Kunizo Arai

Essential Role of the Apoptotic Cell Engulfment Genes draper and ced-6 in Programmed Axon Pruning during Drosophila Metamorphosis

Axon pruning is a common phenomenon in neural circuit development. Previous studies demonstrate that the engulfing action of glial cells is essential in this process. The underlying molecular mechanisms, however, remain unknown. We show that draper (drpr) and ced-6, which are essential for the clearance of apoptotic cells in C. elegans, function in the glial engulfment of larval axons during Drosophila metamorphosis. The drpr mutation and glia-specific knockdown of drpr and ced-6 by RNA interference suppress glial engulfment, resulting in the inhibition of axon pruning. drpr and ced-6 interact genetically in the glial action. Disruption of the microtubule cytoskeleton in the axons to be pruned occurs via ecdysone signaling, independent of glial engulfment. These findings suggest that glial cells engulf degenerating axons through drpr and ced-6. We propose that apoptotic cells and degenerating axons of living neurons are removed by a similar molecular mechanism.

Na,K-ATPase α3 subunit in the goldfish retina during optic nerve regeneration

The goldfish optic nerve can regenerate after injury. To understand the molecular mechanism of optic nerve regrowth, we identified genes whose expression is specifically up‐regulated during the early stage of optic nerve regeneration. A cDNA library constructed from goldfish retina 5 days after transection was screened by differential hybridization with cDNA probes derived from axotomized or normal retina. Of six cDNA clones isolated, one clone was identified as the␣Na,K‐ATPase catalytic subunit α3 isoform by high‐ sequence homology. In northern hybridization, the expression level of the mRNA was significantly increased at 2 days and peaked at 5–10 days, and then gradually decreased and returned to control level by 45 days after optic nerve transection. Both in situ hybridization and immunohistochemical staining have revealed the location of this transient retinal change after optic nerve transection. The increased expression was observed only in the ganglion cell layer and optic nerve fiber layer at 5–20 days after optic nerve transection. In an explant culture system, neurite outgrowth from the retina 7 days after optic nerve transection was spontaneously promoted. A low concentration of ouabain (50–100 nm) completely blocked the spontaneous neurite outgrowth from the lesioned retina. Together, these data indicate that up‐regulation of the Na,K‐ATPase α3 subunit is involved in the regrowth of ganglion cell axons after axotomy.

Changes in NADPH diaphorase expression in the fish visual system during optic nerve regeneration and retinal development

The various functions of nitric oxide (NO) in the nervous system are not fully understood, including its role in neuronal regeneration. The goldfish can regenerate its optic nerve after transection, making it a useful model for studying central nervous regeneration in response to injury. Therefore, we have studied the pattern of NO expression in the retina and optic tectum after optic nerve transection, using NADPH diaphorase histochemistry. NO synthesis was transiently up-regulated in the ganglion cell bodies, peaking during the period when retinal axons reach the tectum, between 20-45 days after optic nerve transection. Enzyme activity in the tectum was transiently down-regulated and then returned to control levels at 60 days after optic nerve transection, during synaptic refinement. To compare NO expression in the developing and regenerating retina, we have looked at NO expression in the developing zebrafish retina. In the developing zebrafish retina the pattern of staining roughly followed the pattern of development with the inner plexiform layer and horizontal cells having the strongest pattern of staining. These results suggest that NO may be involved in the survival of ganglion cells in the regenerating retina, and that it plays a different role in the developing retina. In the tectum, NO may be involved in synaptic refinement.

Neuritogenic activity of a genipin derivative in retinal ganglion cells is mediated by retinoic acid receptor β expression through nitric oxide/S-nitrosylation signaling.

J. Neurochem. (2011) 10.1111/j.1471‐4159.2011.07533.x

Protective action of nipradilol mediated through S-nitrosylation of Keap1 and HO-1 induction in retinal ganglion cells.

Nipradilol (Nip), which has α1- and β-adrenoceptor antagonist and nitric oxide (NO)-donating properties, has clinically been used as an anti-glaucomatous agent in Japan. NO mediates cellular signaling pathways that regulate physiological functions. The major signaling mechanisms mediated by NO are cGMP-dependent signaling and protein S-nitrosylation-dependent signalings. Nip has been described as having neuroprotective effects through cGMP-dependent pathway in retinal ganglion cells (RGCs). However, the effect seems to be partial. On the other hand, whether Nip can prevent cell death through S-nitrosylation is not yet clarified. In this study, we therefore focused on the neuroprotective mechanism of Nip through S-nitrosylation. Nip showed a dramatic neuroprotective effect against oxidative stress-induced death of RGC-5 cells. However, denitro-nipradilol, which does not have NO-donating properties, was not protective against oxidative stress. Furthermore, an NO scavenger significantly reversed the protective action of Nip against oxidative stress. In addition, we demonstrated that α1- or β-adrenoceptor antagonists (prazosin or timolol) did not show any neuroprotective effect against oxidative stress in RGC-5 cells. We also demonstrated that Nip induced the expression of the NO-dependent antioxidant enzyme, heme oxygenase-1 (HO-1). S-nitrosylation of Kelch-like ECH-associated protein by Nip was shown to contribute to the translocation of NF-E2-related factor 2 to the nucleus, and triggered transcriptional activation of HO-1. Furthermore, RGC death and levels of 4-hydroxy-2-nonenal (4HNE) were increased after optic nerve injury in vivo. Pretreatment with Nip significantly suppressed RGC death and accumulation of 4HNE after injury through an HO-1 activity-dependent mechanism. These data demonstrate a novel neuroprotective action of Nip against oxidative stress-induced RGC death in vitro and in vivo.

Requirement of Retinoic Acid Receptor β for Genipin Derivative-Induced Optic Nerve Regeneration in Adult Rat Retina

Like other CNS neurons, mature retinal ganglion cells (RGCs) are unable to regenerate their axons after nerve injury due to a diminished intrinsic regenerative capacity. One of the reasons why they lose the capacity for axon regeneration seems to be associated with a dramatic shift in RGCs’ program of gene expression by epigenetic modulation. We recently reported that (1R)-isoPropyloxygenipin (IPRG001), a genipin derivative, has both neuroprotective and neurite outgrowth activities in murine RGC-5 retinal precursor cells. These effects were both mediated by nitric oxide (NO)/S-nitrosylation signaling. Neuritogenic activity was mediated by S-nitrosylation of histone deacetylase-2 (HDAC2), which subsequently induced retinoic acid receptor β (RARβ) expression via chromatin remodeling in vitro. RARβ plays important roles of neural growth and differentiation in development. However, the role of RARβ expression during adult rat optic nerve regeneration is not clear. In the present study, we extended this hypothesis to examine optic nerve regeneration by IPRG001 in adult rat RGCs in vivo. We found a correlation between RARβ expression and neurite outgrowth with age in the developing rat retina. Moreover, we found that IPRG001 significantly induced RARβ expression in adult rat RGCs through the S-nitrosylation of HDAC2 processing mechanism. Concomitant with RARβ expression, adult rat RGCs displayed a regenerative capacity for optic axons in vivo by IPRG001 treatment. These neuritogenic effects of IPRG001 were specifically suppressed by siRNA for RARβ. Thus, the dual neuroprotective and neuritogenic actions of genipin via S-nitrosylation might offer a powerful therapeutic tool for the treatment of RGC degenerative disorders.

Prescription rate of medications potentially contributing to lower urinary tract symptoms and detection of adverse reactions by prescription sequence symmetry analysis

BackgroundThe lower urinary tract symptoms (LUTS) increases with age and can have a significant effect on the quality of life of the patients. Elderly patients, who are often characterized by a decline in physiological functional and polypharmacy, are susceptible to adverse drug reactions to pharmacotherapy. LUTS can also be a side effect of medication. The purpose of this study was to investigate the possible association between the initiation of LUTS-causing drug therapy and the onset of LUTS.MethodsDrug dispensing data at the individual level were retrieved from the CISA (Platform for Clinical Information Statistical Analysis: http://www.cisa.jp) database. A retrospective study was conducted by reviewing patients with LUTS who were dispensed drugs that increased the risk of LUTS between April 2011 and March 2012. Prescription sequence symmetry analysis (PSSA) was employed to investigate the associations between the dispensing of medicines of LUTS and that of LUTS-causing drugs.ResultsLUTS-causing drugs were frequently dispensed to patients with LUTS. The use of medications potentially contributing to LUTS was associated with polypharmacy [number of prescription drugs:12.13 ± 6.78 (user) vs. 5.67 ± 5.24 (nonuser)] but not patient age [ age: (71.38 ± 13.28 (user) vs. 70.45 ± 14.80 (nonuser)]. Significant adverse drug events were observed the use of donepezil, cyclophosphamide, antiparkinson drugs, antidepressant, diazepam, antipsychotic drugs for peptic ulcer, tiotropium bromide, and opioids.ConclusionsThe use of prescription LUTS-causing drugs was correlated with polypharmacy. The adverse drug events associated with LUTS-causing drugs were highly prevalent in elderly patients. To prevent of adverse drug events in patients with LUTS, pharmacists and physicians should regularly review medication lists and reduce the prescribed medicines.

A simple estimation of peroxisomal degradation with green fluorescent protein - an application for cell cycle analysis.

When nutrients are depleted from the environment, mammalian cells begin to degrade their own cytosol and organelles. This bulk protein degradation is mediated by autophagy. In this study, peroxisomes in living CHO‐K1 cells were visualized by targeting the green fluorescent protein (GFP) tagged with a type 1 peroxisomal targeting signal. The nutrient‐starved condition induced a decay of GFP fluorescence in the peroxisomes and autophagic inhibitors such as 3‐methyladenine suppressed the decay of GFP fluorescence (13–60% of starvation). By double labeling the nuclear DNA and peroxisomal GFP, the autophagy specifically occurred at the G1 phase of the cell cycle and the autophagic inhibitors suppressed the G1 arrest. The vital stain technique with GFP is a very simple and useful marker to quantitatively estimate or to further study peroxisomal degradation.

Nipradilol promotes axon regeneration through S-nitrosylation of PTEN in retinal ganglion cells.

Nipradilol (Nip) is registered as an anti-glaucoma agent. More recently, a protective effect of Nip has been demonstrated in retinal ganglion cells (RGCs) mediated by S-nitrosylation of antioxidative-related Keap1 protein due to its nitric oxide (NO)-donating effect. It also has been reported that Nip promoted axon outgrowth in cat RGCs. However, the detailed mechanism remains unclear. NO physiologically regulates numerous cellular responses through S-nitrosylation of protein at cysteine residues. It has been reported that phosphatase and tensin homologue deleted on chromosome 10 (PTEN) deletion strongly showed axon regeneration after optic nerve injury. PTEN inactivation by S-nitrosylation results in the accumulation of phosphatidylinositol (3, 4, 5) triphosphate (PIP3) and the activation of Akt/mammalian target of rapamycin (mTOR) signaling. The ribosomal S6 kinase 1 (S6K) which can monitor as phospho-S6 (pS6) is one of major target of mTOR. In this study, we investigated the possibility that Nip can promote axon outgrowth in RGCs by Akt/mTOR signaling thorough S-nitrosylation of PTEN.

Investigation for Risk Factor and Preventive Effect of NSAIDs, Opioid on Gemcitabine-induced Vascular Pain

ゲムシタビン塩酸塩(GEM)は,膵臓がん, 胆道がん,肺がん,乳がんなどの治療に用いられ, 良好な治療成績が報告されている .GEM によ る有害事象としては,血小板減少等の重篤な骨髄 抑制が挙げられるが,その毒性は一般的に軽度な ものと考えられている.しかし,GEM に特徴的 な有害事象として,経静脈投与時の血管痛がある. これは,生命を脅かす重篤な有害事象ではないが, GEM 投与中の患者の苦痛が大きい場合もあり, 治療を継続する際の大きな問題となることがあ る. GEM による血管痛の発現頻度は,約 30%との 報告がみられるが ,報告によりその発現頻度 には違いがみられる.また,GEM による血管痛 の予防方法としては,ホットパックを用いて血管 を温める方法や GEM の溶解液を生理食塩液から 5 %ブドウ糖液への変更が行われている .し かし,ホットパックや 5 %ブドウ糖液を使用して も強い血管痛が生じることもあり,血管痛の予防 対策は十分とは言い難い.このため,GEM によ