Chunxiao Liu

Activation of AMPK and inactivation of Akt result in suppression of mTOR-mediated S6K1 and 4E-BP1 pathways leading to neuronal cell death in in vitro models of Parkinson’s disease

Parkinsons disease (PD) is a neurodegenerative disorder characterized by loss of dopaminergic neurons. Dysregulation of mammalian target of rapamycin (mTOR) has been implicated in the pathogenesis of PD. However, the underlying mechanism is incompletely elucidated. Here, we show that PD mimetics (6-hydroxydopamine, N-methyl-4-phenylpyridine or rotenone) suppressed phosphorylation of mTOR, S6K1 and 4E-BP1, reduced cell viability, and activated caspase-3 and PARP in PC12 cells and primary neurons. Overexpression of wild-type mTOR or constitutively active S6K1, or downregulation of 4E-BP1 in PC12 cells partially prevented cell death in response to the PD toxins, revealing that mTOR-mediated S6K1 and 4E-BP1 pathways due to the PD toxins were inhibited, leading to neuronal cell death. Furthermore, we found that the inhibition of mTOR signaling contributing to neuronal cell death was attributed to suppression of Akt and activation of AMPK. This is supported by the findings that ectopic expression of constitutively active Akt or dominant negative AMPKα, or inhibition of AMPKα with compound C partially attenuated inhibition of phosphorylation of mTOR, S6K1 and 4E-BP1, activation of caspase-3, and neuronal cell death triggered by the PD toxins. The results indicate that PD stresses activate AMPK and inactivate Akt, causing neuronal cell death via inhibiting mTOR-mediated S6K1 and 4E-BP1 pathways. Our findings suggest that proper co-manipulation of AMPK/Akt/mTOR signaling may be a potential strategy for prevention and treatment of PD.

Rotenone Induction of Hydrogen Peroxide Inhibits mTOR-mediated S6K1 and 4E-BP1/eIF4E Pathways, Leading to Neuronal Apoptosis

Rotenone, a common pesticide and inhibitor of mitochondrial complex I, induces loss of dopaminergic neurons and consequential aspects of Parkinsons disease (PD). However, the exact mechanism of rotenone neurotoxicity is not fully elucidated. Here, we show that rotenone induced reactive oxygen species (ROS), leading to apoptotic cell death in PC12 cells and primary neurons. Pretreatment with catalase (CAT), a hydrogen peroxide-scavenging enzyme, attenuated rotenone-induced ROS and neuronal apoptosis, implying hydrogen peroxide (H₂O₂) involved, which was further verified by imaging intracellular H₂O₂ using a peroxide-selective probe H2DCFDA. Using thenoyltrifluoroacetone (TTFA), antimycin A, or Mito-TEMPO, we further demonstrated rotenone-induced mitochondrial H₂O₂-dependent neuronal apoptosis. Rotenone dramatically inhibited mTOR-mediated phosphorylation of S6K1 and 4E-BP1, which was also attenuated by CAT in the neuronal cells. Of interest, ectopic expression of wild-type mTOR or constitutively active S6K1, or downregulation of 4E-BP1 partially prevented rotenone-induced H₂O₂ and cell apoptosis. Furthermore, we noticed that rotenone-induced H₂O₂ was linked to the activation of caspase-3 pathway. This was evidenced by the finding that pretreatment with CAT partially blocked rotenone-induced cleavages of caspase-3 and poly (ADP-ribose) polymerase. Of note, zVAD-fmk, a pan caspase inhibitor, only partially prevented rotenone-induced apoptosis in PC12 cells and primary neurons. Expression of mTOR-wt, S6K1-ca, or silencing 4E-BP1 potentiated zVAD-fmk protection against rotenone-induced apoptosis in the cells. The results indicate that rotenone induction of H₂O₂ inhibits mTOR-mediated S6K1 and 4E-BP1/eIF4E pathways, resulting in caspase-dependent and -independent apoptosis in neuronal cells. Our findings suggest that rotenone-induced neuronal loss in PD may be prevented by activating mTOR signaling and/or administering antioxidants.

Rapamycin ameliorates cadmium-induced activation of MAPK pathway and neuronal apoptosis by preventing mitochondrial ROS inactivation of PP2A

Cadmium (Cd) is a highly toxic metal that affects the central nervous system. Recently we have demonstrated that inhibition of mTOR by rapamycin rescues neuronal cells from Cd-poisoning. Here we show that rapamycin inhibited Cd-induced mitochondrial ROS-dependent neuronal apoptosis. Intriguingly, rapamycin remarkably blocked phosphorylation of JNK, Erk1/2 and p38 in neuronal cells induced by Cd, which was strengthened by co-treatment with Mito-TEMPO. Inhibition of JNK and Erk1/2 by SP600125 and U0126, respectively, potentiated rapamycins prevention from Cd-induced apoptosis. Consistently, over-expression of dominant negative c-Jun or MKK1 also potently improved the inhibitory effect of rapamycin on Cd neurotoxicity. Furthermore, pretreatment with SP600125 or U0126, or expression of dominant negative c-Jun or MKK1 enhanced the inhibitory effects of rapamycin or Mito-TEMPO on Cd-induced ROS. Further investigation found that co-treatment with Mito-TEMPO/rapamycin more effectively rescued cells by preventing Cd inactivation of PP2A than treatment with rapamycin or Mito-TEMPO alone. Over-expression of wild-type PP2A reinforced rapamycin or Mito-TEMPO suppression of activated JNK and Erk1/2 pathways, as well as ROS production and apoptosis in neuronal cells in response to Cd. The findings indicate that rapamycin ameliorates Cd-evoked neuronal apoptosis by preventing mitochondrial ROS inactivation of PP2A, thereby suppressing activation of JNK and Erk1/2 pathways. Our results underline that rapamycin may have a potential in preventing Cd-induced oxidative stress and neurodegenerative diseases.

Crosstalk between Ca 2+ signaling and mitochondrial H 2 O 2 is required for rotenone inhibition of mTOR signaling pathway leading to neuronal apoptosis

Rotenone, a neurotoxic pesticide, induces loss of dopaminergic neurons related to Parkinsons disease. Previous studies have shown that rotenone induces neuronal apoptosis partly by triggering hydrogen peroxide (H2O2)-dependent suppression of mTOR pathway. However, the underlying mechanism is not fully understood. Here, we show that rotenone elevates intracellular free calcium ion ([Ca2+]i) level, and activates CaMKII, resulting in inhibition of mTOR signaling and induction of neuronal apoptosis. Chelating [Ca2+]i with BAPTA/AM, preventing extracellular Ca2+ influx using EGTA, inhibiting CaMKII with KN93, or silencing CaMKII significantly attenuated rotenone-induced H2O2 production, mTOR inhibition, and cell death. Interestingly, using TTFA, antimycin A, catalase or Mito-TEMPO, we found that rotenone-induced mitochondrial H2O2 also in turn elevated [Ca2+]i level, thereby stimulating CaMKII, leading to inhibition of mTOR pathway and induction of neuronal apoptosis. Expression of wild type mTOR or constitutively active S6K1, or silencing 4E-BP1 strengthened the inhibitory effects of catalase, Mito-TEMPO, BAPTA/AM or EGTA on rotenone-induced [Ca2+]i elevation, CaMKII phosphorylation and neuronal apoptosis. Together, the results indicate that the crosstalk between Ca2+ signaling and mitochondrial H2O2 is required for rotenone inhibition of mTOR-mediated S6K1 and 4E-BP1 pathways. Our findings suggest that how to control over-elevation of intracellular Ca2+ and overproduction of mitochondrial H2O2 may be a new approach to deal with the neurotoxicity of rotenone.

Rapamycin inhibits BAFF-stimulated cell proliferation and survival by suppressing mTOR-mediated PP2A-Erk1/2 signaling pathway in normal and neoplastic B-lymphoid cells.

B-cell activating factor (BAFF) is involved in not only physiology of normal B cells, but also pathophysiology of aggressive B cells related to malignant and autoimmune diseases. Rapamycin, a lipophilic macrolide antibiotic, has recently shown to be effective in the treatment of human lupus erythematosus. However, how rapamycin inhibits BAFF-stimulated B-cell proliferation and survival has not been fully elucidated. Here, we show that rapamycin inhibited human soluble BAFF (hsBAFF)-induced cell proliferation and survival in normal and B-lymphoid (Raji and Daudi) cells by activation of PP2A and inactivation of Erk1/2. Pretreatment with PD98059, down-regulation of Erk1/2, expression of dominant negative MKK1, or overexpression of wild-type PP2A potentiated rapamycin’s suppression of hsBAFF-activated Erk1/2 and B-cell proliferation/viability, whereas expression of constitutively active MKK1, inhibition of PP2A by okadaic acid, or expression of dominant negative PP2A attenuated the inhibitory effects of rapamycin. Furthermore, expression of a rapamycin-resistant and kinase-active mTOR (mTOR-T), but not a rapamycin-resistant and kinase-dead mTOR-T (mTOR-TE), conferred resistance to rapamycin’s effects on PP2A, Erk1/2 and B-cell proliferation/viability, implying mTOR-dependent mechanism involved. The findings indicate that rapamycin inhibits BAFF-stimulated cell proliferation/survival by targeting mTOR-mediated PP2A-Erk1/2 signaling pathway in normal and neoplastic B-lymphoid cells. Our data highlight that rapamycin may be exploited for preventing excessive BAFF-induced aggressive B-cell malignancies and autoimmune diseases.

Rapamycin prevents cadmium-induced neuronal cell death via targeting both mTORC1 and mTORC2 pathways.

Cadmium (Cd), a toxic environmental contaminant, contributes to neurodegeneration. Rapamycin, a macrocyclic lactone, has shown preventive effect on Cd-induced neuronal cell death. However, the underlying mechanism is not fully understood. Here, we show that rapamycin prevented Cd-induced apoptotic cell death in neuronal cells. Coincidently, rapamycin markedly blocked Cd-induced phosphorylation of Akt, S6K1 and 4E-BP1 in the cells. Expression of a rapamycin-resistant and kinase-active mTOR (S2035T, mTOR-T), but not a rapamycin-resistant and kinase-dead mTOR (S2035T/D2357E, mTOR-TE), conferred resistance to rapamycin inhibition of Cd-induced cell death, implying that the preventive effect of rapamycin on Cd-induced neurotoxicity is mTOR kinase activity-dependent. It appeared that both mTORC1 and mTORC2 were involved in the inhibitory activity of rapamycin, as silencing raptor, rictor or raptor/rictor enhanced rapamycins blockage of Cd-induced cell death. Furthermore, downregulation of S6K1, ectopic expression of constitutively hypophosphorylated 4E-BP1 or dominant negative Akt, or co-treatment with Akt inhibitor also potentiated the rapamycins inhibitory effect. The findings indicate that rapamycin prevents Cd-induced neuronal cell death via suppressing both mTORC1 and mTORC2 pathways. Our results highlight that rapamycin may be exploited for the prevention of Cd-induced neurodegenerative disorders.

Celastrol ameliorates Cd-induced neuronal apoptosis by targeting NOX2-derived ROS-dependent PP5-JNK signaling pathway

Celastrol, a plant‐derived triterpene, has neuroprotective benefit in the models of neurodegenerative disorders that are characterized by overproduction of reactive oxygen species (ROS). Recently, we have reported that cadmium (Cd) activates c‐Jun N‐terminal kinase (JNK) pathway leading to neuronal cell death by inducing ROS inactivation of protein phosphatase 5 (PP5), and celastrol prevents Cd‐activated JNK pathway against neuronal apoptosis. Therefore, we hypothesized that celastrol could hinder Cd induction of ROS‐dependent PP5‐JNK signaling pathway from apoptosis in neuronal cells. Here, we show that celastrol attenuated Cd‐induced expression of NADPH oxidase 2 (NOX2) and its regulatory proteins (p22phox, p40phox, p47phox, p67phox, and Rac1), as well as the generation of ROS in PC12 cells and primary neurons. Also, N‐acetyl‐l‐cysteine, a ROS scavenger, potentiated celastrols inhibition of the events in the cells triggered by Cd, implying neuroprotection by celastrol via blocking Cd‐evoked NOX2‐derived ROS. Further research revealed that celastrol was involved in the regulation of PP5 inactivation and JNK/c‐Jun activation induced by Cd, as celastrol prevented Cd from reducing PP5 expression, and over‐expression of wild‐type PP5 or dominant negative c‐Jun strengthened celastrols inhibition of Cd‐induced phosphorylation of JNK and/or c‐Jun, as well as apoptosis in neuronal cells. Of importance, inhibiting NOX2 with apocynin or silencing NOX2 by RNA interference enhanced the inhibitory effects of celastrol on Cd‐induced inactivation of PP5, activation of JNK/c‐Jun, ROS, and apoptosis in the cells. Furthermore, we noticed that expression of wild‐type PP5 or dominant negative c‐Jun, or pretreatment with JNK inhibitor SP600125 reinforced celastrols suppression of Cd‐induced NOX2 and its regulatory proteins, and consequential ROS in neuronal cells. These findings indicate that celastrol ameliorates Cd‐induced neuronal apoptosis via targeting NOX2‐derived ROS‐dependent PP5‐JNK signaling pathway. Our data highlight a beneficial role of celastrol in the prevention of Cd‐induced oxidative stress and neurodegenerative diseases.

Resveratrol prevents cadmium activation of Erk1/2 and JNK pathways from neuronal cell death via protein phosphatases 2A and 5

Cadmium (Cd), a toxic environmental contaminant, induces neurodegenerative disorders. Resveratrol, a natural product, has been found to exert neuroprotective effects. However, little is known regarding the effect of resveratrol on Cd‐evoked neurotoxicity. Here, we show that resveratrol effectively reversed Cd‐elicited cell viability reduction, morphological change, nuclear fragmentation and condensation, as well as activation of caspase‐3 in neuronal cells, implying neuroprotection against Cd‐poisoning by resveratrol. Further research revealed that both c‐Jun N‐terminal kinase (JNK) and extracellular signal‐regulated kinases 1/2 (Erk1/2) were involved in the inhibitory effect of resveratrol on Cd‐induced cell death, as selective inhibitors of Erk1/2 (U0126) and JNK (SP600125), or over‐expression of dominant negative mitogen‐activated protein kinase kinase 1 (MKK1) or dominant negative c‐Jun potentiated resveratrols prevention of Cd‐induced phosphorylation of JNK and Erk1/2, as well as cell death in neuronal cells. Interestingly, resveratrol potently rescued the cells from Cd‐induced suppression of protein phosphatases 2A (PP2A) and 5 (PP5) activity. Over‐expression of PP2A or PP5 strengthened the inhibitory effects of resveratrol on Cd‐induced activation of Erk1/2 and/or JNK, as well as cell death. The results indicate that resveratrol prevents Cd‐induced activation of Erk1/2 and JNK pathways and neuronal cell death in part via activating PP2A and PP5. Our findings strongly support the notion that resveratrol may serve as a potential therapeutic agent in the prevention of Cd‐induced neurodegenerative diseases.

Rapamycin attenuates BAFF‐extended proliferation and survival via disruption of mTORC1/2 signaling in normal and neoplastic B‐lymphoid cells

B cell activating factor from the TNF family (BAFF) stimulates B‐cell proliferation and survival, but excessive BAFF promotes the development of aggressive B cells leading to malignant and autoimmune diseases. Recently, we have reported that rapamycin, a macrocyclic lactone, attenuates human soluble BAFF (hsBAFF)‐stimulated B‐cell proliferation/survival by suppressing mTOR‐mediated PP2A‐Erk1/2 signaling pathway. Here, we show that the inhibitory effect of rapamycin on hsBAFF‐promoted B cell proliferation/survival is also related to blocking hsBAFF‐stimulated phosphorylation of Akt, S6K1, and 4E‐BP1, as well as expression of survivin in normal and B‐lymphoid (Raji and Daudi) cells. It appeared that both mTORC1 and mTORC2 were involved in the inhibitory activity of rapamycin, as silencing raptor or rictor enhanced rapamycins suppression of hsBAFF‐induced survivin expression and proliferation/viability in B cells. Also, PP242, an mTORC1/2 kinase inhibitor, repressed survivin expression, and cell proliferation/viability more potently than rapamycin (mTORC1 inhibitor) in B cells in response to hsBAFF. Of interest, ectopic expression of constitutively active Akt (myr‐Akt) or constitutively active S6K1 (S6K1‐ca), or downregulation of 4E‐BP1 conferred resistance to rapamycins attenuation of hsBAFF‐induced survivin expression and B‐cell proliferation/viability, whereas overexpression of dominant negative Akt (dn‐Akt) or constitutively hypophosphorylated 4E‐BP1 (4EBP1‐5A), or downregulation of S6K1, or co‐treatment with Akt inhibitor potentiated the inhibitory effects of rapamycin. The findings indicate that rapamycin attenuates excessive hsBAFF‐induced cell proliferation/survival via blocking mTORC1/2 signaling in normal and neoplastic B‐lymphoid cells. Our data underscore that rapamycin may be a potential agent for preventing excessive BAFF‐evoked aggressive B‐cell malignancies and autoimmune diseases.

IL-2, IL-4, IFN-γ or TNF-α enhances BAFF-stimulated cell viability and survival by activating Erk1/2 and S6K1 pathways in neoplastic B-lymphoid cells.

B-cell activating factor of the TNF family (BAFF) has been documented to act as a critical factor in the development of aggressive B lymphocytes and autoimmune diseases. However, the effect of various cytokines on BAFF-elicited neoplastic B-lymphoid cells is not known. In this study, we exhibited that administration of human soluble BAFF (hsBAFF), IL-2, IL-4, IFN-γ, or TNF-α alone increased cell viability and survival in Raji cells concentration-dependently, yet a more robust viability/survival was seen in the cells co-treatment of IL-2, IL-4, IFN-γ, or TNF-α with hsBAFF, respectively. Further research revealed that both Erk1/2 and S6K1 signaling pathways were essential for IL-2, IL-4, IFN-γ, or TNF-α enhancement of the viability/survival in the hsBAFF-stimulated cells, as inhibition of Erk1/2 with U0126 or down-regulation of Erk1/2, or blockage of S6K1 with rapamycin or silencing S6K1, or silencing S6K1/Erk1/2, respectively, reduced the cell viability/survival in the cells treated with/without hsBAFF±IL-2, IL-4, IFN-γ, or TNF-α. These findings indicate that IL-2, IL-4, IFN-γ or TNF-α enhances BAFF-stimulated cell viability/survival by activating Erk1/2 and S6K1 signaling in neoplastic B-lymphoid cells. Our data suggest that modulation of IL-2, IL-4, IFN-γ and/or TNF-α levels, or inhibitors of Erk1/2 or S6K1 may be a new approach to prevent BAFF-induced aggressive B-cell malignancies.