An auxiliary strategy of partial least squares regression in pharmacokinetic/pharmacodynamic studies: A case of application of guhong injection in myocardial ischemia/reperfusion rats.

Guhong injection (GHI) has been applied in the therapy of cardio-cerebrovascular disease in clinic, but there is no report about the pharmacokinetic/pharmacodynamic (PK/PD) research on GHI treating myocardial ischemia/reperfusion (MI/R) injury in rats. In this study, eight compounds of GHI in plasma, including N-acetyl-L-glutamine (NAG), chlorogenic acid (CGA), hydroxysafflor yellow A (HSYA), p-coumaric acid ( pCA), rutin, hyperoside, kaempferol-3-O-rutinoside, and kaempferol-3-O-glucoside, were quantified by LC-MS/MS. We discovered that the values of t1/2ß, k12, V2, and CL2 were larger than those of t1/2α, k21, V1, and CL1 for all compounds. The levels of four biomarkers, creatine kinase-MB (CK-MB), cardiac troponin I (cTn I), ischemia-modified albumin (IMA), and alpha-hydroxybutyrate dehydrogenase (α-HBDH) in plasma were determined by ELISA. The elevated level of these biomarkers induced by MI/R was declined to different degrees via administrating GHI and verapamil hydrochloride (positive control). The weighted regression coefficients of NAG, HSYA, CGA, and pCA in PLSR equations generated from The Unscrambler X software (version 11) were mostly minus, suggesting these four ingredients were positively correlated to the diminution of the level of four biomarkers. Emax and ED50, two parameters in PK/PD equations that were obtained by adopting Drug and Statistics software (version 3.2.6), were almost enlarged with the rise of GHI dosage. Obviously, all analytes were dominantly distributed and eliminated in the peripheral compartment with features of rapid distribution and slow elimination. With the enhancement of GHI dosage, the ingredients only filled in the central compartment if the peripheral compartment was replete. Meanwhile, high-dose of GHI generated the optimum intrinsic activity, but the affinity of compounds with receptors was the worst, which may be caused by the saturation of receptors. Among the eight analytes, NAG, HSYA, CGA, and pCA exhibited superior cardioprotection, which probably served as the pharmacodynamic substance basis of GHI in treating MI/R injury.

Toll-Like Receptor 4 Deficiency Ameliorates Propofol-Induced Impairments of Cognitive Function and Synaptic Plasticity in Young Mice.

Propofol is one of the most used intravenous anesthetic agents, which is widely used in clinical anesthesia induction and maintenance of pediatric patients. Exposure of the developing brain to propofol has been reported to lead to adverse brain changes, which in turn can induce persistent behavioral abnormalities in adulthood. However, the mechanisms by which propofol exposure in the developing brain induces cognitive impairment remain unclear. Here we report that repeated propofol exposure during the second postnatal week impairs spatial learning and memory in young mice. The reduced excitatory synaptic function and synaptogenesis in hippocampal CA1 neurons underlie this cognitive impairment. Propofol exposure specifically activates Toll-like receptor 4 (TLR4)-myeloid differentiation primary response protein 88 (MyD88)-NF-κB signaling pathway. TLR4 deficiency recues propofol exposure-induced synaptic function and cognitive deficits in young mice. Thus, we provide evidence that the activation of the TLR4-mediated pathway by propofol exposure may serve as a crucial trigger for the cognitive impairment in young adulthood caused by repeated exposure to propofol in the developing brain.

Potassium channel Kir 4.1 regulates oligodendrocyte differentiation via intracellular pH regulation.

In humans, loss-of-function mutations of Kcnj10 in SeSAME/EAST syndrome, which encodes the inwardly rectifying K+ channel 4.1 (Kir 4.1), causes progressive neurological decline. Despite its rich expression in oligodendrocyte (OL) lineage cells and an emerging link with demyelinating disease, the function of Kir 4.1 in OLs is unclear. Here we show a novel role of Kir 4.1 in OL development. Kir 4.1 expression is markedly greater in OLs than in OL precursor cells (OPCs), and the down-regulation of Kir 4.1 impairs OL maturation by affecting OPC differentiation. Interestingly, Kir 4.1 regulates the intracellular pH of OPCs and OLs via the Na+ /H+ exchanger, which underlies impeded OPC differentiation by Kir 4.1 inhibition. Furthermore, Kir 4.1 regulates GSK3ß and SOX10, two molecules critical to OPC development. Collectively, our work opens a new avenue to understanding the functions of Kir 4.1 and intracellular pH in OLs.

Correction to: EPAC Negatively Regulates Myelination via Controlling Proliferation of Oligodendrocyte Precursor Cells.

The original version of this article unfortunately contained a mistake.

EPAC Negatively Regulates Myelination via Controlling Proliferation of Oligodendrocyte Precursor Cells.

Increasing evidence suggests that a cyclic adenosine monophosphate (cAMP)-dependent intracellular signal drives the process of myelination. Yet, the signal transduction underlying the action of cAMP on central nervous system myelination remains undefined. In the present work, we sought to determine the role of EPAC (exchange protein activated by cAMP), a downstream effector of cAMP, in the development of the myelin sheath using EPAC1 and EPAC2 double-knockout (EPACdKO) mice. The results showed an age-dependent regulatory effect of EPAC1 and EPAC2 on myelin development, as their deficiency caused more myelin sheaths in postnatal early but not late adult mice. Knockout of EPAC promoted the proliferation of oligodendrocyte precursor cells and had diverse effects on myelin-related transcription factors, which in turn increased the expression of myelin-related proteins. These results indicate that EPAC proteins are negative regulators of myelination and may be promising targets for the treatment of myelin-related diseases.

Gab1 mediates PDGF signaling and is essential to oligodendrocyte differentiation and CNS myelination.

Oligodendrocytes (OLs) myelinate axons and provide electrical insulation and trophic support for neurons in the central nervous system (CNS). Platelet-derived growth factor (PDGF) is critical for steady-state number and differentiation of oligodendrocyte precursor cells (OPCs), but its downstream targets are unclear. Here, we show for the first time that Gab1, an adaptor protein of receptor tyrosine kinase, is specifically expressed in OL lineage cells and is an essential effector of PDGF signaling in OPCs in mice. Gab1 is downregulated by PDGF stimulation and upregulated during OPC differentiation. Conditional deletions of Gab1 in OLs cause CNS hypomyelination by affecting OPC differentiation. Moreover, Gab1 binds to downstream GSK3ß and regulated its activity, and thereby affects the nuclear accumulation of ß-catenin and the expression of a number of transcription factors critical to myelination. Our work uncovers a novel downstream target of PDGF signaling, which is essential to OPC differentiation and CNS myelination.

Leucine-Rich Glioma Inactivated 1 Promotes Oligodendrocyte Differentiation and Myelination via TSC-mTOR Signaling.

Leucine-rich glioma inactivated 1 (Lgi1), a putative tumor suppressor, is tightly associated with autosomal dominant lateral temporal lobe epilepsy (ADLTE). It has been shown that Lgi1 regulates the myelination of Schwann cells in the peripheral nervous system (PNS). However, the function and underlying mechanisms for Lgi1 regulation of oligodendrocyte differentiation and myelination in the central nervous system (CNS) remain elusive. In addition, whether Lgi1 is required for myelin maintenance is unknown. Here, we show that Lgi1 is necessary and sufficient for the differentiation of oligodendrocyte precursor cells and is also required for the maintenance of myelinated fibers. The hypomyelination in Lgi1-/- mice attributes to the inhibition of the biosynthesis of lipids and proteins in oligodendrocytes (OLs). Moreover, we found that Lgi1 deficiency leads to a decrease in expression of tuberous sclerosis complex 1 (TSC1) and activates mammalian target of rapamycin signaling. Together, the present work establishes that Lgi1 is a regulator of oligodendrocyte development and myelination in CNS.

Celecoxib Ameliorates Seizure Susceptibility in Autosomal Dominant Lateral Temporal Epilepsy.

Autosomal dominant lateral temporal epilepsy (ADLTE) is an inherited syndrome caused by mutations in the leucine-rich glioma inactivated 1 (LGI1) gene. It is known that glutamatergic transmission is altered in LGI1 mutant mice, and seizures can be reduced by restoring LGI1 function. Yet, the mechanism underlying ADLTE is unclear. Here, we propose that seizures in male LGI1-/- mice are due to nonsynaptic epileptiform activity in cortical neurons. We examined the intrinsic excitability of pyramidal neurons in the temporal cortex of male LGI1-/- mice and found that the voltage-gated K+ channel Kv1.2 was significantly downregulated. We also found that cytosolic phospholipase A2 (cPLA2)-cyclooxygenase 2 (Cox2) signaling was enhanced in LGI1-/- mice. Interestingly, Cox2 inhibition effectively restored the dysregulated Kv1.2 and reduced the intrinsic excitability of pyramidal neurons. Moreover, in vivo injection of celecoxib, an FDA-approved nonsteroidal anti-inflammatory drug, rescued the defective Kv1.2 (an ∼1.9-fold increase), thereby alleviating the seizure susceptibility and extending the life of LGI1-/- mice by 5 d. In summary, we conclude that LGI1 deficiency dysregulates cPLA2-Cox2 signaling to cause hyperexcitability of cortical pyramidal neurons, and celecoxib is a potential agent to manage human ADLTE.SIGNIFICANCE STATEMENT Haploinsufficiency of the leucine-rich glioma inactivated 1 (LGI1) gene is the major pathogenic basis for ADLTE, an inherited syndrome with no cure to date. Existing studies suggest that altered glutamatergic transmission in the hippocampus causes this disease, but the data are paradoxical. We demonstrate that the loss of LGI1 decreases Kv1.2 expression, enhances intrinsic excitability, and thereby causes epilepsy. Interestingly, for the first time, we show that an FDA-approved drug, celecoxib, rescues the Kv1.2 defect and alleviates seizure susceptibility in LGI1-/- mice, as well as improving their survival. Thus, we suggest that celecoxib is a promising drug for the treatment of ADLTE patients.

Numb deficiency in cerebellar Purkinje cells impairs synaptic expression of metabotropic glutamate receptor and motor coordination.

Protein Numb, first identified as a cell-fate determinant in Drosophila, has been shown to promote the development of neurites in mammals and to be cotransported with endocytic receptors in clathrin-coated vesicles in vitro. Nevertheless, its function in mature neurons has not yet been elucidated. Here we show that cerebellar Purkinje cells (PCs) express high levels of Numb during adulthood and that conditional deletion of Numb in PCs is sufficient to impair motor coordination despite maintenance of a normal cerebellar cyto-architecture. Numb proved to be critical for internalization and recycling of metabotropic glutamate 1 receptor (mGlu1) in PCs. A significant decrease of mGlu1 and an inhibition of long-term depression at the parallel fiber-PC synapse were observed in conditional Numb knockout mice. Indeed, the trafficking of mGlu1 induced by agonists was inhibited significantly in these mutants, but the expression of ionotropic glutamate receptor subunits and of mGlu1-associated proteins was not affected by the loss of Numb. Moreover, transient and persistent forms of mGlu1 plasticity were robustly induced in mutant PCs, suggesting that they do not require mGlu1 trafficking. Together, our data demonstrate that Numb is a regulator for constitutive expression and dynamic transport of mGlu1.

GSK3ß promotes the differentiation of oligodendrocyte precursor cells via ß-catenin-mediated transcriptional regulation.

Oligodendrocytes are generated by the differentiation and maturation of oligodendrocyte precursor cells (OPCs). The failure of OPC differentiation is a major cause of demyelinating diseases; thus, identifying the molecular mechanisms that affect OPC differentiation is critical for understanding the myelination process and repairing after demyelination. Although prevailing evidence shows that OPC differentiation is a highly coordinated process controlled by multiple extrinsic and intrinsic factors, such as growth factors, axon signals, and transcription factors, the intracellular signaling in OPC differentiation is still unclear. Here, we showed that glycogen synthase kinase 3ß (GSK3ß) is an essential positive modulator of OPC differentiation. Both pharmacologic inhibition and knockdown of GSK3ß remarkably suppressed OPC differentiation. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assays and Ki67 staining showed that the effect of GSK3ß on OPC differentiation was not via cell death. Conversely, activated GSK3ß was sufficient to promote OPC differentiation. Our results also demonstrated that the transcription of myelin genes was regulated by GSK3ß inhibition, accompanying accumulated nuclear ß-catenin, and reduced the expression of transcriptional factors that are relevant to the expression of myelin genes. Taken together, our study identified GSK3ß as a profound positive regulator of OPC differentiation, suggesting that GSK3ß may contribute to the inefficient regeneration of oligodendrocytes and myelin repair after demyelination.

Upregulation of glutamate transporter GLT-1 by mTOR-Akt-NF-кB cascade in astrocytic oxygen-glucose deprivation.

Excessive extracellular glutamate leads to neuronal death in central nervous system. Excitatory glutamate transporter subtype 2 (GLT-1) carries bulk of glutamate reuptake in cerebral ischemia. Although GLT-1 expression fluctuates during the period of ischemia, little is known about its regulatory mechanism. Here we show an up-regulation of GLT-1 via mammalian target of rapamycin (mTOR)-Akt-nuclear factor-кB (NF-кB) signaling cascade in oxygen glucose deprivation (OGD). We found that brief rapamycin treatment significantly increased GLT-1 expression in cultured astrocytes. Rapamycin increased phosphorylation of raptor at Ser792 and decreased phosphorylation of rictor at Thr1135, suggesting that both mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) are involved in GLT-1 expression. This conclusion was further confirmed by raptor and rictor disruption experiments. Akt was activated by mTORC1 inhibition and required for GLT-1 expression because triciribine, a specific inhibitor of Akt, blocked the increase of GLT-1 expression. mTOR-Akt cascade then activated NF-кB and increased кB-motif-binding phosphoprotein (KBBP) expression and GLT-1 transcription. We next demonstrated that mTOR-Akt-NF-кB cascade was activated in OGD and subsequently caused the upregulation of GLT-1. Supporting evidence included: (1) inhibition of Akt or NF-кB occluded OGD-induced GLT-1 upregulation; (2) Raptor knock-down plus OGD did not add to the increase of GLT-1 expression; (3) Intact mTORC2 was required for GLT-1 enhancement. In summary, our data first showed that mTOR-Akt-NF-кB cascade played critical roles to up-regulate GLT-1 in OGD. This signaling cascade may work to promote glutamate uptake in brain ischemia and neurodegenerative diseases.

Distinct functions of nuclear distribution proteins LIS1, Ndel1 and NudCL in regulating axonal mitochondrial transport.

Neurons critically depend on the long-distance transport of mitochondria. Motor proteins kinesin and dynein control anterograde and retrograde mitochondrial transport, respectively in axons. The regulatory molecules that link them to mitochondria need to be better characterized. Nuclear distribution (Nud) family proteins LIS1, Ndel1 and NudCL are critical components of cytoplasmic dynein complex. Roles of these Nud proteins in neuronal mitochondrial transport are unknown. Here we report distinct functions of LIS1, Ndel1 and NudCL on axonal mitochondrial transport in cultured hippocampal neurons. We found that LIS1 interacted with kinsein family protein KIF5b. Depletion of LIS1 enormously suppressed mitochondrial motility in both anterograde and retrograde directions. Inhibition of either Ndel1 or NudCL only partially reduced retrograde mitochondrial motility. However, knocking down both Ndel1 and NudCL almost blocked retrograde mitochondrial transport, suggesting these proteins may work together to regulate retrograde mitochondrial transport through linking dynein-LIS1 complex. Taken together, our results uncover novel roles of LIS1, Ndel1 and NudCL in the transport of mitochondria in axons.

Chronic zinc exposure decreases the surface expression of NR2A-containing NMDA receptors in cultured hippocampal neurons.

BACKGROUND: Zinc distributes widely in the central nervous system, especially in the hippocampus, amygdala and cortex. The dynamic balance of zinc is critical for neuronal functions. Zinc modulates the activity of N-methyl-D-aspartate receptors (NMDARs) through the direct inhibition and various intracellular signaling pathways. Abnormal NMDAR activities have been implicated in the aetiology of many brain diseases. Sustained zinc accumulation in the extracellular fluid is known to link to pathological conditions. However, the mechanism linking this chronic zinc exposure and NMDAR dysfunction is poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: We reported that chronic zinc exposure reduced the numbers of NR1 and NR2A clusters in cultured hippocampal pyramidal neurons. Whole-cell and synaptic NR2A-mediated currents also decreased. By contrast, zinc did not affect NR2B, suggesting that chronic zinc exposure specifically influences NR2A-containg NMDARs. Surface biotinylation indicated that zinc exposure attenuated the membrane expression of NR1 and NR2A, which might arise from to the dissociation of the NR2A-PSD-95-Src complex. CONCLUSIONS: Chronic zinc exposure perturbs the interaction of NR2A to PSD-95 and causes the disorder of NMDARs in hippocampal neurons, suggesting a novel action of zinc distinct from its acute effects on NMDAR activity.

AMPA receptors regulate exocytosis and insulin release in pancreatic ß cells.

Ionotropic glutamate receptors (iGluRs) are expressed in islets and insulinoma cells and involved in insulin secretion. However, the exact roles that iGluRs play in ß cells remain unclear. Here, we demonstrated that GluR2-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) were expressed in mouse ß cells. Glutamate application increased both cytosolic calcium and the number of docked insulin-containing granules, which resulted in augmentation of depolarization-induced exocytosis and high-glucose-stimulated insulin release. While glutamate application directly depolarized ß cells, it also induced an enormous depolarization when K(ATP) channels were available. Glutamate application reduced the conductance of K(ATP) channels and increased voltage oscillations. Moreover, actions of AMPARs were absent in Kir6.2 knock-out mice. The effects of AMPARs on K(ATP) channels were mediated by cytosolic cGMP. Taken together, our experiments uncovered a novel mechanism by which AMPARs participate in insulin release.