Neuronal protein NP 185 is developmentally regulated,initially expressed during synaptogenesis,and localized in synaptic terminals

Evidence is presented here that demonstrates the presence of NP185 (AP₃) in neuronal cells, specifically within syn-aptic terminals of the central nervous system and in the peripheral nervous system, particularly in the neuro-muscular junction of adult chicken muscle. Biochemical results obtained in our laboratories indicate that NP185 is associated with brain synaptic vesicles, with clathrin-coated vesicles, and with the synaptosomal plasma membrane. Also, NP185 binds to tubulin and clathrin light chains and the binding is regulated by phosphorylation (Su et al., 1991). Based on these properties and the data reported here, we advance the postulate that NP185 fulfills multiple functions in synaptic terminals. One function is that of a plasma membrane docking or channel protein, another of a signaling molecule for brain vesicles to reach the synaptic terminal region, and a third is that of a recycling molecule by binding to protein components on the lipid bilayer of the synaptic plasma membrane during the process of endocytosis. In support of these premises, a thorough study of NP185 using the developing chick brain, adult mouse brain, and chicken straited muscle was begun by temporally and spatially mapping the expression and localization of NP185 in evolving and mature nerve endings. To achieve these objectives, monoclonal antibodies to NP185 were used for immunocytochemistry in tissue sections of chicken and mouse cerebella. The distribution of NP185 was compared with those of other cytoskeletal and cytoplasmic proteins of axons and synapses, namely synaptophysin, vimentin, neurofilament NF68, and the intermediate filaments of glial cells (GFAP). The data indicate that expression of NP185 temporally coincides with synaptogenesis, and that the distribution of this protein is specific for synaptic terminal buttons of the CNS and the PNS.     

A neuronal protein (NP185) associated with clathrin-coated vesicles. Characterization of NP185 with monoclonal antibodies

Two monoclonal antibodies (S-8G8 and S-6G7) are characterized that react with an abundant neuronal protein associated with brain clathrin-coated vesicles (CCVs). This 185-kDa polypeptide (NP185) is not a transmembrane cargo molecule and is distinguishable from clathrin by several criteria including neuronal specificity, chymotryptic sensitivity, migration during two-dimensional gel electrophoresis, lack of cross-reactivity of S-8G8 or S-6G7 with purified clathrin, and lack of associated clathrin light chains. When 0.9 M NaCl extracts of CCVs were diluted and immunoprecipitated by either S-8G8 or S-6G7, NP185 precipitated as a complex with a fraction of the CCV assembly polypeptides. Immunofluorescence microscopy of PC12 cells cultured in nerve growth factor (NGF) revealed that NP185 was distributed in a punctate manner throughout the mature neurites. Immunoblot analysis of PC12 cell extracts, taken at various times during NGF-induced differentiation, revealed that steady-state accumulation of NP185 reaches significant levels 3 days after the addition of NGF and returns to undetectable levels when NGF is removed from the cultures. Significantly, the quantity of NP185 detected in differentiated PC12 cells exceeded the quantity of clathrin. These data indicate that while NP185 may be a specialized component of neuronal CCVs, its function in neuronal cells cannot be associated exclusively with these organelles.     

Phosphorylation of Tubulin by Casein Kinase II Regulates Its Binding to a Neuronal Protein (NP185) Associated with Brain Coated Vesicles

We recently described a new protein associated exclusively with neuronal clathrin-coated vesicles (CCVs), and characterized two monoclonal antibodies that react with it (S-8G8 and S-6G7). In this report, the association of neuronal protein of 185 kilodaltons (NP185) with CCV kinases and its interaction with tubulin are described. The affinity of NP185 for tubulin is significantly enhanced when tubulin is phosphorylated by CCV-associated casein kinase II. In contrast, phosphorylation of tubulin by a kinase activity associated with purified brain tubulin decreases its affinity for NP185. Together, these data suggest that the interaction of NP185 with tubulin is modulated by protein phosphorylation. Recent evidence has suggested that tubulin is phosphorylated by casein kinase II during neurite development. The enhanced affinity of NP185 for tubulin phosphorylated by casein kinase II could be important for proper intracellular sorting of this protein in the developing neuron.     

Expression of neuronal plasticity markers in hypoglycemia induced brain injury

The expression of neuroplasticity markers was analyzed in four brain regions, namely cerebral hemispheres (CH), cerebellum (CB), brain stem (BS) and diencephalon (DC) from insulin-induced hypoglycemic young adult rats. Significant decrease in neural cell adhesion molecule (NCAM) isoforms and growth-associated protein-43 (GAP-43) was observed following hypoglycemic injury from majority of brain regions studied. The glial fibrillary acidic protein (GFAP) level increased significantly in cerebral hemispheres and diencephalon regions, whereas, synaptophysin level increased in cerebellum, brain stem and diencephalon regions. The selective downregulation of the neuronal plasticity marker proteins (GAP-43 and NCAM), and enhanced expression of GFAP and synaptophysin suggests that in acute hypoglycemia, mechanisms other than energy failure may also contribute to neuronal cell damage in the brain.     

Localization of the Extracellular Matrix Protein SC1 Coincides with Synaptogenesis During Rat Postnatal Development

SC1 is an extracellular matrix protein that belongs to the SPARC family of matricellular molecules. This anti-adhesive protein localizes to synapses in the adult rat brain and has been postulated to modulate synapse shape. In this study, increased levels of SC1 were detected from postnatal days 10–20, with a peak at postnatal day 15, a period of intense synaptogenesis. During this time, increased colocalization of SC1 with the synaptic marker synaptophysin was observed in synapse-rich regions of the cerebellum and the cerebral cortex. These findings indicate that the pattern of SC1 localization coincided with synaptogenesis during rat postnatal development.     

Synapse formation and agrin expression in stratospheroid cultures from embryonic chick retina

Stratospheroids are three-dimensional cellular spheres which develop in vitro through the proliferation and differentiation of retinal neuroepithelial precursor cells. We investigated synapse formation in stratospheroids by analyzing the development of aggregates of synapse-associated molecules and of electron microscopically identifiable synaptic specializations. Our results show that the first aggregates of the GABA(A) receptor, the glycine receptor, and gephyrin appear in the inner plexiform layer after 8 days in culture simultaneously with the development of the first active zones and postsynaptic densities. In contrast, presynaptic molecules including synaptophysin could be detected in the inner plexiform layer before synaptogenesis, suggesting functions for these molecules in addition to neurotransmitter exocytosis at mature synapses. Similar to the retina in vivo, synapses were not found in the nuclear layers of stratospheroids. We also analyzed the isoform pattern, expression, and distribution of the extracellular matrix molecule agrin, a key regulator during formation, maintenance, and regeneration of the neuromuscular junction. In stratospheroids, several agrin isoforms were expressed as highly glycosylated proteins with an apparent molecular weight of approximately 400 kDa, similar to the molecular weight of agrin in the retina in vivo. The expression specifically of the neuronal isoforms of agrin was concurrent with the onset of synaptogenesis. Moreover, the neuronal agrin isoforms were exclusively found in the synapse-containing inner plexiform layer, whereas other agrin isoforms were associated also with the inner limiting membrane and with Müller glial cells. These results show that synapse formation is very similar in stratospheroids and in the retina in vivo, and they suggest an important role for agrin during CNS development.     

Expression Pattern of Synucleins (Non-Aβ Component of Alzheimer's Disease Amyloid Precursor Protein/α-Synuclein) During Murine Brain Development

Abstract: The non-Aβ component of Alzheimer's disease amyloid precursor protein (NACP) is predominantly a neuron-specific presynaptic protein that may play a central role in neurodegeneration because NACP fragments are found in Alzheimer's disease amyloid and a mutation in the NACP gene is associated with familial Parkinson's disease. In addition, NACP may play an important role during synaptogenesis and CNS development. To understand better the patterns of NACP expression during development, we analyzed the levels of this protein as well as the levels of another synaptic protein (synaptophysin) by ribonuclease protection assay, western blotting, and immunocytochemistry in fetal, juvenile, and adult mouse brain. From embryonic day 12 to 15, there was a slight increase, which was then followed by a more dramatic increase at later time points. Immunocytochemical staining for NACP increases throughout these stages as well. Although NACP appeared early in CNS development, synaptophysin levels started to rise at a later stage. These findings support the contention that NACP might be important for CNS development. Furthermore, the cytosolic component of NACP precedes the particulate component in development, indicating that a redistribution of the protein to the membrane fraction may be important for events later in neuronal development and in synaptogenesis.     

Narp and NP1 form heterocomplexes that function in developmental and activity-dependent synaptic plasticity

Narp is a neuronal immediate early gene that plays a role in excitatory synaptogenesis. Here, we report that native Narp in brain is part of a pentraxin complex that includes NP1. These proteins are covalently linked by disulfide bonds into highly organized complexes, and their relative ratio in the complex is dynamically dependent upon the neuron's activity history and developmental stage. Complex formation is dependent on their distinct N-terminal coiled-coil domains, while their closely homologous C-terminal pentraxin domains mediate association with AMPA-type glutamate receptors. Narp is substantially more effective in assays of cell surface cluster formation, coclustering of AMPA receptors, and excitatory synaptogenesis, yet their combined expression results in supraadditive effects. These studies support a model in which Narp can regulate the latent synaptogenic activity of NP1 by forming mixed pentraxin assemblies. This mechanism appears to contribute to both activity-independent and activity-dependent excitatory synaptogenesis.     

A novel intermediate filament-associated protein, NAPA-73, that binds to different filament types at different stages of nervous system development

The antigen recognized by the E/C8-monoclonal antibody is expressed in various avian embryonic cell types known also to express neurofilament (NF) immunoreactivity. To determine whether the E/C8-antigen corresponds to any of the known NF components, we compared their subcellular locations, immunocross-reactivities, and electrophoretic behaviors. We found that the E/C8-antibody binds to NF bundles in electron microscope preparations of neurons, but does not correspond to any of the known NF proteins by immunological or electrophoretic criteria. Immunoadsorption with the monoclonal antibody resulted in co-purification of a 73,000-D protein with one of the known NF proteins in homogenates from 20-d embryonic chick brains, but with vimentin intermediate filament protein in similarly prepared homogenates from 4-d embryonic chicks. We suggest that the E/C8-antigen is an intermediate filament-associated protein that binds to different filament types at different stages of development. We have named it NAPA-73, an acronym for neurofilament-associated protein, avian-specific, 73,000 D, on the basis of its binding specificity in mature neurons.     

Monoclonal antibodies to epitopes on different regions of the 200 000 dalton neurofilament protein. Probes for the geometry of the filament

Neurofilaments in mammalian nervous tissues have three subunit proteins. These subunit proteins have apparent molecular masses of 200 (NF200), 150 (NF150) and 68 (NF68) kD. Biochemical assembly studies have indicated that the NF68 protein forms the core of the filament and that the other two proteins are associated proteins. Electron microscopy immunolocalization studies have been performed previously on isolated filaments and on filaments from neurons in culture, and have confirmed the localization of NF68 as a core filament protein and NF200 as a peripheral protein. We have raised two monoclonal antibodies to the NF200 components. Using immunogold labelled protein A, we have been able to localize these antibodies to tissue sections of adult cerebellum at the EM level. With this method, we have found that one of the monoclonal antibodies (NF2) shows a linear arrangement of gold particles directly on the filament, whereas the second monoclonal antibody (NF111) reacts with the filaments to give a periodic arrangement of gold particles. By immunoblotting against chymotryptic fragments of the NF200 protein, we have found that the mAB-NF111 reacts solely with a 160 kD piece, whereas the other monoclonal antibody reacts with both the 160 kD piece and the 40 kD piece. The latter piece was shown to be associated to the filament by binding studies with iodinated NF68. Thus the EM localization studies and the biochemical studies indicate that the two monoclonal antibodies react with different parts of the NF200 molecule, one binding to a part of the molecule which is located closer to the filament, and one to a more peripheral part of the molecule.     

Expression of Synaptophysin During Postnatal Development of the Mouse Brain

The expression of the synaptic vesicle membrane protein, synaptophysin, was analyzed during postnatal development of the mouse cerebrum using a quantitative immunoblotting procedure. From birth to adulthood, the relative contents of synaptophysin increased 80-fold, reaching a final level of 3.5 micrograms/mg of total protein. The time course of accumulation suggests that synaptophysin expression is correlated with synaptogenesis. Thus synaptophysin may be used as a reliable marker of nerve terminal differentiation.     

Monoclonal antibodies to epitopes on different regions of the 200 00 dalton neurofilament protein : Probes for the geometry of the filament

Neurofilaments in mammalian nervous tissues have three subunit proteins. These subunit proteins have apparent molecular masses of 200 (NF200), 150 (NF150) and 68 (NF68) kD. Biochemical assembly studies have indicated that the NF68 protein forms the core of the filament and that the other two proteins are associated proteins. Electron microscopy immunolocalization studies have been performed previously on isolated filaments and on filaments from neurons in culture, and have confirmed the localization of NF68 as a core filament protein and NF200 as a peripheral protein. We have raised two monoclonal antibodies to the NF200 components. Using immunogold labelled protein A, we have been able to localize these antibodies to tissue sections of adult cerebellum at the EM level. With this method, we have found that one of the monoclonal antibodies (NF2) shows a linear arrangement of gold particles directly on the filament, whereas the second monoclonal antibody (NF111) reacts with the filaments to give a periodic arrangement of gold particles. By immunoblotting against chymotryptic fragments of the NF200 protein, we have found that the mAB-NF111 reacts solely with a 160 kD piece, whereas the other monoclonal antibody reacts with both the 160 kD piece and the 40 kD piece. The latter piece was shown to be associated to the filament by binding studies with iodinated NF68. Thus the EM localization studies and the biochemical studies indicate that the two monoclonal antibodies react with different parts of the NF200 molecule, one binding to a part of the molecule which is located closer to the filament, and one to a more peripheral part of the molecule.     

Isolation of a Potential Neural Stem Cell Line from the Internal Capsule of an Adult Transgenic Rat Brain

A thermosensitive mutation of simian virus 40 large T antigen (LTA) gene, the tsA58 gene, was cloned downstream of the 6-kbp neurofilament light chain promoter in pPOLYIII and injected into the pronucleus of fertilised oocytes of Sprague-Dawley rats to develop a strain harbouring six copies of the transgene. Immunocytochemical staining of hemizygous adult tissues with antibodies to the C-terminus of LTA showed that the inactive form of LTA was expressed only in the fibres of the internal capsule and in the choroid plexus of the brain. Culturing the former region at 33 degrees C, the permissive temperature for LTA, yielded a cell line, NF2C, which produced active LTA and grew at 33 degrees C but which produced only inactive LTA and eventually died at the non-permissive temperature of 39 degrees C. This clonal cell line was heterogeneous at 33 degrees C, producing the precursor neuronal cell marker nestin and the glial-specific markers glial fibrillary acidic protein, vimentin and S100A1, as well as weakly producing the neuronal cell markers 68-kDa neurofilament protein (NF68) and microtubule-associated protein 2 (MAP2) in different subpopulations of cells. However, at 39 degrees C, the cells produced dendritic, neuronal-like processes and elevated levels of NF68 and MAP2, as well as the neuronal markers synaptophysin, neurone-specific enolase, and low levels of tau, all determined by western blotting and immunofluorescent staining. Basic fibroblast growth factor enhanced the growth of the cells at 33 degrees C but also enhanced the formation of dendritic neuronal-like processes at 39 degrees C. It is suggested that NF2C represents a potential stem cell line from adult brain that expresses precursor and glial cell markers at 33 degrees C but undergoes partial differentiation to a neuronal cell phenotype at 39 degrees C.     

A Novel Synaptic Vesicle-Associated Phosphoprotein: SVAPP-120

Generation of antibodies and direct protein sequencing were used to identify and characterize proteins associated with highly purified synaptic vesicles from rat brain. A protein doublet of low abundance of 119 and 124 kDa apparent molecular mass [synaptic vesicle-associated phosphoprotein with a molecular mass of 120 kDa (SVAPP-120)] was identified using polyclonal antibodies. SVAPP-120 was found to copurify with synaptic vesicles and to be enriched in the purified synaptic vesicle fraction to the same extent as synapsin I. Like synapsin I, SVAPP-120 is not an integral membrane protein because it was released from synaptic vesicles by high salt concentrations. This protein was demonstrated to be brain specific, and its distribution in various brain regions paralleled the distribution of synapsin I and synaptophysin. During the postnatal development of the rat cortex and cerebellum, its expression correlated with synaptogenesis. SVAPP-120 was demonstrated to be a phosphoprotein both in vivo and in vitro. It was shown to be phosphorylated on serine and to a lesser extent on threonine residues. These results provide evidence that SVAPP-120 represents a novel synaptic vesicle-associated phosphoprotein. In addition, aldolase, a glycolytic enzyme, and alpha c-adaptin, a clathrin assembly-promoting protein, were identified on purified synaptic vesicles by direct protein sequencing.     

Patterns of synaptophysin expression during development of the inner ear in the chick

The onset of active neural connections between the periphery and the central nervous system is integral to the development of sensory systems. This study presents patterns of synaptogenesis in the chick basilar papilla (i.e., cochlea) by examining the immunohistochemical expression of synaptophysin with a specific monoclonal antibody, SBI 20.10. The initial onset of synaptophysin expression occurs in nerve fibers and ganglion cell bodies at a time when neurites reach the basement membrane of the chick cochlea on embryonic day 6-7 (ED 6-7). By ED 8, synaptophysin positive fibers invade the neural side of the entire length of the cochlea, so that by ED 9-10, fibers are forming multiple terminals on the basolateral ends of retracting receptor or hair cells. In contrast, on the abneural side, immunoreactive terminals are seen first as small, punctate contacts and then as large, synaptophysin positive calyceal endings beneath short hair cells. These terminals are sparse during early development, more numerous by ED 17-19, but still incomplete after 2 weeks posthatching. In comparison, hair cells show synaptophysin immunoreactivity in both supra- and infranuclear regions by ED 11-12, a time when efferent innervation is incomplete. Thus, during development, synaptophysin is expressed at both synaptic and nonsynaptic sites, is relatively selective in its regional distribution, and is expressed in hair cells at a time when auditory function begins. Our results present a framework with which to understand the potential role of synaptophysin in early synaptogenesis of the cochlea.     

Genetic Program of Neuronal Differentiation and Growth Induced by Specific Activation of NMDA Receptors

Glutamate and its receptors are expressed very early during development and may play important roles in neurogenesis, synapse formation and brain wiring. The levels of glutamate and activity of its receptors can be influenced by exogenous factors, leading to neurodevelopmental disorders. To investigate the role of NMDA receptors on gene regulation in a neuronal model, we used primary neuronal cultures developed from embryonic rat cerebri in serum-free medium. Using Affymetrix Gene Arrays, we found that genes known to be involved in neuronal plasticity were differentially expressed 24 h after a brief activation of NMDA receptors. The upregulation of these genes was accompanied by a sustained induction of CREB phosphorylation, and an increase in synaptophysin immunoreactivity. We conclude that NMDA receptor activation elicits expression of genes whose downstream products are involved in the regulation of early phases of the process leading to synaptogenesis and its consolidation, at least in part through sustained CREB phosphorylation. Special issue dedicated to Dr. Anthony Campagnoni.     

The expression pattern and assembly profile of synaptic membrane proteins in ribbon synapses of the developing mouse retina

In the present study, we generated a systematic overview of the expression pattern and assembly profile of synaptic membrane proteins in ribbon synapses of the developing mouse retina. Using indirect immunofluorescence microscopy, we analyzed the spatial and temporal distribution of 11 important membrane and membrane-associated synaptic proteins (syntaxin 1/3, SNAP-25, synaptobrevin 2, synaptogyrin, synaptotagmin I, SV2A, SV2B, Rab3A, clathrin light chains, CSP and neuroligin I) during synaptogenesis. The temporospatial distribution of these synaptic proteins was "normalized" by the simultaneous visualization of the synaptic vesicle protein synaptophysin, which served as an internal reference protein. We found that expression of various synaptic membrane proteins started at different time points and changed progressively during development. At early stages of development synaptic vesicle membrane proteins at extrasynaptic locations did not always colocalize with synaptophysin, indicating that these proteins probably do not reside in the same transport vesicles. Despite a non-synchronized onset of protein expression, clustering and colocalization of all synaptic membrane proteins at ribbon synapses roughly occurred in the same time window (between day 4 after birth, P4, and P5). Thus, the basic synaptic membrane machinery is already present in ribbon synapses before the well-known complete morphological maturation of ribbon synapses between P7 and P12. We conclude that ribbon synapse formation is a multistep process in which the concerted recruitment of synaptic membrane proteins is a relatively early event and clearly not the final step.     

Differential expression of syntaxin-1 and synaptophysin in the developing and adult human retina

Synaptophysin and syntaxin-1 are membrane proteins that associate with synaptic vesicles and presynaptic active zones at nerve endings, respectively. The former is known to be a good marker of synaptogenesis; this aspect, however, is not clear with syntaxin-1. In this study, the expression of both proteins was examined in the developing human retina and compared with their distribution in postnatal to adult retinas, by immunohistochemistry. In the inner plexiform layer, both were expressed simultaneously at 11–12 weeks of gestation, when synaptogenesis reportedly begins in the central retina. In the outer plexiform layer, however, the immunoreactivities were prominent by 16 weeks of gestation. Their expression in both plexiform layers followed a centre-to-periphery gradient. The immunoreactivities for both proteins were found in the immature photoreceptor, amacrine and ganglion cells; however, synaptophysin was differentially localized in bipolar cells and their axons, and syntaxin was present in some horizontal cells. In postnatal-to-adult retinas, synaptophysin immunoreactivity was prominent in photoreceptor terminals lying in the outer plexiform layer; on the contrary, syntaxin-1 was present in a thin immunoreactive band in this layer. In the inner plexiform layer, however, both were homogeneously distributed. Our study suggests that (i) syntaxin-1 appears in parallel with synapse formation; (ii) synaptogenesis in the human retina might follow a centre-to-periphery gradient; (iii) syntaxin-1 is likely to be absent from ribbon synapses of the outer plexiform layer, but may occur at presynaptic terminals of photoreceptor and horizontal cells, as is apparent from its localization in these cells, which is hitherto unreported for any vertebrate retina.     

HP1alpha guides neuronal fate by timing E2F-targeted genes silencing during terminal differentiation

A critical step of neuronal terminal differentiation is the permanent withdrawal from the cell cycle that requires the silencing of genes that drive mitosis. Here, we describe that the alpha isoform of the heterochromatin protein 1 (HP1) protein family exerts such silencing on several E2F-targeted genes. Among the different isoforms, HP1alpha levels progressively increase throughout differentiation and take over HP1gamma binding on E2F sites in mature neurons. When overexpressed, only HP1alpha is able to ensure a timed repression of E2F genes. Specific inhibition of HP1alpha expression drives neuronal progenitors either towards death or cell cycle progression, yet preventing the expression of the neuronal marker microtubule-associated protein 2. Furthermore, we provide evidence that this mechanism occurs in cerebellar granule neurons in vivo, during the postnatal development of the cerebellum. Finally, our results suggest that E2F-targeted genes are packaged into higher-order chromatin structures in mature neurons relative to neuroblasts, likely reflecting a transition from a 'repressed' versus 'silenced' status of these genes. Together, these data present new epigenetic regulations orchestrated by HP1 isoforms, critical for permanent cell cycle exit during neuronal differentiation.