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The persistent expression of a highly polysialylated NCAM in the dentate gyrus of the adult rat
Abstract
The expression of a highly polysialylated form of the neural cell adhesion molecule (NCAM-H), often termed 'embryonic NCAM', has been investigated in the hippocampal formation of developing and adult rats. To determine the immunohistochemical localization of NCAM-H, a monoclonal antibody that reacts with the polysialic acid portion of NCAM-H was used. In the late embryonic and early postnatal periods, immunoreactivity for NCAM-H was found throughout the hippocampal formation, except for the ventricular layer. Thereafter, the immunoreactivity gradually decreased and almost vanished in most parts in the adult. However, a strong immunoreactivity remained on a number of cells in the dentate gyrus of adult rats, particularly in the deepest part of the granular layer. The immunoreactive arborized dendrites, mostly arising from the primary apical pole of the granule cells, were found to enter the molecular layer. The mossy fibers also were positive. Electron-microscopic examination of the hilus portion showed that the immunoreactivity was detected on the plasma membrane of some axons in the mossy fiber bundles. Since postnatal neurogenesis is known to continue into adulthood in the deepest part of the granule cell layer of the dentate gyrus, these results suggest that, in the adult dentate gyrus, NCAM-H is expressed by newly generated granule cells, and that the NCAM-H-expressing new cells may participate in the formation of new neural circuits.
... Across the years, different markers were introduced to detect a wide range of biological processes, from stem cell identity to cell division and from cell migration to neuronal maturation (e.g., doublecortin, nestin, tubulinJ-1, SOX, PAX, ASCL1 genes, NeuN, CNGA3, NeuroD, Calretinin, as well as several markers of cell proliferation; reviewed in [25][26][27]). One of the first molecules used in studies on CNS structural plasticity, including adult neurogenesis, was the polysialylated form of the neural cell adhesion molecule (PSA-NCAM) [28][29][30] since its expression allows a wide range of neuronal changes, reveals cell immaturity, and provides the complete visualization of the cells and their processes (see below). Thirty years after the introduction of this marker, its detection can still provide insights into apparently unsolved controversies on human adult neurogenesis. ...
... In 1991, PSA-NCAM-expressing cells were unexpectedly found in two adult brain regions of rats: an inner part of the granule cell layer in the dentate gyrus, called the subgranular zone (SGZ [29]), and layer II of the piriform cortex [28]. Soon after, two reports performing the complete mapping of PSA-NCAM in the adult rat CNS extended the number of regions expressing this "embryonic" isoform of MCAM (including the olfactory bulb, hypothalamus, mesencephalic central grey, ependyma of the central canal, and superficial laminae of the dorsal horn in the spinal cord [57,58]. ...
Studies on brain plasticity have undertaken different roads, tackling a wide range of biological processes: from small synaptic changes affecting the contacts among neurons at the very tip of their processes, to birth, differentiation, and integration of new neurons (adult neurogenesis). Stem cell-driven adult neurogenesis is an exception in the substantially static mammalian brain, yet, it has dominated the research in neurodevelopmental biology during the last thirty years. Studies of comparative neuroplasticity have revealed that neurogenic processes are reduced in large-brained mammals, including humans. On the other hand, large-brained mammals, with respect to rodents, host large populations of special “immature” neurons that are generated prenatally but express immature markers in adulthood. The history of these “immature” neurons started from studies on adhesion molecules carried out at the beginning of the nineties. The identity of these neurons as “stand by” cells “frozen” in a state of immaturity remained un-detected for long time, because of their ill-defined features and because clouded by research ef-forts focused on adult neurogenesis. In this review article, the history of these cells will be reconstructed, and a series of nuances and confounding factors that have hindered the distinction between newly generated and “immature” neurons will be addressed.
... Recent reports [13,34,109,110] led to the consideration of the possibility that another type of structural plasticity might exist: The occurrence of populations of "persistently immature" neurons that are not newly generated, yet remain in a relatively undifferentiated state in the adult by continuing to express molecular markers of immaturity, such as the polysialylated form of the neural cell adhesion molecule (PSA-NCAM [111,112]) and doublecortin (DCX [113]). These cells were firstly identified in layer II of the piriform and lateral enthorinal cortices of adult rats and mice [111,112] (Figure 3A), namely, the paleocortex or three-layered "allocortex". ...
... Recent reports [13,34,109,110] led to the consideration of the possibility that another type of structural plasticity might exist: The occurrence of populations of "persistently immature" neurons that are not newly generated, yet remain in a relatively undifferentiated state in the adult by continuing to express molecular markers of immaturity, such as the polysialylated form of the neural cell adhesion molecule (PSA-NCAM [111,112]) and doublecortin (DCX [113]). These cells were firstly identified in layer II of the piriform and lateral enthorinal cortices of adult rats and mice [111,112] (Figure 3A), namely, the paleocortex or three-layered "allocortex". They exist in two main morphological types: Small cells, with a 3-9 µm cell soma diameter (type 1 cells) and large cells, with 9-17 µm diameter (type 2 cells; the range depending on the animal species; Figure 3B). ...
Brain plasticity is important for translational purposes since most neurological disorders and brain aging problems remain substantially incurable. In the mammalian nervous system, neurons are mostly not renewed throughout life and cannot be replaced. In humans, the increasing life expectancy explains the increase in brain health problems, also producing heavy social and economic burden. An exception to the “static” brain is represented by stem cell niches leading to the production of new neurons. Such adult neurogenesis is dramatically reduced from fish to mammals, and in large-brained mammals with respect to rodents. Some examples of neurogenesis occurring outside the neurogenic niches have been reported, yet these new neurons actually do not integrate in the mature nervous tissue. Non-newly generated, “immature” neurons (nng-INs) are also present: Prenatally generated cells continuing to express molecules of immaturity (mostly shared with the newly born neurons). Of interest, nng-INs seem to show an inverse phylogenetic trend across mammals, being abundant in higher-order brain regions not served by neurogenesis and providing structural plasticity in rather stable areas. Both newly generated and nng-INs represent a potential reservoir of young cells (a “brain reserve”) that might be exploited for preventing the damage of aging and/or delay the onset/reduce the impact of neurological disorders.
... Thickness of the mossy fiber bundle. The axons of the granule cells, the mossy fibers, can be visualized with PSA-NCAM immunohistochemistry303132. In each sampled section, the area of the region occupied by the mossy fibers in field CA3 was first measured by tracing its contour. ...
... The region of synaptic contact forms a layer called the stratum lucidum (Fig. 1B ). The mossy fiber axons express PSA- NCAM303132, which makes it possible to examine this bundle in sections immunoprocessed for PSA-NCAM. Ts65Dn mice have a reduced number of granule cells in comparison with euploid mice [12], which implies a reduction in the number of mossy fibers reaching the stratum lucidum. ...
Down syndrome (DS) is a high-incidence genetic pathology characterized by severe impairment of cognitive functions, including declarative memory. Impairment of hippocampus-dependent long-term memory in DS appears to be related to anatomo-functional alterations of the hippocampal trisynaptic circuit formed by the dentate gyrus (DG) granule cells - CA3 pyramidal neurons - CA1 pyramidal neurons. No therapies exist to improve cognitive disability in individuals with DS. In previous studies we demonstrated that pharmacotherapy with fluoxetine restores neurogenesis, granule cell number and dendritic morphology in the DG of the Ts65Dn mouse model of DS. The goal of the current study was to establish whether treatment rescues the impairment of synaptic connectivity between the DG and CA3 that characterizes the trisomic condition. Euploid and Ts65Dn mice were treated with fluoxetine during the first two postnatal weeks and examined 45-60 days after treatment cessation. Untreated Ts65Dn mice had a hypotrophyc mossy fiber bundle, fewer synaptic contacts, fewer glutamatergic contacts, and fewer dendritic spines in the stratum lucidum of CA3, the terminal field of the granule cell projections. Electrophysiological recordings from CA3 pyramidal neurons showed that in Ts65Dn mice the frequency of both mEPSCs and mIPSCs was reduced, indicating an overall impairment of excitatory and inhibitory inputs to CA3 pyramidal neurons. In treated Ts65Dn mice all these aberrant features were fully normalized, indicating that fluoxetine can rescue functional connectivity between the DG and CA3. The positive effects of fluoxetine on the DG-CA3 system suggest that early treatment with this drug could be a suitable therapy, possibly usable in humans, to restore the physiology of the hippocampal networks and, hence, memory functions.
... A novel, counterintuitive example of "young" neurons has been introduced by the demonstration that some populations of undifferentiated cells, displaying the same markers of immaturity expressed by newlyborn neurons, do not divide at all (Gómez-Climent et al., 2008;Piumatti et al., 2018). These cells were firstly described in the piriform cortex layer II (Seki and Arai, 1991;Bonfanti et al., 1992), and are currently (provisionally) referred to as "immature" or "dormant" neurons, since they are generated pre-natally, then remaining in a "standby" state of immaturity for long time (Luzzati et al., 2009;Gómez-Climent et al., 2008;Klempin et al., 2011;Bonfanti and Nacher, 2012;König et al., 2016;Rotheneichner et al., 2018;Benedetti and Couillard-Després, 2022). This idea of "young, " non-dividing neurons has evolved slowly through the years, somehow overwhelmed by the emphasis focused on adult neurogenesis (Bonfanti and Seki, 2021). ...
After the discovery of adult neurogenesis (stem cell-driven production of new neuronal elements), it is conceivable to find young, undifferentiated neurons mixed with mature neurons in the neural networks of the adult mammalian brain. This “canonical” neurogenesis is restricted to small stem cell niches persisting from embryonic germinal layers, yet, the genesis of new neurons has also been reported in various parenchymal brain regions. Whichever the process involved, several populations of “young” neurons can be found at different locations of the brain. Across the years, further complexity emerged: (i) molecules of immaturity can also be expressed by non-dividing cells born during embryogenesis, then maintaining immature features later on; (ii) remarkable interspecies differences exist concerning the types, location, amount of undifferentiated neurons; (iii) re-expression of immaturity can occur in aging (dematuration). These twists are introducing a somewhat different definition of neurogenesis than normally assumed, in which our knowledge of the “young” neurons is less sharp. In this emerging complexity, there is a need for complete mapping of the different “types” of young neurons, considering their role in postnatal development, plasticity, functioning, and interspecies differences. Several important aspects are at stake: the possible role(s) that the young neurons may play in maintaining brain efficiency and in prevention/repair of neurological disorders; nonetheless, the correct translation of results obtained from laboratory rodents. Hence, the open question is: how many types of undifferentiated neurons do exist in the brain, and how widespread are they?
... During postnatal mouse brain development, the amounts of polySia remain high during the first week, before rapidly declining between postnatal days 9-17. This decrease continues into adulthood [17][18][19]. In both adult rodents and humans, polySia is selectively expressed in areas where neurogenesis persists [20][21][22]. ...
Polysialic acid (polySia) is a linear homopolymer of α2-8-linked sialic acids that is highly expressed during early stages of mammalian brain development and modulates a multitude of cellular functions. While degree of polymerization (DP) can affect such functions, currently available methods do not accurately characterize this parameter, because of the instability of the polymer. We developed two improved methods to characterize the DP and total polySia content in biological samples. PolySia chains with exposed reducing termini can be derivatized with DMB for subsequent HPLC analysis. However, application to biological samples of polySia-glycoproteins requires release of polySia chains from the underlying glycan, which is difficult to achieve without concurrent partial hydrolysis of the α2-8-linkages of the polySia chain, affecting its accurate characterization. We report an approach to protect internal α2-8sia linkages of long polySia chains, using previously known esterification conditions that generate stable polylactone structures. Such polylactonized molecules are more stable during acid hydrolysis release and acidic DMB derivatization. Additionally, we used the highly specific Endoneuraminidase-NF enzyme to discriminate polysialic acid and other sialic acid and developed an approach to precisely measure the total content of polySia in a biological sample. These two methods provide improved quantification and characterization of polySia.
... Polysialylated neural cell adhesion molecule is one of the glycoforms of NCAM, modified by the addition of polysialic acid (PSA). Polysialic acid reduces the adhesive efficacy of NCAM, thereby participating in negative regulation of cell interactions (Seki and Arai, 1991). It has been shown that developing neurons express PSA-NCAM during their migration, neurite outgrowth and synaptogenesis. ...
Neural stem cells (NSCs) provide promising approaches for investigating embryonic neurogenesis, modeling of the pathogenesis of diseases of the central nervous system, and for designing drug-screening systems. Such cells also have an application in regenerative medicine. The most convenient and acceptable source of NSCs is pluripotent stem cells (embryonic stem cells or induced pluripotent stem cells). However, there are many different protocols for the induction and differentiation of NSCs, and these result in a wide range of neural cell types. This review is intended to summarize the knowledge accumulated, to date, by workers in this field. It should be particularly useful for researchers who are beginning investigations in this area of cell biology.
... Interneurons migrate in the rostral migratory stream (RMS) to the OB (Miragall et al., 1988). The other area is the hippocampus (HP), especially the subgranular zone (SGZ) of the dentate gyrus (DG), which includes the granule cell layer (GCL) and mossy fibers (MF) in the HP (Seki and Arai, 1991b). These two areas are characteristic for their neurogenesis and neural plasticity in adults. ...
Polysialic acid (polySia, PSA) is a unique constituent of the glycocalyx on the surface of bacterial and vertebrate cells. In vertebrates, its biosynthesis is highly regulated, not only in quantity and quality, but also in time and location, which allows polySia to be involved in various important biological phenomena. Therefore, impairments in the expression and structure of polySia sometimes relate to diseases, such as schizophrenia, bipolar disorder, and cancer. Some bacteria express polySia as a tool for protecting themselves from the host immune system during invasion. PolySia is proven to be a biosafe material; polySia, as well as polySia-recognizing molecules, are key therapeutic agents. This review first comprehensive outlines the occurrence, features, biosynthesis, and functions of polySia and subsequently focuses on the related diseases.
... For example, while PSA is expressed broadly in hippocampus and substantia nigra in the brains of 3-day-old rats, punctate PSA immunoreactivity is found in 2-to 3-month-old rats, probably representing synapses [133]. PSA expression is retained in the functionally active adult brain structures: olfactory bulb and piriform cortex in neurons of layer II, which receive input from the olfactory bulb, where neurogenesis and formation of neural circuits take place [129,135]; in the supraoptic and paraventricular nuclei of the hypothalamus and in the neurohypophysis, which undergo neuronal-glial and synaptic rearrangements in response to physiological stimuli [144]; in the hippocampal dentate gyrus, where postnatal neurogenesis continues into adulthood in the deep part of the granule cell layer [145]. At the subcellular level, PSA is often found in synapses, for example in some, but not all spine synapses in the outer third of the molecular layer of the dentate gyrus [146], suggesting that PSA contributes to synaptic activity and plasticity. ...
The success of investigations on the structure and function of the genome (genomics) has been paralleled by an equally awesome progress in the analysis of protein structure and function (proteomics). We propose that the investigation of carbohydrate structures that go beyond a cell’s metabolism is a rapidly developing frontier in our expanding knowledge on the structure and function of carbohydrates (glycomics). No other functional system appears to be suited as well as the nervous system to study the functions of glycans, which had been originally characterized outside the nervous system. In this review, we describe the multiple studies on the functions of LewisX, the human natural killer cell antigen-1 (HNK-1), as well as oligomannosidic and sialic (neuraminic) acids. We attempt to show the sophistication of these structures in ontogenetic development, synaptic function and plasticity, and recovery from trauma, with a view on neurodegeneration and possibilities to ameliorate deterioration. In view of clinical applications, we emphasize the need for glycomimetic small organic compounds which surpass the usefulness of natural glycans in that they are metabolically more stable, more parsimonious to synthesize or isolate, and more advantageous for therapy, since many of them pass the blood brain barrier and are drug-approved for treatments other than those in the nervous system, thus allowing a more ready access for application in neurological diseases. We describe the isolation of such mimetic compounds using not only Western NIH, but also traditional Chinese medical libraries. With this review, we hope to deepen the interests in this exciting field.
... Commonly, DCX hippocampal expression is temporally in-frame with PSA-NCAM expression [40]. The molecule is exceedingly expressed in the brain during development; still, in the adult murine brain, newborn granule cells of the dentate gyrus highly express PSA-NCAM as well [108]. Accordingly, PSA-NCAM is a popular marker to study structural plasticity and neurogenesis in mammals. ...
Adult neurogenesis is a complex physiological process, which plays a central role in maintaining cognitive functions, and consists of progenitor cell proliferation, newborn cell migration, and cell maturation. Adult neurogenesis is susceptible to alterations under various physiological and pathological conditions. A substantial decay of neurogenesis has been documented in Alzheimer’s disease (AD) patients and animal AD models; however, several treatment strategies can halt any further decline and even induce neurogenesis.
Our previous results indicated a potential effect of arginase inhibition, with norvaline, on various aspects of neurogenesis in triple-transgenic mice. To better evaluate this effect, we chronically administer an arginase inhibitor, norvaline, to triple-transgenic and wild-type mice, and apply an advanced immunohistochemistry approach with several biomarkers and bright-field microscopy.
Remarkably, we evidence a significant reduction in the density of neuronal progenitors, which demonstrate a different phenotype in the hippocampi of triple-transgenic mice as compared to wild-type animals. However, norvaline shows no significant effect upon the progenitor cell number and constitution. We demonstrate that norvaline treatment leads to an escalation of the polysialylated neuronal cell adhesion molecule immunopositivity, which suggests an improvement in the newborn neuron survival rate. Additionally, we identify a significant increase in the hippocampal microtubule-associated protein 2 stain intensity. We also explore the molecular mechanisms underlying the effects of norvaline on adult mice neurogenesis and provide insights into their machinery.
... Commonly, DCX hippocampal expression is temporally in-frame with PSA-NCAM expression [40]. The molecule is exceedingly expressed in the brain during development; still, in the adult murine brain, newborn granule cells of the dentate gyrus highly express PSA-NCAM as well [108]. Accordingly, PSA-NCAM is a popular marker to study structural plasticity and neurogenesis in mammals. ...
Adult neurogenesis is a complex physiological process, which plays a central role in maintaining cognitive functions, and consists of progenitor cell proliferation, newborn cell migration, and cell maturation. Adult neurogenesis is susceptible to alterations under various physiological and pathological conditions. A substantial decay of neurogenesis has been documented in Alzheimer's disease (AD) patients and animal AD models; however, several treatment strategies can halt any further decline and even induce neurogenesis. Our previous results indicated a potential effect of arginase inhibition, with norvaline, on various aspects of neurogenesis in triple-transgenic mice. To better evaluate this effect, we chronically administered an arginase inhibitor, norvaline, to triple-transgenic and wild-type mice, and applied an advanced immunohistochemistry approach with several biomarkers and bright-field microscopy. Remarkably, we evidenced a significant reduction in the density of neuronal progenitors, which demonstrate a different phenotype in the hippocampi of triple-transgenic mice as compared to wild-type animals. However, norvaline showed no significant effect upon the progenitor cell number and constitution. We demonstrated that norvaline treatment leads to an escalation of the polysialylated neuronal cell adhesion molecule immunopositivity, which suggests an improvement in the newborn neuron survival rate. Additionally, we identified a significant increase in the hippocampal microtubule-associated protein 2 stain intensity. We also explore the molecular mechanisms underlying the effects of norvaline on adult mice neurogenesis and provide insights into their machinery.
... Commonly, DCX hippocampal expression is temporally in-frame with PSA-NCAM expression [42]. The molecule is exceedingly expressed in the brain during development; still, in the adult murine brain, newborn granule cells of the dentate gyrus highly express PSA-NCAM as well [44]. ...
Adult neurogenesis is a complex physiological process, which plays a central role in maintaining cognitive functions, and consists of progenitor cell proliferation, newborn cell migration, and cell maturation. Adult neurogenesis is susceptible to alterations under various physiological and pathological conditions. A substantial decay of neurogenesis has been documented in Alzheimer’s disease (AD) patients and animal AD models; however, several treatment strategies can halt any further decline and even induce neurogenesis.
Our previous results indicated a potential effect of arginase inhibition, with norvaline, on various aspects of neurogenesis in triple-transgenic mice. To better evaluate this effect, we chronically administer an arginase inhibitor, norvaline, to triple-transgenic and wild-type mice, and apply an advanced immunohistochemistry approach with several biomarkers and bright-field microscopy.
Remarkably, we evidence a significant reduction in the density of neuronal progenitors, which demonstrate a different phenotype in the hippocampi of triple-transgenic mice as compared to wild-type animals. However, norvaline shows no significant effect upon the progenitor cell number and constitution. We demonstrate that norvaline treatment leads to an escalation of the polysialylated neuronal cell adhesion molecule immunopositivity, which suggests an improvement in the newborn neuron survival rate. Additionally, we identify a significant increase in the hippocampal microtubule-associated protein 2 stain intensity. We also explore the molecular mechanisms underlying the effects of norvaline on adult mice neurogenesis and provide insights into their machinery.
... These sections were incubated with primary antibodies in blocking solution overnight at 4°C, and with AlexaFluor-488/-568-conjugated secondary antibodies (1:500, Invitrogen) for 2 h at RT in blocking solution. The following primary antibodies were used: mouse IgM anti-polysialylated form of neural cell adhesion molecule (PSA-NCAM) (1:1000, a kind gift from Dr. Tatsunori Seki, Tokyo Medical University) (Seki and Arai, 1991) and mouse IgG anti-acetylated tubulin (1:11000, Sigma-Aldrich; RRID:AB_477585). Nuclei were stained with Hoechst 33342 (1:5000, Invitrogen). ...
New neurons, referred to as neuroblasts, are continuously generated in the ventricular-subventricular zone of the brain throughout an animal's life. These neuroblasts are characterized by their unique potential for proliferation, formation of chain-like cell aggregates, and long-distance and high-speed migration through the rostral migratory stream (RMS) toward the olfactory bulb (OB), where they decelerate and differentiate into mature interneurons. The dynamic changes of ultrastructural features in postnatal-born neuroblasts during migration are not yet fully understood. Here we report the presence of a primary cilium, and its ultrastructural morphology and spatiotemporal dynamics, in migrating neuroblasts in the postnatal RMS and OB. The primary cilium was observed in migrating neuroblasts in the postnatal RMS and OB in male and female mice and zebrafish, and a male rhesus monkey. Inhibition of intraflagellar transport molecules in migrating neuroblasts impaired their ciliogenesis and rostral migration toward the OB. Serial section transmission electron microscopy revealed that each migrating neuroblast possesses either a pair of centrioles, or a basal body with an immature or mature primary cilium. Using immunohistochemistry, live imaging, and serial block-face scanning electron microscopy, we demonstrate that the localization and orientation of the primary cilium are altered depending on the mitotic state, saltatory migration, and deceleration of neuroblasts. Together, our results highlight a close mutual relationship between spatiotemporal regulation of the primary cilium and efficient chain migration of neuroblasts in the postnatal brain.SIGNIFICANCE STATEMENTImmature neurons (neuroblasts) generated in the postnatal brain have a mitotic potential and migrate in chain-like cell aggregates toward the olfactory bulb. Here we report that migrating neuroblasts possess a tiny cellular protrusion called a primary cilium. Immunohistochemical studies with zebrafish, mouse, and monkey brains suggest that the presence of the primary cilium in migrating neuroblasts is evolutionarily conserved. Ciliogenesis in migrating neuroblasts in the RMS is suppressed during mitosis and promoted after cell cycle exit. Moreover, live imaging and three-dimensional electron microscopy revealed that ciliary localization and orientation change during saltatory movement of neuroblasts. Our results reveal highly organized dynamics in maturation and positioning of the primary cilium during neuroblast migration that underlie saltatory movement of postnatal-born neuroblasts.
... One consists of the precursor cells of interneurons, which are generated from the SVZ of the lateral ventricle and rostral migratory stream (RMS); these interneurons migrate to the OB [59]. The other is the subgranular zone (SGZ) of the dentate gyrus (DG) which includes the granule cell layer, and mossy fibers in the HIP [60]. It is well-known that neurogenesis and neural plasticity are observed in these two areas, including in the adult brain, and therefore, PSA is considered to be an oncodevelopmental antigen. ...
Mental disorders, such as schizophrenia, bipolar disorder, and autism spectrum disorder, are challenging to manage, worldwide. Understanding the molecular mechanisms underlying these disorders is essential and required. Studies investigating such molecular mechanisms are well performed and important findings are accumulating apace. Based on the fact that these disorders are due in part to the accumulation of genetic and environmental risk factors, consideration of multi-molecular and/or multi-system dependent phenomena might be important. Acidic glycans are an attractive family of molecules for understanding these disorders, because impairment of the fine-tuned glycan system affects a large number of molecules that are deeply involved in normal brain function. One of the candidates of this important family of glycan epitopes in the brain is polysialic acid (PSA/polySia). PSA is a well-known molecule because of its role as an oncodevelopmental antigen and is also widely used as a marker of adult neurogenesis. Recently, several reports have suggested that PSA and PSA-related genes are associated with multiple mental disorders. The relationships among PSA, PSA-related genes, and mental disorders are reviewed here.
... Developmental differences in the levels of expression of the TrkB receptor, and in particular its noncatalytic, truncated isoform, therefore may play a role in the cellular specificity of BDNF (Dugich-Djordjevic et al., 1993;Escandon et al., 1994;Knusel et al., 1994;Fryer et al., 1996). Interestingly, increased immunoreactivity for the polysialylated form of neural cell adhesion molecule (NCAM) is evident in developing DGCs (Seki and Arai, 1991;Parent et al., 1999;Seki and Arai, 1999), which might contribute to an increased responsiveness to BDNF, because PSA-NCAM increases the potency of BDNF in activating TrkB (Muller et al., 2000;Vutskits et al., 2001). ...
During limbic epileptogenesis in vivo the dentate granule cells (DGCs) exhibit increased expression of brain-derived neurotrophic factor (BDNF), followed by striking morphologic plasticities, namely the formation of basal dendrites and the sprouting of mossy fibers. We hypothesized that increased expression of BDNF intrinsic to DGCs is sufficient to induce these plasticities. To test this hypothesis, we transfected DGCs in rat hippocampal slice cultures with BDNF or nerve growth factor (NGF) via particle-mediated gene transfer, and we visualized the neuronal processes with cotransfected green fluorescent protein. Transfection with BDNF produced significant increases in axonal branch and basal dendrite number relative to NGF or empty vector controls. Structural changes were prevented by the tyrosine kinase inhibitor K252a. Thus increased expression of BDNF within DGCs is sufficient to induce these morphological plasticities, which may represent one mechanism by which BDNF promotes limbic epileptogenesis.
... The primary antibodies and concentrations included rabbit anti-Prox1 (1:5,000; Millipore, Billerica, MA), mouse IgM anti-PSA (12E3; 1:2,000; a generous gift from Dr. Seki) [18,19], rat anti-BrdU (1:1,000; AbD Serotec, Oxford, UK), mouse anti-Prox1 (1:2,000; Millipore), mouse anti-Glial fibrillary acidic protein (GFAP; 1:1,000; Millipore), mouse anti-beta III Tubulin (TuJ1; 1:500; abcam), and rabbit anti-Neuraminidase1 (NEU1; 1:1,000; Rockland, Philadelphia, PA). The secondary antibodies, goat anti-rabbit IgG Alexa 488 and 594, goat anti-mouse IgG Alexa 594, and goat anti-mouse IgM Alexa 488, were purchased from Life Technologies and used at a concentration of 1:500. ...
Hippocampal granule cells (GCs) are generated throughout the lifetime and are properly incorporated into the innermost region of the granule cell layer (GCL). Hypotheses for the well-regulated lamination of newly generated GCs suggest that polysialic acid (PSA) is present on the GC surface to modulate GC-to-GC interactions, regulating the process of GC migration; however, direct evidence of this involvement is lacking. We show that PSA facilitates the migration of newly generated GCs and that the activity of N-acetyl-α-neuraminidase 1 (NEU1, sialidase 1) cleaves PSA from immature GCs, terminating their migration in the innermost GCL. Developing a migration assay of immature GCs in vitro, we found that the pharmacological depletion of PSA prevents the migration of GCs, whereas the inhibition of PSA degradation with a neuraminidase inhibitor accelerates this migration. We found that NEU1 is highly expressed in immature GCs. The knockdown of NEU1 in newly generated GCs in vivo increased PSA presence on these cells, and attenuated the proper termination of GC migration in the innermost GCL. In conclusion, this study identifies a novel mechanism that underlies the proper lamination of newly generated GCs through the modulation of PSA presence by neuronal NEU1.
... The best-studied adhesion molecules are the polysialic acid-neural cell adhesion molecule (PSA-NCAM), L1 cell adhesion molecule and leucinerich repeat-and Ig domain-containing nogo receptor-interacting protein 1 (LINGO-1) (Fig. 2E). During development, the haemophilic NCAM-NCAM adhesion, i.e. cell-cell interaction, is prevented because all growing nerve fibres in the CNS express the PSA-NCAM [157], persisting in areas of adult brain that exhibit plasticity [158][159][160]. So, in order to occur interactions between OL and neurons and consequently myelination, PSA-NCAM has to be downregulated when neurons are electrically active [161][162][163][164]. ...
Oligodendrocytes are the myelinating cells of the central nervous system that constitute about 5 to 10% of the total glial population. These cells are responsible for myelin sheath production, which is essential not only for the rapid and efficient conduction of the electrical impulses along the axons, but also for preserving axonal integrity. Oligodendrocytes arise from oligodendrocyte progenitor cells that proliferate and differentiate just before and after birth, under a highly-regulated program. Both oligodendrocytes and their precursors are very susceptible to injury by several mechanisms, including excitotoxic damage, oxidative stress and inflammatory events. In this review, we will cover not only several important aspects of oligodendrocyte development and regulatory mechanisms involved in this process, but also some of the most important pathways of injury associated to oligodendrogenesis. In particular, we will also address some neurological disorders along life journey that present impairment in oligodendrocyte function and in myelination during neurodevelopment, such as periventricular leukomalacia, hypoxia/ischemia and hyperbilirubinemia that in turn can potentiate the emergence of neurological and neurodegenerative diseases like schizophrenia, multiple sclerosis and Alzheimer disease.
... Newborn neurons in this region are able to form synaptic connections with CA3 pyramidal neurons, even if they are not yet fully mature [49]. With their distinct structural [49][50][51] and synaptic plasticity [52,53], newborn neurons are poised to play a critical role in forming new memories. The time courses of maturation and functional integration of these neurons are critical for their influences on hippocampal circuits. ...
Adult hippocampal neurogenesis is a process involving the continuous generation of newborn neurons in the hippocampus of adult animals.Mounting evidence has suggested that hippocampal neurogenesis contributes to some forms of hippocampus-dependent learning and memory; however, the detailed mechanism concerning how this small number of newborn neurons could affect learning and memory remains unclear. In this review, we discuss the relationship between adult-born neurons and learning and memory, with a highlight on recently discovered potential roles of neurogenesis in pattern separation and forgetting.
... In particular, in this study, we chose primary hippocampal neurons for metabolic labeling of PSA-NCAMs, because the primary culture would make it possible to follow the important neuronal developments from the early stage, including neuritogenesis, by metabolic engineering. PSA is found in the hippocampal region of the brain and responsible for the tasks related to learning and memory, even during adulthood (35), which would allow for study on the spatial and temporal distributions of PSA-NCAMs. ...
Significance
The roles of cell-surface glycans remain elusive compared with those of proteins or lipids because of their diverse and dynamic nature. Metabolic incorporation of unnatural monosaccharides in the biochemical synthesis of glycans as a chemical reporter has been a successful method to investigate the functions of cell-surface glycans but has also left an issue of cytotoxicity for certain cells. In this work, we developed a tissue-based strategy for metabolic incorporation of a chemical reporter to primary neurons. We let an unnatural monosaccharide be metabolized by hippocampal tissues before dissociation into individual cells, and thereby, we could eliminate cytotoxicity. We used this method to describe, for the first time to our knowledge, the real-time distribution of polysialic acids on the membranes of neurons.
... In this study, we demonstrate for the first time that FLX treatment might also induce dematuration of parvalbumin+ interneurons in the mFC and hippocampal CA3 region, while our finding in the amygdala is consistent with that of a previous study [20]. Consistently, the present study demonstrates that chronic FLX treatment increases the expression of polysialic acidneural cell adhesion molecule (PSA-NCAM), which is a marker for immature neurons and a regulator of neural plasticity [42,43], in the mFC, hippocampus, and amygdala; this is in agreement with previous findings [44,45] (Additional file 1: Figure S7). This suggests that dematuration of parvalbumin+ interneurons is induced by FLX treatment in the mFC, hippocampus, and amygdala, where neural plasticity might be enhanced by FLX treatment. ...
The selective serotonin reuptake inhibitor fluoxetine (FLX) is widely used to treat depression and anxiety disorders, but cellular mechanisms underlying the antidepressant effect of FLX remain largely unknown. The generally accepted effect of chronic FLX treatment is increased adult neurogenesis in the hippocampal dentate gyrus. It was recently demonstrated that FLX treatments can reverse the established neuronal maturation of granule cells in the hippocampal dentate gyrus and of gamma-aminobutyric acidergic (GABAergic) interneurons in the basolateral amygdala. However, it is not clear whether this dematuration effect of FLX occurs in other brain regions. Thus, in this study, we used immunohistological analysis to assess the effect of FLX treatment on GABAergic interneurons in the medial frontal cortex (mFC) and reticular thalamic nucleus (RTN).
Immunofluorescence analysis for perineuronal nets (PNNs), which is a marker of neuronal maturation, and for parvalbumin, calretinin, and somatostatin, which are markers for specific GABAergic interneuron type, showed lower number of parvalbumin-positive (+) cells and PNN+/parvalbumin+ cells in the mFC of FLX-treated mice compared to vehicle-treated mice. However, FLX treatment had no effect on the number of cells expressing calretinin and somatostatin in the mFC. In the RTN, the number of PNN+ cells and parvalbumin+ cells was unaltered by FLX treatments. Furthermore, the number of total GABA+ cells and apoptotic cells in the mFC was similar between vehicle- and FLX-treated mice, suggesting that FLX treatment did not induce cell death in this region. Rather, our findings suggest that the decreased number of parvalbumin+ cells in the mFC was due to a decreased expression of parvalbumin proteins in the interneurons.
This study indicates that FLX decreases the levels of parvalbumin, a mature marker of fast-spiking interneurons, and PNNs in parvalbumin+ interneurons in the mFC, suggesting that FLX treatment induces a dematuration of this type of neurons. Induction of a juvenile-like state in fast-spiking inhibitory interneurons in these regions might be involved in the therapeutic mechanism of this antidepressant drug and/or some of its adverse effects.
... These high electron density cells were localized at the innermost portion in the granule cell layer. The granule cells in the innermost region are known to be newly generated (Kaplan and Hinds 1977; Kuhn et al. 1996; Eriksson et al. 1998) and are also stained by anti-PSA antibody (Seki and Arai 1991). PSA is known to be attached mainly to NCAM, to be highly expressed during neuronal development, and to affect the plasticity of neurons (Rutishauser and Jessell 1988; Landmesser et al. 1990; Seki and Arai 1993; Miller et al. 1994 ). ...
The histochemical distribution of sialoglycoconjugates in the CA1 region in the hippocampus formation of 9-week-old rats and 30-month-old rats was examined using electron microscopy in combination with two lectins, Maackia amurensis lectin, specific for Siaα2–3Gal, and Sambucus sieboldiana agglutinin, specific for Siaα2–6Gal. Each lectin stained the plasma membranes of pyramidal cells, indicating that the Siaα2–3Gal and Siaα2–6Gal groups were expressed on their plasma membranes. These lectins also bound to synapses in the stratum lacunosum moleculare. The staining intensity of the lectins in the synapses in these layers was downregulated in the 30-month-old rats. These results indicated that both the Siaα2–3Gal and Siaα2–6Gal groups are expressed on these synapses and that the expression of these sialyl linkages decreases in the aged brain.
... There are, however, various hotspots of polySia expression in the mature brain. Most prominent, migrating neuroblasts arising from the neurogenic niches of the anterior subventricular zone [72,73,95,96] and early postmitotic granular cell precursors in the subgranular layer of the hippocampal dentate gyrus [97][98][99][100][101][102] are characterized by their high polySia content and have been observed in all mammals including man [103][104][105]. Other major sources of polySia in the adult brain comprise widely spread subsets of interneurons and a population of immature neurons in layer II of the paleocortex. ...
Polymers of sialic acid can be produced by pro- and eukaryotic cells. In vertebrates polysialic acid consists of α2,8-linked N-acetylneuraminic acid and is most prominent during nervous system development. Polysialic acid is produced by two complementary sialyltransferases, ST8SiaII and ST8SiaIV. The major, but not the only, carrier of polysialic acid is the neural cell adhesion molecule (NCAM). In this review we highlight how polySia dictates the interactions of various cell types during development and plasticity of the vertebrate central nervous system on different molecular levels. Recent progress in generating mouse models with differential ablation of the polysialyltransferases or NCAM revealed the dramatic impact of polysialic acid-negative NCAM on brain development and elaborate electrophysiological studies allowed for new insights into the role of polysialic acid in regulating synaptic plasticity and learning. The implications of dysregulated polysialylation for brain disease and neuropsychiatric disorders are discussed.
... The expression of PSA-NCAM within the adult nervous system is essential in multiple facets of neural plasticity, including neurogenesis, synaptic plasticity, and neurite out- growth [10, 30, 31, 97, 98] . Importantly, PSA-NCAM functions across all aspects of hippocampal neurogenesis (proliferation , migration, differentiation, and survival), therefore changes in the polysialylation of newly generated neurons alter neurogenesis as a whole99100101102103104 (seeTable 2). For instance, chronic mild stress reduces the expression of both the core NCAM protein [65] and the addition of the PSA moiety [11,105106107108 in the hippocampus; however these changes are dependent on the type of stressor [109] (seeTable 3). ...
Depression is a devastating and prevalent disease, with profound effects on neural structure and function; however the etiology and neuropathology of depression remain poorly understood. Though antidepressant drugs exist, they are not ideal, as only a segment of patients are effectively treated, therapeutic onset is delayed, and the exact mechanism of these drugs remains to be elucidated. Several theories of depression do exist, including modulation of monoaminergic neurotransmission, alterations in neurotrophic factors, and the upregulation of adult hippocampal neurogenesis, and are briefly mentioned in the review. However none of these theories sufficiently explains the pathology and treatment of depression unto itself. Recently, neural plasticity theories of depression have postulated that multiple aspects of brain plasticity, beyond neurogenesis, may bridge the prevailing theories. The term "neural plasticity" encompasses an array of mechanisms, from the birth, survival, migration, and integration of new neurons to neurite outgrowth, synaptogenesis, and the modulation of mature synapses. This review critically assesses the role of adult hippocampal neurogenesis and the cell adhesion molecule, PSA-NCAM (which is known to be involved in many facets of neural plasticity), in depression and antidepressant treatment.
Transient expression of somatostatin (SST) has been observed in the olfactory epithelium (OE) and nerves of chick embryos. Intense expression of SST in these regions on embryonic days (E) 5–8 coincides with the migration of neurons producing gonadotropin‐releasing hormone (GnRH) from the OE to the forebrain (FB), suggesting that SST plays a role in the development of GnRH neurons. Using in ovo electroporation of small interfering RNA, we found that the suppression of SST mRNA in the olfactory placode (OP) of E3.5 chick embryos significantly reduced the number of GnRH and Islet‐1‐immunoreactive neurons in the nasal region without affecting the entry of GnRH neurons into the FB at E5.5–6. SST knockdown did not lead to changes in the number of apoptotic, proliferating, or HuC/D‐positive neuronal cells in the OE; therefore, it is possible that SST is involved in the neurogenesis/differentiation of GnRH neurons and OP‐derived GnRH‐negative migratory neurons. In whole OP explant cultures, we also found that SST or its analog octreotide treatment significantly increased the number of migratory GnRH neurons and the migratory distance from the explants. The co‐application of an SST antagonist blocked the octreotide‐induced increase in the number of GnRH neurons. Furthermore, the fasciculation of polysialylated neural cell adhesion molecule‐immunoreactive fibers emerging from the explants was dependent on octreotide. Taken together, our results provide evidence that SST exerts facilitatory effects on the development of neurons expressing GnRH or Islet‐1 and on GnRH neuronal migration, in addition to olfactory‐related fiber fasciculation.
During the development of the cerebral cortex, N-cadherin plays a crucial role in facilitating radial migration by enabling cell-to-cell adhesion between migrating neurons and radial glial fibers or Cajar-Reztius cells. ADP ribosylation factor 4 (Arf4) and Arf5, which belong to the class II Arf small GTPase subfamily, control membrane trafficking in the endocytic and secretory pathways. However, their specific contribution to cerebral cortex development remains unclear. In this study, we sought to investigate the functional involvement of class II Arfs in radial migration during the layer formation of the cerebral cortex using mouse embryos and pups. Our findings indicate that knockdown of Arf4, but not Arf5, resulted in the stalling of transfected neurons with disorientation of the Golgi in the upper intermediate zone (IZ) and reduction in the migration speed in both the IZ and cortical plate. Migrating neurons with Arf4 knockdown exhibited cytoplasmic accumulation of N-cadherin, along with disturbed organelle morphology and distribution. Furthermore, supplementation of exogenous N-cadherin partially rescued the migration defect caused by Arf4 knockdown. In conclusion, our results suggest that Arf4 plays a crucial role in regulating radial migration via N-cadherin trafficking during cerebral cortical development.
Significance Statement
In the cortical layer formation, the distribution of N-cadherin on cell surface in migrating neurons is tightly regulated by endosomal trafficking system. However, its molecular detail remained fully understood. Here, we demonstrated that Arf4 small GTPase, a critical regulator of membrane trafficking in the trans-Golgi network (TGN), plays distinct roles from Arf5 in radial migration. We further demonstrated that Arf4 regulates N-cadherin trafficking in and out of the TGN and the contact of migrating neurons with radial fibers. Our results suggest that Arf4 regulates radial migration through N-cadherin trafficking in the TGN.
Cell migration from the olfactory placode (OP) is a well‐known phenomenon wherein various cell types, such as gonadotropin‐releasing hormone (GnRH)‐producing neurons, migrate toward the telencephalon (TEL) during early embryonic development. However, the spatial relationship between early migratory cells and the forebrain is unclear. We examined the early development of whole‐mount chick embryos to observe the three‐dimensional spatial relationship among OP‐derived migratory neurons, olfactory nerve (ON), and TEL. Migratory neurons that express highly polysialylated neural cell adhesion molecule (PSA‐NCAM) emerge from the OP and spread over a relatively wide TEL area at the Hamburger and Hamilton (HH) stage 17. Most migratory neurons form a cellular cord between the olfactory pit and rostral TEL within HH18–20. The earliest axons from the olfactory epithelium (OE) were detected along this neuronal cord using DiI‐labeling at HH21. Furthermore, a few PSA‐NCAM‐positive neurons were dispersed around the cellular cord and over the lateral TEL at HH18. A long cellular cord branch extending to the lateral TEL was transiently observed within HH18–24. These results suggest a novel migratory route of OP‐derived neurons during the early developmental stages. Following GFP vector introduction into the OP of HH13–15 embryos, labeled neurons were detected around and within the lateral TEL at HH23 and HH27. At HH36, labeled cells were observed in the rostral‐lateral TEL, including the olfactory bulb (OB) region. GFP‐labeled and calretinin‐positive neurons were detected in the OB, suggesting that early OP‐derived neurons enter the forebrain and function as interneurons in the OB.
The olfactory placode (OP) of vertebrates generates several classes of migrating cells, including hypothalamic gonadotropin-releasing hormone (GnRH)-producing neurons, which play essential roles in the reproduction system. Previous studies using OP cell labeling have demonstrated that OP-derived non-GnRH cells enter the developing forebrain; however, their final fates and phenotypes are less well understood. In chick embryos, a subpopulation of migratory cells from the OP that is distinct from GnRH neurons transiently expresses somatostatin (SS). We postulated that these cells are destined to develop into brain neurons. In this study, we examined the expression pattern of SS mRNA in the olfactory-forebrain region during development, as well as the destination of OP-derived migratory cells, including SS mRNA-expressing cells. Utilizing the Tol2 genomic integration system to induce long-term fluorescent protein expression in OP cells, we found that OP-derived migratory cells labeled at embryonic day (E) 3 resided in the olfactory nerve and medial forebrain at E17-19. A subpopulation of green fluorescent protein (GFP)-labeled GnRH neurons that remained in the olfactory nerve was considered to comprise terminal nerve neurons. In the forebrain, GFP-labeled cells showed a distribution pattern similar to that of GnRH neurons. A large proportion of GFP-labeled cells expressed the mature neuronal marker NeuN. Among the GFP-labeled cells, the percentage of GnRH neurons was low, while the remaining GnRH-negative neurons either expressed SS mRNA, neuropeptide Y, or calbindin D-28k or did not express any of them. These results indicate that a diverse population of OP-derived neuronal cells, other than GnRH neurons, integrates into the chick medial forebrain.
Neuroplastic alterations are the key processes involved in adaptation and rehabilitation after all neurological injuries and pathologies. Being the central contributor to the developmental and adult neuroplasticity, the polysialylated form of Neural Cell Adhesion Molecule (PSA-NCAM) may prove to be a potential target to facilitate repair/regeneration after CNS injury and disease. Over the years, several experimental approaches have been developed to exploit the therapeutic potential of PSA-NCAM. Broadly, the studies focused on cell-transplantation strategies to alter PSA-NCAM properties at the injury site, injection of peptide based as well as synthetic PSA mimetics directly into the injury site or the application of PSA containing hydrogels and scaffolds as biomaterials. A comprehensive understanding of the PSA-based experimental approaches, as well as their pros and cons, is urgently required for successful implementation of this molecule in therapeutics. The current review, therefore, has been designed to give the readers a thorough account of all the diverse roles of PSA in the adult nervous system and the recent progress that has been made in developing PSA-based therapeutic approaches for neuroregeneration.
Sialic acid (Sia) is involved in many biological activities and commonly occurs as a monosialyl residue at the nonreducing terminal end of glycoconjugates. The loss of activity of UDP-GlcNAc2-epimerase/ManNAc kinase, which is a key enzyme in Sia biosynthesis, is lethal to the embryo, which clearly indicates the importance of Sia in embryogenesis. Occasionally, oligo/polymeric Sia structures such as disialic acid (diSia), oligosialic acid (oligoSia), and polysialic acid (polySia) occur in glycoconjugates. In particular, polySia, a well-known epitope that commonly occurs in neuroinvasive bacteria and vertebrate brains, is one of the most well-known and biologically/neurologically important glycotopes in vertebrates. The biological effects of polySia, especially on neural cell-adhesion molecules, have been well studied, and in-depth knowledge regarding polySia has been accumulated. In addition, the importance of diSia and oligoSia epitopes has been reported. In this chapter, the recent advances in the study of diSia, oligoSia, and polySia residues in glycoproteins in neurology, and their history, definition, occurrence, analytical methods, biosynthesis, and biological functions evaluated by phenotypes of gene-targeted mice, biochemical features, and related diseases are described.
Radial glia (RG) are embryonic neural stem cells (NSCs) that produce neuroblasts and provide fibers that act as a scaffold for neuroblast migration during embryonic development. Although they normally disappear soon after birth, here we found that RG fibers can persist in injured neonatal mouse brains and act as a scaffold for postnatal ventricular-subventricular zone (V-SVZ)-derived neuroblasts that migrate to the lesion site. This injury-induced maintenance of RG fibers has a limited time window during post-natal development and promotes directional saltatory movement of neuroblasts via N-cadherin-mediated cell-cell contacts that promote RhoA activation. Transplanting an N-cadherin-containing scaffold into injured neonatal brains likewise promotes migration and maturation of V-SVZ-derived neuroblasts, leading to functional improvements in impaired gait behaviors. Together these results suggest that RG fibers enable postnatal V-SVZ-derived neuroblasts to migrate toward sites of injury, thereby enhancing neuronal regeneration and functional recovery from neonatal brain injuries.
Adult neurogenesis is now a very popular phenomenon in neuroscience. It is widely accepted that neurons continue to be generated in special regions of the adult brain such as the subventricular zone (SVZ) of the forebrain and the subgranular zone (SGZ) of the dentate granule cell layer. Adult neurogenesis, however, is not a special type of neurogenesis that occurs only in the adult stage, but is a part of the persistent neurogenesis that continues from embryonic to adult stages. Here, we describe the similarities and differences in the properties of progenitors and cellular architecture of proliferative zones from embryonic to adult periods and discuss the elements required for persistent neurogenesis.
Suppressor of cytokine signaling 2 (SOCS2) is a well-established negative regulator of growth hormone signaling that acts on adult hippocampal neurogenesis during ischemic insults. To explore whether SCOS2 is involved in poststroke neurogenesis, we studied the temporal expression of SOCS2 mRNA in the subventricular zone (SVZ) of rats after transient focal cerebral ischemia. We found that SOCS2 expression was upregulated in the SVZ of the infarcted hemisphere. The number of SOCS2-expressing cells was significantly increased in the ipsilateral SVZ compared with that on the contralateral side on days 7-10 after reperfusion, and SOCS2-expressing cells were highly proliferative, coinciding both spatially and temporally with stroke-induced neurogenesis. Almost all SOCS2-expressing cells in the SVZ were colabeled with the neural stem cell markers nestin and musashi1 and the neural/glial progenitor transcription factor Sox-2. In addition, SOCS2 was highly expressed in newly generated neurons that were immunoreactive for polysialic acid-neural cell adhesion molecule, indicating that SOCS2 expression may be persistent during neuronal differentiation. Thus, our data demonstrated that SOCS2 mRNA was highly expressed in proliferating neural stem/precursor cells and postmitotic migratory neuroblasts in the SVZ niche after focal cerebral ischemia, suggesting that SOCS2 may be actively involved in regulating adult neurogenesis induced by ischemic stroke.
Polysialated neural cell adhesion molecule (PSA-NCAM) is a membrane bound glycoprotein widely expressed during nervous system development. While commonly described in the neurogenic niches of the adult human brain, there is limited evidence of its distribution in other brain regions. PSA-NCAM is an important regulator of cell-cell interactions and facilitates cell migration and plasticity. Recent evidence suggests these functions may be altered in neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease. This study provides a detailed description of the PSA-NCAM distribution throughout the human brain and quantitatively compares the staining load in cortical regions and sub-cortical structures between the control, Alzheimer’s, and Parkinson’s disease brain. Our results provide evidence of widespread, yet specific, PSA-NCAM expression throughout the human brain including regions devoid of PSA-NCAM in the rodent brain such as the caudate nucleus and cerebellum. We also detected a significant reduction in PSA-NCAM load in the entorhinal cortex of Alzheimer’s disease cases that was inversely correlated with hyperphosphorylated tau load. These results demonstrate that PSA-NCAM mediated structural plasticity may not be limited to neurogenic niches and is conserved in the aged brain. We also provide evidence that PSA-NCAM is reduced in the entorhinal cortex, a region severely affected by Alzheimer’s disease pathology.
Polysialic acid (polySia, PSA) is a unique and functionally important glycan, particularly in vertebrate brains. It is involved in higher brain functions such as learning, memory, and social behaviors. Recently, an association between several genetic variations and single nucleotide polymorphisms (SNPs) of ST8SIA2/STX, one of two polysialyltransferase genes in vertebrates, and psychiatric disorders, such as schizophrenia (SZ), bipolar disorder (BD), and autism spectrum disorder (ASD), was reported based on candidate gene approaches and genome-wide studies among normal and mental disorder patients. It is of critical importance to determine if the reported mutations and SNPs in ST8SIA2 lead to impairments of the structure and function of polySia, which is the final product of ST8SIA2. To date, however, only a few such forward-directed studies have been conducted. In addition, the molecular mechanisms underlying polySia-involved brain functions remain unknown, although polySia was shown to have an anti-adhesive effect. In this report, we review the relationships between psychiatric disorders and polySia and/or ST8SIA2, and describe a new function of polySia as a regulator of neurologically active molecules, such as brain-derived neurotrophic factor (BDNF) and dopamine, which are deeply involved in psychiatric disorders.
Both cognitive functions and mental health are known to be influenced by diet, although the underlying mechanisms are not well understood. However, there is a consensus that the brain responds to changes in the fatty acid composition of diet, and that simultaneous changes in neural membrane components occur to affect membrane fluidity, gene expression and neuronal functions. For polyunsaturated fatty acids (PUFA), various functions in the neurons have been proposed such as lipid storage, membrane synthesis, β-oxidation, enzyme activity and transcription programs. PUFA have recently emerged as a new class of modulator for the synaptic transmission and plasticity in the brain. As chaperones, fatty acid binding proteins (FABP) facilitate the transport of PUFA to specific compartments in the neurons. Both PUFA and FABP are nowadays known to be major regulators of adult neurogenesis, and circumstantial evidence implicates adult hippocampal neurogenesis in learning, memory and emotions. The survival of newborn neurons is increased by enriched environment and hippocampus-dependent stimuli. The key molecule that can explain synergistic effects of PUFA and stimuli should be brain-derived neurotrophic factor (BDNF), because this molecule is synthesized predominantly in hippocampal neurons for the structural remodeling and synaptic plasticity. In response to exercise, dietary energy restriction, or cognitive stimulation, levels of BDNF are increased in the hippocampus to promote adult neurogenesis. The recent discovery that newborn neurons and glia in the primate hippocampus express the free fatty acid-receptor, G protein-coupled receptor 40 (GPR40), was a minor breakthrough in the research on adult neurogenesis, because PUFA-GPR40 binding can lead to BDNF production via activations of cAMP-response element binding protein (CREB). CREB-dependent gene expression is crucial for a variety of neuronal functions such as learning, memory and emotions through BDNF synthesis. GPR40 may be one of the candidates explaining the effects of PUFA upon neuronal differentiation and synaptogenesis. By paying special attention to available evidence of the “PUFA-GPR40 signaling,” this review aims to understand one of the main cascades of adult neurogenesis.
Polysialic acid (PSA) is one of the most abundant glycopolymer present in embryonic brain, and it is known to be involved in key roles such as plasticity in the central nervous system, cell adhesion, migration and localization of neurotrophins. However, in adult brain, its expression is quite low. The exception to this is in Alzheimer's disease (AD) brain, where significantly increased levels of polysilylated neural cell adhesion molecule (PSA-NCAM) have been reported. Here, we confirm the role of PSA as a metal chelator, allowing it to decrease cytotoxicity caused by high levels of transition metals, commonly found in AD brain, and as a regulator of cell behavior. UV-visible (UV-vis) and circular dichroism (CD) spectroscopy, atomic force microscopy (AFM), and isothermal titration calorimetry (ITC) techniques were used to investigate the assembly of PSA-metals complexes. These PSA-metal complexes exhibited less toxicity compared to free metal ions, and in particular, the PSA-Cu(2+) complex synergistically promoted neurite outgrowth in PC12 cells.
The granule cell layer of the adult dentate gyrus possesses two characteristics of an immature nervous system. The first is that granule cells continue to be generated in the innermost region of the granule cell layer, and newly generated and developing granule cells in the adult express highly polysialylated neural cell adhesion molecule (PSA-NCAM). PSA-NCAM-expressing apical dendrites have dynamically unstable processes such as irregular shafts and many stick-like or fan-shaped fine processes. The second is that radial glia-like cells expressing glial fibrillary acidic protein (GFAP) remain in a similar region of the granular layer. The numbers of PSA-NCAM-expressing granule cells and GFAP-expressing radial glia-like cells show a parallel age-dependent decrease during aging. Moreover, by using confocal laser scanning microscopy and immunoelectron microscopy, we demonstrated that PSA-NCAM-expressing dendrites and GFAP-expressing radial processes are partly in contact with each other, and occasionally the radial glial processes envelop the PSA-NCAM-positive dendritic processes. The temporal and spatial relationship between the two immature elements suggests that the processes of the radial glia-like cells are closely associated with the dendritic growth of the newly generated granule cells in the adult dentate gyrus and that these two immature features of neurons and glia in the dentate gyrus diminish with age. J. Comp. Neurol. 410:503–513, 1999.
Polysialic acid (polySia) is a homopolymer of sialic acid with a degree of polymerization (DP) of 8–400. When present on neural cell adhesion molecule (NCAM), polySia has anti-adhesive effects on cell–cell interactions owing to its bulky polyanionic nature, and is involved in the regulation of neurogenesis and neuronal functions. Recently, we demonstrated that polySia functions not only as an anti-cell adhesion molecule, but also as a reservoir scaffold for brain-derived neurotrophic factor (BDNF) and fibroblast growth factor 2 (FGF2), which are biologically active molecules in neurogenesis. To understand the significance of polySia structure in the reservoir function, we focused on polySia-NCAM biosynthesized by mutated polysialyltransferase (ST8SiaII or STX) that was reported in a schizophrenia patient. The polySia-NCAM biosynthesized by mutant ST8SiaII/STX contained less polySia with shorter chain length and exhibited impaired reservoir function for BDNF and FGF2 as compared with that synthesized by wild-type (wt) ST8SiaII/STX. Our findings suggest that the quantity and quality of polySia on NCAM are important for normal neuronal functioning.
Abstract As an anti-adhesive, a reservoir for key biological molecules, and a modulator of signaling, polysialic acid (polySia) is critical for nervous system development and maintenance, promotes cancer metastasis, tissue regeneration and repair, and is implicated in psychiatric diseases. In this review, we focus on the biosynthesis and functions of mammalian polySia, and the use of polySia in therapeutic applications. PolySia modifies a small subset of mammalian glycoproteins, with the neural cell adhesion molecule, NCAM, serving as its major carrier. Studies show that mammalian polysialyltransferases employ a unique recognition mechanism to limit the addition of polySia to a select group of proteins. PolySia has long been considered an anti-adhesive molecule, and its impact on cell adhesion and signaling attributed directly to this property. However, recent studies have shown that polySia specifically binds neurotrophins, growth factors, and neurotransmitters and that this binding depends on chain length. This work highlights the importance of considering polySia quality and quantity, and not simply its presence or absence, as its various roles are explored. The capsular polySia of neuroinvasive bacteria allows these organisms to evade the host immune response. While this "stealth" characteristic has made meningitis vaccine development difficult, it has also made polySia a worthy replacement for polyetheylene glycol in the generation of therapeutic proteins with low immunogenicity and improved circulating half-lives. Bacterial polysialyltransferases are more promiscuous than the protein-specific mammalian enzymes, and new studies suggest that these enzymes have tremendous therapeutic potential, especially for strategies aimed at neural regeneration and tissue repair.
Anosmin-1 is the glycoprotein encoded by the KAL1 gene and part of the extracellular matrix, which was first identified as defective in human Kallmann syndrome (KS, characterised by hypogonadotropic hypogonadism and anosmia); biochemically it is a cell adhesion protein. The meticulous biochemical dissection of the anosmin-1 domains has identified which domains are necessary for the protein to bind its different partners to display its biological effects. Research in the last decade has unravelled different roles of anosmin-1 during CNS development (axon pathfinding, axonal collateralisation, cell motility and migration), some of them intimately related with the cited KS but not only with this. More recently, anosmin-1 has been identified in other pathological scenarios both within (multiple sclerosis) and outside (cancer, atopic dermatitis) the CNS.
Previously we have demonstrated that intraventricular injection of 6-hydroxydopamine (6-OHDA) results in increased proliferation and de-differentiation of rat cortical astrocytes into progenitor-like cells four days after lesion (Wachter et al., 2010). To find out if these cells express tyrosine hydroxylase (TH), the rate-limiting enzyme in the catecholamine synthesis pathway, we performed immunohistochemistry in the rat cortex following intraventricular injection of 6-OHDA. Four days after injection we demonstrated a strong emergence of TH-positive (TH(+)) somata in the cortices of 6-OHDA lesioned animals. The number of TH(+) cells in the cortex of 6-OHDA-lesioned animals was 15 times higher than in sham operated animals, where virtually no TH(+) somata occurred. Combining TH immunohistochemistry with classical Nissl stain yielded complete congruency, and ∼ 45% of the TH(+) cells co-expressed calretinin, which indicates an interneuron affiliation. There was no co-staining of TH with other interneuron markers or with glial markers such as GFAP and nestin, nor could we find co-localization with the proliferation marker Ki67. However, we found a co-localization of TH with glial progenitor cell markers (Sox2 and S100β) and with PSA-NCAM, which has been shown to be expressed in immature, but not recently generated cortical neurons. Taken together, this study seems to confirm our previous findings with respect to a 6-OHDA-induced expression of neuronal precursor markers in cells of the rat cortex, although the TH(+) cells found in this study are not identical with the potentially de-differentiated astrocytes described recently (Wachter et al., 2010). The detection of cortical cells expressing the catecholaminergic key enzyme TH might indicate a possible compensatory role of these cells in a dopamine depleted system. Future studies are needed to determine whether the TH(+) cells are capable of dopamine synthesis to confirm this hypothesis.
The significance of glycosphingolipids and glycoproteins is discussed in their relation to normal aging and pathological aging, aging with diseases. Healthy myelin that looks stable is found to be gradually degraded and reconstructed throughout life for remodeling. An exciting finding is that myelin P0 protein is located in neurons and glycosylated in aging brains. In pathological aging, the roles of glycosphingolipids and glycoproteins as risk factors or protective agents for Alzheimer's and Parkinson's diseases are discussed. Intensive studies have been performed aiming to remove the risks from and to restore the functional deficits of the brain. Some of them are expected to be translated to therapeutic means.
Every cell in nature carries a rich surface coat of glycans, its glycocalyx, which constitutes the cell's interface with its environment. In eukaryotes, the glycocalyx is composed of glycolipids, glycoproteins, and proteoglycans, the compositions of which vary among different tissues and cell types. Many of the linear and branched glycans on cell surface glycoproteins and glycolipids of vertebrates are terminated with sialic acids, nine-carbon sugars with a carboxylic acid, a glycerol side-chain, and an N-acyl group that, along with their display at the outmost end of cell surface glycans, provide for varied molecular interactions. Among their functions, sialic acids regulate cell-cell interactions, modulate the activities of their glycoprotein and glycolipid scaffolds as well as other cell surface molecules, and are receptors for pathogens and toxins. In the brain, two families of sialoglycans are of particular interest: gangliosides and polysialic acid. Gangliosides, sialylated glycosphingolipids, are the most abundant sialoglycans of nerve cells. Mouse genetic studies and human disorders of ganglioside metabolism implicate gangliosides in axon-myelin interactions, axon stability, axon regeneration, and the modulation of nerve cell excitability. Polysialic acid is a unique homopolymer that reaches >90 sialic acid residues attached to select glycoproteins, especially the neural cell adhesion molecule in the brain. Molecular, cellular, and genetic studies implicate polysialic acid in the control of cell-cell and cell-matrix interactions, intermolecular interactions at cell surfaces, and interactions with other molecules in the cellular environment. Polysialic acid is essential for appropriate brain development, and polymorphisms in the human genes responsible for polysialic acid biosynthesis are associated with psychiatric disorders including schizophrenia, autism, and bipolar disorder. Polysialic acid also appears to play a role in adult brain plasticity, including regeneration. Together, vertebrate brain sialoglycans are key regulatory components that contribute to proper development, maintenance, and health of the nervous system.
In the adult mammalian brain, two neurogenic regions have been characterized, the subventricular zone (SVZ) of the lateral ventricle (LV) and the subgranular zone (SGZ) of the dentate gyrus (DG). Despite remarkable knowledge in rodents, the detailed arrangement of neurogenic regions in most mammals is poorly understood. In this study, we used immunohistochemistry and cell type-specific antibodies to investigate the organization of two germinal regions in the adult ferret, which belongs to the order Carnivora and is widely used as a model animal with a gyrencephalic brain. From the SVZ to the olfactory bulb, doublecortin-positive cells tended to organize in chain-like clusters, which were surrounded by a meshwork of astrocytes. This structure was homologous to the rostral migratory stream (RMS) described in other species. Different from rodents, the horizontal limb of the RMS emerged directly from the LV, and the anterior region of the LV extended rostrally and reached the olfactory bulb. In the DG, glial fibrillary acidic protein-positive cells with long radial processes as well as doublecortin-positive cells were oriented in the SGZ. In both regions, doublecortin-positive cells showed characteristic morphology and were positive for polysialylated-neural cell adhesion molecule, beta-III tubulin, and lamin B1 (intense staining). Proliferating cells were detected in both regions using antibodies against proliferating cell nuclear antigen and phospho-histone H3. These observations demonstrate that the two neurogenic regions in ferrets have similar cellular composition as those of other mammalian species despite anatomical differences in the brain. J. Comp. Neurol., 2013. © 2013 Wiley Periodicals, Inc.
According to the neuroplastic hypothesis of the depression, antidepressant drugs can trigger plastic mechanisms in the adult brain, which may revert the reduction of neuronal plasticity produced by depression. However, there is controversy on the efficacy of these drugs on older patients. With the present study we seek to explore the effects of the antidepressant fluoxetine, a selective serotonin reuptake inhibitor, on neuronal plasticity in the medial prefrontal cortex (mPFC) in middle aged rats. This plasticity may be mediated by the polysialylated form of the neural cell adhesion molecule (PSA-NCAM), which is expressed in the adult mPFC, associated to inhibitory elements. PSA-NCAM expressing interneurons can be found in the mPFC of rodents and humans and they belong to different subpopulations of interneurons, defined by the expression of calcium binding proteins and neuropeptides.
Most of them express serotonin type 3 receptors (5HT3), but not 5HT1A or 5HT2A. Moreover, previous results from our group have shown changes in the expression of PSA-NCAM and synaptophysin (SYN) in this cortical region after chronic treatment with fluoxetine in young rats (3 months old). These effects are mediated by 5HT3 receptors, since their pharmacological blockade prevent the changes induced by fluoxetine. In the present study, we analyze the expression of these and other plasticity related molecules in the same region, such as glutamic acid decarboxylase (GAD67) and vesicular glutamate transporter 1 (VGLUT1) after chronic fluoxetine treatment (14 days) in middle aged rats (8 months old). These rats were injected intraperitoneally daily with a dose of 10 mg/Kg of fluoxetine in standard stabulary conditions. We observed the typical significant changes in the weight of fluoxetine treated rats (p = 0.00057). Using immunohistochemistry and a specialized software, we analyzed the expression of the different molecules and their density of puncta in the mPFC neuropil.
Preliminary results show no significant change between the fluoxetine treated animals and the controls in the expression of PSA-NCAM (p= 0.24 for the puncta analysis and p = 0.43 for fluorescence intensity), SYN (p = 0.6 for puncta analysis and p = 0.71 for fluorescence intensity), GAD67 (p = 0.9 for puncta and p = 0.78 for fluorescence intensity), and VGLUT1 (p = 0.5 for puncta and p = 0.59 for fluorescence intensity) in the mPFC of these middle aged rats. Therefore, the changes in PSA-NCAM expression induced by antidepressants are compromised during aging and, consequently, may result in a reduced plastic response, which is reflected in the absence of changes in the expression of molecules related to general, inhibitory or excitatory neurotransmission. The effects of antidepressants on the expression of molecules related to neuronal structural plasticity in other brain regions of middle aged rats such as the somatosensory cortex, hippocampus or the amygdala, are currently being analyzed. These results invite to reconsider the efficacy of antidepressant drugs in older patients.
Many cell adhesion molecules have a distinct pattern of expression and well defined role in cell-cell recognition. By contrast, NCAM is broadly expressed and perturbations of its function affect many diverse aspects of embryonic development. Evidence has been obtained suggesting that the molecule and its unusual polysialic acid moiety serve not only to contribute to specific interactions, but also to regulate overall cell-cell interaction. In this latter mode the molecule can have both a positive and a negative effect on a wide variety of contact-dependent cellular events during development.
Adult neurogenesis can only be observed in some specific brain regions. One of these areas is the dentate gyrus of the hippocampal formation. The progenitor cells located in the subgranular layer of the dentate gyrus proliferate, differentiate, and give rise to young neurons that can become integrated into existing neuronal circuits. Under physiological conditions, hippocampal neurogenesis is linked to hippocampal-dependent learning, whereas deficits in adult hippocampal neurogenesis have been shown to correlate with disturbances in spatial learning and memory. This review summarizes the phenomenon of adult hippocampal neurogenesis and the use of suitable markers for the investigation of adult hippocampal neurogenesis. In addition, we focused on the disturbances in neurogenesis that can be seen in depression. Interestingly, several antidepressants have been found to be capable of increasing the rate of hippocampal neurogenesis. Based on that, it can be speculated that factors, which directly or indirectly increase the rate of hippocampal neurogenesis, may be helpful in the treatment of depression.
Polysialic acids (polysia)" are a structurally diverse family of' linear carbohydratc chains that consist of N-acetylneuraminic acid (NeuSAc) or N-glycolylneuraminic acid (NeuSGc) residues, usually joined internally by a2,8-, a2,9-, or alternating a2,8-/a2,9-ketosidic linkages. 3 -D e o x y -~ -g ~ ~ ~ c e r o -~ -g a ~ a c t o -2 -nonulosonic acid (KDN) is a unique deaminated form of Sia, and polyKDN chains share many properties in common with polySia (Table I). The finding of poly(NeuSAc), poly(NeuSGc), poly(NeuSAc,NeuSGc), poly(KDN) chains and their partially 0-acetylated and 0-lactylated forms in salmonid fish egg glycoproteins demon-strates the natural occurrence of multiple forms of these unique sugar chains. The polySia glycotope covalently modifies cell surface glycoconjugates on *Al?h~-evi;~l~onh used i l l this ch;lp[cr: Sii~, hizllic ;lcid; NeuSAc. N-;~cc~yl~icur;~minic acid; NcuS<;c, N-glycolylncuraminic acid; polySia, a2,8-linked polysialic acid; KDN, 3-deoxy-~-g/ycero-~-pnlacto-2-lon nu lo sonic acid; LPS, lipopolysaccharide; GSL, glycosphingolipid; polyST, CMP-sialic acid:polyu2,8-sialosyl sialytransferase; Endo-N, endo-N-acylneuraminidase; DP, degree of polymer-iz;~tion; 4-MU-NeuSAc, 4-methylumbelliferyl NeuSAc; 4-MU-KDN. 4-methylumbclliferyl KDN.
Glial fibrillary acidic protein was localized at the electron microscope level in the cerebellum of adult mice by indirect immunoperoxidase histology. In confirmation of previous studies at the light microscope level, the antigen was detectable in astrocytes and their processes, but not in neurons or their processes, or in oligodendroglia. Astrocytic processes were stained in white matter, in the granular layet surrounding synaptic glomerular complexes, and in the molecular layer in the form of radially oriented fibers and of sheaths surrounding Purkinje cell dendrites. Astrocytic endfeet impinging on meninges and perivascular membranes were also antigen positive. In astrocytic perikarya and processes, the immunohistochemical reaction product appears both as a diffuse cytoplasmic label and as elongated strands, which by their distribution and frequency could be considered glial filaments.
Three-month-old rats were injected intraperitoneally with [3H]thymidine (4.3 microcuries per gram of body weight) and allowed to survive for 30 days. Radioautography of 1-micrometer sections revealed labeled cells in the granular layers of dentate gyrus and olfactory bulb; these were confirmed as neurons by electron microscopy of reembedded 1-micrometer sections.
We have undertaken a quantitative analysis of the mouse olfactory bulb to address several major questions concerning the development of neural circuitry in the postnatal mammalian brain. These are: (1) To what degree are new elements and circuits added during maturation? (2) How long do such processes go on? and (3) Does postnatal development involve a net addition of circuits and their constituent elements, or is there elimination of some portion of an initial surfeit? Using male mice of known age, weight, and length, we measured the overall size of the bulb, the numbers of processing units (glomeruli) within the bulb, the extent and complexity of postsynaptic dendrites within the glomeruli, and the number of synapses in different regions of the bulb. Between birth and the time mice reach sexual maturity at 6-7 weeks of age, the bulb increases in size by a factor of 8, the number of glomeruli by a factor of 4-5, the length of mitral cell dendritic branches by a factor of 11, and the number of glomerular and extraglomerular synapses by factors of 90 and 170, respectively. Each of these parameters increases steadily from birth, in concert with the enlargement of the olfactory mucosa, the overall growth of the brain, and indeed, of the entire animal. We found no evidence of an initial surfeit of processing units, dendritic branches, or synapses. Further elaboration of neural circuitry by each of these measures is also apparent from the time of sexual maturity until the animals reach their full adult size at about 10-12 weeks of age. The developmental strategy in this part of the mouse brain evidently involves prolonged construction that persists until the growth of the body is complete. This ongoing elaboration of neural circuitry in the postnatal mammalian brain may be relevant to understanding a number of unexplained developmental phenomena, including critical periods, the ability of the juvenile brain to recover from injuries that would cause severe and permanent deficits in older animals, and the special ability of the maturing brain to encode large amounts of new information.
We have compared the expression of the neural cell adhesion molecule (N-CAM) and the neuron-glial cell adhesion molecule (Ng-CAM) during histogenesis of the chick nervous system. Data from immunohistochemistry and photometry were combined to construct maps of the overall distribution and dynamics of CAM appearance and disappearance. Each CAM appeared in a characteristic spatial and temporal pattern in various areas during cell movement, fiber outgrowth, tract formation, and myelination. N-CAM was more uniformly distributed than Ng-CAM and was present on all neural cell bodies and processes of the CNS and PNS. In the adult, the staining pattern of N-CAM remained similar to that in the embryo, although the staining intensity was diminished. During embryonic development, Ng-CAM was expressed on extending neurites and migrating neurons. The appearance Ng-CAM in the CNS was correlated particularly with times of cell migration in spinal cord and cerebellum, and in regions undergoing neurite extension, such as the developing white matter of the spinal cord, the optic nerve, and the medial longitudinal fasciculus. Cell bodies not undergoing migration were negative for Ng-CAM. In the adult CNS, Ng-CAM was markedly decreased in myelinated fiber tracts like the white matter of the spinal cord but persisted in unmyelinated regions such as the olfactory bulb. In contrast, in the PNS (for example, the dorsal root ganglion and sciatic nerve), Ng-CAM appeared early on both cell bodies and neurites, and it continued to be present on both in the adult, even in the presence of myelin. Maps comparing the relative distribution of Ng-CAM and N-CAM showed dynamic reversals as the nervous system developed and, as a result, the pattern of CAM expression was markedly different in embryos and adults. This difference appears to reflect changes in the roles of selective adhesion and of the two neuronal CAMs at different times of development.
A cumulative labelling protocol using 5-bromo-2'-deoxyuridine (BUdR) was followed to determine: (1) the growth fraction (i.e., the proportion of cells that comprise the proliferating population), (2) the length of the cell cycle, and (3) the length of the DNA-synthetic phase (S-phase) for proliferative cells in the dentate gyrus of the mouse. On postnatal day 20 (P20), C57BL/6J mice were injected with BUdR at two hour intervals for a total period of 12 hours. Animals were sacrificed at selected intervals, and the brains were processed for immunohistochemistry using a monoclonal antibody directed against single-stranded DNA containing BUdR. The numbers of BUdR-labelled and unlabelled cells in sections through the hilus of the dentate gyrus were counted. The number of BUdR-labelled cells increased linearly from an initial value of about 12% of the total number of cells to a maximum value of just over 24% of the total. These findings indicate that, at P20, a maximum of 24.2 +/- 1.2% of the cells in the dentate hilus are part of the proliferating population. The calculated length of the cell cycle of the cells comprising the intrahilar proliferative zone was estimated to be 16.1 +/- 0.8 h. The length of the S-phase was estimated at 8.0 +/- 0.4 h. In addition, mathematical analysis, using one and two population models, indicates that over 90% of the proliferating cells in the dentate hilus at this age comprise a single population at least in terms of the lengths of the cell cycle and the S-phase.(ABSTRACT TRUNCATED AT 250 WORDS)
The cellular and subcellular localization of the neural cell adhesion molecules L1 and N-CAM was studied by pre- and postembedding immunoelectron microscopic labeling procedures in the developing mouse cerebellar cortex. The salient features of the study are: L1 displays a previously unrecognized restricted expression by particular neuronal cell types (i.e., it is expressed by granule cells but not by stellate and basket cells) and by particular subcellular compartments (i.e., it is expressed on axons but not on dendrites or cell bodies of Purkinje cells). L1 is always expressed on fasciculating axons and on postmitotic, premigratory, and migrating granule cells at sites of neuron-neuron contact, but never at contact sites between neuron and glia, thus strengthening the view that L1 is not involved in granule cell migration as a neuron-glia adhesion molecule. While N-CAM antibodies reacting with the three major components of N-CAM (180, 140, and 120 kD) show a rather uniform labeling of all cell types, antibodies to the 180-kD component (N-CAM180) stain only the postmigratory granule cell bodies supporting the notion that N-CAM180, the N-CAM component with the longest cytoplasmic domain, is not expressed before stable cell contacts are formed. Furthermore, N-CAM180 is only transiently expressed on Purkinje cell dendrites. N-CAM is present in synapses on both pre- and post-synaptic membranes. L1 is expressed only preterminally and not in the subsynaptic membranes. These observations indicate an exquisite degree of fine tuning in adhesion molecule expression during neural development and suggest a rich combinatorial repertoire in the specification of cell surface contacts.
The neural cell adhesion molecule, N-CAM, is a cell surface glycoprotein found on embryonic and adult neurons and on a variety of ectodermal and mesodermal tissues in very early embryos. During development, it shows local variations in prevalence at the cell surface as well as conversion from an embryonic form (E form) with high sialic acid content to an adult form (A form) with lesser amounts of this sugar. This E leads to A conversion occurs on different schedules in different brain regions, and it has been hypothesized that both the conversion and the prevalence changes are related to early regulation of pattern formation and connectivity. In order to identify precisely the consequences of these mechanisms of local cell surface modulation of N-CAM, an assay was developed to measure the rate of aggregation either of vesicles reconstituted from lipid and purified N-CAM or of native brain membrane vesicles. In both preparations, aggregation was greater than 95% inhibitable by specific anti-(N-CAM) Fab' fragments. The rates of aggregation of reconstituted N-CAM vesicles and native brain vesicles were found to be inversely related to the sialic acid content of their N-CAM molecules, with full desialylation resulting in about a 4-fold increase in rate over E-form N-CAM. Intermediate rates were obtained both with A-form N-CAM (which contains only one-third of the sialic acid content of E-form N-CAM) and with partially desialylated E-form N-CAM. The rate of coaggregation of reconstituted vesicles containing E-form N-CAM with reconstituted vesicles containing A-form N-CAM was also intermediate, implying that desialylation did not change the nature of (N-CAM)-(N-CAM) binding but only its rate. Even larger alterations in vesicle aggregation rate were seen when the amount of N-CAM per vesicle was altered. A 2-fold increase in the N-CAM-to-lipid ratio of reconstituted vesicles resulted in a greater than 30-fold increase in their rate of aggregation. Moreover, desialylation did not cause a further increase in the rate of aggregation of these already rapidly aggregating vesicles. These results in a model system demonstrate the large range of binding rates that are obtainable by various forms of local surface modulation of N-CAM. They are consistent with the proposal that similar alterations affecting (N-CAM)-mediated cell adhesion in vivo may be major factors in pattern formation during development of the nervous system.
N-CAM, the neural cell adhesion molecule, has been found at a number of regions in the early (1-5 days) chicken embryo by fluorescent antibody techniques. These regions appear to be those concerned with induction of the primary developmental axis (neural plate, neural tube, notochord, somites) or those in which later inductive events occur (neural crest cells, optic, otic, and pharyngeal placodes, cardiac mesoderm, mesonephric primordium, limb buds). The staining patterns in the latter group of regions are highly dynamic and transient and are limited to the epithelial components of the placodes and to the precursors of mesonephric tubules. In neural crest cells, N-CAM appears early, disappears during migration of the cells on fibronectin, and reappears at sites where ganglia are formed. In other regions of the nervous system, particularly those related directly to the neural tube, the N-CAM molecule is stained at all stages. The results raise the possibility that adhesion mediated by N-CAM plays a primary role in early embryogenesis as well as in later histogenesis.
A neural cell adhesion molecule (N-CAM) was purified in milligram quantities from detergent extracts of embryonic chick brain membranes. N-CAM has an unusual carbohydrate content and structure, is polydisperse in solution, and is associated with proteolytic activity leading to its spontaneous cleavage. The carbohydrate composition of N-CAM includes 13 mol of sialic acid but only 1.4 mol of galactose/100 mol of amino acids, suggesting the presence of a sialic acid to protein linkage not previously observed in higher organisms. N-CAM appears to be an integral membrane protein in that its extraction from membranes required detergent. Although soluble, the purified molecule was aggregated (Mr = 0.5 to 1.2 X 10(6)) and polydisperse in detergent-free solutions. N-CAM from brain also migrated as a broad but continuously stained region from Mr = 200,000 to Mr = 250,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis; the molecule from retina was similar but had a somewhat faster mobility. Desialation of N-CAM did not significantly change its behavior in solution, but converted both brain and retinal N-CAM to components migrating on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as material of about Mr = 140,000. Despite the apparent heterogeneity, amino acid sequence analysis and comparison of proteolytic fragments suggest that all forms of the glycoprotein are derived from the same polypeptide chain. On prolonged incubation at neutral pH, N-CAM undergoes apparent proteolysis to yield a polypeptide that contains little sialic acid and has a Mr = 65,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a separate sialic acid-rich component, and a variety of small peptides. The 65,000-dalton polypeptide appeared to contain all of the antigenic determinants of intact N-CAM that neutralize the adhesion-blocking ability of anti-retinal cell Fab' fragments.
We have previously identified a cell surface glycoprotein of the mouse nervous system named brain cell surface protein-2 (BSP-2). Here we report that this antigen is not a single, discrete entity, but a family of antigenically and structurally related molecules. Three components of 180, 140, and 120 K were characteristic for more mature nervous tissues. Adult cerebral cortex contained the 140-K and 120-K antigens, adult spinal cord only the 120-K, and dorsal root ganglia from young mice mainly the 180-K component. Very different forms of the antigen that migrated as a diffuse zone from 180-250-K in SDS-polyacrylamide gels were found in immature nervous tissues. A molecule different from the previous ones was found in a neuroblastoma line. Evidence is presented that the structural diversity of BSP-2 is due to differences in glycosylation. This result indicates that cell type- and developmental stage-specific glycoprotein patterns previously found in the nervous system may in part be due to different glycosylation of identical polypeptides. The finding that a neural cell surface protein may be glycosylated in different ways has important implications for the generation of cell surface specificity.
We have found in the adult rat that the persistent expression of a highly polysialylated neural cell adhesion molecule (NCAM-H) that is generally specific to developing tissues, remains restrictively in the cells of the deepest portion of the dentate granular layer. Since the granule cells are known to continue to be generated in this region during the adult period, we have tried to determine whether NCAM-H is expressed by newly generated granule cells. Immunoelectron microscopic observation revealed that about half of the NCAM-H-expressing cells had the features of dentate granule cells, and that the rest of these cells appeared to be immature cells. Double immunostaining for NCAM-H and glial fibrillary acidic protein (GFAP) revealed that the NCAM-H-expressing cells differed from GFAP-positive glial cells. In rats injected with 5-bromo-2'-deoxyuridine (BrdU) at post-natal day 35, double immunostaining for NCAM-H and BrdU demonstrated that the BrdU-labeled cells expressed NCAM-H at 12 d after the injection but not at 80 d. These results provide the first direct evidence that NCAM-H is expressed transiently by newly generated granule cells that may add new neuronal circuits to the adult hippocampal formation.
Polysialic acid, referring specifically to linear homopolymers of α2-8-linked N-acetylneuraminic acid (abbreviated here as PSA), is a remarkable carbohydrate structure. The basis for this statement begins with some surprising general observations: (1) the abundant presence of this structure in three very different biological contexts [bacterial capsules (Finne, 1985; Troy, 1979), fish eggs (Kitajima et al., 1986; Inoue et al., 1987), and surfaces of a variety of vertebrate cells (Finne, 1985; Margolis and Margolis, 1983; Chuong and Edelman, 1984; Finne et al., 1987)], (2) the nearly complete restriction of vertebrate PSA to a single cell surface protein [the neural cell adhesion molecule (NCAM)] (Hoffman et al., 1982; Finne et al., 1983), and (3) the fact that in each of these situations the carbohydrate appears to form a space or barrier around a cell. In this chapter I will focus on the molecular, cell, and tissue biology of NCAM PSA, with particular emphasis on the hypothesis (Rutishauser et al., 1988) that variations in this carbohydrate during development serve as an overall regulator of cell—cell and possibly cell—matrix interactions.
Glial fibrillary acidic protein was localized at the electron microscope level in the cerebellum of adult mice by indirect immunoperoxidase histology. In confirmation of previous studies at the light microscope level, the antigen was detectable in astrocytes and their processes, but not in neurons or their processes, or in oligodendroglia. Astrocytic processes were stained in white matter, in the granular layet surrounding synaptic glomerular complexes, and in the molecular layer in the form of radially oriented fibers and of sheaths surrounding Purkinje cell dendrites. Astrocytic endfeet impinging on meninges and perivascular membranes were also antigen positive. In astrocytic perikarya and processes, the immunohistochemical reaction product appears both as a diffuse cytoplasmic label and as elongated strands, which by their distribution and frequency could be considered glial filaments.
Adhesive mechanisms have been implicated in several morphogenetic processes during development. In the last decade several molecules mediating specific adhesion between cells and between cells and their environment have been characterized. Possible roles in morphogenesis have been established for some of these molecules. In the brain, four cell-cell adhesion molecules have been characterized: NCAM, L1, the myelin-associated glycoprotein and N-cadherin. Furthermore, a cell-substrate adhesion molecule, cytotactin, and its proteoglycan ligand have been described. We here review the data on structure, localization, developmental regulation and function of these molecules in brain.Copyright © 1989 S. Karger AG, Basel
The expression of the two adhesion molecules L1 and N-CAM and their shared carbohydrate epitope recognized by monoclonal antibody L2, was studied during development of the embryonic mouse neocortex by immunohistology at light- and electron-microscopic levels between embryonic days 9 and 18. Throughout this time period N-CAM is expressed in all layers of the telencephalic anlage. L1 antigen shows a more restricted expression than N-CAM. It is not detectable at day 9. From day 10 onward it is expressed on young neurons in the marginal zone, but not in the ventricular layer. At embryonic day 13 L1 antigen appears also in the intermediate zone on afferent fibers from subcortical structures and on migrating neurons. Neuronal cell bodies in the cortical plate and subplate express L1 antigen only transiently on embryonic days 13–16. These observations suggest that L1 antigen does not play a prominent role in the initiation of neuronal migration in the ventricular zone, but could be functional during later stages of migration and in the aggregation of neuronal cell bodies at their final position in the cortical plate. The L2 epitope also shows a more restricted expression than N-CAM during the time period studied. Similar to L1 antigen, it first appears at embryonic day 10 in the marginal zone and remains undetectable in the ventricular layer also at later stages. In the marginal zone the L2 epitope is strongly expressed on neuroepithelial endfeet at the basal lamina. The basal lamina itself is L2 epitope-negative. From embryonic day 10 onward the L2 epitope is most strongly expressed in the marginal zone and subplate and more weakly in the cortical plate and intermediate zone. In the subplate it is not only associated with the surface membrane, but also with the extracellular matrix. These observations support previous biochemical data which show that the L2 epitope is not present on all N-CAM molecules of the embryonic or adult forms and suggest that the independent regulation or L2 epitope expression may have functional implications during development.
Much of our present understanding of the cellular mechanisms of learning and memory derives from studies on the hippocampus in which long-term potentiation (LTP) of synaptic transmission is produced by a train of high-frequency electrical stimulation1 or by potassium channel blockers2. The hippocampus is also a seizure-prone region and recent studies have revealed that brief seizure episodes produce remarkably long-lasting changes which are reminiscent of ‘classical’ LTP3. A brief seizure episode also sets in motion a cascade of events that includes changes in gene expression, sprouting of fibres and the establishment of new synaptic contacts. This paper reviews this use-dependent structural rearrangement of the neuronal network and discusses its possible role in epilepsy and as a model of plasticity in the adult nervous system.
The differentiation of granule cell dendrites in the dentate gyrus of the hippocampal region was studied in a series of developing fetal and postnatal rhesus monkeys whose brains were processed by the rapid Golgi method. The total combined lengths of dendrites, the total number of dendritic spines, and their density on the proximal, middle, and distal thirds of the dendritic shafts were determined at embryonic days 58, 95, 120, 153, term (165), postnatal days 3, 20, 60, 150, 365, and adults. At all ages examined, granule cells exhibited various levels of maturation with the more differentiated cells being situated in the superficial strata of the granular layer and the less mature cells lying in progressively deeper positions, thus conforming to the outside-to-inside spatiotemporal gradient of their genesis. Quantitative analysis shows that, in this primate, hippocampal granule cells differentiate mainly in the second half of gestation with all measured parameters attaining mature values by the time of birth. However, the analysis also reveals a transient phase of exuberant postnatal development which involves excessive dendritic branching, regional changes in dendritic length, overproduction of dendritic spines, and redistribution of spines within the molecular layer. After reaching peak values around the middle of the first year of life, these parameters decrease and in adult monkeys fall back to the neonatal level
Rats were tested in a new spatial discrimination procedure which measured working memory. Following preoperative testing, lesions were placed to disrupt each of the major extrinsic fiber connections of the hippocampal formation. Destruction of the entorhinal area, body of the fimbria-fornix anterior to hippocampus, septum, or postcommisural fornix produced a severe and consistent impairment in performance. Analysis of error patterns indicated that when animals with limbic lesions made errors, they were likely to make these errors in the same sequence as the original choices. These data support the hypothesis that the hippocampus has an important role in the processing of information about spatial location, and that normal performance on this task requires an intact hippocampal circuitry.
The dentate gyrus of the rat contains about 600,000 granule cells. These small neurons are generated over a prolonged period from the 14th day of gestation until some time after the second postnatal week. The majority of the cells pass through their last phase of DNA synthesis in the postnatal period, and during the peak period of cell generation, between the fifth and seventh days after birth, up to 50,000 granule cells are formed each day. Contrary to earlier reports, most of the cells pass through their last mitotic division either within the stratum granulosum itself, or within the hilar region of the developing gyrus. The precursor population of cells in the hilar region must therefore constitute a pool of true neuroblasts. The origin of this pool of cells has not been definitely established but it seems probable that its cells are derived from the neuroepithelium lining the lateral ventricle adjacent to the region from which the hippocampal pyramidal cells are generated. Examination of the final location of granule cells labeled at different stages reveals three distinct morphogenetic gradients in the gyrus. The cells in the dorsal blade tend to be formed earlier than those in the ventral blade; cells in the more caudal (or temporal) portions of the gyrus are generated earlier than those in more rostral (or septal) regions; and in all regions the more superficial neurons in the stratum granulosum are formed earlier than the deeper granule cells. The bearing of some of these findings on the development and organization of the connections of the dentate gyrus is discussed.
Single units were recorded from the CA1 field of the hippocampus in the freely-moving rat. They were classified as place units, displace units or others. Place units were defined as those for which the rat's position on the maze was a necessary condition for maximal unit firing. Some of these place units (misplace units) fired maximally when the animal sniffed in a place, either because it found something new there or failed to find something which was usually there. Displace units increased their rates during behaviors associated with theta activity in the hippocampal slow waves. In general these were behaviors which changed the rat's position relative to the environment. The influence of various environmental manipulations (e.g., turning off the room lights) on the firing pattern of the place units was tested and the results suggest that they were not responding to a simple sensory stimulus nor to a specific motor behavior. Nor could the unit firing be due purely to motivational or incentive factors. The results are interpreted as strong support for the cognitive map theory of hippocampal function.
To gain insight into the cellular and molecular mechanisms underlying neurogenesis in adult mouse olfactory bulb, several adhesion molecules expressed by glial cells and neurons were investigated. In the germinal zone of the olfactory bulb, the subependymal layer of the rostral region of the lateral ventricles, two adhesion molecules are detectable that are characteristic of early morphogenetic events: J1/tenascin and the polysialylated form, the so-called embryonic form, of N-CAM. The polysialylated form of N-CAM is expressed by most cells in the subependymal layer, and by some astrocytes and neurons in the granular layer adjacent to the subependymal layer. This suggests that bipotential precursor cells retain expression of the embryonic form during their migration from the subependymal layer and during the first stages of differentiation into neurons and glia.
Expression of the polysialylated form of N-CAM is also retained in monolayer cultures of six-day-old olfactory bulbs, 55 days after seedingin vitro. J1/tenascin was detectable in the subependymal layer using two monoclonal antibodies. The immunostaining pattern was different between the two antibodies and more restricted to the subependymal layer than when staining with polyclonal J1 antibodies was performed, indicating that J1/tenascin exists in distinct isoforms.
Finally, our observations suggest that, in the adult olfactory bulb, L1 is not only a neuron-neuron adhesion molecule, but it may also be involved in neuron-glia interactions, since it is found at contact sites between these two cell types. L1, therefore, may be a neuron-glia adhesion molecule in some parts of the CNS, while it is not in others.
The expression of a highly polysialylated form of the neural cell adhesion molecule (NCAM-H) has been investigated in the neocortex and piriform cortex of the developing and the adult rat by using a monoclonal antibody 12E3, which has been found to recognize the polysialic acid portion of NCAM-H. Immunoblot analysis of the cortical homogenates showed that NCAM-H was temporarily expressed during the late embryonic and early postnatal stages. Further, immunohistochemical observations revealed that NCAM-H appeared at embryonic day 13 (E13) in the plexiform primordium in horizontally-oriented cells, probably Cajal-Retzius cells, which are the first neurons to differentiate. During the late embryonic stage, the marginal zone, subplate, and intermediate zone strongly stained, whereas the ventricular zone stained weakly. After birth, the NCAM-H expression was progressively attenuated from a week onwards, and almost vanished in the adult neocortex. In the primordium of the piriform cortex, NCAM-H immunoreactivity also became positive at E13. The time sequences of the NCAM-H expression in these neurons were similar to those of the neurons in the neocortical area. In the piriform cortex, however, the expression remained in a number of neurons in the layer II, which receives a large number of olfactory fibers from the olfactory bulb, where prolonged neurogenesis and construction of neural circuits take place in adulthood. These results suggest that NCAM-H not only plays an important role in the developing rat cortex, but also may be involved in some functions related to reorganization in the adult piriform cortex.
Progress in the investigation of cell-cell interactions in tissue formation has been particularly rapid in recent years (for review see Rutishauser and Jessell, 1988). A fundamental hypothesis throughout this work has been that these interactions reflect specific molecular complementarity mediated by distinct components at the cell surface. In addition, it is generally assumed that the developmental control of these events occurs through regulation of expression of the molecules directly involved.
We have used monolayers of control 3T3 cells and 3T3 cells transfected with a cDNA encoding human N-CAM as a culture substrate for embryonic chick retinal ganglion cells (RGCs). At embryonic day 6 (E6), but not at E11, RGCs extended longer neurites on monolayers of N-CAM-transfected cells. This loss of RGC responsiveness was not associated with substantial changes in the level of N-CAM expression on RGC growth cones. The neurite outgrowth response from E6 RGCs could be inhibited by removal of N-CAM from the monolayer, by removal of alpha 2-8-linked polysialic acid from neuronal N-CAM, or by antibodies that bind exclusively to chick (neuronal) N-CAM. In contrast, the response was not dependent on neuronal beta 1 integrin function. These data provide substantive evidence for a homophilic binding mechanism directly mediating N-CAM-dependent neurite outgrowth, and suggest that changes in polysialic acid expression on neuronal N-CAM may modulate N-CAM-dependent axonal growth during development.
The expression of the highly polysialylated form of the neural cell adhesion molecule (N-CAM)--the so-called embryonic N-CAM (E-N-CAM)--was investigated in the developing and adult mouse cerebellar cortex by immunohistology and immunocytology at the light and electron microscopic levels. E-N-CAM was never (from embryonic day 14 to postnatal day 15) detectable in the germinal zone of neuroblasts destined to form or forming the external granular layer and was only observed once small cerebellar interneurons had become postmitotic before the beginning of granule cell migration. Granule cells expressed E-N-CAM on cell bodies, axons, and leading and trailing processes also during migration but ceased to reveal detectable levels of E-N-CAM at the end of migration after having reached their final position in the internal granular layer. Other cerebellar cell types, such as Purkinje cells, Bergmann glia, astrocytes, oligodendrocytes, and most prominently, stellate and basket cells, also expressed E-N-CAM, but became E-N-CAM-negative during the third and fourth postnatal weeks, coinciding with overt cessation of cerebellar histogenesis. Thus, except for neuroblasts, E-N-CAM appeared characteristic of growing and moving cellular structures, in agreement with the notion that the highly polysialylated form of N-CAM is less adhesive than the adult form.
The neural cell adhesion molecule (NCAM) is thought to be involved in several important events during CNS vertebrate development. This study provides additional information concerning the biochemical determination and anatomical localization of NCAM transcripts. Using S1 nuclease protection assays (S1-NPAs), NCAM transcripts in brain appear highest at birth, with NCAM messenger levels reduced some 20-fold by adulthood. By use of in situ hybridization, NCAM mRNA is demonstrated to be developmentally regulated in the cerebellum and hippocampus. The in situ hybridization findings, in addition to providing results to compare with past studies of NCAM immunolocalization, reveal that NCAM expression in dentate gyrus granule cells and cerebellar Purkinje cells is correlated with the final stages of axonal growth, e.g., synaptic stabilization. In situ hybridization demonstrates a developmental outside-to-inside gradient of NCAM transcripts in the dentate gyrus. Neurological mutant mice, reeler and stagger, provide evidence that NCAM expression is normal in the brain regions investigated, and does not correlate with the developmental perturbations present in these strains.
The role of polysialic acid (PSA) during initial innervation of chick muscle was examined. Previously, the adhesion molecules L1 and N-CAM were shown to be important in balancing axon-axon and axon-muscle adhesion during this process. Here we demonstrate developmental changes in the pattern of innervation that are not correlated with levels of L1 or N-CAM expression, but rather with the amount of PSA at the axon surface. Removal of PSA by a specific endoneuraminidase (Endo-N) increased axon fasciculation and reduced nerve branching. In contrast, the nerve trunk defasciculation and increased branching produced by neuromuscular activity blockade were associated with an increase in axonal PSA levels. Furthermore, Endo-N prevented these inactivity-induced effects on branching. Together these results illustrate the potential of PSA as a regulator of cell-cell interactions and provide a direct example of a molecular link between the morphogenic effects of adhesion-mediated and synaptic activity-dependent processes.
The expression of the cell adhesion molecules L1, J1 and N-CAM and their shared carbohydrate L2 was studied in the embryonic cerebral cortex of normal and reeler mutant mice using light and electron microscopic immunocytochemistry. Apart from a general delay in their appearance in the reeler cortex, the 4 antigens were present with a cellular distribution in both genotypes reflecting the anatomical characteristics of normal and mutant phenotypes. The cell surface glycoprotein L1 was exclusively expressed by neurons, particularly axons, but was never detected at sites of neuron-glia contact. L1 was accumulated in the marginal zone and subplate of the normal cortex and in the homologous layers of the reeler cortex. The secreted glycoprotein J1 was found on glia and neurons. Although initially present in regions of fiber outgrowth, J1 became characteristically excluded from the large fiber tracts at later stages. J1 mapped in the marginal zone and subcortical plate of the normal cortex and in the corresponding layers of the mutant cortex. N-CAM had a more ubiquitous distribution and was present in ventricular zones, particularly at early stages, as well as on glia and neurons and large fiber tracts at later developmental stages. The distribution of the L2 epitope was quite similar to that of the J1 molecule but remained present on large fiber tracts, like N-CAM and L1, also at later developmental stages. These comparative observations in normal and reeler mutant mice lend support to previous suggestions that L1, together with N-CAM, may play a role in the aggregation of neuronal cell bodies after migration and in the fasciculation of developing fiber bundles. They also point to a possible function of the extracellular matrix component J1 in the guidance or support of fiber outgrowth in large fiber tracts.
Adhesive mechanisms have been implicated in several morphogenetic processes during development. In the last decade several molecules mediating specific adhesion between cells and between cells and their environment have been characterized. Possible roles in morphogenesis have been established for some of these molecules. In the brain, four cell-cell adhesion molecules have been characterized: NCAM, L1, the myelin-associated glycoprotein and N-cadherin. Furthermore, a cell-substrate adhesion molecule, cytotactin, and its proteoglycan ligand have been described. We here review the data on structure, localization, developmental regulation and function of these molecules in brain.
A monoclonal antibody raised against the capsular polysaccharides of meningococcus B was used for immunohistochemical studies in the rat brain, with particular focus on the substantia nigra. This antibody recognizes polysialic acid residues specifically associated with the neuronal-cell adhesion molecule, and reacts with the highly sialylated embryonic neuronal-cell adhesion molecule, but not with the weakly sialylated adult form of the molecule. Immunoreactivity to this monoclonal antibody was intense and widespread in the brain of 1-10-day-old hooded rats. Immunolabeling was associated with cell membranes and present in the intersomata space. In sections from 16- and 25-day-old rats, marked heterogeneity in the level of immunostaining appeared among individual brain nuclei. Areas devoid of labeling with the anti-meningococcus antibody still expressed immunoreactivity to a polyclonal anti-neuronal-cell adhesion molecule antibody. This suggests that the loss of immunostaining with the monoclonal antibody did not correspond to a loss of expression of neuronal-cell adhesion molecule, but to a maturation from the embryonic to the adult form of the molecule, occurring at different rates in various brain regions. In 2-month-old rats, immunolabeling with the monoclonal antibody was still present in discrete brain areas, including the substantia nigra, suggesting that the presence of highly sialylated neuronal-cell adhesion molecule outlasts post-natal development in those brain regions. It is proposed that neuronal-cell adhesion molecule associated polysialic residues may play a role in neuronal plasticity in restricted areas of the adult brain.
The expression of the neural adhesion molecules L1 and N-CAM has been studied in the embryonic and early postnatal olfactory system of the mouse in order to gain insight into the function of these molecules during development of a neural structure which retains neuronal turnover capacities throughout adulthood. N-CAM was slightly expressed and L1 was not significantly expressed in the olfactory placode on Embryonic Day 9, the earliest stage tested. Rather, N-CAM was strongly expressed in the mesenchyme underlying the olfactory placode. In the developing nasal pit, L1 and N-CAM were detectable in the developing olfactory epithelium, but not in regions developing into the respiratory epithelium. At early developmental stages, expression of the so-called embryonic form of N-CAM (E-N-CAM) coincides with the expression of N-CAM, whereas at later developmental stages and in the adult it is restricted to a smaller number of sensory cell bodies and axons, suggesting that the less adhesive embryonic form is characteristic of morphogenetically dynamic neuronal structures. Moreover, E-N-CAM is highly expressed at contact sites between olfactory axons and their target cells in the glomeruli of the olfactory bulb. L1 and N-CAM 180, the component of N-CAM that accumulates at cell contacts by interaction with the cytoskeleton are detectable as early as the first axons extend toward the primordial olfactory bulb. L1 remains prominent throughout development on axonal processes, both at contacts with other axons and with ensheathing cells. Contrary to N-CAM 180 which remains detectable on differentiating sensory neuronal cell bodies, L1 is only transiently expressed on these and is no longer detectable on primary olfactory neuronal cell bodies in the adult. Furthermore, whereas throughout development L1 has a molecular form similar to that seen in other parts of the developing and adult central nervous systems, N-CAM and, in particular, N-CAM 180 retain their highly sialylated form at least partially throughout all ages studied. These observations suggest that E-N-CAM and N-CAM 180 are characteristic of developmentally active structures and L1 may not only be involved in neurite outgrowth, but also in stabilization of contacts among fasciculating axons and between axons and ensheathing cells, as it has previously been found in the developing peripheral nervous system.
The neural cell adhesion molecule (NCAM) can influence a number of diverse intercellular events, including junctional communication,
the association of axons with pathways and targets, and signals that alter levels of neurotransmitter enzymes. These pleiotropic
effects appear to reflect the ability of NCAM to regulate membrane-membrane contact required to initiate specific interactions
between other molecules. Such regulation can occur through changes in either NCAM expression or the molecule's content of
polysialic acid (PSA). When NCAM with a low PSA content is expressed, adhesion is increased and contact-dependent events are
triggered. In contrast, the large excluded volume of NCAM PSA can inhibit cell-cell interactions through hindrance of overall
membrane apposition.
The expression of the neural cell adhesion molecules N-CAM and L1 was investigated in the olfactory system of the mouse using immunocytochemical and immunochemical techniques. In the olfactory epithelium, globose basal cells and olfactory neurons were stained by the polyclonal N-CAM antibody reacting with all three components of N-CAM (N-CAM total) in their adult and embryonic states. Dark basal cells and supporting cells were not found positive for N-CAM total. The embryonic form of N-CAM (E-N-CAM) was only observed on the majority of globose basal cells, the precursor cells of olfactory neurons, and some neuronal elements, probably immature neurons, since they were localized adjacent to the basal cell layer. Differentiated neurons in the olfactory epithelium did not express E-N-CAM. In contrast to N-CAM total, the 180-kDa component of N-CAM (N-CAM180) and E-N-CAM, L1 was not detectable on cell bodies in the olfactory epithelium. L1 and N-CAM180 were strongly expressed on axons leaving the olfactory epithelium. Olfactory axons were also labeled by antibodies to N-CAM180 and L1 in the lamina propria and the nerve fiber and glomerular layers of the olfactory bulb, but only some axons showed a positive immunoreaction for E-N-CAM. Ensheathing cells in the olfactory nerve were observed to bear some labeling for N-CAM total, L1, and N-CAM180, but not E-N-CAM. In the olfactory bulb, L1 was not present on glial cells. In contrast, N-CAM180 was detectable on some glia and N-CAM total on virtually all glia. Glia in the nerve fiber layer were labeled by E-N-CAM antibody only at the external glial limiting membrane. In the glomerular layer, E-N-CAM expression was particularly pronounced at contacts between olfactory axons and target cells. The presence of E-N-CAM in the adult olfactory epithelium and bulb was confirmed by Western blot analysis. The continued presence of E-N-CAM in adulthood on neuronal precursor cells, a subpopulation of olfactory axons, glial cells at the glia limitans, and contacts between olfactory axons and their target cells indicates the retention of embryonic features in the mammalian olfactory system, which may underlie its remarkable regenerative capacity.
The anatomical organization and development of the hippocampal mossy fiber system has been reviewed with special reference to its organization in the common laboratory rat. The mossy fibers originate from the granule cells of the dentate granular layer and the few granule cells found scattered in the dentate molecular layer and hilus. Via a complex system of collaterals the mossy fibers terminate on several types of neurons in the hilus, e.g. the basket cells and the mossy cells. Upon leaving the hilus to pass into Ammon's horn, the mossy fibers converge to form a distinct band of fibers that terminates on the proximal part of the apical and basal dendrites of the pyramidal and basket cells of the regio inferior. In some mammalian species the mossy fibers may continue into the adjacent part of the regio superior. Despite differences in the number of granule cells and pyramidal cells at different septotemporal levels this organization is relatively uniform along the septotemporal extent of the hippocampus. During development the mossy fibers grow out in a sequential manner that matches the pattern of neurogenesis and the aggregation of the cells of origin. From the level at which they originate, the fibers diverge along the septotemporal axis in such a way that the oldest granule cells have the most extensive projections. The adult topographic organization, which is already apparent at the earliest developmental stages, is thus formed in a stepwise fashion. It is concluded that the organization of the hippocampal mossy fibers indicates that neuronal specificity should not be explained by cellular recognition alone, but rather as the cumulated product of the preceding sequence of developmental events that include neurogenesis, migration, aggregation and directed axonal outgrowth.
Ten-day-old Sprague-Dawley rats were injected intraperitoneally with 3H-thymidine (3H-TdR) and allowed to survive until postnatal day (P) 40, P120, P200, P300 or P450. Following the preparation of the tissue for autoradiography, the location of the labeled neurons within the granule cell layer of the dentate gyrus was determined at two different septo-temporal levels. At P40 the labeled cells in the suprapyramidal blade of the dentate gyrus were found only in the deep part of the granule cell layer, where it borders on the hilus, and more than four cell diameters from the molecular layer. The latter relationship remained constant at each of the five ages studied, but between P40 and P120 the average distance from the labeled granule cells to the hilus almost doubled. At the middle of the temporal portion of the dentate gyrus this trend continues with age, so that by P450 the labeled neurons, though no nearer to the molecular layer than at earlier stages, were found, on average, in the middle of the granule cell layer, more-or-less halfway between the hilus and the molecular layer. Near the middle of the septal half of the dentate gyrus a similar pattern was seen at P40 and P120 but thereafter the labeled cells within the granule cell layer remained at about the same distance from the hilus.(ABSTRACT TRUNCATED AT 250 WORDS)
The fine structure of the dentate gyrus of the rat has been studied in glutaraldehyde perfused material. With the exception of the short axon association cells, the major components of each of the three layers of the gyrus have been identified, and a detailed account is given of the synaptic organization of the dentate granule cells.
The granule cells have most of the features typical of small neurons and although they contain no large aggregates of granular endoplasmic reticular they exhibit a variety of cisternal specializations. In the granule cell layer numerous axo-somatic and axo-dendritic synapses have been observed, and on occasion a single presynaptic fiber has been seen to contact the soma of one cell and a proximal dendrite of another. In the inner part of the molecular layer the majority of synapses are upon the main dendritic shafts of the granule cells but many also contact short dendritic spines. In the outer two-thirds of this layer most presynaptic fibers end upon long dendritic spines some of which are characterized by a prominent projection from the subsynaptic surface into the presynaptic process. The unmyelinated axons of the granule cells establish many en passant contacts with dendrites and spines of CA4 pyramids before ending in large presynaptic bags which may contain a number of large agranular vesicles (up to 2,000 Å in diameter) and vesicles with dense cores.
A brain cell surface protein (BSP-2) was isolated from mice of different ages by affinity chromatography using a monoclonal antibody. Analysis of glycopeptides obtained after pronase digestion revealed that the embryonal and neonatal forms of the antigen contained an unusually high proportion of sialic acid, which decreased during development. Methylation analysis of native and neuraminidase treated glycopeptides indicated that the sialic acid occurred as alpha 2-8 bound polysialosyl units, similar to those of the recently described developmentally regulated polysialosyl glycopeptides of rat brain. Furthermore, the carbohydrate and amino acid composition, and electrophoretic mobility of BSP-2 antigen correspond to those reported for a neural cell adhesion molecule (N-CAM).
Interactions between neural cell surfaces seem to be of prime importance during neuroontogenesis, and responsible for the guidance of migrating neuroblasts and growing axons and for the formation of synapses. Little is known about the underlying molecular mechanisms, but most hypotheses imply the existence of cell-surface molecules that mediate the formation of transient or permanent bonds between neural cells. Recently, a membrane glycoprotein called neural cell adhesion molecule (N-CAM) has been characterized in chick and rodent nervous tissue that appears to act as a ligand in adhesion among neural cell bodies or neurites. We have identified a mouse neural surface glycoprotein, named BSP-2 (ref. 7), which by criteriaof electrophoretic migration, developmental changes, amino acid and sugar composition seems to be closely related or identical to N-CAM. Both BSP-2 (refs 8, 9) and N-CAM undergo conversion from an embryonic to an adult form during brain development and it has been suggested that this transition changes the adhesive properties or the binding specificity of the molecule. Using a neuroblastoma line to study functional differences between embryonic and adult BSP-2/N-CAM molecules, we show here that liposomes bearing adult BSP-2 but not those bearing the embryonic form adhere to neuroblastoma cells, demonstrating that the two forms do indeed possess different binding properties.
Volumetric estimates of the total number of granule cells in rats 30, 120, 200, and 365 days old increase linearly by approximately 35 to 43 percent between 1 month and 1 year. Total volume of the granular layer also grows linearly during that time. These results demonstrate a numerical increase in a neuronal population during adulthood in the mammalian brain.
Expression of highly polysialylated NCAM in the neocortex and piriform cortex of the developing and adult rat Highly polysialylateo NCAM is expressed in newly generated granule cells of the adult dentate gyrus Cell adhesion molecules in early chicken embryogenesis
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Occurrence of a2-8 linked polysialosyl units in a neural cell adhesion molecule
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The organization and development of the hippocampal mossy fiber system
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NCAM gene expression during the development of cerebellum and dentate gyrus in the mouse
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