Dysregulated bioenergetics: a key regulator of joint inflammation.

OBJECTIVES: This study examines the relationship between synovial hypoxia and cellular bioenergetics with synovial inflammation. METHODS: Primary rheumatoid arthritis synovial fibroblasts (RASF) were cultured with hypoxia, dimethyloxalylglycine (DMOG) or metabolic intermediates. Mitochondrial respiration, mitochondrial DNA mutations, cell invasion, cytokines, glucose and lactate were quantified using specific functional assays. RASF metabolism was assessed by the XF24-Flux Analyzer. Mitochondrial structural morphology was assessed by transmission electron microscopy (TEM). In vivo synovial tissue oxygen (tpO2 mmHg) was measured in patients with inflammatory arthritis (n=42) at arthroscopy, and markers of glycolysis/oxidative phosphorylation (glyceraldehyde 3-phosphate dehydrogenase (GAPDH), PKM2, GLUT1, ATP) were quantified by immunohistology. A subgroup of patients underwent contiguous MRI and positron emission tomography (PET)/CT imaging. RASF and human dermal microvascular endothelial cells (HMVEC) migration/angiogenesis, transcriptional activation (HIF1α, pSTAT3, Notch1-IC) and cytokines were examined in the presence of glycolytic inhibitor 3-(3-Pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO). RESULTS: DMOG significantly increased mtDNA mutations, mitochondrial membrane potential, mitochondrial mass, reactive oxygen species and glycolytic RASF activity with concomitant attenuation of mitochondrial respiration and ATP activity (all p<0.01). This was coupled with altered mitochondrial morphology. Hypoxia-induced lactate levels (p<0.01), which in turn induced basic fibroblast growth factor (bFGF) secretion and RASF invasiveness (all p<0.05). In vivo glycolytic markers were inversely associated with synovial tpO2 levels <20 mm Hg, in contrast ATP was significantly reduced (all p<0.05). Decrease in GAPDH and GLUT1 was paralleled by an increase in in vivo tpO2 in tumour necrosis factor alpha inhibitor (TNFi) responders. Novel PET/MRI hybrid imaging demonstrated close association between metabolic activity and inflammation. 3PO significantly inhibited RASF invasion/migration, angiogenic tube formation, secretion of proinflammatory mediators (all p<0.05), and activation of HIF1α, pSTAT3 and Notch-1IC under normoxic and hypoxic conditions. CONCLUSIONS: Hypoxia alters cellular bioenergetics by inducing mitochondrial dysfunction and promoting a switch to glycolysis, supporting abnormal angiogenesis, cellular invasion and pannus formation.

Microglia-derived IL-1ß triggers p53-mediated cell cycle arrest and apoptosis in neural precursor cells.

Neurogenesis persists in the adult brain and can contribute to learning and memory processes and potentially to regeneration and repair of the affected nervous system. Deregulated neurogenesis has been observed in neuropathological conditions including neurodegenerative diseases, trauma and stroke. However, the survival of neural precursor cells (NPCs) and newly born neurons is adversely affected by the inflammatory environment that arises as a result of microglial activation associated with injury or disease processes. In the present study, we have investigated the mechanisms by which microglia affect NPC proliferation and survival. Importantly, we demonstrate that interleukin-1ß (IL-1ß) produced by lipopolysaccharide/interferon-γ-activated microglia is necessary to induce cell cycle arrest and apoptosis in NPCs in vitro. Mechanistically, we show that IL-1ß activates the tumor suppressor p53 through an oxidative stress-dependent mechanism resulting in p53-mediated induction of the cyclin-dependent kinase inhibitor p21 and the proapoptotic Bcl-2 (B-cell lymphoma-2) family members Puma (p53-upregulated modulator of apoptosis) and Noxa. Furthermore, we demonstrate that cell cycle arrest and apoptosis induced by recombinant IL-1ß or activated microglia is attenuated in p53-deficient NPCs. Finally, we have determined that IL-1ß induces NPC death via the p53-dependent induction of Puma leading to the activation of a Bax (Bcl-2-associated X protein)-mediated mitochondrial apoptotic pathway. In summary, we have elucidated a novel role for p53 in the regulation of NPC proliferation and survival during neuroinflammatory conditions that could be targeted to promote neurogenesis and repair in a number of neurological conditions.

Microglia-derived TNFα induces apoptosis in neural precursor cells via transcriptional activation of the Bcl-2 family member Puma.

Neuroinflammation is a common feature of acute neurological conditions such as stroke and spinal cord injury, as well as neurodegenerative conditions such as Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis. Previous studies have demonstrated that acute neuroinflammation can adversely affect the survival of neural precursor cells (NPCs) and thereby limit the capacity for regeneration and repair. However, the mechanisms by which neuroinflammatory processes induce NPC death remain unclear. Microglia are key mediators of neuroinflammation and when activated to induce a pro-inflammatory state produce a number of factors that could affect NPC survival. Importantly, in the present study we demonstrate that tumor necrosis factor α (TNFα) produced by lipopolysaccharide-activated microglia is necessary and sufficient to trigger apoptosis in mouse NPCs in vitro. Furthermore, we demonstrate that microglia-derived TNFα induces NPC apoptosis via a mitochondrial pathway regulated by the Bcl-2 family protein Bax. BH3-only proteins are known to play a key role in regulating Bax activation and we demonstrate that microglia-derived TNFα induces the expression of the BH3-only family member Puma in NPCs via an NF-κB-dependent mechanism. Specifically, we show that NF-κB is activated in NPCs treated with conditioned media from activated microglia and that Puma induction and NPC apoptosis is blocked by the NF-κB inhibitor BAY-117082. Importantly, we have determined that NPC apoptosis induced by activated microglia-derived TNFα is attenuated in Puma-deficient NPCs, indicating that Puma induction is required for NPC death. Consistent with this, we demonstrate that Puma-deficient NPCs exhibit an ∼13-fold increase in survival as compared with wild-type NPCs following transplantation into the inflammatory environment of the injured spinal cord in vivo. In summary, we have identified a key signaling pathway that regulates neuroinflammation induced apoptosis in NPCs in vitro and in vivo that could be targeted to promote regeneration and repair in diverse neurological conditions.

APAF1 is a key transcriptional target for p53 in the regulation of neuronal cell death.

p53 is a transcriptional activator which has been implicated as a key regulator of neuronal cell death after acute injury. We have shown previously that p53-mediated neuronal cell death involves a Bax-dependent activation of caspase 3; however, the transcriptional targets involved in the regulation of this process have not been identified. In the present study, we demonstrate that p53 directly upregulates Apaf1 transcription as a critical step in the induction of neuronal cell death. Using DNA microarray analysis of total RNA isolated from neurons undergoing p53-induced apoptosis a 5-6-fold upregulation of Apaf1 mRNA was detected. Induction of neuronal cell death by camptothecin, a DNA-damaging agent that functions through a p53-dependent mechanism, resulted in increased Apaf1 mRNA in p53-positive, but not p53-deficient neurons. In both in vitro and in vivo neuronal cell death processes of p53-induced cell death, Apaf1 protein levels were increased. We addressed whether p53 directly regulates Apaf1 transcription via the two p53 consensus binding sites in the Apaf1 promoter. Electrophoretic mobility shift assays demonstrated p53-DNA binding activity at both p53 consensus binding sequences in extracts obtained from neurons undergoing p53-induced cell death, but not in healthy control cultures or when p53 or the p53 binding sites were inactivated by mutation. In transient transfections in a neuronal cell line with p53 and Apaf1 promoter-luciferase constructs, p53 directly activated the Apaf1 promoter via both p53 sites. The importance of Apaf1 as a p53 target gene in neuronal cell death was evaluated by examining p53-induced apoptotic pathways in primary cultures of Apaf1-deficient neurons. Neurons treated with camptothecin were significantly protected in the absence of Apaf1 relative to those derived from wild-type littermates. Together, these results demonstrate that Apaf1 is a key transcriptional target for p53 that plays a pivotal role in the regulation of apoptosis after neuronal injury.

Helper-dependent adenovirus vectors: their use as a gene delivery system to neurons.

Recombinant adenovirus vectors have provided a major advance in gene delivery systems for post-mitotic neurons. However, the use of these first generation vectors has been limited due to the onset of virally mediated effects on cellular function and viability. In the present study we have used primary cultures of cerebellar granule neurons to examine the efficacy and cytotoxic effects of a helper-dependent adenovirus vector (hdAd) in comparison with a first generation vector. Our results demonstrate that the hdAd system provides equally efficient infectivity with significantly reduced toxicity in comparison to first generation vectors. Neurons transduced with a high titre of a first generation vector exhibited a time-dependent shut down in global protein synthesis and impaired physiological function as demonstrated by a loss of glutamate receptor responsiveness. This was followed by an increase in the fraction of TUNEL-positive cells and a loss of neuronal survival. In contrast, hdAds could be used at titres that transduce >85% of neurons with little cytotoxic effect: cellular glutamate receptor responses and rates of protein synthesis were indistinguishable from uninfected controls. Furthermore, cell viability was not significantly affected for at least 7 days after infection. At excessive viral titres, however, infection with hdAd did cause moderate but significant changes in cell function and viability in primary neuronal cultures. Thus, while these vectors are remarkably improved over first generation vectors, these also have limitations with respect to viral effects on cellular function and viability. Gene Therapy (2000) 7, 1200-1209.

Induction and modulation of cerebellar granule neuron death by E2F-1.

Growing evidence suggests that certain cell cycle regulators also mediate neuronal death. Of relevance, cyclin D1-associated kinase activity is increased and the retinoblastoma protein (Rb), a substrate of the cyclin D1-Cdk4/6 complex, is phosphorylated during K(+) deprivation-evoked death of cerebellar granule neurons (CGNs). Cyclin-dependent kinase (CDK) inhibitors block this death, suggesting a requirement for the cyclin D1/Cdk4/6-Rb pathway. However, the downstream target(s) of this pathway are not well defined. The transcription factor E2F-1 is regulated by Rb and is reported to evoke death in proliferating cells when overexpressed. Accordingly, we examined whether E2F-1 was sufficient to evoke death of CGNs and whether it was required for death evoked by low K(+). We show that adenovirus-mediated expression of E2F-1 in CGNs results in apoptotic death, which is independent of p53, dependent upon Bax, and associated with caspase 3-like activity. In addition, we demonstrate that levels of E2F-1 mRNA and protein increase during K(+) deprivation-evoked death. The increase in E2F-1 protein is blocked by the CDK inhibitor flavopiridol. Finally, E2F-1-deficient neurons are modestly resistant to death induced by low K(+). These results indicate that E2F-1 expression is sufficient to promote neuronal apoptosis and that endogenous E2F-1 modulates the death of CGNs evoked by low K(+).

Two pathways for the induction of apoptosis in human lymphocytes.

PURPOSE: To assess the roles of cell membranes and DNA as targets in radiation-induced apoptosis. MATERIALS AND METHODS: Peripheral blood lymphocytes from normal human donors were exposed to different types of apoptosis-inducing agents. Several measures of apoptosis were used to compare the kinetics of the processes induced, as well as to correlate the processes with DNA damage and membrane oxidation. RESULTS: Two kinetically distinct processes were observed. DNA-damaging agents, such as ionizing radiation, bleomycin, cisplatin and the topoisomerase inhibitor m-amsacrine, induced apoptosis by a kinetically slow process initiated by DNA damage and dependent on protein synthesis, but which did not correlate with membrane oxidation. Conversely, the agents t-butyl hydroperoxide and cumene hydroperoxide induced apoptosis by a kinetically fast process independent of protein synthesis and which did correlate with membrane oxidation. CONCLUSIONS: Slowly repaired or unrepairable DNA lesions, such as some of those produced by ionizing radiation exposure, trigger apoptosis by a kinetically slow process. This slow apoptotic pathway is distinct from a fast process not induced by radiation but which is induced by membrane-oxidizing agents.

Apoptosis and the adaptive response in human lymphocytes.

PURPOSE: To determine whether the sensitivity of human lymphocytes for apoptosis induced by either a membrane oxidizing agent or a DNA damaging agent is modified by an adaptive response. MATERIALS AND METHODS: Peripheral blood lymphocytes from normal human donors were exposed to low doses of the DNA damaging agent gamma-radiation, or the membrane oxidizing agent t-butyl hydroperoxide (t-BuOOH), incubated for various times and then tested for their sensitivity to induction of apoptosis by a subsequent exposure to a high dose of either agent. Apoptosis was measured using a fluorescent assay of DNA unwinding or a terminal deoxynucleotide transferase assay. RESULTS: The results show that Go lymphocytes pre-exposed to an adapting dose of radiation or DNA strand breaking agent are not protected but can become sensitized to subsequent apoptosis induced by radiation (a kinetically slow process). Inter- and intraindividual variations were observed. However, neither pre-exposure to radiation nor to a membrane oxidizing agent sensitized lymphocytes from any donor to apoptosis induced by a membrane oxidizing agent (a kinetically fast process). CONCLUSIONS: Since an increase in the elimination of genetically damaged cells by apoptosis could reduce the risk of cancer from exposure to radiation or other DNA damaging agents, this cellular sensitization for apoptosis may represent a novel adaptive response mechanism.

Bax-dependent caspase-3 activation is a key determinant in p53-induced apoptosis in neurons.

p53 is a pivotal molecule regulating the death of neurons both after acute injury and during development. The molecular mechanisms by which p53 induces apoptosis in neuronal cells, however, are not well understood. We have shown previously that adenovirus-mediated p53 gene delivery to neurons was sufficient to induce apoptosis. In the present study we have examined the molecular mechanism by which p53 evokes neuronal cell death. Adenovirus-mediated delivery of p53 to cerebellar granule neurons resulted in caspase-3 (CPP32) activation followed by terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) staining and loss of viability as determined by an MTT survival assay. To determine whether Bax is essential for caspase-3 activation, p53 was expressed in Bax-deficient cells. Bax null neurons did not exhibit caspase-3 activation in response to p53 and were protected from apoptosis. To determine whether Bax-dependent caspase-3 activation was required in p53-mediated neuronal cell death, caspase-3-deficient neurons were examined. Our results indicate that caspase-3-deficient neurons exhibit a remarkable delay in apoptosis and a dramatic decrease in TUNEL-positive cells. These studies demonstrate that p53-induced cell death in postmitotic neurons involves a Bax-dependent caspase-3 activation, suggesting that these molecules are important determinants in neuronal cell death after injury.

Modification of radiation-induced apoptosis in radiation- or hyperthermia-adapted human lymphocytes.

We have investigated the influence of the cellular adaptive response to ionizing radiation on radiation-induced apoptosis in human cells. The adaptive response is believed to be a protective mechanism that confers resistance to the detrimental effects of ionizing radiation and that can be induced by different agents, including hyperthermia and radiation. We have used fluorescence analysis of DNA unwinding (FADU) to assay the induction of apoptosis in human peripheral blood lymphocytes by ionizing radiation. Using the FADU assay, we have observed the initial radiation-induced DNA damage, its subsequent disappearance due to enzymatic repair, and its time- and dose-dependent reappearance. We believe this reappearance of DNA damage to be indicative of the DNA fragmentation event associated with apoptosis. This interpretation has been supported at the individual cell level using an in situ terminal deoxynucleotidyl transferase (TDT) assay (Apoptag, Oncor Inc.), which detects the 3'-hydroxyl ends of fragmented DNA, and by fluorescence analysis of nuclear morphology in Hoechst 33258 stained cells. Pretreatment of cells with low-dose gamma-radiation (0.1 Gy) or mild hyperthermia (40 degrees C for 30 min) altered the extent of radiation-induced (3 Gy) apoptosis. Both pretreatments sensitized lymphocytes to become apoptotic after the 3-Gy radiation exposure. This sensitization may represent an adaptive response mechanism that reduces the risk that genetically damaged cells will proliferate. The ability to modify the probability of radiation-induced apoptosis may lower the cancer risk from a radiation exposure.