A facile three-step total synthesis of tanshinone I.

A facile synthetic approach for total synthesis of tanshinone I has been accomplished. The key precursor is a novel compound, epoxy phenanthraquinone. And this synthesis of tanshinone I is achieved in only three simple stages, which include Diels-Alder reaction, Δ(2)-Weitz-Scheffer-type epoxidation, and Feist-Bénary reaction from commercially available styrene.

Phosphorylation of Tyrosine 1070 at the GluN2B Subunit Is Regulated by Synaptic Activity and Critical for Surface Expression of N-Methyl-D-aspartate (NMDA) Receptors.

The number and subunit composition of synaptic N-methyl-d-aspartate receptors (NMDARs) play critical roles in synaptic plasticity, learning, and memory and are implicated in neurological disorders. Tyrosine phosphorylation provides a powerful means of regulating NMDAR function, but the underling mechanism remains elusive. In this study we identified a tyrosine site on the GluN2B subunit, Tyr-1070, which was phosphorylated by a proto-oncogene tyrosine-protein (Fyn) kinase and critical for the surface expression of GluN2B-containing NMDARs. The phosphorylation of GluN2B at Tyr-1070 was required for binding of Fyn kinase to GluN2B, which up-regulated the phosphorylation of GluN2B at Tyr-1472. Moreover, our results revealed that the phosphorylation change of GluN2B at Tyr-1070 accompanied the Tyr-1472 phosphorylation and Fyn associated with GluN2B in synaptic plasticity induced by both chemical and contextual fear learning. Taken together, our findings provide a new mechanism for regulating the surface expression of NMDARs with implications for synaptic plasticity.

A novel phosphorylation site of N-methyl-D-aspartate receptor GluN2B at S1284 is regulated by Cdk5 in neuronal ischemia.

N-methyl-D-aspartate receptors (NMDARs) are a key player in synaptic and several neurological diseases, such as stroke. Phosphorylation of NMDAR subunits at their cytoplasmic carboxyl termini has been considered to be an important mechanism to regulate the receptor function. Cyclin-dependent kinase 5 (Cdk5) has been demonstrated to be responsible for regulating phosphorylation and function of NMDARs. Besides, it is also suggested that Cdk5 is involved in ischemic insult. In the present study, we showed that GluN2B subunit serine 1284 at its cytoplasmic carboxyl termini was regulated by Cdk5 in neuronal ischemia. Interestingly, both oxygen glucose deprivation (OGD) in cultured hippocampal neurons and transient global ischemia in mice induce dramatic changes in the phosphorylated level of GluN2B at S1284. However, no significant changes in the phosphorylation of this site are found neither in chemical LTP stimulation in cultured hippocampal neurons nor fear conditioning in adult mice. Taken together, our study identified NMDAR GluN2B S1284 as a novel phosphorylation site regulated by Cdk5 with implication in neuronal ischemia.

An increase in synaptic NMDA receptors in the insular cortex contributes to neuropathic pain.

Neurons in the insular cortex are activated by acute and chronic pain, and inhibition of neuronal activity in the insular cortex has analgesic effects. We found that in a mouse model in which peripheral nerve injury leads to the development of neuropathic pain, the insular cortex showed changes in synaptic plasticity, which were associated with a long-term increase in the amount of synaptic N-methyl-d-aspartate receptors (NMDARs), but not that of extrasynaptic NMDARs. Activation of cyclic adenosine monophosphate (cAMP)-dependent signaling enhanced the amount of synaptic NMDARs in acutely isolated insular cortical slices and increased the surface localization of NMDARs in cultured cortical neurons. We found that the increase in the amount of NMDARs required phosphorylation of the NMDAR subunit GluN2B at Tyr(1472) by a pathway involving adenylyl cyclase subtype 1 (AC1), protein kinase A (PKA), and Src family kinases. Finally, injecting NMDAR or GluN2B-specific antagonists into the insular cortex reduced behavioral responses to normally nonnoxious stimuli in the mouse model of neuropathic pain. Our results suggest that activity-dependent plasticity takes place in the insular cortex after nerve injury and that inhibiting the increase in NMDAR function may help to prevent or treat neuropathic pain.

Adaptor protein APPL1 couples synaptic NMDA receptor with neuronal prosurvival phosphatidylinositol 3-kinase/Akt pathway.

It is well known that NMDA receptors (NMDARs) can both induce neurotoxicity and promote neuronal survival under different circumstances. Recent studies show that such paradoxical responses are related to the receptor location: the former to the extrasynaptic and the latter to the synaptic. The phosphoinositide 3-kinase (PI3K)/Akt kinase cascade is a key pathway responsible for the synaptic NMDAR-dependent neuroprotection. However, it is still unknown how synaptic NMDARs are coupled with the PI3K/Akt pathway. Here, we explored the role of an adaptor protein-adaptor protein containing pH domain, PTB domain, and leucine zipper motif (APPL1)-in this signal coupling using rat cortical neurons. We found that APPL1 existed in postsynaptic densities and associated with the NMDAR complex through binding to PSD95 at its C-terminal PDZ-binding motif. NMDARs, APPL1, and the PI3K/Akt cascade formed a complex in rat cortical neurons. Synaptic NMDAR activity increased the association of this complex, induced activation of the PI3K/Akt pathway, and consequently protected neurons against starvation-induced apoptosis. Perturbing APPL1 interaction with PSD95 by a peptide comprising the APPL1 C-terminal PDZ-binding motif dissociated the PI3K/Akt pathway from NMDARs. Either the peptide or lentiviral knockdown of APPL1 blocked synaptic NMDAR-dependent recruitment and activation of PI3K/Akt pathway, and consequently blocked synaptic NMDAR-dependent neuroprotection. These results suggest that APPL1 contributes to connecting synaptic NMDARs with the intracellular PI3K/Akt cascade and the downstream prosurvival signaling pathway in rat cortical neurons.

Transmembrane region of N-methyl-D-aspartate receptor (NMDAR) subunit is required for receptor subunit assembly.

N-Methyl-D-aspartate receptors (NMDARs), one of three main classes of ionotropic glutamate receptors, play major roles in synaptic plasticity, synaptogenesis, and excitotoxicity. Unlike non-NMDA receptors, NMDARs are thought to comprise obligatory heterotetrameric complexes mainly composed of GluN1 and GluN2 subunits. When expressed alone in heterogenous cells, such as HEK293 cells, most of the NMDAR subunits can neither leave the endoplasmic reticulum (ER) nor be expressed in the cell membrane because of the ER retention signals. Only when NMDARs are heteromerically assembled can the ER retention signals be masked and NMDARs be expressed in the surface membrane. However, the mechanisms underlying NMDAR assembly remain poorly understood. To identify regions in subunits that mediate this assembly, we made a series of truncated or chimeric cDNA constructs. Using FRET measurement in living cells combined with immunostaining and coimmunoprecipitation analysis, we examined the assembly-determining domains of NMDAR subunits. Our results indicate that the transmembrane region of subunits is necessary for the assembly of NMDAR subunits, both for the homodimer and the heteromer.