Houng-Chi Liou

Autophagy protects neuron from Aβ-induced cytotoxicity

Autophagy is a degradation pathway for the turnover of dysfunctional organelles or aggregated proteins in cells. Extracellular accumulation of β-amyloid peptide has been reported to be a major cause of Alzheimers disease (AD) and large numbers of autophagic vacuoles accumulate in the brain of AD patient. However, how autophagic process is involved in Aβ-induced neurotoxicity and how Aβ peptide is transported into neuron and metabolized is still unknown. In order to study the role of autophagic process in Aβ-induced neurotoxicity, EGFP-LC3 was over-expressed in SH-SY5Y cells (SH-SY5Y/pEGFP-LC3). It was found that treatment with Aβ25-35, Aβ1-42 or serum-starvation induced strong autophagy response in SH-SY5Y/pEGFP-LC3. Confocal double-staining image showed that exogenous application of Aβ1-42 in medium caused the co-localization of Aβ1-42 with LC3 in neuronal cells. Concomitant treatment of Aβ with a selective α7nAChR antagonist, α-bungarotoxin (α-BTX), enhanced Aβ-induced neurotoxicity in SH-SY5Y cells. On the other hand, nicotine (nAChR agonist) enhanced the autophagic process and also inhibited cell death following Aβ application. In addition, nicotine but not α-BTX increased primary hippocampal neuronal survival following Aβ treatment. Furthermore, using Atg7 siRNA to inhibit autophagosome formation in an early step or α7nAChR siRNA to knockdown α7nAChR significantly enhanced Aβ-induced neurotoxicity. Confocal double-staining image shows that nicotine treatment in the presence of Aβ enhanced the co-localization of α7nAChR with autophagosomes. These results suggest that α7nAChR may act as a carrier to bind with eAβ and internalize into cytoplasm and further inhibit Aβ-induced neurotoxicity via autophagic degradation pathway. Our results suggest that autophagy process plays a neuroprotective role against Aβ-induced neurotoxicity. Defect in autophagic regulation or Aβ-α7nAChR transport system may impair the clearance of Aβ and enhance the neuronal death.

Overexpression of Heme Oxygenase-1 Protects Dopaminergic Neurons against 1-Methyl-4-Phenylpyridinium-Induced Neurotoxicity

Heme oxygenase-1 (HO-1) is up-regulated in response to oxidative stress and catalyzes the degradation of pro-oxidant heme to carbon monoxide (CO), iron, and bilirubin. Intense HO-1 immunostaining in the Parkinsonian brain is demonstrated, indicating that HO-1 may be involved in the pathogenesis of Parkinsonism. We here locally injected adenovirus containing human HO-1 gene (Ad-HO-1) into rat substantia nigra concomitantly with 1-methyl-4-phenylpyridinium (MPP+). Seven days after injection of MPP+ and Ad-HO-1, the brain was isolated for immunostaining and for measurement of dopamine content and inflammatory cytokines. It was found that overexpression of HO-1 significantly increased the survival rate of dopaminergic neurons; reduced the production of tumor necrosis factor α (TNF-α) and interleukin-1β (IL-1β) in substantia nigra; antagonized the reduction of striatal dopamine content induced by MPP+; and also up-regulated brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) expression in substantia nigra. Apomorphine-induced rotation after MPP+ treatment was also inhibited by Ad-HO-1. On the other hand, inhibition of HO enzymatic activity by zinc protoporphyrin-IX facilitated the MPP+-induced rotatory behavior and enhanced the reduction of dopamine content. HO-1 overexpression also protected dopaminergic neurons against MPP+-induced neurotoxicity in midbrain neuron-glia cocultures. Overexpression of HO-1 increased the expression of BDNF and GDNF in astrocytes and BDNF in neurons. Our results indicate that HO-1 induction exerts neuroprotection both in vitro and in vivo. Pharmacological or genetic approaches targeting HO-1 may represent a promising and novel therapeutic strategy in treating Parkinsonism.

Inhibition of Hypoxia-Induced Increase of Blood-Brain Barrier Permeability by YC-1 through the Antagonism of HIF-1α Accumulation and VEGF Expression

Cerebral microvascular endothelial cells form the anatomical basis of the blood-brain barrier (BBB), and the tight junctions of the BBB are critical for maintaining brain homeostasis and low permeability. Ischemia/reperfusion is known to damage the tight junctions of BBB and lead to permeability changes. Here we investigated the protective role of 3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole (YC-1), against chemical hypoxia and hypoxia/reoxygenation (H/R)-induced BBB hyperpermeability using adult rat brain endothelial cell culture (ARBEC). YC-1 significantly decreased CoCl2- and H/R-induced hyperpermeability of fluorescein isothiocyanate (FITC)-dextran in cell culture inserts. It was found that the decrease and disorganization of tight junction protein zonular occludens-1 (ZO-1) in response to CoCl2, and H/R was antagonized by YC-1. The protection of YC-1 may result from the inhibition of HIF-1α accumulation and production of its downstream target vascular endothelial growth factor (VEGF). VEGF alone significantly increased FITC-dextran permeability and down-regulated mRNA and protein levels of ZO-1 in ARBECs. We further used animal model to examine the effect of YC-1 on BBB permeability after cerebral ischemia/reperfusion. It was found that YC-1 significantly protected the BBB against ischemia/reperfusion-induced injury. Taken together, these results indicate that YC-1 may inhibit HIF-1α accumulation and VEGF production, which in turn protect BBB from injury caused by hypoxia.

Enhancement of Fibronectin Synthesis and Fibrillogenesis by BMP-4 in Cultured Rat Osteoblast†

The skeletal extracellular matrix produced by osteoblasts contains the glycoprotein fibronectin (Fn), which regulates the adhesion, differentiation, and function of osteoblasts. Fn fibrillogenesis is involved in the process of bone mineralization. Bone morphogenetic proteins (BMPs) can be isolated from organic bone matrix and are able to initiate de novo cartilage and bone formation. In this study, the effect of BMP‐4 on Fn fibrillogenesis in cultured rat osteoblasts was examined. BMP‐4 enhanced Fn synthesis and extracellular Fn assembly in primary cultured osteoblasts. In addition, the extracellular assembly of Fn from exogenously applied soluble human Fn was also increased by BMP‐4. It has been reported that α5β1 integrin is related to Fn fibrillogenesis. The synthesis of both α5 and β1 integrins was upregulated by BMP‐4. Immunocytochemistry showed that the clustering of α5 and β1 integrins was also increased by BMP‐4. BMP‐4 increased fibril formation of Fn and the adhesion of osteoblasts onto Fn matrix, which was inhibited by disintegrin triflavin and Gly‐Arg‐Gly‐Asp‐Ser (GRGDS) peptide. Phosphorylation of extracellular signal‐regulated kinase (ERK) and focal adhesion kinase (FAK) was increased by BMP‐4. Enhancement of extracellular Fn fibrillogenesis and the mRNA expression of β1 integrin by BMP‐4 were inhibited by ERK kinase (MEK) inhibitor PD98059. These results suggest that the enhancement of extracellular Fn fibrillogenesis by BMP‐4 in cultured osteoblasts is related to the increase of the synthesis of Fn and clustering of α5 and β1 integrins. ERK is involved in the signaling pathway of BMP‐4 in regulating Fn fibrillogenesis in osteoblasts.

YC-1 attenuates LPS-induced proinflammatory responses and activation of nuclear factor-κB in microglia

An inflammatory response in the central nervous system mediated by the activation of microglia is a key event in the early stages of the development of neurodegenerative diseases. LPS has been reported to cause marked microglia activation. It is very important to develop drugs that can inhibit microglia activation and neuroinflammation. Here, we investigated the inhibitory effect of YC‐1, a known activator of soluble guanylyl cyclase, against LPS‐induced inflammatory responses in microglia.

The mechanism of heme oxygenase-1 action involved in the enhancement of neurotrophic factor expression

Heme oxygenase-1 (HO-1) is up-regulated in response to oxidative stress and catalyzes the degradation of pro-oxidant heme to carbon monoxide (CO), iron and bilirubin. Bilirubin is a potent antioxidant and neuroprotectant. Neurotrophic factors of BDNF and GDNF also play important roles in survival and morphological differentiation of dopaminergic neurons. We have previously found that HO-1 induction by adenovirus containing human HO-1 gene (Ad-HO-1) in substantia nigra of rat increases BDNF and GDNF expression. We here further examined the possible mechanism of HO-1 action involved in the enhancement of neurotrophic factor expression. Treatment of anti-BDNF/GDNF antibody significantly enhanced dopaminergic neuronal death, whereas Ad-HO-1 co-treatment was able to antagonize the apoptosis-inducing effect of these antibodies. The confocal imaging shows that HO-1 induction appeared in dopaminergic neuron, astrocyte and microglia at 24 h after injecting Ad-HO-1. HO-1 induced-BDNF/GDNF mRNA expression in substantia nigra was 26/21 folds of that of the contralateral Ad-injected side. The downstream product bilirubin increased GDNF expression through ERK and PI3K-Akt pathways, and also enhanced NFkappaB (p65 and p50) nuclear translocation in glia-enriched cultures. In addition, bilirubin also enhanced BDNF expression through similar pathway in cortical neuron-enriched cultures. We also examined the effect of another HO-1 product, CO, by using CO donor. [Ru(CO)3Cl2]2 increased neurotrophic factor expression via sGC-PKG pathway in both neuron and glia. These results indicate that the downstream products of HO-1, i.e. bilirubin and CO, modulate BDNF and GDNF expression in neuron and astrocyte.

A forward loop between glioma and microglia: glioma-derived extracellular matrix-activated microglia secrete IL-18 to enhance the migration of glioma cells.

The mediators and cellular effectors of inflammation are important constituents of the local environment of tumors. In some occasions, oncogenic changes induce an inflammatory microenvironment that promotes the progression of tumors. In gliomas, the presence of microglia may represent tumor‐related inflammation and microglia activation, and subsequent inflammatory responses may influence tumor growth and metastasis. Here, we found that C6 glioma—but not primary astrocyte‐derived extracellular matrix (ECM) could activate microglia, including primary microglia and BV‐2 cell line, and activated microglia‐secreted interleukin (IL)‐18, a potent inflammatory cytokine of the IL‐1 family, to promote C6 migration. In addition, by coating purified ECM components, it was found that secretion of IL‐18 by activated microglia was enhanced when microglia encountered with fibronectin and vitronectin. Furthermore, IL‐18‐induced C6 migration and microfilament disassembly were antagonized by iNOS inhibitor, guanylate cyclase inhibitor, and protein kinase G inhibitor. Taken together, these results indicate that IL‐18 secreted by microglia, which was activated by C6 glioma‐derived ECM, enhanced migration of C6 glioma through NO/cGMP pathway. J. Cell. Physiol. 227: 558–568, 2012.

Potentiation of spontaneous acetylcholine release from motor nerve terminals by glutamate in Xenopus tadpoles

Extracellular application of glutamate (100 microM) increased the spontaneous secretion of acetylcholine, as well as the amplitude and decay time of miniature endplate potentials at developing neuromuscular synapses in Xenopus tadpoles. Kainate, quisqualate and N-methyl-D-aspartate (100 microM each) increased miniature endplate potential frequency by 26-, 13- and four-fold, respectively. The rank order of efficacy at 100 microM was kainate > quisqualate > N-methyl-D-aspartate > glutamate. The effect of kainate on miniature endplate potential frequency was inhibited by 6-cyano-2,3-dihydroxy-7-nitroquinoxaline (20 microM), but not by (+/-)-2-amino-5-phosphonovalerate (20 microM). Treatment with the voltage-dependent Ca2+ channel blockers verapamil (10 microM), Cd2+ (100 microM) or omega-conotoxin (1 microM) inhibited the potentiating action of kainate on miniature endplate potential frequency. On the other hand, 1S,3R-1-aminocyclopentane-1,3-dicarboxylate (300 microM), a glutamate metabotropic receptor agonist, inhibited the spontaneous acetylcholine release, which was antagonized by the application of 2-amino-3-phosphonopropionate (500 microM). The potentiating effect of glutamate receptor agonists on the miniature endplate potential frequency declined or disappeared in older Xenopus tadpoles. Quisqualate (100 microM) and N-methyl-D-aspartate (100 microM) but not kainate (30 microM) increased the amplitude and decay time of miniature endplate potential, whereas 1S, 3R-1-aminocyclopentane-1, 3-dicarboxylate (300 microM) only increased the decay time of miniature endplate potentials. These results suggest that there are kainate/quisqualate and N-methyl-D-aspartate receptors existing in the motor nerve terminals of younger Xenopus tadpoles and the activation of these receptors potentiates spontaneous acetylcholine release through increasing Ca2+ influx. Our data suggest that the presynaptic glutamate receptors on cholinergic terminals may be involved in feedback regulation of acetylcholine secretion at earlier embryonic stages.

Involvement of 15‐lipoxygenase in the inflammatory arthritis

15‐Lipoxygenase (15‐LOX) is involved in many pathological processes. The aim of this study is to examine the role of 15‐LOX in the matrix metalloproteinase (MMP) expression and inflammatory arthritis. It was found that treatment of 15‐LOX downstream product of 15‐(S)‐HETE (15‐S‐hydroxyeicosatetraenoic acid) increased the mRNA and protein levels of MMP‐2 in rheumatoid arthritis synovial fibroblast (RASF) derived from rheumatoid arthritis patients. The enhancement effect of 15‐(S)‐HETE was antagonized by the addition of LY294002 (PI3K inhibitor) and PDTC (NF‐κB inhibitor). Treatment of 15‐(S)‐HETE increased the phosphorylation of AKT, nuclear translocation of p65 and the breakdown of IκBα. TNF‐α and IL‐1β are the key cytokines involved in arthritis and also increase the activity of MMP‐2 in RASF, which was antagonized by pretreatment with 15‐LOX inhibitor PD146176 or knockdown of 15‐LOX. It was also found that these two cytokines increased the expression of 15‐LOX in RASF. Treatment of glucocorticoid but not NSAIDs inhibited 15‐(S)‐HETE‐induced expression of MMP‐2. In comparison with wild‐type mice, adjuvant‐induced arthritis and MMP‐2 expression in synovial membrane were markedly inhibited in 15‐LOX knockout (KO) mice. These results indicate that 15‐LOX plays an important role in the disease progression of arthritis and may be involved in the inflammatory action induced by TNF‐α and IL‐1β. 15‐LOX is thus a good target for developing drugs in the treatment of inflammatory arthritis. J. Cell. Biochem. 113: 2279–2289, 2012.

Inhibition of neuropathic pain by a potent disintegrin—triflavin

Injury to peripheral nerves may result in severe and intractable neuropathic pain. Many efforts have been focused on the elucidation of the mechanisms of neuropathic pain. It was found here that integrin plays an important role in the induction of neuropathic pain and treatment of disintegrin is able to attenuate neuropathic pain. The rats were induced hyperalgesia by tightly ligating the L5 spinal nerve and cut just distal to the ligature on one side. Mechanical and thermal stimuli were applied in the middle dermatome of the hind paw. Epidural administration of triflavin (TFV), an arginine-glycine-aspartic acid (RGD) containing disintegrin, inhibited hyperalgesia induced by either mechanical or thermal stimulation. Immunohistochemistry showed that the sprouting of sympathetic nerves into DRG by neuropathic surgery was markedly inhibited by TFV. Beta 1 integrin mRNA of L5 DRG increased immediately 1 day after tight ligation and cut of L5 spinal nerve. However, beta 1 integrin mRNA in uninjured L4 DRG increased later on Day 3 after surgery. On the other hand, alpha-CGRP precursor mRNA decreased in ipsilateral L5 DRG but increased in L4 DRG after neuropathic surgery. Immunohistochemistry shows that beta 3 integrins of L5 as well as L4 increased in response to neuropathic surgery and administration of triflavin antagonized the increasing action. These results suggest that there is interaction between injured and uninjured neurons and the induction of neuropathic pain is related to neuronal sprouting. Disintegrin is able to inhibit neuronal sprouting and the induction of hyperalgesia induced by peripheral nerve injury and may thus be a new category of drugs to be developed for the treatment of neuropathic pain.