Tatsuo Shiba

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.

Disruption of ion-trafficking system in the cochlear spiral ligament prior to permanent hearing loss induced by exposure to intense noise: possible involvement of 4-hydroxy-2-nonenal as a mediator of oxidative stress.

Noise-induced hearing loss is at least in part due to disruption of endocochlear potential, which is maintained by various K+ transport apparatuses including Na+, K+-ATPase and gap junction-mediated intercellular communication in the lateral wall structures. In this study, we examined the changes in the ion-trafficking-related proteins in the spiral ligament fibrocytes (SLFs) following in vivo acoustic overstimulation or in vitro exposure of cultured SLFs to 4-hydroxy-2-nonenal, which is a mediator of oxidative stress. Connexin (Cx)26 and Cx30 were ubiquitously expressed throughout the spiral ligament, whereas Na+, K+-ATPase α1 was predominantly detected in the stria vascularis and spiral prominence (type 2 SLFs). One-hour exposure of mice to 8 kHz octave band noise at a 110 dB sound pressure level produced an immediate and prolonged decrease in the Cx26 expression level and in Na+, K+-ATPase activity, as well as a delayed decrease in Cx30 expression in the SLFs. The noise-induced hearing loss and decrease in the Cx26 protein level and Na+, K+-ATPase activity were abolished by a systemic treatment with a free radical-scavenging agent, 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl, or with a nitric oxide synthase inhibitor, Nω-nitro-L-arginine methyl ester hydrochloride. In vitro exposure of SLFs in primary culture to 4-hydroxy-2-nonenal produced a decrease in the protein levels of Cx26 and Na+, K+-ATPase α1, as well as Na+, K+-ATPase activity, and also resulted in dysfunction of the intercellular communication between the SLFs. Taken together, our data suggest that disruption of the ion-trafficking system in the cochlear SLFs is caused by the decrease in Cxs level and Na+, K+-ATPase activity, and at least in part involved in permanent hearing loss induced by intense noise. Oxidative stress-mediated products might contribute to the decrease in Cxs content and Na+, K+-ATPase activity in the cochlear lateral wall structures.

Lithium Promotes Neuronal Repair and Ameliorates Depression-Like Behavior following Trimethyltin-Induced Neuronal Loss in the Dentate Gyrus

Lithium, a mood stabilizer, is known to ameliorate the stress-induced decrease in hippocampal neurogenesis seen in animal models of stress-related disorders. However, it is unclear whether lithium has beneficial effect on neuronal repair following neuronal damage in neuronal degenerative diseases. Here, we evaluated the effect of in vivo treatment with lithium on the hippocampal neuronal repair in a mouse model of trimethyltin (TMT)-induced neuronal loss/self-repair in the hippocampal dentate gyrus (such mice referred to as “impaired animals”) [Ogita et al. (2005) J Neurosci Res 82: 609–621]. The impaired animals had a dramatically increased number of 5-bromo-2′-deoxyuridine (BrdU)-incorporating cells in their dentate gyrus at the initial time window (days 3 to 5 post-TMT treatment) of the self-repair stage. A single treatment with lithium produced no significant change in the number of BrdU-incorporating cells in the dentate granule cell layer and subgranular zone on day 3 post-TMT treatment. On day 5 post-TMT treatment, however, BrdU-incorporating cells were significantly increased in number by lithium treatment for 3 days. Most interestingly, chronic treatment (15 days) with lithium increased the number of BrdU-incorporating cells positive for NeuN or doublecortin in the dentate granule cell layer of the impaired animals, but not in that of naïve animals. The results of a forced swimming test revealed that the chronic treatment with lithium improved the depression-like behavior seen in the impaired animals. Taken together, our data suggest that lithium had a beneficial effect on neuronal repair following neuronal loss in the dentate gyrus through promoted proliferation and survival/neuronal differentiation of neural stem/progenitor cells in the subgranular zone.

Beneficial effect of cilostazol‐mediated neuronal repair following trimethyltin‐induced neuronal loss in the dentate gyrus

Cilostazol acts as an antiplatelet agent and has other pleiotropic effects based on phosphodiesterase‐3‐dependent mechanisms. We evaluated whether cilostazol would have a beneficial effect on neuronal repair following hippocampal neuronal damage by using a mouse model of trimethyltin (TMT)‐induced neuronal loss/self‐repair in the hippocampal dentate gyrus [Ogita et al. (2005) J Neurosci Res 82:609−621]; these mice will hereafter be referred to as impaired animals. A single treatment with cilostazol (10 mg/kg, i.p.) produced no significant change in the number of 5‐bromo‐2′‐deoxyuridine (BrdU)‐incorporating cells in the dentate granule cell layer (GCL) or subgranular zone on day 3 after TMT treatment. However, chronic treatment with cilostazol on days 3–15 posttreatment resulted in an increase in the number of BrdU‐incorporating cells in the dentate GCL of the impaired animals, and these cells were positive for neuronal nuclear antigen or doublecortin. Cilostazol was effective in elevating the level of phosphorylated cyclic adrenosine monophosphate response element‐binding protein (pCREB) in the dentate gyrus of impaired animals. The results of a forced swimming test revealed that the chronic treatment with cilostazol improved the depression‐like behavior seen in the impaired animals. In the cultures of hippocampal neural stem/progenitor cells, exposure to cilostazol produced not only enhancement of proliferation activity but also elevation of pCREB levels. Taken together, our data suggest that cilostazol has a beneficial effect on neuronal repair following neuronal loss in the dentate gyrus through promotion of proliferation and/or neuronal differentiation of neural progenitor cells in the subgranular zone.

Opposing roles of glucocorticoid receptor and mineralocorticoid receptor in trimethyltin-induced cytotoxicity in the mouse hippocampus

The organotin trimethyltin (TMT) is known to cause neuronal degeneration in the murine brain. Earlier studies indicate that TMT-induced neuronal degeneration is enhanced by adrenalectomy and prevented by exogenous glucocorticoid. The aim of this study was to investigate the regulation of TMT neuroxicity by corticosterone receptors including type I (mineralocorticoid receptor, MR) and type II (glucocorticoid receptor, GR) in adult mice. The systemic injection of TMT at the dose of 2.0 or 2.8 mg/kg produced a marked elevation in the level of plasma corticosterone that was both dose and time dependent. The MR agonist aldosterone had the ability to exacerbate TMT cytotoxicity in the dentate granule cell layer, whereas its antagonist spironolactone protected neurons from TMT cytotoxicity there. In contrast, the GR antagonist mifepristone exacerbated the TMT cytotoxicity. Taken together, our data suggest TMT cytotoxicity is oppositely regulated by GR and MR signals, being exacerbated by MR activation in adult mice.

Ryanodine receptor regulates proliferative activity in neural stem/progenitor cells derived from subventricular zone of adult mice

Subventricular zone (SVZ) of adult brain has neural stem/progenitor cells (NPCs) that generate new neurons throughout life. In the NPCs, intracellular Ca2+ is known to play a critical role in regulating different stages of early brain development and neurogenesis. To elucidate participation of Ca2+ signaling pathway in proliferation of the NPCs, we evaluated the effect of ryanodine receptor (RyR) inhibitor dantrolene on proliferative activity in the NPCs derived from the SVZ of adult mice. Cells were prepared from the SVZ of 5-week-old Std-ddY male mice and then primarily cultured in DMEM/F12 medium with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) for 8 days in vitro (DIV). After replating, the cells were secondarily cultured for 5 DIV under the same conditions in the absence or presence of dantrolene for a period of 8–13 DIV. In the presence of both EGF and bFGF, marked round spheres were formed, continued to grow, and proliferate to form large neurospheres. In addition, most cells obtained from the neurospheres were immunoreactive to nestin. To determine the expression of RyR subunits in the NPCs, we performed RT-PCR analysis using total RNA prepared from the NPCs. Although there exist 3 subunits of RyR in the NPCs, RyR3 was the highest level of 3 subunits of RyR. ELISA for 5′-bromo2′-deoxyuridine revealed that a marked decrease in the proliferative activity was seen by treatment with dantrolene in a dose-dependent manner (2, 10, 20 or 50 M). However, dantrolene was ineffective in releasing lactate dehydrogenase into the culture medium. Moreover, treatment with dantrolene resulted in a marked reduction in STAT3 and Pax6 mRNA in NPCs. These results suggest that Ca2+ release from the endoplasmic reticulum via the RyR would positively regulate proliferative activity in NPCs of adult mouse SVZ.

Characterization of cells newly generated after granule cell loss in the hippocampal dentate gyrus of mice

Subventricular zone (SVZ) of adult brain has neural stem/progenitor cells (NPCs) that generate new neurons throughout life. In the NPCs, intracellular Ca2+ is known to play a critical role in regulating different stages of early brain development and neurogenesis. To elucidate participation of Ca2+ signaling pathway in proliferation of the NPCs, we evaluated the effect of ryanodine receptor (RyR) inhibitor dantrolene on proliferative activity in the NPCs derived from the SVZ of adult mice. Cells were prepared from the SVZ of 5-week-old Std-ddY male mice and then primarily cultured in DMEM/F12 medium with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) for 8 days in vitro (DIV). After replating, the cells were secondarily cultured for 5 DIV under the same conditions in the absence or presence of dantrolene for a period of 8–13 DIV. In the presence of both EGF and bFGF, marked round spheres were formed, continued to grow, and proliferate to form large neurospheres. In addition, most cells obtained from the neurospheres were immunoreactive to nestin. To determine the expression of RyR subunits in the NPCs, we performed RT-PCR analysis using total RNA prepared from the NPCs. Although there exist 3 subunits of RyR in the NPCs, RyR3 was the highest level of 3 subunits of RyR. ELISA for 5′-bromo2′-deoxyuridine revealed that a marked decrease in the proliferative activity was seen by treatment with dantrolene in a dose-dependent manner (2, 10, 20 or 50 M). However, dantrolene was ineffective in releasing lactate dehydrogenase into the culture medium. Moreover, treatment with dantrolene resulted in a marked reduction in STAT3 and Pax6 mRNA in NPCs. These results suggest that Ca2+ release from the endoplasmic reticulum via the RyR would positively regulate proliferative activity in NPCs of adult mouse SVZ.