Young-Ji Shiao

The Neuroprotective Effects of Phytoestrogens on Amyloid β Protein-induced Toxicity Are Mediated by Abrogating the Activation of Caspase Cascade in Rat Cortical Neurons

Amyloid β protein (Aβ) elicits a toxic effect on neurons in vitro and in vivo. In present study we attempt to elucidate the mechanism by which Aβ confers its neurotoxicity. The neuroprotective effects of phytoestrogens on Aβ-mediated toxicity were also investigated. Cortical neurons treated with 5 μm Aβ-(25–35) for 40 h decreased the cell viability by 45.5 ± 4.6% concomitant with the appearance of apoptotic morphology. 50 μm kaempferol and apigenin decreased the Aβ-induced cell death by 81.5 ± 9.4% and 49.2 ± 9.9%, respectively. Aβ increased the activity of caspase 3 by 10.6-fold and to a lesser extent for caspase 2, 8, and 9. The Aβ-induced activation of caspase 3 and release of cytochromec showed a biphasic pattern. Apigenin abrogated Aβ-induced cytochrome c release, and the activation of caspase cascade. Kaempferol showed a similar effect but to a less extent. Kaempferol was also capable of eliminating Aβ-induced accumulation of reactive oxygen species. These two events accounted for the remarkable effect of kaempferol on neuroprotection. Quercetin and probucol did not affect the Aβ-mediated neurotoxicity. However, they potentiated the protective effect of apigenin. Therefore, these results demonstrate that Aβ elicited activation of caspase cascades and reactive oxygen species accumulation, thereby causing neuronal death. The blockade of caspase activation conferred the major neuroprotective effect of phytoestrogens. The antioxidative activity of phytoestrogens also modulated their neuroprotective effects on Aβ-mediated toxicity.

Mechanism mediating oligomeric Aβ clearance by naïve primary microglia

The accumulation of soluble oligomeric amyloid-β peptide (oAβ) proceeds the formation of senile plaques and contributes to synaptic and memory deficits in Alzheimers disease (AD). The mechanism of mediating microglial oAβ clearance remains unclear and thought to occur via scavenger receptors (SRs) in microglia. SRs respond to their ligands in a subtype-specific manner. Therefore, we sought to identify the specific subtypes of SRs that mediate oAβ internalization and proteases that degrade oAβ species in naïve primary microglia. The component of oAβ species were characterized by western blot analysis, analytical ultracentrifugation analysis, and atomic force microscopy. The oAβ species remained soluble in the medium and microglial lysates during incubation at 37 °C. SR-A, but not CD36, mediated oAβ internalization in microglia as suggested by the use of subtype-specific neutralizing antibodies and small interfering RNAs (siRNAs). Immunoprecipitation analysis showed that oAβ interacted with SR-A on the plasma membrane. After internalization, over 40% of oAβ vesicles were trafficked toward lysosomes and degraded by cysteine proteases, including cathepsin B. The inhibitors of proteasome, neprilysin, matrix metalloproteinases, and insulin degrading enzyme failed to protect internalized oAβ from degradation. Our study suggests that SR-A and lysosomal cathepsin B are critical in microglial oAβ clearance, providing insight into how microglia are involved in the clearance of oAβ and their roles in the early stages of AD.

TGF-β1 blockade of microglial chemotaxis toward Aβ aggregates involves SMAD signaling and down-regulation of CCL5

BackgroundOveractivated microglia that cluster at neuritic plaques constantly release neurotoxins, which actively contribute to progressive neurodegeneration in Alzheimers disease (AD). Therefore, attenuating microglial clustering can reduce focal neuroinflammation at neuritic plaques. Previously, we identified CCL5 and CCL2 as prominent chemokines that mediate the chemotaxis of microglia toward beta-amyloid (Aβ)aggregates. Although transforming growth factor-β1 (TGF-β1) has been shown to down-regulate the expression of chemokines in activated microglia, whether TGF-β1 can reduce the chemotaxis of microglia toward neuritic plaques in AD remains unclear.MethodsIn the present study, we investigated the effects of TGF-β1 on Aβ-induced chemotactic migration of BV-2 microglia using time-lapse recording, transwell assay, real-time PCR, ELISA, and western blotting.ResultsThe cell tracing results suggest that the morphological characteristics and migratory patterns of BV-2 microglia resemble those of microglia in slice cultures. Using this model system, we discovered that TGF-β1 reduces Aβ-induced BV-2 microglial clustering in a dose-dependent manner. Chemotactic migration of these microglial cells toward Aβ aggregates was significantly attenuated by TGF-β1. However, these microglia remained actively moving without any reduction in migration speed. Pharmacological blockade of TGF-β1 receptor I (ALK5) by SB431542 treatment reduced the inhibitory effects of TGF-β1 on Aβ-induced BV-2 microglial clustering, while preventing TGF-β1-mediated cellular events, including SMAD2 phosphorylation and CCL5 down-regulation.ConclusionsOur results suggest that TGF-β1 reduces Aβ-induced microglial chemotaxis via the SMAD2 pathway. The down-regulation of CCL5 by TGF-β1 at least partially contributes to the clustering of microglia at Aβ aggregates. The attenuating effects of SB431542 upon TGF-β1-suppressed microglial clustering may be mediated by restoration of CCL5 to normal levels. TGF-β1 may ameliorate microglia-mediated neuroinflammation in AD by preventing activated microglial clustering at neuritic plaques.

Caspase 3 involves in neuroplasticity, microglial activation and neurogenesis in the mice hippocampus after intracerebral injection of kainic acid

BackgroundThe roles of caspase 3 on the kainic acid-mediated neurodegeneration, dendritic plasticity alteration, neurogenesis, microglial activation and gliosis are not fully understood. Here, we investigate hippocampal changes using a mouse model that receive a single kainic acid-intracerebral ventricle injection. The effects of caspase 3 inhibition on these changes were detected during a period of 1 to 7 days post kainic acid injection.ResultNeurodegeneration was assessed by Fluoro-Jade B staining and neuronal nuclei protein (NeuN) immunostaining. Neurogenesis, gliosis, neuritic plasticity alteration and caspase 3 activation were examined using immunohistochemistry. Dendritic plasticity, cleavvage-dependent activation of calcineurin A and glial fibrillary acidic protein cleavage were analyzed by immunoblotting. We found that kainic acid not only induced neurodegeneration but also arouse several caspase 3-mediated molecular and cellular changes including dendritic plasticity, neurogenesis, and gliosis. The acute caspase 3 activation occurred in pyramidal neurons as well as in hilar interneurons. The delayed caspase 3 activation occurred in astrocytes. The co-injection of caspase 3 inhibitor did not rescue kainic acid-mediated neurodegeneration but seriously and reversibly disturb the structural integrity of axon and dendrite. The kainic acid-induced events include microglia activation, the proliferation of radial glial cells, neurogenesis, and calcineurin A cleavage were significantly inhibited by the co-injection of caspase 3 inhibitor, suggesting the direct involvement of caspase 3 in these events. Alternatively, the kainic acid-mediated astrogliosis is not caspase 3-dependent, although caspase 3 cleavage of glial fibrillary acidic protein occurred.ConclusionsOur results provide the first direct evidence of a causal role of caspase 3 activation in the cellular changes during kainic acid-mediated excitotoxicity. These findings may highlight novel pharmacological strategies to arrest disease progression and control seizures that are refractory to classical anticonvulsant treatment.

Falcarindiol impairs the expression of inducible nitric oxide synthase by abrogating the activation of IKK and JAK in rat primary astrocytes

1 The effects of falcarindiol on the expression of inducible nitric oxide synthase (iNOS) induced by lipopolysaccharide/interferon‐γ (LPS/IFN‐γ) in rat primary astrocytes were investigated. The molecular mechanisms underlying falcarindiol that confers its effect on iNOS expression were also elucidated. 2 Falcarindiol abrogated the LPS/IFN‐γ‐mediated induction of iNOS by about 80%. Falcarindiol attenuated the induction of iNOS in a concentration‐dependent manner. 3 The inhibitory effect of falcarindiol on iNOS induction was attributable to decrease in the protein content and the mRNA level of iNOS. 4 Treatment with 50 μM of falcarindiol for 30 min decreased LPS/IFN‐γ‐induced nuclear factor‐κB (NF‐κB) activation by 32%. 5 Treatment with 50 μM of falcarindiol for 60 min diminished the LPS/IFN‐γ‐mediated activation of IκB kinase‐α (IKK‐α) and IKK‐β by 28.2 and 29.7%, respectively. 6 Falcarindiol modulated the nuclear translocation of signal transducer and activator of transcription 1 (Stat1) in a time‐dependent manner. Falcarindiol (50 μM) decreased the tyrosine phosphorylation of janus kinase 1 (JAK1) by 84.8% at 5 min. Falcarindiol also abrogated the tyrosine phoshorylation of JAK2 by 82.3% at 10 min. 7 The present study demonstrates that falcarindiol attenuated the activation of IKK and JAK contributing to the blockade of activation of NF‐κB and Stat1, thereby leading to the suppression of iNOS expression.

Erinacine S, a Rare Sesterterpene from the Mycelia of Hericium erinaceus

A new sesterterpene, erinacine S, and one cyathane diterpene xyloside, erinacine A, were isolated from the ethanol extract of the mycelia of Hericium erinaceus. Their structures were elucidated by spectroscopic and X-ray analysis. A 30-day oral course of erinacines A and S attenuated Aβ plaque burden in the brains of 5-month-old female APP/PS1 transgenic mice. Moreover, erinacines A and S significantly increased the level of insulin-degrading enzyme in cerebral cortex.

Impaired cognition and cerebral glucose regulation are associated with astrocyte activation in the parenchyma of metabolically stressed APPswe/PS1dE9 mice

Although metabolic syndrome was suggested to be a risk factor for Alzheimers disease (AD), the role of metabolic stress in the initiation of AD pathology remains unclear. In this study, metabolic stress was induced by a high-fat diet and low-dose injection of streptozotocin (HFSTZ) before the appearance of senile plaques in APP/PS1 transgenic mice. We found that, HFSTZ treatment exacerbated amyloid beta burden and astrocyte activation in the vicinity of plaques. Moreover, we observed an upregulation of astrocytic S100B expression in the brain parenchyma of HFSTZ-treated APP/PS1 mice concurrent with increased interleukin-6 expression in cerebral microvascular cells. To determine the impact of HFSTZ treatment on brain function, we performed [(18)F]fludeoxyglucose-positron emission tomography and analyzed nesting behavior. HFSTZ treatment impaired nest construction and cerebral glucose metabolism in several brain regions of APP/PS1 mice during the early stage of AD. These results suggest that HFSTZ-induced peripheral metabolic stress may contribute to vascular inflammation and astrocyte reactivity in the parenchyma and may impair activity of daily living skill and cerebral glucose metabolism in APP/PS1 mice.

Enlargement of Aβ aggregates through chemokine-dependent microglial clustering

The number of microglia surrounding senile plaques is correlated with the size of plaques in Alzheimers disease (AD). It is unclear whether more microglia are passively recruited toward larger senile plaques or, conversely, microglia recruited to senile plaques directly contribute to the growth of plaques. In this study, BV-2 microglia were used to delineate the role of microglia in the growth of plaques using time-lapse recording. Aggregated beta amyloid peptide (Abeta)-induced BV-2 microglia to form clusters. The recruitment of BV-2 microglia bearing membrane-adhered Abeta enlarged preexisting Abeta aggregates. The receptors involved in the microglial uptake of Abeta, including integrin, formyl peptide like receptor 1, and scavenger receptors, also mediated the microglial clustering. Neutralization antibodies against chemokines significantly attenuated Abeta-induced microglial clustering and the enlargement of Abeta aggregates. Our results reveal a novel role of microglia in directly increasing the size of Abeta aggregates and suggest the targeting of Abeta-mediated microglial chemotactic migration in developing therapeutic interventions for AD.

Tournefolic acid B methyl ester attenuates glutamate‐induced toxicity by blockade of ROS accumulation and abrogating the activation of caspases and JNK in rat cortical neurons

The effects of nine polyphenolic compounds on glutamate‐mediated toxicity were investigated. The underlying mechanisms by which a polyphenolic compound confers its effect were also elucidated. Treatment of cortical neurons with 50 µm glutamate for 24 h decreased cell viability by 45.8 ± 7.9%, and 50 µm of tournefolic acid B methyl ester attenuated glutamate‐induced cell death by 46.8 ± 17.8%. Glutamate increased the activity of caspase 35.2‐fold, and to a similar extent for caspase 2, 6, 8 and 9. Tournefolic acid B methyl ester abrogated glutamate‐induced activation of caspase 2, 3, 6 and 9 by about 70%, and to a lesser extent for caspase 8. Treatment with glutamate for 1 h elevated reactive oxygen species (ROS) by 208.3 ± 21.3%. Tournefolic acid B methyl ester eliminated the glutamate‐induced accumulation of ROS. Glutamate increased the phosphorylation of p54‐c‐jun N‐terminal kinase (JNK) concomitantly with activation of the endogenous antioxidant defense system. Tournefolic acid B methyl ester at 50 µm diminished the activity of p54‐JNK in control and glutamate‐treated cells, coinciding with the abolishment of the glutamate‐triggered antioxidant defense system. Therefore, tournefolic acid B methyl ester blocked the activation of the caspase cascade, eliminated ROS accumulation and abrogated the activation of JNK, thereby conferring a neuroprotective effect on glutamate‐mediated neurotoxicity.

Multiplex Brain Proteomic Analysis Revealed the Molecular Therapeutic Effects of Buyang Huanwu Decoction on Cerebral Ischemic Stroke Mice

Stroke is the second-leading cause of death worldwide, and tissue plasminogen activator (TPA) is the only drug used for a limited group of stroke patients in the acute phase. Buyang Huanwu Decoction (BHD), a traditional Chinese medicine prescription, has long been used for improving neurological functional recovery in stroke. In this study, we characterized the therapeutic effect of TPA and BHD in a cerebral ischemia/reperfusion (CIR) injury mouse model using multiplex proteomics approach. After the iTRAQ-based proteomics analysis, 1310 proteins were identified from the mouse brain with <1% false discovery rate. Among them, 877 quantitative proteins, 10.26% (90/877), 1.71% (15/877), and 2.62% (23/877) of the proteins was significantly changed in the CIR, BHD treatment, and TPA treatment, respectively. Functional categorization analysis showed that BHD treatment preserved the integrity of the blood–brain barrier (BBB) (Alb, Fga, and Trf), suppressed excitotoxicity (Grm5, Gnai, and Gdi), and enhanced energy metabolism (Bdh), thereby revealing its multiple effects on ischemic stroke mice. Moreover, the neurogenesis marker doublecortin was upregulated, and the activity of glycogen synthase kinase 3 (GSK-3) and Tau was inhibited, which represented the neuroprotective effects. However, TPA treatment deteriorated BBB breakdown. This study highlights the potential of BHD in clinical applications for ischemic stroke.