Proteasome inhibitors induce apoptosis by superoxide anion generation via NADPH oxidase 5 in human neuroblastoma SH-SY5Y cells.

The ubiquitin-proteasome system (UPS) is a major proteolytic system that plays an important role in the regulation of various cell processes, such as cell cycle, stress response, and transcriptional regulation, especially in neurons, and dysfunction of UPS is considered to be a cause of neuronal cell death in neurodegenerative diseases. However, the mechanism of neuronal cell death caused by UPS dysfunction has not yet been fully elucidated. In this study, we investigated the mechanism of neuronal cell death induced by proteasome inhibitors using human neuroblastoma SH-SY5Y cells. Z-Leu-D-Leu-Leu-al (MG132), a proteasome inhibitor, induced apoptosis in SH-SY5Y cells in a concentration- and time-dependent manner. Antioxidants N-acetylcysteine and EUK-8 attenuated MG132-induced apoptosis. Apocynin and diphenyleneiodonium, inhibitors of NADPH oxidase (NOX), an enzyme that produces superoxide anions, also attenuated MG132-induced apoptosis. It was also found that MG132 treatment increased the expression of NOX5, a NOX family member, and that siRNA-mediated silencing of NOX5 and BAPTA-AM, which inhibits NOX5 by chelating calcium, suppressed MG132-induced apoptosis and production of reactive oxygen species in SH-SY5Y cells. These results suggest that MG132 induces apoptosis in SH-SY5Y cells through the production of superoxide anion by NOX5.

Schwann cell derived-peroxiredoxin protects motor neurons against hydrogen peroxide-induced cell death in mouse motor neuron cell line NSC-34.

Schwann cells and oligodendrocytes secrete proteins that promote neuron survival, but their role in amyotrophic lateral sclerosis (ALS) is unclear. To address this question, we evaluated the effect of molecules secreted by Schwann cells on reactive oxygen species (ROS)-induced motor neuronal cell death. We observed that in motor neuron cell line NSC-34 cultures, the conditioned medium (CM) from Schwann cell line YST-1 (YST-1 CM) cultures had a protective effect against hydrogen peroxide-induced cell death. However, this protective effect of YST-1 CM was abolished by removing peroxiredoxin 1-4 (PRDX1-4) from the CM. We found that the expression of PRDX1 mRNA was markedly downregulated in the lumbar spinal cord of the superoxide dismutase 1 (SOD1)G93A mouse model of ALS. We also found that transient transfection of YST-1 cells with G93A SOD1 resulted in reduced PRDX1 mRNA expression. Additionally, in the mutant transfected cells, YST-1 CM showed decreased neuroprotective effect against hydrogen peroxide-induced NSC-34 cell death compared to those transfected with WT SOD1. Our results suggest that Schwann cells protect motor neurons from oxidative stress by secreting PRDX1 and that the reduction of PRDX secreted from Schwann cells contributes to increased ROS and associated motor neuronal death in ALS.

An Increase in Peroxiredoxin 6 Expression Induces Neurotoxic A1 Astrocytes in the Lumbar Spinal Cord of Amyotrophic Lateral Sclerosis Mice Model.

Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disease with selective degeneration of motor neurons. It has been reported that an increase in the levels of inflammatory cytokines and glial cells such as reactive astrocytes is closely involved in the pathological progression of ALS. Recently, the levels of neuropathic cytotoxic (A1) astrocytes among reactive astrocytes have reportedly increased in the central nervous system of ALS mice, which induce motor neuron degeneration through the production of inflammatory cytokines and secretion of neuropathic factors. Hence, elucidating the induction mechanism of A1 astrocytes in ALS is important to understand the mechanism of disease progression in ALS. In this study, we observed that the expression of peroxiredoxin 6 (PRDX6), a member of the peroxiredoxin family, was markedly upregulated in astrocytes of the lumbar spinal cord of SOD1G93A mice model for ALS. Additionally, when PRDX6 was transiently transfected into the mouse astrocyte cell line C8-D1A and human astrocytoma cell line U-251 MG, the mRNA expression of complement C3 (a marker for A1 astrocyte phenotype) and inflammatory cytokines was increased. Furthermore, the mRNA expression of C3 and inflammatory cytokine was increased in C8-D1A and U-251 MG cells stably expressing PRDX6, and the increased mRNA expression was significantly suppressed by MJ33 (lithium[1-hexadecoxy-3-(2,2,2-trifluoroethoxy) propan-2-yl] methyl phosphate), an inhibitor of the phospholipase A2 activity of PRDX6. Our results suggest that the expression of PRDX6 in astrocytes plays an important role in the induction of A1 astrocytes and expression of inflammatory cytokines in the ALS mice model.

Dopamine inhibits the expression of proinflammatory cytokines of microglial cells through the formation of dopamine quinone in the mouse striatum.

We previously reported that dopamine (DA) attenuated lipopolysaccharide (LPS)-induced expression of proinflammatory cytokines through the formation of DA quinone (DAQ) in murine microglial cell line BV-2 and primary murine microglial cells. To reveal whether DA inhibits the expression of proinflammatory cytokines of microglial cells through the formation of DAQ in the central nervous system (CNS), in this study, we examined the effect of DAQ on LPS-induced mRNA expression of proinflammatory cytokines in C57BL/6 mouse brain under two experimental conditions: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration and l-dopa/carbidopa administration. Acute MPTP administration reduced the number of tyrosine hydroxylase-positive cells in the substantia nigra, and decreased the level of quinoprotein, an indicator of DAQ formation, in the striatum. Real-time RT-PCR analysis revealed that intraperitoneal administration of LPS increased the mRNA levels of proinflammatory cytokines, including tumor-necrosis factor-α and interleukin-1ß, in the striatum. These increases were enhanced in MPTP-treated mice. On the other hand, l-dopa/carbidopa administration increased the level of quinoprotein, attenuated the LPS-induced mRNA expression of proinflammatory cytokines, and reduced the LPS-induced increase in the number of microglial cells in the striatum. These results suggest that DA attenuate the expression of proinflammatory cytokines in microglia through the formation of DAQ in the CNS.

Noradrenaline protects neurons against H2 O2 -induced death by increasing the supply of glutathione from astrocytes via ß3 -adrenoceptor stimulation.

Oxidative stress has been implicated in a variety of neurodegenerative disorders, such as Alzheimer's and Parkinson's disease. Astrocytes play a significant role in maintaining survival of neurons by supplying antioxidants such as glutathione (GSH) to neurons. Recently, we found that noradrenaline increased the intracellular GSH concentration in astrocytes via ß3 -adrenoceptor stimulation. These observations suggest that noradrenaline protects neurons from oxidative stress-induced death by increasing the supply of GSH from astrocytes to neurons via the stimulation of ß3 -adrenoceptor in astrocytes. In the present study, we examined the protective effect of noradrenaline against H2 O2 -induced neurotoxicity using two different mixed cultures: the mixed culture of human astrocytoma U-251 MG cells and human neuroblastoma SH-SY5Y cells, and the mouse primary cerebrum mixed culture of neurons and astrocytes. H2 O2 -induced neuronal cell death was significantly attenuated by pretreatment with noradrenaline in both mixed cultures but not in single culture of SH-SY5Y cells or in mouse cerebrum neuron-rich culture. The neuroprotective effect of noradrenaline was inhibited by SR59230A, a selective ß3 -adrenoceptor antagonist, and CL316243, a selective ß3 -adrenoceptor agonist, mimicked the neuroprotective effect of noradrenaline. DL-buthionine-[S,R]-sulfoximine, a GSH synthesis inhibitor, negated the neuroprotective effect of noradrenaline in both mixed cultures. MK571, which inhibits the export of GSH from astrocytes mediated by multidrug resistance-associated protein 1, also prevented the neuroprotective effect of noradrenaline. These results suggest that noradrenaline protects neurons against H2 O2 -induced death by increasing the supply of GSH from astrocytes via ß3 -adrenoceptor stimulation.

Co-Microencapsulation of Islets and MSC CellSaics, Mosaic-Like Aggregates of MSCs and Recombinant Peptide Pieces, and Therapeutic Effects of Their Subcutaneous Transplantation on Diabetes.

The subcutaneous transplantation of microencapsulated islets has been extensively studied as a therapeutic approach for type I diabetes. However, due to the lower vascular density and strong inflammatory response in the subcutaneous area, there have been few reports of successfully normalized blood glucose levels. To address this issue, we developed mosaic-like aggregates comprised of mesenchymal stem cells (MSCs) and recombinant peptide pieces called MSC CellSaics, which provide a continuous release of angiogenic factors and anti-inflammatory cytokines. Our previous report revealed that the diabetes of immunodeficient diabetic model mice was reversed by the subcutaneous co-transplantation of the MSC CellSaics and rat islets. In this study, we focused on the development of immune-isolating microcapsules to co-encapsulate the MSC CellSaics and rat islets, and their therapeutic efficiency via subcutaneous transplantation into immunocompetent diabetic model mice. As blood glucose level was monitored for 28 days following transplantation, the normalization rate of the new immuno-isolating microcapsules was confirmed to be significantly higher than those of the microcapsules without the MSC CellSaics, and the MSC CellSaics transplanted outside the microcapsules (p < 0.01). Furthermore, the number of islets required for the treatment was reduced. In the stained sections, a larger number/area of blood vessels was observed around the new immuno-isolating microcapsules, which suggests that angiogenic factors secreted by the MSC CellSaics through the microcapsules function locally for their enhanced efficacy.

Endogenous Apelin Is Protective Against Age-Associated Loss of Retinal Ganglion Cells in Mice.

Age-associated loss of retinal ganglion cells (RGCs) causes visual deficits, but there is not yet any therapeutic agent to prevent the loss of these cells. Herein, we report that apelin, an endogenous peptide ligand of APJ receptor, is protective against the age-related loss of RGCs in mice. The mRNA expression of apelin was reduced in the retina of old mice compared with that in young mice, whereas retinal APJ expression increased with age. Immunofluorescence staining showed that APJ was present in RGCs and their surrounding cells expressed apelin. In addition, both functional and histological analyses demonstrated that apelin deficiency accelerated the loss of RGCs associated with age in mice. These results suggest that endogenous apelin plays a protective role against the degeneration of RGCs and that the apelinergic axis may be a new target for preventing age-related visual impairment.

Systemic Administration of an Apelin Receptor Agonist Prevents NMDA-Induced Loss of Retinal Neuronal Cells in Mice.

Glutamate excitotoxicity via N-methyl-D-aspartate (NMDA) receptors is thought to be a factor involved in the loss of retinal neuronal cells, including retinal ganglion cells, in retinal diseases such as diabetic retinopathy and acute angle closure glaucoma. Herein we report the protective effect of systemic administration of ML233, an apelin receptor agonist, against retinal neuronal cell death induced by the intravitreal injection of NMDA into mice. Intraperitoneal administration of ML233 prevented the NMDA-induced reduction in the amplitude of scotopic threshold responses (STR), which mainly reflect the activity of the retinal ganglion cells. Immunohistochemical staining showed that ML233 inhibited the NMDA-induced loss of retinal ganglion cells and amacrine cells. In addition, ML233 suppressed the breakdown of spectrin αII, a neuronal cytoskeleton protein cleaved by calpain activation, in the retina after intravitreal injection of NMDA. Intraperitoneal administration of ML233 increased the phosphorylation of Akt, a potent anti-apoptotic protein in neurons, in the retina. Furthermore, oral administration of ML233 protected against the decrease in the STR amplitudes and the loss of retinal ganglion cells caused by NMDA. These results suggest that systemic administration of ML233 protected retinal neurons from NMDA receptor-mediated excitotoxicity and that drugs activating the apelin receptor may be a new candidate for preventing the progression of these retinal diseases.

Dopamine attenuates lipopolysaccharide-induced expression of proinflammatory cytokines by inhibiting the nuclear translocation of NF-κB p65 through the formation of dopamine quinone in microglia.

Many reports have indicated that dopamine has immunomodulatory effects on peripheral immune cells. The purpose of this study was to reveal the immunomodulatory effect of dopamine on the expression of proinflammatory cytokines in microglial cells, which are the immune cells of the central nervous system. In murine microglial cell line BV-2 cells, pretreatment with dopamine for 24 h attenuated the lipopolysaccharide (LPS)-induced expression of proinflammatory cytokines such as tumor-necrosis factor-α, interleukin-1ß, and interleukin-6. Neither (5R)-8-chloro-3-methyl-5-phenyl-1,2,4,5-tetrahydro-3-benzazepin-7-ol; hydrochloride (SCH-23390) nor sulpiride, which are dopamine D1-like and D2-like receptor antagonists, respectively, affected the attenuation of LPS-induced expression of cytokines by dopamine. In addition, pretreatment with neither (-)-(6aR,12bR)-4,6,6a,7,8,12b-Hexahydro-7-methylindolo[4,3-a]phenanthridin (CY208-243) nor bromocriptine, dopamine D1-like and D2-like receptor agonists, respectively, was effective in doing so. However, N-acetylcysteine (NAC), which inhibits dopamine oxidation to dopamine quinone, did inhibit this attenuated expression. Dopamine increased the level of quinoproteins, and this increase was inhibited by NAC. Western blot and immunocytochemical analyses revealed that dopamine inhibited LPS-induced nuclear translocation of nuclear factor-kappa B (NF-κB) p65. Dopamine also attenuated the expression of cytokines and the nuclear translocation of NF-κB p65 induced by LPS in mouse microglial cells in primary culture. These results suggest that dopamine attenuated LPS-induced expression of cytokines by inhibiting the nuclear translocation of NF-κB p65 through the formation of dopamine quinone in microglial cells.

An apelin receptor antagonist prevents pathological retinal angiogenesis with ischemic retinopathy in mice.

Pathological retinal angiogenesis is caused by the progression of ischemic retinal diseases and can result in retinal detachment and irreversible blindness. This neovascularization is initiated from the retinal veins and their associated capillaries and involves the overgrowth of vascular endothelial cells. Since expression of the apelin receptor (APJ) is restricted to the veins and proliferative endothelial cells during physiological retinal angiogenesis, in the present study, we investigated the effect of APJ inhibition on pathological retinal angiogenesis in a mouse model of oxygen-induced retinopathy (OIR). In vitro experiments revealed that ML221, an APJ antagonist, suppressed cultured-endothelial cell proliferation in a dose-dependent manner. Intraperitoneal administration of ML221 inhibited pathological angiogenesis but enhanced the recovery of normal vessels into the ischemic regions in the retina of the OIR model mice. ML221 did not affect the expression levels of vascular endothelial growth factor (VEGF) and its receptor (VEGFR2) in the retina. APJ was highly expressed in the endothelial cells within abnormal vessels but was only detected in small amounts in morphologically normal vessels. These results suggest that APJ inhibitors selectively prevent pathological retinal angiogenesis and that the drugs targeting APJ may be new a candidate for treating ischemic retinopathy.

Apelin protects against NMDA-induced retinal neuronal death via an APJ receptor by activating Akt and ERK1/2, and suppressing TNF-α expression in mice.

Glutamate excitotoxicity mediated by N-methyl-d-aspartate (NMDA) receptors is an important cause of retinal ganglion cell death in glaucoma. To elucidate whether apelin protects against retinal neuronal cell death, we examined protective effects of exogenous and endogenous apelin on neuronal cell death induced by intravitreal injection of NMDA in the retinas of mice. An intravitreal injection of NMDA induced neuronal cell death in both the retinal ganglion cell layer and inner nuclear layer, and reduced the amplitudes of scotopic threshold response (STR) in electroretinography studies. Both cell death and STR amplitudes decrease induced by NMDA were prevented by a co-injection of [Pyr1]-apelin-13, and were facilitated by apelin deficiency. The neuroprotective effects of [Pyr1]-apelin-13 were blocked by an apelin receptor APJ antagonist, and by inhibitors of Akt and extracellular signal-regulated kinase 1/2 signaling pathways. Additionally, an intravitreal injection of tumor necrosis factor-α (TNF-α) neutralizing antibody prevented NMDA-induced retinal neuronal cell death, and exogenous and endogenous apelin suppressed NMDA-induced upregulation of TNF-α in the retina. These results suggest that apelin protects neuronal cells against NMDA-induced death via an APJ receptor in the retina, and that apelin may have beneficial effects in the treatment of glaucoma.

Introduction to a new cell transplantation platform via recombinant peptide petaloid pieces and its application to islet transplantation with mesenchymal stem cells.

Cell death cluster in transplanted cells remains a critical obstacle for regeneration strategies. This study describes a novel platform for cell transplantation (CellSaic) consisting of human mesenchymal stem cells (hMSCs) and petaloid pieces of recombinant peptide (RCP), which can prevent cell death by arranging the cells in a mosaic. When hMSC CellSaics were subcutaneously implanted into NOD/SCID mice, hMSC CellSaics prevented cell death and accelerated angiogenesis in the graft, compared to the findings obtained on solely implanting cell spheroids. Additionally, we examined the application of CellSaic for subcutaneous cotransplantation of 200 rat islets with 2 × 10(5) hMSCs into diabetic mice. As the results of blood glucose levels at 1 m, the islet-only group was 398 ± 30 mg/dl and the islets with hMSCs group were 180 ± 65 mg/dl. On the other hand, the islets with hMSCs CellSaic group showed 129 ± 15 mg/dl and significantly improved glucose tolerance (P < 0.05). Additionally, we showed that the surface texture of the RCP petaloid pieces played an important role in graft survival and angiogenesis. It is anticipated that CellSaic will be used as a new platform for cell transplantation and tissue regeneration.

Dopamine inhibits lipopolysaccharide-induced nitric oxide production through the formation of dopamine quinone in murine microglia BV-2 cells.

Dopamine (DA) has been suggested to modulate functions of glial cells including microglial cells. To reveal the regulatory role of DA in microglial function, in the present study, we investigated the effect of DA on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in murine microglial cell line BV-2. Pretreatment with DA for 24 h concentration-dependently attenuated LPS-induced NO production in BV-2 cells. The inhibitory effect of DA on LPS-induced NO production was not inhibited by SCH-23390 and sulpiride, D1-like and D2-like DA receptor antagonists, respectively. In addition, pretreatment with (-)-(6aR,12bR)-4,6,6a,7,8,12b-Hexahydro-7-methylindolo[4,3-a]phenanthridin (CY 208-243) and bromocriptine, D1-like and D2-like DA receptor agonists, respectively, did not affect the LPS-induced NO production. N-Acetylcysteine, which inhibits DA oxidation, completely inhibited the effect of DA. Tyrosinase, which catalyzes the oxidation of DA to DA quionone (DAQ), accelerated the inhibitory effect of DA on LPS-induced NO production. These results suggest that DA attenuates LPS-induced NO production through the formation of DAQ in BV-2 cells.

Noradrenaline increases intracellular glutathione in human astrocytoma U-251 MG cells by inducing glutamate-cysteine ligase protein via ß3-adrenoceptor stimulation.

Glutathione (GSH) plays a critical role in protecting cells from oxidative damage. Since neurons rely on the supply of GSH from astrocytes to maintain optimal intracellular GSH concentrations, the GSH concentration of astrocytes is important for the survival of neighboring neurons against oxidative stress. The neurotransmitter noradrenaline is known to modulate the functions of astrocytes and has been suggested to have neuroprotective properties in neurodegenerative diseases. To elucidate the mechanisms underlying the neuroprotective properties of noradrenaline, in this study, we investigated the effect of noradrenaline on the concentrations of intracellular GSH in human U-251 malignant glioma (MG; astrocytoma) cells. Treatment of the cells with noradrenaline for 24h concentration-dependently increased their intracellular GSH concentration. This increase was inhibited by a non-selective ß-adrenoceptor antagonist propranolol and by a selective ß3-adrenoceptor antagonist SR59230A, but not by a non-selective α-adrenoceptor antagonist phenoxybenzamine, or by a selective ß1-adrenoceptor antagonist atenolol or by a selective ß2-adrenoceptor antagonist butoxamine. In addition, the selective ß3-adrenoceptor agonist CL316243 increased the intracellular GSH in U-251 MG cells. Treatment of the cells with noradrenaline (10µM) for 24h increased the protein level of the catalytic subunit of glutamate-cysteine ligase (GCLc), the rate-limiting enzyme of GSH synthesis; and this increase was inhibited by SR59230A. These results thus suggest that noradrenaline increased the GSH concentration in astrocytes by inducing GCLc protein in them via ß3-adrenoceptor stimulation.

Laser-induced choroidal neovascularization in mice attenuated by deficiency in the apelin-APJ system.

PURPOSE: To investigate the role of the apelin-APJ system in the development of choroidal neovascularization (CNV). METHODS: Experimental CNV was induced by laser photocoagulation in wild-type (WT), apelin-deficient (apelin-KO), and apelin receptor (APJ)-deficient (APJ-KO) mice. The gene expression levels of angiogenic or inflammatory factors were determined by quantitative real-time reverse transcription-polymerase chain reaction. APJ expression in CNV lesions was examined by immunohistochemistry. The sizes of the CNV lesions in the three mouse models were measured and compared histologically using isolectin B4 staining. Macrophage recruitment was measured by flow cytometric analysis. Proliferation of endothelial cells was determined using the alamar Blue assay. RESULTS: Laser photocoagulation significantly increased expression of apelin and APJ in the retina-retinal pigment epithelium (RPE) complex. APJ immunoreactive cells were found in the CNV lesions and colocalized with platelet endothelial cell adhesion molecule-1, an endothelial cell marker. The sizes of the CNV lesions in apelin-KO or APJ-KO mice decreased significantly compared with those in the WT mice. Macrophages in the RPE complex of the apelin-KO mice, in which gene expression of the inflammatory factors was almost equal to that in WT mice, were recruited as a result of laser photocoagulation to the same degree as in WT mice. In addition, apelin small and interfering RNA (siRNA) suppressed proliferation of endothelial cells independently of vascular endothelial growth factor (VEGF) receptor 2 signaling, while VEGF increased expression of apelin and APJ in human umbilical vein endothelial cells. CONCLUSIONS: The results suggested that the apelin-APJ system contributes to CNV development partially independent of the VEGF pathway.

Inhibition of apelin expression switches endothelial cells from proliferative to mature state in pathological retinal angiogenesis.

The recruitment of mural cells such as pericytes to patent vessels with an endothelial lumen is a key factor for the maturation of blood vessels and the prevention of hemorrhage in pathological angiogenesis. To date, our understanding of the specific trigger underlying the transition from cell growth to the maturation phase remains incomplete. Since rapid endothelial cell growth causes pericyte loss, we hypothesized that suppression of endothelial growth factors would both promote pericyte recruitment, in addition to inhibiting pathological angiogenesis. Here, we demonstrate that targeted knockdown of apelin in endothelial cells using siRNA induced the expression of monocyte chemoattractant protein-1 (MCP-1) through activation of Smad3, via suppression of the PI3K/Akt pathway. The conditioned medium of endothelial cells treated with apelin siRNA enhanced the migration of vascular smooth muscle cells, through MCP-1 and its receptor pathway. Moreover, in vivo delivery of siRNA targeting apelin, which causes exuberant endothelial cell proliferation and pathological angiogenesis through its receptor APJ, led to increased pericyte coverage and suppressed pathological angiogenesis in an oxygen-induced retinopathy model. These data demonstrate that apelin is not only a potent endothelial growth factor, but also restricts pericyte recruitment, establishing a new connection between endothelial cell proliferation signaling and a trigger of mural recruitment.

Apelin deficiency accelerates the progression of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the selective loss of motor neurons. Recent studies have implicated that chronic hypoxia and insufficient vascular endothelial growth factor (VEGF)-dependent neuroprotection may lead to the degeneration of motor neurons in ALS. Expression of apelin, an endogenous ligand for the G protein-coupled receptor APJ, is regulated by hypoxia. In addition, recent reports suggest that apelin protects neurons against glutamate-induced excitotoxicity. Here, we examined whether apelin is an endogenous neuroprotective factor using SOD1(G93A) mouse model of ALS. In mouse CNS tissues, the highest expressions of both apelin and APJ mRNAs were detected in spinal cord. APJ immunoreactivity was observed in neuronal cell bodies located in gray matter of spinal cord. Although apelin mRNA expression in the spinal cord of wild-type mice was not changed from 4 to 18 weeks age, that of SOD1(G93A) mice was reduced along with the paralytic phenotype. In addition, double mutant apelin-deficient and SOD1(G93A) displayed the disease phenotypes earlier than SOD1(G93A) littermates. Immunohistochemical observation revealed that the number of motor neurons was decreased and microglia were activated in the spinal cord of the double mutant mice, indicating that apelin deficiency pathologically accelerated the progression of ALS. Furthermore, we showed that apelin enhanced the protective effect of VEGF on H(2)O(2)-induced neuronal death in primary neurons. These results suggest that apelin/APJ system in the spinal cord has a neuroprotective effect against the pathogenesis of ALS.

Caspase-4 directly activates caspase-9 in endoplasmic reticulum stress-induced apoptosis in SH-SY5Y cells.

The present study investigated the function of caspase-4 in endoplasmic reticulum (ER) stress-induced apoptosis in human neuronal cell line SH-SY5Y. Tunicamycin, which is known to induce ER stress, activated both caspase-9 and caspase-4, and the activation of caspase-4 preceded that of caspase-9. The caspase-4 inhibitor LEVD-CHO suppressed both the apoptosis and caspase-9 activation. In addition, human recombinant active caspase-4 cleaved wild type and D330A mutant substituted Asp-330 for alanine of human recombinant procaspase-9, but did not cleave D315A mutant substituted Asp-315 for alanine. These results suggest that caspase-4 directly activates caspase-9 by the processing of procaspase-9 at Asp-315 in ER stress-induced neuronal apoptosis.

Caspase-4 Directly Activates Caspase-9 in Endoplasmic Reticulum Stress-Induced Apoptosis in SH-SY5Y Cells.

The present study investigated the function of caspase-4 in endoplasmic reticulum (ER) stress-induced apoptosis in human neuronal cell line SH-SY5Y. Tunicamycin, which is known to induce ER stress, activated both caspase-9 and caspase-4, and the activation of caspase-4 preceded that of caspase-9. The caspase-4 inhibitor LEVD-CHO suppressed both the apoptosis and caspase-9 activation. In addition, human recombinant active caspase-4 cleaved wild type and D330A mutant substituted Asp-330 for alanine of human recombinant procaspase-9, but did not cleave D315A mutant substituted Asp-315 for alanine. These results suggest that caspase-4 directly activates caspase-9 by the processing of procaspase-9 at Asp-315 in ER stress-induced neuronal apoptosis.

Apelin is a crucial factor for hypoxia-induced retinal angiogenesis.

OBJECTIVE: To investigate the role of endogenous apelin in pathological retinal angiogenesis. METHODS AND RESULTS: The progression of ischemic retinal diseases, such as diabetic retinopathy, is closely associated with pathological retinal angiogenesis, mainly induced by vascular endothelial growth factor (VEGF) and erythropoietin. Although antiangiogenic therapies using anti-VEGF drugs are effective in treating retinal neovascularization, they show a transient efficacy and cause general adverse effects. New therapeutic target molecules are needed to resolve these issues. It was recently demonstrated that the apelin/APJ system, a newly deorphanized G protein-coupled receptor system, is involved in physiological retinal vascularization. Retinal angiography and mRNA expression were examined during hypoxia-induced retinal angiogenesis in a mouse model of oxygen-induced retinopathy. Compared with age-matched control mice, retinal apelin expression was dramatically increased during the hypoxic phase in oxygen-induced retinopathy model mice. APJ was colocalized in proliferative cells, which were probably endothelial cells of the ectopic vessels in the vitreous body. Apelin deficiency hardly induced hypoxia-induced retinal angiogenesis despite the upregulation of VEGF and erythropoietin mRNA in oxygen-induced retinopathy model mice. Apelin small and interfering RNA suppressed the proliferation of endothelial cells independent of the VEGF/VEGF receptor 2 signaling pathway. CONCLUSIONS: These results suggest that apelin is a prerequisite factor for hypoxia-induced retinal angiogenesis.