Zhanyang Yu

Effects of neuroglobin overexpression on mitochondrial function and oxidative stress following hypoxia/reoxygenation in cultured neurons

Neuroglobin (Ngb) is a recently discovered tissue globin with a high affinity for oxygen that is widely and specifically expressed in neurons of vertebrate central and peripheral nervous systems. Our laboratory and others have shown Ngb overexpression can protect neurons against hypoxic/ischemic insults, but the underlying mechanisms remain poorly understood. In this study, we examined the effects of Ngb overexpression on mitochondrial function, oxidative stress, and neurotoxicity in primary cortical neurons following hypoxia/reoxygenation (H/R). Ngb‐overexpressing transgenic neurons (Ngb‐Tg) were significantly protected against H/R‐induced cell death. Rates of decline in ATP levels, MTT reduction, and mitochondrial membrane potential were significantly ameliorated in Ngb‐Tg neurons. Furthermore, Ngb overexpression reduced superoxide anion generation after H/R, whereas glutathione levels were significantly improved compared with WT controls. Taken together, these data suggest that Ngb is neuroprotective against hypoxia, in part by improving mitochondria function and decreasing oxidative stress.

Annexin A2 combined with low-dose tPA improves thrombolytic therapy in a rat model of focal embolic stroke

Recent studies showed that soluble annexin A2 dramatically increases tissue plasminogen activator (tPA)-mediated plasmin generation in vitro, and reduces thrombus formation in vivo. Here, we hypothesize that combining annexin A2 with tPA can significantly enhance thrombolysis efficacy, so that lower doses of tPA can be applied in ischemic stroke to avoid neurotoxic and hemorrhagic complications. In vitro activity assays confirmed tPA-specific amplification of plasmin generation by recombinant annexin A2. In a rat focal embolic stroke model, combination therapy with tPA and recombinant annexin A2 protein at 2 h post-ischemia decreased the effective dose required for tPA by four-fold and reduced brain infarction. Combining annexin A2 with tPA also lengthened the time window for thrombolysis. Compared with tPA (10 mg/kg) alone, the combination of annexin A2 (5 mg/kg) plus low-dose tPA (2.5 mg/kg) significantly enhanced fibrinolysis, attenuated mortality, brain infarction, and hemorrhagic transformation, even when administered at 4 h post-ischemia. Combination with recombinant annexin A2, the effective thrombolytic dose of tPA can be decreased. As a result, brain hemorrhage and infarction are reduced, and the time window for stroke reperfusion prolonged. Our present findings provide a promising new approach for enhancing tPA-based thrombolytic stroke therapy.

Increased nuclear apoptosis-inducing factor after transient focal ischemia: a 12/15-lipoxygenase- dependent organelle damage pathway

12/15-lipoxygenase (12/15-LOX) contributes to acute neuronal injury and edema formation in mouse models of middle cerebral artery occlusion (MCAO). The apoptosis-inducing factor (AIF) is implicated in caspase-independent forms of apoptosis, and has been linked to ischemic neuronal cell death. We show here that increased AIF in the peri-ischemic cortex of mouse colocalizes with 12/15-LOX after 2 h of MCAO. The 12/15-LOX inhibitor baicalein prevents the increase and nuclear localization of AIF, suggesting this pathway may be partially responsible for the neuroprotective qualities of baicalein. Using an established cell line model of neuronal oxidative stress, we show that 12/15-LOX activated after glutathione depletion leads to AIF translocation to the nucleus, which is abrogated by the 12/15-LOX inhibitor baicalein (control: 19.3%±6.8% versus Glutamate: 64.0%±8.2% versus glutamate plus baicalein: 11.4%±2.2%). Concomitantly, resident proteins of the ER are dispersed throughout the cell (control: 31.0%±8.4% versus glutamate: 70.0%±5.5% versus glutamate plus baicalein: 8.0%±2.7%), suggesting cell death through organelle damage. Taken together, these findings show that 12/15-LOX and AIF are sequential actors in a common cell death pathway that may contribute to stroke-induced brain damage.

Neuroglobin-overexpression Alters Hypoxic Response Gene Expression in Primary Neuron Culture Following Oxygen Glucose Deprivation

Neuroglobin (Ngb) is a tissue globin specifically expressed in neurons. Our laboratory and others have shown that Ngb overexpression protects neurons against hypoxia/ischemia, but the underlying mechanisms remain poorly understood. Recent studies demonstrate that hypoxia/ischemia induces a multitude of spatially and temporally regulated responses in gene expression, and initial evidence suggested that Ngb might function in altering biological processes of gene expression. In this study, we asked how Ngb may help regulate genes responsive to hypoxia. Expression of hypoxic response genes following oxygen-glucose deprivation (OGD) was examined using mRNA arrays in neuroglobin-overexpressing transgenic (Ngb-Tg) and wild type (WT) mouse neurons. From a total of 113 genes on the microarray, mRNA expression of 65 genes was detected. Under rest condition, 14 genes were downregulated in Ngb-Tg neurons compared to WT. In WT neurons, after 4-h OGD followed by 4-h reoxygenation (O4/R4), 20 genes were significantly downregulated, and only Fos mRNA was significantly increased. However, out of the 20 downregulated genes in WT neurons, 12 of them were no longer significantly changed in Ngb-Tg neurons: Add1, Arnt2, Camk2g, Cstb, Dr1, Epas1, Gna11, Hif1a, Il6st, Khsrp, Mars and Rara. Among these 12 genes, 8 (Add1, Camk2g, Cstb, Dr1, Epas1, Gna11, Hif1a, Khsrp) were already reduced in Ngb-Tg neurons compared to WT under rest conditions. Additionally, three genes that initially showed no changes in WT neurons (Ctgf, Egfr and Pea15) were downregulated after OGD in the Ngb-Tg neurons. These findings suggest that Ngb overexpression modulates mRNA expression of multiple hypoxic response genes in the early phase after OGD/reoxygenation. Further studies on these gene networks and interactions may lead to better understanding of Ngb in signaling pathways that contribute to neuroprotection.

Neuroglobin overexpression inhibits oxygen–glucose deprivation-induced mitochondrial permeability transition pore opening in primary cultured mouse cortical neurons

Neuroglobin (Ngb) is an endogenous neuroprotective molecule against hypoxic/ischemic brain injury, but the underlying mechanisms remain largely undefined. Our recent study revealed that Ngb can bind to voltage-dependent anion channel (VDAC), a regulator of mitochondria permeability transition (MPT). In this study we examined the role of Ngb in MPT pore (mPTP) opening following oxygen-glucose deprivation (OGD) in primary cultured mouse cortical neurons. Co-immunoprecipitation (Co-IP) and immunocytochemistry showed that the binding between Ngb and VDAC was increased after OGD compared to normoxia, indicating the OGD-enhanced Ngb-VDAC interaction. Ngb overexpression protected primary mouse cortical neurons from OGD-induced neuronal death, to an extent comparable to mPTP opening inhibitor, cyclosporine A (CsA) pretreatment. We further measured the role of Ngb in OGD-induced mPTP opening using Ngb overexpression and knockdown approaches in primary cultured neurons, and recombinant Ngb exposure to isolated mitochondria. Same as CsA pretreatment, Ngb overexpression significantly reduced OGD-induced mPTP opening markers including mitochondria swelling, mitochondrial NAD(+) release, and cytochrome c (Cyt c) release in primary cultured neurons. Recombinant Ngb incubation significantly reduced OGD-induced NAD(+) release and Cyt c release from isolated mitochondria. In contrast, Ngb knockdown significantly increased OGD-induced neuron death, and increased OGD-induced mitochondrial NAD(+) release and Cyt c release as well, and these outcomes could be rescued by CsA pretreatment. In summary, our results demonstrated that Ngb overexpression can inhibit OGD-induced mPTP opening in primary cultured mouse cortical neurons, which may be one of the molecular mechanisms of Ngbs neuroprotection.

A Rat Model of Studying Tissue-Type Plasminogen Activator Thrombolysis in Ischemic Stroke With Diabetes

Background and Purpose— Poststroke hyperglycemia and diabetes mellitus are associated with lower thrombolytic efficacy and an increased risk of postischemic cerebral hemorrhage. We aimed to develop a rodent model of thrombolysis in diabetic stroke that mimics the clinical situation. Method— Male 6-week Type I diabetic rats (14 weeks old) were subjected to embolic focal stroke and treated with tissue-type plasminogen activator at 1.5 hours. Reperfusion and 24-hour neurological outcomes were measured and compared with nondiabetic control rats. Results— Diabetic rats exhibited resistance to thrombolytic reperfusion, larger infarction volumes, and increased intracerebral hemorrhage. Conclusions— This animal model would be relevant to future studies investigating pathophysiological mechanisms and in developing new therapeutic approaches to enhance the efficacy of tissue-type plasminogen activator thrombolysis in stroke patients with diabetes or poststroke hyperglycemia.

Neuroprotective roles and mechanisms of neuroglobin

Abstract Objective: The objectives of this work were to update and summarize recent experimental works on neuroglobin, mainly focus on its neuroprotective effects and the mechanisms. Methods: The literature was reviewed using PubMed database, and some of the recent findings from our laboratory were included. Results: Neuroglobin is a recently discovered tissue globin with a high affinity for oxygen and is widely and specifically expressed in neurons of vertebrates central and peripheral nervous systems. Investigations in the past several years have advanced our knowledge on the functions and mechanisms of neuroglobin, but many issues remain unclear. Emerging reports have shown that overexpression of neuroglobin confers neuroprotection against neuronal hypoxia or ischemia-induced damage in cultured neurons and in cerebral ischemic animal models. Accumulating findings suggest several possible neuroprotective roles and mechanisms including ligand binding and oxygen sensing, modulation of cell signaling pathways and maintenance of mitochondria function. Conclusion: Emerging experimental works suggest that neuroglobin is neuroprotective against hypoxic/ischemic insults, probably via ligand binding and oxygen sensing, modulation of cell signaling pathways and maintenance of mitochondria function.

Neuroglobin, a Novel Target for Endogenous Neuroprotection against Stroke and Neurodegenerative Disorders

Brain neurons and tissues respond to sublethal injury by activating endogenous protective pathways. Recently, following the failure of a large number of clinical trials for protective strategies against stroke that aim to inhibit a specific ischemia response pathway, endogenous neuroprotection has emerged as a more promising and hopeful strategy for development of therapeutics against stroke and neurodegenerative disorders. Neuroglobin (Ngb) is an oxygen-binding globin protein that is highly and specifically expressed in brain neurons. Accumulating evidence have clearly demonstrated that Ngb is an endogenous neuroprotective molecule against hypoxic/ischemic and oxidative stress-related insults in cultured neurons and animals, as well as neurodegenerative disorders such as Alzheimer’s disease, thus any pharmacological strategy that can up-regulate endogenous Ngb expression may lead to novel therapeutics against these brain disorders. In this review, we summarize recent studies about the biological function, regulation of gene expression, and neuroprotective mechanisms of Ngb. Furthermore, strategies for identification of chemical compounds that can up-regulate endogenous Ngb expression for neuroprotection against stroke and neurodegenerative disorders are discussed.

Mitochondrial distribution of neuroglobin and its response to oxygen–glucose deprivation in primary-cultured mouse cortical neurons

Neuroglobin (Ngb) is a new member of the globin family and a novel endogenous neuroprotective molecule, but its neuroprotective mechanisms remain largely undefined. Previous studies suggest Ngb is both physically and functionally related to mitochondria, however without direct evidence. Our recent discovery has shown that Ngb can physically interact with a number of mitochondrial proteins. In this study we aimed to define the physical interaction between Ngb and mitochondria by determining whether there is a mitochondrial distribution of Ngb under both physiological-resting and pathological oxygen-glucose deprivation (OGD) conditions. Western blot for the first time revealed a small portion of Ngb was physically localized in mitochondria, and the relative mitochondrial Ngb level was significantly increased after OGD in primary-cultured mouse cortical neurons, indicating a translocation of Ngb into mitochondria. Complementary approaches including confocal imaging and immuno-electron microscopy confirmed Ngb distribution in mitochondria under both basal-resting condition and OGD. Inhibitors of mitochondria permeability transition pore (mPTP) and Voltage-Dependent Anion Channel (VDAC) blocked OGD-induced increase of mitochondrial Ngb level, demonstrating a possible role of mPTP in Ngbs mitochondrial translocation. We further found that Ngb overexpression-conferred neuroprotection was correlated with increased mitochondrial Ngb level, suggesting the mitochondria distribution of Ngb is clearly associated with and may contribute to Ngbs neuroprotection.

Neuroglobin is an endogenous neuroprotectant for retinal ganglion cells against glaucomatous damage.

Neuroglobin (NGB), a newly discovered member of the globin superfamily, may regulate neuronal survival under hypoxia or oxidative stress. Although NGB is greatly expressed in retinal neurons, the biological functions of NGB in retinal diseases remain largely unknown. We investigated the role of NGB in an experimental model of glaucoma, a neurodegenerative disorder that usually involves elevation of intraocular pressure (IOP). Elevated IOP is thought to induce oxidative stress in retinal ganglion cells (RGCs), thereby causing RGC death and, eventually, blindness. We found that NGB plays a critical role in increasing RGC resistance to ocular hypertension and glaucomatous damage. Elevation of IOP stimulated a transient up-regulation of endogenous NGB in RGCs. Constitutive overexpression of NGB in transgenic mice prevented RGC damage induced by glutamate cytotoxicity in vitro and/or by chronic IOP elevation in vivo. Moreover, overexpression of NGB attenuated ocular hypertension-induced superoxide production and the associated decrease in ATP levels in mice, suggesting that NGB acts as an endogenous neuroprotectant to reduce oxidative stress and improve mitochondrial function, thereby promoting RGC survival. Thus, NGB may modulate RGC susceptibility to glaucomatous neural damage. Manipulating the expression and bioactivity of NGB may represent a novel therapeutic strategy for glaucoma.