Blocking mPTP on Neural Stem Cells and Activating the Nicotinic Acetylcholine Receptor α7 Subunit on Microglia Attenuate Aβ-Induced Neurotoxicity on Neural Stem Cells

Neurochemical Research - β-Amyloid (Aβ) can stimulate microglia to release a variety of proinflammatory cytokines and induce neurotoxicity. Nicotine has been reported to inhibit...     

The α5 Subunit Regulates the Expression and Function of α4*-Containing Neuronal Nicotinic Acetylcholine Receptors in the Ventral-Tegmental Area

Human genetic association studies have shown gene variants in the α5 subunit of the neuronal nicotinic receptor (nAChR) influence both ethanol and nicotine dependence. The α5 subunit is an accessory subunit that facilitates α4* nAChRs assembly in vitro. However, it is unknown whether this occurs in the brain, as there are few research tools to adequately address this question. As the α4*-containing nAChRs are highly expressed in the ventral tegmental area (VTA) we assessed the molecular, functional and pharmacological roles of α5 in α4*-containing nAChRs in the VTA. We utilized transgenic mice α5+/+(α4YFP) and α5-/-(α4YFP) that allow the direct visualization and measurement of α4-YFP expression and the effect of the presence (α5+/+) and absence of α5 (-/-) subunit, as the antibodies for detecting the α4* subunits of the nAChR are not specific. We performed voltage clamp electrophysiological experiments to study baseline nicotinic currents in VTA dopaminergic neurons. We show that in the presence of the α5 subunit, the overall expression of α4 subunit is increased significantly by 60% in the VTA. Furthermore, the α5 subunit strengthens baseline nAChR currents, suggesting the increased expression of α4* nAChRs to be likely on the cell surface. While the presence of the α5 subunit blunts the desensitization of nAChRs following nicotine exposure, it does not alter the amount of ethanol potentiation of VTA dopaminergic neurons. Our data demonstrates a major regulatory role for the α5 subunit in both the maintenance of α4*-containing nAChRs expression and in modulating nicotinic currents in VTA dopaminergic neurons. Additionally, the α5α4* nAChR in VTA dopaminergic neurons regulates the effect of nicotine but not ethanol on currents. Together, the data suggest that the α5 subunit is critical for controlling the expression and functional role of a population of α4*-containing nAChRs in the VTA.     

Elucidation of Molecular Impediments in the α6 Subunit for in Vitro Expression of Functional α6β4* Nicotinic Acetylcholine Receptors

Explorations into the α6-containing nicotinic acetylcholine receptors (α6* nAChRs) as putative drug targets have been severely hampered by the inefficient functional expression of the receptors in heterologous expression systems. In this study, the molecular basis for the problem was investigated through the construction of chimeric α6/α3 and mutant α3 and α6 subunits and functional characterization of these co-expressed with β4 or β4β3 subunits in tsA201 cells in a fluorescence-based assay and in Xenopus oocytes using two-electrode voltage clamp electrophysiology. Substitution of a small C-terminal segment in the second intracellular loop or the Phe²²³ residue in transmembrane helix 1 of α6 with the corresponding α3 segment or residue was found to enhance α6β4 functionality in tsA201 cells significantly, in part due to increased cell surface expression of the receptors. The gain-of-function effects of these substitutions appeared to be additive since incorporation of both α3 elements into α6 resulted in assembly of α6β4* receptors exhibiting robust functional responses to acetylcholine. The pharmacological properties exhibited by α6β4β3 receptors comprising one of these novel α6/α3 chimeras in oocytes were found to be in good agreement with those from previous studies of α6* nAChRs formed from other surrogate α6 subunits or concatenated subunits and studies of other heteromeric nAChRs. In contrast, co-expression of this α6/α3 chimera with β2 or β2β3 subunits in oocytes did not result in efficient formation of functional receptors, indicating that the identified molecular elements in α6 could be specific impediments for the expression of functional α6β4* nAChRs.     

Nicotine Deteriorates the Osteogenic Differentiation of Periodontal Ligament Stem Cells through α7 Nicotinic Acetylcholine Receptor Regulating wnt Pathway

Aims

Cigarette smoking is one of the high risk factors of adult chronic periodontitis and nicotine is the well established toxic substance in cigarette. However, the mechanism of nicotine induced periodontitis is still unknown. Here we studied whether nicotine impaired the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) through activating α7 nicotinic acetylcholine receptor (α7 nAChR).

Methods

hPDLSCs with multi differentiation potential and surface makers for mesenchymal stem cells were harvested by limiting dilution technique. The level of mineralized nodule formation was assessed by alizarin red S staining. Expression level of ostegenic related genes and proteins were detected by real-time PCR and western blot analysis. The expression of α7 nAChR and its downstream signaling pathway were examined by western blot. The role of the receptor and related signaling pathway in nicotine impairing the osteogenic potential of hPDLSCs were also studied in different levels.

Results

Nicotine deteriorated the ostegenic differentiation of hPDLSCs in a dose dependent manner. Activation of α7 nAChR by nicotine treatment activated wnt/β-catenin signaling pathway, leading to osteogenic deficiency of hPDLSCs. Blockage of α7 nAChR and wnt pathway inhibitor treatment rescued nicotine induced osteogenic differentiation deficiency.

Conclusions

These data suggested that nicotine activated α7 nAChR expressed on PDLSCs and further activated wnt signaling downstream, thus deteriorating the osteogenic potential of PDLSCs. The impairment of osteogenic differentiation of PDLSCs by nicotine might lead to cigarette smoking related periodontitis.     

Protective Effect of Paeoniflorin on Aβ25–35-Induced SH-SY5Y Cell Injury by Preventing Mitochondrial Dysfunction

Alzheimer’s disease (AD) is a major neurodegenerative brain disorder affecting about 14 million people worldwide. Aβ-induced cell injury is a crucial cause of neuronal loss in AD, thus the suppression of which might be useful for the treatment of this disease. In this study, we aimed to evaluate the effect of paeoniflorin (PF), a monoterpene glycoside isolated from aqueous extract of Radix Paeoniae Alba, on Aβ_25–35-induced cytotoxicity in SH-SY5Y cells. The results showed PF could attenuate or restore the viability loss, apoptotic increase, and ROS production induced by Aβ_25–35 in SH-SY5Y cells. In addition, PF strikingly inhibited Aβ_25–35-induced mitochondrial dysfunction, which includes decreased mitochondrial membrane potential, increased Bax/Bcl-2 ratio, cytochrome c release and activity of caspase-3 and caspase-9. Therefore, our study provided the first experimental evidence that PF could modulate ROS production and apoptotic mitochondrial pathway in model of neuron injury in vitro and which might provide new insights into its application toward Alzheimer’s disease therapy.     

Resveratrol Protects SH-SY5Y Cells Against Oleic Acid-Induced Glucolipid Metabolic Dysfunction and Cell Injuries Via the Wnt/β-Catenin Signalling Pathway

Neurochemical Research - Resveratrol (RES) is a polyphenol with diverse beneficial biological and pharmacological activities, and our previous results have demonstrated its neuroprotective effects...     

GABAA Receptor α1 Subunit Mutation A322D Associated with Autosomal Dominant Juvenile Myoclonic Epilepsy Reduces the Expression and Alters the Composition of Wild Type GABAA Receptors

A GABA_A receptor (GABA_AR) α1 subunit mutation, A322D (AD), causes an autosomal dominant form of juvenile myoclonic epilepsy (ADJME). Previous studies demonstrated that the mutation caused α1(AD) subunit misfolding and rapid degradation, reducing its total and surface expression substantially. Here, we determined the effects of the residual α1(AD) subunit expression on wild type GABA_AR expression to determine whether the AD mutation conferred a dominant negative effect. We found that although the α1(AD) subunit did not substitute for wild type α1 subunits on the cell surface, it reduced the surface expression of α1β2γ2 and α3β2γ2 receptors by associating with the wild type subunits within the endoplasmic reticulum and preventing them from trafficking to the cell surface. The α1(AD) subunit reduced surface expression of α3β2γ2 receptors by a greater amount than α1β2γ2 receptors, thus altering cell surface GABA_AR composition. When transfected into cultured cortical neurons, the α1(AD) subunit altered the time course of miniature inhibitory postsynaptic current kinetics and reduced miniature inhibitory postsynaptic current amplitudes. These findings demonstrated that, in addition to causing a heterozygous loss of function of α1(AD) subunits, this epilepsy mutation also elicited a modest dominant negative effect that likely shapes the epilepsy phenotype.     

A Human-Specific α7-Nicotinic Acetylcholine Receptor Gene in Human Leukocytes: Identification,Regulation and the Consequences of CHRFAM7A Expression

The human genome contains a variant form of the α7-nicotinic acetylcholine receptor (α7nAChR) gene that is uniquely human. This CHRFAM7A gene arose during human speciation and recent data suggests that its expression alters ligand tropism of the normally homopentameric human α7-AChR ligand-gated cell surface ion channel that is found on the surface of many different cell types. To understand its possible significance in regulating inflammation in humans, we investigated its expression in normal human leukocytes and leukocyte cell lines, compared CHRFAM7A expression to that of the CHRNA7 gene, mapped its promoter and characterized the effects of stable CHRFAM7A overexpression. We report here that CHRFAM7A is highly expressed in human leukocytes but that the levels of both CHRFAM7A and CHRNA7 mRNAs were independent and varied widely. To this end, mapping of the CHRFAM7A promoter in its 5′-untranslated region (UTR) identified a unique 1-kb sequence that independently regulates CHRFAM7A gene expression. Because overexpression of CHRFAM7A in THP1 cells altered the cell phenotype and modified the expression of genes associated with focal adhesion (for example, FAK, P13K, Akt, rho, GEF, Elk1, CycD), leukocyte transepithelial migration (Nox, ITG, MMPs, PKC) and cancer (kit, kitL, ras, cFos cyclinD1, Frizzled and GPCR), we conclude that CHRFAM7A is biologically active. Most surprisingly however, stable CHRFAM7A overexpression in THP1 cells upregulated CHRNA7, which, in turn, led to increased binding of the specific α7nAChR ligand, bungarotoxin, on the THP1 cell surface. Taken together, these data confirm the close association between CHRFAM7A and CHRNA7 expression, establish a biological consequence to CHRFAM7A expression in human leukocytes and support the possibility that this human-specific gene might contribute to, and/or gauge, a human-specific response to inflammation.     

Adhesion of Lymphoma Cells to Fibronectin: Differential Use of α4β1and α5β1Integrins and Stimulation by the 9EG7 mAb against the Murine β1Integrin Subunit

Murine ESb and MDAY-D2 lymphoma cells are highly metastatic, in particular to the liver, and are highly invasive in hepatocyte cultures. This may involve adhesion to hepatocyte surface-associated fibronectin (Kemperman et al., 1994, Cell Adh. and Communic. 2:45). Both ESb and MDAY-D2 cells express the fibronectin receptor α₄β₁, and MDAY-D2 cells in addition also α₅β₁. Yet, adhesion of ESb cells to fibronectin was low, and MDAY-D2 cells did not adhere at all, but adhesion of both cells was stimulated by phorbol myristate acetate (PMA) and Mn²⁺. In ESb cells, this adhesion was mediated by α₄β₁. In MDAY-D2 cells, however, only α₅β₁was involved, despite β₄β₁levels similar to ESb cells. The α₄β₁integrin was functional since it mediated adhesion of MDAY-D2 cells to VCAM-1. An α₅β₁-negative variant of MDAY-D2 adhered to fibronectin and this was mediated by α₅β₁. These results indicate that α₄β₁function in these cells is suppressed in the presence of α₅β₁. Adhesion of ESb cells to hepatocytes was inhibited by anti-α₄antibody, but only by 30%, and fibronectin adhesion was found to have no role in the interaction of MDAY-D2 cells with hepatocytes. This suggests that α₄β₁and α₅β₁function is not activated during this interaction.

The 9EG7 antibody against mouse β₁integrin was described to inhibit β₁integrins (Lenter et al., 1993, Proc. Natl. Acad. Sci. USA, 90, 9051). In contrast, we observed that β₁stimulated Printegrin function: Adhesion of ESb and MDAY-D2 cells not only to fibronectin, but also to laminin was induced or enhanced.     

Expression of a Highly Antigenic and Native-Like Folded Extracellular Domain of the Human α1 Subunit of Muscle Nicotinic Acetylcholine Receptor,Suitable for Use in Antigen Specific Therapies for Myasthenia Gravis

We describe the expression of the extracellular domain of the human α1 nicotinic acetylcholine receptor (nAChR) in lepidopteran insect cells (i-α1-ECD) and its suitability for use in antigen-specific therapies for Myasthenia Gravis (MG). Compared to the previously expressed protein in P. pastoris (y-α1-ECD), i-α1-ECD had a 2-fold increased expression yield, bound anti-nAChR monoclonal antibodies and autoantibodies from MG patients two to several-fold more efficiently and resulted in a secondary structure closer to that of the crystal structure of mouse α1-ECD. Our results indicate that i-α1-ECD is an improved protein for use in antigen-specific MG therapeutic strategies.     

Fluorescence Resonance Energy Transfer (FRET) Indicates That Association with the Type I Ryanodine Receptor (RyR1) Causes Reorientation of Multiple Cytoplasmic Domains of the Dihydropyridine Receptor (DHPR) α1S Subunit

The skeletal muscle dihydropyridine receptor (DHPR) in the t-tubular membrane serves as the Ca²⁺ channel and voltage sensor for excitation-contraction (EC) coupling, triggering Ca²⁺ release via the type 1 ryanodine receptor (RyR1) in the sarcoplasmic reticulum (SR). The two proteins appear to be physically linked, and both the α_1S and β_1a subunits of the DHPR are essential for EC coupling. Within α_1S, cytoplasmic domains of importance include the I-II loop (to which β_1a binds), the II-III and III-IV loops, and the C terminus. However, the spatial relationship of these domains to one another has not been established. Here, we have taken the approach of measuring FRET between fluorescent proteins inserted into pairs of α_1S cytoplasmic domains. Expression of these constructs in dyspedic (RyR1 null) and dysgenic (α_1S null) myotubes was used to test for function and targeting to plasma membrane/SR junctions and to test whether the presence of RyR1 caused altered FRET. We found that in the absence of RyR1, measureable FRET occurred between the N terminus and C terminus (residue 1636), and between the II-III loop (residue 626) and both the N and C termini; the I-II loop (residue 406) showed weak FRET with the II-III loop but not with the N terminus. Association with RyR1 caused II-III loop FRET to decrease with the C terminus and increase with the N terminus and caused I-II loop FRET to increase with both the II-III loop and N terminus. Overall, RyR1 appears to cause a substantial reorientation of the cytoplasmic α_1S domains consistent with their becoming more closely packed.     

A Functional Role for Specific Spliced Variants of the α7β1 Integrin in Acetylcholine Receptor Clustering

The clustering of acetylcholine receptors (AChR) on skeletal muscle fibers is an early event in the formation of neuromuscular junctions. Recent studies show that laminin as well as agrin can induce AChR clustering. Since the α7β1 integrin is a major laminin receptor in skeletal muscle, we determined if this integrin participates in laminin and/or agrin-induced AChR clustering. The alternative cytoplasmic domain variants, α7A and α7B, and the extracellular spliced forms, α7X1 and α7X2, were studied for their ability to engage in AChR clustering. Immunofluorescence microscopy of C2C12 myofibers shows that the α7β1 integrin colocalizes with laminin-induced AChR clusters and to a much lesser extent with agrin-induced AChR clusters. However, together laminin and agrin promote a synergistic response and all AChR colocalize with the integrin. Laminin also induces the physical association of the integrin and AChR. High concentrations of anti-α7 antibodies inhibit colocalization of the integrin with AChR clusters as well as the enhanced response promoted by both laminin and agrin. Engaging the integrin with low concentrations of anti-α7 antibody initiates cluster formation in the absence of agrin or laminin. Whereas both the α7A and α7B cytoplasmic domain variants cluster with AChR, only those isoforms containing the α7X2 extracellular domain were active. These results demonstrate that the α7β1 integrin has a physiologic role in laminin-induced AChR clustering, that alternative splicing is integral to this function of the α7 chain, and that laminin, agrin, and the α7β1 integrin interact in a common or convergent pathway in the formation of neuromuscular junctions.     

Lateral Diffusion,Function, and Expression of the Slow Channel Congenital Myasthenia Syndrome αC418W Nicotinic Receptor Mutation with Changes in Lipid Raft Components

Lipid rafts, specialized membrane microdomains in the plasma membrane rich in cholesterol and sphingolipids, are hot spots for a number of important cellular processes. The novel nicotinic acetylcholine receptor (nAChR) mutation αC418W, the first lipid-exposed mutation identified in a patient that causes slow channel congenital myasthenia syndrome was shown to be cholesterol-sensitive and to accumulate in microdomains rich in the membrane raft marker protein caveolin-1. The objective of this study is to gain insight into the mechanism by which lateral segregation into specialized raft membrane microdomains regulates the activable pool of nAChRs. We performed fluorescent recovery after photobleaching (FRAP), quantitative RT-PCR, and whole cell patch clamp recordings of GFP-encoding Mus musculus nAChRs transfected into HEK 293 cells to assess the role of cholesterol and caveolin-1 (CAV-1) in the diffusion, expression, and functionality of the nAChR (WT and αC418W). Our findings support the hypothesis that a cholesterol-sensitive nAChR might reside in specialized membrane microdomains that upon cholesterol depletion become disrupted and release the cholesterol-sensitive nAChRs to the pool of activable receptors. In addition, our results in HEK 293 cells show an interdependence between CAV-1 and αC418W that could confer end plates rich in αC418W nAChRs to a susceptibility to changes in cholesterol levels that could cause adverse drug reactions to cholesterol-lowering drugs such as statins. The current work suggests that the interplay between cholesterol and CAV-1 provides the molecular basis for modulating the function and dynamics of the cholesterol-sensitive αC418W nAChR.     

Resistance to Inhibitors of Cholinesterase 3 (Ric-3) Expression Promotes Selective Protein Associations with the Human α7-Nicotinic Acetylcholine Receptor Interactome

The α7-nicotinic acetylcholine receptor (α7-nAChR) is a ligand-gated ion channel widely expressed in vertebrates and is associated with numerous physiological functions. As transmembrane ion channels, α7-nAChRs need to be expressed on the surface of the plasma membrane to function. The receptor has been reported to associate with proteins involved with receptor biogenesis, modulation of receptor properties, as well as intracellular signaling cascades and some of these associated proteins may affect surface expression of α7-nAChRs. The putative chaperone resistance to inhibitors of cholinesterase 3 (Ric-3) has been reported to interact with, and enhance the surface expression of, α7-nAChRs. In this study, we identified proteins that associate with α7-nAChRs when Ric-3 is expressed. Using α-bungarotoxin (α-bgtx), we isolated and compared α7-nAChR-associated proteins from two stably transfected, human tumor-derived cell lines: SH-EP1-hα7 expressing human α7-nAChRs and the same cell line further transfected to express Ric-3, SH-EP1-hα7-Ric-3. Mass spectrometric analysis of peptides identified thirty-nine proteins that are associated with α7-nAChRs only when Ric-3 was expressed. Significantly, and consistent with reports of Ric-3 function in the literature, several of the identified proteins are involved in biological processes that may affect nAChR surface expression such as post-translational processing of proteins, protein trafficking, and protein transport. Additionally, proteins affecting the cell cycle, the cytoskeleton, stress responses, as well as cyclic AMP- and inositol triphosphate-dependent signaling cascades were identified. These results illuminate how α-bgtx may be used to isolate and identify α7-nAChRs as well as how the expression of chaperones such as Ric-3 can influence proteins associating with α7-nAChRs. These associating proteins may alter activities of α7-nAChRs to expand their functionally-relevant repertoire as well as to affect biogenesis and membrane trafficking of α7-nAChRs.     

HSV-2-Driven Increase in the Expression of α4β7 Correlates with Increased Susceptibility to Vaginal SHIVSF162P3 Infection

The availability of highly susceptible HIV target cells that can rapidly reach the mucosal lymphoid tissues may increase the chances of an otherwise rare transmission event to occur. Expression of α₄β₇ is required for trafficking of immune cells to gut inductive sites where HIV can expand and it is expressed at high level on cells particularly susceptible to HIV infection. We hypothesized that HSV-2 modulates the expression of α₄β₇ and other homing receptors in the vaginal tissue and that this correlates with the increased risk of HIV acquisition in HSV-2 positive individuals. To test this hypothesis we used an in vivo rhesus macaque (RM) model of HSV-2 vaginal infection and a new ex vivo model of macaque vaginal explants. In vivo we found that HSV-2 latently infected RMs appeared to be more susceptible to vaginal SHIV_SF162P3 infection, had higher frequency of α₄β₇ ^high CD4⁺ T cells in the vaginal tissue and higher expression of α₄β₇ and CD11c on vaginal DCs. Similarly, ex vivo HSV-2 infection increased the susceptibility of the vaginal tissue to SHIV_SF162P3. HSV-2 infection increased the frequencies of α₄β₇ ^high CD4⁺ T cells and this directly correlated with HSV-2 replication. A higher amount of inflammatory cytokines in vaginal fluids of the HSV-2 infected animals was similar to those found in the supernatants of the infected explants. Remarkably, the HSV-2-driven increase in the frequency of α₄β₇ ^high CD4⁺ T cells directly correlated with SHIV replication in the HSV-2 infected tissues. Our results suggest that the HSV-2-driven increase in availability of CD4⁺ T cells and DCs that express high levels of α₄β₇ is associated with the increase in susceptibility to SHIV due to HSV-2. This may persists in absence of HSV-2 shedding. Hence, higher availability of α₄β₇ positive HIV target cells in the vaginal tissue may constitute a risk factor for HIV transmission.     

The Role of Calpain-Myosin 9-Rab7b Pathway in Mediating the Expression of Toll-Like Receptor 4 in Platelets: A Novel Mechanism Involved in α-Granules Trafficking

Toll-like receptors (TLRs) plays a critical role in innate immunity. In 2004, Aslam R. and Shiraki R. first determined that murine and human platelets express functional TLRs. Additionally, Andonegui G. demonstrated that platelets express TLR4, which contributes to thrombocytopenia. However, the underlying mechanisms of TLR4 expression by platelets have been rarely explored until now. The aim of this study was to identify the mechanism of TLR4 expression underlying thrombin treatment. The human washed platelets were used in this study. According to flowcytometry and western blot analysis, the surface levels of TLR4 were significantly enhanced in thrombin-activated human platelets and decreased by TMB-8, calpeptin, and {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075"}}U73122, but not Y27632 (a Rho-associated protein kinase ROCK inhibitor) indicating that thrombin-mediated TLR4 expression was modulated by PAR/PLC pathway, calcium and calpain. Co-immunoprecipitation (co-IP) assay demonstrated that the interaction between TLR4 and myosin-9 (a substrate of calpain) was regulated by calpain; cleavage of myosin-9 enhanced TLR4 expression in thrombin treated platelets. Transmission electron microscope data indicated that human platelets used α-granules to control TLR4 expression; the co-IP experiment suggested that myosin-9 did not coordinate with Rab7b to negatively regulate TLR4 trafficking in thrombin treated platelets. In summary, phospholipase Cγ-calpain-myosin 9-Rab7b axis was responsible for the mechanism underlying the regulation of TLR4 containing α-granules trafficking in thrombin-stimulated platelets, which was involved in coagulation.     

Protective Effects of Hesperidin Against Amyloid-β (Aβ) Induced Neurotoxicity Through the Voltage Dependent Anion Channel 1 (VDAC1)-Mediated Mitochondrial Apoptotic Pathway in PC12 Cells

Amyloid-β (Aβ) is known to exert cytotoxic effects by inducing mitochondrial dysfunction. Additionally, the mitochondrial voltage-dependent anion channel 1 (VDAC1), which is involved in the release of apoptotic proteins with possible relevance in Alzheimer’s disease (AD) neuropathology, plays an important role in maintaining mitochondrial function and integrity. However, the application of therapeutic drugs, especially natural products in (AD) therapy via VDAC1-regulated mitochondrial apoptotic pathway has not aroused extensive attention. In the present study, we investigated neuroprotective effects of hesperidin, a bioactive flavonoid compound, on Aβ_25–35-induced neurotoxicity in PC12 cells and also examined the potential cellular signalling mechanism. Our results showed that treatment with hesperidin significantly inhibited Aβ_25–35-induced apoptosis by reversing Aβ-induced mitochondrial dysfunction, including the mitochondrial permeability transition pore opening, intracellular free calcium increase and reactive oxygen species production. Further study indicated that hesperidin can increase the level of VDAC1 phosphorylation through enhancing the activity of the glycogen synthasekinase-3β and decrease the level of hexokinaseI in mitochondrial, resulting in mitochondrial release of cytochrome c. Furthermore, hesperidin inhibited mitochondria-dependent downstream caspase-mediated apoptotic pathway, such as that involving caspase-9 and caspase-3. These results demonstrate that hesperidin can protect Aβ-induced neurotoxicity via VDAC1-regulated mitochondrial apoptotic pathway, and they raise the possibility that hesperidin could be developed into a clinically valuable treatment for AD and other neuronal degenerative diseases associated with mitochondrial dysfunction.     

SOD3 Ameliorates Aβ<Subscript>25–35</Subscript>-Induced Oxidative Damage in SH-SY5Y Cells by Inhibiting the Mitochondrial Pathway

This study was designed to investigate the protective effects of extracellular superoxide dismutase (SOD3) against amyloid beta (Aβ_25–35)-induced damage in human neuroblastoma SH-SY5Y cells and to elucidate the mechanisms responsible for this beneficial effect. SH-SY5Y cells overexpressing SOD3 were generated by adenoviral vector-mediated infection and Aβ_25–35 was then added to the cell culture system to establish an in vitro model of oxidative stress. Cell viability, the generation of intracellular reactive oxygen species (ROS), the expression and activity of antioxidant enzymes, the levels of lipid peroxidation malondialdehyde (MDA), the expression of mitochondrial apoptosis-related genes and calcium images were examined. Following Aβ_25–35 exposure, SOD3 overexpression promoted the survival of SH-SY5Y cells, decreased the production of ROS, decreased MDA and calcium levels, and decreased cytochrome c, caspase-3, caspase-9 and Bax gene expression. Furthermore, SOD3 overexpression increased the expression and activity of antioxidant enzyme genes and Bcl-2 expression. Together, our data demonstrate that SOD3 ameliorates Aβ_25–35-induced oxidative damage in neuroblastoma SH-SY5Y cells by inhibiting the mitochondrial pathway. These data provide new insights into the functional actions of SOD3 on oxidative stress-induced cell damage.