Apoptotic cell death influences the signaling activity of the amyloid precursor protein through ShcA and Grb2 adaptor proteins in neuroblastoma SH-SY5Y cells

The amyloid precursor protein (APP) is an ubiquitous receptor-like molecule involved in the pathogenesis of Alzheimer's disease (AD). APP and some of its C-terminal proteolytic fragments (CTFs) have been shown to be phosphorylated and to interact with cytosolic phosphotyrosine binding (PTB) domain containing proteins involved in cell signaling and vesicular transport. Among others, the interaction between tyrosine-phosphorylated CTFs and ShcA-Grb2 adaptors is highly enhanced in AD brain. Here we have identified in SH-SY5Y neuroblastoma cells an interaction between APP holoprotein and the adaptor Grb2. Upon activation of apoptotic cell death this interaction is rapidly degraded, APP is partially cleaved and the complex APP/Grb2 is replaced by a new complex between CTFs and ShcA that still involves Grb2. The formation of these complexes is regulated by beta-site APP-cleaving enzyme 1 and influences the phosphorylation of mitogen-activated protein kinase p44/42 extracellular signal-regulated kinase as well as the level of apoptotic death of the cells. These data suggest a dual role in cell signaling for APP and its CTFs in neuroblastoma cells, in a manner similar to that previously reported for other tyrosine kinase receptor, through a tightly regulated coupling with alternative intracellular adaptors to control the signaling of the cell.     

Acetylcholinesterase inhibitors increase ADAM10 activity by promoting its trafficking in neuroblastoma cell lines

Acetylcholinesterase inhibitors (AChEIs) are the only currently available drugs for treating Alzheimer's Disease (AD). Some authors have suggested a function of AChEIs not only in the induction of AChE overproduction and alternative splicing shifts but also a possible role of these drugs in amyloid metabolism beyond their well-known symptomatic effect. Here, we investigate the mechanisms of action of the AChEI donepezil on APP (amyloid precursor protein) metabolism and on the activity/trafficking of the alpha-secretase candidate ADAM 10, in differentiated human neuroblastoma cells (SH-SY5Y). In these cells, the activity of AChE is significantly decreased after 2 h of donepezil treatment. Further, SH-SY5Y cells released significantly more sAPPalpha into the medium, whereas total APP levels in cell lysates were unchanged. Interestingly, treated cells showed increased ADAM 10 levels in membrane compartments. This effect was prevented by pretreatment with tunicamycin or brefeldin, suggesting that donepezil affects trafficking and/or maturation of ADAM 10; additionally, this pretreatment significantly decreased sAPPalpha levels. Pre-incubation with atropine decreased release of sAPPalpha significantly but did not revert ADAM 10 activity to control levels further suggesting that donepezil acts not solely through a purely receptor mediated pathway. These findings indicate that donepezil exerts multiple mechanisms involving processing and trafficking of key proteins involved in AD pathogenesis.     

Interaction of Double-stranded RNA-dependent Protein Kinase (PKR) with the Death Receptor Signaling Pathway in Amyloid �� (A��)-treated Cells and in APPSLPS1 Knock-in Mice

For 10 years, research has focused on signaling pathways controlling translation to explain neuronal death in Alzheimer Disease (AD). Previous studies demonstrated in different cellular and animal models and AD patients that translation is down-regulated by the activation of double-stranded RNA-dependent protein kinase (PKR). Among downstream factors of PKR, the Fas-associated protein with a death domain (FADD) and subsequent activated caspase-8 are responsible for PKR-induced apoptosis in recombinant virus-infected cells. However, no studies have reported the role of PKR in death receptor signaling in AD. The aim of this project is to determine physical and functional interactions of PKR with FADD in amyloid-β peptide (Aβ) neurotoxicity and in APP_SLPS1 KI transgenic mice. In SH-SY5Y cells, results showed that Aβ42 induced a large increase in phosphorylated PKR and FADD levels and a physical interaction between PKR and FADD in the nucleus, also observed in the cortex of APP_SLPS1 KI mice. However, PKR gene silencing or treatment with a specific PKR inhibitor significantly prevented the increase in pT⁴⁵¹-PKR and pS¹⁹⁴-FADD levels in SH-SY5Y nuclei and completely inhibited activities of caspase-3 and -8. The contribution of PKR in neurodegeneration through the death receptor signaling pathway may support the development of therapeutics targeting PKR to limit neuronal death in AD.     

The consequences of reducing expression of the alpha7 nicotinic receptor by RNA interference and of stimulating its activity with an alpha7 agonist in SH-SY5Y cells indicate that this receptor plays a neuroprotective role in connection with the pathogenesis of Alzheimer's disease

In order to examine the neuroprotective effects of the alpha7 nicotinic receptor (nAChR) in relationship to the pathogenesis of Alzheimer's disease (AD), neuroblastoma (SH-SY5Y) cells were transfected with small interference RNAs (siRNAs) that targets specifically towards alpha7 nAChR or exposed to 20microM 3-[2,4-dimethoxybenzylidene] anabaseine (DMXB), a selective agonist of this same receptor. The levels of alpha7 nAChR mRNA and protein were measured by RT-PCR and Western blotting, respectively. The levels of the alpha-form of secreted amyloid precursor protein (alphaAPPs), total APP and the extracellular signal-regulated kinase 1/2 (ERK1/2) were also determined by Western blotting. SH-SY5Y cells transfected with siRNA or exposed to DMXB were then treated with 1microM Abeta(25-35), following which the levels of lipid peroxidation and rate of reduction of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] were characterized by utilizing spectrophotometric procedures. Compared to controls, SH-SY5Y cells transfected with siRNA expressed the decreases in the levels of alpha7 nAChR mRNA and protein by 81% and 69% lower levels, respectively; exhibited reduced levels of the alphaAPPs and ERK1/2 proteins; and demonstrated enhanced lipid peroxidation and a decreased rate of MTT reduction. In cells exposed to DMXB, the level of alpha7 nAChR protein was elevated by 23%, with no alteration in the content of the corresponding mRNA; the levels of the alphaAPPs and ERK1/2 proteins were increased. Inhibition of the expression of the alpha7 nAChR gene enhanced the toxicity exerted by Abeta, whereas stimulation of this receptor attenuated this toxicity exerted. These findings indicate that alpha7 nAChR may play a significant neuroprotective role by enhancing cleavage of APP by alpha-secretase, regulating signal transduction, improving antioxidant defenses and inhibiting the toxicity of Abeta, which is connected with the pathogenesis of AD.     

Adiponectin is Protective against Oxidative Stress Induced Cytotoxicity in Amyloid-Beta Neurotoxicity

Beta-amyloid (Aβ ) neurotoxicity is important in Alzheimer’s disease (AD) pathogenesis. Aβ neurotoxicity causes oxidative stress, inflammation and mitochondrial damage resulting in neuronal degeneration and death. Oxidative stress, inflammation and mitochondrial failure are also pathophysiological mechanisms of type 2 diabetes (T₂DM) which is characterized by insulin resistance. Interestingly, T₂DM increases risk to develop AD which is associated with reduced neuronal insulin sensitivity (central insulin resistance). We studied the potential protective effect of adiponectin (an adipokine with insulin-sensitizing, anti-inflammatory and anti-oxidant properties) against Aβ neurotoxicity in human neuroblastoma cells (SH-SY5Y) transfected with the Swedish amyloid precursor protein (Sw-APP) mutant, which overproduced Aβ with abnormal intracellular Aβ accumulation. Cytotoxicity was measured by assay for lactate dehydrogenase (LDH) released upon cell death and lysis. Our results revealed that Sw-APP transfected SH-SY5Y cells expressed both adiponectin receptor 1 and 2, and had increased AMP-activated protein kinase (AMPK) activation and enhanced nuclear factor-kappa B (NF-κB) activation compared to control empty-vector transfected SH-SY5Y cells. Importantly, adiponectin at physiological concentration of 10 µg/ml protected Sw-APP transfected SH-SY5Y cells against cytotoxicity under oxidative stress induced by hydrogen peroxide. This neuroprotective action of adiponectin against Aβ neurotoxicity-induced cytotoxicity under oxidative stress involved 1) AMPK activation mediated via the endosomal adaptor protein APPL1 (adaptor protein with phosphotyrosine binding, pleckstrin homology domains and leucine zipper motif) and possibly 2) suppression of NF-κB activation. This raises the possibility of novel therapies for AD such as adiponectin receptor agonists.     

Loading into Nanoparticles Improves Quercetin's Efficacy in Preventing Neuroinflammation Induced by Oxysterols

Chronic inflammatory events appear to play a fundamental role in Alzheimer''s disease (AD)-related neuropathological changes, and to result in neuronal dysfunction and death. The inflammatory responses observed in the AD brain include activation and proliferation of glial cells, together with up-regulation of inflammatory mediators and of free radicals. Along with glial cells, neurons themselves can also react and contribute to neuroinflammatory changes in the AD brain, by serving as sources of inflammatory mediators. Because excess cholesterol cannot be degraded in the brain, it must be excreted from that organ as cholesterol oxidation products (oxysterols), in order to prevent its accumulation. Among risk factors for this neurodegenerative disease, a mechanistic link between altered cholesterol metabolism and AD has been suggested; oxysterols appear to be the missing linkers between the two, because of their neurotoxic effects. This study shows that 24-hydroxycholesterol, 27-hydroxycholesterol, and 7β-hydroxycholesterol, the three oxysterols potentially implicated in AD pathogenesis, induce some pro-inflammatory mediator expression in human neuroblastoma SH-SY5Y cells, via Toll-like receptor-4/cyclooxygenase-2/membrane bound prostaglandin E synthase (TLR4/COX-2/mPGES-1); this clearly indicates that oxysterols may promote neuroinflammatory changes in AD. To confirm this evidence, cells were incubated with the anti-inflammatory flavonoid quercetin; remarkably, its anti-inflammatory effects in SH-SY5Y cells were enhanced when it was loaded into β-cyclodextrin-dodecylcarbonate nanoparticles, versus cells pretreated with free quercetin. The goal of loading quercetin into nanoparticles was to improve its permeation across the blood-brain barrier into the brain, and its bioavailability to reach target cells. The findings show that this drug delivery system might be a new therapeutic strategy for preventing or reducing AD progression.     

Oxidative stress induced by beta-amyloid peptide(1-42) is involved in the altered composition of cellular membrane lipids and the decreased expression of nicotinic receptors in human SH-SY5Y neuroblastoma cells

The neurotoxic effects and influence of beta-amyloid peptide (Aβ)_1–42 on membrane lipids and nicotinic acetylcholine receptors (nAChRs) in human SH-SY5Y neuroblastoma cells were investigated in parallel. Exposure of the cultured cells to varying concentrations of Aβ_1–42 evoked a significantly decrease in cellular reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5,diphenyl tetrazolium bromide), together with enhanced lipid peroxidation and protein oxidation. Significant reductions in the total contents of phospholipid and unbiquinone-10, as well as in the levels of the 3 and 7 subunit proteins of nAChRs were detected in cells exposed to Aβ_1–42. In contrast, such treatment had no effect on the total cellular content of cholesterol. Among these alterations, increased lipid peroxidation and decreased levels of cellular phospholipids were most sensitive to Aβ_1–42, occurring at lower concentrations. In addition, when SH-SY5Y cells were pretreated with the antioxidant Vitamin E, prior to the addition of Aβ_1–42, these alterations in neurotoxicity, oxidative stress, composition of membrane lipids and expression of nAChRs were partially prevented. These findings suggest that stimulation of lipid peroxidation by Aβ may be involved in eliciting the alterations in membrane lipid composition and the reduced expression of nAChRs associated with the pathogenesis of AD.     

Inhibiting gene expression of alpha3 nicotinic receptor in SH-SY5Y cells with the effects on APP metabolism and antioxidation in Alzheimer's disease

In order to examine the effects of alpha3 nicotinic acetylcholine receptor (nAChR) in connection with the pathogenesis of Alzheimer's disease (AD), neuroblastoma (SH-SY5Y) cells were transfected with small interference RNAs (siRNAs) that target specifically towards alpha3 nAChR. The expressions of alpha3 nAChR mRNA and protein were measured by real-time PCR and Western blotting, respectively. The levels of the alpha-form of secreted amyloid precursor protein (alphaAPPs) and total-APP were determined by Western blotting. SH-SY5Y cells transfected with siRNA were then treated with 1muM beta-amyloid peptide (Abeta)(1-42), following which the levels of lipid peroxidation, the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), and the reduction rate of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] were characterized by utilizing spectrophotometric procedures. As compared to controls, SH-SY5Y cells transfected with siRNA expressed the decreases in the levels of alpha3 nAChR mRNA and protein by 98% and 66% lower levels, respectively; exhibited reduced level of the alphaAPPs; and demonstrated enhanced lipid peroxidation, decreased rate of MTT reduction, and declined activities of SOD and GSH-Px. Inhibited gene expression of the alpha3 nAChR enhanced the toxicity exerted by Abeta. These results indicate that alpha3 nAChR may improve cleavage of APP by alpha-secretase, enhance antioxidation and inhibit the toxicity of Abeta, suggesting that the receptor might play an important role in AD.     

SIRT2 interferes with autophagy-mediated degradation of protein aggregates in neuronal cells under proteasome inhibition

Abnormal protein aggregates have been suggested as a common pathogenesis of many neurodegenerative diseases. Two well-known protein degradation pathways are responsible for protein homeostasis by balancing protein biosynthesis and degradative processes: the ubiquitin–proteasome system (UPS) and autophagy-lysosomal system. UPS serves as the primary route for degradation of short-lived proteins, but large-size protein aggregates cannot be degraded by UPS. Autophagy is a unique cellular process that facilitates degradation of bulky protein aggregates by lysosome. Recent studies have demonstrated that autophagy plays a crucial role in the pathogenesis of neurodegenerative diseases characterized by abnormal protein accumulation, suggesting that regulation of autophagy may be a valuable therapeutic strategy for the treatment of various neurodegenerative diseases. Sirtuin-2 (SIRT2) is a class III histone deacetylase that is expressed abundantly in aging brain tissue. Here, we report that SIRT2 increases protein accumulation in murine cholinergic SN56 cells and human neuroblastoma SH-SY5Y cells under proteasome inhibition. Overexpression of SIRT2 inhibits lysosome-mediated autophagic turnover by interfering with aggresome formation and also makes cells more vulnerable to accumulated protein-mediated cytotoxicity by MG132 and amyloid beta. Moreover, MG132-induced accumulation of ubiquitinated proteins and p62 as well as cytotoxicity are attenuated in siRNA-mediated SIRT2-silencing cells. Taken together, these results suggest that regulation of SIRT2 could be a good therapeutic target for a range of neurodegenerative diseases by regulating autophagic flux.     

The prevention of the staurosporine-induced apoptosis by Bcl-XL, but not by Bcl-2 or caspase inhibitors, allows the extensive differentiation of human neuroblastoma cells

Staurosporine is one of the best apoptotic inducers in different cell types including neuroblastomas. In this study we have compared the efficiency and final outcome of three different anti-apoptotic strategies in staurosporine-treated SH-SY5Y human neuroblastoma cells. At staurosporine concentrations up to 500 nm, z-VAD.fmk a broad-spectrum, noncompetitive inhibitor of caspases, reduced apoptosis in SH-SY5Y cells. At higher concentrations, z-VAD.fmk continued to inhibit caspases and the apoptotic phenotype but not cell death which seems to result from oxidative damage. Stable over-expression of Bcl-2 in SH-SY5Y protected cells from death at doses of staurosporine up to 1 microm. At higher doses, cytochrome c release from mitochondria occurred, caspases were activated and cells died by apoptosis. Therefore, we conclude that Bcl-2 increased the threshold for apoptotic cell death commitment. Over-expression of Bcl-X(L) was far more effective than Bcl-2. Bcl-X(L) transfected cells showed a remarkable resistance staurosporine-induced cytochrome c release and associated apoptotic changes and survived for up to 15 days in 1 microm staurosporine. In these conditions, SH-SY5Y displayed a remarkable phenotype of neuronal differentiation as assessed by neurite outgrowth and expression of neurofilament, Tau and MAP-2 neuronal specific proteins.     

Protective effect of Pycnogenol in human neuroblastoma SH-SY5Y cells following acrolein-induced cytotoxicity

Oxidative stress is one of the hypotheses involved in the etiology of Alzheimer's disease (AD). Considerable attention has been focused on increasing the intracellular glutathione (GSH) levels in many neurodegenerative diseases, including AD. Pycnogenol (PYC) has antioxidant properties and stabilizes intracellular antioxidant defense systems including glutathione levels. The present study investigated the protective effects of PYC on acrolein-induced oxidative cell toxicity in cultured SH-SY5Y neuroblastoma cells. Decreased cell survival in SH-SY5Y cultures treated with acrolein correlated with oxidative stress, increased NADPH oxidase activity, free radical production, protein oxidation/nitration (protein carbonyl, 3-nitrotyrosine), and lipid peroxidation (4-hydroxy-2-nonenal). Pretreatment with PYC significantly attenuated acrolein-induced cytotoxicity, protein damage, lipid peroxidation, and cell death. A dose-response study suggested that PYC showed protective effects against acrolein toxicity by modulating oxidative stress and increasing GSH. These findings provide support that PYC may provide a promising approach for the treatment of oxidative stress-related neurodegenerative diseases such as AD.     

Proteasome Inhibitors Alter Levels of Intracellular Peptides in HEK293T and SH-SY5Y Cells

The proteasome cleaves intracellular proteins into peptides. Earlier studies found that treatment of human embryonic kidney 293T (HEK293T) cells with epoxomicin (an irreversible proteasome inhibitor) generally caused a decrease in levels of intracellular peptides. However, bortezomib (an antitumor drug and proteasome inhibitor) caused an unexpected increase in the levels of most intracellular peptides in HEK293T and SH-SY5Y cells. To address this apparent paradox, quantitative peptidomics was used to study the effect of a variety of other proteasome inhibitors on peptide levels in HEK293T and SH-SY5Y cells. Inhibitors tested included carfilzomib, MG132, MG262, MLN2238, AM114, and clasto-Lactacystin β-lactone. Only MG262 caused a substantial elevation in peptide levels that was comparable to the effect of bortezomib, although carfilzomib and MLN2238 elevated the levels of some peptides. To explore off-target effects, the proteosome inhibitors were tested with various cellular peptidases. Bortezomib did not inhibit tripeptidyl peptidase 2 and only weakly inhibited cellular aminopeptidase activity, as did some of the other proteasome inhibitors. However, potent inhibitors of tripeptidyl peptidase 2 (butabindide) and cellular aminopeptidases (bestatin) did not substantially alter the peptidome, indicating that the increase in peptide levels due to proteasome inhibitors is not a result of peptidase inhibition. Although we cannot exclude other possibilities, we presume that the paradoxical increase in peptide levels upon treatment with bortezomib and other inhibitors is the result of allosteric effects of these compounds on the proteasome. Because intracellular peptides are likely to be functional, it is possible that some of the physiologic effects of bortezomib and carfilzomib arise from the perturbation of peptide levels inside the cell.     

Neuroprotective Effects of Phenolic and Carboxylic Acids on Oxidative Stress-Induced Toxicity in Human Neuroblastoma SH-SY5Y Cells

An increase in oxidative stress is a key factor responsible for neurotoxicity induction and cell death leading to neurodegenerative diseases including Parkinson’s and Alzheimer’s diseases. Plant phenolics exert diverse bioactivities i.e., antioxidant, anti-inflammatory, and neuroprotective effects. Herein, phenolic compounds, namely protocatechuic aldehyde (PCA) constituents of Hydnophytum formicarum Jack. including vanillic acid (VA) and trans-ferulic acid (FA) found in Spilanthes acmella Murr., were explored for anti-neurodegenerative properties using an in vitro model of oxidative stress-induced neuroblastoma SH-SY5Y cells. Exposure of the neuronal cells with H₂O₂ resulted in the decrease of cell viability, but increasing in the level of reactive oxygen species (ROS) together with morphological changes and inducing cellular apoptosis. SH-SY5Y cells pretreated with 5 µM of PCA, VA, and FA were able to attenuate cell death caused by H₂O₂-induced toxicity, as well as decreased ROS level and apoptotic cells after 24 h of treatment. Pretreated SH-SY5Y cells with phenolic compounds also helped to upregulate H₂O₂-induced depletion of the expressions of sirtuin-1 (SIRT1) and forkhead box O (FoxO) 3a as well as induce the levels of antioxidant (superoxide dismutase (SOD) 2 and catalase) and antiapoptotic B-cell lymphoma 2 (Bcl-2) proteins. The findings suggest that these phenolics might be promising compounds against neurodegeneration.     

Involvement of prostaglandin E2 in production of amyloid-beta peptides both in vitro and in vivo

Amyloid-beta peptides (Abeta), generated by proteolysis of the beta-amyloid precursor protein (APP) by beta- and gamma-secretases, play an important role in the pathogenesis of Alzheimer disease (AD). Inflammation is also believed to be integral to the pathogenesis of AD. Here we show that prostaglandin E(2) (PGE(2)), a strong inducer of inflammation, stimulates the production of Abeta in cultured human embryonic kidney (HEK) 293 or human neuroblastoma (SH-SY5Y) cells, both of which express a mutant type of APP. We have demonstrated using subtype-specific agonists that, of the four main subtypes of PGE(2) receptors (EP(1-4)), EP(4) receptors alone or EP(2) and EP(4) receptors together are responsible for this PGE(2)-stimulated production of Abeta in HEK293 or SH-SY5Y cells, respectively. An EP(4) receptor antagonist suppressed the PGE(2)-stimulated production of Abeta in HEK293 cells. This stimulation was accompanied by an increase in cellular cAMP levels, and an analogue of cAMP stimulated the production of Abeta, demonstrating that increases in the cellular level of cAMP are responsible for the PGE(2)-stimulated production of Abeta. Immunoblotting experiments and direct measurement of gamma-secretase activity suggested that PGE(2)-stimulated production of Abeta is mediated by activation ofgamma-secretase but not of beta-secretase. Transgenic mice expressing the mutant type of APP showed lower levels of Abeta in the brain, when they were crossed with mice lacking either EP(2) or EP(4) receptors, suggesting that PGE(2)-mediated activation of EP(2) and EP(4) receptors is involved in the production of Abeta in vivo and in the pathogenesis of AD.     

A high-throughput drug screen targeted to the 5'untranslated region of Alzheimer amyloid precursor protein mRNA

The authors employed a novel approach to identify therapeutics effective in Alzheimer disease (AD). The 5'untranslated region (5'UTR) of the mRNA of AD amyloid precursor protein (APP) is a significant regulator of the levels of the APP holoprotein and amyloid beta (Abeta) peptide in the central nervous system. The authors generated stable neuroblastoma SH-SY5Y transfectants that express luciferase under the translational control of the 146-nucleotide APP mRNA 5'UTR and green fluorescent protein (GFP) driven by a viral internal ribosomal entry site. Using a high-throughput screen (HTS), they screened for the effect of 110,000 compounds obtained from the library of the Laboratory for Drug Discovery on Neurodegeneration (LDDN) on the APP mRNA 5'UTR-controlled translation of the luciferase reporter. This screening yielded several nontoxic specific inhibitors of APP mRNA 5'UTR-driven luciferase that had no effect on the GFP expression in the stable SH-SY5Y transfectants. Moreover, these compounds either did not inhibit or inhibited to a much lower extent the expression of the luciferase reporter regulated by a prion protein (PrP) mRNA 5'UTR, used as an alternative mRNA structure to counterscreen APP mRNA 5'UTR in stably transfected SH-SY5Y cell lines. The hits obtained from this robust, specific, and highly quantitative HTS will be characterized to identify agents that may be developed into useful future therapeutic agents to limit APP translation and Abeta production for AD.     

Hydrogen sulphide: a novel inhibitor of hypochlorous acid-mediated oxidative damage in the brain?

Hydrogen sulphide (H(2)S) is a cytotoxic gas that has recently been proposed as a novel neuromodulator. Endogenous levels of H(2)S in the brain range between 50 and 160 microM, and considerably lower H(2)S levels are reported in the brains of Alzheimer's disease (AD) patients. Levels of myeloperoxidase (MPO), an enzyme that catalyses the formation of the oxidant hypochlorous acid (HOCl), are elevated in the prefrontal cortex, hippocampal microglia, and neurons of AD patients where MPO co-localised with beta-amyloid plaques. Recently 3-chlorotyrosine, a bio-marker for MPO activity (and HOCl production), was shown to be elevated threefold in hippocampal proteins from AD patients. Since H(2)S and HOCl are important mediators in brain function and disease, we investigated the effects of H(2)S on HOCl-mediated damage to bio-molecules and to cultured human SH-SY5Y cells. H(2)S significantly inhibited HOCl-mediated inactivation of alpha(1)-antiproteinase and protein oxidation to a comparable extent to reduced glutathione. H(2)S also inhibited HOCl-induced cytotoxicity, intracellular protein oxidation, and lipid peroxidation in SH-SY5Y cells. These data suggest that H(2)S has the potential to act as an inhibitor of HOCl-mediated processes in vivo and that the potential antioxidant action of H(2)S deserves further study, especially since extracellular GSH levels in the brain are very low.     

Characterization of chronic low-level proteasome inhibition on neural homeostasis

Increasing evidence suggests that proteasome inhibition plays a causal role in promoting the neurodegeneration and neuron death observed in multiple disorders, including Alzheimer's disease (AD) and Parkinson's disease (PD). The ability of severe and acute inhibition of proteasome function to induce neuron death and neuropathology similar to that observed in AD and PD is well documented. However, at present the effects of chronic low-level proteasome inhibition on neural homeostasis has not been elucidated. In order to determine the effects of chronic low-level proteasome inhibition on neural homeostasis, we conducted studies in individual colonies of neural SH-SY5Y cells that were isolated following continual exposure to low concentrations (100 nm) of the proteasome inhibitor MG115. Clonal cell lines appeared morphologically similar to control cultures but exhibited significantly different rates of both proliferation and differentiation. Elevated levels of protein oxidation and protein insolubility were observed in clonal cell lines, with all clonal cell lines being more resistant to neural death induced by serum withdrawal and oxidative stress. Interestingly, clonal cell lines demonstrated evidence for increased macroautophagy, suggesting that chronic low-level proteasome inhibition may cause an excessive activation of the lysosomal system. Taken together, these data indicate that chronic low-level proteasome inhibition has multiple effects on neural homeostasis, and suggests that studying the effects of chronic low-level proteasome inhibition may be useful in understanding the relationship between protein oxidation, protein insolubility, proteasome function, macroautophagy and neural viability in AD and PD.     

Parkin attenuates manganese-induced dopaminergic cell death

Manganese as environmental factor is considered to cause parkinsonism and induce endoplasmic reticulum stress-mediated dopaminergic cell death. We examined the effects of manganese on parkin, identified as the gene responsible for familial Parkinson's disease, and the role of parkin in manganese-induced neuronal cell death. Manganese dose-dependently induced cell death of dopaminergic SH-SY5Y and CATH.a cells and cholinergic Neuro-2a cells, and that the former two cell types were more sensitive to manganese toxicity than Neuro-2a cells. Moreover, manganese increased the expression of endoplasmic reticulum stress-associated genes, including parkin, in SH-SY5Y cells and CATH.a cells, but not in Neuro-2a cells. Treatment with manganese resulted in accumulation of parkin protein in SH-SY5Y cells and its redistribution to the perinuclear region, especially aggregated Golgi complex, while in Neuro-2a cells neither expression nor redistribution of parkin was noted. Manganese showed no changes in proteasome activities in either cell. Transient transfection of parkin gene inhibited manganese- or manganese plus dopamine-induced cell death of SH-SY5Y cells, but not of Neuro-2a cells. Our results suggest that the attenuating effects of parkin against manganese- or manganese plus dopamine-induced cell death are dopaminergic cell-specific compensatory reactions associated with its accumulation and redistribution to perinuclear regions but not with proteasome system.     

Transglutaminase 2 interaction with small heat shock proteins mediate cell survival upon excitotoxic stress

Transglutaminase 2 has been postulated to be involved in the pathogenesis of central nervous system neurodegenerative disorders. However, its role in neuronal cell death remains to be elucidated. Excitotoxicity is a common event underlying neurodegeneration. We aimed to evaluate the protein targets for transglutaminase 2 in cell response to NMDA-induced excitotoxic stress, using SH-SY5Y neuroblastoma cells which express high tranglutaminase 2 levels upon retinoic acid-driven differentiation toward neurons. NMDA-evoked calcium increase led to transglutaminase 2 activation that mediated cell survival, as at first suggested by the exacerbation of NMDA toxicity in the presence of R283, a synthetic competitive inhibitor of transglutaminase active site. Assays of R283-mediated transglutaminase inhibition showed the involvement of enzyme activity in NMDA-induced reduction in protein basal levels of pro-apoptotic caspase-3 and the stress protein Hsp20. However, this occurred in a way different from protein cross-linking, given that macromolecular assemblies were not observed in our experimental conditions for both proteins. Co-immunoprecipitation experiments provided evidence for the interaction, in basal conditions, between transglutaminase 2 and Hsp20, as well as between Hsp20 and Hsp27, a major anti-apoptotic protein promoting caspase-3 inactivation and degradation. NMDA treatment disrupted both these interactions that were restored upon transglutaminase 2 inhibition with R283. These results suggest that transglutaminase 2 might be protective against NMDA-evoked excitotoxic insult in neuronal-like SH-SY5Y cells in a way, independent from transamidation that likely involves its interaction with the complex Hsp20/Hsp27 playing a pro-survival role.