The precise molecular mechanism underlying arsenic trioxide (As 2 O 3 )-induced apoptosis is a subject of extensive study. Here, we show that clinically relevant doses of As 2 O 3 can induce typical apoptosis in IM-9, a multiple myeloma cell line, in a Bcl-2 inhibitable manner. We confirmed that As 2 O 3 directly induced cytochrome c (cyto c) release from isolated mouse liver mitochondria via the mitochondrial permeability transition pore, and we further identified the voltage-dependent anion channel (VDAC) as a biological target of As 2 O 3 responsible for eliciting cyto c release in apoptosis. First, pretreatment of the isolated mitochondria with an anti-VDAC antibody specifically prevented As 2 O 3 -induced cyto c release. Second, in proteoliposome experiments, VDAC by itself was sufficient to mediate As 2 O 3 -induced cyto c release, which could be specifically inhibited by Bcl-X L . Third, As 2 O 3 induced mitochondria membrane potential (DWm) reduction and cyto c release only in the VDAC-expressing, but not in the VDAC-deficient yeast strain. Finally, we found that As 2 O 3 induced the increased expression and homodimerization of VDAC in IM-9 cells, but not in Bcl-2 overexpressing cells, suggesting that VDAC homodimerization could potentially determine its gating capacity to cyto c, and Bcl-2 blockage of VDAC homodimerization represents a novel mechanism for its inhibition of apoptosis.

The imbalance between the synthesis of reactive oxygen species and their elimination by antioxidant defense systems results in macromolecular damage and disruption of cellular redox signaling, affecting cardiac structure and function, thus contributing to contractile dysfunction, myocardial hypertrophy, and fibrosis in chronic heart failure [chronic heart failure (CHF)]. The Kelch-like ECH-associated protein 1-nuclear factor erythroid 2-related factor 2 (Nrf2) pathway is an important antioxidant defense mechanism and is closely associated with oxidative stress-mediated cardiac remodeling in CHF. In the present study, we investigated the regulation of myocardial Nrf2 in the postmyocardial infarction (post-MI) state. Six weeks post-MI, Nrf2 protein was downregulated in the heart, resulting in a decrease of Nrf2-targeted antioxidant enzymes, whereas paradoxically the transcription of Nrf2 was increased, suggesting that translational inhibition of Nrf2 may contribute to the dysregulation in CHF. We therefore hypothesized that microRNAs may be involved in the translational repression of Nrf2 mRNA in the setting of CHF. Using quantitative real-time PCR analysis, we found that three microRNAs, including microRNA-27a, microRNA-28-3p, and microRNA-34a, were highly expressed in the left ventricle of infarcted hearts compared with other organs. Furthermore, in vitro analysis revealed that cultured cardiac myocytes and fibroblasts expressed these three microRNAs in response to TNF-α stimulation. These microRNAs were preferentially incorporated into exosomes and secreted into the extracellular space in which microRNA-enriched exosomes mediated intercellular communication and Nrf2 dysregulation. Taken together, these results suggest that increased local microRNAs induced by MI may contribute to oxidative stress by the inhibition of Nrf2 translation in CHF. NEW & NOTEWORTHY The results of this work provide a novel mechanism mediated by microRNA-enriched exosomes, contributing to the nuclear factor erythroid 2-related factor 2 dysregulation and subsequent oxidative stress. Importantly, these new findings will provide a promising strategy to improve the therapeutic efficacy through targeting nuclear factor erythroid 2-related factor 2-related microRNAs in the chronic heart failure state, which show potentially clinical applications.

Neurogenesis, tied to the proliferation, migration and differentiation of neural progenitor cells (NPC) is affected during neurodegenerative diseases, but how neurogenesis is affected during HIV-1 associated dementia (HAD) has not been fully addressed. Here we test the hypothesis that HIV-1-infected and/or immune-activated brain macrophages affect NPC proliferation and differentiation through the regulation of cytokines. We showed that human monocyte-derived macrophages (MDM) conditioned medium (MCM) induces a dose dependent increase in NPC proliferation. Conditioned media from lipopolysaccharide (LPS)-activated MDM (LPS-MCM) or HIV-infected MCM (HIV-MCM) induced a profound increase in NPC proliferation. HIV-infected and LPS-activated MCM (HIV+LPS-MCM) induced the most robust increase in NPC proliferation. Moreover, LPS-MCM and HIV+LPS-MCM decreased beta-III-tubulin and increased GFAP expression, demonstrating an induction of gliogenesis and inhibition of neurogenesis. The increase of NPC proliferation and gliogenesis correlated with increases in production of TNF-alpha by infected/activated MDM. Although both IL-1beta and TNF-alpha induced NPC proliferation and gliogenesis, these effects were only partially abrogated by soluble TNF-alpha receptors R1 and R2 (TNF-R1R2), but not by the IL-1 receptor antagonist (IL-1ra). This indicated that the HIV-1-infected/LPS-activated MCM-mediated effects were, in part, through TNF-alpha. These observations were confirmed in severe combined immunodeficient (SCID) mice with HIV-1 encephalitis (HIVE). In these HIVE mice, NPC injected with HIV-infected MDM showed more astrocyte differentiation and less neuronal differentiation compared to NPC injection alone. These observations demonstrated that HIV-1-infected and immune-activated MDM could affect neurogenesis through induction of NPC proliferation, inhibition of neurogenesis, and activation of gliogenesis.

We identify calcium-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors on human neural progenitor cells (NPCs) and present a physiological role in neurogenesis. RNA editing of the GluR2 subunit at the Q/R site is responsible for making most AMPA receptors impermeable to calcium. Because a single-point mutation could eliminate the need for editing at the Q/R site and Q/R-unedited GluR2 exists during embryogenesis, the Q/R-unedited GluR2 subunit presumably has some important actions early in development. Using calcium imaging, we found that NPCs contain calcium-permeable AMPA receptors, whereas NPCs differentiated to neurons and astrocytes express calcium-impermeable AMPA receptors. We utilized reverse-transcription polymerase chain reaction and BbvI digestion to demonstrate that NPCs contain Q/R-unedited GluR2, and differentiated cells contain Q/R-edited GluR2 subunits. This is consistent with the observation that the nuclear enzyme responsible for Q/R-editing, adenosine deaminase (ADAR2), is increased during differentiation. Activation of calcium-permeable AMPA receptors induces NPCs to differentiate to the neuronal lineage and increases dendritic arbor formation in NPCs differentiated to neurons. AMPA-induced differentiation of NPCs to neurons is abrogated by overexpression of ADAR2 in NPCs. This elucidates the role of AMPA receptors as inductors of neurogenesis and provides a possible explanation for why the Q/R editing process exists.

Impairment of microglial functions, such as phagocytosis and/or dysregulation of immune responses, has been implicated as an underlying factor involved in the pathogenesis of various neurodegenerative disorders. Our previous studies have demonstrated that long intergenic noncoding RNA (lincRNA)-Cox2 expression is influenced by nuclear factor κB (NF-κB) signaling and serves as a coactivator of transcriptional factors to regulate the expression of a vast array of immune-related genes in microglia. Extracellular vesicles (EVs) have been recognized as primary facilitators of cell-to-cell communication and cellular regulation. Herein, we show that EVs derived from astrocytes exposed to morphine can be taken up by microglial endosomes, leading, in turn, to activation of Toll-like receptor 7 (TLR7) with a subsequent upregulation of lincRNA-Cox2 expression, ultimately resulting in impaired microglial phagocytosis. This was further validated in vivo, wherein inhibition of microglial phagocytic activity was also observed in brain slices isolated from morphine-administrated mice compared with control mice. Additionally, we also showed that intranasal delivery of EVs containing lincRNA-Cox2 siRNA (small interfering RNA) was able to restore microglial phagocytic activity in mice administered morphine. These findings have ramifications for the development of EV-loaded RNA-based therapeutics for the treatment of various disorders involving functional impairment of microglia.

Bax is activated and translocated onto mitochondria to mediate cytochrome c release and apoptosis. The molecular mechanisms of Bax activation during apoptosis remain a subject of debate. We addressed the question of whether reactive oxygen species could directly activate Bax for its subsequent translocation and apoptosis. Using the SW480 human colon adenocarcinoma cell line stably expressing Bax fused to GFP, we showed that H 2 O 2 induces Bax conformational change, mitochondrial translocation, and subsequent oligomerization at mitochondria. We found that H 2 O 2 -induced Bax activation is dependent on the conserved cysteine residue 62 of Bax. Mutation of cysteine 62, but not cysteine 126, to serine or alanine abolished its activation by H 2 O 2 but not other death stimuli, both in SW480 and Bax-deficient HCT116 cells, whereas wild type Bax sensitizes these cells to apoptosis. Cysteines of Bax could chemically react with H 2 O 2 . Mutation of Bax BH3 domain in the presence of cysteine 62 also abolished Bax proapoptotic activity. We conclude that reactive oxygen species could be a direct signal for Bax activation by reacting with cysteine residues. Our results identify a critical role of cysteine 62 in oxidative stress-induced Bax activation and subsequent apoptosis.

Methamphetamine (METH) induces neurodegeneration through damage and apoptosis of dopaminergic nerve terminals and striatal cells, presumably via cross-talk between the endoplasmic reticulum and mitochondria-dependent death cascades. However, the effects of METH on neural progenitor cells (NPC), an important reservoir for replacing neurons and glia during development and injury, remain elusive. Using a rat hippocampal NPC (rhNPC) culture, we characterized the METH-induced mitochondrial fragmentation, apoptosis, and its related signaling mechanism through immunocytochemistry, flow cytometry, and Western blotting. We observed that METH induced rhNPC mitochondrial fragmentation, apoptosis, and inhibited cell proliferation. The mitochondrial fission protein dynamin-related protein 1 (Drp1) and reactive oxygen species (ROS), but not calcium (Ca2+) influx, were involved in the regulation of METH-induced mitochondrial fragmentation. Furthermore, our results indicated that dysregulation of ROS contributed to the oligomerization and translocation of Drp1, resulting in mitochondrial fragmentation in rhNPC. Taken together, our data demonstrate that METH-mediated ROS generation results in the dysregulation of Drp1, which leads to mitochondrial fragmentation and subsequent apoptosis in rhNPC. This provides a potential mechanism for METH-related neurodegenerative disorders, and also provides insight into therapeutic strategies for the neurodegenerative effects of METH.

This study explores the roles of Bax and other Bcl-2 family members play in arsenic trioxide (As 2 O 3 )-induced apoptosis. We showed that As 2 O 3 treatment triggered Bax conformational change and subsequent translocation from cytosol to mitochondria to form various multimeric homo-oligomers in IM-9 cells. On the other hand, human leukemic Jurkat cells deficient in Bax showed dramatically reduced apoptosis in response to As 2 O 3 . Stable overexpression of Bcl-2 in IM-9 cells (IM-9/Bcl-2) inhibited As 2 O 3 -mediated Bax activation and apoptosis, and this inhibition could be partially averted by cellpermeable Bid-Bcl-2 homology (BH)3 peptide. Meanwhile, Bax conformational change and oligomerization induced by As 2 O 3 were not inhibited by the pancaspase inhibitor z-VAD-fmk, although Bid cleavage could be completely abolished. Bax activation by As 2 O 3 seemed to require stress-induced intracellular reactive oxygen species (ROS), since the ROS scavengers (N-acetyl-Lcysteine and lipoic acid) could completely block the conformational change and translocation of Bax from cytosol to mitochondria. These data suggest that As 2 O 3 might exert the cell killing in part by inducing Bax activation through a Bcl-2-suppressible pathway in hematopoietic cells that is caspase independent and intracellular ROS regulated.