Rebecca A. Kirkland

Bax, Reactive Oxygen, and Cytochrome c Release in Neuronal Apoptosis

Half of all neurons produced during embryogenesis undergo apoptotic death shortly before birth or soon thereafter. Two cell culture models have been used extensively to investigate the cellular and molecular mechanisms underlying apoptosis during neuronal development: (a) sympathetic neurons deprived of their required neurotrophic factor, nerve growth factor, and (b) cerebellar granule neurons deprived of serum in low-potassium medium. A dramatic increase in mitochondrial-derived reactive oxygen species (ROS) occurs during the apoptotic death of both of these cell types. These ROS lie downstream from the proapoptotic protein, Bax. Bax normally resides in the cytoplasm, but translocates to the outer mitochondrial membrane during apoptosis. Once associated with mitochondria, Bax causes release of apoptogenic factors from the mitochondria into the cytoplasm, thus inducing or augmenting the apoptotic cascade. Although there is much controversy about the exact mechanism by which Bax causes release of these factors, recent evidence suggests that the Bax-induced ROS are critical for this release to occur in both sympathetic and cerebellar granule neurons. Because Bax is critical for the apoptotic death of many other types of neurons, it is likely that increased ROS is important for the death of these cells as well.

Rapid Activation of Antioxidant Defenses by Nerve Growth Factor Suppresses Reactive Oxygen Species during Neuronal Apoptosis: Evidence for a Role in Cytochrome c Redistribution

Depriving mouse sympathetic neurons of nerve growth factor (NGF) causes their apoptotic death. A Bax-dependent increase of mitochondrial-derived reactive oxygen species (ROS) begins in these cells soon after NGF withdrawal. We investigated the effects on these ROS of adding NGF to cultures of NGF-deprived neurons. ROS levels were monitored with the fluorescent, redox-sensitive dyes CM-H2DCFDA and MitoSOX Red. The intensity of the former dye increases when it is oxidized by H2O2 and free radicals downstream of H2O2. MitoSOX Red is relatively insensitive to oxidation by H2O2 but is sensitive to oxidation by superoxide (O2.−). Withdrawing NGF increased CM-H2DCFDA intensity, indicating elevated H2O2-associated ROS. Re-exposure of cells deprived of NGF to NGF resulted in rapid suppression of these ROS. Neurons deprived of NGF also had increased MitoSOX Red intensities. Readdition of NGF had no effect on MitoSOX Red fluorescence. The suppression of CM-H2DCFDA-detected ROS by NGF was caused by a rapid activation of glutathione redox cycling. The most likely explanation for these findings is that mitochondria increased O2.− production after NGF withdrawal. The O2.− was converted to H2O2 by dismutation, and the H2O2 was detoxified by accelerated glutathione redox cycling. Our previous work shows that H2O2 induces cytochrome c to be released from mitochondria in NGF-supported sympathetic neurons, whereas antioxidants that detoxify H2O2 block cytochrome c redistribution in NGF-deprived neurons. Readdition of NGF also immediately inhibits cytochrome c release. We present evidence that this inhibition is mediated by the rapid activation of glutathione redox cycling by NGF.

Bax Regulates Production of Superoxide in Both Apoptotic and Nonapoptotic Neurons: Role of Caspases

A Bax- and, apparently, mitochondria-dependent increase in superoxide (O2·−) and other reactive oxygen species (ROS) occurs in apoptotic superior cervical ganglion (SCG) and cerebellar granule (CG) neurons. Here we show that Bax also lies upstream of ROS produced in nonapoptotic neurons and present evidence that caspases partially mediate the pro-oxidant effect of Bax. We used the O2·−-sensitive dye MitoSOX to monitor O2·− in neurons expressing different levels of Bax and mitochondrial superoxide dismutase (SOD2). Basal and apoptotic O2·− levels in both SCG and CG neurons were reduced in SOD2 wild-type (WT) cells having lower Bax concentrations. Apoptotic and nonapoptotic neurons from Bax-WT/SOD2-null but not Bax-null/SOD2-null mice had increased O2·− levels. A caspase inhibitor inhibited O2·− in both apoptotic and nonapoptotic SCG neurons. O2·− production increased when WT, but not Bax-null, SCG neurons were permeabilized and treated with active caspase 3. There was no apoptosis and little increase in O2·− in SCG neurons from caspase 3-null mice exposed to an apoptotic stimulus. O2·− levels in nonapoptotic caspase 3-null SCG neurons were lower than in WT cells but not as low as in caspase inhibitor-treated cells. These data indicate that Bax lies upstream of most O2·− produced in neurons, that caspase 3 is required for increased O2·− production during neuronal apoptosis, that caspase 3 is partially involved in O2·− production in nonapoptotic neurons, and that other caspases may also be involved in Bax-dependent O2·− production in nonapoptotic cells.

Bax affects production of reactive oxygen by the mitochondria of non-apoptotic neurons

Depriving sympathetic neurons in cell culture of nerve growth factor (NGF) causes their apoptotic death. Bax-induced release of cytochrome c from mitochondria and the subsequent activation of cytosolic caspases are central to this death. A Bax-dependent increase of mitochondrial-derived reactive oxygen species (ROS) that is an important component of the apoptotic cascade in these cells begins soon after NGF withdrawal. Here we report that Bax can also influence mitochondrial production of ROS in non-apoptotic sympathetic neurons. We determined ROS levels by using confocal microscopy to monitor changes in the fluorescence intensity of a redox-sensitive dye loaded into single cells. ROS levels were similar in NGF-replete bax wild-type neurons and neurons from which bax had been deleted. To enhance any effects that Bax might have on ROS levels in NGF-replete cells we exposed cultures to the ATP synthase inhibitor, oligomycin. This treatment hyperpolarizes mitochondrial membrane potential (DeltaPsi(m)), an event that can favor increased ROS production. NGF-replete neurons from mice in which bax had been deleted had much higher levels of mitochondrial-derived ROS when treated with oligomycin than did bax wild-type cells. Oligomycin treatment also caused greater hyperpolarization of DeltaPsi(m) in bax-deleted cells than in wild-type cells. These findings indicate that Bax can affect mitochondrial ROS production in non-apoptotic neurons and may do so by altering DeltaPsi(m).

Prooxidant effects of NGF withdrawal and MEK inhibition in sympathetic neurons.

An increase of mitochondrial-derived reactive oxygen species (ROS) occurs in nerve growth factor (NGF)-deprived sympathetic neurons undergoing apoptotic death. It has been reported that NGF suppresses increased ROS production by the mitochondria in these cells through a mitogen-activated protein kinase kinase (MEK)/mitogen-activated protein (MAP) kinase pathway because NGF withdrawal inactivates this pathway and the MEK inhibitor, PD98059, increases ROS in the presence of NGF. We show here that treating rat sympathetic neurons in cell culture with PD98059 greatly decreased cellular concentrations of reduced glutathione (GSH), a major cellular antioxidant. Therefore, it is likely that this inhibitor induces a cellular prooxidant state in NGF-maintained sympathetic neurons primarily by decreasing GSH concentration rather than by causing increased mitochondrial ROS production. These data suggest that the MEK/MAP kinase signaling pathway regulates cellular GSH concentration.

Blueberry Supplementation Influences the Gut Microbiota, Inflammation, and Insulin Resistance in High-Fat-Diet–Fed Rats

Background Gut microbiota dysbiosis has been linked to obesity-associated chronic inflammation. Microbiota manipulation may therefore affect obesity-related comorbidities. Blueberries are rich in anthocyanins, which have anti-inflammatory properties and may alter the gut microbiota. Objective We hypothesized that blueberry supplementation would alter the gut microbiota, reduce systemic inflammation, and improve insulin resistance in high-fat (HF)-diet-fed rats. Methods Twenty-four male Wistar rats (260-270 g; n = 8/group) were fed low-fat (LF; 10% fat), HF (45% fat), or HF with 10% by weight blueberry powder (HF_BB) diets for 8 wk. LF rats were fed ad libitum, whereas HF and HF_BB rats were pair-fed with diets matched for fiber and sugar contents. Glucose tolerance, microbiota composition (16S ribosomal RNA sequencing), intestinal integrity [villus height, gene expression of mucin 2 (Muc2) and β-defensin 2 (Defb2)], and inflammation (gene expression of proinflammatory cytokines) were assessed. Results Blueberry altered microbiota composition with an increase in Gammaproteobacteria abundance (P < 0.001) compared with LF and HF rats. HF feeding led to an ∼15% decrease in ileal villus height compared with LF rats (P < 0.05), which was restored by blueberry supplementation. Ileal gene expression of Muc2 was ∼150% higher in HF_BB rats compared with HF rats (P < 0.05), with expression in the LF group not being different from that in either the HF or HF_BB groups. Tumor necrosis factor α (Tnfa) and interleukin 1β (Il1b) gene expression in visceral fat was increased by HF feeding when compared with the LF group (by 300% and 500%, respectively; P < 0.05) and normalized by blueberry supplementation. Finally, blueberry improved markers of insulin sensitivity. Hepatic insulin receptor substrate 1 (IRS1) phosphorylation at serine 307:IRS1 ratio was ∼35% higher in HF rats compared with LF rats (P < 0.05) and HF_BB rats. Conclusion In HF-diet-fed male rats, blueberry supplementation led to compositional changes in the gut microbiota associated with improvements in systemic inflammation and insulin signaling.

Tumor progression locus 2 differentially regulates IFNγ and IL-17 production by effector CD4+ T cells in a T cell transfer model of colitis.

Autoimmune diseases are approaching epidemic levels, estimated to affect 5–8% of the population. A number of autoimmune diseases are believed to be driven by autoreactive T cells, specifically by T helper 1 (Th1) cells and T helper 17 (Th17) cells. One molecule gaining interest as a therapeutic target is the serine-threonine kinase, Tpl2, which promotes expression of proinflammatory mediators. We previously demonstrated that Tpl2 regulates Th1 differentiation, secretion of the inflammatory cytokine IFNγ, and host defense against the intracellular parasite Toxoplasma gondii. The goal of this study was to determine whether Tpl2 also regulates Th1 or Th17 differentiation in vivo in a model of colitis associated with mixed Th1/Th17 pathology. In vitro, Tpl2−/− naïve CD4 T cells were significantly impaired in IL-17A secretion under traditional Th17 inducing conditions. Reduced IL-17A secretion correlated with increased expression of FoxP3, a transcription factor known to antagonize RORγt function. In a murine T cell transfer model of colitis, transfer of Tpl2−/− T cells resulted in reduced proportions of CD4 T cells expressing IFNγ, but not IL-17A, compared to that induced by wild type T cells. Further studies revealed that IL-17A differentiation induced by IL-6 and IL-23, cytokines implicated in driving Th17 differentiation in vivo, was unaffected by Tpl2 deficiency. Collectively, these results implicate Tpl2 in TGF-β-induced FoxP3 expression. Additionally, they underscore the contribution of Tpl2 to Th1 immunopathology specifically, which suggests that Tpl2 inhibitors may selectively target Th1-based inflammation.

Bax and caspases regulate increased production of mitochondria-derived reactive species in neuronal apoptosis: LACK of A role for depletion of cytochrome c from the mitochondrial electron transport chain

A Bax-dependent increase of reactive oxygen species (ROS) and other reactive species (RS) occurs after withdrawing NGF from mouse sympathetic neurons in cell culture. Possible mechanisms underlying the increased ROS/RS are leakage of electrons from the mitochondrial electron transport chain secondary to caspase cleavage of respiratory complexes or leakage secondary to depletion of cytochrome c from the chain. We previously demonstrated that deletion of Bax or caspase 3 from these cells reduces ROS/RS production to near baseline levels indicating a central role for both Bax and caspase 3 in generating the ROS/RS. Here we depleted cytochrome c to a similar level in neurons from wild type and bax hemizygous or knockout mice by NGF withdrawal or treatment with H2O2. Death was prevented with a caspase inhibitor that caused a partial reduction of ROS/RS levels but did not completely prevent the ROS/RS increase. ROS/RS was highest in bax wild-type cells, lowest in bax knockout cells, and at an intermediate level in the bax hemizygous cells. These and our previous findings indicate that Bax and caspase 3 are necessary for the increased ROS/RS after withdrawing NGF from these cells and that little or none of the increased ROS/RS are secondary to a depletion of cytochrome c from the electron transport chain.

Rate of neurite outgrowth in sympathetic neurons is highly resistant to suppression of protein synthesis: Role of protein degradation/synthesis coupling

Neurites projecting to their target tissues during embryogenesis are subject to many perturbations that could influence their rate of growth. For example, environmental influences such as supply of neurotrophic factor or electrical activity profoundly influence the rate of neuronal protein synthesis. Because accumulation of protein is necessary for outgrowth to proceed normally, a perturbation in protein synthesis could cause a net change in the rate of accumulation of proteins with the result that neurite outgrowth rate increases or decreases. That neurite outgrowth does not normally seem to be subject to such perturbations suggests involvement of a homeostatic system controlling the rate of outgrowth. Consistent with this hypothesis, we show here that the rate of growth of neurites of sympathetic neurons is highly resistant to decreased rates of protein synthesis. Chronic suppression of protein synthesis by 60% had no significant effect on neurite outgrowth over a 2-day period while complete suppression halted it almost immediately. By the 3rd day of exposure, 60% suppression slowed outgrowth. Sustained suppression of protein synthesis rate by 33% had no effect on rate of outgrowth even after 7 days. We show that the ability of the growing neurites to resist protein synthesis suppression appears to be caused, at least in part, by a parallel decrease in the rate of protein degradation. The result of this coupling between degradation and synthesis is that proteins can continue to accumulate even when protein synthesis rate decreases, allowing normal rates of neurite outgrowth.

Tumor Progression Locus 2 (Tpl2) Activates the Mammalian Target of Rapamycin (mTOR) Pathway, Inhibits Forkhead Box P3 (FoxP3) Expression, and Limits Regulatory T Cell (Treg) Immunosuppressive Functions.

The serine/threonine kinase tumor progression locus 2 (Tpl2, also known as Map3k8/Cot) is a potent inflammatory mediator that drives the production of TNFα, IL-1β, and IFNγ. We previously demonstrated that Tpl2 regulates T cell receptor (TCR) signaling and modulates T helper cell differentiation. However, very little is known about how Tpl2 modulates the development of regulatory T cells (Tregs). Tregs are a specialized subset of T cells that express FoxP3 and possess immunosuppressive properties to limit excess inflammation. Because of the documented role of Tpl2 in promoting inflammation, we hypothesized that Tpl2 antagonizes Treg development and immunosuppressive function. Here we demonstrate that Tpl2 constrains the development of inducible Tregs. Tpl2−/− naïve CD4+ T cells preferentially develop into FoxP3+ inducible Tregs in vitro as well as in vivo in a murine model of ovalbumin (OVA)-induced systemic tolerance. Treg biasing of Tpl2−/− T cells depended on TCR signal strength and corresponded with reduced activation of the mammalian target of rapamycin (mTOR) pathway. Importantly, Tpl2−/− Tregs have basally increased expression of FoxP3 and immunosuppressive molecules, IL-10 and cytotoxic T lymphocyte-associated protein 4 (CTLA-4). Furthermore, they were more immunosuppressive in vivo in a T cell transfer model of colitis, as evidenced by reduced effector T cell accumulation, systemic production of inflammatory cytokines, and colonic inflammation. These results demonstrate that Tpl2 promotes inflammation in part by constraining FoxP3 expression and Treg immunosuppressive functions. Overall, these findings suggest that Tpl2 inhibition could be used to preferentially drive Treg induction and thereby limit inflammation in a variety of autoimmune diseases.