Huey-Jen Tsay

cAMP/PKA Regulates Osteogenesis, Adipogenesis and Ratio of RANKL/OPG mRNA Expression in Mesenchymal Stem Cells by Suppressing Leptin

Background Mesenchymal stem cells (MSCs) are a pluripotent cell type that can differentiate into adipocytes, osteoblasts and other cells. The reciprocal relationship between adipogenesis and osteogenesis was previously demonstrated; however, the mechanisms remain largely unknown. Methods and Findings We report that activation of PKA by 3-isobutyl-1 methyl xanthine (IBMX) and forskolin enhances adipogenesis, the gene expression of PPARγ2 and LPL, and downregulates the gene expression of Runx2 and osteopontin, markers of osteogenesis. PKA activation also decreases the ratio of Receptor Activator of the NF-κB Ligand to Osteoprotegerin (RANKL/OPG) gene expression – the key factors of osteoclastogenesis. All these effects are mediated by the cAMP/PKA/CREB pathway by suppressing leptin, and may contribute to PKA stimulators-induced in vivo bone loss in developing zebrafish. Conclusions Using MSCs, the center of a newly proposed bone metabolic unit, we identified cAMP/PKA signaling, one of the many signaling pathways that regulate bone homeostasis via controlling cyto-differentiation of MSCs and altering RANKL/OPG gene expression.

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.

Movement disorder and neuromuscular change in zebrafish embryos after exposure to caffeine

Though caffeine is broadly distributed in many plants and foods, little is known about the teratogenic effects of caffeine during early embryonic development. Here, we used zebrafish as a model to test toxicity and teratogenicity since they have transparent eggs, making the organogenesis of zebrafish embryos easier to observe. When the exposure doses of caffeine were less than 150 ppm (17.5, 35, 50, 100 and 150 ppm), the zebrafish embryos exhibited no significant differences in survival rates after comparison with vehicle-control (0 ppm) group. As the exposure dosages increased, the survival rates decreased. No embryos survived after treatment with 300 ppm caffeine or higher dosages. The most evident change in embryos treated with caffeine was a shorter body length (vehicle-control: 3.26+/-0.01 mm, n=49; vs 150 ppm of caffeine: 2.67+/-0.03 mm, n=50). In addition, caffeine-treated embryos exhibited significantly reduced tactile sensitivity frequencies of touch-induced movement (vehicle-control: 9.93+/-0.77 vs 17.5-150 ppm caffeine: 5.37+/-0.52-0.10+/-0.06). Subtle changes are easily observed by staining with specific monoclonal antibodies F59, Znp1 and Zn5 to detect morphological changes in muscle fibers, primary motor axons and secondary motor axon projections, respectively. Our data show that the treatment of caffeine leads to misalignment of muscle fibers and motor neuron defects, especially secondary motor neuron axonal growth defects.

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.

Inactivation of zebrafish mrf4 leads to myofibril misalignment and motor axon growth disorganization

Mrf4 is a basic helix‐loop‐helix (bHLH) transcription factor associated with myogenesis. Two mrf4 transcripts, mrf4_tv1 and mrf4_tv2, were identified in zebrafish generated by alternative splicing. To study their biological functions, we separately injected the Mrf4‐morpholinos, including MO1 (mrf4_tv1:mrf4_tv2 knockdown), MO2+MO3 (mrf4_tv1:mrf4_tv2 knockdown), MO3 (mrf4_tv1 knockdown), and MO4 (mrf4_tv2 knockdown), into zebrafish embryos to observe mrf4 gene knockdown phenotypes. No phenotypic abnormalities were observed following injection with 0.5 ng of MO1 but those injected with 4.5, 9, or 13.5 ng displayed curved‐body phenotypes, such as indistinct somite boundaries, and a lack of uniformly sized cell blocks. Similar results were also observed in the (MO2+MO3)‐, MO3‐, and MO4‐injected groups. To further investigate the molecular mechanisms that lead to curved‐body phenotypes, we stained embryos with α‐bungrotoxin and specific monoclonal antibodies F59, Znp1, and Zn5 to detect morphological changes in acetyl‐choline receptor (AChR) clusters, muscle fibers, common path of the primary neurons, and secondary neurons axonal projections, respectively. Our results show that the muscle fibers of mrf4_(tv1:tv2)‐morphant aligned disorderly and lost their integrity and attachment, while the defects became milder in either mrf4_tv1‐morphant or mrf4_tv2‐morphant. On the other hand, reduced axonal projections and AChR clusters were found in both mrf4_tv2‐morphant and mrf4_(tv1:tv2)‐morphant but distributed normally in the mrf4_tv1‐morphant. We conclude that Mrf4_tv2 is involved in alignment of muscle fibers, and Mrf4_tv1 might have cooperative function with Mrf4_tv2 in muscle fiber alignment, without affecting the muscle‐nerve connection. Developmental Dynamics 237:1043–1050, 2008.

Heatstroke induces c-fos expression in the rat hypothalamus.

We induced heat stress in urethane-anesthetized rats (the animals were exposed to an ambient temperature at 42 degrees C), and monitored their colon temperature, mean arterial pressure and local cerebral blood flow. Rats 0, 20, 40 or 80 min after heat stress were sacrificed for determination of c-fos mRNA and protein expression in the paraventricular nucleus (PVN), supraoptic nucleus (SON) and preoptic nucleus (PON). The heatstroke, which appears as profound decreases in both mean arterial pressure and local cerebral blood flow and increases in colon temperature, is produced 80 min after heat stress. We show the c-fos mRNA and protein is strongly induced in all these nuclei of rat hypothalamus after the onset of heatstroke. We conclude that c-fos expression in the hypothalamus during rat heatstroke is associated with hyperthermia, arterial hypotension and cerebral ischemia.

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.

ATP-stimulated c-fos and zif268 mRNA expression is inhibited by chemical hypoxia in a rat brain-derived type 2 astrocyte cell line, RBA-2.

The stimulus‐transcriptional coupling during ischemia/hypoxia was examined for ATP‐stimulated expression of immediate early genes (IEGs; c‐fos, zif268, c‐myc and nur77) in a rat brain‐derived type 2 astrocyte cell line, RBA‐2. Incubation of cells with 1 mM of extracellular ATP stimulated time‐dependent expression of c‐fos and zif268. ATP induced the largest increases in zif268 mRNA and a lesser one in c‐fos mRNA. ATP also induced a slight increase in nur77 mRNA but was ineffective in inducing c‐myc expression in these cells. Brief exposure of cells to potassium cyanide to simulate chemical hypoxia induced 9‐fold and 7‐fold transient increases in c‐fos and zif268 expression, respectively, but did not affect c‐myc or nur77 expression. When cyanide and ATP were added together, the expression of c‐fos and zif268 expression was inhibited, and the effect was mimicked by simulating chemical hypoxia with sodium azide. To elucidate the mechanism involved, the effect of cyanide on ATP‐stimulated increases in intracellular Ca2+ concentrations, [Ca2+]i, and phospholipase D (PLD) activities were measured. Cyanide induced an increase in [Ca2+]i and further enhanced the ATP‐stimulated increases in [Ca2+]i and PLD activities. Nevertheless, metabolic inhibitor, iodoacetate, blocked the ATP‐induced c‐fos and partially inhibited zif268 expression, and deprivation of cells with glucose also inhibited the ATP‐induced c‐fos expression. Taken together, these results demonstrate that both extracellular ATP and chemical hypoxia induce c‐fos and zif268 expression in RBA‐2 type 2 astrocytes. The chemical hypoxia inhibited ATP‐stimulated c‐fos and zif268 expression is not due to alterations in Ca2+ and PLD signaling, and is at least partially related to metabolic disturbance in these cells. J. Cell. Biochem. 77:323–332, 2000.

Expression of zebrafish Hoxa1a in neuronal cells of the midbrain and anterior hindbrain

The expression pattern of zebrafish hoxa1a mRNA during embryonic development was studied. Herein, we show that hoxa1a mRNA is expressed in the ventral region of both the midbrain and anterior hindbrain during the developmental period from the pharyngula to the protruding-mouth stages via whole-mount in situ hybridization. Furthermore, double-labeling with anti-zHu antibody confirms that the zebrafish hoxa1a gene is expressed in neuronal cells. The observed temporal and spatial distributions of zebrafish hoxa1a mRNA differ greatly from the expression patterns of zebrafish hoxb1a and hoxb1b paralagous genes. In addition, in embryos injected with mouse ihh mRNA, hoxa1a-expressing cells increase in number with a dorsalized expression pattern in the midbrain.

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.