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PubMed1998

PDZ motifs in PTP-BL and RIL bind to internal protein segments in the LIM domain protein RIL.

The specificity of protein-protein interactions in cellular signaling cascades is dependent on the sequence and intramolecular location of distinct amino acid motifs. We used the two-hybrid interaction trap to identify proteins that can associate with the PDZ motif-rich segment in the protein tyrosine phosphatase PTP-BL. A specific interaction was found with the Lin-11, Isl-1, Mec-3 (LIM) domain containing protein RIL. More detailed analysis demonstrated that the binding specificity resides in the second and fourth PDZ motif of PTP-BL and the LIM domain in RIL. Immunohistochemistry on various mouse tissues revealed a submembranous colocalization of PTP-BL and RIL in epithelial cells. Remarkably, there is also an N-terminal PDZ motif in RIL itself that can bind to the RIL-LIM domain. We demonstrate here that the RIL-LIM domain can be phosphorylated on tyrosine in vitro and in vivo and can be dephosphorylated in vitro by the PTPase domain of PTP-BL. Our data point to the presence of a double PDZ-binding interface on the RIL-LIM domain and suggest tyrosine phosphorylation as a regulatory mechanism for LIM-PDZ associations in the assembly of multiprotein complexes. These findings are in line with an important role of PDZ-mediated interactions in the shaping and organization of submembranous microenvironments of polarized cells.

Amino Acid Sequence↗
PubMed2003

Induction of megakaryocytes to synthesize and store a releasable pool of human factor VIII.

von Willebrand factor (VWF) is a complex plasma glycoprotein that modulates platelet adhesion at the site of a vascular injury, and it also serves as a carrier protein for factor (F)VIII. As megakaryocytes are the only hematopoietic lineage to naturally synthesize and store VWF within alpha-granules, this study was performed to determine if expression of a FVIII transgene in megakaryocytes could lead to trafficking and storage of FVIII with VWF in platelet alpha-granules. Isolex selected CD34+ cells from human G-CSF mobilized peripheral blood cells (PBC) and murine bone marrow were transduced with a retrovirus encoding the B-domain deleted form of human FVIII (BDD-FVIII). Cells were then induced with cytokines to form a population of multiple lineages including megakaryocytes. Chromogenic analysis of culture supernatant from FVIII-transduced human cells demonstrated synthesis of functional FVIII. Treatment of cells with agonists of platelet activation (ADP, epinephrine, and thrombin receptor-activating peptide) resulted in the release of VWF antigen and active FVIII into the supernatant from transduced cells. Immunofluorescence analysis of cultured human and murine megakaryocytes revealed a punctate pattern of staining for FVIII that was consistent with staining for VWF. Electron microscopy of transduced megakaryocytes using immunogold-conjugated antibodies colocalized FVIII and VWF within the alpha-granules. FVIII retained its association with VWF in human platelets isolated from the peripheral blood of NOD/SCID mice at 2-6 weeks post-transplant of transduced human PBC. These results suggest feasibility for the development of a locally inducible secretory pool of FVIII in platelets of patients with hemophilia A.

Animals↗
PubMed2006

Diabetes-induced mechanical hyperalgesia involves spinal mitogen-activated protein kinase activation in neurons and microglia via N-methyl-D-aspartate-dependent mechanisms.

Molecular mechanisms underlying diabetes-induced painful neuropathy are poorly understood. We have demonstrated, in rats with streptozotocin-induced diabetes, that mechanical hyperalgesia, a common symptom of diabetic neuropathy, was correlated with an early increase in extracellular signal-regulated protein kinase (ERK), p38, and c-Jun N-terminal kinase (JNK) phosphorylation in the spinal cord and dorsal root ganglion at 3 weeks after induction of diabetes. This change was specific to hyperalgesia because nonhyperalgesic rats failed to have such an increase. Immunoblot analysis showed no variation of protein levels, suggesting a post-translational regulation of the corresponding kinases. In diabetic hyperalgesic rats, immunocytochemistry revealed that all phosphorylated mitogen-activated protein kinases (MAPKs) colocalized with both the neuronal (NeuN) and microglial (OX42) cell-specific markers but not with the astrocyte marker [glial fibrillary acidic protein (GFAP)] in the superficial dorsal horn-laminae of the spinal cord. In these same rats, a 7-day administration [5 microg/rat/day, intrathecal (i.t.)] of 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U0126), 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB203580), and anthra(1,9-cd)pyrazol-6(2H)-one (SP600125), which inhibited MAPK kinase, p38, and JNK, respectively, suppressed mechanical hyperalgesia, and decreased phosphorylation of the kinases. To characterize the cellular events upstream of MAPKs, we have examined the role of the NMDA receptor known to be implicated in pain hypersensitivity. The prolonged blockade of this receptor during 7 days by (5R, 10S)-(+)-5-methyl-10, 11-dihydro-5H-dibenzo[a,d]-cyclohepten-5-10-imine hydrogen maleate (MK801; 5 microg/rat/day, i.t.), a noncompetitive NMDA receptor antagonist, reversed hyperalgesia developed by diabetic rats and blocked phosphorylation of all MAPKs. These results demonstrate for the first time that NMDA receptor-dependent phosphorylation of MAPKs in spinal cord neurons and microglia contribute to the establishment and longterm maintenance of painful diabetic hyperalgesia and that these kinases represent potential targets for pain therapy.

Animals↗
PubMed2002

A novel and conserved protein-protein interaction domain of mammalian Lin-2/CASK binds and recruits SAP97 to the lateral surface of epithelia.

Mammalian Lin-2 (mLin-2)/CASK is a membrane-associated guanylate kinase (MAGUK) and contains multidomain modules that mediate protein-protein interactions important for the establishment and maintenance of neuronal and epithelial cell polarization. The importance of mLin-2/CASK in mammalian development is demonstrated by the fact that mutations in mLin-2/CASK or SAP97, another MAGUK protein, lead to cleft palate in mice. We recently identified a new protein-protein interaction domain, called the L27 domain, which is present twice in mLin-2/CASK. In this report, we further define the binding of the L27C domain of mLin-2/CASK to the L27 domain of mLin-7 and identify the binding partner for L27N of mLin-2/CASK. Biochemical analysis reveals that this L27N domain binds to the N terminus of SAP97, a region that was previously reported to be essential for the lateral membrane recruitment of SAP97 in epithelia. Our colocalization studies, using dominant-negative mLin-2/CASK, show that the association with mLin-2/CASK is crucial for lateral localization of SAP97 in MDCK cells. We also report the identification of a novel isoform of Discs Large, a Drosophila melanogaster orthologue of SAP97, which contains a region highly related to the SAP97 N terminus and which binds Camguk, a Drosophila orthologue of mLin-2/CASK. Our data identify evolutionarily conserved protein-protein interaction domains that link mLin-2/CASK to SAP97 and account for their common phenotype when mutated in mice.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Hypoxia-induced expression of complement receptor type 1 (CR1, CD35) in human vascular endothelial cells.

Reoxygenation of hypoxic human umbilical vein endothelial cells (HUVECs) increases protein expression of the complement regulators CD46 and CD55. As the receptor for C3b is known to be present on injured bovine endothelial cells, we investigated whether hypoxia or inflammatory mediators induce complement receptor type 1 (CR1; CD35) expression on HUVECs. CR1 protein expression increased 3.7 +/- 0. 6-fold as measured by ELISA on HUVECs following hypoxia (48 h, 1% O2). Colocalization of CD35 and von Willebrand factor by confocal microscopy confirmed that CD35 was predominantly intracellular. Lipopolysaccharide or tumor necrosis factor-alpha also significantly increased HUVEC CR1 protein expression. Western blot analysis of neutrophil or hypoxic HUVEC lysates revealed a 221-kDa CR1 band under nonreducing conditions. RT-PCR of hypoxic HUVEC mRNA revealed a single band that, after sequencing, was identified as CD35. In situ hybridization of hypoxic HUVECs, but not normoxic HUVECs or fibroblasts, demonstrated increased CD35 mRNA. Hypoxic HUVECs bound immune complexes and acted as a cofactor for factor I-mediated cleavage of C3b. Thus hypoxia induces functional HUVEC CR1 expression.

Antigen-Antibody Complex↗
PubMed1992

Colocalization and release of angiotensin and renin in renal cortical cells.

Angiotensin is generated within the kidney, but the precise loci for the formation of angiotensin I (ANG I) and angiotensin II (ANG II) have not been demonstrated. We performed electron microscopy immunocytochemistry in kidney sections of 10-day-old (newborn) and adult Wistar-Kyoto (WKY) rats using specific antibodies to renin, ANG I, ANG II, and angiotensinogen (AO). Renin, ANG I, ANG II, and AO were present in juxtaglomerular (JG) cells. Renin was largely confined to cytoplasmic granules; ANG I and ANG II were colocalized to these granules but also were present in the cytoplasm; AO was distributed throughout the cytoplasm. AO also was present in a renal cortical distribution in proximal tubular cells. Northern blot analysis demonstrated AO mRNA in total kidney and liver but not in renal microvessels. Using the reverse hemolytic plaque assay, we demonstrated release of ANG I and renin from individual renocortical cells of adult WKY rats. Under control conditions, the number of releasing cells was 11 +/- 1 for ANG I and 10 +/- 1 for renin. Addition of rat renin inhibitor (RI) (1 x 10(-5) M), which inhibited renin activity in the medium from 37 to 9 pg ANG I.ml-1.h-1, did not alter ANG I plaque number. Addition of rat AO increased ANG I plaque number to 17 +/- 2 (P less than 0.05). Incubation with both RI and AO prevented the increase in ANG I plaque number obtained with AO alone. Enalapril treatment (7 days; n = 5) increased the number of plaque-forming cells to 22 +/- 2 for ANG I (P less than 0.0005) and to 39 +/- 7 for renin (P less than 0.001). The results suggest an intracellular location for AO and angiotensin and release of renin and ANG I by renal cortical cells and suggest that released angiotensin is produced intracellularly and that secretion of ANG I is augmented by converting enzyme inhibition.

Angiotensin I↗
PubMed2005

Expression of the Opa1 mitochondrial protein in retinal ganglion cells: its downregulation causes aggregation of the mitochondrial network.

PURPOSE: Mutations in the mitochondrial dynamin-related GTPase OPA1 cause autosomal dominant optic atrophy (ADOA), but the pathophysiology of this disease is unknown. As a first step in functional studies, this study was conducted to evaluate the expression of Opa1 in whole retina and in isolated retinal ganglion cells (RGCs) and to test the effects of Opa1 downregulation in cultured RGCs. METHODS: Opa1 mRNA isoforms from total retina and from RGCs freshly isolated by immunopanning were determined by RT-PCR. Protein expression was examined by immunohistochemistry and Western blot with antibodies against Opa1 and cytochrome c, and the mitochondrial network was visualized with a mitochondrial marker. Short interfering (si)RNA targeting OPA1 mRNAs were transfected to cultured RGCs and mitochondrial network phenotypes were followed for 15 days, in comparison with those of cerebellar granule cells (CGCs). RESULTS: Opa1 expression did not predominate in rat postnatal RGCs as found by immunohistochemistry and Western blot analysis. The pattern of mRNA isoforms was similar in whole retina and RGCs. After a few days in culture, isolated RGCs showed fine mitochondrial punctiform structures in the soma and neurites that colocalized with cytochrome c and Opa1. Opa1 knockdown in RGCs induced mitochondrial network aggregation at a higher rate than in CGCs. CONCLUSIONS: Results suggest that the level of expression and the mRNA isoforms do not underlie the vulnerability of RGCs to OPA1 mutations. However, aggregation of the mitochondrial network induced by the downregulation of Opa1 appears more frequent in RGCs than in control CGCs.

Animals↗
PubMed1997

Intact insulin stimulation of skeletal muscle blood flow, its heterogeneity and redistribution, but not of glucose uptake in non-insulin-dependent diabetes mellitus.

We tested the hypothesis that defects in insulin stimulation of skeletal muscle blood flow, flow dispersion, and coupling between flow and glucose uptake contribute to insulin resistance of glucose uptake in non-insulin-dependent diabetes mellitus (NIDDM). We used positron emission tomography combined with [15O]H2O and [18F]-2-deoxy--glucose and a Bayesian iterative reconstruction algorithm to quantitate mean muscle blood flow, flow heterogeneity, and their relationship to glucose uptake under normoglycemic hyperinsulinemic conditions in 10 men with NIDDM (HbA1c 8.1+/-0.5%, age 43+/-2 yr, BMI 27.3+/-0.7 kg/m2) and in 7 matched normal men. In patients with NIDDM, rates of whole body (35+/-3 vs. 44+/-3 micromol/kg body weight.min, P < 0.05) and femoral muscle (71+/-6 vs. 96+/-7 micromol/kg muscle.min, P < 0.02) glucose uptake were significantly decreased. Insulin increased mean muscle blood flow similarly in both groups, from 1.9+/-0.3 to 2.8+/-0.4 ml/100 g muscle.min in the patients with NIDDM, P < 0.01, and from 2.3+/-0.3 to 3.0+/-0.3 ml/100 g muscle.min in the normal subjects, P < 0.02. Pixel-by-pixel analysis of flow images revealed marked spatial heterogeneity of blood flow. In both groups, insulin increased absolute but not relative dispersion of flow, and insulin-stimulated but not basal blood flow colocalized with glucose uptake. These data provide the first evidence for physiological flow heterogeneity in human skeletal muscle, and demonstrate that insulin increases absolute but not relative dispersion of flow. Furthermore, insulin redirects flow to areas where it stimulates glucose uptake. In patients with NIDDM, these novel actions of insulin are intact, implying that muscle insulin resistance can be attributed to impaired cellular glucose uptake.

Adult↗
PubMed2001

Fetal antigen 1 in healthy adults and patients with pituitary disease: relation to physiological, pathological, and pharmacological GH levels.

Immunohistochemical analysis of the distribution of human fetal antigen 1 (FA1) in adult human tissues has demonstrated a strong association between FA1 and (neuro)endocrine structures. In the anterior pituitary gland FA1 was colocalized with GH, and the present study was performed to evaluate a possible relationship between GH and FA1. FA1 and GH levels were measured during a 24-h period at 20-min intervals. In contrast to the known GH peaks during 24-h sampling, there was no detectable FA1 peak. The FA1 responses to placebo were not significantly different from the responses to the combination of pyridostigmine and GHRH. No significant difference was found between basal FA1 (nanograms per ml) levels [median (minimum-maximum)] in healthy adults [n = 40; 28.6 ng/ml (12.5-72.0)], acromegalic patients [n = 11; 31.0 ng/ml (21.6-56.3)], and patients with GH deficiency [n = 22; 32.1 ng/ml (13.4-108.7)]. FA1 levels were significantly reduced, in the six of seven acromegalic GH responders to octreotide, from [median (minimum-maximum)] 30.6 ng/ml (20.0-43.1) to 20.3 (13.9-30.2; P < 0.02). There was no significant change during placebo. FA1 levels were significantly increased compared with placebo values during 3 months of GH therapy. The increase in FA1 levels was significantly higher than the change during placebo (P < 0.003). In conclusion, a common secretory and stimulatory pathway for FA1 and GH in healthy adults has been ruled out. However, we found that pharmacologically induced changes in GH levels during weeks to months had a corresponding direct or indirect effect on FA1 levels in patients with GH deficiency or acromegaly. However, a direct effect of octreotide on FA1 levels, independent of GH levels, has not been ruled out.

Acromegaly↗
PubMed2006

Novel function of beta-arrestin2 in the nucleus of mature spermatozoa.

A growing number of proteins originally found in endocytic structures of the plasma membrane appear to be able to traffic into the nucleus, but the cellular function of this translocation remains unclear. We have found that beta-arrestin2, which typically shows a cytoplasmic localization owing to constitutive nuclear export, appears in the nucleus after stimulation of the G-protein-coupled odorant receptor hOR17-4. In the nucleus, beta-arrestin2 was involved in transcriptional regulation as shown by a Gal4-based transactivation assay. Moreover, we discovered that beta-arrestin2 and hOR17-4, a receptor known to have a role in sperm-egg communication, colocalize in the midpiece of mature human spermatozoa. Stimulation of hOR17-4 in spermatozoa induced PKA-dependent translocation of beta-arrestin2 to the nucleus and nuclear accumulation of phosphorylated MAPKs. Analysis of the interaction partners of beta-arrestin2 indicates that odorant receptor signaling in spermatozoa may be important for the regulation of gene expression during the early processes of fertilization.

Aldehydes↗
PubMed1994

Targeting of chimeric G alpha i proteins to specific membrane domains.

Heterotrimeric guanine nucleotide-regulatory (G) proteins are associated with a variety of intracellular membranes and specific plasma membrane domains. In polarized epithelial LLC-PK1 cells we have shown previously that endogenous G alpha i-2 is localized on the basolateral plasma membrane, whereas G alpha i-3 is localized on Golgi membranes. The targeting of these highly homologous G alpha i proteins to distinct membrane domains was studied by the transfection and expression of chimeric G alpha i proteins in LLC-PK1 cells. Chimeric cDNAs were constructed from the cDNAs for G alpha i-3 and G alpha i-2 and introduced into a pMXX eukaryotic expression vector containing a mouse metallothionein-I promoter. Stably transfected cell lines were produced that expressed either G alpha i-2/3 or G alpha i-3/2 chimeric proteins. Chimeric and endogenous G alpha i proteins were detected in cells using specific carboxy-terminal peptide antibodies. Immunofluorescence staining was used to localize endogenous and chimeric G alpha i proteins in LLC-PK1 cells. The staining of chimeric proteins was detected as an increased intensity of staining on membranes containing endogenous G alpha i proteins. Using confocal microscopy and image analysis we localized G alpha i-2 to a specific sub-domain of the lateral membrane of polarized cells, the chimeric G alpha i-3/2 protein was then shown to colocalize with endogenous G alpha i-2 in the same lateral plasma membrane domain. The chimeric G alpha i-2/3 protein colocalized with endogenous G alpha i-3 on Golgi membranes in LLC-PK1 cells. These results show that chimeric G alpha i proteins were targeted to the same membrane domains as endogenous G alpha i proteins and the specificity of their membrane targeting was conferred by the carboxy-terminal end of the proteins. These data provide the first evidence for specific targeting information contained in the carboxy termini of G alpha i proteins, which appears to be independent of amino-terminal membrane attachment sites in these proteins.

Amino Acid Sequence↗
PubMed1996

Intracellular targeting of GLUT4 in transfected insulinoma cells: evidence for association with constitutively recycling vesicles distinct from synaptophysin and insulin vesicles.

In adipocytes and muscle cells, the GLUT4 glucose transporter isoform is present in intracellular vesicles which continuously recycle between an intracytoplasmic location and the plasma membrane. It is not clear whether the GLUT4-vesicles represent a specific kind of vesicle or resemble typical secretory granules or synaptic-like microvesicles. To approach this question, we expressed GLUT4 in the beta cell line RINm5F and determined its intracellular localization by subcellular fractionation and by immunofluorescence and immunoelectron microscopy. GLUT4 was not found in insulin granules but was associated with a subpopulation of smooth-surface vesicles present in the trans-Golgi region and in vesicular structures adjacent to the plasma membrane. In the trans-Golgi region, GLUT4 did not colocalize with synaptophysin or TGN38. Incubation of the cells with horseradish peroxidase (HRP) led to colocalization of HRP and GLUT4 in some endosomal structures adjacent to the plasma membrane and in occasional trans-Golgi region vesicles. When cells were incubated in the presence of Bafilomycin A, analysis by confocal microscopy revealed GLUT4 in numerous large spots present throughout the cytoplasm, many of which costained for TGN38 and synaptophysin. By immunoelectron microscopy, numerous endosomes were observed which stained strongly for GLUT4. Together our data demonstrate that ectopic expression of GLUT4 in insulinoma cells reveals the presence of a subset of vesicular structures distinct from synaptic-like vesicles and insulin secretory granules. Furthermore, they indicate that GLUT4 constitutively recycles between the plasma membrane and its intracellular location by an endocytic route also taken by TGN38 and synaptophysin.

Animals↗
PubMed2003

Expression of functional tyrosine kinase B receptors by rhythmically active respiratory neurons in the pre-Bötzinger complex of neonatal mice.

Genetic loss of brain-derived neurotrophic factor (BDNF) severely disrupts brainstem control of respiratory rhythmogenesis in newborn mice; however, the sites at which BDNF acts to regulate respiratory rhythmogenesis are unknown. Using immunochemical and multiplex RT-PCR analysis in mouse brainstem slices, we report that the BDNF receptor, Tyrosine kinase B (TrkB), is strongly expressed in the pre-Bötzinger complex (PBC), the presumed site for rhythm generation, and colocalizes with neurokinin 1 (NK1), a marker of neurons critical for breathing. The period of the respiratory rhythm generated by PBC neurons in vitro was increased by 30% after BDNF treatment (100 ng/ml) and not by nerve growth factor (100 ng/ml) or BDNF (100 ng/ml) in the presence of the tyrosine kinase inhibitor K252a (200 nm). Both synaptic and voltage-dependent properties of PBC neurons were modified by BDNF. Synaptic currents underlying spontaneous rhythmic bursts and glutamate-evoked currents were enhanced by 66 and 33%, respectively. BDNF reduced the Ih current amplitude in rhythmic neurons by 46% and shifted its activation curve by -17 mV. All neurons expressing TrkB mRNA (n = 8) also expressed mRNAs for the Ih current [hyperpolarization-activated cyclic nucleotide-sensitive cation nonselective channel (HCN1)], and three of four NK1-positive neurons coexpressed TrkB and HCN mRNA. Six of 16 PBC neurons expressed BDNF mRNA, supporting the possibility of autocrine and paracrine actions of BDNF within the respiratory pattern generator. Our data demonstrate that BDNF can modulate respiratory network activity through TrkB signaling in rhythmic PBC neurons.

Animals↗
PubMed1995

Experimental autoimmune encephalomyelitis. Qualitative and semiquantitative differences in heat shock protein 60 expression in the central nervous system.

The factors that contribute to disease progression in multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), are not known. In this study, we have tested the hypothesis that inflammation in the central nervous system (CNS) is associated with altered expression of heat shock proteins (hsp) that may function as a target in the development of chronic disease. In normal mouse spinal cord, hsp 60 immunoreactivity was localized exclusively to its constitutive site in mitochondria. In animals with acute EAE, lesions were accentuated by increased expression of hsp 60, primarily by infiltrating cells. In chronic EAE, hsp 60 was found predominantly on CNS components, particularly oligodendrocytes, and astrocytes. In both acute and chronic lesions, hsp 60 was located in the cytoplasm of cells, but in non-lesion areas, it was mitochondrial. By semiquantitative Western blotting, hsp 60 in normal mouse spinal cord (n = 6) was 0.99 +/- 0.09% of total protein. In acute EAE (n = 6), the value was 1.08 +/- 0.1%, and in chronic EAE (n = 8), 1.39 +/- 0.39%. FACS analysis showed that a subpopulation of CD3+ cells expressed hsp 60 on the cell surface, an observation confirmed by cell surface labeling and immunoprecipitation. In chronic EAE lesions, gamma delta T cells colocalized with areas of increased hsp 60 immunoreactivity. The results support the conclusion that development of autoimmune-directed inflammation in the CNS is associated with differences in hsp 60 expression that may act as an additional target and/or modulator of the immune response.

Animals↗
PubMed1996

Organization of AMPA receptor subunits at a glutamate synapse: a quantitative immunogold analysis of hair cell synapses in the rat organ of Corti.

Sensitive and high-resolution immunocytochemical procedures were used to investigate the spatial organization of AMPA receptor subunits (GluR1-4) at the synapse between the inner hair cells and the afferent dendrites in the rat organ of Corti. This is a synapse with special functional properties and with a presynaptic dense body that defines the center of the synapse and facilitates its morphometric analysis. A quantitative postembedding immunocytochemical analysis was performed on specimens that had been embedded in a metachrylate resin at low temperature after freeze substitution. Single- and double-labeling procedures indicated that GluR2/3 and GluR4 subunits were colocalized throughout the postsynaptic density, with a maximum distance of 300 nm from the presynaptic body and with higher concentrations peripherally than centrally. No receptor immunolabeling was found at extrasynaptic membranes, but some GluR4 subunits appeared to be expressed presynaptically. The synapses between outer hair cells and afferent dendrites were devoid of labeling. The present data indicate that AMPA receptor subunits are inserted into the postsynaptic membrane in a very precise manner and that their density increases on moving away from the center of the synapse.

Animals↗
PubMed2003

Distribution of gephyrin in the human brain: an immunohistochemical analysis.

Gephyrin is an ubiquitously expressed protein that, in the central nervous system, generates a protein scaffold to anchor inhibitory neurotransmitter receptors in the postsynaptic membrane. It was first identified as a protein component of the glycine receptor complex. Recent studies have demonstrated that gephyrin is colocalized with several subtypes of GABA(A) receptors and is part of postsynaptic GABA(A) receptor clusters. Here, we describe a study of the regional and cellular distribution of gephyrin in the human brain, determined by immunohistochemical localisation at the light and confocal laser scanning microscopic levels. At the regional level, gephyrin immunoreactivity was observed in most of the major brain regions examined. The most intense staining was in the cerebral cortex, hippocampus and caudate-putamen, in various brainstem nuclei with more moderate levels in the thalamus and cerebellum. At the cellular level gephyrin immunoreactivity was present on the plasma membranes of the soma and dendrites of pyramidal neurons throughout the various cortical regions examined. In the hippocampus, intense staining was observed on the granule cells of the dentate gyrus, and neurons of the CA1 and CA3 regions showed intense punctate gephyrin staining on their apical dendrites and cell bodies. Gephyrin immunoreactivity was also observed on neurons in the thalamus, globus pallidus and substantia nigra. In the putamen intense labelling of the striosomes was observed; most of the medium-sized neurons in the caudate-putamen were weakly labelled and many large neurons of the striatum were conspicuously stained. Many of the brainstem nuclei, notably the dorsal motor nucleus of the vagus, hypoglossal nucleus, trigeminal nucleus and inferior olive were all labelled with gephyrin. The spinal cord also showed high levels of gephyrin immunoreactivity. Our results demonstrate that the anchoring protein gephyrin is ubiquitously present in the human brain. We therefore suggest that gephyrin may have a central organizer role in assembling and stabilizing inhibitory postsynaptic membranes in human brain and is similar in function to those observed in the rodent brain. These findings contribute towards elucidating the role of gephyrin in the human brain.

Aged↗
PubMed2001

Atypical mouse cerebellar development is caused by ectopic expression of the forkhead box transcription factor HNF-3beta.

To assess the role of hepatocyte nuclear factor-3beta (HNF-3beta) in hepatocyte-specific gene transcription, we reported the characterization of the liver phenotype with transgenic mice in which the -3-kb transthyretin (TTR) promoter functioned to increase HNF-3beta expression. During breeding of the TTR-HNF-3beta transgenic mice we noticed that they displayed severe ataxia. In this study, we describe the analysis of our transgenic cerebellar phenotype and demonstrate that ectopic expression of HNF-3beta disrupted cerebellar morphogenesis and caused reduction in cerebellar size. In postnatal cerebellum, the HNF-3beta transgene expression pattern is colocalized to glial fibrillary acidic protein-positive cerebellar astrocytes and Bergmann glial cells. As a result of protracted expression, the transgenic cerebella are impaired in terms of astrocyte dispersal and formation of Bergmann glial cell processes. This caused a disruption in neuronal cell migration to the cortical laminar layers and Purkinje dendritic arbor maturation, thus leading to diminished foliation. Differential hybridization of cDNA arrays was used to identify altered expression of cerebellar genes, which is consistent with the observed defect in transgenic cerebellar morphogenesis and size as well as glial maturation. These include diminished expression of the brain lipid-binding protein, which is required for glial morphological differentiation, and the basic helix-loop-helix NeuroD/Beta2 and homeodomain Engrailed-2 transcription factors, which are required for normal cerebellar morphogenesis and foliation. Undetectable levels of ataxia telangiectasia (ATM), which is required for proper development of the Purkinje dendritic arbor, were found in postnatal transgenic cerebella. Furthermore, the transgenic cerebella displayed levels of insulin-like growth factor binding protein-1 elevated to 22 times greater than those measured for wild-type cerebella, an elevation consistent with the reduction in transgenic cerebellar size.

Animals↗
PubMed1993

Colocalization of low- and high-affinity NGF receptors on PC12 cells, C6 glioma cells and dorsal root ganglion neurons.

The biological responsiveness of neural cells to nerve growth factor (NGF) appears to require expression and ligand binding to both the low-affinity NGF receptor (LNGFR) and the proto-oncogene product trk, the latter being a receptor tyrosine kinase. Immunolocalization of the LNGFR and the high-affinity component of the NGF receptor, trk (HNGFR) was studied by electron microscopic morphometric analysis on cultured PC12 pheochromocytoma cells, C6 glioma cells and neonatal rat dorsal root ganglia neurons using a double immunogold labeling technique. Two receptor-specific antibodies, anti-LNGFR monoclonal antibody 192-IgG and a polyclonal antibody against the 14 carboxy-terminal amino acids of the Trk protein, were utilized in conjunction with immunoglobulin conjugated to colloidal gold particles of different sizes. All cells treated with NGF (50 ng/ml) displayed significant colocalization of LNGFR/HNGFR-like immunoreactivity. Gold particles associated with LNGFR (LNGFR-like immunoreactivity) were frequently seen near 2 or 3 (or more) particles delineating the HNGFR on all cell surfaces. Positive Trk-like immunoreactivity (HNGFR) thus seems to localize in close proximity to LNGFRs in at least these cell types.

Animals↗