of the pro-apoptotic activity of GSK3b. Hence, the activation of NR2B and the Ser9-independent inhibition of GSK3b are two newly identified elements of the signaling network that mediates the pro-survival effects of NMDA. Keywords: cell death, extrasynaptic, glutamate receptors, N-methyl-D-aspartate receptor subunit 2B, survival. During development, neuronal survival is supported by multiple signals, including peptide trophic factors and neurotransmitters (Oppenheim 1991;Snider 1994;Verhage et al. 2000). In the central nervous system (CNS), an important pro-survival signal is delivered to neurons by the excitatory neurotransmitter, glutamate. N-Methyl-D-aspartate (NMDA) subtype of ionotropic glutamate receptors (NMDAR) is the principal mediator of glutamate trophic activity. The role of NMDAR in survival support was initially reported in cultured cerebellar granule neurons (CGN). Exogenous NMDA prevented the reduction of CGN survival that was triggered by exposure to suboptimal KCl concentrations (Balazs et al. 1988a;Balazs et al. 1988b). NMDA suppressed CGN apoptosis that followed placement in low KCl media (Yan et al. 1994

Background Binge, as well as chronic, alcohol consumption affects global histone acetylation leading to changes in gene expression. It is becoming increasingly evident that these histone associated epigenetic modifications play an important role in the development of alcohol-mediated hepatic injury. Methods C57BL/6 mice were gavaged 3 times (12 h intervals) with ethanol (4.5 g/kg). Hepatic histone deacetylase (Hdac) mRNAs were assessed by qRT-PCR. Total HDAC activity was estimated by a colorimetric HDAC activity/inhibition assay. Histone acetylation levels were evaluated by Western blot. Liver steatosis and injury were evaluated by histopathology, plasma ALT activity, and liver triglyceride accumulation. Fatty acid synthase (Fas) and carnitine palmitoyl transferase 1a (Cpt1a) expression were also examined. HDAC 9 association with Fas promoter was analyzed. Results Binge alcohol exposure resulted in alterations of hepatic Hdac mRNA levels. Down-regulation of HDAC Class I (Hdac 1), Class II (Hdac 7, 9, 10), Class IV (Hdac 11), and up-regulation of HDAC Class I (Hdac 3) gene expression were observed. Correspondent to the decrease in HDAC activity an increase in hepatic histone acetylation was observed. These molecular events were associated with microvesicular hepatic steatosis and injury characterized by increased hepatic triglycerides (48.02±3.83 vs 19.90±3.48 mg/g liver, p<0.05) and elevated plasma ALT activity (51.98±6.91 vs 20.8±0.62 U/L, p<0.05). Hepatic steatosis was associated with an increase in FAS and a decrease in Cpt1a mRNA and protein expression. Fas promoter analysis revealed that binge ethanol treatment decreased HDAC 9 occupancy at the Fas promoter resulting in its transcriptional activation. Conclusions Deregulation of hepatic Hdac expression likely plays a major role in the binge alcohol-induced hepatic steatosis and liver injury by affecting lipogenesis and fatty acid β-oxidation.

The alternative splicing of the mek5 gene gives rise to two isoforms. MEK5␤ lacks an extended N terminus present in MEK5␣. Comparison of their activities led us to identify a novel mitogen-activated protein kinase (MAPK) docking site in the N terminus of MEK5␣ that is distinct from the consensus motif identified in the other MAPK kinases. It consists of a cluster of acidic residues at position 61 and positions 63 to 66. The formation of the MEK5/extracellular signal-regulated kinase 5 (ERK5) complex is critical for MEK5 to activate ERK5, to increase transcription via MEF2, and to enhance cellular survival in response to osmotic stress. Certain mutations in the ERK5 docking site that prevent MEK5/ERK5 interaction also abrogate the ability of MEKK2 to bind and activate MEK5. However, the identification of MEK5␣ mutants with selective binding defect demonstrates that the MEK5/ERK5 interaction does not rely on the binding of MEK5␣ to MEKK2 via their respective PB1 domains. Altogether these results establish that the N terminus of MEK5␣ is critical for the specific organization of the components of the ERK5 signaling pathway.The mitogen-activated protein kinase (MAPK) signaling pathways regulate numerous physiological processes during development and pathogenesis (10). They consist of the sequential activation of protein kinases that include a MAPK, a MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK or MKK) and a MEK kinase (MEKK) (10). At least four MAPK subfamilies have been identified: ERK1/2, ERK5, c-Jun NH 2 -terminal protein kinases (JNKs), and p38 MAPKs. MAPK activators include MEK1 and MEK2 for ERK1/2, MEK5 for ERK5, MKK4 and MKK7 for JNKs, and MKK3 and MKK6 for p38 MAPKs. MEKs/MKKs activate MAPK by dual phosphorylation on threonine (T) and tyrosine (Y) residues within a T-X-Y motif. A conserved cluster of two to five basic residues identified in the N terminus of MEK1 and -2 and MKK3, -4, -6, and -7 constitutes a consensus MAPK binding site that is necessary for the transmission of the signal (1, 28). A similar motif has been identified by sequence homology in MEK5, but its function as a docking site for ERK5 has not been proven (28).The ERK5 signaling pathway regulates by phosphorylation the activity of a number of transcription factors including myocyte enhancer factor 2 (MEF2) (12, 13). Consistent with its role in stimulating gene expression through the regulation of MEF2 activity, ERK5 contributes in vitro to regulating muscle differentiation and neuronal survival (6,16,24). The analysis of mutant mice in which the erk5 gene can be conditionally deleted has provided in vivo genetic evidence that ERK5 mediates the survival of endothelial cells (9). The loss of endothelial survival may be responsible for the cardiovascular defects observed in erk5 Ϫ/Ϫ and mek5 Ϫ/Ϫ embryos (21,25,29,32). Being twice the size (815 amino acids) of the other MAPKs, ERK5 possesses a unique C-terminal tail that contains a MEF2-interacting domain and a potent transcriptional activation domain (11). The C-terminal tail...