A pro-drug of the green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) prevents differentiated SH-SY5Y cells from toxicity induced by 6-hydroxydopamine

Regular consumption of green tea benefits people in prevention from cardiovascular disorders, obesity as well as neurodegenerative diseases. (-)-Epigallocatechin-3-gallate (EGCG) is regarded as the most biologically active catechin in green tea. However, the stability and bioavailability of EGCG are restricted. The purpose of the present study was to investigate whether a pro-drug, a fully acetylated EGCG (pEGCG), could be more effective in neuroprotection in Parkinsonism mimic cellular model. Retinoic acid (RA)-differentiated neuroblastoma SH-SY5Y cells were pre-treated with different concentrations of EGCG and pEGCG for 30 min and followed by incubation of 25 μM 6-hydroxydopamine (6-OHDA) for 24 h. We found that a broad dosage range of pEGCG (from 0.1 to 10 μM) could significantly reduce lactate dehydrogenase release. Likewise, 10 μM of pEGCG was effective in reducing caspase-3 activity, while EGCG at all concentrations tested in the model failed to attenuate caspase-3 activity induced by 6-OHDA. Furthermore, Western-blot analysis showed that Akt could be one of the specific signaling pathways stimulated by pEGCG in neuroprotection. It was demonstrated that 25 μM of 6-OHDA significantly suppressed the phosphorylation level of Akt. Only pEGCG at 10 μM markedly increased its phosphorylation level compared to 6-OHDA alone. Taken together, as pEGCG has higher stability and bioavailbility for further investigation, it could be a potential neuroprotective agent and our current findings may offer certain clues for optimizing its application in future. © 2009 Elsevier Ireland Ltd. All rights reserved.postprin

Significance of molecular signaling for protein translation control in neurodegenerative diseases

It has long been known that protein synthesis is inhibited in neurological disorders. Protein synthesis includes protein transcription and translation. While many studies about protein transcription have been done in the last decade, we are just starting to understand more about the impact of protein translation. Protein translation control can be accomplished at the initiation or elongation steps. In this review, we will focus on translation control at initiation. Neurons have long neurites in which proteins have to be transported from the cell body to the end of the neurite. Since supply of proteins cannot meet the need of neuronal activity at the spine, protein locally translated at the spine will be a good solution to replace the turnover of proteins. Therefore, local protein translation is an important mechanism to maintain normal neuronal functions. In this notion, we have to separate the concept of global and local protein translation control. Both global and local protein translation control modulate normal neuronal functions from development to cognitive functions. Increasing lines of evidence show that they also play significant roles in neurodegenerative diseases, e.g. neuronal apoptosis, synaptic degeneration and autophagy. We summarize all the evidence in this review and focus on the control at initiation. The new live-cell imaging technology together with photoconvertible fluorescent probes allows us to investigate newly translated proteins in situ. Protein translation control is another line to modulate neuronal function in neuron-neuron communication as well as in response to stress in neurodegenerative diseases. Copyright © 2007 S. Karger AG.link_to_subscribed_fulltex

Characterizing the neuroprotective effects of alkaline extract of Lycium barbarum on β-amyloid peptide neurotoxicity

Lycium barbarum is an oriental medicinal herb that has long been used for its anti-aging and cell-protective properties. Previous studies have shown that aqueous extracts from L. barbarum exhibit neuroprotection via inhibiting pro-apoptotic signaling pathways. Other active components can also be accomplished by novel alkaline extraction method, which may give different profiles of water-soluble components. We hypothesize that another active component obtained by alkaline extraction method exerts different biological mechanisms to protect neurons. In this study, we aim to examine the neuroprotective effects from the alkaline extract of L. barbarum, namely LBB, to attenuate β-amyloid (Aβ) peptide neurotoxicity. Primary cortical neurons were exposed to Aβ-peptides inducing apoptosis and neuronal cell death. Pretreatment of LBB significantly reduced the level of lactate dehydrogenase (LDH) release and the activity of caspase-3 triggered by Aβ. "Wash-out" procedures did not reduce its neuroprotective effects, suggesting that LBB may not bind directly to Aβ. We have further isolated three subfractions from LBB, namely LBB-0, LBB-I and LBB-II. LBB-I and LBB-II showed differential neuroprotective effects. Western blot analysis demonstrated that LBB-I and LBB-II markedly enhanced the phosphorylation of Akt. Taken together, our results suggested that the glycoconjugate isolated from novel alkaline extraction method can open up a new avenue for drug discovery in neurodegenerative diseases. © 2007 Elsevier B.V. All rights reserved.link_to_subscribed_fulltex

Antagonizing β-amyloid peptide neurotoxicity of the anti-aging fungus Ganoderma lucidum

Ganoderma lucidum (Leyss. ex Fr.) Karst. (Lingzhi) is a medicinal fungus used clinically in many Asian countries to promote health and longevity. Synaptic degeneration is another key mode of neurodegeneration in Alzheimer's disease (AD). Recent studies have shown the loss of synaptic density proteins in each individual neuron during the progression of AD. It was recently reported that β-amyloid (Aβ) could cause synaptic dysfunction and contribute to AD pathology. In this study, we reported that aqueous extract of G. lucidum significantly attenuated Aβ-induced synaptotoxicity by preserving the synaptic density protein, synaptophysin. In addition, G. lucidum aqueous extract antagonized Aβ-triggered DEVD cleavage activities in a dose-dependent manner. Further studies elucidated that phosphorylation of c-Jun N-terminal kinase, c-Jun, and p38 MAP kinase was attenuated by G. lucidum in Aβ-stressed neurons. Taken together, the results prove a hypothesis that anti-aging G. lucidum can prevent harmful effects of the exterminating toxin Aβ in AD. © 2007 Elsevier B.V. All rights reserved.link_to_subscribed_fulltex

Disruptions of endoplasmic reticulum and mitochondria prime mTOR suppression in low molecular weight Aβ-induced autophagy

Poster Presentations: P1-239BACKGROUND: Alzheimer’s disease (AD) is a progressive neurodegenerative disorder which pathological hallmarks associate with the deposition of insoluble beta-amyloid (Aβ) plaques in various regions of the brain. Reports suggested that various soluble Aβ species can induce neurotoxicity and are capable in inducing autophagy, the type II programmed cell death, with elusive mechanism of activation. METHODS: Primary cultures of rat embryonic cortical neurons were prepared and transfected with LC3-, DFCP-1-, endoplasmic reticulum (ER) retention signal (KDEL)-, and mitochondrial retention signal expressing plasmids. Neurons were treated with low molecular weight (LMW) Aβ. Protein lysates were collected afterwards for Western-blot analysis. Autophagosome, omegosome, lysosome and the morphology of the ER and mitochondria were examined by confocal microscopy. Quantitative data was analyzed by one way analysis of variance (ANOVA) followed by Student Newman Keul test according to the statistical program SigmaStat® (Jandel Scientific) to compare the level significance. A p-value less than 0.05 was regarded as significant, at *p < 0.05. RESULTS: In the present study, we demonstrated that low molecular weight Aβ induced autophagic vacuole and omegosome formation which showed partial colocalization in cortical neurons. Aβ induced AMP-activated protein kinase (AMPK) but did not cause significant suppression on mTOR and its downstream target p70 S6 kinase (p70S6K) simultaneously. On the other hand, low MW Aβ caused morphological damages in both ER and mitochondria at early time points. Organelle damage coincided with up-regulations in autophagy and lysosomal machinery. CONCLUSIONS: Our results suggest that low MW Aβ first confers its toxicity on the intracellular organelles by causing structural damages to initiate autophagy. Dysregulated cellular metabolism due to mitochondrial damage activates AMPK, which suppresses mTOR at later time points to exert synergistic effects on autophagy regulation

Characterization of the effects of anti-aging medicine Fructus lycii on β-amyloid peptide neurotoxicity

Alzheimer's disease (AD) is an age-related neurodegenerative disease. There are increasing lines of evidence showing that the molecular signaling pathways in aged cells are altered so that cells are susceptible to injury. We and other laboratories have demonstrated the significant involvement of double-stranded RNA-dependent protein kinase (PKR) in β-amyloid (Aβ) peptide neurotoxicity and in AD. Fructus lycii (the fruit of Lycium barbarum) has long been used in oriental medicine as an anti-aging agent. Our previous studies demonstrated that the aqueous extract isolated from L. barbarum exhibited significant protection on cultured neurons against harmful chemical toxins such as Aβ and dithiothreitol. We also showed that the polysaccharide-containing extract (LBP) from L. barbarum exhibited neuroprotective effects in the retina against ocular hypertension in a laser-induced glaucoma animal model. In this study, we aimed to investigate whether LBP can elicit neuroprotection to neurons stressed by Aβ peptides. Furthermore, we planned to isolate and identify the neuroprotective agent from LBP using chromatographic methods. Our results showed that pretreatment of LBP effectively protected neurons against Aβ-induced apoptosis by reducing the activity of both caspase-3 and -2, but not caspase-8 and -9. A new arabino-galactan-protein (LBP-III) was isolated from LBP and attenuated Aβ peptide-activated caspase-3-like activity. LBP-III markedly reduced the phosphorylation of PKR triggered by Aβ peptide. Since the phosphorylation state of PKR increased with age, reduction of its phosphorylation triggered by Aβ peptide may implicate that LBP-III from Fructus lycii is a potential neuroprotective agent in AD. As herbal medicine has received increasing attention for the treatment of AD, our study will open a window for the development of a neuroprotective agent for anti-aging from Chinese medicine.link_to_subscribed_fulltex

Effects of all-trans-retinoic acid on human SH-SY5Y neuroblastoma as in vitro model in neurotoxicity research

Human neuroblastoma SH-SY5Y is a dopaminergic neuronal cell line which has been used as an in vitro model for neurotoxicity experiments. Although the neuroblastoma is usually differentiated by all-trans-retinoic acid (RA), both RA-differentiated and undifferentiated SH-SY5Y cells have been used in neuroscience research. However, the changes in neuronal properties triggered by RA as well as the subsequent responsiveness to neurotoxins have not been comprehensively studied. Therefore, we aim to re-evaluate the differentiation property of RA on this cell line. We hypothesize that modulation of signaling pathways and neuronal properties during RA-mediated differentiation in SH-SY5Y cells can affect their susceptibility to neurotoxins. The differentiation property of RA was confirmed by showing an extensive outgrowth of neurites, increased expressions of neuronal nuclei, neuron specific enolase, synaptophysin and synaptic associated protein-97, and decreased expression of inhibitor of differentiation-1. While undifferentiated SH-SY5Y cells were susceptible to 6-OHDA and MPP+, RA-differentiation conferred SH-SY5Y cells higher tolerance, potentially by up-regulating survival signaling, including Akt pathway as inhibition of Akt removed RA-induced neuroprotection against 6-OHDA. As a result, the real toxicity cannot be revealed in RA-differentiated cells. Therefore, undifferentiated SH-SY5Y is more appropriate for studying neurotoxicity or neuroprotection in experimental Parkinson's disease research. © 2008 Elsevier Inc. All rights reserved.link_to_subscribed_fulltex

Temporal relationship of autophagy and apoptosis in neurons challenged by low molecular weight β-amyloid peptide

Alzheimer's disease (AD) is an aging-related progressive neurodegenerative disorder. Previous studies suggested that various soluble Aβ species are neurotoxic and able to activate apoptosis and autophagy, the type I and type II programmed cell death, respectively. However, the sequential and functional relationships between these two cellular events remain elusive. Here we report that low molecular weight Aβ triggered cleavage of caspase 3 and poly (ADP-ribose) polymerase to cause neuronal apoptosis in rat cortical neurons. On the other hand, Aβ activated autophagy by inducing autophagic vesicle formation and autophagy related gene 12 (ATG12), and up-regulated the lysoso-mal machinery for the degradation of autophagosomes. Moreover, we demonstrated that activation of autophagy by Aβ preceded that of apoptosis, with death associated protein kinase phosphorylation as the potential molecular link. More importantly, under Aβ toxicity, neurons exhibiting high level of autophagosome formation were absent of apoptotic features, and inhibition of autophagy by 3-methylade-nine advanced neuronal apoptosis, suggesting that autophagy can protect neurons from Aβ-induced apoptosis. © 2011 The Authors Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.link_to_subscribed_fulltex

Determinants of daytime blood pressure in relation to obstructive sleep apnea in men

This study investigated the roles of different potential pathophysiological mechanisms in the determination of blood pressure in relation to obstructive sleep apnea. The study was designed as a cross-sectional study. Consecutive healthy male subjects who were to undergo polysomnography were recruited. Demographic and anthropometric data were collected. Blood pressure measurements were taken in the evening before sleep and the next morning on waking. Overnight urinary samples for catecholamines and fasting blood for cortisol, insulin, glucose, and lipids were taken. Ninety-four men were analyzed, with a mean age of 43.7 ± 9.3 years and mean apnea-hypopnea index (AHI) of 27.5 ± 26.2 events/h. Sixty-nine patients (73%) had obstructive sleep apnea (AHI ≥5). Urinary catecholamines were positively correlated with severity of sleep apnea, independent of obesity. Blood pressure measurements correlated with age, obesity, severity of sleep apnea, and urinary catecholamines. Regression analysis showed that sleep indices and urinary catecholamines were independent determinants of morning systolic and diastolic blood pressure, respectively, while total cholesterol and waist circumference were respective additional factors. Urinary catecholamines and waist circumference were determinants of evening blood pressure, with morning cortisol being an additional determinant for diastolic blood pressure. Obstructive sleep apnea and related sympathetic activity contributed significantly to the determination of daytime blood pressure in overweight middle-aged men without overt cardiometabolic diseases, and other contributing factors include abdominal obesity, total cholesterol, and cortisol levels. © 2009 Springer Science+Business Media, LLC.link_to_subscribed_fulltex

Low molecular weight Aβ induces collapse of endoplasmic reticulum

The endoplasmic reticulum (ER) is a dynamic multifunction organelle that is responsible for Ca2+ homeostasis, protein folding, post-translational modification, protein degradation, and transportation of nascent proteins. Disruption of ER architecture might affect the normal physiology of the cell. In yeast, expansion of the ER is observed under unfolded protein response (UPR) and subsequently induces autophagy initiated from the ER. Here, we found that soluble low molecular weight of Aβ disrupted the anchoring between ER and microtubules (MT) and induced collapse of ER. In addition, it decreased the stability of MT. Subsequently, low molecular weight Aβ triggered autophagy and enhanced lysosomal degradation, as shown by electron microscopy and live-cell imaging. Dysfunction of ER can be further proved in postmortem AD brain and transgenic mice bearing APP Swedish mutation by immunohistochemical analysis of calreticulin. Treatment with Taxol, a MT-stabilizing agent, could partially inhibit collapse of the ER and induction of autophagy. The results show that Aβ-induced disruption of MT can affect the architecture of the ER. Collapse/aggregation of the ER may play an important role in Aβ peptide-triggered neurodegenerative processes. © 2009 Elsevier Inc. All rights reserved.link_to_subscribed_fulltex