Masanori Yoneyama

Regulation of neuronal differentiation by N-methyl-D-aspartate receptors expressed in neural progenitor cells isolated from adult mouse hippocampus.

In vitro culture of neural progenitor cells isolated from adult murine hippocampus according to the Percoll density gradient method resulted in formation of round spheres not immunoreactive to microtubule‐associated protein‐2 (MAP‐2) or glial fibrillary acidic protein in the presence of basic fibroblast growth factor within 12 days in vitro (DIV). Reverse‐transcription PCR analysis revealed constitutive expression in these neurospheres of different subunits required for assembly of functional heteromeric N‐methyl‐D‐aspartate (NMDA) receptor channels. Immunocytochemical analysis confirmed expression of NR1, NR2A, and NR2B subunits in neurospheres cultured for 4–12 DIV. Brief (5 min) exposure to NMDA induced marked expression of c‐Fos, Fos‐B, Fra‐2, and c‐Jun proteins in neurospheres cultured for 12 DIV 2 hr later. The NMDA receptor antagonist dizocilpine markedly inhibited expression of both c‐Jun and c‐Fos proteins in NMDA‐exposed neurospheres. Sustained exposure to NMDA not only markedly inhibited neurosphere formation by 12 DIV when exposed from 4–12 DIV, but also resulted in facilitation of subsequent differentiation of neurospheres exposed to all‐trans retinoic acid to cells immunoreactive to both neuron‐specific enolase and neuronal nuclei, in addition to MAP‐2, as revealed by Western blot and immunocytochemistry analyses. These results suggest that functional heteromeric NMDA receptors may be expressed constitutively in neural progenitor cells before differentiation to play a crucial role in commitment and differentiation to neurons in adult murine hippocampus.

Chronic restraint stress impairs neurogenesis and hippocampus‐dependent fear memory in mice: possible involvement of a brain‐specific transcription factor Npas4

J. Neurochem. (2010) 114, 1840–1851.

Regeneration of granule neurons after lesioning of hippocampal dentate gyrus: Evaluation using adult mice treated with trimethyltin chloride as a model

The hippocampal dentate gyrus in adult animals is known to contain neural progenitors that proliferate and differentiate into neurons in response to brain injury. Little has been observed, however, on regeneration of the granule cell layer of the dentate gyrus that has been directly injured. Using trimethyltin (TMT)‐treated mice as an in vivo model, we evaluated the ability of this layer to regenerate after injury. The administration of TMT induced neuronal death in the dentate gyrus selectively 2 days later, with recovery of granule neurons on day 14 and thereafter. At an early stage (days 2–5) after the damage by TMT treatment, 5‐bromo‐2′‐deoxyuridine (BrdU) incorporation into at least two different types of cells was facilitated in the dentate gyrus: BrdU‐positive/neuronal nuclear antigen (NeuN)‐negative cells were found predominantly in the subgranular zone and granule cell layer, whereas BrdU‐positive/NeuN‐positive cells were numerous in the dentate molecular layer and hilus. In addition, expression of proliferating cell nuclear antigen, nestin, NeuroD3, and doublecortin, which are markers for proliferating cells and neural progenitors/neuronal precursors, was extremely enhanced in the dentate gyrus at the early stage after treatment. Double staining revealed that BrdU was colocalized with nestin and doublecortin in the subgranular zone. Behavioral analysis revealed that TMT‐induced cognition impairment was ameliorated by day 14 after the treatment. Taken together, our data indicate that the hippocampal dentate gyrus itself is capable of regenerating the neuronal cell layer through rapid enhancement of neurogenesis after injury.

Endogenous reactive oxygen species are essential for proliferation of neural stem/progenitor cells.

It is widely thought that accumulation of reactive oxygen species (ROS) causes injury to cells. In this study, we investigated the effect of endogenous ROS on the proliferation of neural stem/progenitor cells derived from the hippocampus of embryonic mice. The cells were treated with free radical-scavenging agents [3-methyl-1-phenyl-2-pyrazolin-5-one (edaravone) or 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (tempol)], an NADPH oxidase inhibitor (apocynin), catalase, a nitric oxide synthase inhibitor [N(omega)-nitro-L-arginine methyl ester hydrochloride (L-NAME)] or a peroxynitrite generator (SIN-1) during the culture period. Edaravone and tempol had the ability to decrease endogenous ROS in the cells exposed for periods from 1 to 24h, with attenuation of the proliferation activity of the cells during culture. Apocynin and L-NAME were also effective in attenuating cell proliferation but not cellular damage. Conversely, SIN-1 was capable of promoting the proliferation activity. However, catalase had no effect on the proliferation activity of the cells during culture. Furthermore, tempol significantly decreased the level of NFkappaB p65, phospho-cyclic AMP response element-binding protein, and beta-catenin within the nucleus of the cells. These data suggest that endogenous ROS and nitric oxide are essential for the proliferation of embryonic neural stem/progenitor cells.

Possible regulation by N-methyl-d-aspartate receptors of proliferative progenitor cells expressed in adult mouse hippocampal dentate gyrus.

An immunohistochemical technique was employed to analyze mechanisms underlying modulation by N‐methyl‐d‐aspartate (NMDA) receptors of proliferation of neural progenitor cells in adult mouse brain. The systemic administration of NMDA at 100 mg/kg resulted in marked expression of c‐Fos, Fra‐2 and c‐Jun proteins in the granule cell layers of the dentate gyrus in murine hippocampus 2 h later, followed by a significant reduction of the incorporation of 5‐bromo‐2′‐deoxyuridine (BrdU) in a manner sensitive to the antagonist dizocilpine 2 days after administration. The administration of NMDA also suppressed constitutive expression of both nestin and proliferating cell nuclear antigen (PCNA) in the dentate granule cells 2 days later, without markedly affecting cell viability for up to 8 weeks after administration. In the subventricular zone and olfactory bulb, however, NMDA failed to affect either the incorporation of BrdU or the expression of nestin and PCNA. The NR1 subunit was highly expressed in the dentate gyrus in addition to the stratum oriens in the hippocampus, but not in the subventricular zone and olfactory bulb. These results suggest that NMDA receptors may play a role crucial for maintenance of the integrity and function of proliferative neural progenitor cells through expression of the nuclear transcription factor activator protein‐1 in granule cells of the dentate gyrus in adult mouse brain.

In vivo depletion of endogenous glutathione facilitates trimethyltin-induced neuronal damage in the dentate gyrus of mice by enhancing oxidative stress

Acute treatment with trimethyltin chloride (TMT) produces neuronal damage in the hippocampal dentate gyrus of mice. We investigated the in vivo role of glutathione in mechanisms associated with TMT-induced neural cell damage in the hippocampus by examining mice depleted of endogenous glutathione by prior treatment with 2-cyclohexen-1-one (CHO). In the hippocampus of animals treated with CHO 1h beforehand, a significant increase was seen in the number of single-stranded DNA-positive cells in the dentate gyrus when determined on day 2 after the injection of TMT at a dose of 2.0 mg/kg. Immunoblot analysis revealed that CHO treatment induced a significant increase in the phosphorylation of c-Jun N-terminal kinase in the cytosolic and nuclear fractions obtained from the dentate gyrus at 16 h after the TMT injection. There was also a concomitant increase in the level of phospho-c-Jun in the cytosol at 16 h after the injection. Expectedly, lipid peroxidation was increased by TMT in the hippocampus, and was enhanced by the CHO treatment. Moreover, CHO treatment facilitated behavioral changes induced by TMT. Taken together, our data indicate that TMT-induced neuronal damage is caused by activation of cell death signals induced at least in part by oxidative stress. We conclude that endogenous glutathione protectively regulates neuronal damage induced by TMT by attenuating oxidative stress.

Modulation of cellular proliferation and differentiation through GABAB receptors expressed by undifferentiated neural progenitor cells isolated from fetal mouse brain

In this study, we have attempted to evaluate the possible role of metabotropic GABAB receptors (GABABR) expressed by neural progenitor cells prepared from neocortex of embryonic Std‐ddY mice. Immunocytochemical analysis confirmed the validity of isolation procedures of neural progenitors, while round spheres were formed with clustered cells during culture with epidermal growth factor (EGF) for 10 days. A reverse transcription polymerase chain reaction analysis revealed constitutive expression of GABAAR, GABABR, and GABACR subtypes in undifferentiated progenitors and neurospheres formed within 10 days. Exposure to GABA led to concentration‐dependent increases in the total area and proliferation activity of neurospheres at 10–300 µM, while the GABABR agonist baclofen at 100 µM significantly increased the size of neurospheres expressing both GABABR1 and GABABR2 subunits in a manner sensitive to a GABABR antagonist. By contrast, a significant decrease was seen in the total areas of neurospheres prepared from mice deficient of the GABABR1 subunit. In neurospheres of GABABR1‐null mice, a significant increase was induced in the number of cells immunoreactive for a glial marker protein, with a concomitant decrease in that of a neuronal marker protein, upon spontaneous differentiation after the removal of EGF. These results suggest that GABABR may be functionally expressed by neural progenitor cells to preferentially promote the commitment toward a neuronal lineage after the activation of cellular proliferation toward self‐replication in the developing mouse brain. J. Cell. Physiol. 216: 507–519, 2008.

Role of endogenous pituitary adenylate cyclase-activating polypeptide in adult hippocampal neurogenesis

Hippocampal neurogenesis occurs throughout life in mammals and has pivotal roles in brain functions. An enriched environment stimulates hippocampal neurogenesis, but the exact mechanisms are still unclear. The present study investigated the role of pituitary adenylate cyclase-activating polypeptide (PACAP) in adult hippocampal neurogenesis under standard or enriched rearing conditions. Rearing in the enriched conditions from 4-weeks old for 4-weeks increased the survival of newly divided cells in the subgranular zone and granule cell layer of the dentate gyrus of wild-type and PACAP-knockout (PACAP-/-) mice. The increase in the survival in the granule cell layer was less in PACAP-/- mice than in the wild-type mice. In contrast, the proliferation of newly divided cells in mice reared in the standard and enriched conditions did not differ between the wild-type and PACAP-/- mice. Regarding the differentiation of newborn cells in the dentate gyrus, most of the newly divided cells exhibited the neuronal phenotype in both the wild-type and PACAP-/- mice under standard and enriched conditions. These findings suggest that endogenous PACAP is partly involved in the survival of the enriched environment-induced generation, but not in the basal rate, of newborn cells in the dentate gyrus of the adult hippocampus.

Activation of GABAA receptors facilitates astroglial differentiation induced by ciliary neurotrophic factor in neural progenitors isolated from fetal rat brain

Immunocytochemical analysis confirmed the validity of isolation procedures of neural progenitors capable of self‐replication and differentiation from discrete fetal rat brain structures. A reverse transcription‐polymerase chain reaction analysis revealed the expression of particular GABAA receptor (GABAAR), GABABR‐1 and GABACR, but not GABABR‐2, subunits in neocortical cells before commitment. Sustained exposure to the GABAAR agonist muscimol at 100 μmol/L led to significant increases in the mitochondrial activity and the total areas of neocortical neurospheres formed during the cultivation for 12 days in a manner sensitive to a GABAAR antagonist, with lactate dehydrogenase release being unchanged. Moreover, prior sustained exposure to muscimol significantly facilitated the subsequent expression of an astroglial marker protein in cells differentiated by ciliary neurotrophic factor (CNTF) toward an astroglial lineage, with a concomitant decrease in the neuronal marker protein expression, in an antagonist‐sensitive manner on Western blotting analysis. However, muscimol failed to significantly affect the expression of both marker proteins in cells differentiated in either the presence or absence of all‐trans‐retinoic acid. These results suggest that prior activation of GABAAR may preferentially facilitate the commitment by CNTF of neural progenitor cells toward an astroglial lineage after simulation of the self‐replication activity in the developing rat brain.

In Vivo Acute Treatment With Trimethyltin Chloride Causes Neuronal Degeneration in the Murine Olfactory Bulb and Anterior Olfactory Nucleus by Different Cascades in Each Region

Our earlier study demonstrated that in vivo acute treatment with trimethyltin chloride (TMT) produces severe neuronal damage in the dentate gyrus and cognition impairment in mice. In the present study, we assessed whether TMT was capable of causing neuronal degeneration in the olfactory bulb (OB) and anterior olfactory nucleus (AON) of the mouse brain. An intraperitoneal injection of TMT at the dose of 2.8 mg/kg led to a dramatic increase in the number of degenerating cells, which were reactive with antibody against single‐stranded DNA, in the granule cell layer (GCL) of the OB and AON 1 day and 2 days later, respectively. TMT treatment produced a marked translocation of phospho‐c‐Jun‐N‐terminal kinase from the cytoplasm to the nucleus in the AON. Expectedly, a marked increase in phospho‐c‐Jun‐positive cells was seen in the AON after the treatment. In addition to the AON, the mitral cell layer of the olfactory bulb showed the presence of phospho‐c‐Jun‐positive cells after the treatment. However, the GCL had no cells positive for either phospho‐c‐Jun‐N‐terminal kinase or phospho‐c‐Jun at any time after the treatment with TMT. Similarly, TMT‐induced nuclear translocation of the lysosomal enzyme deoxyribonuclease II was seen in the AON, but not in the GCL. On the other hand, TMT elicited the expression of activated caspase 3 in the GCL but not in the AON. Taken together, our results suggest that TMT is capable of causing neuronal degeneration in the murine OB and AON through different cascades in the two structures.