PSA-NCAM in postnatally generated immature neurons of the olfactory bulb: a crucial role in regulating p75 expression and cell survival

In the mammalian brain, ongoing neurogenesis via the rostral migratory stream (RMS) maintains neuronal replacement in the olfactory bulb throughout life. Mechanisms that regulate the final number of new neurons in this system include proliferation, migration and apoptosis. Here we show that the polysialylated isoforms of the neural cell adhesion molecule (PSA-NCAM) act as a pro-survival molecule in immature newborn neurons. Confocal microscopic analysis revealed a threefold increase in TUNEL-positive cells in the subventricular zone (SVZ) and the RMS of transgenic animals lacking the gene encoding NCAM (NCAM(-/-)), as compared with wild types. The enhanced apoptotic cell death occurred specifically in the population of mCD24-positive newborn neurons, but not in GFAP-positive astrocytes. Using in vitro cultures of purified SVZ-derived neurons, we demonstrate that the loss or inactivation of PSA on NCAM, as well as the deletion of NCAM, lead to reduced survival in response to neurotrophins including BDNF and NGF. These changes in cell survival are accompanied by an upregulation of p75 neurotrophin receptor expression in vitro as well as in vivo. Furthermore, the negative effects of PSA-NCAM inactivation on cell survival could be prevented by the pharmacological blockade of the p75 receptor-signaling pathway. We propose that PSA-NCAM may promote survival by controlling the expression of the p75 receptor in developing neurons.     

Polysialic acid directs tumor cell growth by controlling heterophilic neural cell adhesion molecule interactions

Polysialic acid (PSA), a carbohydrate polymer attached to the neural cell adhesion molecule (NCAM), promotes neural plasticity and tumor malignancy, but its mode of action is controversial. Here we establish that PSA controls tumor cell growth and differentiation by interfering with NCAM signaling at cell-cell contacts. Interactions between cells with different PSA and NCAM expression profiles were initiated by enzymatic removal of PSA and by ectopic expression of NCAM or PSA-NCAM. Removal of PSA from the cell surface led to reduced proliferation and activated extracellular signal-regulated kinase (ERK), inducing enhanced survival and neuronal differentiation of neuroblastoma cells. Blocking with an NCAM-specific peptide prevented these effects. Combinatorial transinteraction studies with cells and membranes with different PSA and NCAM phenotypes revealed that heterophilic NCAM binding mimics the cellular responses to PSA removal. In conclusion, our data demonstrate that PSA masks heterophilic NCAM signals, having a direct impact on tumor cell growth. This provides a mechanism for how PSA may promote the genesis and progression of highly aggressive PSA-NCAM-positive tumors.     

多聚唾液酸(PSA)及其修饰的神经粘附分子(PSA-NCAM)对肿瘤及细胞信号通路的影响

神经粘附分子(Neural cell adhesion molecule, NCAM)是免疫球蛋白家族中的一员,在细胞粘附和细胞通信,尤其是神经系统的生长和塑型中起重要作用。而多聚唾液酸(Polysialic acid, PSA)则是控制NCAM粘附能力形成与神经系统分化的重要因素。研究发现,多种肿瘤细胞中存在PSA以及多聚唾液酸化的神经粘附分子(PSA-NCAM)再表达的现象,预示PSA及PSA-NCAM与多种肿瘤细胞的粘附性、迁移性和侵袭性等特性密切相关,影响肿瘤细胞的生长与转移,并通过介导多种细胞信号通路影响癌症的发生与发展。文章综述了NCAM以及PSA对癌症的发生与发展、预后的作用及其功能对细胞下游信号传导的影响。     

Targeting epileptogenesis-associated induction of neurogenesis by enzymatic depolysialylation of NCAM counteracts spatial learning dysfunction but fails to impact epilepsy development

Polysialylation is a post-translational modification of the neural cell adhesion molecule (NCAM), which in the adult brain promotes structural changes in regions of neurogenesis and neuroplasticity. Because a variety of plastic changes including neurogenesis have been suggested to be functionally involved in the pathophysiology of epilepsies, it is of specific interest to define the impact of the polysialic acid (PSA)-NCAM system on development of this disease and associated comorbidities. Therefore, we studied the impact of transient enzymatic depolysialylation of NCAM on the pathophysiology in an electrically induced rat post-status epilepticus (SE) model. Loss of PSA counteracted the SE-induced increase in neurogenesis in a significant manner. This effect of endoneuraminidase (endoN) treatment on hippocampal neurogenesis did not impact the subsequent development of spontaneous seizures. In contrast, transient lack of PSA during SE and in the early phase of epileptogenesis exhibited a cognition sparing effect as revealed in the Morris water maze paradigm. In conclusion, our data do not support a central role of neurogenesis in the development of a hyperexcitable epileptic network. However, in view of the cognition-sparing effect, the transient modulation of the PSA-NCAM system seems to allow beneficial long-term disease modification, which might be mediated by the partial normalization of neurogenesis.     

Distinctive PSA-NCAM and NCAM Hallmarks in Glutamate-Induced Dendritic Atrophy and Synaptic Disassembly

Dendritic and synapse remodeling are forms of structural plasticity that play a critical role in normal hippocampal function. Neural cell adhesion molecule (NCAM) and its polysialylated form (PSA-NCAM) participate in neurite outgrowth and synapse formation and plasticity. However, it remains unclear whether they contribute to dendritic retraction and synaptic disassembly. Cultured hippocampal neurons exposed to glutamate (5 µM) showed a reduced MAP-2 (+) area in the absence of neuronal death 24 h after the insult. Concomitantly, synapse loss, revealed by decreased synaptophysin and post-synaptic density-95 cluster number and area, together with changes in NCAM and PSA-NCAM levels were found. Dendritic atrophy and PSA-NCAM reduction proved NMDA-receptor dependent. Live-imaging experiments evidenced dendritic atrophy 4 h after the insult; this effect was preceded by smaller NCAM clusters (1 h) and decreased surface and total PSA-NCAM levels (3 h). Simultaneously, total NCAM cluster number and area remained unchanged. The subsequent synapse disassembly (6 h) was accompanied by reductions in total NCAM cluster number and area. A PSA mimetic peptide prevented both the dendritic atrophy and the subsequent synaptic changes (6 h) but had no effect on the earliest synaptic remodeling (3 h). Thus, NCAM-synaptic reorganization and PSA-NCAM level decrease precede glutamate-induced dendritic atrophy, whereas the NCAM level reduction is a delayed event related to synapse loss. Consequently, distinctive stages in PSA-NCAM/NCAM balance seem to accompany glutamate-induced dendritic atrophy and synapse loss.     

Polysialic acid regulates the clustering, migration, and neuronal differentiation of progenitor cells in the adult hippocampus

Newborn cells of the adult dentate gyrus in the hippocampus are characterized by their abundant expression of polysialic acid (PSA), a carbohydrate attached to the neural cell adhesion molecule (NCAM). PSA+ newborn cells of the dentate gyrus form clusters with proliferating neural progenitor cells, migrate away from these clusters, and terminally differentiate. To identify the roles of PSA in the development of adult progenitors of the dentate gyrus, we injected endoneuraminidase N (endoN) into the hippocampus of adult rats to specifically cleave PSA from NCAM. Two days later, we administered the mitotic marker, 5-bromo-2'-deoxyuridine (BrdU). Three days after BrdU injection, BrdU+ cells were found inside and outside the clusters of newborn cells. In endoN-treated animals, the total number of BrdU+ cells was not changed but significantly more BrdU+ cells were present within clusters, suggesting that PSA normally facilitates the migration of progenitors away from the clusters. Seven days post-BrdU injection, endoN-treated animals had significantly more BrdU+ cells which were also positive for the mature neuronal nuclear marker NeuN compared with controls, indicating that the loss of PSA from progenitor cells increases neuronal differentiation. This report is the first demonstration that PSA is involved in controlling the spatio-temporal neuronal maturation of adult hippocampal progenitors in the normal brain. In vitro, the removal of PSA from adult-derived neural progenitors significantly enhanced neuronal differentiation, strengthening our in vivo findings and indicating that PSA removal on isolated progenitor cells, apart from a complex in vivo environment, induces neuronal maturation.     

Polysialic acid is required for dopamine D2 receptor-mediated plasticity involving inhibitory circuits of the rat medial prefrontal cortex

Decreased expression of dopamine D2 receptors (D2R), dysfunction of inhibitory neurotransmission and impairments in the structure and connectivity of neurons in the medial prefrontal cortex (mPFC) are involved in the pathogenesis of schizophrenia and major depression, but the relationship between these changes remains unclear. The polysialylated form of the neural cell adhesion molecule (PSA-NCAM), a plasticity-related molecule, may serve as a link. This molecule is expressed in cortical interneurons and dopamine, via D2R, modulates its expression in parallel to that of proteins related to synapses and inhibitory neurotransmission, suggesting that D2R-targeted antipsychotics/antidepressants may act by affecting the plasticity of mPFC inhibitory circuits. To understand the role of PSA-NCAM in this plasticity, rats were chronically treated with a D2R agonist (PPHT) after cortical PSA depletion. PPHT-induced increases in GAD67 and synaptophysin (SYN) neuropil expression were blocked when PSA was previously removed, indicating a role for PSA-NCAM in this plasticity. The number of PSA-NCAM expressing interneuron somata also increased after PPHT treatment, but the percentages of these cells belonging to different interneuronal subpopulations did not change. Cortical pyramidal neurons did not express PSA-NCAM, but puncta co-expressing this molecule and parvalbumin could be found surrounding their somata. PPHT treatment increased the number of PSA-NCAM and parvalbumin expressing perisomatic puncta, but decreased the percentage of parvalbumin puncta that co-expressed SYN. PSA depletion did not block these effects on the perisomatic region, but increased further the number of parvalbumin expressing puncta and increased the percentage of puncta co-expressing SYN and parvalbumin, suggesting that the polysialylation of NCAM may regulate perisomatic inhibition of mPFC principal neurons. Summarizing, the present results indicate that dopamine acting on D2R influences structural plasticity of mPFC interneurons and point to PSA-NCAM as a key player in this remodeling.     

Activity-dependent mobilization of the adhesion molecule polysialic NCAM to the cell surface of neurons and endocrine cells.

The alpha-2,8-linked sialic acid polymer (PSA) on the neural cell adhesion molecule (NCAM) is an important regulator of cell surface interactions. We have examined the translocation of PSA-NCAM to the surface of cultured cortical neurons and insulin secreting beta cells under different conditions of cell activity. Endoneuraminidase N, an enzyme that specifically cleaves PSA chains, was used to remove pre-existing PSA from the plasma membrane and the re-expression of the molecule was monitored by immunocytochemistry. Punctate PSA immunostaining was restored on the surface of 68% of neurons within 1 h. This recovery was almost completely prevented by tetrodotoxin, suggesting that spontaneous electrical activity is required. K+ depolarization (50 mM) allowed recovery of PSA surface staining in the presence of tetrodotoxin and this effect required the presence of extracellular Ca2+. Rapid redistribution of PSA-NCAM to the surface of beta cells was observed under conditions that stimulate insulin secretion. Ca2+ channel inhibition decreased both PSA-NCAM expression and insulin secretion to control, non-stimulated levels. Finally, subcellular fractionation of an insulin-secreting cell line showed that the secretory vesicle fraction is highly enriched in PSA-NCAM. These results suggest that PSA-NCAM can be translocated to the cell surface via regulated exocytosis. Taken together, our results provide unprecedented evidence linking cell activity and PSA-NCAM expression, and suggest a mechanism for rapid modulation of cell surface interactions.     

Polysialic acid controls NCAM-induced differentiation of neuronal precursors into calretinin-positive olfactory bulb interneurons

Understanding the mechanisms that regulate neurogenesis is a prerequisite for brain repair approaches based on neuronal precursor cells. One important regulator of postnatal neurogenesis is polysialic acid (polySia), a post-translational modification of the neural cell adhesion molecule NCAM. In the present study, we investigated the role of polySia in differentiation of neuronal precursors isolated from the subventricular zone of early postnatal mice. Removal of polySia promoted neurite induction and selectively enhanced maturation into a calretinin-positive phenotype. Expression of calbindin and Pax6, indicative for other lineages of olfactory bulb interneurons, were not affected. A decrease in the number of TUNEL-positive cells indicated that cell survival was slightly improved by removing polySia. Time lapse imaging revealed the absence of chain migration and low cell motility, in the presence and absence of polySia. The changes in survival and differentiation, therefore, could be dissected from the well-known function of polySia as a promoter of precursor migration. The differentiation response was mimicked by exposure of cells to soluble or substrate-bound NCAM and prevented by the C3d-peptide, a synthetic ligand blocking NCAM interactions. Moreover, a higher degree of differentiation was observed in cultures from polysialyltransferase-depleted mice and after NCAM exposure of precursors from NCAM-knockout mice demonstrating that the NCAM function is mediated via heterophilic binding partners. In conclusion, these data reveal that polySia controls instructive NCAM signals, which direct the differentiation of subventricular zone-derived precursors towards the calretinin-positive phenotype of olfactory bulb interneurons.     

Dietary Restriction Enhances Kainate-Induced Increase in NCAM While Blocking the Glial Activation in Adult Rat Brain

In the present study effect of dietary restriction (DR) on neuronal plasticity markers neural cell adhesion molecule (NCAM) and its polysialylated form PSA-NCAM and astrocytic marker glial fibrillary acidic protein (GFAP) was assessed following brain injury by intraperitoneal injection of kainic acid or physiological saline in adult male wistar rats. After 7-day recovery period, rats were sacrificed to study the NCAM-ir, PSA-NCAM-ir, and GFAP-ir in all the groups with immunohistofluorescence and immunoblotting. We noticed increase in NCAM and PSA-NCAM expression after KA excitotoxicity, and DR enhanced this increase in NCAM and PSA-NCAM expression. A marked increase in NCAM and PSA-NCAM-ir was observed in CA3 region of hippocampus, subgranular region and hilus of dentate gyrus, hypothalamus, and piriform cortex in both vehicle treated as well KA-treated DR rats as compared to vehicle and KA-treated AL rats, respectively. Whenever, CNS is damaged it undergoes an injury response called reactive gliosis. Our study confirmed the neuroprotective role of DR as evident from attenuation of GFAP-ir and enhanced levels of neuronal plasticity markers NCAM and PSA-NCAM. The potential beneficial role of DR regimen in attenuating KA-induced reactive astrogliosis and enhancing expression of neuronal plasticity markers may point the way to new strategies of intervention therapy by DR that will facilitate recovery from ageing and disease related neuronal dysfunction and enhance restorative processes by modulating astrogliosis.     

Relationships between neuronal cell adhesion molecule and LHRH neurons in the urodele brain: a developmental immunohistochemical study.

Polysialic acid (PSA), a homopolymer attached to neural cell adhesion molecule (NCAM) is considered a major hallmark of vertebrate cell migration. We studied the distribution of PSA-NCAM by immunohistochemistry, during brain development, in two urodele amphibians, Pleurodeles waltl and the neotenic newt Ambystoma mexicanum. In both species a gradual increase of immunolabelling was observed throughout the brain from developmental stage 30 to stage 52. At the onset of metamorphosis, some differences became evident: in Pleurodeles immunostaining was gradually restricted to the olfactory system while in Ambystoma, PSA-NCAM maintained a more extended distribution (for example throughout the telencephalic walls) suggesting, for the brain of this latter species, a rather preserved neuronal plasticity. The aim of the present work was to correlate the above described PSA-NCAM-immunoreactivity (IR) with the distribution of luteinizing hormone-releasing hormone (LH-RH) containing neurons, which represent a well known example of neural elements migrating from the olfactory placode. LHRH-IR, undetectable till stage 30, was later found together with PSA-NCAM-IR in both the olfactory system and septo-hypothalamic areas. Such observations further support a role of PSA in providing a migration route toward the establishment of a part, at least, of the urodele LHRH system. The possible functional meaning of the LHRH-containing neurons localized between dorsal and ventral thalamus of Ambystoma, never reported before in this area, almost devoid of PSA-NCAM-IR, is discussed.     

Real time imaging of human progenitor neurogenesis

Human neural progenitors are increasingly being employed in drug screens and emerging cell therapies targeted towards neurological disorders where neurogenesis is thought to play a key role including developmental disorders, Alzheimer's disease, and depression. Key to the success of these applications is understanding the mechanisms by which neurons arise. Our understanding of development can provide some guidance but since little is known about the specifics of human neural development and the requirement that cultures be expanded in vitro prior to use, it is unclear whether neural progenitors obey the same developmental mechanisms that exist in vivo. In previous studies we have shown that progenitors derived from fetal cortex can be cultured for many weeks in vitro as undifferentiated neurospheres and then induced to undergo neurogenesis by removing mitogens and exposing them to supportive substrates. Here we use live time lapse imaging and immunocytochemical analysis to show that neural progenitors use developmental mechanisms to generate neurons. Cells with morphologies and marker profiles consistent with radial glia and recently described outer radial glia divide asymmetrically and symmetrically to generate multipolar intermediate progenitors, a portion of which express ASCL1. These multipolar intermediate progenitors subsequently divide symmetrically to produce CTIP2(+) neurons. This 3-cell neurogenic scheme echoes observations in rodents in vivo and in human fetal slice cultures in vitro, providing evidence that hNPCs represent a renewable and robust in vitro assay system to explore mechanisms of human neurogenesis without the continual need for fresh primary human fetal tissue. Knowledge provided by this and future explorations of human neural progenitor neurogenesis will help maximize the safety and efficacy of new stem cell therapies by providing an understanding of how to generate physiologically-relevant cell types that maintain their identities when placed in diagnostic or transplantation environments.     

PSA-NCAM: Synaptic functions mediated by its interactions with proteoglycans and glutamate receptors

Dynamic regulation of glycosylation of the neural cell adhesion molecule (NCAM) by an unusual large negatively charged polysialic acid (PSA) is the major prerequisite for correct formation of brain circuitries during development and for normal synaptic plasticity, learning and memory in the adult. Traditionally, PSA is viewed as a de-adhesive highly hydrated molecule, which interferes with cell adhesion and promotes cellular/synaptic dynamics by steric hindrance. Analysis of synaptic functions of PSA-NCAM highlighted additional features of this molecule. First, PSA promotes interaction of NCAM with heparan sulfate proteoglycans and thus stimulates synaptogenesis. Second, PSA-NCAM modulates glutamate receptors: it restrains activity of extrasynaptic GluN2B-containing NMDA receptors and facilitates activity of a subset of AMPA receptors. Perturbation in polysialylation and/or NCAM expression in mouse models recapitulates many symptoms of human brain disorders such as schizophrenia, depression, anxiety and Alzheimer's disease.     

Alteration of neural tissue structure by expression of polysialic acid induced by viral delivery of PST polysialyltransferase

The expression of polysialic acid (PSA) on neural cell adhesion molecule (NCAM) is known to attenuate cell-cell interactions. During neural development the widespread expression of PSA-NCAM creates permissive conditions for the migration of neuronal and glial precursors and the guidance and targeting of axons. NCAM polysialylation can occur via either of two specific sialyltransferases, ST8SiaII (STX) and ST8SiaIV (PST), and the purpose of this study was to determine if retroviral delivery of either PST or STX could induce PSA expression in vivo and thereby alter tissue plasticity. Retroviruses expressing GFP-PST or GFP-STX were injected into embryonic retina, and development was evaluated by examining neuroepithelial structure, the expression of markers for specific cell types, cellular proliferation, and apoptosis. Chick retina was chosen because it down-regulates PSA early in its development and has a highly stereotyped program of morphogenesis. Retroviral expression of PST induced PSA expression in retina and resulted in severe but localized alterations in retinal morphogenesis, including an early disruption of radial glial cell morphology, highly disorganized retinal layers, and invasion of pigmented cells into the neural retina. In contrast, retroviral delivery of STX did not induce PSA expression or affect morphogenesis. These findings demonstrate that expression of PSA is sufficient to promote morphological alterations in a relatively nonplastic neural tissue.     

Adult neurogenesis and modulation of neural circuit function

A growing body of evidence indicates that adult neurogenesis is involved in the modulation of certain types of hippocampus-dependent memory. Recent studies suggest that newly born neurons play a key role in pattern separation mediated by the dentate gyrus, in systems consolidation, through which memory becomes progressively independent of the hippocampus, and in social memory-based reproductive behavior. Furthermore, neural activity and learning are now thought to regulate the proliferation of neuronal precursors as well as the survival and apoptosis of new neurons. Moreover, these processes also affect the development of the dendritic arbor and dendritic spines of new neurons, thereby modulating the integration of adult-born neurons into the functional neural circuit.     

Polysialylation increases lateral diffusion of neural cell adhesion molecule in the cell membrane

Polysialic acid (PSA) is a polymer of N-acetylneuraminic acid residues added post-translationally to the membrane-bound neural cell adhesion molecule (NCAM). The large excluded volume created by PSA polymer is thought to facilitate cell migration by decreasing cell adhesion. Here we used live cell imaging (spot fluorescence recovery after photobleaching and fluorescence correlation spectroscopy) combined with biochemical approaches in an attempt to uncover a link between cell motility and the impact of polysialylation on NCAM dynamics. We show that PSA regulates specifically NCAM lateral diffusion and this is dependent on the integrity of the cytoskeleton. However, whereas the glial-derivative neurotrophic factor chemotactic effect is dependent on PSA, the molecular dynamics of PSA-NCAM is not directly affected by glial-derivative neurotrophic factor. These findings reveal a new intrinsic mechanism by which polysialylation regulates NCAM dynamics and thereby a biological function like cell migration.     

Development of neural stem cell in the adult brain

New neurons are continuously generated in the dentate gyrus of the mammalian hippocampus and in the subventricular zone of the lateral ventricles throughout life. The origin of these new neurons is believed to be from multipotent adult neural stem cells. Aided by new methodologies, significant progress has been made in the characterization of neural stem cells and their development in the adult brain. Recent studies have also begun to reveal essential extrinsic and intrinsic molecular mechanisms that govern sequential steps of adult neurogenesis in the hippocampus and subventricular zone/olfactory bulb, from proliferation and fate specification of neural progenitors to maturation, navigation, and synaptic integration of the neuronal progeny. Future identification of molecular mechanisms and physiological functions of adult neurogenesis will provide further insight into the plasticity and regenerative capacity of the mature central nervous system.