Zhengping Yu

Exposure to 1800 MHz radiofrequency radiation induces oxidative damage to mitochondrial DNA in primary cultured neurons

Increasing evidence indicates that oxidative stress may be involved in the adverse effects of radiofrequency (RF) radiation on the brain. Because mitochondrial DNA (mtDNA) defects are closely associated with various nervous system diseases and mtDNA is particularly susceptible to oxidative stress, the purpose of this study was to determine whether radiofrequency radiation can cause oxidative damage to mtDNA. In this study, we exposed primary cultured cortical neurons to pulsed RF electromagnetic fields at a frequency of 1800 MHz modulated by 217 Hz at an average special absorption rate (SAR) of 2 W/kg. At 24 h after exposure, we found that RF radiation induced a significant increase in the levels of 8-hydroxyguanine (8-OHdG), a common biomarker of DNA oxidative damage, in the mitochondria of neurons. Concomitant with this finding, the copy number of mtDNA and the levels of mitochondrial RNA (mtRNA) transcripts showed an obvious reduction after RF exposure. Each of these mtDNA disturbances could be reversed by pretreatment with melatonin, which is known to be an efficient antioxidant in the brain. Together, these results suggested that 1800 MHz RF radiation could cause oxidative damage to mtDNA in primary cultured neurons. Oxidative damage to mtDNA may account for the neurotoxicity of RF radiation in the brain.

Overexpression of Tfam protects mitochondria against β‐amyloid‐induced oxidative damage in SH‐SY5Y cells

There is strong evidence that β‐amyloid (Aβ) causes oxidative stress and induces mitochondrial dysfunction in the pathogenesis of Alzheimer’s disease. Mitochondrial transcription factor A (Tfam) has multiple roles in the maintenance of mtDNA. To study the protective roles of Tfam against amyloid neurotoxicity, we established SH‐SY5Y cell lines stably overexpressing Tfam and exposed them to 10 μm Aβ1‐42 for 24 h. We found that Tfam overexpression attenuated Aβ1‐42‐induced cell viability damage and apoptosis. In addition, Tfam overexpression significantly suppressed the increase in excess reactive oxygen species and reversed the reduction in cytochrome c oxidase activity and ATP production induced by Aβ1‐42. Furthermore, overexpression of ΔC‐Tfam, which has no functional domain for stimulating mtDNA transcription but can still maintain the mtDNA nucleoid formation and mtDNA copy number, also exhibited protective effects against Aβ1‐42 cytotoxicity in SH‐SY5Y cells. Together, our data suggest that Tfam overexpression protects mitochondria against Aβ‐induced oxidative damage in SH‐SY5Y cells. These beneficial effects may be attributable to the roles of Tfam in maintaining mtDNA nucleoid formation and mtDNA copy number.

Exposure to 1800 MHz radiofrequency electromagnetic radiation induces oxidative DNA base damage in a mouse spermatocyte-derived cell line

Whether exposure to radiofrequency electromagnetic radiation (RF-EMR) emitted from mobile phones can induce DNA damage in male germ cells remains unclear. In this study, we conducted a 24h intermittent exposure (5 min on and 10 min off) of a mouse spermatocyte-derived GC-2 cell line to 1800 MHz Global System for Mobile Communication (GSM) signals in GSM-Talk mode at specific absorption rates (SAR) of 1 W/kg, 2 W/kg or 4 W/kg. Subsequently, through the use of formamidopyrimidine DNA glycosylase (FPG) in a modified comet assay, we determined that the extent of DNA migration was significantly increased at a SAR of 4 W/kg. Flow cytometry analysis demonstrated that levels of the DNA adduct 8-oxoguanine (8-oxoG) were also increased at a SAR of 4 W/kg. These increases were concomitant with similar increases in the generation of reactive oxygen species (ROS); these phenomena were mitigated by co-treatment with the antioxidant α-tocopherol. However, no detectable DNA strand breakage was observed by the alkaline comet assay. Taking together, these findings may imply the novel possibility that RF-EMR with insufficient energy for the direct induction of DNA strand breaks may produce genotoxicity through oxidative DNA base damage in male germ cells.

Melatonin protects against Nickel-induced neurotoxicity in vitro by reducing oxidative stress and maintaining mitochondrial function.

Abstract:  Nickel is a potential neurotoxic pollutant. Oxidative stress is supposed to be involved in the mechanism underlying nickel‐induced neurotoxicity. Melatonin has efficient protective effects against various oxidative damages in nervous system. The purpose of this study was to investigate whether melatonin could efficiently protect against neurotoxicity induced by nickel. Here, we exposed primary cultured cortical neurons and mouse neuroblastoma cell lines (neuro2a) to different concentrations of nickel chloride (NiCl2) (0.125, 0.25, 0.5, and 1 mm) for 12 hr or 0.5 mm NiCl2 for various periods (0, 3, 6, 12, and 24 hr). We found that nickel significantly increased reactive oxygen species production and caused the loss of cell viability both in cortical neurons and neuro2a cells. In addition, nickel exposure obviously inhibited the mitochondrial function, disrupted the mitochondrial membrane potential (ΔΨm), reduced ATP production, and decreased mitochondrial DNA (mtDNA) content. However, each of these oxidative damages was efficiently attenuated by melatonin pretreatment. These protective effects of melatonin may be attributable to its roles in reducing oxidative stress and improving mitochondrial function in nickel‐treated nerve cells. Our results suggested that melatonin may have great pharmacological potential in protecting against the adverse effects of nickel in the nervous system.

A TRPC1-mediated increase in store-operated Ca2+ entry is required for the proliferation of adult hippocampal neural progenitor cells

Adult hippocampal neurogenesis plays an important role in brain function and neurological diseases. Adult neural progenitor cell (aNPC) proliferation is a critical first step in hippocampal neurogenesis. However, the mechanisms that modulate aNPC proliferation have not been fully identified. Ample evidence has demonstrated that cell proliferation is dependent on the intracellular Ca(2+) concentration. We hypothesized that store-operated Ca(2+) channels (SOCs), which are ubiquitously expressed in all cell types, participate in aNPC proliferation. We found that store-operated Ca(2+) entry (SOCE) was involved in the proliferation of aNPCs and that 2-APB, Gd(3+) and SKF96365, antagonists of SOCE and canonical transient receptor potential (TRPC), respectively, inhibited the increase in SOCE and aNPC proliferation. We therefore analyzed the expression of TRPCs in aNPCs and showed that TRPC1 is the most significantly upregulated member under proliferative conditions. Interestingly, knockdown of TRPC1 and using an antibody against TRPC1 markedly reduced the degree of SOCE and aNPC proliferation. In parallel, we observed the suppression of aNPC proliferation was found to be associated with cell cycle arrest in G0/G1 phase. Furthermore, gene expression microarray analysis revealed a selective up- or downregulation of 10 genes in aNPCs following TRPC1 silencing. Knockdown of Orai1 or STIM1 also induced a significant inhibition of SOCE and proliferation in aNPCs, and all three proteins were colocalized in the plasma membrane region of cells. Together, these results indicate that SOCE represents a principal mechanism regulating the proliferation of aNPCs and that TRPC1 is an essential component of this pathway. This discovery may be important in improving adult hippocampal neurogenesis and treating cognitive deficits.

Protective effects of lycopene against amyloid β-induced neurotoxicity in cultured rat cortical neurons.

The neurotoxicity of amyloid β (Aβ) has been implicated as a critical cause in the pathogenesis of Alzheimers disease (AD). Among antioxidant phytochemicals derived from fruit and vegetables, lycopene has recently received considerable attention for its potent protective properties already demonstrated in several models of oxidative damage. The present study aims to investigate whether lycopene could provide protective effects against Aβ-induced neurotoxicity in primary cultured rat cortical neurons. The cultured cortical neurons were pretreated with different dose of lycopene for 4h, followed by the challenge with 25 μM Aβ(25-35) for 24h. The results showed that pretreatment with lycopene efficiently attenuated Aβ(25-35)-induced neurotoxicity, as evidenced by the improved cell viability and the decreased apoptotic rate. In addition, lycopene inhibited the reactive oxygen species generation and mitochondrial membrane potential depolarization caused by Aβ(25-35). Lycopene also restored the levels of proapoptotic Bax, antiapoptotic Bcl-2, and inhibited caspase-3 activation. These beneficial effects may contribute to the protection against Aβ-induced neurotoxicity. Together, our results suggest that the natural antioxidant lycopene has potential for neuroprotection and therefore, may be a promising candidate for AD treatment.

The role of the JAK2-STAT3 pathway in pro-inflammatory responses of EMF-stimulated N9 microglial cells

BackgroundIn several neuropathological conditions, microglia can become overactivated and cause neurotoxicity by initiating neuronal damage in response to pro-inflammatory stimuli. Our previous studies have shown that exposure to electromagnetic fields (EMF) activates cultured microglia to produce tumor necrosis factor (TNF)-α and nitric oxide (NO) through signal transduction involving the activator of transcription STAT3. Here, we investigated the role of STAT3 signaling in EMF-induced microglial activation and pro-inflammatory responses in more detail than the previous study.MethodsN9 microglial cells were treated with EMF exposure or a sham treatment, with or without pretreatment with an inhibitor (Pyridone 6, P6) of the Janus family of tyrosine kinases (JAK). The activation state of microglia was assessed via immunoreaction using the microglial marker CD11b. Levels of inducible nitric oxide synthase (iNOS), TNF-α and NO were measured using real-time reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA) and the nitrate reductase method. Activation of JAKs and STAT3 proteins was evaluated by western blotting for specific tyrosine phosphorylation. The ability of STAT3 to bind to DNA was detected with an electrophoresis mobility shift assay (EMSA).ResultsEMF was found to significantly induce phosphorylation of JAK2 and STAT3, and DNA-binding ability of STAT3 in N9 microglia. In addition, EMF dramatically increased the expression of CD11b, TNF-α and iNOS, and the production of NO. P6 strongly suppressed the phosphorylation of JAK2 and STAT3 and diminished STAT3 activity in EMF-stimulated microglia. Interestingly, expression of CD11b as well as gene expression and production of TNF-α and iNOS were suppressed by P6 at 12 h, but not at 3 h, after EMF exposure.ConclusionsEMF exposure directly triggers initial activation of microglia and produces a significant pro-inflammatory response. Our findings confirm that the JAK2-STAT3 pathway may not mediate this initial microglial activation but does promote pro-inflammatory responses in EMF-stimulated microglial cells. Thus, the JAK2-STAT3 pathway might be a therapeutic target for reducing pro-inflammatory responses in EMF-activated microglia.

Mortalin overexpression attenuates beta-amyloid-induced neurotoxicity in SH-SY5Y cells

Amyloid-beta peptide (Aβ) is shown to be toxic to the mitochondria and implicates this organelle in the pathogenesis of Alzheimers disease. Previous studies suggest that targeting mitochondria for protection may be a useful strategy to reduce Aβ-induced neurotoxicity. Mortalin is the mitochondrial located member of the heat shock protein 70 family, which serves as a major mitochondrial molecular chaperone and plays a key role in mitochondrial import of proteins. Several studies have demonstrated the protective potential of Hsp75 overexpression against apoptosis induced by various forms of stresses. To investigate whether mortalin overexpression could provide protective effects on Aβ toxicity, SH-SY5Y cells were used to transfect human mortalin gene and then treated with Aβ(1-42) for 24h. It is found that overexpression of mortalin efficiently attenuated Aβ(1-42)-induced cell viability damage and apoptosis. Additionally, inhibition of mortalin expression by mortalin-specific siRNA oligonucleotides sensitized SH-SY5Y cells to Aβ(1-42)-induced neurotoxicity. Furthermore, mortalin overexpression significantly inhibited the Aβ(1-42)-induced depolarization of mitochondrial membrane potential, reversed the Aβ(1-42)-induced reduction in cytochrome c oxidase activity and ATP generation, and suppressed the Aβ(1-42)-induced reactive oxygen species accumulation and lipid peroxidation. Together, our results suggest that mortalin can afford protection against Aβ(1-42)-induced neurotoxicity in SH-SY5Y cells. These beneficial effects of mortalin overexpression may be attributable to its roles in maintaining mitochondrial function and reducing oxidative stress.

Lycopene protects against trimethyltin-induced neurotoxicity in primary cultured rat hippocampal neurons by inhibiting the mitochondrial apoptotic pathway

Lycopene is a potent free radicals scavenger with demonstrated protective efficacy in several experimental models of oxidative damage. Trimethyltin (TMT) is an organotin compound with neurotoxic effects on the hippocampus and other limbic structures and is used to model neurodegenerative diseases targeting these brain areas. Oxidative stress is widely accepted as a central pathogenic mechanism of TMT-mediated neurotoxicity. The present study investigated whether the plant carotene lycopene protects against TMT-induced neurotoxicity in primary cultured rat hippocampal neurons. Lycopene pretreatment improved cell viability in TMT-treated hippocampal neurons and inhibited neuronal apoptosis. Microfluorometric imaging revealed that lycopene inhibited the accumulation of mitochondria-derived reactive oxygen species (ROS) during TMT exposure. Moreover, lycopene ameliorated TMT-induced activation of the mitochondrial permeability transition pore (mPTP) and the concomitant depolarization of the mitochondrial membrane potential (ΔΨ(m)). Consequently, cytochrome c release from the mitochondria and ensuing caspase-3 activation were markedly reduced. These findings reveal that lycopene protects against TMT-induced neurotoxicity by inhibiting the mitochondrial apoptotic pathway. The anti-apoptotic effect of lycopene on hippocampal neurons highlights the therapeutic potential of plant-derived antioxidants against neurodegenerative diseases.

Inhibition of mitochondrial permeability transition pore opening is involved in the protective effects of mortalin overexpression against beta-amyloid-induced apoptosis in SH-SY5Y cells.

Mortalin (mtHsp70) is a mitochondrial heat shock protein critical for maintaining the functional integrity of mitochondrial proteins. Our previous study demonstrated that mortalin overexpression protected against Aβ-induced neurotoxicity through a mitochondria-dependent mechanism, but the molecular details remained unclear. Recent biochemical studies implicate opening of the mitochondrial permeability transition pore (mPTP) in Aβ-mediated mitochondrial dysfunction. The present study investigated the effect of mortalin overexpression on Aβ-induced mPTP activation and ensuing neuronal apoptosis. Mortalin overexpression inhibited mPTP activation and protected SH-SY5Y neurons against Aβ-induced apoptosis. Compared to controls, neurons overexpressing mortalin also demonstrated superior intracellular free calcium regulation, lower mitochondrial reactive oxygen species generation, and decreased Bax/Bcl-2 ratios in response to Aβ treatment. Mortalin overexpression suppressed activation of the mitochondrial apoptotic cascade as demonstrated by inhibition of cytochrome c release and caspase-3 activation. Our results indicate that the cytoprotective efficacy of mortalin under Aβ-induced stress is mediated, at least in part, by inhibition of mPTP opening. Demonstration of the neuroprotective action of mortalin provides additional insights into the pathogenic mechanisms of Aβ toxicity and defines possible molecular targets for therapeutic intervention.