Receptor Beta-subunit Research Articles

The glucose-lowering agent sodium tungstate increases the levels and translocation of GLUT4 in L6 myotubes through a mechanism associated with ERK1/2 and MEF2D

The aim of this study was to investigate the action of the glucose-lowering compound sodium tungstate on glucose transport in muscle myotubes and to unravel the molecular events underlying the effects observed. We studied the effects of tungstate on 2-deoxy-D: -glucose uptake, levels and translocation of the glucose transporters GLUT4 and GLUT1, and Glut4 (also known as Slc2a4) promoter activity. We also measured the modifications of individual components of the signalling pathways involved in the effects observed. Tungstate increased 2-deoxy-D: -glucose uptake in differentiated L6 myotubes through an increase in the total amount and translocation of GLUT4 transporter. The effects on glucose uptake were additive to those of insulin. Tungstate activated transcription of the Glut4 promoter, as shown by an increase in Glut4 mRNA, and by a promoter reporter assay. The assay of deletions of the Glut4 promoter indicated that the effect of tungstate is mediated by the myocyte enhancer factor 2 (MEF2)-binding domain. Accordingly, MEF2 levels and DNA binding activities were increased in response to the treatment. Tungstate-induced glucose uptake and GLUT4 transcriptional activation were dependent on the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), while no changes were observed in the phosphorylation state of the beta subunit of the insulin receptor, in the phosphatidylinositol 3-kinase pathway or in the activation of 5'AMP-activated protein kinase. Tungstate activates glucose uptake in myotubes through a novel ERK1/2-dependent mechanism. This effect is exerted by an increase in the content and translocation of the GLUT4 transporter. This is the first report of a glucose-lowering compound activating Glut4 transcription through an ERK1/2-dependent increase in MEF2 levels.

Structure-Based Rationale for Interleukin 5 Receptor Antagonism

Human interleukin 5 (IL5) is the major hematopoietin that stimulates the proliferation, migration and activation of eosinophils and is implicated in the pathogenesis of inflammatory and other myeloproliferative diseases. IL5 functions through the signaling of a common receptor subunit beta (beta c), in a receptor activation process that requires initial recruitment of an IL5 specific receptor subunit alpha (IL5Ralpha), for cytokine presentation to beta c. Important advances have been made to understand molecular mechanisms of cytokine recognition and receptor antagonism. Mutational studies indicate that a pair of charge complementary regions play an essential role in specific interaction between IL5Ralpha and IL5. Moreover, peptide studies with the IL5 system have identified a cyclic peptide inhibitor, AF17121, which binds specifically to IL5Ralpha by mimicking the cytokine. A key receptor-recognition pharmacophore has been identified in this peptide inhibitor, and sites of inhibitor recognition can be proposed in the homology-deduced structural model of IL5Ralpha. These results provide an experimental platform to derive enhanced-potency peptidomimetic inhibitors. Such inhibitors have potential use as tools to evaluate the role of eosinophilia in disease and as potential leads to antagonists to treat hyper-eosinophilic diseases such as eosinophilic esophagitis, asthma and chronic myeloproliferative leukemias.

Possible role of Angiotensin II Receptor (AT2) in regulation of Insulin Receptor signaling

Renin angiotensin system plays an important role in the development of metabolic syndrome, primarily characterized by insulin resistance, which poses a potential risk for other factors like obesity, hypertension etc. The actions of angiotensin II mediated through the AT1 receptor are well studied, but the role of the AT2 receptor is not clearly established. We have shown that the AT2 can interact with the beta subunit of Insulin Receptor (IR) in yeast two‐hybrid assay. To further evaluate the effects of AT2 on the signaling mechanism of IR, we studied the interaction between the two proteins in Chinese Hamster Ovary (CHO) cells and the human breast cancer (MCF‐7) cells. In our studies using CHO cells, we showed that transient transfection of the AT2 could result in AT2‐IR complex formation and lack of phosphorylation of IR‐beta. However, a truncated AT2 in which C‐terminal cytoplasmic region was deleted did not show interaction with the IR‐beta. MCF‐7 cells constitutively express the AT1 receptor. We have seen that starving these cells for 6 hours or more changes the expression pattern and the levels of the AT1 goes down while the levels of the AT2 increases. These cells expressing the AT2 receptor show lack of phosphorylation of the IR‐beta. Based on these data, we plan to study the proteomics in these cells to further understand the cross talk between angiotensin II and insulin signaling at a cellular and molecular level.

The role of GABAA receptors in the control of transient lower oesophageal sphincter relaxations in the dog

Transient lower oesophageal sphincter relaxations (TLESRs) are triggered by activation of mechanosensitive gastric vagal afferents and are the major cause of gastroesophageal reflux and therefore an important target for therapeutic intervention in gastroesophageal reflux disease (GERD). Activation of the metabotropic GABA(B) receptor has shown to inhibit TLESRs. The aim of the present study was to assess the role of the ionotropic GABA(A) receptor in the regulation of TLESRs. TLESRs were quantified using Dentsleeve manometry in dogs, and GABA(A) agonists were given i.v. prior to gastric distension. Immunohistochemistry and RT-PCR were used to localize GABA(A) receptors in the dog nodose ganglion, the source of vagal afferents which initiate TLESRs. The prototypical GABA(A) agonist muscimol produced a dose-dependent inhibition of TLESRs ranging from 19 to 56%. The two other GABA(A) agonists evaluated, isoguvacine and 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridin-3-ol (THIP), as well as the GABA(A) positive allosteric modulator diazepam, had no major effects on TLESRs. Evaluation of higher doses was limited by emesis (THIP and isoguvacine) or restlessness/sedation (diazepam). Of the predominant GABA(A) receptor subunits (alpha, beta and gamma components), alpha and beta but not gamma were detected in the dog nodose ganglion by RT-PCR, while immunohistochemistry in addition demonstrated nerve fibres expressing the gamma subunit. The present observations demonstrate that GABA(A) receptors exert an inhibitory control of TLESRs. These results warrant further studies using GABA(A) isoform-selective agonists to define the identity of receptors involved.

Pirh2 interacts with and ubiquitylates signal recognition particle receptor β subunit

Pirh2 is a RING finger type ubiquitin ligase which ubiquitylates various proteins including p53, p27(Kip1), HDAC1, and epsilon-COP. In this study, we identified signal recognition particle receptor beta subunit (SRbeta), an integral membrane protein of the endoplasmic reticulum (ER), as a novel Pirh2-interacting protein by yeast two-hybrid screening. We confirmed that Pirh2 interacted with SRbeta in mammalian cells. An immunofluorescent staining revealed that Pirh2 colocalized with SRbeta in the ER. Pirh2 poly-ubiquitylated SRbeta in an intact RING finger domain-dependent manner in vivo and in vitro. Unexpectedly, different from other Pirh2 substrates, neither overexpression of Pirh2 nor depletion of cellular Pirh2 affected SRbeta protein stability. Pirh2 preferentially utilized lysine residues 6 and 29 of the ubiquitin to mediate the formation of polyubiquitin chains on SRbeta. These results suggest that Pirh2 may regulate SRbeta function by mediating poly-ubiquitylation of SRbeta without affecting the stability of SRbeta protein per se.

Ameliorating Effects of Sulfonylurea Drugs on Insulin Resistance in Otsuka Long-Evans Tokushima Fatty Rats

OLETF (Otsuka Long-Evans Tokushima Fatty) rats are characterized by obesity-related insulin resistance, which is a phenotype of type 2 diabetes. Sulfonylurea drugs or benzoic acid derivatives as inhibitors of the ATP-sensitive potassium (K(ATP)) channel are commercially available to treat diabetes. The present study compared sulfonylurea drugs (glimepiride and gliclazide) with one of benzoic acid derivatives (repaglinide) in regard to their long-term effect on ameliorating insulin sensitivity in OLETF rats. Each drug was dissolved and fed with drinking water from 29 weeks of age. On high glucose loading at 45 weeks of age, response of blood glucose recovery was the greatest in the group treated with glimepiride. On immunohistochemistry analysis for the Kir6.2 subunit of K(ATP) channels, insulin receptor beta-subunits, and glucose transporters (GLUT) type 2 and 4 in liver, fat and skeletal muscle tissues, the sulfonylurea drugs (glimepiride and gliclazide) were more effective than repaglinide in recovery from their decreased expressions in OLETF rats. From these results, it seems to be plausible that K(ATP)-channel inhibitors containing sulfonylurea moiety may be much more effective in reducing insulin resistance than those with benzoic acid moiety. In contrast to gliclazide, non-tissue selectivity of glimepiride on K(ATP) channel inhibition may further strengthen an amelioration of insulin sensitivity unless considering other side effects.

The contribution of transcription factor IRF1 to the interferon-γ–interleukin 12 signaling axis and TH1 versus TH-17 differentiation of CD4+ T cells

Interleukin-12 (IL-12) and interferon-gamma (IFN-gamma) drive T helper type 1 (T(H)1) differentiation, but the mechanisms underlying the regulation of the complicated gene networks involved in this differentiation are not fully understood. Here we show that the IFN-gamma-induced transcription factor IRF1 was essential in T(H)1 differentiation by acting on Il12rb1, the gene encoding the IL-12 receptor beta1 subunit (IL-12Rbeta1). IRF1 directly interacted with and activated the Il12rb1 promoter in CD4+ T cells. Notably, the IRF1-dependent induction of IL-12Rbeta1 was essential for IFN-gamma-IL-12 signaling but was dispensable for IL-23-IL-17 signaling. Because both IL-12 and IL-23 bind to and transmit signals through IL-12Rbeta1, our data suggest that distinct thresholds of IL-12Rbeta1 expression are required for T(H)1 versus T(H)-17 differentiation.

Identification of Host Proteins Associated with Retroviral Vector Particles by Proteomic Analysis of Highly Purified Vector Preparations

The Moloney murine leukemia virus (MMLV) belongs to the Retroviridae family of enveloped viruses, which is known to acquire minute amounts of host cellular proteins both on the surface and inside the virion. Despite the extensive use of retroviral vectors in experimental and clinical applications, the repertoire of host proteins incorporated into MMLV vector particles remains unexplored. We report here the identification of host proteins from highly purified retroviral vector preparations obtained by rate-zonal ultracentrifugation. Viral proteins were fractionated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in-gel tryptic digested, and subjected to liquid chromatography/tandem mass spectrometry analysis. Immunogold electron microscopy studies confirmed the presence of several host membrane proteins exposed at the vector surface. These studies led to the identification of 27 host proteins on MMLV vector particles derived from 293 HEK cells, including 5 proteins previously described as part of wild-type MMLV. Nineteen host proteins identified corresponded to intracellular proteins. A total of eight host membrane proteins were identified, including cell adhesion proteins integrin beta1 (fibronectin receptor subunit beta) and HMFG-E8, tetraspanins CD81 and CD9, and late endosomal markers CD63 and Lamp-2. Identification of membrane proteins on the retroviral surface is particularly attractive, since they can serve as anchoring sites for the insertion of tags for targeting or purification purposes. The implications of our findings for retrovirus-mediated gene therapy are discussed.

Runx proteins are involved in regulation of CD122, Ly49 family and IFN-γ expression during NK cell differentiation

Runx family proteins play indispensable roles in the development of various hematopoietic lineage cells. However, their function in NK cells is still uncertain. We found that NK cells and CD8 T cells dominantly express Runx3 protein, whereas NKT cells and CD4 T cells express Runx1. Reverse transcription-PCR analysis revealed that Runx3 expression is initiated at the NK precursor stage and is maintained along the course of NK cell differentiation. In order to examine their role in the earlier stage of NK cell development, we introduced Runx dominant-negative (Runx dn) form into Lin(-)c-kit(+)Sca-1(+) hematopoietic stem cells, which were applied to NK cell-inducing culture. Post-cultured cells showed a decreased expression of IL-2/IL-15 common receptor beta subunit (CD122), consistent with another finding that Runx binds to promoter region of CD122 gene. To examine the Runx function in the later developmental stage, we used transgenic mouse, in which Runx dn form is expressed in immature and mature NK cells. This mouse showed decreased expressions of NK maturation markers, such as Ly49 family, Mac-1 and CD43, whereas IFN-gamma production was greatly enhanced. These findings suggest that Runx proteins, especially Runx3, play multiple roles in NK cell differentiation.

Identification, Coassembly, and Activity of γ-Aminobutyric Acid Receptor Subunits in Renal Proximal Tubular Cells

Although the properties and functions of GABA(A) receptors in the mammalian central nervous system have been well studied, the presence and significance of GABA(A) receptors in non-neural tissues are less clear. The goal of this study was to examine the expression of GABA(A) receptor alpha(1), alpha(2), alpha(4), alpha(5), beta(1), gamma(1), gamma(2), and delta subunits in the kidney and to determine whether these subunits coassemble to form an active renal epithelial cell GABA(A) receptor. Using reverse transcriptase products from RNA isolated from rat and rabbit kidney cortex and brain or cerebellum through polymerase chain reaction (PCR) and sequencing of the PCR products, we revealed that rat kidney cortex contained the alpha(1), alpha(5), beta(1), gamma(1), and gamma(2) subunits and that they were similar to the neuronal subunits. Sequencing of the PCR products revealed that the rabbit kidney cortex contained the alpha(1) and gamma(2) subunits and that they were similar to their neuronal counterparts. Immunoprecipitation and immunoblot studies using GABA(A) receptor subunit-specific antibodies and detergent-solubilized rat kidney cortex membranes identified a GABA(A) receptor complex containing alpha(5), beta(1), and gamma(1). Isolated rat renal proximal tubular cells exhibited GABA-mediated, picrotoxin-sensitive (36)Cl(-) uptake. These studies demonstrate the presence of numerous GABA(A) receptor subunits in the kidneys of two species, the assembly of the subunits into at least one novel receptor complex, and an active GABA(A) receptor in renal proximal tubular cells.

Construction of SH-EP1-alpha4beta2-hAPP695 cell line and effects of nicotinic agonists on beta-amyloid in the cells.

(1) Nicotinic acetylcholine receptors in central nervous system are thought to be new targets for Alzheimer's disease. However, the most involved nicotinic receptor subtype in Alzheimer's disease is unclear. alpha4beta2 receptor is the most widely spread subtype in brain, involving in several important aspects of cognitive and other functions. We constructed cell line by transfecting human amyloid precursor protein (695) gene into SH-EP1 cells which have been transfected with human nicotinic receptor alpha4 subunit and beta2 subunit gene, to observe effects of alpha4beta2 receptors activation on beta-amyloid, expecting to provide a new cell line for drug screening and research purpose. (2) Liposome transfection was used to express human amyloid precursor protein (695) gene in SH-EP1-alpha4beta2 cells. Function of the transfected alpha4beta2 receptors was tested by patch clamp. Effects of nicotine and epibatidine (selective alpha4beta2 nicotinic receptor agonist) on beta-amyloid were detected by Western blot and ELISA. Effects of nicotine and epibatidine on amyloid precursor protein (695) mRNA level were measured using real-time PCR. (3) Human amyloid precursor protein (695) gene was stably expressed in SH-EP1-alpha4beta2 cells; Nicotine (1 muM) and epibatidine (0.1 muM) decreased intracellular and secreted beta-amyloid in the cells; and activation of alpha4beta2 receptors did not affect amyloid precursor protein (695) mRNA level. (4) These results suggest that the constructed cell line, expressing both amyloid precursor protein (695) gene and human nicotinic receptor alpha4 subunit and beta2 subunit gene, might be useful for screening specific nicotinic receptor agonists against Alzheimer's disease. Alteration of Abeta level induced by activation of alpha4beta2 nAChR in our study might occur at a post-translational level.

GABAA α6-Containing Receptors Are Selectively Compromised in Cerebellar Granule Cells of the Ataxic Mouse, Stargazer

Stargazer mice fail to express the gamma2 isoform of transmembrane alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate (AMPA) receptor regulatory proteins that has been shown to be absolutely required for the trafficking and synaptic targeting of excitatory AMPA receptors in adult murine cerebellar granule cells. Here we show that 30 +/- 6% fewer inhibitory gamma-aminobutyric acid, type A (GABA(A)), receptors were expressed in adult stargazer cerebellum compared with controls because of a specific loss of GABA(A) receptor expression in the cerebellar granule cell layer. Radioligand binding assays allied to in situ immunogold-EM analysis and furosemide-sensitive tonic current estimates revealed that expression of the extrasynaptic (alpha6betaxdelta) alpha6-containing GABA(A) receptor were markedly and selectively reduced in stargazer. These observations were compatible with a marked reduction in expression of GABA(A) receptor alpha6, delta (mature cerebellar granule cell-specific proteins), and beta3 subunit expression in stargazer. The subunit composition of the residual alpha6-containing GABA(A) receptors was unaffected by the stargazer mutation. However, we did find evidence of an approximately 4-fold up-regulation of alpha1betadelta receptors that may compensate for the loss of alpha6-containing GABA(A) receptors. PCR analysis identified a dramatic reduction in the steady-state level of alpha6 mRNA, compatible with alpha6 being the primary target of the stargazer mutation-mediated GABA(A) receptor abnormalities. We propose that some aspects of assembly, trafficking, targeting, and/or expression of extrasynaptic alpha6-containing GABA(A) receptors in cerebellar granule cells are selectively regulated by AMPA receptor-mediated signaling.

Rapid Preparation of a Plasma Membrane Fraction from Adipocytes and Muscle Cells: Application to Detection of Translocated Glucose Transporter 4 on the Plasma Membrane

The aim of this study was to establish a rapid preparation of plasma membrane from adipocytes and muscle cells to detect translocated glucose transporter (GLUT) 4. A plasma membrane fraction was prepared by sequential centrifugation with buffer containing detergents, and its purity was estimated by detecting insulin receptor beta-subunit (IRbeta). After insulin stimulus, GLUT4 translocation was observed in 3T3-L1 adipocytes and L6 myotubes. It was found that IRbeta and GLUT4 levels on the plasma membrane decreased in adipose and muscle with intake of a 29% lard diet for 14 weeks. Hence, this method should be useful for rapid preparation of the plasma membrane fraction.

Early Evolution of Ionotropic GABA Receptors and Selective Regimes Acting on the Mammalian-Specific Theta and Epsilon Subunits

BackgroundThe amino acid neurotransmitter GABA is abundant in the central nervous system (CNS) of both invertebrates and vertebrates. Receptors of this neurotransmitter play a key role in important processes such as learning and memory. Yet, little is known about the mode and tempo of evolution of the receptors of this neurotransmitter. Here, we investigate the phylogenetic relationships of GABA receptor subunits across the chordates and detail their mode of evolution among mammals.Principal FindingsOur analyses support two major monophyletic clades: one clade containing GABAA receptor α, γ, and ε subunits, and another one containing GABAA receptor ρ, β, δ, θ, and π subunits. The presence of GABA receptor subunits from each of the major clades in the Ciona intestinalis genome suggests that these ancestral duplication events occurred before the divergence of urochordates. However, while gene divergence proceeded at similar rates on most receptor subunits, we show that the mammalian-specific subunits θ and ε experienced an episode of positive selection and of relaxed constraints, respectively, after the duplication event. Sites putatively under positive selection are placed on a three-dimensional model obtained by homology-modeling.ConclusionsOur results suggest an early divergence of the GABA receptor subunits, before the split from urochordates. We show that functional changes occurred in the lineages leading to the mammalian-specific subunit θ, and we identify the amino acid sites putatively responsible for the functional divergence. We discuss potential consequences for the evolution of mammals and of their CNS.

A Conserved Tyrosine in the β2Subunit M4 Segment Is a Determinant of γ-Aminobutyric Acid Type A Receptor Sensitivity to Propofol

The gamma-aminobutyric acid type A receptor (GABAA-R) beta subunits are critical targets for the actions for several intravenous general anesthetics, but the precise nature of the anesthetic binding sites are unknown. In addition, little is known about the role the fourth transmembrane (M4) segment of the receptor plays in receptor function. The aim of this study was to better define the propofol binding site on the GABAA-R by conducting a tryptophan scan in the M4 segment of the beta2 subunit. Seven tryptophan mutations were introduced into the C-terminal end of the M4 segment of the GABAA-R beta2 subunit. GABAA-R subunit complementary DNAs were transfected into human embryonic kidney 293 cells grown on glass coverslips. After transfection (36-72 h), coverslips were transferred to a perfusion chamber to assay receptor function. Cells were whole cell patch clamped and exposed to GABA, propofol, etomidate, and pregnenolone. Chemicals were delivered to the cells using two 10-channel infusion pumps and a rapid solution exchanger. All tryptophan mutations were well tolerated, and with one exception, all resulted in minimal changes in receptor activation by GABA. One mutation, beta2(Y444W), selectively suppressed the ability of propofol to enhance receptor function while retaining normal sensitivity to etomidate and pregnenolone. This is the first report of a mutation that selectively reduces propofol sensitivity without altering the action of etomidate. The reduction in propofol sensitivity is consistent with the loss of a hydrogen bond within the propofol binding site. These results also suggest a possible orientation of the propofol molecule within its binding site.

Validation of an Extracerebral Reference Region Approach for the Quantification of Brain Nicotinic Acetylcholine Receptors in Squirrel Monkeys with PET and 2-18F-Fluoro-A-85380

The aim of the present study was to explore the applicability of an extracerebral reference region for the quantification of cerebral receptors with PET. Male squirrel monkeys underwent quantitative PET studies of cerebral nicotinic acetylcholine receptors (nAChRs) with 2-(18)F-fluoro-A-85380 (2-FA). Data from dynamic PET scans were analyzed with various compartment- and non-compartment-based models, including a simplified reference tissue model (SRTM). Nondisplaceable volume-of-distribution (VDnd) values were determined in regions of interest after the blockade of 2-FA-specific binding by nicotine infusion. Binding potential values, estimated with the cerebellum and muscle as reference regions, were compared and the reproducibility of measurements was determined. One- and 2-tissue-compartment modeling and linear graphic analysis provided similar total volume-of-distribution (VD(T)) values for each studied region. VD(T) values were high in the thalamus, intermediate in the cortex and midbrain, and low in the cerebellum and muscle, consistent with the distribution pattern of nAChR containing alpha(4) and beta(2) receptor subunits (alpha(4)beta(2)*). The administration of nicotine at 2 mg/kg/d via an osmotic pump resulted in a nearly complete saturation of 2-FA-specific binding and led to very small changes in volumes of distribution in the cerebellum and muscle (-9% +/- 4% [mean +/- SEM] and 0% +/- 6%, respectively), suggesting limited specific binding of the radioligand in these areas. VD(T) measured in muscle in 15 monkeys was reasonably constant (3.0 +/- 0.2, with a coefficient of variation of 8%). VDnd in studied brain regions exceeded VD(T) in muscles by a factor of 1.3. With this factor and with muscle as a reference region, BP* values calculated for studied brain regions with the SRTM were in good agreement with those obtained with the cerebellum as a reference region. Significant correlations were observed between BP* values estimated with these 2 approaches. The reproducibilities of BP* measurements obtained with the 2 methods were comparable, with coefficients of variation of less than 11% and 13% for the thalamus and the cortex, respectively. These results suggest that the accurate quantification of nAChRs can be performed with 2-FA and a reference region outside the brain, providing a novel approach for the quantification of brain receptors when no suitable cerebral reference region is available.

Study of antidiabetic activity of a new ultralow-dose antibody preparation on the model of streptozotocin diabetes in rats

Antidiabetic activity of a new ultralow-dose preparation of antibodies to insulin receptor beta-subunit was revealed on the model of streptozotocin diabetes in rats: treatment with this preparation in a daily dose of 2.5 ml/kg (for 50 days through a gastric tube) normalized blood glucose level, restored glucose tolerance in the oral glucose test and body weight gain, and improved animal survival. By its hypoglycemic activity and effect on glucose tolerance the preparation was not inferior to classical drugs for the treatment of types 1 and 2 diabetes mellitus insulin (12 U/kg) and glybenclamide (8 mg/kg).

Gene expression of myogenic regulatory factors, nicotinic acetylcholine receptor subunits, and GAP‐43 in skeletal muscle following denervation in a rat model

Neuromuscular junction destabilization following nerve injury contributes to irreversible functional impairment. Myogenic Regulatory Factors (MRF's) including myoblast determination factor (MyoD), MRF-4, Myogenin, and myogenic factors-5 (myf-5), and Growth-associated protein 43 KDa (GAP43) regulate gene expression of nicotinic acetylcholine receptor (nAChR) subunits (alpha, beta, delta, gamma, and epsilon). We hypothesized that nerve injury induces altered gene expression of MRF's, nAChRs, and GAP-43 in the skeletal muscle which destabilize neuromuscular junctions. The tibial nerve was transected in 42 juvenile male Sprague-Dawley rats. Denervated and contralateral control gastrocnemius m. mRNA for nAChR subunits, MRF's, and GAP-43 were determined by real time reverse transcription polymerase chain reaction (real time RT-PCR). After transection, muscle mass decreased for 1 year with a nadir of 75% at 3 months. Alpha, gamma, and epsilon subunit genes increased by 3 and peaked at 7 days before returning to control levels (P < 0.05). Beta subunits and GAP-43 tended to increase. Delta subunits peaked at 3 days returning to control levels by 30 days. By one month, most of the nAChR subunits had returned to control levels. Alpha, beta, gamma, and delta subunit expression remained significantly lower than control up to 1 year later (P < 0.05). MRF4, Myogenin, and MyoD expression paralleled that of alpha, gamma, and epsilon nAChR subunits (P < 0.05). Gene expression of nAChR alpha, gamma, delta and epsilon subunits was biphasic in the first month after nerve injury, similar to that of MRF's. nAChR subunits and MRF's may play a critical role in neuromuscular junction stability.

Retrograde activation of STAT3 by leukemia inhibitory factor in sympathetic neurons

Leukemia inhibitory factor (LIF) is a member of the interleukin-6 family of cytokines and signals through the glycoprotein 130 and LIF receptor beta subunits. Binding of cytokines to these subunits activates multiple signaling cascades, including the Janus kinase (Jak)/signal transducers and activators of transcription (STAT) pathway. We used compartmentalized cultures of sympathetic neurons and immunocytochemical analyses of STAT3 to examine the mechanisms involved in retrograde signaling of LIF from distal neurites (DN) to cell bodies. Addition of LIF to the DN of these neurons triggers the activation and nuclear translocation of STAT3. Inhibition of Jak activity in the cell bodies prevented LIF-induced retrograde activation of STAT3, while block of Jak activity in the DN had no effect on the appearance of activated STAT3 in the nucleus. These results show that the transport of activated Jak is not the main mechanism mediating retrograde signaling. Although there is an increase in phosphorylated STAT3 in the neurites after distal stimulation, the transport of activated STAT3 is not necessary for retrograde signaling. Our results are consistent with a signaling endosome model for retrograde signaling, in which the LIF/glycoprotein 130/LIF receptor/Jak complex is internalized and transported to activate STAT3 in the cell body.

The Neuregulin-1 Receptor ErbB4 Controls Glutamatergic Synapse Maturation and Plasticity

Neuregulin-1 (NRG1) signaling participates in numerous neurodevelopmental processes. Through linkage analysis, nrg1 has been associated with schizophrenia, although its pathophysiological role is not understood. The prevailing models of schizophrenia invoke hypofunction of the glutamatergic synapse and defects in early development of hippocampal-cortical circuitry. Here, we show that the erbB4 receptor, as a postsynaptic target of NRG1, plays a key role in activity-dependent maturation and plasticity of excitatory synaptic structure and function. Synaptic activity leads to the activation and recruitment of erbB4 into the synapse. Overexpressed erbB4 selectively enhances AMPA synaptic currents and increases dendritic spine size. Preventing NRG1/erbB4 signaling destabilizes synaptic AMPA receptors and leads to loss of synaptic NMDA currents and spines. Our results indicate that normal activity-driven glutamatergic synapse development is impaired by genetic deficits in NRG1/erbB4 signaling leading to glutamatergic hypofunction. These findings link proposed effectors in schizophrenia: NRG1/erbB4 signaling perturbation, neurodevelopmental deficit, and glutamatergic hypofunction.