NOX2-mediated Ferroptosis Contributes To Learning And Memory Deficits In Rotenone-treated Mice

BackgroundRotenone（Rot）is a natural compound derived from plants and is widely used as an insecticide,and fish killer in agriculture.Epidemiological studies have shown that long-term low-dose exposure to neurotoxic Rot can cause dopamine（DA）neuronal degeneration and increase the risk of Parkinson’s disease（PD）.Synaptic loss and neuronal death are important causes of neurodegenerative diseases such as PD.Recently,increasing reports have shown that Rot exposure leads to neurodegeneration and cognitive dysfunction in the hippocampus and cortex of mice.However,the molecular mechanisms by which Rot induces cognitive dysfunction are not well understood.Phagocyte NADPH oxidase 2（NOX2）is an activity-dependent enzyme complex that is widely expressed in immune cells such as microglia.Increased NOX2 activity leads to neuronal degeneration and cognitive dysfunction and memory impairment.Overexpression of NOX2 significantly reduced the sensitivity of ferroptosis inhibitors,suggesting that NOX2-mediated lipid peroxidation may be a target of ferroptosis.Previous studies have revealed a close link between altered iron homeostasis and neurodegeneration.Ferroptosis is an iron-dependent non-apoptotic form of cell death that is thought to be implicated in the pathogenesis of a variety of neurological diseases,including PD.Ferroptosis is associated with two main biochemical features,namely iron homeostasis imbalance and lipid peroxidation.Recombinant Solute Carrier Family 7,Member 11（SLC7A11）,and glutathione peroxidase 4（GPX4）are considered central regulators of ferroptosis,and reduced levels of SLC7A11 and GPX4 are markers of cellular ferroptosis.It was shown that GPX4 decreased and lipid peroxidation levels and expression of gp91^phoxand p47^phox,two subunits of NOX2,increased in the hippocampus of PD mice.But whether NOX2 via ferroptosis and thus participates in the molecular mechanisms underlying the association between Rot-induced cognitive decline is unknown.ObjectivesTherefore,we used Rot exposed mice to investigate the effect of Rot on cognitive impairment in mice;meanwhile,adeno-associated virus（AAV）was used to knock down the expression of NOX2 in the brain of mice to investigate the effect of NOX2 activity on cognitive impairment in Rot mice.Further,we investigated the role of ferroptosis in the hippocampus and cortex of Rot-exposed mice by detecting changes in iron content,lipid peroxidation,and ferroptosis,and clarified the role of NOX2-regulated ferroptosis in Rot-induced cognitive impairment.Methods1.Stereotaxic apparatus:Sixty-four male C57BL/6J mice were randomly divided into 4 groups according to body weight:AAV-CD68-shCtrl Con group,AAV-CD68-shCtrl Rot group,AAV-CD68-shNOX2 Con group,and AAV-CD68-shNOX2 Rot group,with 16 mice in each group.Adeno-associated virus（AAV）knocked down NOX2 gene expression in mice by stereotaxic injection into the brain.2.Rot exposure model:At the end of the brain stereotaxic experiment,Rot exposure treatment was performed.Mice in the AAV-CD68-shCtrl Rot and AAV-CD68-shNOX2 Rot groups were exposed to Rot（1.5 mg/kg）by intraperitoneal injection once a day（5 m L/kg）for 21 days.AAV-CD68-shCtrl Con and AAV-CD68-shNOX2 Con groups received the same dose of vehicle.3.Western blot assay:NOX2 activity assay:Subunit gp91^phox,p47^phoxcontent,and p47^phoxphosphorylation levels.4.Immunohistochemical staining:（1）Detection of activation levels of microglia and astrocytes:Iba-1（microglial activation marker）,GFAP（a marker of astrocyte activation）antibodies（2）hippocampal and cortical neuronal injury,synaptic loss detection:Neu-N（neuronal nuclear antigen）,PSD95（postsynaptic density protein 95）antibodies.5.Immunofluorescence staining:Microglia Assay:Iba-1.6.Morris water maze（MWM）test:localization navigation experiment and space exploration experiment.7.Kit detection:（1）tissue iron content detection kit（2）Prussian blue staining kit（3）lipid peroxidation MDA,GSH kit.（4）NOX2 activity assay:NAPD+/NADPH kit.8.qRT-PCR was performed to detect:（1）mRNA levels of iron content-related factors DMTI,IRP1,IRP2,and ferritin.（2）mRNA levels of ferroptosis-related factors GPX4,COX2,ACSL4,and SLC7A11.（3）Changes in mRNA levels of inflammation-related factors TNF-α,INOS,and IL-1β.Results1.Rot increased NOX2 activity in the hippocampus and cortex of mice:Western blot was performed in the hippocampus and cortex of mice exposed to Con and Rot by gp91^phoxantibody and p47^phoxphosphorylated antibody.The results showed that the phosphorylation level of p47^phoxand gp91^phoxexpression level was significantly increased in the Rot exposed group compared with the Con group;the ratio of NADP+and NADPH was significantly increased in the Rot group compared with the Con group.2.Knockdown of NOX2 mitigated synaptic loss and neuronal damage in Rot-induced mice:immunohistochemical staining was performed using PSD95 and Neu-N antibodies.The results showed that compared with the AV-CD68-shCtrl Con group,the PSD95 color of each region of the hippocampus（DG,CA1,CA2,CA3）and cortex was significantly lighter and the number of Neu-N was significantly reduced in the AAV-CD68-shCtrl Rot group.The levels of PSD95 and Neu-N were significantly increased in the AAV-CD68-shNOX2 Rot group compared with the AAV-CD68-shCtrl Rot group.3.Knockdown of NOX2 reduced cognitive dysfunction in Rot-induced mice:MWM results showed that escape latency and total distance were significantly increased in Rot group mice compared with Con,and the difference was statistically significant.In the spatial exploration test,the Rot group showed a significant decrease in the number of passes through the platform region,a significant increase in the time to first cross the platform,and a significant decrease in the percentage of target quadrants compared to the Con.Compared with the AAV-CD68-shCtrl Rot group,the AAV-CD68-shNOX2 Rot group had a significantly increased number of platform crossings,significantly decreased time to first platform crossing,and no significant difference in the percentage of target quadrants.4.Knockdown of NOX2 decreased iron content in the hippocampus and cortex of mice induced by Rot:the results showed that compared with the AAV-CD68-shCtrl Con group,the Prussian blue staining color of the hippocampus and cortex of mice in the AAV-CD68-shCtrl Rot group changed significantly brown（positive staining results）,and the tissue iron content increased;compared with the AAV-CD68-shCtrl Rot group,the brown color of mice in the AAV-CD68-shCtrl Rot group decreased significantly,and the tissue iron content decreased.Compared with AAV-CD68-shCtrl Con group,the contents of DMT1,ferritin,IRP1 and IRP2 in hippocampus and cortex of AAV-CD68-shCtrl Rot group were significantly increased,and compared with AAV-CD68-shCtrl Rot group,the contents of DMT1,ferritin,IRP1 and IRP2 in hippocampus and cortex of AAV-CD68-shNOX2 Rot group were significantly decreased.5.Knockdown of NOX2 decreased Rot-induced lipid peroxidation in the hippocampus and cortex of mice:The results showed that compared with the AAV-CD68-shCtrl Con group,the content of MDA and the ratio of GSSH/GSH in the hippocampus and cortex of the AAV-CD68-shCtrl Rot group were significantly higher,and the content of Total GSH was significantly reduced.Compared with the AAV-CD68-shCtrl Rot group,the content of MDA and the ratio of GSSH/GSH in the hippocampus and cortex of the AAV-CD68-shNOX2 Rot group was significantly decreased,and the content of Total GSH was significantly increased.6.NOX2 knockdown reduced Rot-induced ferroptosis in hippocampal and cortical neurons of mice:qRT-PCR detected ferroptosis signature factors in the hippocampus and cortex of mice in each group.The results showed that compared with the AAV-CD68-shCtrl Con group,the contents of GPX4 and SLC7A11 in the hippocampus and cortex of the AAV-CD68-shCtrl Rot group were significantly decreased,and the contents of COX2 and ACSL4 were significantly increased.Compared with the AAV-CD68-shCtrl Rot group,the contents of GPX4 and SLC7A11 in the hippocampus and cortex of the AAV-CD68-shNOX2 Rot group were significantly increased,and the contents of COX2 and ACSL4 were significantly decreased.7.Knockdown of NOX2 decreased the activation of microglia and astrocytes in the hippocampus and cortex of mice induced by Rot:Compared with the AAV-CD68-shCtrl Con group,microglia and astrocytes in the hippocampus and cortex of the AAV-CD68-shCtrl Rot group showed cell body hypertrophy,the color was significantly deepened,the processes around the cell body became thicker and more,Iba-1 and GFAP immunostaining was enhanced,and the optical density value analysis was significantly increased.Compared with the results of hippocampal and cortical staining in AAV-CD68-shCtrl Rot mice,Iba-1 and GFAP immunostaining became lighter,and optical density values were significantly lower in AAV-CD68-shNOX2 Rot mice.8.Knockdown of NOX2 reduced the production of proinflammatory factors in the hippocampus and cortex of mice induced by Rot:mRNA levels of TNF-α,INOS,and IL-1βwere significantly increased in the AAV-CD68-shCtrl Rot group compared with the hippocampus and cortex of mice in the AAV-CD68-shCtrl Con group.Changes in mRNA levels of TNF-α,INOS,and IL-1βwere significantly lower in the AAV-CD68-shNOX2 Rot group compared with the AAV-CD68-shCtrl Rot group.Conclusions1.Rot induces neuronal damage,synaptic loss,and learning and memory impairment in mice.2.Knockdown of NOX2 expression attenuates learning and memory impairment in Rot mice.3.NOX2 may promote Rot-induced learning and memory impairment in mice by regulating ferroptosis.