Characterization of novel glycoprotein components of synaptic membranes and postsynaptic densities, gp65 and gp55, with a monoclonal antibody

A monoclonal antibody, mab SMgp65, which recognises two major glycoprotein components of isolated forebrain synaptic subfractions has been raised. The mab has been used to study the cellular and subcellular localisation of these novel glycoproteins and for the partial characterisation of both molecular species. Western blots show that the mab reacts with two diffuse glycoprotein bands (gp) of apparent Mr 65,000, gp65, and 55,000, gp55. Both glycoproteins are membrane-bound, only detectable in CNS tissue and exist solely in a concanavalin A (con A) binding form. Digestion with endoglycosidase H lowers the Mr of both glycoproteins by some 5-7 kDa. Gp65 and gp55 are enriched in synaptic membrane (SM), light membrane (LM) and microsomal fractions. However, whilst gp65 is enriched in isolated postsynaptic densities (psds) gp55 is conspicuously absent from this fraction. Regional distribution studies show a marked variation in the level of gp65. Gp65 is concentrated in several forebrain regions notably cerebral cortex, hippocampus and striatum, is present only in low levels in cerebellum and is barely detectable in pons and medulla. In contrast gp55 is present in all regions studied, but is most concentrated in cerebellum. Immunocytochemical studies show intense staining of regions rich in gp65, but no staining of regions deficient in this glycoprotein. This suggests that the mab recognises gp65, but not gp55 in fixed tissue sections. Exposure of tissue sections to Triton X-100 increases the intensity of gp65-like immunoreactivity, but does not alter its pattern of subcellular distribution. Higher resolution studies show the immunoreactivity to be localised to subsets of neurites, many being axonal. The reaction deposits also extend into the synaptic region of the immunoreactive neurones. Cultured cerebellar granule cells, but not astrocytes express gp55. The results are discussed in terms of the molecular properties and localisation of these two novel glycoproteins.     

Characterisation of a novel glycoprotein (AvGp50) in the avian nervous system, with a monoclonal antibody.

A size fractionated lentil lectin-positive fraction derived from a deoxycholate extract of 1-day-old chick forebrain membranes was used to generate a series of monoclonal antibodies (Mabs) against neural antigens. One of these, MabSA1.7 recognises a glycoprotein which is enriched in synaptic plasma membranes, designated AvGp50. Polyacrylamide gel electrophoresis and Western blots show that AvGp50 is comprised of at least two glycoforms, with M(r)s of 53 kDa and 49 kDa respectively. AvGp50 is nervous system specific and most abundantly expressed in the forebrain, tecta and cerebellum where its pattern of expression is developmentally regulated. Immunohistochemical data localises AvGp50 to regions characterised by highly concentrated synapses, in particular, the molecular and granule cell layers of the cerebellum and in the inner and outer plexiform layers in the retina. Solubilization of the protein with the detergent Triton X-100 shows that AvGp50 is predominantly a cytoskeletally associated glycoprotein. However, when a synaptic plasma membrane fraction was treated with Triton X-114, AvGp50 partitioned into the detergent phase. Digestion of the protein with N-glycanase cleaved five N-linked carbohydrate side chains and reduced the molecular weight to approximately 34 and 31 kDa. Removal of the carbohydrate side chains led to an almost complete loss of recognition of the 34 kDa glycoform by the MabSA1.7, suggesting that the monoclonal antibody recognises a carbohydrate rather than peptide epitope.     

Neurite Outgrowth Inhibitor of Gliotic Brain Tissue. Mode of Action and Cellular Localization, Studied with Specific Monoclonal Antibodies

Membranes from injured adult rat brain express a heparan/chondroitin sulphate proteoglycan that inhibits neurite outgrowth in vitro. We have developed monoclonal antibodies (Mabs) against this proteoglycan, two of which were characterized and used for the study of the inhibitor mode of action and localization in normal and injured adult brain. The antibodies recognized a molecule of apparent molecular weight 200 kDa in Western blots of injured brain membranes. One of the Mabs blocked both the inhibition of neurite outgrowth and the growth cone collapse activity, associated with the proteoglycan. In adult brain, inhibitor immunoreactivity was found predominantly in neurons but, after a lesion, it was associated mainly with reactive glial cells. The localization of neurite outgrowth inhibitors in reactive glia supports the idea that gliotic tissue is largely responsible for the failure of axonal regeneration in mammalian CNS     

Variable membrane glycoproteins in different growth cone populations.

The question of whether growth cones generated by different neurons contain distinctive membrane glycoproteins was examined. Growth cone particles (GCPs) were isolated from specific regions of fetal or early postnatal brain, and their membrane proteins were analyzed by 2D gel electrophoresis and Western blotting, using WGA as a probe. These blots were compared to those generated by synaptosomes from adult brain. The patterns reveal a number of WGA-binding glycoproteins that are uniformly present in these subcellular fractions and others that are found in GCPs from selected brain regions only. The results indicate, therefore, substantial pattern diversity for the different, restricted growth cone populations. Some of the WGA-binding glycoproteins seen in GCPs disappear with increasing age and are absent from synaptosomes, while others seem to become more prominent. One of the glycoprotein complexes present in all GCP and synaptosome fractions analyzed is gp93. It has an apparent molecular weight of 90-97 kDa and exhibits unusually high heterogeneity in GCPs from whole fetal brain. The gp93 complex covers a pI range from about 4.9 to about 6.4 and consists of at least 12 different species, probably isoelectric variants. In GCPs from different brain regions, the sets of gp93 species observed are different and characteristic. Neuraminidase digestion shifts the gp93 pattern to a more neutral pI but simplifies it only partially, indicating that variable sialic acid content explains the molecular diversity to some extent. Thus, gp93 is a glycoprotein complex whose members are expressed and/or posttranslationally processed differentially in different growth cone populations. Such a glycoprotein family may be involved in selective cell-cell recognition.     

Distribution of calcium/calmodulin-dependent kinase 2 in the brain of Apteronotus leptorhynchus.

Antibodies directed against the mammalian alpha and beta subunits of calcium/calmodulin-dependent kinase 2 (CaMK2) and brain dissection were used for immunoblot analysis of these proteins in various brain regions of Apteronotus leptorhynchus. Western blots revealed that the CaMK2alpha antibody labeled a single band of the expected molecular mass (approximately 50 kDa) for this enzyme in rat cortex and electric fish brain. CaMK2alpha was enriched in fish forebrain and hypothalamus and also strongly expressed in midbrain sensory areas. Western blots revealed that CaMK2beta antibodies labeled bands in an appropriate molecular mass range (approximately 58-64 kDa) for this enzyme in mammalian cortex and electric fish brain. However, a higher molecular mass band (approximately 80 kDa) was also labeled; because all these bands were eliminated by preadsorbtion with the CaMK2-derived peptide antigen, they may all represent CaMK2beta-like isoforms. We mapped the brain distribution of CaMK2 isoforms with emphasis on the electrosensory system. CaMK2alpha was present at high density in dorsal forebrain, hypothalamic nuclei, torus semicircularis, and tectum. It was also enriched in discrete fiber tracts in forebrain, diencephalon, and rhombencephalon. CaMK2beta-like isoforms were enriched in ventral forebrain, hypothalamic nuclei, torus semicircularis and the reticular formation. Unlike CaMK2alpha, CaMK2beta -like isoforms were predominantly present in cell bodies and rarely found in fiber tracts or neuropil. In the electrosensory lateral line lobe, CaMK2alpha was restricted to specific feedback fibers, i.e., tractus stratum fibrosum and its terminal field in the ventral molecular layer. In contrast, CaMK2beta-like isoforms were enriched in somata and dendrites of pyramidal cells and granular interneurons.     

Light and electron microscopic analysis of KChIP and Kv4 localization in rat cerebellar granule cells

Potassium channels are key determinants of neuronal excitability. We recently identified KChIPs as a family of calcium binding proteins that coassociate and colocalize with Kv4 family potassium channels in mammalian brain (An et al. [2000] Nature 403:553). Here, we used light microscopic immunohistochemistry and multilabel immunofluorescence labeling, together with transmission electron microscopic immunohistochemistry, to examine the subcellular distribution of KChIPs and Kv4 channels in adult rat cerebellum. Light microscopic immunohistochemistry was performed on 40-microm free-floating sections using a diaminobenzidine labeling procedure. Multilabel immunofluorescence staining was performed on free-floating sections and on 1-microm ultrathin cryosections. Electron microscopic immunohistochemistry was performed using an immunoperoxidase pre-embedding labeling procedure. By light microscopy, immunoperoxidase labeling showed that Kv4.2, Kv4.3, and KChIPs 1, 3, and 4 (but not KChIP2) were expressed at high levels in cerebellar granule cells (GCs). Kv4.2 and KChIP1 were highly expressed in GCs in rostral cerebellum, whereas Kv4.3 was more highly expressed in GCs in caudal cerebellum. Immunofluorescence labeling revealed that KChIP1 and Kv4.2 are concentrated in somata of cerebellar granule cells and in synaptic glomeruli that surround synaptophysin-positive mossy fiber axon terminals. Electron microscopic analysis revealed that KChIP1 and Kv4.2 immunoreactivity is concentrated along the plasma membrane of cerebellar granule cell somata and dendrites. In synaptic glomeruli, KChIP1 and Kv4.2 immunoreactivity is concentrated along the granule cell dendritic membrane, but is not concentrated at postsynaptic densities. Taken together, these data suggest that A-type potassium channels containing Kv4.2 and KChIP1, and perhaps also KChIP3 and 4, play a critical role in regulating postsynaptic excitability at the cerebellar mossy-fiber/granule cell synapse.     

Major differences in the concanavalin A binding glycoproteins of postsynaptic densities from rat forebrain and cerebellum

A highly purified fraction of postsynaptic densities (PSDs) was isolated from rat forebrain and cerebellum. The glycoproteins in the PSDs from the two brain regions were compared by probing nitrocellulose blots with concanavalin A. This approach revealed the presence of 8, previously undetected PSD glycoproteins and a 230K M_r glycoprotein unique to forebrain PSDs and 260K M_r glycoprotein unique to cerebellar PSDs. There were also variations in the relative abundances of glycoproteins common to both brain regions. These observations may indicate that individual PSDs differ in their complement of major glycoproteins.     

Cellular and subcellular localization of a newly identified member of the protein 4.1 family, brain 4.1, in the cerebellum of adult and postnatally developing rats

For obtaining a deeper insight into the properties of a newly characterized member of the protein 4.1 family, brain 4.1, the cellular and subcellular localization was investigated in the cerebellar cortex of adult and postnatally developing rats. Fluorescent immunohistochemical observations showed that brain 4.1 localized predominantly to glomeruli in the granular layer and throughout the molecular layer in adult rat cerebellar cortex. Analysis of subcellular localization of brain 4.1 by immuno-electron microscopy further demonstrated that presynaptic terminals of mossy fibers and parallel fibers, cytoplasm of granule cells and cytoplasm and/or processes of glial cells contained brain 4.1 while postsynaptic regions of the dendrites of granule cells and Purkinje cells, axons and myelin sheaths did not. Thus, one of the major subcellular destination of brain 4.1 was presynaptic terminal in the cerebellum. This was further supported by the fact that the immunostaining pattern of brain 4.1 in the cerebellum changed in a similar way to that of a synaptic terminal marker, synaptophysin during the postnatal development. Immunoblot analysis also demonstrated that contents of brain 4.1 isoforms varied in parallel with the changes of the immunostaining pattern. Biochemical analysis confirmed the presence of brain 4.1 at synaptic terminals, but there was no obvious correlation between each isoform and its subcellular localization. These results suggested that brain 4.1 is involved in the formation and maintenance of synapse as a membrane skeletal component at presynaptic terminals in the cerebellum.     

The Alzheimer disease-related calcium-binding protein Calmyrin is present in human forebrain with an altered distribution in Alzheimer's as compared to normal ageing brains

The EF-hand calcium binding protein Calmyrin (also called CIB-1) was shown to interact with presenilin-2 (PS-2), suggesting that this interaction might play a role in the pathogenesis of Alzheimer's disease (AD). Here we have investigated the distribution of Calmyrin in normal human and AD brain. In normal brain Calmyrin immunoreactivity was unevenly distributed with immunostaining in pyramidal neurones and interneurones of the palaeo-cortex and neocortex, cerebellar granule cells and hypothalamic neurones of the paraventricular, ventromedial and arcuate nuclei. Moderate immunoreactivity was present in hippocampal pyramidal cells and stronger in dentate gyrus neurones. Thalamic and septal neurones were devoid of immunoreactivity. No apparent differences were visible between stainings of brain sections from younger and older nondemented patients. In AD brain a substantial loss of Calmyrin-immunopositive neurones was observed in all regions, especially in cortical areas. Still immunoreactive neurones, however, displayed stronger staining that was especially concentrated in perinuclear regions. Calmyrin immunosignals were in part associated with diffuse and senile plaques. Thus, although protein levels of Calmyrin are low in human forebrain, its cellular localization as well as its altered distribution in AD brain suggest that it may be involved in the pathogenesis of AD.     

Protein and glycoprotein composition of synaptic junctions prepared from discrete synaptic regions and different species

Synaptic junction (SJ) were isolated by subcellular fractionation from different areas of the steer brain and from the brains of different species (steer, rat, chicken and human) for the purpose of comparing their protein and glycoprotein composition. The synaptic junction fractions from different brain regions and species were of comparable morphological purity. Analysis of the polypeptide composition of isolated synaptic junction fractions via SDS polyacrylamide gel electrophoresis showed that the major polypeptides were represented in all junctional fractions independent of their source. Tubulin, actin, the major 52,000 Mr postsynaptic density protein and a group of proteins with a molecular weight of 200-250,000 Mr were all similarly represented. Most other components were also similar but quantitative differences were found for a few polypeptides. Interspecies differences were more prevalent than those between different brain areas of the same species. The protein compositions of different brain areas were similar even when an area consisting of primarily one neuronal type was compared to areas containing a mixture of neuronal types. However, two-dimensional gel electrophoresis revealed distinct (but usually minor) polypeptides in enriched quantities in one or more brain areas. Tryptic peptide maps were carried out on the major postsynaptic density protein of different species. These maps showed a high degree of conservation in this protein's primary structure among all species studied. The glycoproteins of isolated synaptic junctions which bind the plant lectin concanavalin A (Con A) were also examined. To identify individual Con A binding components, SJ fractions were solubilized and constituent glycoproteins were separated by SDS gel electrophoresis. Gels were then incubated in 125I-Con A. The glycoproteins which bound Con A in gels were few in number and were not the major Coomassie blue staining bands. The great majority of the Con A binding glycoproteins were similar between species and among brain areas of the same species.     

Transient increase of synapsin-I immunoreactivity in the mossy fiber layer of the hippocampus after transient forebrain ischemia in the mongolian gerbil

Synapsin-I is a vesicular phosphoprotein, which regulates neurotransmitter release, neurite development, and maturation of synaptic contacts during normal development and following various brain lesions in adulthood. In the present study, we have examined by immunohistochemistry possible modifications in the expression of synapsin-I in the hippocampus of Mongolian gerbils after transient forebrain ischemia. The animals were subjected to 5 min of transient forebrain ischemia through bilateral common carotid occlusion, and were examined at different time-points post-ischemia. Transient forebrain ischemia produces cell death of the majority of CA1 pyramidal neurons of the hippocampus and polymorphic hilar neurons of the dentate gyrus. This is followed by reactive changes, including synaptic reorganization and modifications in the expression of synaptic proteins, which provide the molecular bases of synaptic plasticity. Transient decrease of synapsin-I immunoreactivity was observed in the inner zone of the molecular layer of the dentate gyrus, thus suggesting denervation and posterior reinervation in this area. In addition, a strong increase in synapsin-I immunoreactivity was observed in the hilus of the dentate gyrus and in the mossy fiber layer of the hippocampus at 2, 4 and 7 days after ischemia. Parallel increases in synaptophysin immunoreactivity were not observed, thus suggesting a selective induction of synapsin-I after ischemia. The present results indicate that synapsin-I participates in the reactive response of granule cells to transient forebrain ischemia in the hippocampus of the gerbil, and suggest a role for this protein in the plastic adaptations of the hippocampus following injury.     

Immunohistochemical localization of N-methyl-D-aspartate receptor subunits in the adult murine hippocampal formation: evidence for a unique role of the NR2D subunit

NMDA receptors were immunopurified from adult mouse forebrain and screened by immunoblotting. NR1 was co-associated with NR2A, NR2B and NR2D but not NR2C, nor was NR2C detected in adult mouse hippocampal membranes. The anatomical distribution of NR1, 2A, 2B and 2D was mapped in the adult murine hippocampal formation. NR1-like immunoreactivity was localised to cell bodies of pyramidal neurons, granule cells and hilar cells of the dentate gyrus. Apical dendrites of the CA subfields and hilar cells were also immunopositive. NR2A- and NR2B-like immunoreactivity essentially co-localised with that of NR1 implying co-assembly of all three subunits in this brain structure. NR2D-like immunoreactivity was distinct, being totally excluded from pyramidal, granule and hilar cell bodies. Strong, punctate staining was restricted to the oriens layer of CA1 and the stratum lucidum of CA3 consistent with labelling of presynaptic receptors. Less intense staining was also observed in the internal third of the molecular layer of the dentate gyrus.     

Distribution of Thy-1 in the avian nervous system: Immunohistochemical and absorption analyses with a monoclonal antibody

A monoclonal antibody against chicken Thy-1 has been used to study the histochemical localisation of Thy-1 in chicken nervous and lymphoid tissues and to quantitate the relative amounts of Thy-1 in different brain subregions, subcellular fractions and non-neural tissues. An indirect ELISA using chicken brain membranes as a target established that the highest levels of Thy-1 were present in chicken forebrain, followed by midbrain, brainstem, spinal cord, cerebellum, retina and sciatic nerve. Analyses of subcellular fractions of chicken forebrain revealed a generalised localisation of Thy-1 on membranes comprising both the junctional and extrajunctional components of the synaptosome. Consistent with this finding are the immunohistochemical studies on cryostat sections where Thy-1 was localised to certain axonal and synaptic regions of chicken nervous tissue. Strong monoclonal antibody binding was found in the molecular layer and white matter of chicken cerebellar sections with fibrous staining on the axons running through the granule cell layer. No continuous staining could be seen on the perikaryal membranes of Purkinje cells or granule cells and no staining was present within the cell bodies. The Bergmann glia of the cerebellum were Thy-1-negative. The monoclonal antibody showed preferential binding to the inner plexiform and optic fibre layers of the chicken retina, suggesting a retinal ganglion cell localisation for chicken Thy-1, as has been suggested for the rat and mouse homologues. Surprisingly the lymphocytes of both the bursa and thymus gland were Thy-1-negative, however some extracellular staining was observed of interlobular connective tissue of the bursa.     

Status epilepticus-induced changes in the subcellular distribution and activity of calcineurin in rat forebrain

This study was performed to determine the effect of prolonged status epilepticus on the activity and subcellular location of a neuronally enriched, calcium-regulated enzyme, calcineurin. Brain fractions isolated from control animals and rats subjected to pilocarpine-induced status epilepticus were subjected to differential centrifugation. Specific subcellular fractions were tested for both calcineurin activity and enzyme content. Significant, status epilepticus-induced increases in calcineurin activity were found in homogenates, nuclear fractions, and crude synaptic membrane-enriched fractions isolated from both cortex and hippocampus. Additionally, significant increases in enzyme levels were observed in crude synaptic fractions as measured by Western analysis. Immunohistochemical studies revealed a status epilepticus-induced increase in calcineurin immunoreactivity in dendritic structures of pyramidal neurons of the hippocampus. The data demonstrate a status epilepticus-induced increase in calcineurin activity and concentration in the postsynaptic region of forebrain pyramidal neurons.     

Comparison of the immunocytochemical distribution of the phosphoprotein B-50 in the cerebellum and hippocampus of immature and adult rat brain

In this study we compare the distribution of the phosphoprotein B-50 in two regions of immature and adult rat brain using affinity-purified antibodies to B-50. In the cerebellum of the 8-day-old rat we observed distinct patterns of distribution of B-50 immunoreactivity (BIR) in the premigratory zone and the developing molecular layer, likely associated with outgrowing parallel and climbing fibers contacting Purkinje cells in the internal granular layer and in axons coursing through the cerebellar medulla. In contrast, in adult cerebellum, a sparcer distribution of BIR as punctuate deposits is observed in the molecular layer, outlining dendritic trees and the perikarya of neurons. At relatively lower density BIR is found dispersed between the cells of the granular layer and along fibers in the white matter. In the immature hippocampal formation, fibers penetrating between unstained cells of the stratum pyramidale and the subiculum, and neuropil areas are immunostained. In the adult rat a graded immunostaining pattern corresponding to the laminar structure of the hippocampal formation is found with high density of BIR in the strata oriens, radiatum, parts of stratum lacunosum molecular and in the stratum molecular adjoining the field of the proximal apical dendrites of the granule cells. BIR appears to be absent from the proximal part of the mossy fiber pathway. In neuropil areas of adult hippocampus and cerebellum BIR is fairly restricted to dot-like deposits indicating a synaptic localization. This is in correspondence with our previous ultrastructural findings. The present observations in developing brain of B-50-like components in fibers, as well, suggest that B-50 (and/or B-50-like precursors) are involved in neurite outgrowth.     

Distribution of adenylate cyclase in the brain of Apteronotus leptorhynchus as revealed by forskolin binding.

An antibody directed against an isoform of the rat regulatory subunit of protein kinase A and brain dissection was used for immunoblot analysis of this protein in various brain regions of Apteronotus leptorhynchus. Western blots revealed that the antibody labeled a band of the expected molecular mass (approximately 53 kDa) for this enzyme in mammalian cortex and electric fish brain, suggesting that this protein is also found in fish brains. The 53-kDa band was enriched in fish forebrain. [3H]Forskolin binding was used as a marker for the distribution of adenylate cyclase. [3H]Forskolin binding was nearly completely displaced by excess cold forskolin; specific [3H]forskolin binding sites were heterogenously distributed with relatively high densities in some gray matter regions and low densities in fiber tracts. A high density of [3H]forskolin binding sites was found in the dorsal forebrain with lower densities in most ventral forebrain nuclei. Moderate binding densities were observed in the preoptic and hypothalamic areas with the exception of the nucleus tuberis anterior, which had high levels. The thalamus and midbrain had low levels of binding. The cerebellar molecular layer had dense binding, in contrast to the granule cell layer where binding was low. In the electrosensory lateral line lobe (ELL), there was moderate binding in the dorsal and ventral molecular layers, which contain feedback inputs; the cellular layers of the ELL had low binding densities.     

Distribution of protein kinase C in the brain of Apteronotus leptorhynchus as revealed by phorbol ester binding.

An antibody to the mammalian protein kinase C alpha (PKCalpha) subunit and brain dissection was used for immunoblot analysis of this protein in various brain regions of Apteronotus leptorhynchus. Western blots revealed that the antibody labeled a band of the expected molecular mass (approximately 80 kDa) for this enzyme in mammalian cortex and electric fish brain, suggesting that this protein is also found in gymnotiform brain. The 80-kDa band was enriched in fish forebrain and cerebellum compared with hypothalamus and brainstem areas. [3H]Phorbol 12,13-dibutyrate ([3H]PDBu) binding was used as a marker for the distribution of protein kinase C (PKC). [3H]PDBu binding was nearly completely displaced by excess cold PDBu; specific [3H]PDBu binding sites were heterogenously distributed with high densities in some gray matter regions and negligible densities in fiber tracts. A very high density of [3H]PDBu binding sites were found in the dorsal forebrain with far lower densities in most ventral forebrain nuclei. Low binding densities were observed in preoptic and hypothalamic areas with the exception of the nucleus diffusus and nucleus tuberis anterior. The thalamus and midbrain also had only low levels of binding. The cerebellar molecular layer had dense binding, in contrast to the granule cell layer where binding was negligible. In the electrosensory lateral line lobe (ELL), there was moderate binding in the dorsal molecular layer, which contains cerebellar parallel fibers; the other layers of the ELL had far lower binding densities.