Efficacies of S-nitrosoglutathione (GSNO) and GSNO reductase inhibitor in SARS-CoV-2 spike protein induced acute lung disease in mice.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which initially surfaced in late 2019, often triggers severe pulmonary complications, encompassing various disease mechanisms such as intense lung inflammation, vascular dysfunction, and pulmonary embolism. Currently, however, there's no drug addressing all these mechanisms simultaneously. This study explored the multi-targeting potential of S-nitrosoglutathione (GSNO) and N6022, an inhibitor of GSNO reductase (GSNOR) on markers of inflammatory, vascular, and thrombotic diseases related to COVID-19-induced acute lung disease. For this, acute lung disease was induced in C57BL/6 mice through intranasal administration of recombinant SARS-CoV-2 spike protein S1 domain (SP-S1). The mice exhibited fever, body weight loss, and increased blood levels and lung expression of proinflammatory cytokines (e.g., TNF-α and IL-6) as well as increased vascular inflammation mediated by ICAM-1 and VCAM-1 and lung infiltration by immune cells (e.g., neutrophils, monocytes, and activated cytotoxic and helper T cells). Further, the mice exhibited increased lung hyperpermeability (lung Evans blue extravasation) leading to lung edema development as well as elevated blood coagulation factors (e.g., fibrinogen, thrombin, activated platelets, and von Willebrand factor) and lung fibrin deposition. Similar to the patients with COVID-19, male mice showed more severe disease than female mice, along with higher GSNOR expression in the lungs. Optimization of GSNO by treatment with exogenous GSNO or inhibition of GSNOR by N6022 (or GSNO knockout) protects against SP-S1-induced lung diseases in both genders. These findings provide evidence for the potential efficacies of GSNO and GSNOR inhibitors in addressing the multi-mechanistic nature of SARS-CoV-2 SP-associated acute-lung disease.

Neuroprotective effects of Alda-1 mitigate spinal cord injury in mice: involvement of Alda-1-induced ALDH2 activation-mediated suppression of reactive aldehyde mechanisms.

Spinal cord injury (SCI) is associated with high production and excessive accumulation of pathological 4-hydroxy-trans-2-nonenal (4-HNE), a reactive aldehyde, formed by SCI-induced metabolic dysregulation of membrane lipids. Reactive aldehyde load causes redox alteration, neuroinflammation, neurodegeneration, pain-like behaviors, and locomotion deficits. Pharmacological scavenging of reactive aldehydes results in limited improved motor and sensory functions. In this study, we targeted the activity of mitochondrial enzyme aldehyde dehydrogenase 2 (ALDH2) to detoxify 4-HNE for accelerated functional recovery and improved pain-like behavior in a male mouse model of contusion SCI. N-(1,3-benzodioxol-5-ylmethyl)-2,6-dichlorobenzamide (Alda-1), a selective activator of ALDH2, was used as a therapeutic tool to suppress the 4-HNE load. SCI was induced by an impactor at the T9-10 vertebral level. Injured animals were initially treated with Alda-1 at 2 hours after injury, followed by once-daily treatment with Alda-1 for 30 consecutive days. Locomotor function was evaluated by the Basso Mouse Scale, and pain-like behaviors were assessed by mechanical allodynia and thermal algesia. ALDH2 activity was measured by enzymatic assay. 4-HNE protein adducts and enzyme/protein expression levels were determined by western blot analysis and histology/immunohistochemistry. SCI resulted in a sustained and prolonged overload of 4-HNE, which parallels with the decreased activity of ALDH2 and low functional recovery. Alda-1 treatment of SCI decreased 4-HNE load and enhanced the activity of ALDH2 in both the acute and the chronic phases of SCI. Furthermore, the treatment with Alda-1 reduced neuroinflammation, oxidative stress, and neuronal loss and increased adenosine 5'-triphosphate levels stimulated the neurorepair process and improved locomotor and sensory functions. Conclusively, the results provide evidence that enhancing the ALDH2 activity by Alda-1 treatment of SCI mice suppresses the 4-HNE load that attenuates neuroinflammation and neurodegeneration, promotes the neurorepair process, and improves functional outcomes. Consequently, we suggest that Alda-1 may have therapeutic potential for the treatment of human SCI. Animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of MUSC (IACUC-2019-00864) on December 21, 2019.

Vascular and immunopathological role of Asymmetric Dimethylarginine (ADMA) in Experimental Autoimmune Encephalomyelitis.

Asymmetric dimethylarginine (ADMA) is an endogenous nitric oxide synthase (NOS) inhibitor/uncoupler inducing vascular pathology. Vascular pathology is an important factor for the development and progression of CNS pathology of MS, yet the role of ADMA in MS remains elusive. Patients with multiple sclerosis (MS) are reported to have elevated blood levels of ADMA, and mice with experimental autoimmune encephalomyelitis (EAE, an animal model of MS) generated by auto-immunization of myelin oligodendrocyte glycoprotein (MOG) and blood-brain barrier (BBB) disruption by pertussis toxin also had increased blood ADMA levels in parallel with induction of clinical disease. To explore the role of ADMA in EAE pathogenesis, EAE mice were treated with a daily dose of ADMA. It is of special interest that ADMA treatment enhanced the BBB disruption in EAE mice and exacerbated the clinical and CNS disease of EAE. ADMA treatment also induced the BBB disruption and EAE disease in MOG-immunized mice even without pertussis toxin treatment, suggesting the role of ADMA in BBB dysfunction in EAE. T-cell polarization studies also documented that ADMA treatment promotes TH 1- and TH 17-mediated immune responses but without affecting Treg-mediated immune response in EAE mice as well as in in vitro T-cell culture. Taken together, these data, for the first time, document the vascular and immunopathogenic roles of ADMA in EAE, thus pointing to the potential of ADMA-mediated mechanism as a new target of potential therapy for MS.

Regulation of B cell functions by S-nitrosoglutathione in the EAE model.

B cells play both protective and pathogenic roles in T cell-mediated autoimmune diseases by releasing regulatory vs. pathogenic cytokines. B cell-depleting therapy has been attempted in various autoimmune diseases but its efficacy varies and can even worsen symptoms due to depletion of B cells releasing regulatory cytokines along with B cells releasing pathogenic cytokines. Here, we report that S-nitrosoglutathione (GSNO) and GSNO-reductase (GSNOR) inhibitor N6022 drive upregulation of regulatory cytokine (IL-10) and downregulation of pathogenic effector cytokine (IL-6) in B cells and protected against the neuroinflammatory disease of experimental autoimmune encephalomyelitis (EAE). In human and mouse B cells, the GSNO/N6022-mediated regulation of IL-10 vs. IL-6 was not limited to regulatory B cells but also to a broad range of B cell subsets and antibody-secreting cells. Adoptive transfer of B cells from N6022 treated EAE mice or EAE mice deficient in the GSNOR gene also regulated T cell balance (Treg > Th17) and reduced clinical disease in the recipient EAE mice. The data presented here provide evidence of the role of GSNO in shifting B cell immune balance (IL-10 > IL-6) and the preclinical relevance of N6022, a first-in-class drug targeting GSNOR with proven human safety, as therapeutics for autoimmune disorders including multiple sclerosis.

GSNOR and ALDH2 alleviate traumatic spinal cord injury.

Traumatic spinal cord injury (SCI) enhances the activity of S-nitrosoglutathione reductase (GSNOR) and inhibits the mitochondrial aldehyde dehydrogenase 2 (ALDH2) activity, resulting in prolonged and sustained pain and functional deficits. This study's objective was to test the hypotheses that GSNOR's specific inhibitor N6022 mitigates pain and improves functional recovery in a mouse model of SCI. Furthermore, the degree of recovery is enhanced and the rate of recovery is accelerated by an ALDH2 activator Alda-1. Using both wild-type and GSNOR-/- mice, the SCI model deployed for groups was contusion at the T9-T10 vertebral level. The enzymatic activity of GSNOR and ALDH2 was measured, and the expression of GSNOR and ALDH2 was determined by western blot analysis. Functional improvements in experimental animals were assessed with locomotor, sensorimotor, and pain-like behavior tests. Wild-type SCI animals had enhanced GSNOR activity and decreased ALDH2 activity, leading to neurovascular dysfunction, edema, and worsened functional outcomes, including locomotor deficits and pain. Compared to wild-type SCI mice, GSNOR-/- mice had better functional outcomes. Monotherapy with either GSNOR inhibition by N6022 or enhanced ALDH2 activity by Alda-1 correlated well with functional recovery and lessened pain. However, combination therapy provided synergistic pain-relieving effects and more significant functional recovery compared with monotherapy. Conclusively, dysregulations in GSNOR and ALDH2 are among the causative mechanisms of SCI injury. Either inhibiting GSNOR or activating ALDH2 ameliorates SCI. Combining the specific inhibitor of GSNOR (N6022) with the selective activator of ALDH2 (Alda-1) provides greater protection to the neurovascular unit and confers greater functional recovery. The study is novel, and the combination therapy (N6022 + Alda-1) possesses translational potential.

Detoxification of Reactive Aldehydes by Alda-1 Treatment Ameliorates Experimental Autoimmune Encephalomyelitis in Mice.

Reactive aldehydes are generated as a toxic end-product of lipid peroxidation under inflammatory oxidative stress condition which is a well-established phenomenon in the pathogenesis of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Alda-1, a selective agonist of mitochondrial aldehyde dehydrogenase 2 (ALDH2), is known to detoxify the reactive aldehydes. In this study, we investigated the effect of Alda-1 on CNS myelin pathology associated with reactive aldehydes and mitochondrial/peroxisomal dysfunctions in a mouse model of EAE. Daily treatment of EAE mice with Alda-1, starting at the peak of disease, ameliorated the clinical manifestation of disease along with the improvement of motor functions. Accordingly, Alda-1 treatment improved demyelination and neuroaxonal degeneration in EAE mice. EAE mice had increased levels of reactive aldehyde species, such as 4-hydroxynonenal (4-HNE), malondialdehyde (MDA), and acrolein (ACL) in the spinal cords and these levels were significantly reduced in Alda-1-treated EAE mice. Furthermore, Alda-1 treatment improved the loss of mitochondrial (OXPHOS) and peroxisomal (PMP70 and catalase) proteins as well as mitochondrial/peroxisomal proliferation factors (PGC-1α and PPARs) in the spinal cords of EAE mice. Taken together, this study demonstrates the therapeutic efficacy of ALDH2-agonist Alda-1 in the abatement of EAE disease through the detoxification of reactive aldehydes, thus suggesting Alda-1 as a potential therapeutic intervention for MS.

Targeting GSNOR for functional recovery in a middle-aged mouse model of stroke.

The nitric oxide (NO) metabolome and the NO metabolite-based neurovascular protective pathways are dysregulated after stroke. The major NO metabolite S-nitrosoglutahione (GSNO) is essential for S-nitrosylation-based signaling events and the inhibition of S-nitrosoglutahione (GSNO)-metabolizing enzyme GSNO reductase (GSNOR) provides protective effects following cardiac ischemia. However, the role of GSNOR and GSNOR inhibition-mediated increased GSNO/S-nitrosylation is not understood in neurovascular diseases such as stroke. Because age is the major risk factor of stroke and recovery in aged stroke patients is low and slow, we investigated the efficacy of GSNOR inhibition using a GSNOR selective inhibitor N6022 in a clinically relevant middle-aged cerebral ischemia and reperfusion (IR) mouse model of stroke. N6022 (5 mg/kg; iv) treatment of IR mice at 2 h after reperfusion followed by the treatment of the same dose daily for 3 days reduced the infarct volume and decreased the neurological score. Daily treatment of IR animals with N6022 for 2 weeks significantly improved neurological score, brain infarctions/atrophy, survival rate, motor (measured by cylinder test) and cognitive (evaluated by novel object recognition test) functions which paralleled the decreased activity of GSNOR, reduced levels of peroxynitrite and decreased neurological score. These results are the first evidence of a new pathway for the treatment of stroke via the inhibition of GSNOR. Based on the efficacy of N6022 in the stroke animal model and its use in human therapeutic studies without toxicity, we submit that GSNOR is a druggable target, and N6022 is a promising drug candidate for human stroke therapy.

Asymmetric dimethylarginine exacerbates cognitive dysfunction associated with cerebrovascular pathology.

Asymmetric dimethylarginine (ADMA), an endogenous inhibitor and uncoupler of nitric oxide synthase, has gained attention as a risk factor for cardiac disease, metabolic syndrome, and cerebrovascular disease. In this study, we investigated the role of systemic ADMA overburden in cerebromicrovascular pathology associated with cognitive dysfunction using APPSwDI transgenic mice expressing human ß-amyloid precursor protein Swedish (Tg-SwDI), a model of cerebrovascular ß-amyloidosis. To induce systemic overburden of ADMA, Tg-SwDI mice were treated with a daily dose of exogenous ADMA. ADMA treatment resulted in elevated ADMA levels in the blood and brain of Tg-SwDI mice. ADMA treatment induced the brain nitrosative stress and inflammation as well as enhanced the brain Aß deposition and cognitive impairment in Tg-SwDI mice. However, ADMA treatment had no such effects on wild type mice. ADMA treatment also exacerbated brain microvascular pathology in Tg-SwDI mice as observed by increased blood-brain barrier dysfunction, loss of tight junction proteins, increased endothelial stress fibers, and decreased microvessel density in the brain. In addition, similar observations were made in cultured human brain microvessel endothelial cells, where ADMA in the presence of VEGF-induced endothelial cell signaling for F-actin stress fiber inducing endothelial barrier dysfunction. Overall, these data document the potential role of ADMA in the cognitive pathology under conditions of cerebrovascular ß-amyloidosis.

S-Nitrosoglutathione Mimics the Beneficial Activity of Endothelial Nitric Oxide Synthase-Derived Nitric Oxide in a Mouse Model of Stroke.

BACKGROUND: The nitric oxide (NO)-producing activity of endothelial nitric oxide synthase (eNOS) plays a significant role in maintaining endothelial function and protecting against the stroke injury. However, the activity of the eNOS enzyme and the metabolism of major NO metabolite S-nitrosoglutathione (GSNO) are dysregulated after stroke, causing endothelial dysfunction. We investigated whether an administration of exogenous of GSNO or enhancing the level of endogenous GSNO protects against neurovascular injury in wild-type (WT) and eNOS-null (endothelial dysfunction) mouse models of cerebral ischemia-reperfusion (IR). METHODS: Transient cerebral ischemic injury was induced by middle cerebral artery occlusion (MCAO) for 60 minutes in male adult WT and eNOS null mice. GSNO (0.1 mg/kg body weight, intravenously) or N6022 (GSNO reductase inhibitor, 5.0 mg/kg body weight, intravenously) was administered 30 minutes before MCAO in preinjury and at the reperfusion in postinjury studies. Brain infarctions, edema, and neurobehavioral functions were evaluated at 24 hours after the reperfusion. RESULTS: eNOS-null mice had a higher degree (P< .05) of injury than WT. Pre- or postinjury treatment with either GSNO or N6022 significantly reduced infarct volume, improved neurological and sensorimotor function in both WT and eNOS-null mice. CONCLUSION: Reduced brain infarctions and edema, and improved neurobehavioral functions by pre- or postinjury GSNO treatment of eNOS knock out mice indicate that GSNO can attenuate IR injury, likely by mimicking the eNOS-derived NO-dependent anti-ischemic and anti-inflammatory functions. Neurovascular protection by GSNO/N6022 in both pre- and postischemic injury groups support GSNO as a promising drug candidate for the prevention and treatment of stroke injury.

Regulation of endothelial barrier integrity by redox-dependent nitric oxide signaling: Implication in traumatic and inflammatory brain injuries.

Nitric oxide (NO) synthesized by eNOS plays a key role in regulation of endothelial barrier integrity but underlying cell signaling pathway is not fully understood at present. Here, we report opposing roles of two different redox-dependent NO metabolites; peroxynitrite (ONOO-) vs. S-nitrosoglutathione (GSNO), in cell signaling pathways for endothelial barrier disruption. In cultured human brain microvessel endothelial cells (hBMVECs), thrombin induced F-actin stress fiber formation causes barrier disruption via activating eNOS. Thrombin induced eNOS activity participated in cell signaling (e.g. RhoA and calcium influx mediated phosphorylation of myosin light chain) for F-actin stress fiber formation by increasing ONOO- levels. On the other hand, thrombin had no effect on intracellular levels of S-nitrosoglutathione (GSNO), another cellular NO metabolite. However, exogenous GSNO treatment attenuated the thrombin-induced cell signaling pathways for endothelial barrier disruption, thus suggesting the role of a shift of NO metabolism (GSNO vs. ONOO-) toward ONOO- synthesis in cell signaling for endothelial barrier disruption. Consistent with these in vitro studies, in animal models of traumatic brain injury and experimental autoimmune encephalomyelitis (EAE), ONOO- scavenger treatment as well as GSNO treatment were effective for attenuation of BBB leakage, edema formation, and CNS infiltration of mononuclear cells. Taken together, these data document that eNOS-mediated NO production and following redox-dependent NO metabolites (ONOO- vs. GSNO) are potential therapeutic target for CNS microvascular disease (traumatic and inflammatory) pathologies.

Amelioration of spinal cord injury in rats by blocking peroxynitrite/calpain activity.

BACKGROUND: Spinal cord injury (SCI) is one of the leading causes of disability and chronic pain. In SCI-induced pathology, homeostasis of the nitric oxide (NO) metabolome is lost. Major NO metabolites such as S-nitrosoglutathione (GSNO) and peroxynitrite are reported to play pivotal roles in regulating the activities of key cysteine proteases, calpains. While peroxynitrite (a metabolite of NO and superoxide) up regulates the activities of calpains leading to neurodegeneration, GSNO (a metabolite of NO and glutathione) down regulates the activities of calpains leading to neuroprotection. In this study, effect of GSNO on locomotor function and pain threshold and their relationship with the levels of peroxynitrite and the activity of calpain in the injured spinal cord were investigated using a 2-week rat model of contusion SCI. RESULTS: SCI animals were initially treated with GSNO at 2 h after the injury followed by a once daily dose of GSNO for 14 days. Locomotor function was evaluated by "Basso Beattie and Bresnahan (BBB) locomotor rating scale" and pain by mechanical allodynia. Peroxynitrite level, as expression of 3-nitrotyrosine (3-NT), calpain activity, as the degradation products of calpain substrate alpha II spectrin, and nNOS activity, as the expression phospho nNOS, were measured by western blot analysis. Treatment with GSNO improved locomotor function and mitigated pain. The treatment also reduced the levels of peroxynitrite (3-NT) and decreased activity of calpains. Reduced levels of peroxynitrite resulted from the GSNO-mediated inhibition of aberrant activity of neuronal nitric oxide synthase (nNOS). CONCLUSIONS: The data indicates that higher levels of 3-NT and aberrant activities of nNOS and calpains correlated with SCI pathology and functional deficits. Treatment with GSNO improved locomotor function and mitigated mechanical allodynia acutely post-injury. Because GSNO shows potential to ameliorate experimental SCI, we discuss implications for GSNO therapy in clinical SCI research.

Combined treatment with GSNO and CAPE accelerates functional recovery via additive antioxidant activities in a mouse model of TBI.

Traumatic brain injury (TBI) is the major cause of physical disability and emotional vulnerability. Treatment of TBI is lacking due to its multimechanistic etiology, including derailed mitochondrial and cellular energy metabolism. Previous studies from our laboratory show that an endogenous nitric oxide (NO) metabolite S-nitrosoglutathione (GSNO) provides neuroprotection and improves neurobehavioral function via anti-inflammatory and anti-neurodegenerative mechanisms. To accelerate the rate and enhance the degree of recovery, we investigated combining GSNO with caffeic acid phenethyl ester (CAPE), a potent antioxidant compound, using a male mouse model of TBI, controlled cortical impact in mice. The combination therapy accelerated improvement of cognitive and depressive-like behavior compared with GSNO or CAPE monotherapy. Separately, both GSNO and CAPE improved mitochondrial integrity/function and decreased oxidative damage; however, the combination therapy had greater effects on Drp1 and MnSOD. Additionally, while CAPE alone activated AMPK, this activation was heightened in combination with GSNO. CAPE treatment of normal animals also significantly increased the expression levels of pAMPK, pACC (activation of AMPK substrate ACC), and pLKB1 (activation of upstream to AMPK kinase LKB1), indicating that CAPE activates AMPK via LKB1. These results show that while GSNO and CAPE provide neuroprotection and improve functional recovery separately, the combination treatment invokes greater recovery by significantly improving mitochondrial functions and activating the AMPK enzyme. Both GSNO and CAPE are in human consumption without any known adverse effects; therefore, a combination therapy-based multimechanistic approach is worthy of investigation in human TBI.

Regulation of IL-10 and IL-17 mediated experimental autoimmune encephalomyelitis by S-nitrosoglutathione.

In this study, we investigated IL-10 and IL-17 specific immunomodulatory potential of S-nitrosoglutathione (GSNO), a physiological nitric oxide carrier molecule, in experimental autoimmune encephalomyelitis (EAE). In active EAE model, GSNO treatment attenuated EAE severity and splenic CD4+ T cells isolated from these mice exhibited decreased IL-17 expression without affecting the IFN-γ expression compared to the cells from untreated EAE mice. Similarly, adoptive transfer of these cells to nave mice resulted in reduction in IL-17 expression in the spinal cords of recipient mice with milder EAE severity. CD4+ T cells isolated from GSNO treated EAE mice, as compared to untreated EAE mice, still expressed lower levels of IL-17 under TH17 skewing conditions, but expressed similar levels of IFN-γ under TH1 skewing condition. Interestingly, under both TH17 and TH1 skewing condition, CD4+ T cells isolated from GSNO treated EAE mice, as compared to untreated EAE mice, expressed higher levels of IL-10 and adoptive transfer of these TH17 and TH1 skewed cells seemingly exhibited milder EAE disease. In addition, adoptive transfer of CD4+ T cells from GSNO treated EAE mice to active EAE mice also ameliorated EAE disease with induction of spinal cord expression of IL-10 and reduction in of IL-17, thus suggesting the participation of IL-10 mechanism in GSNO mediated immunomodulation. GSNO treatment of mice passively immunized with CD4+ T cells either from GSNO treated EAE mice or untreated mice further ameliorated EAE disease, supporting efficacy of GSNO for prophylaxis and therapy in EAE. Overall, these data document a modulatory role of GSNO in IL-17/IL-10 axis of EAE and other autoimmune diseases.

Pathology of nNOS-Expressing GABAergic Neurons in Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is the most common form of dementia that is often accompanied by mood and emotional disturbances and seizures. There is growing body of evidence that neurons expressing γ-aminobutyric acid (GABA) play an important role in regulation of cognition, mood, and emotion as well as seizure susceptibility, but participation of GABAergic neuronal pathology in Alzheimer's disease (AD) is not understood well at present. Here, we report that transgenic mice expressing human amyloid precursor protein Swedish-Dutch-Iowa mutant (APPSweDI) exhibit early loss of neurons expressing GAD67, a GABA-synthesizing enzyme, in advance of the loss of pyramidal neurons in hippocampal CA1 region. The loss of GAD67+ neurons in APPSweDI mice accompanied with decreased spatial cognition as well as increased anxiety-like behaviors and kainic acid-induced seizure susceptibility at early phase. In the hippocampal CA1 region, GAD67+ neurons expressed high basal levels of neuronal nitric oxide synthase (nNOS) and nitrosative stress (nitrotyrosine). Similarly, GAD67+ neurons in primary cortical and hippocampal neuron cultures also expressed high basal levels of nNOS and degenerated in response to lower Aß concentrations due to their high basal levels of nitrosative stress. Given the role of GABAergic neurons in cognitive and neuropsychiatric functions, this study reports the role of nNOS-mediated nitrosative stress in dysfunction of GABAergic neurons and its potential participation in early development of cognitive and neuropsychiatric symptoms in AD.

S-nitrosoglutathione reductase (GSNOR) inhibitor as an immune modulator in experimental autoimmune encephalomyelitis.

We previously reported that S-nitrosoglutathione (GSNO), an endogenous nitric oxide carrier, attenuated TH17-mediated immune responses in experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS). Cellular GSNO homeostasis is regulated via its synthesis by reaction between nitric oxide and glutathione and its enzymatic catabolism by GSNO reductase (GSNOR). In this study, we evaluated potential of reversible inhibitor of GSNOR (N6022) in comparison with exogenous GSNO in immunopathogenesis of EAE. Daily treatment of EAE mice with N6022 or exogenous GSNO significantly attenuated the clinical disease of EAE, but N6022 treatment showed greater efficacy than GSNO. Both N6022 and exogenous GSNO treatments increased the spleen levels of GSNO, as documented by increased protein-associated S-nitrosothiols, and inhibited polarization and CNS effector function of proinflammatory TH17 cells while inducing the polarization and CNS effector function of anti-inflammatory CD4+ CD25+ FOXP3- regulatory T (Treg) cells. Moreover, N6022 further attenuated TH1 while inducing TH2 and CD4+ CD25+ FOXP3+ Treg in their polarization and CNS effector functions. Similar to GSNO, the N6022 treatment protected against the EAE disease induced demyelination. However, neither exogenous GSNO nor N6022 treatment did not cause significant systemic lymphopenic effect as compared to FTY720. Taken together, these data document that optimization of cellular GSNO homeostasis by GSNOR inhibitor (N6022) in NO metabolizing cells attenuates EAE disease via selective inhibition of pro-inflammatory subsets of CD4+ cells (TH1/TH17) while upregulating anti-inflammatory subsets of CD4+ cells (TH2/Treg) without causing lymphopenic effects and thus offers a potential treatment option for MS/EAE.

Combination therapy of lovastatin and AMP-activated protein kinase activator improves mitochondrial and peroxisomal functions and clinical disease in experimental autoimmune encephalomyelitis model.

Recent studies report that loss and dysfunction of mitochondria and peroxisomes contribute to the myelin and axonal damage in multiple sclerosis (MS). In this study, we investigated the efficacy of a combination of lovastatin and AMP-activated protein kinase (AMPK) activator (AICAR) on the loss and dysfunction of mitochondria and peroxisomes and myelin and axonal damage in spinal cords, relative to the clinical disease symptoms, using a mouse model of experimental autoimmune encephalomyelitis (EAE, a model for MS). We observed that lovastatin and AICAR treatments individually provided partial protection of mitochondria/peroxisomes and myelin/axons, and therefore partial attenuation of clinical disease in EAE mice. However, treatment of EAE mice with the lovastatin and AICAR combination provided greater protection of mitochondria/peroxisomes and myelin/axons, and greater improvement in clinical disease compared with individual drug treatments. In spinal cords of EAE mice, lovastatin-mediated inhibition of RhoA and AICAR-mediated activation of AMPK cooperatively enhanced the expression of the transcription factors and regulators (e.g. PPARα/ß, SIRT-1, NRF-1, and TFAM) required for biogenesis and the functions of mitochondria (e.g. OXPHOS, MnSOD) and peroxisomes (e.g. PMP70 and catalase). In summary, these studies document that oral medication with a combination of lovastatin and AICAR, which are individually known to have immunomodulatory effects, provides potent protection and repair of inflammation-induced loss and dysfunction of mitochondria and peroxisomes as well as myelin and axonal abnormalities in EAE. As statins are known to provide protection in progressive MS (Phase II study), these studies support that supplementation statin treatment with an AMPK activator may provide greater efficacy against MS.

GSNO promotes functional recovery in experimental TBI by stabilizing HIF-1α.

Traumatic brain injury (TBI) causes sustained disability due to compromised neurorepair mechanisms. Crucial to neurorepair and functional recovery following both TBI and stroke is hypoxia-inducible factor-1 alpha (HIF-1α). Based on reports that HIF-1α could be stabilized via S-nitrosylation, we tested the hypothesis that the S-nitrosylating agent S-nitrosoglutathione (GSNO) would stabilize HIF-1α, thereby stimulating neurorepair mechanisms and aiding in functional recovery. TBI was induced by controlled cortical impact (CCI) in adult rats. GSNO (0.05mg/kg) was administered at two hours after CCI. The treatment was repeated daily until the 14th day after CCI. Functional recovery was assessed by motor and cognitive functions, and the recovery was compared with the expression of HIF-1α. The mechanisms of GSNO-mediated S-nitrosylation of HIF-1α were determined using brain endothelial cells. While non-treated TBI animals showed sustained neurobehavioral deficits, GSNO treatment of TBI improved neurobehavioral functions. GSNO also increased the expression of HIF-1α and VEGF. The beneficial effects of GSNO on neurobehavioral functions in TBI animals were blocked by treatment with the HIF-1α inhibitor 2-methoxyestradiol (2-ME). The stimulatory effect of GSNO on VEGF was reversed not only by 2-ME but also by the denitrosylating agent dithiothreitol, confirming our hypothesis that GSNO's benefits are mediated by the stabilization of HIF-1α via S-nitrosylation. GSNO's S-nitrosylation of HIF-1α was further confirmed using a biotin switch assay in endothelial cells. The data provide evidence that GSNO treatment of TBI aids functional recovery through stabilizing HIF-1α via S-nitrosylation. GSNO is a natural component of the human brain/body, and its exogenous administration has not shown adverse effects in humans. Therefore, the translational potential of GSNO therapy in TBI is high.

Hypoxia inducible factor-1 alpha stabilization for regenerative therapy in traumatic brain injury.

Mild traumatic brain injury (TBI), also called concussion, initiates sequelae leading to motor deficits, cognitive impairments and subtly compromised neurobehaviors. While the acute phase of TBI is associated with neuroinflammation and nitroxidative burst, the chronic phase shows a lack of stimulation of the neurorepair process and regeneration. The deficiency of nitric oxide (NO), the consequent disturbed NO metabolome, and imbalanced mechanisms of S-nitrosylation are implicated in blocking the mechanisms of neurorepair processes and functional recovery in the both phases. Hypoxia inducible factor-1 alpha (HIF-1α), a master regulator of hypoxia/ischemia, stimulates the process of neurorepair and thus aids in functional recovery after brain trauma. The activity of HIF-1α is regulated by NO via the mechanism of S-nitrosylation of HIF-1α. S-nitrosylation is dynamically regulated by NO metabolites such as S-nitrosoglutathione (GSNO) and peroxynitrite. GSNO stabilizes, and peroxynitrite destabilizes HIF-1α. Exogenously administered GSNO was found not only to stabilize HIF-1α and to induce HIF-1α-dependent genes but also to stimulate the regeneration process and to aid in functional recovery in TBI animals.

Regulation of STAT3 and NF-κB activations by S-nitrosylation in multiple myeloma.

Numerous reports suggest that aberrant activations of STAT3 and NF-κB promote survival and proliferation of multiple myeloma (MM) cells. In the present report, we demonstrate that a synthetic S-nitrosothiol compound, S-nitroso-N-acetylcysteine (SNAC), inhibits proliferation and survival of multiple MM cells via S-nitrosylation-dependent inhibition of STAT3 and NF-κB. In human MM cells (e.g. U266, H929, and IM-9 cells), SNAC treatment increased S-nitrosylation of STAT3 and NF-κB and inhibited their activities. Consequently, SNAC treatment resulted in MM cell cycle arrest at G1/S check point and inhibited their proliferation. SNAC also decreased the expression of cell survival factors and increased the activities of caspases, thus increased sensitivity of MM cells to melphalan, a chemotherapeutic agent for MM. In U266 xenografted mice, SNAC treatment decreased the activity of STAT3 and reduced the growth of human CD138 positive cells (U266 cells) in the bone marrow and also reduced their production of human IgE into the serum. Taken together, these data document the S-nitrosylation mediated inhibition of MM cell proliferation and cell survival via inhibition of STAT3 and NF-κB pathways and its efficacy in animal model of MM.

Biochemical, cell biological, pathological, and therapeutic aspects of Krabbe's disease.

Krabbe's disease (KD; also called globoid cell leukodystrophy) is a genetic disorder involving demyelination of the central (CNS) and peripheral (PNS) nervous systems. The disease may be subdivided into three types, an infantile form, which is the most common and severe; a juvenile form; and a rare adult form. KD is an autosomal recessive disorder caused by a deficiency of galactocerebrosidase activity in lysosomes, leading to accumulation of galactoceramide and neurotoxic galactosylsphingosine (psychosine [PSY]) in macrophages (globoid cells) as well as neural cells, especially in oligodendrocytes and Schwann cells. This ultimately results in damage to myelin in both CNS and PNS with associated morbidity and mortality. Accumulation of PSY, a lysolipid with detergent-like properties, over a threshold level could trigger membrane destabilization, leading to cell lysis. Moreover, subthreshold concentrations of PSY trigger cell signaling pathways that induce oxidative stress, mitochondrial dysfunction, apoptosis, inflammation, endothelial/vascular dysfunctions, and neuronal and axonal damage. From the time the "psychosine hypothesis" was proposed, considerable efforts have been made in search of an effective therapy for lowering PSY load with pharmacological, gene, and stem cell approaches to attenuate PSY-induced neurotoxicity. This Review focuses on the recent advances and prospective research for understanding disease mechanisms and therapeutic approaches for KD. © 2016 Wiley Periodicals, Inc.