Tatsunori Seki

GABAergic Excitation Promotes Neuronal Differentiation in Adult Hippocampal Progenitor Cells

Hippocampal activity influences neurogenesis in the adult dentate gyrus; however, little is known about the involvement of the hippocampal circuitry in this process. In the subgranular zone of the adult dentate gyrus, neurogenesis involves a series of differentiation steps from radial glia-like stem/progenitor (type-1) cells, to transiently amplifying neuronal progenitor (type-2) cells, to postmitotic neurons. In this study, we conducted GFP-targeted recordings of progenitor cells in fresh hippocampal slices from nestin-GFP mice and found that neuronal progenitor (type-2) cells receive active direct neural inputs from the hippocampal circuitry. This input was GABAergic but not glutamatergic. The GABAergic inputs depolarized type-2 cells because of their elevated [Cl(-)](i). This excitation initiated an increase of [Ca(2+)](i) and the expression of NeuroD. A BrdU-pulse labeling study with GABA(A)-R agonists demonstrated the promotion of neuronal differentiation via this GABAergic excitation. Thus, it appears that GABAergic inputs to hippocampal progenitor cells promote activity-dependent neuronal differentiation.

Distribution and possible roles of the highly polysialylated neural cell adhesion molecule (NCAM-H) in the developing and adult central nervous system

The neural cell adhesion molecule (NCAM) is a cell surface glycoprotein which is thought to mediate cell adhesion and recognition. During developmental stages, NCAM is highly polysialylated (NCAM-H) by a unique alpha-2,8-linked polysialic acid chain (PSA), and this PSA portion of NCAM-H has been found to be closely associated with various developmental processes of the nervous system. Further, recent immunohistochemical investigations have revealed that even in the adult nervous system, a persistent PSA expression has been found confined to several regions: the olfactory bulb, the piriform cortex, the hippocampal dentate gyrus, the hypothalamus, some nuclei of the medulla and the dorsal horn of the spinal cord, which are related directly or indirectly to sensory systems. Moreover, in the dentate gyrus and olfactory bulb the expression is connected with adult neurogenesis that may add new neuronal circuits to the adult neural tissue. Therefore, the possible role of NCAM-H in the central nervous system may be associated not only with neural development, but also with adult functions, such as the processing system of sensory information and neuronal plasticity.

Expression of highly polysialylated NCAM in the neocortex and piriform cortex of the developing and the adult rat

SummaryThe 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.

Neuron‐Specific Phosphorylation of Alzheimer's β‐Amyloid Precursor Protein by Cyclin‐Dependent Kinase 5

Abstract: The mature form of Alzheimers β‐amyloid precursor protein (APP) is phosphorylated specifically at Thr668 in neurons. In mature neurons, phosphorylated APP is detected in neurites, with dephosphorylated APP being found mostly in the cell body. In vitro, active cyclin‐dependent kinase 5 (Cdk5) phosphorylated the cytoplasmic domain of APP at Thr668. Treatment of mature neurons with an antisense oligonucleotide to Cdk5 suppressed Cdk5 expression and significantly diminished the level of phosphorylated APP. The expression of APP was unaffected in antisense‐treated neurons. These results indicate that in neurons APP is phosphorylated by Cdk5, and that this may play a role in its localization.

Expression patterns of immature neuronal markers PSA-NCAM, CRMP-4 and NeuroD in the hippocampus of young adult and aged rodents.

Neurogenesis is known to continue in the adult hippocampus of mammals, including humans. The present experiments were undertaken to examine the nature of developing neurons generated in the dentate gyrus of young and older rodents using immature neuronal markers such as highly polysialylated neural cell adhesion molecules (PSA‐NCAM), collapsin response‐mediated protein‐4 (CRMP‐4) and NeuroD. Most PSA‐expressing cells are simultaneously positive for CRMP‐4 and NeuroD in young rats. More than half of the PSA‐positive cells were also positive for mature neuronal markers such as NeuN and MAP2, although the intensity of the immunoreactivities was relatively weak. BrdU analysis revealed that CRMP‐4 is expressed for a longer period than PSA in BrdU‐labeled neurons. The number of immature neurons expressing PSA, NeuroD or CRMP‐4 decreased in older rodents, but no qualitative difference was found in the expression patterns of these molecular markers between young and older rodents. These results suggest not only that immunohistochemistry, using a combination of these immature and mature neuronal markers, is helpful for clarifying the developmental state of newly generated neurons, but also that newly generated neurons in young adult and older rodents have similar properties.

The ontogeny of luteinizing hormone-releasing hormone (LHRH) producing neurons in the chick embryo : Possible evidence for migrating LHRH neurons from the olfactory epithelium expressing a highly polysialylated neural cell adhesion molecule

The development of neurons expressing luteinizing hormone-releasing hormone (LHRH) has been studied immunohistochemically in the chick embryo from the 3.5 embryonic day (ED) to the day of hatching. At ED-3.5, LHRH-immunoreactive neurons were first detected in the medial epithelium of the olfactory pit, but their appearance in the brain was delayed to ED-4.5. On EDs-6-7, cords of the LHRH-immunoreactive cells extended across the nasal septum towards the ventromedial forebrain with the olfactory nerve. By double staining for LHRH and, a highly polysialylated form of neural cell adhesion molecule (NCAM-H), the LHRH-positive neurons in the olfactory-forebrain system were found strongly NCAM-H-positive. At ED-8, a marked decrease in the number of LHRH-positive cells in the olfactory epithelium and a concomitant increase in the LHRH-positive cells in the forebrain area were noted. From ED-11 to the day of hatching, the majority of LHRH-positive neurons tended to move into their usual adult position, whereas the LHRH-positive cells had almost disappeared in the olfactory epithelium. No LHRH-immunoreactive neurons were found strongly positive to NCAM-H. These results suggest that LHRH neurons originate from the olfactory placode, then as they develop they migrate across the nasal septum and enter the forebrain with the olfactory nerve. The close association of NCAM-H with the developing LHRH neurons raises the possibility that NCAM-H plays some role in guiding the migrating LHRH neurons from the olfactory epithelium to the forebrain.

Roles of disrupted-in-schizophrenia 1-interacting protein girdin in postnatal development of the dentate gyrus.

Disrupted-In-Schizophrenia 1 (DISC1), a susceptibility gene for major psychiatric disorders, regulates neuronal migration and differentiation during mammalian brain development. Although roles for DISC1 in postnatal neurogenesis in the dentate gyrus (DG) have recently emerged, it is not known how DISC1 and its interacting proteins govern the migration, positioning, and differentiation of dentate granule cells (DGCs). Here, we report that DISC1 interacts with the actin-binding protein girdin to regulate axonal development. DGCs in girdin-deficient neonatal mice exhibit deficits in axonal sprouting in the cornu ammonis 3 region of the hippocampus. Girdin deficiency, RNA interference-mediated knockdown, and inhibition of the DISC1/girdin interaction lead to overextended migration and mispositioning of the DGCs resulting in profound cytoarchitectural disorganization of the DG. These findings identify girdin as an intrinsic factor in postnatal development of the DG and provide insights into the critical role of the DISC1/girdin interaction in postnatal neurogenesis in the DG.

Hippocampal adult neurogenesis occurs in a microenvironment provided by PSA-NCAM-expressing immature neurons.

Neurons continue to be generated in the adult hippocampus. In the present study, the early developmental processes of newly generated neurons in the adult rat hippocampus were examined by confocal laser scanning microscopy using a combination of bromodeoxyuridine (BrdU) labeling and immunohistochemistry for highly polysialylated neural cell adhesion molecule (PSA‐NCAM) and NeuroD, which are markers for immature neurons, and glial fibrillary acidic protein (GFAP). Rats were injected with BrdU and 2 hours, 1, 3, and 7 days after the injection, the hippocampus was processed for immunohistochemistry. One day after the injection, BrdU‐labeled cells were found frequently in clusters consisting of dividing cells, putative undifferentiated cells, NeuroD‐positive differentiated neurons, and GFAP‐positive astrocytes. Three days later, BrdU‐labeled cells were loosely aggregated and BrdU‐positive fragmented nuclei were sometimes observed, suggesting that apoptosis occurred in the clusters. These BrdU‐labeled nuclei were frequently associated in various ways with the processes of immature PSA‐positive granule cells. They are positioned along PSA‐positive apical and basal dendrites or surrounded by these processes. By 7 days after the injection, the number of the clusters was reduced and the BrdU‐labeled cells had developed dendrites. These cell‐to‐cell associations support the hypothesis that the clustering and a microenvironment provided by the PSA‐expressing immature neurons contribute to the early developmental events of adult neurogenesis, such as proliferation, differentiation, apoptosis, and neurophilic migration in the adult hippocampus.

Temporal and spacial relationships between PSA-NCAM-expressing, newly generated granule cells, and radial glia-like cells in the adult dentate gyrus

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.

Possibility for neurogenesis in substantia nigra of parkinsonian brain.

Recent studies of enhanced hippocampal neurogenesis by antidepressants suggest enhancement of neurogenesis is a potentially effective therapy in neurodegenerative diseases. In this study, we evaluated nigral neurogenesis in animals and autopsy brains including patients with Parkinsons disease (PD). First, proliferating cells in substantia nigra were labeled with retroviral transduction of green fluorescent protein, which is an efficient method to label neuronal stem cells. Subsequent differentiation of labeled cells was followed; many transduced cells became microglia, but no differentiation into tyrosine hydroxylase–positive neurons was detected at 4 weeks after injection, in both intact rodents and those treated with 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine. Second, polysialic acid (PSA)–like immunoreactivity, indicative of newly differentiated neurons, was detected in the substantia nigra of rodent, primate, and human midbrains. A large number of PSA‐positive cells were detected in the substantia nigra pars reticulata of some patients with PD. In rats and a macaque monkey, the dopamine‐depleted hemispheres showed more PSA staining than the intact side. A small number of tyrosine hydroxylase–positive cells were PSA‐positive. Our results suggest enhanced neural reconstruction in PD, which may be important in the design of new therapies against the progression of PD. Ann Neurol 2005