Possible involvement of VEGF signaling system in rescuing effect of endogenous acetylcholine on NMDA-induced long-lasting hippocampal cell damage in organotypic hippocampal slice cultures

In our previous study, elevation of endogenous acetylcholine (ACh) by tacrine (THA) rescued NMDA-induced long-lasting hippocampal cell damage via muscarinic M₁ receptors. However, the detailed molecular mechanism underlying the effect of ACh is unclear. This study investigated possible involvement of the VEGF signaling system in the rescuing effect of ACh on N-methyl-d-aspartate (NMDA)-induced long-lasting hippocampal cell damage using organotypic hippocampal slice cultures (OHSCs). As previously reported, NMDA pretreatment caused long-lasting hippocampal cell damage in OHSCs in a manner reversible by treatment with THA. The protein kinase C (PKC) inhibitor Ro31-8220, but not the extracellular signal-regulated kinase (ERK) inhibitor U0126, dose-dependently and almost completely abolished the effect of THA. The rescuing effect of THA was also partially but significantly blocked by Ki8751, a selective inhibitor of type 2 vascular endothelial growth factor (VEGF) receptor (VEGFR-2) tyrosine kinase. NMDA pretreatment elevated the expression level of HIF1α, whereas it decreased the expression of VEGF-A. Moreover, NMDA pretreatment reduced the level of phosphorylated VEGFR-2 without apparently affecting the level of VEGFR-2 or β-actin. These NMDA pretreatment-induced changes were significantly attenuated by THA treatment. Immunohistochemical analysis conducted 6 days after NMDA pretreatment revealed that VEGF-A and VEGFR-2 were mainly expressed on astrocytes and neurons, respectively, in OHSCs. In OHSCs pretreated with NMDA, THA treatment induced a morphological and activation-related change in astrocytes expressing VEGF-A. The present results demonstrate that endogenous acetylcholine plays a rescuing role towards excitotoxicity-induced long-lasting hippocampal cell damage in part via paracrine VEGF signaling between astrocytes and hippocampal neurons or autocrine VEGF signaling in hippocampal neurons in OHSCs.     

Microglial Toll-like receptor 2 contributes to kainic acid-induced glial activation and hippocampal neuronal cell death

Recent studies indicate that Toll-like receptors (TLRs), originally identified as infectious agent receptors, also mediate sterile inflammatory responses during tissue damage. In this study, we investigated the role of TLR2 in excitotoxic hippocampal cell death using TLR2 knock-out (KO) mice. TLR2 expression was up-regulated in microglia in the ipsilateral hippocampus of kainic acid (KA)-injected mice. KA-mediated hippocampal cell death was significantly reduced in TLR2 KO mice compared with wild-type (WT) mice. Similarly, KA-induced glial activation and proinflammatory gene expression in the hippocampus were compromised in TLR2 KO mice. In addition, neurons in organotypic hippocampal slice cultures (OHSCs) from TLR2 KO mouse brains were less susceptible to KA excitotoxicity than WT OHSCs. This protection is partly attributed to decreased expression of proinflammatory genes, such as TNF-α and IL-1β in TLR2 KO mice OHSCs. These data demonstrate conclusively that TLR2 signaling in microglia contributes to KA-mediated innate immune responses and hippocampal excitotoxicity.     

Cross-talk between human mesenchymal stem/progenitor cells (MSCs) and rat hippocampal slices in LPS-stimulated cocultures: the MSCs are activated to secrete prostaglandin E2

Mesenchymal stem/progenitor cells (MSCs) improve functional outcome in a number of disease models through suppression of inflammation. However, their effects on neuroinflammation are unknown. In this study, we show that MSCs suppress endotoxin-induced glial activation in organotypic hippocampal slice cultures (OHSCs). Lipopolysaccharide-stimulated OHSCs activated MSCs to increase the expression of cyclo-oxygenase-2 and produce prostaglandin E2. MSC-derived prostaglandin E2, then suppressed pro-inflammatory cytokine production by the OHSCs. Together, the results suggest the potential anti-inflammatory mechanism of MSCs in models of disease and support earlier observations that MSCs may offer a therapy for neuroinflammation produced by trauma or disease.     

Insulin in the Brain: Sources, Localization and Functions

Historically, insulin is best known for its role in peripheral glucose homeostasis, and insulin signaling in the brain has received less attention. Insulin-independent brain glucose uptake has been the main reason for considering the brain as an insulin-insensitive organ. However, recent findings showing a high concentration of insulin in brain extracts, and expression of insulin receptors (IRs) in central nervous system tissues have gathered considerable attention over the sources, localization, and functions of insulin in the brain. This review summarizes the current status of knowledge of the peripheral and central sources of insulin in the brain, site-specific expression of IRs, and also neurophysiological functions of insulin including the regulation of food intake, weight control, reproduction, and cognition and memory formation. This review also considers the neuromodulatory and neurotrophic effects of insulin, resulting in proliferation, differentiation, and neurite outgrowth, introducing insulin as an attractive tool for neuroprotection against apoptosis, oxidative stress, beta amyloid toxicity, and brain ischemia.     

Ahsg-fetuin blocks the metabolic arm of insulin action through its interaction with the 95-kD β-subunit of the insulin receptor

We previously have shown that Ahsg, a liver glycoprotein, inhibits insulin receptor (InsR) tyrosine kinase (TK) activity and the ERK1/2 mitogenic signaling arm of insulin signaling. Here we show that Ahsg blocks insulin-stimulated GLUT4 translocation and Akt activation in intact cells (mouse myoblasts). Furthermore, Ahsg inhibits InsR autophosphorylation of highly-purified insulin holoreceptors in a cell-free, ATP-dependent system, with an IC₅₀ within the range of single-chain Ahsg concentrations in human serum. Binding of ¹²⁵I-insulin to living cells overexpressing the InsR shows a dissociation constant (K_D) of 250 pM, unaltered in the presence of 300 nM Ahsg. A mutant InsR cDNA encoding the signal peptide, the β-subunit and the furin processing site, but deleting the α-subunit, was stably expressed in HEK293 cells. Treatment with peroxovanadate, but not insulin, dramatically increased the 95 kD β-subunit tyrosine phosphoryation. The level of tyrosine phosphorylation of the 95-kD β-subunit can be driven down sharply by treatment of living HEK293 transfectant cells with physiological doses of Ahsg. Treatment of myogenic cells with Ahsg blunts insulin-stimulated InsR autophosphorylation and AKT phosphorylation. Taken together, we show that Ahsg antagonizes the metabolic functions initiated by InsR activation without interference in insulin binding. The experiments suggest a direct interaction of Ahsg with the InsR ectodomain β-subunit in a mode that does not significantly alter the high-affinity binding of insulin to the holoreceptor's two complementing α-subunits.     

Appearance of nerve growth factor receptors on cultured neural crest cells

Light microscopic radioautography of differentiating quail neural crest cultures (1 to 2 weeks after explanation) incubated with Iodine-125-labeled nerve growth factor (125I-NGF) revealed that approximately 35% of the cells bound NGF. The binding was specific and saturable; it was blocked by an excess of nonradioactive NGF, and was not detected following incubation with biologically inactive 125I-NGF. In addition, the binding did not appear to be blocked or diminished by insulin. Cell cultures prepared from somites or notochord showed no specific binding of 125I-NGF. Melanocytes comprised approximately 10% of the cell population in these cultures and appeared to be unlabeled. The subpopulation of cells with NGF receptors that were morphologically similar to other non-melanocyte unlabeled cells present in the neural crest cultures are probably the targets of the factor during differentiation and development. In contrast, there was no evidence of 125I-NGF binding by premigratory neural crest (adherent to the isolated neural tube) or by early migratory neural crest cells (24 hr after explantation). Both of these types of neural crest cells are relatively undifferentiated. The cells of the neural tube were also unlabeled. The binding of 125I-NGF to differentiating neural crest cells was not noticeably diminished by a brief pretreatment with trypsin or Dispase, enzymes used in the isolation of neural tubes. Hence, the absence of NGF receptors on premigratory neural crest and early migratory neural crest cultures was not due to enzymatic alterations of the receptor. It seems, therefore, that receptors for NGF appear on neural crest cells during the time when these cells are acquiring their phenotypic characteristics.     

BACE2 plays a role in the insulin receptor trafficking in pancreatic ß-cells

BACE1 (β-site amyloidogenic cleavage of precursor protein-cleaving enzyme 1) is a β-secretase protein that plays a central role in the production of the β-amyloid peptide in the brain and is thought to be involved in the Alzheimer's pathogenesis. In type 2 diabetes, amyloid deposition within the pancreatic islets is a pathophysiological hallmark, making crucial the study in the pancreas of BACE1 and its homologous BACE2 to understand the pathological mechanisms of this disease. The objectives of the present study were to characterize the localization of BACE proteins in human pancreas and determine their function. High levels of BACE enzymatic activity were detected in human pancreas. In normal human pancreas, BACE1 was observed in endocrine as well as in exocrine pancreas, whereas BACE2 expression was restricted to β-cells. Intracellular analysis using immunofluorescence showed colocalization of BACE1 with insulin and BACE2 with clathrin-coated vesicles of the plasma membrane in MIN6 cells. When BACE1 and -2 were pharmacologically inhibited, BACE1 localization was not altered, whereas BACE2 content in clathrin-coated vesicles was increased. Insulin internalization rate was reduced, insulin receptor β-subunit (IRβ) expression was decreased at the plasma membrane and increased in the Golgi apparatus, and a significant reduction in insulin gene expression was detected. Similar results were obtained after specific BACE2 silencing in MIN6 cells. All these data point to a role for BACE2 in the IRβ trafficking and insulin signaling. In conclusion, BACE2 is hereby presented as an important enzyme in β-cell function.     

Changes in Insulin Binding to Developing Embryonic Chick Neural Retina Cells

Specific cell surface insulin binding to embryonic chick neural retina cells has been demonstrated in vivo. Kinetics of insulin binding as well as hormonal specificity were similar to those reported for other vertebrate cells and tissues, both neural and nonneural. When surface insulin binding to retinal cells was studied as a function of embryonic age, a developmental relationship was observed. Scatchard analysis revealed that the number of cell surface insulin receptors decreased approximately 75% between days 10 and 16 of embryonic development. Receptor affinities remained fairly constant for this period.     

Levetiracetam protects hippocampal neurons in culture against hypoxia-induced injury

Many experimental studies indicate that some antiepileptic drugs possess neuroprotective properties in varied models of neuronal injury. Levetiracetam is a second-generation antiepileptic drug with a novel mechanism of action. In the present study, we evaluated the putative neuroprotective effect of levetiracetam on primary hippocampal cultures at seven day in vitro. Cell death was induced by incubation of neural cultures in hypoxic conditions over 24 hours. Neuronal injury was assessed by morphometric investigation of death/total ratio of neurons in light microscopy using Trypan blue staining and by evaluation of lactate dehydrogenase (LDH) release in the culture medium. Our results indicate that pre-conditioning of hippocampal cultures with high concentrations of levetiracetam (100 μM and 300 μM) protects neurons against hypoxia-induced death. Two-fold higher number of neurons remained viable as compared to control cultures without drug. Lack of neuroprotective action of the drug on hippocampal neural cultures was observed, when a low concentration (10 μM) of levetiracetam was used.     

Mutations in the inverted repeats of Tn3 affect binding of transposase and transposition immunity.

In order to better understand the interaction between the inverted repeats (IRs) of the transposon Tn3 and Tn3 transposase, we have looked at the effects of mutations within the IRs on binding of transposase and transposition immunity. Binding of transposase to mutated IRs was measured using a site-specific nitrocellulose filter binding assay and by DNase I protection studies. Transposition immunity was measured in vivo using a transposition mating-out assay. The most important determinants for binding of transposase are present within the inside 21 base-pairs of the IR and several single base-pair mutations significantly reduce binding. Base-pair mutations which do not effect binding have strong negative effects on transposition immunity indicating that simple binding of transposase to the IR is not sufficient for the establishment of transposition immunity.     

Oral pioglitazone ameliorates fructose-induced peripheral insulin resistance and hippocampal gliosis but not restores inhibited hippocampal adult neurogenesis

Diet-associated insulin resistance (IR) is intimately correlated with the progression of metabolic syndrome and hippocampal dysfunction. Pioglitazone (PIO), a selective peroxisome proliferator-activated receptor gamma (PPARγ) agonist, has been applied to enhance insulin sensitivity. With limited permeability to blood-brain-barrier, it is unclear that whether oral PIO available to cure both the peripheral IR and the impairment in the hippocampus. We evaluated the levels of peripheral and hippocampal IR via the homeostatic model assessment of insulin resistance and hippocampal IRS-1/Akt phosphorylation, respectively, of Wistar Kyoto rats fed with a regular chew or high fructose diet (HFD) for 12 weeks. Gavage with PIO (30 mg/kg/day, 2 weeks) significantly reduced the peripheral IR and reversed the level of hippocampal PPARγ. Moreover, HFD-activated microglia and astrocyte were effectively relieved by PIO. The suppressed brain-derived neurotrophic factor, CaMKIIα, and postsynaptic density protein 95 in the hippocampus were effectively reversed by PIO. However, the hippocampal IR and inhibition of adult neurogenesis in dentate gyrus were not restored by PIO. Together, PIO oral application may reverse the HFD-induced peripheral IR and maintain the existed neuronal circuit by ameliorating glial activation and enhancing synaptic density through BDNF but failed to restore adult neurogenesis in the hippocampus.     

Chronic reduction of insulin receptors in the ventromedial hypothalamus produces glucose intolerance and islet dysfunction in the absence of weight gain

Insulin is believed to regulate glucose homeostasis mainly via direct effects on the liver, muscle, and adipose tissues. The contribution of insulin's central nervous system effects to disorders of glucose metabolism has received less attention. To evaluate whether postnatal reduction of insulin receptors (IRs) within the ventromedial hypothalamus (VMH), a brain region critical for glucose sensing, contributes to disorders of peripheral glucose metabolism, we microinjected a lentiviral vector expressing an antisense sequence to knockdown IRs or a control lentiviral vector into the VMH of nonobese nondiabetic rats. After 3-4 mo, we assessed 1) glucose tolerance, 2) hepatic insulin sensitivity, and 3) insulin and glucagon secretion, using the glucose clamp technique. Knockdown of IRs locally in the VMH caused glucose intolerance without altering body weight. Increments of plasma insulin during a euglycemic clamp study failed to suppress endogenous glucose production and produced a paradoxical rise in plasma glucagon in the VMH-IR knockdown rats. Unexpectedly, these animals also displayed a 40% reduction (P < 0.05) in insulin secretion in response to an identical hyperglycemic stimulus (～220 mg/dl). Our data demonstrate that chronic suppression of VMH-IR gene expression is sufficient to impair glucose metabolism as well as α-cell and β-cell function in nondiabetic, nonobese rats. These data suggest that insulin resistance within the VMH may be a significant contributor to the development of type 2 diabetes.     

Brain insulin receptors and spatial memory. Correlated changes in gene expression, tyrosine phosphorylation, and signaling molecules in the hippocampus of water maze trained rats

Evidence accumulated from clinical and basic research has indirectly implicated the insulin receptor (IR) in brain cognitive functions, including learning and memory (Wickelgren, I. (1998) Science 280, 517-519). The present study investigates correlative changes in IR expression, phosphorylation, and associated signaling molecules in the rat hippocampus following water maze training. Although the distribution of IR protein matched that of IR mRNA in most forebrain regions, a dissociation of the IR mRNA and protein expression patterns was found in the cerebellar cortex. After training, IR mRNA in the CA1 and dentate gyrus of the hippocampus was up-regulated, and there was increased accumulation of IR protein in the hippocampal crude synaptic membrane fraction. In the CA1 pyramidal neurons, changes in the distribution pattern of IR in particular cellular compartments, such as the nucleus and dendritic regions, was observed only in trained animals. Although IR showed a low level of in vivo tyrosine phosphorylation, an insulin-stimulated increase of in vitro Tyr phosphorylation of IR was detected in trained animals, suggesting that learning may induce IR functional changes, such as enhanced receptor sensitivity. Furthermore, a training-induced co-immunoprecipitation of IR with Shc-66 was detected, along with changes in in vivo Tyr phosphorylation of Shc and mitogen-activated protein kinase, as well as accumulation of Shc-66, Shc-52, and Grb-2 in hippocampal synaptic membrane fractions following training. These findings suggest that IR may participate in memory processing through activation of its receptor Tyr kinase activity, and they suggest possible engagement of Shc/Grb-2/Ras/mitogen-activated protein kinase cascades.     

Functional modulation of voltage-dependent sodium channel expression by wild type and mutated C121W-β1 subunit

Voltage dependent sodium channels are membrane proteins essential for cell excitability. They are composed by a pore-forming α-subunit, encoded in mammals by up to 9 different genes, and 4 different ancillary β-subunits. The expression pattern of the α subunit isoforms confers the distinctive functional and pharmacological properties to different excitable tissues. β subunits are important modulators of channel function and expression. Mutation C121W of the β1-subunit causes an autosomal dominant epileptic syndrome without cardiac symptoms. The C121W mutation may act by a dominant-competition, modifying the expression of α-subunit proteins. To test this hypothesis, we transfected GH3 cells, from neuro-ectoderm origin, with wild-type or mutant β1 subunits and compared them to native cells. To examine the tissue specificity of the C121W-β1 mutation, we compared the effects of the mutation on neural cells with those of H9C2 cells of cardiac origin. We found that in GH3 cells the over-expression of the β1 subunit augments the α subunit mRNA and protein levels, while in the H9C2 cells the enhanced level of β1 subunit not only increases but also qualitatively modifies the sodium channel α isoform expression pattern. Interestingly, the introduction of the epileptogenic C121W-β1 subunit does not alter the sodium channel isoform composition of GH3 cells, while produces additional changes in the α-subunit expression pattern of H9C2 cells. Electrophysiological measurements confirm these molecular results. The expression differences observed could be correlated to the tissue-specific regulatory action of the β1 subunit and to the nervous system specificity of the C121W mutation. Our findings could be helpful for the comprehension of the molecular mechanism of generalised epileptic with febrile seizures plus in patients with identified β1 subunit mutations.     

Dissociation of the insulin receptor from caveolae during TNFα-induced insulin resistance and its recovery by D-PDMP

Previously, we demonstrated that an inhibitor of ganglioside biosynthesis, d-PDMP, could restore impaired insulin signaling in tumor necrosis factor α (TNFα)-treated adipocytes by blocking the increase of GM3 ganglioside. Here, we analyzed the interaction between insulin receptor (IR) and GM3 in the plasma membranes using immunoelectron microscopy. In normal adipocytes, most GM3 molecules localized at planar and non-caveolar regions. Approximately 19% of IR molecules were detected in caveolar regions. The relative ratio of IRs associated with caveolae in TNFα-treated adipocytes was decreased to one-fifth of that in normal adipocytes, but this decrease was restored by d-PDMP. Thus, we could obtain direct evidence that insulin resistance is a membrane microdomain disorder caused by aberrant expression of ganglioside.     

Actin filament binding by a monomeric IQGAP1 fragment with a single calponin homology domain

IQGAP1 is a homodimeric protein that reversibly associates with F-actin, calmodulin, activated Cdc42 and Rac1, CLIP-170, beta-catenin, and E-cadherin. Its F-actin binding site includes a calponin homology domain (CHD) located near the N-terminal of each subunit. Prior studies have implied that medium- to high-affinity F-actin binding (5-50 microM K(d)) requires multiple CHDs located either on an individual polypeptide or on distinct subunits of a multimeric protein. For IQGAP1, a series of six tandem IQGAP coiled-coil repeats (IRs) located past the C-terminal of the CHD of each subunit support protein dimerization and, by extension, the IRs or an undefined subset of them were thought to be essential for F-actin binding mediated by its CHDs. Here we describe efforts to determine the minimal region of IQGAP1 capable of binding F-actin. Several truncation mutants of IQGAP1, which contain progressive deletions of the IRs and CHD, were assayed for F-actin binding in vitro. Fragments that contain both the CHD and at least one IR could bind F-actin and, as expected, removal of all six IRs and the CHD abolished binding. Unexpectedly, a fragment called IQGAP1(2-210), which contains the CHD, but lacks IRs, could bind actin filaments. IQGAP1(2-210) was found to be monomeric, to bind F-actin with a K(d) of approximately 47 microM, to saturate F-actin at a molar ratio of one IQGAP1(2-210) per actin monomer, and to co-localize with cortical actin filaments when expressed by transfection in cultured cells. These collective results identify the first known example of high-affinity actin filament binding mediated by a single CHD.