Oxidative stress results in apoptosis of neuronal cells, leading to neurodegenerative disorders. However, the underlying molecular mechanism remains to be elucidated. Here, we show that hydrogen peroxide (H₂O₂), a major oxidant generated when oxidative stress occurs, induced apoptosis of neuronal cells (PC12 cells and primary murine neurons), by inhibiting the mammalian target of rapamycin (mTOR)-mediated phosphorylation of ribosomal p70 S6 kinase (S6K1) and eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1). N-acetyl-L-cysteine (NAC), a scavenger of reactive oxygen species (ROS), blocked H₂O₂ inhibition of mTOR signaling. Ectopic expression of wild-type (wt) mTOR, constitutively active S6K1 or downregulation of 4E-BP1 partially prevented H₂O₂ induction of apoptosis. Furthermore, we identified that H₂O₂ induction of ROS inhibited the upstream kinases, Akt and phosphoinositide-dependent kinase 1 (PDK1), but not the type I insulin-like growth factor receptor (IGFR), and activated the negative regulator, AMP-activated protein kinase α (AMPKα), but not the phosphatase and tensin homolog (PTEN) in the cells. Expression of a dominant negative AMPKα or downregulation of AMPKα1 conferred partial resistance to H₂O₂ inhibition of phosphorylation of S6K1 and 4E-BP1, as well as cell viability, indicating that H₂O₂ inhibition of mTOR signaling is at least in part through activation of AMPK. Our findings suggest that AMPK inhibitors may be exploited for prevention of H₂O₂-induced neurodegenerative diseases.

Cadmium (Cd), a toxic environmental contaminant, induces oxidative stress, leading to neurodegenerative disorders. Recently we have demonstrated that Cd induces neuronal apoptosis in part by activation of the mitogen-activated protein kineses (MAPK) and mammalian target of rapamycin (mTOR) pathways. However, the underlying mechanism remains elusive. Here we show that Cd elevated intracellular calcium ion ([Ca²⁺]_i) level in PC12, SH-SY5Y cells and primary murine neurons. BAPTA/AM, an intracellular Ca²⁺ chelator, abolished Cd-induced [Ca²⁺]_i elevation, and blocked Cd activation of MAKPs including extracellular signal-regulated kinase 1/2 (Erk1/2), c-Jun N-terminal kinase (JNK) and p38, and mTOR-mediated signaling pathways, as well as cell death. Pretreatment with the extracellular Ca²⁺ chelator EGTA also prevented Cd-induced [Ca²⁺]_i elevation, MAPK/mTOR activation, as well as cell death, suggesting that Cd-induced extracellular Ca²⁺ influx plays a critical role in contributing to neuronal apoptosis. In addition, calmodulin (CaM) antagonist trifluoperazine (TFP) or silencing CaM attenuated the effects of Cd on MAPK/mTOR activation and cell death. Furthermore, Cd-induced [Ca²⁺]_i elevation or CaM activation resulted in induction of reactive oxygen species (ROS). Pretreatment with BAPTA/AM, EGTA or TFP attenuated Cd-induced ROS and cleavage of caspase-3 in the neuronal cells. Our findings indicate that Cd elevates [Ca²⁺]_i, which induces ROS and activates MAPK and mTOR pathways, leading to neuronal apoptosis. The results suggest that regulation of Cd-disrupted [Ca²⁺]_i homeostasis may be a new strategy for prevention of Cd-induced neurodegenerative diseases.

Ribosomal DNA is one of the most variable regions in the human genome with respect to copy number. Despite the importance of rDNA for cellular function, we know virtually nothing about what governs its copy number, stability, and sequence in the mammalian genome due to challenges associated with mapping and analysis. We applied computational and droplet digital PCR approaches to measure rDNA copy number in normal and cancer states in human and mouse genomes. We find that copy number and sequence can change in cancer genomes. Counterintuitively, human cancer genomes show a loss of copies, accompanied by global copy number co-variation. The sequence can also be more variable in the cancer genome. Cancer genomes with lower copies have mutational evidence of mTOR hyperactivity. The PTEN phosphatase is a tumor suppressor that is critical for genome stability and a negative regulator of the mTOR kinase pathway. Surprisingly, but consistent with the human cancer genomes, hematopoietic cancer stem cells from a Pten^-/- mouse model for leukemia have lower rDNA copy number than normal tissue, despite increased proliferation, rRNA production, and protein synthesis. Loss of copies occurs early and is associated with hypersensitivity to DNA damage. Therefore, copy loss is a recurrent feature in cancers associated with mTOR activation. Ribosomal DNA copy number may be a simple and useful indicator of whether a cancer will be sensitive to DNA damaging treatments. The ribosomal DNA encodes the RNAs needed to make ribosomes for protein synthesis and cellular proliferation. However, ribosomal DNA has been excluded from most mammalian genome-wide studies due to challenges associated with its analysis. We find that both the sequence and copy number of the ribosomal DNA can change in human cancer genomes. mTOR is a kinase that senses the nutritional environment and is often over-active in cancer. Given mutational evidence for mTOR activation in the human cancer genomes with loss of ribosomal DNA copies, we analyzed ribosomal DNA in hematopoietic stem cells derived from mice under conditions of mTOR activation. Like the human cancer genomes, the ribosomal DNA copy number contracts in mTOR activated hematopoietic stem cells relative to normal stem cells. Loss is associated with high rates of cellular proliferation, rRNA production, and protein synthesis, but compromised survival in the presence of DNA damage. Contractions are a recurrent feature in cancer genomes with overactive mTOR and may predict sensitivity to DNA damaging chemotherapeutics. Ribosomal DNA may be altered in other disease contexts.

The mammalian target of rapamycin (mTOR) functions in cells at least as two complexes, mTORC1 and mTORC2. Intensive studies have focused on the roles of mTOR in the regulation of cell proliferation, growth, and survival. Recently we found that rapamycin inhibits type I insulin-like growth factor (IGF-1)-stimulated lamellipodia formation and cell motility, indicating involvement of mTOR in regulating cell motility. This study was set to further elucidate the underlying mechanism. Here we show that rapamycin inhibited protein synthesis and activities of small GTPases (RhoA, Cdc42, and Rac1), crucial regulatory proteins for cell migration. Disruption of mTORC1 or mTORC2 by down-regulation of raptor or rictor, respectively, inhibited the activities of these proteins. However, only disruption of mTORC1 mimicked the effect of rapamycin, inhibiting their protein expression. Ectopic expression of rapamycin-resistant and constitutively active S6K1 partially prevented rapamycin inhibition of RhoA, Rac1, and Cdc42 expression, whereas expression of constitutively hypophosphorylated 4E-BP1 (4EBP1-5A) or down-regulation of S6K1 by RNA interference suppressed expression of the GTPases, suggesting that both mTORC1-mediated S6K1 and 4E-BP1 pathways are involved in protein synthesis of the GTPases. Expression of constitutively active RhoA, but not Cdc42 and Rac1, conferred resistance to rapamycin inhibition of IGF-1-stimulated lamellipodia formation and cell migration. The results suggest that rapamycin inhibits cell motility at least in part by down-regulation of RhoA protein expression and activity through mTORC1-mediated S6K1 and 4E-BP1-signaling pathways.