Lina Du

Intra-mitochondrial poly-ADP-ribosylation contributes to NAD+ depletion and cell death induced by oxidative stress

Poly(ADP-ribosylation), primarily via poly(ADP-ribose) polymerase-1 (PARP-1), is a pluripotent cellular process important for maintenance of genomic integrity and RNA transcription in cells. However, during conditions of oxidative stress and energy depletion, poly(ADP-ribosylation) paradoxically contributes to mitochondrial failure and cell death. Although it has been presumed that poly(ADP-ribosylation) within the nucleus mediates this pathologic process, PARP-1 and other poly(ADP-ribosyltransferases) are also localized within mitochondria. To this end, the presence of PARP-1 and poly(ADP-ribosylation) were verified within mitochondrial fractions from primary cortical neurons and fibroblasts. Inhibition of poly(ADP-ribosylation) within the mitochondrial compartment preserved transmembrane potential (ΔΨ m ), NAD+content, and cellular respiration, prevented release of apoptosis-inducing factor, and reduced neuronal cell death triggered by oxidative stress. Treatment with liposomal NAD+ also preserved ΔΨ m and cellular respiration during oxidative stress. Furthermore, inhibition of poly(ADP-ribosylation) prevented intranuclear localization of apoptosis-inducing factor and protected neurons from excitotoxic injury; and PARP-1 null fibroblasts were protected from oxidative stress-induced cell death. Collectively these data suggest that poly(ADP-ribosylation) compartmentalized to the mitochondria can be converted from a homeostatic process to a mechanism of cell death when oxidative stress is accompanied by energy depletion. These data implicate intra-mitochondrial poly(ADP-ribosylation) as an important therapeutic target for central nervous system and other diseases associated with oxidative stress and energy failure.

Intranuclear localization of apoptosis‐inducing factor (AIF) and large scale dna fragmentation after traumatic brain injury in rats and in neuronal cultures exposed to peroxynitrite

Programmed cell death occurs after ischemic, excitotoxic, and traumatic brain injury (TBI). Recently, a caspase‐independent pathway involving intranuclear translocation of mitochondrial apoptosis‐inducing factor (AIF) has been reported in vitro; but whether this occurs after acute brain injury was unknown. To address this question adult rats were sacrificed at various times after TBI. Western blot analysis on subcellular protein fractions demonstrated intranuclear localization of AIF in ipsilateral cortex and hippocampus at 2–72 h. Immunocytochemical analysis showed AIF labeling in neuronal nuclei with DNA fragmentation in the ipsilateral cortex and hippocampus. Immunoelectronmicroscopy verified intranuclear localization of AIF in hippocampal neurons after TBI, primarily in regions of euchromatin. Large‐scale DNA fragmentation (∼50 kbp), a signature event in AIF‐mediated cell death, was detected in ipsilateral cortex and hippocampi by 6 h. Neuron‐enriched cultures exposed to peroxynitrite also demonstrated intranuclear AIF and large‐scale DNA fragmentation concurrent with impaired mitochondrial respiration and cell death, events that are inhibited by treatment with a peroxynitrite decomposition catalyst. Intranuclear localization of AIF and large‐scale DNA fragmentation occurs after TBI and in neurons under conditions of oxidative/nitrosative stress, providing the first evidence of this alternative mechanism by which programmed cell death may proceed in neurons after brain injury.

Starving Neurons Show Sex Difference in Autophagy

Sex-dependent differences in adaptation to famine have long been appreciated, thought to hinge on female versus male preferences for fat versus protein sources, respectively. However, whether these differences can be reduced to neurons, independent of typical nutrient depots, such as adipose tissue, skeletal muscle, and liver, was heretofore unknown. A vital adaptation to starvation is autophagy, a mechanism for recycling amino acids from organelles and proteins. Here we show that segregated neurons from males in culture are more vulnerable to starvation than neurons from females. Nutrient deprivation decreased mitochondrial respiration, increased autophagosome formation, and produced cell death more profoundly in neurons from males versus females. Starvation-induced neuronal death was attenuated by 3-methyladenine, an inhibitor of autophagy; Atg7 knockdown using small interfering RNA; or l-carnitine, essential for transport of fatty acids into mitochondria, all more effective in neurons from males versus females. Relative tolerance to nutrient deprivation in neurons from females was associated with a marked increase in triglyceride and free fatty acid content and a cytosolic phospholipase A2-dependent increase in formation of lipid droplets. Similar sex differences in sensitivity to nutrient deprivation were seen in fibroblasts. However, although inhibition of autophagy using Atg7 small interfering RNA inhibited cell death during starvation in neurons, it increased cell death in fibroblasts, implying that the role of autophagy during starvation is both sex- and tissue-dependent. Thus, during starvation, neurons from males more readily undergo autophagy and die, whereas neurons from females mobilize fatty acids, accumulate triglycerides, form lipid droplets, and survive longer.

Selectively increasing inducible heat shock protein 70 via TAT‐protein transduction protects neurons from nitrosative stress and excitotoxicity

Induction of heat shock protein 70 (Hsp70) via sublethal stress protects neurons from subsequent lethal injuries. Here we show that specific and efficient intracellular transduction of Hsp70 can be achieved utilizing an 11 amino acid leading sequence from human immunodeficiency virus (TAT‐Hsp70) in primary neuronal cultures. Western blot and immunohistochemistry demonstrated intracellular accumulation of Hsp70 in insoluble protein fractions and mitochondrial compartments. We then examined the effects of Hsp70 overexpression using TAT‐Hsp70 in models of nitrosative and excitotoxic neuronal death in vitro. Neurons were pre‐incubated with 300 nm TAT‐Hsp 70 overnight, then exposed to either peroxynitrite (ONOO–) or glutamate. TAT‐Hsp70 maintained cellular respiration, inhibited extracellular lactate dehydrogenase release, and/or reduced cell death assessed by flow cytometry vs. vehicle, wild‐type Hsp70, and TAT‐β‐galactosidase controls. Hsp70 transduction using a TAT fusion protein is an effective method to selectively increase Hsp70 in neurons and is sufficient to provide neuroprotection from nitrosative stress and excitotoxicity. Further study is needed to confirm whether TAT‐Hsp70 is protective in in vivo models of brain injury.

Polynitroxylated pegylated hemoglobin: A novel neuroprotective hemoglobin for acute volume-limited fluid resuscitation after combined traumatic brain injury and hemorrhagic hypotension in mice

Objective:Resuscitation of hemorrhagic hypotension after traumatic brain injury is challenging. A hemoglobin-based oxygen carrier may offer advantages. The novel therapeutic hemoglobin-based oxygen carrier, polynitroxylated pegylated hemoglobin (PNPH), may represent a neuroprotective hemoglobin-based oxygen carrier for traumatic brain injury resuscitation. Hypotheses:1) PNPH is a unique non-neurotoxic hemoglobin-based oxygen carrier in neuronal culture and is neuroprotective in in vitro neuronal injury models. 2) Resuscitation with PNPH would require less volume to restore mean arterial blood pressure than lactated Ringers or Hextend and confer neuroprotection in a mouse model of traumatic brain injury plus hemorrhagic hypotension. Design:Prospective randomized, controlled experimental study. Setting:University center. Measurements and Main Results:In rat primary cortical neuron cultures, control bovine hemoglobin was neurotoxic (lactate dehydrogenase release; 3-[4,5-dimethylthiazol-2-yl-]-2,5-diphenyltetrazolium bromide assay) at concentrations from 12.5 to 0.625 &mgr;M, whereas polyethylene glycol-conjugated hemoglobin showed intermediate toxicity. PNPH was not neurotoxic (p < .05 vs. bovine hemoglobin and polyethylene glycol hemoglobin; all concentrations). PNPH conferred neuroprotection in in vitro neuronal injury (glutamate/glycine exposure and neuronal stretch), as assessed via lactate dehydrogenase and 3-[4,5-dimethylthiazol-2-yl-]-2,5-diphenyltetrazolium bromide (all p < .05 vs. control). C57BL6 mice received controlled cortical impact followed by hemorrhagic hypotension (2 mL/100 g, mean arterial blood pressure ∼35–40 mm Hg) for 90 min. Mice were resuscitated (mean arterial blood pressure >50 mm Hg for 30 min) with lactated Ringers, Hextend, or PNPH, and then shed blood was reinfused. Mean arterial blood pressures, resuscitation volumes, blood gasses, glucose, and lactate were recorded. Brain sections at 7 days were examined via hematoxylin and eosin and Fluoro-Jade C (identifying dying neurons) staining in CA1 and CA3 hippocampus. Resuscitation with PNPH or Hextend required less volume than lactated Ringers (both p < .05). PNPH but not Hextend improved mean arterial blood pressure vs. lactated Ringers (p < .05). Mice resuscitated with PNPH had fewer Fluoro-Jade C positive neurons in CA1 vs. Hextend and lactated Ringers, and CA3 vs. Hextend (p < .05). Conclusions:PNPH is a novel neuroprotective hemoglobin-based oxygen carrier in vitro and in vivo that may offer unique advantages for traumatic brain injury resuscitation.

Deciphering of Mitochondrial Cardiolipin Oxidative Signaling in Cerebral Ischemia-Reperfusion

It is believed that biosynthesis of lipid mediators in the central nervous system after cerebral ischemia-reperfusion starts with phospholipid hydrolysis by calcium-dependent phospholipases and is followed by oxygenation of released fatty acids (FAs). Here, we report an alternative pathway whereby cereberal ischemia-reperfusion triggered oxygenation of a mitochondria-specific phospholipid, cardiolipin (CL), is followed by its hydrolysis to yield monolyso-CLs and oxygenated derivatives of fatty (linoleic) acids. We used a model of global cerebral ischemia-reperfusion characterized by 9 minutes of asphyxia leading to asystole followed by cardiopulmonary resuscitation in postnatal day 17 rats. Global ischemia and cardiopulmonary resuscitation resulted in: (1) selective oxidation and hydrolysis of CLs, (2) accumulation of lyso-CLs and oxygenated free FAs, (3) activation of caspase 3/7 in the brain, and (4) motor and cognitive dysfunction. On the basis of these findings, we used a mitochondria targeted nitroxide electron scavenger, which prevented CL oxidation and subsequent hydrolysis, attenuated caspase activation, and improved neurocognitive outcome when administered after cardiac arrest. These data show that calcium-independent CL oxidation and subsequent hydrolysis represent a previously unidentified pathogenic mechanism of brain injury incurred by ischemia-reperfusion and a clinically relevant therapeutic target.

Ischemia-induced autophagy contributes to neurodegeneration in cerebellar Purkinje cells in the developing rat brain and in primary cortical neurons in vitro.

Increased autophagy/mitophagy is thought to contribute to cerebellar dysfunction in Purkinje cell degeneration mice. Intriguingly, cerebellar Purkinje cells are highly vulnerable to hypoxia-ischemia (HI), related at least in part to their high metabolic activity. Whether or not excessive or supraphysiologic autophagy plays a role in Purkinje cell susceptibility to HI is unknown. Accordingly, we evaluated the role of autophagy in the cerebellum after global ischemia produced by asphyxial cardiac arrest in postnatal day (PND) 16-18 rats, using siRNA-targeted inhibition of Atg7, necessary for microtubule-associated protein light chain 3-II (LC3-II) and Atg12-Atg5 complex formation. Two days before a 9min asphyxial cardiac arrest or sham surgery, Atg7 or control siRNA was injected intracisternally to target the cerebellum. Treatment with Atg7 siRNA: 1) reduced Atg7 protein expression in the cerebellum by 56%; 2) prevented the typical ischemia-induced formation of LC3-II in the cerebellum 24h after asphyxial cardiac arrest; 3) improved performance on the beam-balance apparatus on days 1-5; and 4) increased calbindin-labeled Purkinje cell survival assessed on day 14. Improved Purkinje cell survival was more consistent in female vs. male rats, and improved beam-balance performance was only seen in female rats. Similar responses to Atg7 siRNA i.e. reduced autophagy and neurodegeneration vs. control siRNA were seen when exposing sex-segregated green fluorescent protein-LC3 tagged mouse primary cortical neurons to oxygen glucose deprivation in vitro. Thus, inhibition of autophagy after global ischemia in PND 16-18 rats leads to increased survival of Purkinje cells and improved motor performance in a sex-dependent manner.

The nuclear splicing factor RNA binding motif 5 promotes caspase activation in human neuronal cells, and increases after traumatic brain injury in mice

Splicing factors (SFs) coordinate nuclear intron/exon splicing of RNA. Splicing factor disturbances can cause cell death. RNA binding motif 5 (RBM5) and 10 (RBM10) promote apoptosis in cancer cells by activating detrimental alternative splicing of key death/survival genes. The role(s) of RBM5/10 in neurons has not been established. Here, we report that RBM5 knockdown in human neuronal cells decreases caspase activation by staurosporine. In contrast, RBM10 knockdown augments caspase activation. To determine whether brain injury alters RBM signaling, we measured RBM5/10 protein in mouse cortical/hippocampus homogenates after controlled cortical impact (CCI) traumatic brain injury (TBI) plus hemorrhagic shock (CCI+HS). The RBM5/10 staining was higher 48  to 72 hours after injury and appeared to be increased in neuronal nuclei of the hippocampus. We also measured levels of other nuclear SFs known to be essential for cellular viability and report that splicing factor 1 (SF1) but not splicing factor 3A (SF3A) decreased 4  to 72 hours after injury. Finally, we confirm that RBM5/10 regulate protein expression of several target genes including caspase-2, cellular FLICE-like inhibitory protein (c-FLIP), LETM1 Domain-Containing Protein 1 (LETMD1), and amyloid precursor-like protein 2 (APLP2) in neuronal cells. Knockdown of RBM5 appeared to increase expression of c-FLIP(s), LETMD1, and APLP2 but decrease caspase-2.

Unmasking Sex-Based Disparity in Neuronal Metabolism

Both classic and emerging literature point to sex-based disparity in neuronal metabolism. While detectable under baseline conditions, this phenomenon appears to be exaggerated or sometimes unmasked in neurons by cellular stress. A complex sex-dependent response to nutrient deprivation, excitotoxicity, oxidative/nitrositive stress, oxygen-glucose deprivation, and chemical toxicity has been observed in neurons in vitro, as well as after various insults including ischemic or traumatic brain injury in vivo. Importantly, sex-based disparity in response to diverse therapeutics has been seen in neurons in culture, contemporary animal models of brain injury, and in human disease. These have clear implications for pharmacological design of therapeutics targeting central nervous system diseases involving both males and females, and preclinical testing of promising agents.

Synthesis and Evaluation of a Mitochondria-Targeting Poly(ADP-ribose) Polymerase-1 Inhibitor

The poly(ADP-ribose) polymerase (PARP) family of enzymes plays a crucial role in cellular and molecular processes including DNA damage detection and repair and transcription; indeed, PARP inhibitors are under clinical evaluation as chemotherapeutic adjuncts given their capacity to impede genomic DNA repair in tumor cells. Conversely, overactivation of PARP can lead to NAD+ depletion, mitochondrial energy failure, and cell death. Since PARP activation facilitates genomic but impedes mitochondrial DNA repair, nonselective PARP inhibitors are likely to have opposing effects in these cellular compartments. Herein, we describe the synthesis and evaluation of the mitochondria-targeting PARP inhibitor, XJB-veliparib. Attachment of the hemigramicidin S pentapeptide isostere for mitochondrial targeting using a flexible linker at the primary amide site of veliparib did not disrupt PARP affinity or inhibition. XJB-veliparib was effective at low nanomolar concentrations (10-100 nM) and more potent than veliparib in protection from oxygen-glucose deprivation (OGD) in primary cortical neurons. Both XJB-veliparib and veliparib (10 nM) preserved mitochondrial NAD+ after OGD; however, only XJB-veliparib prevented release of NAD+ into cytosol. XJB-veliparib (10 nM) appeared to inhibit poly(ADP-ribose) polymer formation in mitochondria and preserve mitochondrial cytoarchitecture after OGD in primary cortical neurons. After 10 nM exposure, XJB-veliparib was detected by LC-MS in mitochondria but not nuclear-enriched fractions in neurons and was observed in mitoplasts stripped of the outer mitochondrial membrane obtained from HT22 cells. XJB-veliparib was also effective at preventing glutamate-induced HT22 cell death at micromolar concentrations. Importantly, in HT22 cells exposed to H2O2 to produce DNA damage, XJB-veliparib (10 μM) had no effect on nuclear DNA repair, in contrast to veliparib (10 μM) where DNA repair was retarded. XJB-veliparib and analogous mitochondria-targeting PARP inhibitors warrant further evaluation in vitro and in vivo, particularly in conditions where PARP overactivation leads to mitochondrial energy failure and maintenance of genomic DNA integrity is desirable, e.g., ischemia, oxidative stress, and radiation exposure.