Knockdown of apoptosis signal-regulating kinase 1 affects ischaemia-induced astrocyte activation and glial scar formation.

Reactive astrocytes play an essential role in determining the tissue response to ischaemia. Formation of a glial scar can block the neuronal outgrowth that is required for restoration of damaged tissue. Therefore, regulation of astrocyte activation is important; however, the mediator of this process has not been fully elucidated. Apoptosis signal-regulating kinase 1 (ASK1) is an early responder to oxidative stress, and plays a pivotal role in the intracellular signalling pathway of apoptosis, inflammation, and differentiation. To confirm whether ASK1 mediates astrocyte activation and leads to glial scar formation after cerebral ischaemia, we conducted in vivo and in vitro experiments. C57BL/6 mice were subjected to occlusion of the middle cerebral artery, and astrocyte cultures were exposed to oxygen-glucose deprivation. After silencing of ASK1 , astrocyte-associated genes were downregulated, as seen with the use of microarrays. The glial fibrillary acidic protein (GFAP) level was decreased, and correlated with the reduction in the ASK1 level. In astrocytes, reduction in the ASK1 level decreased the activity of the p38 pathway, and the levels of transcription factors for GFAP and GFAP transcripts after hypoxia. In the chronic phase, ASK1 depletion reduced glial scar formation and conserved neuronal structure, which may lead to better functional recovery. These data suggest that ASK1 may be an important mediator of ischaemia-induced astrocyte activation and scar formation, and could provide a potential therapeutic target for treatment after ischaemic stroke.

Differential caspase activity in the cortex and striatum with chronic infusion of 3-nitropropionic acid.

Systemic administration of 3-nitropropionic acid (3-NP) facilitates the development of select striatal lesions, and some reports provide clues about this pathology. In this study, we investigated the relationship between reduced levels of brain-derived neurotrophic factor (BDNF) in lesioned brain regions and caspase activity, as well as involvement of apoptosis signal-regulating kinase 1 (ASK1) in caspase activation. We analyzed apoptotic cell death, BDNF distribution, caspase-3 activity, caspase-6 activity, ASK1 expression level, and active ASK1 in the cortex and striatum. There were different levels and distributions of these factors within each sub-region. Caspase-6 activity was reduced with down-regulation of ASK1 in the cortex. BDNF protein levels did not decrease in the cortex, but there was replenishment of severely reduced BDNF in the striatum. The present study suggests that an increase in ASK1 in the damaged cortex is related to caspase-6 activation and is involved in cortical depletion of BDNF in the striatum. Furthermore, with systemic infusion of 3-NP, differential expression of ASK1 in the cortex and striatum suggests that this kinase may modulate caspase activation and striatal degeneration.

Statins Promote Long-Term Recovery after Ischemic Stroke by Reconnecting Noradrenergic Neuronal Circuitry.

Inhibitors of HMG-CoA reductase (statins), widely used to lower cholesterol in coronary heart and vascular disease, are effective drugs in reducing the risk of stroke and improving its outcome in the long term. After ischemic stroke, cardiac autonomic dysfunction and psychological problems are common complications related to deficits in the noradrenergic (NA) system. This study investigated the effects of statins on the recovery of NA neuron circuitry and its function after transient focal cerebral ischemia (tFCI). Using the wheat germ agglutinin (WGA) transgene technique combined with the recombinant adenoviral vector system, NA-specific neuronal pathways were labeled, and were identified in the locus coeruleus (LC), where NA neurons originate. NA circuitry in the atorvastatin-treated group recovered faster than in the vehicle-treated group. The damaged NA circuitry was partly reorganized with the gradual recovery of autonomic dysfunction and neurobehavioral deficit. Newly proliferated cells might contribute to reorganizing NA neurons and lead anatomic and functional recovery of NA neurons. Statins may be implicated to play facilitating roles in the recovery of the NA neuron and its function.

Apoptosis signal-regulating kinase-1 aggravates ROS-mediated striatal degeneration in 3-nitropropionic acid-infused mice.

Apoptosis signal-regulating kinase-1 (ASK1), an early signaling element in the cell death pathway, has been suggested to participate in the pathology of neurodegenerative diseases, which may be associated with environmental factors that impact the diseases. Although it is not entirely elucidated, 3-nitropropionic acid (3-NP) provokes mitochondrial dysfunction and selectively forms striatal lesions similar to those found in Huntington's disease. The current study investigated whether ASK1 is involved in striatal pathology following chronic systemic infusion of 3-NP. The results show that ASK1 acts as a primary mediator of there active oxygen species (ROS) cell death signal cascade in the 3-NP-damaged striatal region by disrupting the positive feedback cycle. In 3-NP-infused striatal lesions, ROS increased ASK1. Superoxide dismutase transgenic (SOD-tg) mice reduced ASK1by scavenging ROS, and reduction of ASK1leads to a reduction in cell death. However, ASK1 down-regulation in 3-NP infusion mice also decreased striatal cell death without scavenging ROS. In contrast decreasing cell death by si-ASK1 treatment along with 3-NP in both SOD tg and wild-type mice (wt), cell death rebounded when ASK1 peptide was added to SOD tg mice. The present study suggests that ROS-inducing ASK1 may be an important step in the pathogenesis of 3-NP infused striatal lesions in murine brains.

Cerebroprotective effects of red ginseng extract pretreatment against ischemia-induced oxidative stress and apoptosis.

Panax ginseng C.A. Meyer has been traditionally used as a medicinal plant and has beneficial effects due to pharmacological properties. Although ginseng is thought to be protective under abnormal conditions, the effects of pretreatment with red ginseng (RG) extract on ischemic stroke have not been fully elucidated. We investigated the protective effects of RG extract after focal cerebral ischemia in mice. Crude RG extract (360 mg/kg) was administered intraperitoneally for 2 weeks. Mice were then subjected to occlusion of the middle cerebral artery for 1 hour, followed by reperfusion for 4 and 24 hours. Pretreatment with RG extract followed by ischemia/reperfusion (I/R) resulted in significant reduction of oxidized hydroethidine signals in ischemic areas. At 4 and 24 hours after I/R, the number of 8-hydroxyguanosine and apoptosis signal-regulating kinase 1 (ASK1)-positive cells decreased in the ischemic penumbra as seen using immunofluorescent staining. Western blotting showed that RG efficiently attenuated the protein levels of activated ASK1 in the ischemic penumbra. Consequently, DNA fragmentation and the infarct volume were reduced by RG extract pretreatment 24 hours after I/R. Also, RG extract resulted in better performance in rotarod test after I/R. Thus, RG pretreatment demonstrates a protective effect at suppressing ischemia-induced oxidative stress and apoptosis in ischemic lesions. Pretreatment with crude RG extract may be an effective strategy for preventing brain injury after an ischemic stroke.

The osteogenic effects of sponges synthesized with biomaterials and nano-hydroxyapatite.

Artificial bone substitutes have been developed using various biomaterials for use in medicine. Silk fibroin (SF) displays excellent mechanical properties and cell compatibility. Nonetheless, the mechanical properties of silk fibroin scaffolds used in artificial bone substitutes are weaker than those of natural bone, and silk fibroin is deficient as an osteogenic agent. This limits their effectiveness in bone tissue engineering. We added nano-hydroxyapatite (nHAp) particles to an existing cell-based artificial bone substitute with a silk fibroin scaffold, which will improve its mechanical properties and osteogenic efficacy, leading to significant bone regeneration. The mechanical characters of silk fibroin modifying with nHAp were measured by Atomic Force Microscopy Analysis, dispersive x-ray spectroscopy, Porosity measurement, and Microcomputed Tomography. The proliferation and toxicity of a fibroin/dextran/collagen sponge (FDS) containing nHAp were evaluatedin vitro, and its osteogenic efficacy was evaluated using nude mouse and rabbit radius defect models. The defect area was repaired and showed callus formation of new bone in the rabbit radius defect models of the nHAp-FDS-treated group, whereas the defect area was unchanged in the FDS-treated group. The nHAp-FDS manufactured in this study showed significant bone regeneration owing to the synergistic effects of the components, such as those due to the broad range of pore sizes in the sponge and protein adsorbability of the nHAp, which could be suggested as a better supportive material for bone tissue engineering.

Injectable Click Fibroin Bioadhesive Derived from Spider Silk for Accelerating Wound Closure and Healing Bone Fracture.

Wound closure is a critical step in postoperative wound recovery. Substantial advancements have been made in many different means of facilitating wound closure, including the use of tissue adhesives. Compared to conventional methods, such as suturing, tissue bioadhesives better accelerate wound closure. However, several existing tissue adhesives suffer from cytotoxicity, inadequate tissue adhesive strength, and high costs. In this study, a series of bioadhesives was produced using non-swellable spider silk-derived silk fibroin protein and an outer layer of swellable polyethylene glycol and tannic acid. The gelation time of the spider silk-derived silk fibroin protein bioadhesive is less than three minutes and thus can be used during rapid surgical wound closure. By adding polyethylene glycol (PEG) 2000 and tannic acid as co-crosslinking agents to the N-Hydroxysuccinimide (NHS), and 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) reaction, the adhesive strength of the bioadhesive became 2.5 times greater than that of conventional fibrin glue adhesives. Silk fibroin bioadhesives do not show significant cytotoxicity in vitro compared with other bioadhesives. In conclusion, silk fibroin bioadhesive is promising as a new medical tool for more effective and efficient surgical wound closure, particularly in bone fractures.

Regulation of PDGF signalling and vascular remodelling by peroxiredoxin II.

Platelet-derived growth factor (PDGF) is a potent mitogenic and migratory factor that regulates the tyrosine phosphorylation of a variety of signalling proteins via intracellular production of H2O2 (refs 1, 2-3). Mammalian 2-Cys peroxiredoxin type II (Prx II; gene symbol Prdx2) is a cellular peroxidase that eliminates endogenous H2O2 produced in response to growth factors such as PDGF and epidermal growth factor; however, its involvement in growth factor signalling is largely unknown. Here we show that Prx II is a negative regulator of PDGF signalling. Prx II deficiency results in increased production of H2O2, enhanced activation of PDGF receptor (PDGFR) and phospholipase Cgamma1, and subsequently increased cell proliferation and migration in response to PDGF. These responses are suppressed by expression of wild-type Prx II, but not an inactive mutant. Notably, Prx II is recruited to PDGFR upon PDGF stimulation, and suppresses protein tyrosine phosphatase inactivation. Prx II also leads to the suppression of PDGFR activation in primary culture and a murine restenosis model, including PDGF-dependent neointimal thickening of vascular smooth muscle cells. These results demonstrate a localized role for endogenous H2O2 in PDGF signalling, and indicate a biological function of Prx II in cardiovascular disease.

Decisive role of apurinic/apyrimidinic endonuclease/Ref-1 in initiation of cell death.

The apurinic/apyrimidinic endonuclease/redox effector factor-1 (APE/Ref-1) is involved in the base excision repair of apurinic/apyrimidinic sites induced by oxidative DNA damage. APE/Ref-1 was decreased by kainic acid (KA) injury in a time-dependent manner at the level of proteins, not transcripts. We investigated whether alteration of APE/Ref-1 amounts would influence hippocampal cell fate, survival or death, after KA injury. Overexpression of APE/Ref-1 using adenovirus and restoration of APE small peptides significantly reduced KA-induced hippocampal cell death. Both silencing of APE/Ref-1 by siRNA and inhibition of endonuclease by an antibody significantly increased caspase-3 activity and apoptotic cell death triggered from the early time after exposure to KA. These findings suggest that cell death is initiated by reducing APE/Ref-1 protein and inhibiting its repair function in spite of enough protein amounts. In conclusion, APE/Ref-1 may be a regulator of cell death initiation, and APE small peptides could provide molecular mechanism-based therapies for neuroprotection in progressive excitotoxic neuronal damage.

Selective striatal cell loss is ameliorated by regulated autophagy of the cortex.

AIMS: The harmful cellular environment leads to brain damage, and each brain subregion exhibits a differential vulnerability to its effects. This study investigated the causes of selectively striatal cell loss in systemic 3-nitropropionic acid (3-NP) infused mice. MAIN METHODS: This study was performed in the neuronal cell line, primary neuron, cultured mouse brain, and mice brain tissues. The 3-NP solution was delivered using an osmotic mini-pump system for 7 days. ROS in brain tissue were detected and evaluated with the signals of CM-H2DCFDA for total cellular ROS and MitoSOX Red for mitochondrial ROS. Cellular ROS and the functional status of mitochondria were assessed with a detection kit and analyzed using flow cytometry. To quantify oxidative damaged DNA, apurinic/apyrimidinic (AP) site numbers in DNA were measured. The protein expression level was assessed using Western blotting, and immunohistochemistry was performed. Cleaved caspase-3 activities were measured by using an enzyme-linked immunosorbent assay (ELISA) kit. KEY FINDINGS: By 3-NP, mitochondrial dysfunction was higher in the striatum than in the cortex, and mitochondria-derived ROS levels were higher in the striatum than in the cortex. However, autophagy that may restore the energy depletion resulting from mitochondrial dysfunction occurred comparably less in the striatum than in the cortex. Inhibition of ASK1 by NQDI1 regulates MAPK signaling, apoptosis, and autophagy. Regulated autophagy of the cortex improved non-cell autonomously striatal damaged condition. SIGNIFICANCE: This study illustrated that the different vulnerabilities of the brain subregions, striatum or cortex, against 3-NP are rooted in different mitochondria-derived ROS amounts and autophagic capacity.

WITHDRAWN: The potential role of Ask1 in caspase-6-related huntingtin cleavage and striatal vulnerability.

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Preconditioning with chronic cerebral hypoperfusion reduces a focal cerebral ischemic injury and increases apurinic/apyrimidinic endonuclease/redox factor-1 and matrix metalloproteinase-2 expression.

Atherosclerosis may cause severe stenosis of the arteries supplying the brain, which induces chronic cerebral hypoperfusion. Although an infarction often occurs in the chronically hypoperfused brain area, it has been uncertain whether the stroke severity is attenuated or increased when further decrease of blood flow occurs. To test the hypothesis that chronic cerebral hypoperfusion is protective against the subsequent severe ischemia, we examined the effect of chronic cerebral hypoperfusion on brains subjected to acute focal ischemia. Spontaneous hypertensive rats were subjected to middle cerebral artery occlusion/reperfusion four weeks after bilateral common carotid artery ligation (BCAL) or sham operation. The rats with BCAL had smaller infarctions, determined by 2,3,5-triphenyltetrazolium hydrochloride staining, and less severe neurologic deficits than those with sham operation. The number of DNA-damaged cells, examined by the in situ nick translation study, was significantly reduced in animals with BCAL. Immunoreactivity for apurinic/apyrimidinic endonuclease/redox factor-1, which plays a role in cellular defense mechanism, was markedly increased in those with BCAL. Indirect evidence of extracellular matrix remodeling, which might be associated with adaptive arteriogenesis or angiogenesis, was obtained in the form of increased matrix metalloproteinase-2 activity in them. These findings provide experimental evidence that chronic cerebral hypoperfusion would be protective against subsequent severe ischemic insults.

Minocycline inhibits caspase-dependent and -independent cell death pathways and is neuroprotective against hippocampal damage after treatment with kainic acid in mice.

Although minocycline has been generally thought to have neuroprotective properties, the neuroprotective role of minocycline has not been investigated in the animal model of epilepsy. In this study, we investigated whether minocycline is neuroprotective against kainic acid (KA)-induced cell death through the caspase-dependent or -independent mitochondrial apoptotic pathways. Adult male ICR mice were subjected to seizures by intrahippocampal KA injection with vehicle or with minocycline. For cell death analysis, TdT-mediated dUTP-biotin nick end labeling and cresyl-violet staining were performed. Western blot analysis and immunofluorescent staining for cytochrome c and apoptosis-inducing factor (AIF) were performed. Cell death was reduced in minocycline-treated mice. Cytosolic translocation of cytochrome c and subsequent activation of caspase-3 were diminished by minocycline treatment. AIF nuclear translocation and subsequent large-scale DNA fragmentation were also reduced in minocycline-treated mice. Thus, this study suggests that minocycline inhibits both caspase-dependent and -independent apoptotic pathways and may be neuroprotective against hippocampal damage after KA treatment.

Apoptosis signal-regulating kinase 1 mediates striatal degeneration via the regulation of C1q.

Apoptosis signal-regulating kinase-1 (ASK1), an early signaling element in the cell death pathway, has been hypothesized to participate in the pathology of neurodegenerative diseases. The systemic administration of 3-nitropropionic acid (3-NP) facilitates the development of selective striatal lesions. However, it remains unclear whether specific neurons are selectively targeted in 3-NP-infused striatal degeneration. Recently, it has been proposed that complement-mediated synapse elimination may be reactivated aberrantly in the pathology of neurodegenerative diseases. We hypothesized that ASK1 is involved in striatal astrocyte reactivation; reactive astrocyte secretes molecules detrimental to neuron; and striatal neurons are more susceptible to these factors. Our results indicate that striatal astrocyte is reactivated and ASK1 level increases after 3-NP general and chronic infusion. Reactive striatal astrocyte increases TGF-beta differentially to cortex and striatum. ASK1 may be involved in regulation of astrocyte TGF-beta and it is linked to the C1q level in spatial and temporal, and moreover in the earlier stage of progressing striatal neuronal loss. Conclusively the present study suggests that ASK1 mediates 3-NP toxicity and regulates C1q level through the astrocyte TGF-beta. And also it may suggest that C1q level may be a surrogate of prediction marker representing neurodegenerative disease progress before developing behavioral impairment.

Peroxiredoxin II preserves cognitive function against age-linked hippocampal oxidative damage.

Reactive oxygen species (ROS), routinely produced in biological reactions, contribute to both normal aging and age-related decline in cognitive function. However, little is known regarding the involvement of specific antioxidants in the underlying mechanism(s). Here, we examined if peroxiredoxin II (Prx II) scavenges intracellular ROS that cause age-dependent mitochondrial decay in hippocampal CA1 pyramidal neurons and subsequent impairment of learning and memory. Age-dependent mitochondrial ROS generation and long-term potentiation (LTP) decline were more prominent in hippocampal neurons in Prx II(-/-) than in wild-type mice. Additionally, Prx II(-/-) mice failed to activate synaptic plasticity-related cellular signaling pathways involving CREB, CaMKII, and ERK, or to maintain functional integrity of their mitochondria. Dietary vitamin E alleviated Prx II deficiency-related deficits, including mitochondrial decay and CREB signaling, resulting in restoration of the abrupt cognitive decline in aged Prx II(-/-) mice. These results suggest that Prx II help maintain hippocampal synaptic plasticity against age-related oxidative damage.

Korean Addenbrooke's Cognitive Examination Revised (K-ACER) for differential diagnosis of Alzheimer's disease and subcortical ischemic vascular dementia.

AIM: Sensitive, specific neuropsychological screening tests, such as the Addenbrooke's Cognitive Examination Revised (ACE-R), are essential for dementia diagnosis. We aimed to validate the use of the Korean version of ACE-R (K-ACER) to differentiate Alzheimer's disease (AD) from subcortical ischemic vascular dementia (SIVD). METHODS: Standard tests for dementia screening were applied to 156 subjects (84 controls, 30 AD, 42 SIVD), and total and sub-domain scores on the K-ACER, as well as the sub-domain ratio (VLOM), were compared. RESULTS: The reliability of the K-ACER was very good (α-coefficient 0.84), and cut-off score for dementia was determined (cut-off value 78, sensitivity 0.93, specificity 0.95). The likelihood ratio for dementia was calculated as between 78 and 82. At a cut-off of 78, the likelihood of dementia was 18.6:1. Although a comparison of K-ACER scores between AD and SIVD patients revealed significant differences in verbal fluency, language domain and VLOM ratio, sensitivity and specificity for differential diagnosis between AD and SVID proved less accurate. CONCLUSION: The K-ACER is a rapid, sensitive and specific dementia screening test. Though sub-domains of items may be useful for differentiating between AD and SIVD, sensitivity and specificity is less accurate than dementia screening itself.

Neuronal death/survival signaling pathways in cerebral ischemia.

Cumulative evidence suggests that apoptosis plays a pivotal role in cell death in vitro after hypoxia. Apoptotic cell death pathways have also been implicated in ischemic cerebral injury in in vivo ischemia models. Experimental ischemia and reperfusion models, such as transient focal/global ischemia in rodents, have been thoroughly studied and the numerous reports suggest the involvement of cell survival/death signaling pathways in the pathogenesis of apoptotic cell death in ischemic lesions. In these models, reoxygenation during reperfusion provides a substrate for numerous enzymatic oxidation reactions. Oxygen radicals damage cellular lipids, proteins and nucleic acids, and initiate cell signaling pathways after cerebral ischemia. Genetic manipulation of intrinsic antioxidants and factors in the signaling pathways has provided substantial understanding of the mechanisms involved in cell death/survival signaling pathways and the role of oxygen radicals in ischemic cerebral injury. Future studies of these pathways may provide novel therapeutic strategies in clinical stroke.