Chie Sugiyama

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.

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.

In vivo activation of c-Jun N-terminal kinase signaling cascade prior to granule cell death induced by trimethyltin in the dentate gyrus of mice.

The systemic administration of trimethyltin (TMT, 2.8 mg/kg, i.p.) induced granule cell death in the mouse dentate gyrus selectively 2 days later. The administration of TMT not only enhanced activator protein-1 DNA binding, along with an increase in expression of c-Jun and Fra-2, in the hippocampus 1 day later, but also facilitated phosphorylation of c-Jun N-terminal kinase (JNK) within the cytosol and nucleus. There was also a concomitant increase in the level of phosphorylated JNK kinase (MKK4/SEK1) in the cytosol 16-24 h after the administration. Moreover, TMT markedly elevated endogenous levels of both phosphorylated c-Jun and phosphorylated activating transcription factor-2 (ATF-2), in addition to activating JNK activity in the nuclear extracts obtained 16-24 h post-administration. Immunohistochemical analysis revealed that whereas Fra-2 and phosphorylated ATF-2 were expressed in the CA1 pyramidal cell layer predominantly, phosphorylated c-Jun was observed in both the CA1 pyramidal and dentate granule cell layers after TMT administration. Taken together, our data indicate that TMT activates the JNK pathway in the hippocampus prior to neuronal cell death. The prior activation of this pathway could be at least in part involved in the TMT-induced neural damage seen in the dentate granule cells of mice.

In vivo treatment with the K+ channel blocker 4-aminopyridine protects against kainate-induced neuronal cell death through activation of NMDA receptors in murine hippocampus

Activation of NMDA receptors has been shown to induce either neuronal cell death or neuroprotection against excitotoxicity in cultured neurons in vitro. To elucidate in vivo neuroprotective role of NMDA receptors, we investigated the effects of activation of NMDA receptors by endogenous glutamate on kainate-induced neuronal damage to the mouse hippocampus in vivo. The systemic administration of the K+ channel blocker 4-aminopyridine (4-AP, 5 mg/kg, i.p.) induced expression of c-Fos in the hippocampal neuronal cell layer, which expression was completely abolished by the noncompetitive NMDA receptor antagonist MK-801, thus indicating that the administration of 4-AP would activate NMDA receptors in the hippocampal neurons. The prior administration of 4-AP at 1 h to 1 day before significantly prevented kainate-induced pyramidal cell death in the hippocampus and expression of pyramidal cells immunoreactive with an antibody against single-stranded DNA. Further immunohistochemical study on deoxyribonuclease II revealed that the pretreatment with 4-AP led to complete abolition of deoxyribonuclease II expression induced by kainate in the CA1 and CA3 pyramidal cells. The neuroprotection mediated by 4-AP was blocked by MK-801 and by the adenosine A1 antagonist 8-cyclopenthyltheophylline. Taken together, in vivo activation of NMDA receptors is capable of protecting against kainate-induced neuronal damage through blockade of DNA fragmentation induced by deoxyribonuclease II in the murine hippocampus.

High susceptibility of cortical neural progenitor cells to trimethyltin toxicity: Involvement of both caspases and calpain in cell death

Neural progenitor cells play an essential role in both the developing embryonic nervous system and in the adult brain, where the capacity for self-renewal would be important for normal brain functions. In the present study, we used embryonic cortical neural progenitor cells to investigate the effects of trimethyltin chloride (TMT) on the survival of neural progenitor cells. In cultures of cortical neural progenitor cells, the formation of round neurospheres was observed in the presence of epidermal growth factor and basic fibroblast growth factor within 9 days in vitro. The neurospheres were then harvested for subsequent replating and culturing for assessment of cell viability in either the presence or absence of TMT at the concentration of 5microM. Lasting exposure to TMT produced not only nuclear condensation in the cells in a time-dependent manner over a period of 6-24h, but also the release of lactate dehydrogenase into the culture medium. Immunoblot and immunocytochemical analyses revealed that TMT had the ability to activate both caspase-3 and calpain, as well as to cause nuclear translocation of deoxyribonuclease II, which is located within cytoplasm in intact cells. Additionally, treatment with a calpain inhibitor [trans-epoxysuccinyl-l-leucylamido-(4-guanidino) butane] and a caspase inhibitor [Z-Val-Ala-Asp(OMe)-CH2F] produced a significant reduction in damaged cells induced by TMT. Taken together, our data indicate that neural progenitor cells are highly susceptible to TMT in undergoing cell death via the activation of 2 parallel pathways, ones involving calpain and the other, caspase-3.

Xenobiotic response element binding enriched in both nuclear and microsomal fractions of rat cerebellum

Xenobiotic response element (XRE) is a core nucleotide sequence at the upstream of inducible target genes for the transcription factor aryl hydrocarbon receptor (AhR) that is responsible for signal transduction of exogenous environmental pollutants in eukaryotic cells. Immunoblotting analysis revealed the constitutive expression of AhR‐related proteins in rat liver and brain, while specific binding of a radiolabelled probe containing XRE was detected in nuclear preparations of both liver and brain on gel retardation electrophoresis. Among discrete rat brain structures examined, cerebellum exhibited the highest XRE binding with less potent binding in hypothalamus, midbrain, medulla‐oblongata, hippocampus, cerebral cortex and striatum. In contrast to liver and hippocampus, cerebellum also contained unusually higher XRE binding in microsomal fractions than that in either nuclear or mitochondrial fractions. Limited proteolysis by V8 protease did not markedly affect XRE binding in cerebellar nuclear extracts, with concomitant diminution of that in hepatic and hippocampal nuclear extracts. In primary cultured cerebellar neurons, indigo was effective in significantly increasing XRE binding only when determined immediately after sustained exposure for 120 min in the presence of high potassium chloride. These results suggest the abundance of as‐yet unidentified proteins with high affinity for XRE and responsiveness to indigo in both nuclear and microsomal fractions of rat cerebellum.

Altered expression of heat shock protein 110 family members in mouse hippocampal neurons following trimethyltin treatment in vivo and in vitro.

The heat shock protein (Hsp) 110 family is composed of HSP105, APG-1, and APG-2. As the response of these proteins to neuronal damage is not yet fully understood, in the present study, we assessed their expression in mouse hippocampal neurons following trimethyltin chloride (TMT) treatment in vivo and in vitro. Although each of these three Hsps had a distinct regional distribution within the hippocampus, a low level of all of them was observed in the granule cell layer of the dentate gyrus in naïve animals. TMT was effective in markedly increasing the level of these Hsps in the granule cell layer, at least 16h to 4days after the treatment. In the dentate granule cell layer on day 2 after TMT treatment, HSP105 was expressed mainly in the perikarya of NeuN-positive cells (intact neurons); whereas APG-1 and APG-2 were predominantly found in NeuN-negative cells (damaged neurons as evidenced by signs of cell shrinkage and condensation of chromatin). Assessments using primary cultures of mouse hippocampal neurons exposed to TMT revealed that whereas HSP105 was observed in intact neurons rather than in damaged neurons, APG-1 and APG-2 were detected in both damaged neurons and intact neurons. Taken together, our data suggest that APG-1 and APG-2 may play different roles from HSP105 in neurons damaged by TMT.

Acoustic overstimulation facilitates the expression of glutamate–cysteine ligase catalytic subunit probably through enhanced DNA binding of activator protein-1 and/or NF-κB in the murine cochlea

Glutamate-cysteine ligase (GCL), previously known as gamma-glutamylcysteine synthetase, is the rate-limiting enzyme for GSH synthesis. The expression of GCL is mediated by activator protein-1 (AP-1) and nuclear factor-kappa B (NF-kappaB), which are known to participate in stress-induced apoptotic pathways in neuronal cells. In this study, we investigated the changes in the level of these transcription factors as well as of GCL catalytic subunit in the cochlea in response to acoustic overstimulation. Nuclear extracts were prepared from the cochlear at various time points after intense noise exposure (4kHz octave band, 125dB sound pressure level, 5h), and then determined DNA binding activity of the transcription factors. AP-1 DNA binding was markedly increased 2-12h after the noise exposure, with a peak at 2h after the exposure. NF-kappaB DNA binding was also increased immediately after the exposure. Semi-quantitative RT-PCR revealed that the catalytic subunit of GCL mRNA was elevated in the cochlea 2-24h post the exposure. Further immunohistochemical study revealed that increased level of GCL catalytic subunit observed at least in the spiral ganglion cells after the exposure. These results suggest that intense noise exposure facilitates the expression of GCL catalytic subunit in the cochlea possibly through the activation of transcription factors including AP-1 and NF-kappaB.

Trimethyltin initially activates the caspase 8/caspase 3 pathway for damaging the primary cultured cortical neurons derived from embryonic mice

The organotin trimethyltin (TMT) is well known to cause neuronal damage in the central nervous system. To elucidate the mechanisms underlying the toxicity of TMT toward neurons, we prepared primary cultures of neurons from the neocortex of mouse embryos. A continuous exposure to TMT produced a decrease in cell viability as well as an increase in the number of cells with nuclear condensation/shrinkage at the exposure time window up to 24 hr. In addition to the events at the early time window, lactate dehydrogenase released was significantly elevated at the later exposure time from 36 to 48 hr. With a 3‐hr exposure to TMT, a significant increase was observed in the activity of caspase 8, but not in that of caspase 9. TMT exposure produced no elevation in the level of cytochrome c released from mitochondria until 12 hr of exposure, with a significant facilitation of cytochrome c release at the exposure times of 16 and 24 hr. After the activation of caspase 8 by TMT exposure, caspase 3 activation and nuclear translocation of caspase‐activated DNase were caused by exposure for 6 hr or longer. However, nuclear DNase II was elevated at the later time window of exposure. A caspase inhibitor completely prevented TMT from damaging the cells in any time window. Taken together, our data are the first demonstration that TMT toxicity is initially caused by activation of the caspase 8/caspase 3 pathway for nuclear translocation of DNases in cortical neurons in primary culture.

Altered expression of DJ-1 in the hippocampal cells following in vivo and in vitro neuronal damage induced by trimethyltin

Trimethyltin chloride (TMT) is known to produce neuronal damage in the dentate gyrus at least in part via oxidative stress. DJ-1, an oncogene product, is known to act as an anti-oxidant to prevent neuronal damage in dopaminergic neurons. The aim of this study was to determine the alterations in DJ-1 expression in the hippocampal cells of mice after in vivo and in vitro treatment with TMT. In naïve animals, DJ-1 was ubiquitously expressed in the hippocampus, in which the CA1 pyramidal cell layer and dentate granule cell layer had lower and higher levels of it, respectively. An intraperitoneal injection of TMT at the dose of 2.8 mg/kg produced DJ-1 up-regulation in the CA1 pyramidal cell layer, CA3 stratum lucidum, dentate molecular layer, and dentate hilus, but not in the dentate granule cell layer, on day 3-5 post-treatment. Temporary depletion of endogenous glutathione by the prior subcutaneous injection of 2-cyclohexen-1-one was effective in facilitating neuronal damage and DJ-1 up-regulation in the dentate gyrus induced by an intraperitoneal injection of TMT at the dose of 2.0 mg/kg. In primary cultures of mouse hippocampal cells, DJ-1 was present in neurons, but not in astrocytes. TMT treatment produced a dramatic expression of DJ-1 in the astrocytes in the cultures. Taken together, our data suggest that the DJ-1 protein is positively regulated in response to oxidative stress induced by TMT.