Activation mechanisms of the first sphingosine‐1‐phosphate receptor

Activation of the first sphingosine‐1‐phosphate receptor (S1PR₁) promotes permeability of the blood brain barrier, astrocyte and neuronal protection, and lymphocyte egress from secondary lymphoid tissues. Although an agonist often activates the S1PR₁, the receptor exhibits high levels of basal activity. In this study, we performed long‐timescale molecular dynamics and accelerated molecular dynamics (aMD) simulations to investigate activation mechanisms of the ligand‐free (apo) S1PR₁. In the aMD enhanced sampling simulations, we observed four independent events of activation, which is characterized by close interaction between Y311^7.53 and Y221^5.58 and increased distance between the intracellular ends of transmembrane (TM) helices 3 and 6. Although TM helices TM3, TM6, TM5 and, TM7 are associated with GPCR activation, we discovered that their movements are not necessarily correlated during activation. Instead, TM5 showed a decreased correlation with each of these regions during activation. During activation of the apo receptor, Y221^5.58 and Y311^7.53 became more solvated, because a water channel formed in the intracellular pocket. Additionally, a lipid molecule repeatedly entered the receptor between the extracellular ends of TM1 and TM7, providing important insights into the pathway of ligand entry into the S1PR₁.     

Decreases in plasma membrane Ca2+‐ATPase in brain synaptic membrane rafts from aged rats

Precise regulation of free intracellular Ca²⁺ concentrations [Ca²⁺]_i is critical for normal neuronal function, and alterations in Ca²⁺ homeostasis are associated with brain aging and neurodegenerative diseases. One of the most important proteins controlling [Ca²⁺]_i is the plasma membrane Ca²⁺‐ATPase (PMCA), the high‐affinity transporter that fine tunes the cytosolic nanomolar levels of Ca²⁺. We previously found that PMCA protein in synaptic plasma membranes (SPMs) is decreased with advancing age and the decrease in enzyme activity is much greater than that in protein levels. In this study, we isolated raft and non‐raft fractions from rat brain SPMs and used quantitative mass spectrometry to show that the specialized lipid microdomains in SPMs, the rafts, contain 60% of total PMCA, comprised all four isoforms. The raft PMCA pool had the highest specific activity and this decreased progressively with age. The reduction in PMCA protein could not account for the dramatic activity loss. Addition of excess calmodulin to the assay did not restore PMCA activity to that in young brains. Analysis of the major raft lipids revealed a slight age‐related increase in cholesterol levels and such increases might enhance membrane lipid order and prevent further loss of PMCA activity.     

Vitamin B6 prevents excessive inflammation by reducing accumulation of sphingosine‐1‐phosphate in a sphingosine‐1‐phosphate lyase–dependent manner

Vitamin B6 is necessary to maintain normal metabolism and immune response, especially the anti‐inflammatory immune response. However, the exact mechanism by which vitamin B6 plays the anti‐inflammatory role is still unclear. Here, we report a novel mechanism of preventing excessive inflammation by vitamin B6 via reduction in the accumulation of sphingosine‐1‐phosphate (S1P) in a S1P lyase (SPL)‐dependent manner in macrophages. Vitamin B6 supplementation decreased the expression of pro‐inflammatory cytokines by suppressing nuclear factor‐κB and mitogen‐activated protein kinases signalling pathways. Furthermore, vitamin B6–reduced accumulation of S1P by promoting SPL activity. The anti‐inflammatory effects of vitamin B6 were inhibited by S1P supplementation or SPL deficiency. Importantly, vitamin B6 supplementation protected mice from lethal endotoxic shock and attenuated experimental autoimmune encephalomyelitis progression. Collectively, these findings revealed a novel anti‐inflammatory mechanism of vitamin B6 and provided guidance on its clinical use.     

The effect of the sphingosine‐1‐phosphate analogue FTY720 on atrioventricular nodal tissue

The sphingosine‐1‐phosphate (S1P) receptor modulator, fingolimod (FTY720), has been used for the treatment of patients with relapsing forms of multiple sclerosis, but atrioventricular (AV) conduction block have been reported in some patients after the first dose. The underlying mechanism of this AV node conduction blockade is still not well‐understood. In this study, we hypothesize that expression of this particular arrhythmia might be related to a direct effect of FTY720 on AV node rather than a parasympathetic mimetic action. We, therefore, investigated the effect of FTY720 on AV nodal, using in vitro rat model preparation, under both basal as well as ischaemia/reperfusion conditions. We first look at the expression pattern of S1P receptors on the AV node using real‐time PCR. Although all three S1P receptor isoforms were expressed in AVN tissues, S1P1 receptor isoform expression level was higher than S1P2 and S1P3. The effect of 25 nM FTY720 on cycle length (CL) was subsequently studied via extracellular potentials recordings. FTY720 caused a mild to moderate prolongation in CL by an average 9% in AVN (n = 10, P < 0.05) preparations. We also show that FTY720 attenuated both ischaemia and reperfusion induced AVN rhythmic disturbance. To our knowledge, these remarkable findings have not been previously reported in the literature, and stress the importance for extensive monitoring period in certain cases, especially in patients taking concurrently AV node blocker agents.     

Activity‐dependent release of tissue plasminogen activator from the dendritic spines of hippocampal neurons revealed by live‐cell imaging

Tissue plasminogen activator (tPA) has been implicated in a variety of important cellular functions, including learning‐related synaptic plasticity and potentiating N‐methyl‐D ‐aspartate (NMDA) receptor‐dependent signaling. These findings suggest that tPA may localize to, and undergo activity‐dependent secretion from, synapses; however, conclusive data supporting these hypotheses have remained elusive. To elucidate these issues, we studied the distribution, dynamics, and depolarization‐induced secretion of tPA in hippocampal neurons, using fluorescent chimeras of tPA. We found that tPA resides in dense‐core granules (DCGs) that traffic to postsynaptic dendritic spines and that can remain in spines for extended periods. We also found that depolarization induced by high potassium levels elicits a slow, partial exocytotic release of tPA from DCGs in spines that is dependent on extracellular Ca⁺² concentrations. This slow, partial release demonstrates that exocytosis occurs via a mechanism, such as fuse‐pinch‐linger, that allows partial release and reuse of DCG cargo and suggests a mechanism that hippocampal neurons may rely upon to avoid depleting tPA at active synapses. Our results also demonstrate release of tPA at a site that facilitates interaction with NMDA‐type glutamate receptors, and they provide direct confirmation of fundamental hypotheses about tPA localization and release that bear on its neuromodulatory functions, for example, in learning and memory. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

GSK3α, not GSK3β, drives hippocampal NMDAR‐dependent LTD via tau‐mediated spine anchoring

Glycogen synthase kinase‐3 (GSK3) is an important signalling protein in the brain and modulates different forms of synaptic plasticity. Neuronal functions of GSK3 are typically attributed to one of its two isoforms, GSK3β, simply because of its prevalent expression in the brain. Consequently, the importance of isoform‐specific functions of GSK3 in synaptic plasticity has not been fully explored. We now directly address this question for NMDA receptor‐dependent long‐term depression (LTD) in the hippocampus. Here, we specifically target the GSK3 isoforms with shRNA knock‐down in mouse hippocampus and with novel isoform‐selective drugs to dissect their roles in LTD. Using electrophysiological and live imaging approaches, we find that GSK3α, but not GSK3β, is required for LTD. The specific engagement of GSK3α occurs via its transient anchoring in dendritic spines during LTD induction. We find that the major GSK3 substrate, the microtubule‐binding protein tau, is required for this spine anchoring of GSK3α and mediates GSK3α‐induced LTD. These results link GSK3α and tau in a common mechanism for synaptic depression and rule out a major role for GSK3β in this process.     

Testosterone regulates the expression and functional activity of sphingosine‐1‐phosphate receptors in the rat corpus cavernosum

The bioactive lipid sphingosine‐1‐phosphate (S1P) regulates smooth muscle (SM) contractility predominantly via three G protein‐coupled receptors. The S1P1 receptor is associated with nitric oxide (NO)‐mediated SM relaxation, while S1P2 & S1P3 receptors are linked to SM contraction via activation of the Rho‐kinase pathway. This study is to determine testosterone (T) modulating the expression and functional activity of S1P receptors in corpus cavernosum (CC). Adult male Sprague‐Dawley rats were randomly divided into three groups: sham‐operated controls, surgical castration and T supplemented group. Serum S1P levels were detected by high‐performance liquid chromatography. The expression of S1P1‐3 receptors and sphingosine kinases was detected by real‐time RT‐PCR. In vitro organ bath contractility and in vivo intracavernous pressure (ICP) measurement were also performed. T deprivation significantly decreased ICP rise. Meanwhile, surgical castration induced a significant increase in serum S1P level and the expression of S1P2‐3 receptors by twofold (P < 0.05) but a decrease in the expression of S1P1 receptor. Castration also augmented exogenous phenylephrine (PE), S1P, S1P1,3 receptor agonist FTY720‐P contractility and S1P2‐specific antagonist JTE013 relaxation effect. T supplemented could restore the aforementioned changes. We provide novel data that castration increased serum S1P concentration and up‐regulated the expression of S1P2‐3 receptors in CC. Consistently, agonizing S1P receptors induced CCSM contraction and antagonizing mediated relaxation were augmented. This provides the first clear evidence that S1P system dysregulation may contribute to hypogonadism‐related erectile dysfunction (ED), and S1P receptors may be expected as a potential target for treating ED.     

SCFFbx2‐E3‐ligase‐mediated degradation of BACE1 attenuates Alzheimer’s disease amyloidosis and improves synaptic function

BACE1 (β‐secretase) plays a central role in the β‐amyloidogenesis of Alzheimer’s disease (AD). The ubiquitin–proteasome system, a major intracellular protein quality control system, has been implicated recently in BACE1 metabolism. We report that the SCF^Fbx2‐E3 ligase is involved in the binding and ubiquitination of BACE1 via its Trp 280 residue of F‐box‐associated domain. Physiologically, we found that Fbx2 was expressed in various intracellular organelles in brain neurons and that BACE1 is colocalized with Fbx2 and the amyloid precursor protein (APP), mainly at the early endosome and endoplasmic reticulum. The former are believed to be the major intracellular compartments where the APP is cleaved by BACE1 and β‐amyloid is produced. Importantly, we found that overexpression of Fbx2 in the primary cortical and hippocampal neurons derived from Tg2576 transgenic mice significantly promoted BACE1 degradation and reduced β‐amyloid production. In the search for specific endogenous modulators of Fbx2 expression, we found that PPARγ coactivator‐1α (PGC‐1α) was capable of promoting the degradation of BACE1 through a mechanism involving Fbx2 gene expression. Interestingly, we found that the expression of both Fbx2 and PGC‐1α was significantly decreased in the brains of aging Tg2576 mice. Our in vivo studies using a mouse model of AD revealed that exogenous adenoviral Fbx2 expression in the brain significantly decreased BACE1 protein levels and activity, coincidentally reducing β‐amyloid levels and rescuing synaptic deficits. Our study is the first to suggest that promoting Fbx2 in the brain may represent a novel strategy for the treatment of AD.     

Phosphatidylinositol 4‐phosphate on Rab7‐positive autophagosomes revealed by the freeze‐fracture replica labeling

Phosphatidylinositol 4‐phophate (PtdIns(4)P) is an essential signaling molecule in the Golgi body, endosomal system, and plasma membrane and functions in the regulation of membrane trafficking, cytoskeletal organization, lipid metabolism and signal transduction pathways, all mediated by direct interaction with PtdIns(4)P‐binding proteins. PtdIns(4)P was recently reported to have functional roles in autophagosome biogenesis. LC3 and GABARAP subfamilies and a small GTP‐binding protein, Rab7, are localized on autophagosomal membranes and participate at each stage of autophagosome formation and maturation. To better understand autophagosome biogenesis, it is essential to determine the localization of PtdIns(4)P and to examine its relationship with LC3 and GABARAP subfamilies and Rab7. To analyze PtdIns(4)P distribution, we used an electron microscopy technique that labels PtdIns(4)P on the freeze‐fracture replica of intracellular biological membranes, which minimizes the possibility of artificial perturbation because molecules in the membrane are physically immobilized in situ. Using this technique, we found that PtdIns(4)P is localized on the cytoplasmic, but not the luminal (exoplasmic), leaflet of the inner and outer membranes of autophagosomes. Double labeling revealed that PtdIns(4)P mostly colocalizes with Rab7, but not with LC3B, GABARAP, GABARAPL1 and GABARAPL2. Rab7 plays essential roles in autophagosome maturation and in autophagosome‐lysosome fusion events. We suggest that PtdIns(4)P is localized to the cytoplasmic leaflet of the autophagosome at later stages, which may illuminate the importance of PtdIns(4)P at the later stages of autophagosome formation.      

Structural basis of intramitochondrial phosphatidic acid transport mediated by Ups1‐Mdm35 complex

Ups1 forms a complex with Mdm35 and is critical for the transport of phosphatidic acid (PA) from the mitochondrial outer membrane to the inner membrane. We report the crystal structure of the Ups1‐Mdm35‐PA complex and the functional characterization of Ups1‐Mdm35 in PA binding and transfer. Ups1 features a barrel‐like structure consisting of an antiparallel β‐sheet and three α‐helices. Mdm35 adopts a three‐helical clamp‐like structure to wrap around Ups1 to form a stable complex. The β‐sheet and α‐helices of Ups1 form a long tunnel‐like pocket to accommodate the substrate PA, and a short helix α2 acts as a lid to cover the pocket. The hydrophobic residues lining the pocket and helix α2 are critical for PA binding and transfer. In addition, a hydrophilic patch on the surface of Ups1 near the PA phosphate‐binding site also plays an important role in the function of Ups1‐Mdm35. Our study reveals the molecular basis of the function of Ups1‐Mdm35 and sheds new light on the mechanism of intramitochondrial phospholipid transport by the MSF1/PRELI family proteins.     

Low levels of methyl β‐cyclodextrin disrupt GluA1‐dependent synaptic potentiation but not synaptic depression

Methyl‐β‐cyclodextrin (MβCD) is a reagent that depletes cholesterol and disrupts lipid rafts, a type of cholesterol‐enriched cell membrane microdomain. Lipid rafts are essential for neuronal functions such as synaptic transmission and plasticity, which are sensitive to even low doses of MβCD. However, how MβCD changes synaptic function, such as N‐methyl‐d ‐aspartate receptor (NMDA‐R) activity, remains unclear. We monitored changes in synaptic transmission and plasticity after disrupting lipid rafts with MβCD. At low concentrations (0.5 mg/mL), MβCD decreased basal synaptic transmission and miniature excitatory post‐synaptic current without changing NMDA‐R‐mediated synaptic transmission and the paired‐pulse facilitation ratio. Interestingly, low doses of MβCD failed to deplete cholesterol or affect α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor (AMPA‐R) and NMDA‐R levels, while clearly reducing GluA1 levels selectively in the synaptosomal fraction. Low doses of MβCD decreased the inhibitory effects of NASPM, an inhibitor for GluA2‐lacking AMPA‐R. MβCD successfully decreased NMDA‐R‐mediated long‐term potentiation but did not affect the formation of either NMDA‐R‐mediated or group I metabotropic glutamate receptor‐dependent long‐term depression. MβCD inhibited de‐depression without affecting de‐potentiation. These results suggest that MβCD regulates GluA1‐dependent synaptic potentiation but not synaptic depression in a cholesterol‐independent manner.

     

Tau inhibits PKA by nuclear proteasome‐dependent PKAR2α elevation with suppressed CREB/GluA1 phosphorylation

Intraneuronal accumulation of wild‐type tau plays a key role in Alzheimer's disease, while the mechanisms underlying tauopathy and memory impairment remain unclear. Here, we report that overexpressing full‐length wild‐type human tau (hTau) in mouse hippocampus induces learning and memory deficits with remarkably reduced levels of multiple synapse‐ and memory‐associated proteins. Overexpressing hTau inhibits the activity of protein kinase A (PKA) and decreases the phosphorylation level of cAMP‐response element binding protein (CREB), GluA1, and TrkB with reduced BDNF mRNA and protein levels both in vitro and in vivo. Simultaneously, overexpressing hTau increased PKAR2α (an inhibitory subunit of PKA) in nuclear fraction and inactivated proteasome activity. With an increased association of PKAR2α with PA28γ (a nuclear proteasome activator), the formation of PA28γ‐20S proteasome complex remarkably decreased in the nuclear fraction, followed by a reduced interaction of PKAR2α with 20S proteasome. Both downregulating PKAR2α by shRNA and upregulating proteasome by expressing PA28γ rescued hTau‐induced PKA inhibition and CREB dephosphorylation, and upregulating PKA improved hTau‐induced cognitive deficits in mice. Together, these data reveal that intracellular tau accumulation induces synapse and memory impairments by inhibiting PKA/CREB/BDNF/TrkB and PKA/GluA1 signaling, and deficit of PA28γ‐20S proteasome complex formation contributes to PKAR2α elevation and PKA inhibition.