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1.
Transl Psychiatry ; 4: e372, 2014 Mar 18.
Article in English | MEDLINE | ID: mdl-24643163

ABSTRACT

Recent genome-wide association studies have pointed to single-nucleotide polymorphisms (SNPs) in genes encoding the neuronal calcium channel CaV1.2 (CACNA1C; rs1006737) and the presynaptic active zone protein Piccolo (PCLO; rs2522833) as risk factors for affective disorders, particularly major depression. Previous neuroimaging studies of depression-related endophenotypes have highlighted the role of the subgenual cingulate cortex (CG25) in negative mood and depressive psychopathology. Here, we aimed to assess how recently associated PCLO and CACNA1C depression risk alleles jointly affect memory-related CG25 activity as an intermediate phenotype in clinically healthy humans. To investigate the combined effects of rs1006737 and rs2522833 on the CG25 response, we conducted three functional magnetic resonance imaging studies of episodic memory formation in three independent cohorts (N=79, 300, 113). An epistatic interaction of PCLO and CACNA1C risk alleles in CG25 during memory encoding was observed in all groups, with carriers of no risk allele and of both risk alleles showing higher CG25 activation during encoding when compared with carriers of only one risk allele. Moreover, PCLO risk allele carriers showed lower memory performance and reduced encoding-related hippocampal activation. In summary, our results point to region-specific epistatic effects of PCLO and CACNA1C risk variants in CG25, potentially related to episodic memory. Our data further suggest that genetic risk factors on the SNP level do not necessarily have additive effects but may show complex interactions. Such epistatic interactions might contribute to the 'missing heritability' of complex phenotypes.


Subject(s)
Calcium Channels, L-Type/genetics , Cytoskeletal Proteins/genetics , Depressive Disorder, Major/genetics , Epistasis, Genetic/genetics , Gyrus Cinguli/physiopathology , Memory, Episodic , Neuropeptides/genetics , Adult , Functional Neuroimaging , Hippocampus/physiopathology , Humans , Magnetic Resonance Imaging , Phenotype , Polymorphism, Single Nucleotide
2.
Transl Psychiatry ; 1: e15, 2011 Jul 05.
Article in English | MEDLINE | ID: mdl-22832518

ABSTRACT

Dopaminergic projections to the prefrontal cortex support higher-order cognitive functions, and are critically involved in many psychiatric disorders that involve memory deficits, including schizophrenia. The role of prefrontal dopamine in long-term memory, however, is still unclear. We used an imaging genetics approach to examine the hypothesis that dopamine availability in the prefrontal cortex selectively affects the ability to suppress interfering memories. Human participants were scanned via functional magnetic resonance imaging while practicing retrieval of previously studied target information in the face of interference from previously studied non-target information. This retrieval practice (RP) rendered the non-target information less retrievable on a later final test-a phenomenon known as retrieval-induced forgetting (RIF). In total, 54 participants were genotyped for the catechol-O-methyltransferase (COMT) Val(108/158)Met polymorphism. The COMT Val(108/158)Met genotype showed a selective and linear gene-dose effect on RIF, with the Met allele, which leads to higher prefrontal dopamine availability, being associated with greater RIF. Mirroring the behavioral pattern, the functional magnetic resonance imaging data revealed that Met allele carriers, compared with Val allele carriers, showed a greater response reduction in inhibitory control areas of the right inferior frontal cortex during RP, suggesting that they more efficiently reduced interference. These data support the hypothesis that the cortical dopaminergic system is centrally involved in the dynamic control of human long-term memory, supporting efficient remembering via the adaptive suppression of interfering memories.


Subject(s)
Dopamine/physiology , Memory, Long-Term/physiology , Mental Recall/physiology , Prefrontal Cortex/physiology , Adult , Humans , Inhibition, Psychological , Young Adult
3.
Mol Cell Neurosci ; 19(3): 459-75, 2002 Mar.
Article in English | MEDLINE | ID: mdl-11906216

ABSTRACT

Caldendrin is the founder member of a recently discovered family of calmodulin-like proteins, which are highly abundant in brain. In this study we examined the organization of the murine and human caldendrin gene as well as the expression pattern of transcripts for caldendrin and two novel splice variants. In addition the distribution of caldendrin in rat brain has been assessed by immunohistochemistry. Caldendrin is localized to the somatodendritic compartment of a subpopulation of mainly principal neurons in brain regions with a laminar organization and is present only at a subset of mature excitatory synapses. Caldendrin immunoreactivity (IR) is tightly associated with the cortical cytoskeleton, enriched in the postsynaptic density (PSD) fraction, and associates late during development with the synaptic cytomatrix. The expression is highly heterogenous within cortex, with highest levels of caldendrin IR in layer III of the piriform and layer II/III of the somatosensory cortex. The segregated cortical distribution to areas, which represent the most important primary sensory systems of the rodent brain, may reflect different requirements for dendritic Ca2+-signaling in these neurons. The presence of caldendrin in the PSD of distinct synapses may have important implications for Ca2+-modulated processes of synaptic plasticity.


Subject(s)
Alternative Splicing/genetics , Calcium-Binding Proteins/genetics , Cerebral Cortex/cytology , Nerve Tissue Proteins/genetics , Neurons/physiology , Amino Acid Sequence , Animals , Antibody Specificity , Calcium-Binding Proteins/analysis , Calcium-Binding Proteins/immunology , Cerebral Cortex/chemistry , Dendrites/chemistry , Dendrites/ultrastructure , Gene Expression/physiology , Humans , Male , Microscopy, Immunoelectron , Molecular Sequence Data , Nerve Tissue Proteins/analysis , Nerve Tissue Proteins/immunology , Neurons/chemistry , Neurons/ultrastructure , RNA, Messenger/analysis , Rats , Rats, Sprague-Dawley , Synapses/physiology
4.
Mol Cell Biol ; 21(17): 5970-8, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11486035

ABSTRACT

Neurocan is a component of the extracellular matrix in brain. Due to its inhibition of neuronal adhesion and outgrowth in vitro and its expression pattern in vivo it was suggested to play an important role in axon guidance and neurite growth. To study the role of neurocan in brain development we generated neurocan-deficient mice by targeted disruption of the neurocan gene. These mice are viable and fertile and have no obvious deficits in reproduction and general performance. Brain anatomy, morphology, and ultrastructure are similar to those of wild-type mice. Perineuronal nets surrounding neurons appear largely normal. Mild deficits in synaptic plasticity may exist, as maintenance of late-phase hippocampal long-term potentiation is reduced. These data indicate that neurocan has either a redundant or a more subtle function in the development of the brain.


Subject(s)
Brain/growth & development , Chondroitin Sulfate Proteoglycans/physiology , Extracellular Matrix Proteins/physiology , Nerve Tissue Proteins/physiology , Animals , Brain/embryology , Brain/pathology , Brevican , Chondroitin Sulfate Proteoglycans/genetics , Extracellular Matrix Proteins/genetics , Gene Expression Regulation, Developmental , Hippocampus/physiology , Lectins, C-Type , Mice , Mice, Inbred C57BL , Mice, Knockout , Nerve Tissue Proteins/genetics , Neurocan , Neuronal Plasticity , Synapses/physiology , Tenascin/genetics , Up-Regulation
5.
Invest Ophthalmol Vis Sci ; 42(8): 1907-14, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11431460

ABSTRACT

PURPOSE: To examine the expression and localization of the neuroplastins (np), two synapse-enriched members of the immunoglobulin (Ig) superfamily of cell-adhesion molecules, in the developing and adult retina and optic nerve. METHODS: Expressions of the two isoforms np55 and np65 and carboxyl-terminal splice variants were investigated by immunocytochemistry, Western blot analysis, RT-PCR, and in situ hybridization. RESULTS: Immunoreactivity for both neuroplastins was confined to the two synaptic layers of the retina: the inner (IPL) and outer plexiform layer (OPL). Significant overlap was found in staining at synaptic structures with synaptophysin. A large proportion of immunoreactivity for both isoforms, however, was of perisynaptic origin. In situ hybridization studies were suggestive of a pre- and postsynaptic localization of np65 in the OPL. Transcripts for np55 were already present at birth in the inner retina, but the hybridization signals increased during postnatal development. Np65 transcripts and immunosignals appeared at later developmental ages, concomitant with synapse formation in the OPL. Several C-terminal neuroplastin cDNA clones harbor an insert of 12 bp, coding for four amino acids (DDEP) in the intracellular domain of neuroplastins. Splice isoforms containing the insert exhibited a developmental expression pattern similar to that of np55; however, both neuroplastins could harbor the C-terminal insert. Neuroplastins were also detected in optic nerve homogenates. RT-PCR and blockade of axonal transport by nerve crush confirmed transcript and protein expression in optic nerve tissue. CONCLUSIONS: The findings suggest a role for neuroplastins in cell adhesion in the plexiform layers during histogenesis, as well as in maintenance of connections between specific cellular structures.


Subject(s)
Immunoglobulins/genetics , Membrane Glycoproteins/genetics , Nerve Tissue Proteins/genetics , Retina/metabolism , Animals , Blotting, Western , DNA Primers/chemistry , Fluorescent Antibody Technique, Indirect , Gene Expression , Immunoglobulins/metabolism , In Situ Hybridization , Male , Membrane Glycoproteins/metabolism , Nerve Tissue Proteins/metabolism , Optic Nerve/metabolism , Protein Isoforms , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Retina/growth & development , Reverse Transcriptase Polymerase Chain Reaction
6.
J Comp Neurol ; 428(4): 616-29, 2000 Dec 25.
Article in English | MEDLINE | ID: mdl-11077416

ABSTRACT

The extracellular matrix glycoprotein tenascin-R (TN-R), colocalizing with hyaluronan, phosphacan, and aggregating chondroitin sulphate proteoglycans in the white and grey matter, is accumulated in perineuronal nets that surround different types of neurons in many brain regions. To characterize the role of TN-R in the formation of perineuronal nets, we studied their postnatal development in wild-type mice and in a TN-R knock-out mutant by using the lectin Wisteria floribunda agglutinin and an antibody to nonspecified chondroitin sulphate proteoglycans as established cytochemical markers. We detected the matrix components TN-R, hyaluronan, phosphacan, neurocan, and brevican in the perineuronal nets of cortical and subcortical regions. In wild-type mice, lectin-stained, immature perineuronal nets were first seen on postnatal day 4 in the brainstem and on day 14 in the cerebral cortex. The staining intensity of these nets for TN-R, hyaluronan, phosphacan, neurocan, and brevican was extremely weak or not distinguishable from that of the surrounding neuropil. However, all markers showed an increase in staining intensity of perineuronal nets reaching maximal levels between postnatal days 21 and 40. In TN-R-deficient animals, the perineuronal nets tended to show a granular component within their lattice-like structure at early stages of development. Additionally, the staining intensity in perineuronal nets was reduced for brevican, extremely low for hyaluronan and neurocan, and virtually no immunoreactivity was detectable for phosphacan. The granular configuration of perineuronal nets became more predominant with advancing age of the mutant animals, indicating the continued abnormal aggregation of chondroitin sulphate proteoglycans complexed with hyaluronan. As shown by electron microscopy in the cerebral cortex, the disruption of perineuronal nets was not accompanied by apparent changes in the synaptic structure on net-bearing neurons. The regional distribution patterns and the temporal course of development of perineuronal nets were not obviously changed in the mutant. We conclude that the lack of TN-R initially and continuously disturbs the molecular scaffolding of extracellular matrix components in perineuronal nets. This may interfere with the development of the specific micromilieu of the ensheathed neurons and adjacent glial cells and may also permanently change their functional properties.


Subject(s)
Animals, Wild/metabolism , Brain/growth & development , Brain/metabolism , Extracellular Matrix/metabolism , Mice, Knockout/metabolism , Neurons/metabolism , Tenascin/deficiency , Age Factors , Animals , Animals, Wild/anatomy & histology , Brain/ultrastructure , Brevican , Chondroitin Sulfate Proteoglycans/metabolism , Extracellular Matrix/ultrastructure , Female , Hyaluronic Acid/metabolism , Lectins , Lectins, C-Type , Male , Mice , Mice, Knockout/anatomy & histology , Nerve Tissue Proteins/metabolism , Neurocan , Neurons/ultrastructure , Receptor-Like Protein Tyrosine Phosphatases, Class 5 , Tenascin/genetics
7.
Cell Tissue Res ; 298(1): 21-32, 1999 Oct.
Article in English | MEDLINE | ID: mdl-10555536

ABSTRACT

Caldendrin is a novel calcium-binding protein confined to the somatodendritic compartment of neurons. Here we have studied the expression pattern of caldendrin in the rat retina. First we assessed the distribution of caldendrin transcripts in the adult and developing retina by in situ hybridization. In the adult retina, transcripts are expressed mainly in the inner half of the inner nuclear layer (INL) and to a lesser extent in the ganglion cell layer (GCL). During development labeling of the inner part of the cytoblast layer, where amacrine cells reside, is already present at postnatal day 1 (P1). The intensity of hybridization signal in this sublamina of the developing INL increases up to P8, whereas significant labeling in the GCL was first found at P14, coinciding with eye opening. Immunodetection with a polyclonal antibody revealed intensive staining of cells in the inner retina, which are presumably mainly amacrine and significantly fewer bipolar and ganglion cells. All parvalbumin-containing All amacrines were immunopositive for caldendrin. Colocalization with calbindin was found in cone bipolar cells, the majority of AII amacrines, and calbindin-positive cells in the GCL. In the GCL, caldendrin was also colocalized with calretinin-immunopositive cells. Most caldendrin-positive amacrine cells in the adult rat retina were glycinergic and only a few were GABAergic. In retinal flat mounts, it was confirmed that less than 10% of retrogradely labeled retinal ganglion cells (RGC) are caldendrin-positive. Caldendrin immunoreactivity does not colocalize with tyrosine hydroxylase, VIP, substance P and somatostatin immunoreactivity. In summary, caldendrin expression is regulated differentially in retinal cell types during development and is restricted to a subpopulation of amacrine, bipolar, and ganglion cells, suggesting specific functions in the developing and mature retina.


Subject(s)
Calcium-Binding Proteins/metabolism , Nerve Tissue Proteins/metabolism , Retina/cytology , Retina/metabolism , Retinal Ganglion Cells/metabolism , Animals , Calcium-Binding Proteins/chemistry , Calcium-Binding Proteins/genetics , Choline O-Acetyltransferase/metabolism , Cytoskeleton/metabolism , EF Hand Motifs , Gene Expression Regulation, Developmental , Glycine/metabolism , Immunohistochemistry , In Situ Hybridization , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/genetics , Neurons/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Retina/growth & development , gamma-Aminobutyric Acid/metabolism
8.
Brain Res Mol Brain Res ; 69(2): 232-41, 1999 Jun 08.
Article in English | MEDLINE | ID: mdl-10366744

ABSTRACT

The expression of c-fos, c-jun, jun-b, jun-d, srf and pc4 mRNA was examined after partial optic nerve crush in the adult rat retina by in situ hybridization. Optic nerve injury led exclusively to the upregulation of c-jun, with cellular label indicative for c-jun mRNA in the retinal ganglion cell layer after two days, three days and one week post-injury. This expression pattern was in accordance with the appearance of c-Jun immunoreactivity in retinal flat mounts. Injection of an antisense but not a missense oligonucleotide against c-jun after partial crush resulted in a reduced number of connected retinal ganglion cells (RGCs) as shown by retrograde labeling. Prelabeling of RGCs with fluorogold before optic nerve section and subsequent antisense targeting against c-jun, however, led to a slightly higher number of surviving but axotomized RGCs. C-Jun antibody staining of retinal whole mounts pre- or postlabeled after crush by intracollicular administration of fluorogold showed strong c-Jun immunoreactivity in connected RGCs and also in a population of disconnected RGCs. Double labeling with an antibody directed against the transcription factor ATF-2 revealed strong co-expression of c-Jun and ATF-2 in connected RGCs but not in axotomized cells. Taken together these data indicate that both RGCs in continuity and those in discontinuity with the superior colliculus respond both equally to the noxious stimulus with c-Jun expression. Moreover, the co-expression of c-Jun with high levels of ATF-2 appears to be essential for either the continuity or survival of RGCs which remain connected with their target. In disconnected RGCs, however, low levels of ATF-2 and the co-expression of c-Jun may be related to cell death.


Subject(s)
Axons/metabolism , Gene Expression Regulation/physiology , Nerve Tissue Proteins/genetics , Optic Nerve Injuries , Retinal Ganglion Cells/metabolism , Activating Transcription Factor 2 , Animals , Axotomy , Cell Survival/physiology , Cyclic AMP Response Element-Binding Protein/genetics , In Situ Hybridization , Nerve Crush , Neural Pathways/physiology , Oligonucleotides, Antisense , Optic Nerve/metabolism , Optic Nerve/ultrastructure , Proto-Oncogene Proteins c-jun/genetics , Rats , Rats, Inbred Strains , Retinal Ganglion Cells/cytology , Transcription Factors/genetics
9.
J Neurotrauma ; 16(2): 153-63, 1999 Feb.
Article in English | MEDLINE | ID: mdl-10098960

ABSTRACT

We have investigated time course and characteristics of retinal ganglion cell (RGC) death after partial optic nerve injury. In situ end labeling of DNA fragments with the terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine (dUTP)-biotin nick end labeling (TUNEL) method revealed the presence of apoptotic cells on as early as 5 days postcrush with a very high number of TUNEL-positive cells 1 week postinjury. At the ultrastructural level, features of apoptosis were clearly present in the ganglion cell layer at this time point. Moreover, TUNEL-positive cells could be identified as retinal ganglion cells by retrograde labeling with fluorogold. In addition, DNA laddering characteristic for apoptosis was found 1 week postinjury. A considerable number of TUNEL-labeled cells was still found after 2 weeks postinjury. Retinal whole mounts prepared at postlesion days 2-5, however, revealed that many cell bodies with ruptured membranes as evidenced by nucleosomal Sytox staining were present. These cells were also identified as retinal ganglion cells by retrograde labeling with fluorogold. Moreover, at this early stages of RGC degeneration necrotic cellular profiles could be detected by electron microscopic analysis. Thus, evidence is provided that necrosis and apoptosis follow a distinctly different time course after partial optic nerve injury.


Subject(s)
Apoptosis , Brain Injuries/pathology , Necrosis , Nerve Degeneration/physiopathology , Optic Nerve Diseases/pathology , Retinal Ganglion Cells/pathology , Animals , Disease Models, Animal , Disease Progression , In Situ Nick-End Labeling , Male , Rats , Rats, Inbred Strains , Time Factors
10.
Restor Neurol Neurosci ; 14(2): 127-34, 1999 Jan 01.
Article in English | MEDLINE | ID: mdl-22387508

ABSTRACT

In the past few years we established the partial crush of the optic nerve as an in vivo model system for the study of signaling pathways involved in molecular plasticity after axonal injury. The simplicity of this model at the cellular level allows decisive questions to be anwsered whilst functional aspects of visual information processing can be studied in parallel. A major advantage of a partial optic nerve crush model is the opportunity to directly compare different cell populations: (i) the rapidly degenerating retinal ganglion cells (RGC), (ii) the axotomized RGC population that eventually dies over the period of the next few weeks, (iii) the axotomized RGC population surviving for a long time in the retina without an axon and (iv) the surviving RGC population that maintains axonal connections to their brain targets. Thus, differential aspects of post-lesion plasticity can be analyzed. Using this axonal injury model we investigated the expression of immediate early genes, glutamate receptors, and other differentially expressed genes that we identified with a combined subtractive hybridization and suppression polymerase chain reaction (PCR) screen. Moreover, we characterized time course of cell death, the astroglia response of the retina and optic nerve as well as the topography of anterograde and retrograde axonal transport.

11.
J Neurosci ; 18(20): 8278-91, 1998 Oct 15.
Article in English | MEDLINE | ID: mdl-9763472

ABSTRACT

Cellular-specific splicing of the retinal NMDAR1 receptor (NR1) and expression of NMDAR2 receptor (NR2) subunits in response to optic nerve injury was investigated by in situ hybridization in adult rats. A controlled optic nerve crush led to a clear alteration in the expression of alternatively spliced NR1 variants in the retinal ganglion cell layer (GCL). The NR1-2b and NR1-4b isoforms were preferentially expressed between 2 d and 1 week after injury, whereas expression for all other isoforms remained either unchanged or decreased to barely detectable levels within 4 weeks. Cellular silver grain density for NR2 subunits also declined in the GCL after trauma. To directly test the hypothesis that NR1b expression is crucial for cell survival after axonal trauma, we administered intraocularly an antisense oligonucleotide against the NR1b isoform 2 and 3 d after injury. This led to a drastic loss of retrogradely labeled retinal ganglion cells (RGCs). Antisense targeting clearly reduced retinal NR1 protein levels, as judged by Western blot analysis, but had no effect on the cell number in control retinas. These findings point toward injury-specific changes in alternative splicing of the NR1 receptor, which are crucial for the survival of RGCs after partial axonal trauma. We therefore propose that this reflects an adaptive, rather than a pathogenic, cellular response to neurotrauma.


Subject(s)
Alternative Splicing/physiology , Axons/physiology , Optic Nerve Injuries , Receptors, N-Methyl-D-Aspartate/genetics , Retinal Ganglion Cells/cytology , Animals , Antisense Elements (Genetics) , Blotting, Western , Cell Death/physiology , Cell Survival/physiology , Gene Expression/physiology , Horseradish Peroxidase , Image Processing, Computer-Assisted , In Situ Hybridization , Isomerism , Male , Nerve Crush , Oligonucleotide Probes , Optic Nerve/cytology , Optic Nerve/physiology , RNA, Messenger/analysis , Rats , Rats, Inbred Strains , Receptors, N-Methyl-D-Aspartate/chemistry , Retinal Ganglion Cells/chemistry , Retinal Ganglion Cells/ultrastructure , Reverse Transcriptase Polymerase Chain Reaction
12.
Eur J Neurosci ; 10(5): 1621-30, 1998 May.
Article in English | MEDLINE | ID: mdl-9751135

ABSTRACT

Brevican is a member of the aggrecan/versican family of proteoglycans. In contrast to the other family members, brevican occurs both as soluble isoforms secreted into the extracellular space and membrane-bound isoforms which are anchored to the cell surface via a glycosylphosphatidylinositol (GPI) moiety. Expression of both variants, which are encoded by two differentially processed transcripts from the same gene, is confined to the nervous system. In the current study, we have used in situ hybridization to examine the cellular sites of synthesis for both mRNAs during postnatal development of the rat brain. Whereas the 3.6-kb transcript encoding secreted brevican displays a widespread distribution in grey matter structures, including cerebellar and cerebral cortex, hippocampus and thalamic nuclei with silver grains accumulating over neuronal cell bodies, the smaller transcript (3.3 kb) encoding GPI-anchored isoforms appears to be largely confined to white matter tracts and diffusely distributed glial cells. This expression pattern is further confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) experiments with RNA from different glial cell cultures, and by biochemical data demonstrating that the crude membrane fraction from isolated optic nerve contains high amounts of phosphatidylinositol-specific phospholipase C (PI-PLC)-sensitive brevican immunoreactivity. During ontogenetic development, both brevican transcripts are generally up-regulated. However, the expression of glypiated brevican is delayed by about 1 week, compared with the expression of the secreted isoform. This late appearance of GPI-linked brevican, its predominant expression in glial cells and its tight association with brain myelin fractions suggest a functional role in neuroglia.


Subject(s)
Alternative Splicing , Brain Chemistry/physiology , Chondroitin Sulfate Proteoglycans/metabolism , Glycosylphosphatidylinositols , Nerve Tissue Proteins/metabolism , RNA, Messenger/analysis , Animals , Brevican , Cells, Cultured , Cerebellum/chemistry , Cerebral Cortex/chemistry , Hippocampus/chemistry , Immunohistochemistry , In Situ Hybridization , Lectins, C-Type , Neuroglia/chemistry , Rats
13.
J Biol Chem ; 273(33): 21324-31, 1998 Aug 14.
Article in English | MEDLINE | ID: mdl-9694893

ABSTRACT

Using antibodies against synaptic protein preparations, we cloned the cDNA of a new Ca2+-binding protein. Its C-terminal portion displays significant similarity with calmodulin and contains two EF-hand motifs. The corresponding mRNA is highly expressed in rat brain, primarily in cerebral cortex, hippocampus, and cerebellum; its expression appears to be restricted to neurons. Transcript levels increase during postnatal development. A recombinant C-terminal protein fragment binds Ca2+ as indicated by a Ca2+-induced mobility shift in SDS-polyacrylamide gel electrophoresis. Antisera generated against the bacterial fusion protein recognize a brain-specific protein doublet with apparent molecular masses of 33 and 36 kDa. These data are confirmed by in vitro translation, which generates a single 36-kDa polypeptide, and by the heterologous expression in 293 cells, which yields a 33/36-kDa doublet comparable to that found in brain. On two-dimensional gels, the 33-kDa band separates into a chain of spots plausibly due to differential phosphorylation. This view is supported by in situ phosphorylation studies in hippocampal slices. Most of the immunoreactivity is detectable in cytoskeletal preparations with a further enrichment in the synapse-associated cytomatrix. These biochemical data, together with the ultra-structural localization in dendrites and the postsynaptic density, strongly suggest an association with the somato-dendritic cytoskeleton. Therefore, this novel Ca2+-binding protein was named caldendrin.


Subject(s)
Calcium-Binding Proteins/metabolism , Dendrites/metabolism , Nerve Tissue Proteins/metabolism , Neurons/metabolism , Amino Acid Sequence , Animals , Base Sequence , Calcium-Binding Proteins/genetics , Cloning, Molecular , DNA, Complementary , Molecular Sequence Data , Nerve Tissue Proteins/genetics , Protein Kinase C/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Subcellular Fractions/metabolism , Substrate Specificity
14.
J Biol Chem ; 270(45): 27206-12, 1995 Nov 10.
Article in English | MEDLINE | ID: mdl-7592978

ABSTRACT

cDNA clones encoding proteins related to the aggrecan/versican family of proteoglycan core proteins have been isolated with antisera against rat brain synaptic junctions. Two sets of overlapping cDNAs have been characterized that differ in their 3'-terminal regions. Northern analyses with probes derived from unique regions of each set were found to hybridize with two brain-specific transcripts of 3.3 and 3.6 kilobases (kb). The 3.6-kb transcript encodes a polypeptide that exhibits 82% sequence identity with bovine brevican and is thought to be the rat ortholog of brevican. Interestingly, the polypeptide deduced from the open reading frame of the 3.3-kb transcript is truncated just carboxyl-terminal of the central domain of brevican and instead contains a putative glypiation signal. Antibodies raised against a bacterially expressed glutathione S-transferase-brevican fusion protein have been used to show that both soluble and membrane-bound brevican isoforms exist. Treatment of the crude membrane fraction and purified synaptic plasma membranes with phosphatidylinositol-specific phospholipase C revealed that isoforms of brevican are indeed glycosylphosphatidylinositol-anchored to the plasma membrane. Moreover, digestions with chondroitinase ABC have indicated that rat brevican, like its bovine ortholog, is a conditional chondroitin sulfate proteoglycan. Immunohistochemical studies have shown that brevican is widely distributed in the brain and is localized extracellularly. During postnatal development, amounts of both soluble and phosphatidylinositol-specific phospholipase C-sensitive isoforms increase, suggesting a role for brevican in the terminally differentiating and the adult nervous system.


Subject(s)
Brain/metabolism , Chondroitin Sulfate Proteoglycans/metabolism , Glycosylphosphatidylinositols/metabolism , Nerve Tissue Proteins/metabolism , Amino Acid Sequence , Animals , Base Sequence , Brevican , Cattle , Cell Line , Cell Membrane/metabolism , Chondroitin Sulfate Proteoglycans/genetics , Cloning, Molecular , DNA, Complementary/genetics , Gene Expression Regulation, Developmental , Humans , Immunohistochemistry , Lectins, C-Type , Molecular Sequence Data , Nerve Tissue Proteins/genetics , Phosphatidylinositol Diacylglycerol-Lyase , Phosphoinositide Phospholipase C , Phosphoric Diester Hydrolases/metabolism , RNA/genetics , RNA/metabolism , Rats , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/metabolism , Solubility , Tissue Distribution , Transfection
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