Chronic cerebral hypoperfusion (CCH), featuring in most of the Alzheimer's disease spectrum, plays a detrimental role in brain amyloid-β (Aβ) homeostasis, cerebrovascular morbidity, and cognitive decline; therefore, early management of cerebrovascular pathology is considered to be important for intervention in the impending cognitive decline. S-nitrosoglutathione (GSNO) is an endogenous nitric oxide carrier modulating endothelial function, inflammation, and neurotransmission. Therefore, the effect of GSNO treatment on CCH-associated neurocognitive pathologies was determined in vivo by using rats with permanent bilateral common carotid artery occlusion (BCCAO), a rat model of chronic cerebral hypoperfusion. We observed that rats subjected to permanent BCCAO showed a significant decrease in learning/memory performance and increases in brain levels of Aβ and vascular inflammatory markers. GSNO treatment (50 μg/kg/day for 2 months) significantly improved learning and memory performance of BCCAO rats and reduced the Aβ levels and ICAM-1/VCAM-1 expression in the brain. Further, in in vitro cell culture studies, GSNO treatment also decreased the cytokine-induced proinflammatory responses, such as activations of NFκB and STAT3 and expression of ICAM-1 and VCAM-1 in endothelial cells. In addition, GSNO treatment increased the endothelial and microglial Aβ uptake. Additionally, GSNO treatment inhibited the β-secretase activity in primary rat neuron cell culture, thus reducing secretion of Aβ, suggesting GSNO mediated mechanisms in anti-inflammatory and anti-amyloidogenic activities. Taken together, these data document that systemic GSNO treatment is beneficial for improvement of cognitive decline under the conditions of chronic cerebral hypoperfusion and suggests a potential therapeutic use of GSNO for cerebral hypoperfusion associated mild cognitive impairment in Alzheimer's disease.
Alzheimer's disease; amyloid-β; bilateral common carotid artery occlusion; cerebral hypoperfusion; inflammation; S-nitrosylation; S-nitrosoglutathione
Krabbe disease is a lethal, demyelinating condition caused by genetic deficiency of galactocerebrosidase (GALC) and resultant accumulation of its cytotoxic substrate, psychosine (galactosylsphingosine), primarily in oligodendrocytes (OLs). Psychosine is generated by galactosylation of sphingosine by UDP-galactose:ceramide galactosyltransferase (CGT), a galactosylceramide synthesizing enzyme which is primarily expressed in OLs. The expression of CGT and the synthesis of galactosyl-sphingolipids are associated with the terminal differentiation of OL, but little is known about the participation of endogenous psychosine accumulation in OL differentiation under GALC deficient conditions. In this study, we report that accumulation of endogenous psychosine under GALC deficient Krabbe conditions impedes OL differentiation process both by decreasing the expression of myelin lipids and protein and by inducing the cell death of maturating OLs. The psychosine pathology under GALC deficient conditions involves participation of secretory phospholipase A2 (sPLA2) activation and increase in its metabolites, as evidenced by attenuation of psychosine-induced pathology by treatment with pharmacological inhibitor of sPLA2 7,7-dimethyleicosadienoic acid (DEDA). These observations suggest for potential therapeutic efficacy of sPLA2 inhibitor in Krabbe disease.
X-linked adrenoleukodystrophy (X-ALD) is a peroxisomal disorder caused by mutations in the ABCD1 gene. Accumulation of very long chain fatty acids (VLCFA) that have been attributed to reduced peroxisomal VLCFA β-oxidation activity are the hallmark of the disease. Overexpression of ABCD2 gene, the closest homolog of ABCD1, has been shown to compensate for ABCD1, thus correcting the VLCFA derrangement. The accumulation of VLCFA leads to a neuroinflammatory disease process associated with demyelination of the cerebral white matter. The present study underlines the importance of caffeic acid phenethyl ester (CAPE) in inducing the expression of ABCD2 (ALDRP), and normalizing the peroxisomal β-oxidation as well as the levels of saturated and monounsaturated VLCFAs in cultured human skin fibroblasts of X-ALD patients. The expression of ELOVL1, the single elongase catalyzing the synthesis of both saturated VLCFA (C26:0) and mono-unsaturated VLCFA (C26:1), was also reduced by CAPE treatment. Importantly, CAPE upregulated Abcd2 expression and peroxisomal β-oxidation and lowered the VLCFA levels in Abcd1-deficient U87 astrocytes and B12 oligodendrocytes. In addition, using Abcd1/Abcd2-silenced mouse primary astrocytes we examined the effects of CAPE in VLCFA-induced inflammatory response. CAPE treatment decreased the inflammatory response as the expression of inducible nitric oxide synthase, inflammatory cytokine, and activation of NF-κB in Abcd1/Abcd2-silenced mouse primary astrocytes was reduced. The observations indicate that CAPE corrects both the metabolic disease of VLCFA as well as secondary inflammatory disease; therefore, it may be a potential drug candidate to be tested for X-ALD therapy in humans.
X-ALD; Peroxisomes; very long chain fatty acids; glia; nitric oxide; cytokines; ALDP; ABCD2
Lumbar spinal stenosis (LSS) is the leading cause of morbidity and mortality worldwide. LSS pathology is associated with secondary injury caused by inflammation, oxidative damage and cell death. Apart from laminectomy, pharmacological therapy targeting secondary injury is limited. Statins are FDA-approved cholesterol-lowering drug. They also show pleiotropic anti-inflammatory, antioxidant and neuroprotective effects. To investigate the therapeutic efficacy of simvastatin in restoring normal locomotor function after cauda equina compression (CEC) in a rat model of LSS, CEC injury was induced in rats by implanting silicone gels into the epidural spaces of L4 and L6. Experimental group was treated with simvastatin (5 mg/kg body weight), while the injured (vehicle) and sham operated (sham) groups received vehicle solution. Locomotor function in terms of latency on rotarod was measured for 49 days and the threshold of pain was determined for 14 days. Rats were sacrificed on day 3 and 14 and the spinal cord and cauda equina fibers were extracted and studied by histology, immunofluorescence, electron microscopy (EM) and TUNEL assay. Simvastatin aided locomotor functional recovery and enhanced the threshold of pain after the CEC. Cellular Infiltration and demyelination decreased in the spinal cord from the simvastatin group. EM revealed enhanced myelination of cauda equina in the simvastatin group. TUNEL assay showed significantly decreased number of apoptotic neurons in spinal cord from the simvastatin group compared to the vehicle group. Simvastatin hastens the locomotor functional recovery and reduces pain after CEC. These outcomes are mediated through the neuroprotective and anti-inflammatory properties of simvastatin. The data indicate that simvastatin may be a promising drug candidate for LSS treatment in humans.
Spinal stenosis; Simvastatin; Cauda equina; Demyelination; g-ratio
We earlier documented that lovastatin (LOV)-mediated inhibition of small Rho GTPases activity protects vulnerable oligodendrocytes (OLs) in mixed glial cell cultures stimulated with Th1 cytokines and in a murine model of multiple sclerosis (MS). However, the precise mechanism of OL protection remains unclear. We here employed genetic and biochemical approaches to elucidate the underlying mechanism that protects LOV treated OLs from Th1 (tumor necrosis factor-α) and Th17 (interleukin-17) cytokines toxicity in in vitro. Cytokines enhanced the reactive oxygen species (ROS) generation and mitochondrial membrane depolarization with corresponding lowering of glutathione (reduced) level in OLs and that were reverted by LOV. In addition, the expression of ROS detoxifying enzymes (catalase and superoxide-dismutase 2) and the transactivation of peroxisome proliferators-activated receptor (PPAR)-α/-β/-γ including PPAR-γ coactivator-1α were enhanced by LOV in similarly treated OLs. Interestingly, LOV-mediated inhibition of small Rho GTPases, i.e., RhoA and cdc42, and Rho-associated kinase (ROCK) activity enhanced the levels of PPAR ligands in OLs via extracellular signal regulated kinase (1/2)/p38 mitogen-activated protein kinase/cytoplasmic phospholipase 2/cyclooxygenase-2 signaling cascade activation. Small hairpin RNA transfection-based studies established that LOV mainly enhances PPAR-α and less so of PPAR-β and PPAR-γ transactivation that enhances ROS detoxifying defense in OLs. In support of this, the observed LOV-mediated protection was lacking in PPAR-α-deficient OLs exposed to cytokines. Collectively, these data provide unprecedented evidence that LOV-mediated inhibition of the Rho–ROCK signaling pathway boosts ROS detoxifying defense in OLs via PPAR-α-dependent mechanism that has implication in neurodegenerative disorders including MS.
lovastatin; EAE/MS; oligodendrocyte progenitors; PPAR-α; RhoA-ROCK; survival; differentiation
Multiple Sclerosis (MS) is an incurable central nervous system (CNS) demyelinating disease affecting several million people worldwide. Due to the multifactorial and complex pathology of MS, FDA approved drugs often show limited efficacy inpatients. We earlier documented that both lovastatin (cholesterol lowering drug) and metformin (anti-diabetic drug) attenuate experimental autoimmune encephalomyelitis (EAE), a widely used model of MS via different mechanisms of action. Since combination therapy of two or more agents has advantage over monotherapy, we here assessed the therapeutic efficacy of metformin and lovastatin combination in EAE. We found that suboptimal doses of these drugs in combination had additive effect to attenuate established EAE in treated animals than their individual treatments. Histological, immunohistochemistry and western blotting analyses revealed that the observed demyelination and axonal loss as evident from reduced levels of myelin and neurofilament proteins in the spinal cords of EAE animals were attenuated by treatment with these drugs in combination. Accordingly, the observed infiltration of myelin reactive T cells (CD4 and CD8) and macrophages (CD68) as well as the increased expression of their signatory cytokines in the spinal cords of EAE animals were attenuated by this regimen as revealed by enzyme-linked immune-sorbent assay and real-time PCR analyses. In the periphery, this regimen biased the class of elicited anti-myelin basic protein immunoglobulins from IgG2a to IgG1 and IgG2b, suggesting a Th1 to Th2 shift which was further supported by the increased expression of their signatory cytokines in EAE animals. Taken together, these data imply that metformin and lovastatin combination attenuates T-cell autoimmunity and neurodegeneration in treated EAE animals thereby suggesting that the oral administration of these FDA approved drugs in combination has potential to limit MS pathogenesis.
Multiple sclerosis; Experimental autoimmune encephalomyelitis; Lovastatin; Metformin; T-cell autoimmunity; Neuroprotection
The hallmark of stroke injury is endothelial dysfunction leading to blood brain barrier (BBB) leakage and edema. Among the causative factors of BBB disruption are accelerating peroxynitrite formation and the resultant decreased bioavailability of nitric oxide (NO). S-nitrosoglutathione (GSNO), an S-nitrosylating agent, was found not only to reduce the levels of peroxynitrite but also to protect the integrity of BBB in a rat model of cerebral ischemia and reperfusion (IR). A treatment with GSNO (3 μmol/kg) after IR reduced 3-nitrotyrosine levels in and around vessels and maintained NO levels in brain. This mechanism protected endothelial function by reducing BBB leakage, increasing the expression of Zonula occludens-1 (ZO-1), decreasing edema, and reducing the expression of MMP-9 and E-selectin in the neurovascular unit. An administration of the peroxynitrite-forming agent 3-morpholino sydnonimine (3 μmol/kg) at reperfusion increased BBB leakage and decreased the expression of ZO-1, supporting the involvement of peroxynitrite in BBB disruption and edema. Mechanistically, the endothelium-protecting action of GSNO was invoked by reducing the activity of NF-κB and increasing the expression of S-nitrosylated proteins. Taken together, the results support the ability of GSNO to improve endothelial function by reducing nitroxidative stress in stroke.
ischemia reperfusion; GSNO; S-nitrosylation; peroxynitrite; BBB; endothelial dysfunction
Amenorrhea following spinal cord injury (SCI) has been well documented. There has been little research on the underlying molecular mechanisms and therapeutics.
The purpose of the present study was to investigate the effect of GSNO in ameliorating SCI-induced amenorrhea through affecting the expression of CX43, NFkB, and ERβ protein.
SCI was induced in female SD rats at the T9-T10 level. Estrous stage was determined by vaginal smear. GSNO (50 μg/kg body weight) was gavage fed daily. Animals were sacrificed on day 7 and 14 post SCI. Ovaries were fixed for histological and biochemical studies. Expression levels of ERβ, CX-43, and NFkB were analyzed by Western blot and immunofluorescence.
Main Outcome Measures
GSNO hastens resumption of the estrous cycle following SCI-induced transient arrest.
Resumption of estrous cycle was hastened by GSNO. Atretic and degenerating follicles seen in the ovary of SCI rats on day 14 post-SCI were decreased in GSNO treated animals. The increased CX43 expression observed with SCI ovary was decreased by GSNO. ERβ expression decreased significantly on day 7 and 14 post-SCI and was restored with GSNO treatment. Following SCI, NFkB expression was increased in the ovarian follicles and the expression was reduced with GSNO administration. The number of terminal deoxynucleotidyl transferase-mediated biotinylated uridine triphosphate (UTP) nick end labeling positive follicular and luteal cells was increased after SCI. GSNO-treated animals had significantly fewer apoptotic cells in the ovary.
SCI-induced amenorrhea is accompanied by an increase in CX43 expression and a decrease in ERβ expression. SCI animals treated with GSNO resumed the estrous cycle significantly earlier. These results indicate a potential therapeutic value for GSNO in treating amenorrhea among SCI patients.
Spinal Cord Injury; Amenorrhea; Sexual Function; Estrous Cycle
Fatty liver or hepatic steatosis is a common health problem associated with abnormal liver function and increased susceptibility to ischemia/reperfusion injury. The objective of this study was to investigate the effect of the fatty acid synthase inhibitor cerulenin on hepatic function in steatotic ob/ob mice. Different dosages of cerulenin were administered intraperitoneally to ob/ob mice for 2 to 7 days. Body weight, serum AST/ALT, hepatic energy state, and gene expression patterns in ob/ob mice were examined. We found that cerulenin treatment markedly improved hepatic function in ob/ob mice. Serum AST/ALT levels were significantly decreased and hepatic ATP levels increased in treated obese mice compared to obese controls, accompanied by fat depletion in the hepatocyte. Expression of peroxisome proliferator-activated receptors α and γ and uncoupling protein 2 were suppressed with cerulenin treatment and paralleled changes in AST/ALT levels. Hepatic glutathione content were increased in some cases and apoptotic activity in the steatotic livers was minimally changed with cerulenin treatment. In conclusion, these results demonstrate that fatty acid synthase blockade constitutes a novel therapeutic strategy for altering hepatic steatosis at non-stressed states in obese livers.
Lumbar spinal stenosis (LSS) causes ischemia, inflammation, demyelination and results in dysfunction of the cauda equina (CE), leading to pain and locomotor functional deficits. We investigated whether exogenous administration of S-nitrosoglutathione (GSNO), an endogenous redox modulating anti-neuroinflammatory agent, hastens functional recovery in a CE compression (CEC) rat model. CEC was induced in adult female rats by the surgical implantation of two silicone blocks within the epidural spaces of L4-L6 vertebrae. GSNO (50 μg/kg body weight) was administered by gavage 1 h after the injury, and the treatment was continued daily thereafter. GSNO induced change in the pain threshold was evaluated for four days after the compression. Tissue analyses and locomotor function evaluation were carried out at two weeks and four weeks after the CEC respectively. GSNO significantly improved motor function in CEC rats as evidenced by an increased latency on rotarod compared with vehicle-treated CEC rats. CEC induced hyperalgesia was decreased by GSNO. GSNO also increased the expression of VEGF, reduced cellular infiltration (H&E staining) and apoptotic cell death (TUNEL assay), and hampered demyelination (LFB staining and g-ratio). These data demonstrate that administration of GSNO after CEC decreased inflammation, hyperalgesia and cell death leading to improved locomotor function of CEC rats. The therapeutic potential of GSNO observed in the present study with CEC rats suggests that GSNO is a candidate drug to test in LSS patients.
LSS; VEGF; demyelination; g-ratio; neuroprotection
In X-ALD, mutation/deletion of ALD gene (ABCD1) and the resultant very long chain fatty acid (VLCFA) derangement has dramatically opposing effects in astrocytes and oligodendrocytes. While loss of Abcd1 in astrocytes produces a robust inflammatory response, the oligodendrocytes undergo cell death leading to demyelination in X-linked adrenoleukodystrophy (X-ALD). The mechanisms of these distinct pathways in the two cell types are not well understood. Here, we investigated the effects of Abcd1-knockdown and the subsequent alteration in VLCFA metabolism in human U87 astrocytes and rat B12 oligodendrocytes. Loss of Abcd1 inhibited peroxisomal β-oxidation activity and increased expression of VLCFA synthesizing enzymes, elongase of very long chain fatty acids (ELOVLs) (1 and 3) in both cell types. However, higher induction of ELOVL's in Abcd1-deficient B12 oligodendrocytes than astrocytes suggests that ELOVL pathway may play a prominent role in oligodendrocytes in X-ALD. While astrocytes are able to maintain the cellular homeostasis of anti-apoptotic proteins, Abcd1-deletion in B12 oligodendrocytes downregulated the anti-apototic (Bcl-2 and Bcl-xL) and cell survival (phospho-Erk1/2) proteins, and upregulated the pro-apoptotic proteins (Bad, Bim, Bax and Bid) leading to cell loss. These observations provide insights into different cellular signaling mechanisms in response to Abcd1-deletion in two different cell types of CNS. The apoptotic responses were accompanied by activation of caspase-3 and caspase-9 suggesting the involvement of mitochondrial-caspase-9-dependent mechanism in Abcd1-deficient oligodendrocytes. Treatment with histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) corrected the VLCFA derangement both in vitro and in vivo, and inhibited the oligodendrocytes loss. These observations provide a proof-of principle that HDAC inhibitor SAHA may have a therapeutic potential for X-ALD.
In most cases of neonatal hypoxic-ischemic encephalopathy, the exact timing of the hypoxic-ischemic event is unknown, and we have few reliable biomarkers to precisely identify the phase of injury or recovery in an individual patient. However, it is becoming increasingly clear that for neuroprotection in neonates to succeed, an understanding of the phase of injury is important to ascertain. In addition, in utero antecedents of chronic hypoxia, hypoxic preconditioning, intrauterine infection, and fetal gender may change the expected time course of injury. Neuroprotective interventions, such as hypothermia and N-acetylcysteine, currently have efficacy in human and animal studies only if instituted early in the inflammatory cascade. While these cascades are currently being investigated, molecular mechanisms of recovery have received little attention and may ultimately reveal a window for therapeutic intervention that is much longer than current paradigms.
Stroke disability stems from insufficient neurorepair mechanisms. Improvement of functions has been achieved through rehabilitation or therapeutic agents. Therefore, we combined exercise with a neurovascular protective agent, S-nitrosoglutathione (GSNO), to accelerate functional recovery.
Stroke was induced by middle cerebral artery occlusion for 60 min followed by reperfusion in adult male rats. Animals were treated with vehicle (IR group), GSNO (0.25 mg/kg, GSNO group), rotarod exercise (EX group) and GSNO plus exercise (GSNO+EX group). The groups were studied for 14 days to determine neurorepair mechanisms and functional recovery.
Treated groups showed reduced infarction, decreased neuronal cell death, enhanced neurotrophic factors, and improved neurobehavioral functions. However, the GSNO+EX showed greater functional recovery (p<0.05) than the GSNO or the EX group. A GSNO sub group, treated 24 hours after IR, still showed motor function recovery (p<0.001). The protective effect of GSNO or exercise was blocked by the inhibition of Akt activity.
GSNO and exercise aid functional recovery by stimulating neurorepair mechanisms. The improvements by GSNO and exercise depend mechanistically on the Akt pathway. A combination of exercise and GSNO shows greater functional recovery. Improved recovery with GSNO, even administered 24 hours post-IR, demonstrates its clinical relevance.
GSNO; IR; motor exercise; neurorepair; neurobehavior; rehabilitation; S-nitrosylation; stroke
Th1 cytokine-induced loss of oligodendrocytes (OLs) is associated with axonal loss in CNS demyelinating diseases such as multiple sclerosis (MS), which contributes to neurological disabilities in affected individuals. Recent studies indicated that, in addition to Th1-phenotype cytokines including tumor necrosis factor (TNF)-α, Th17 phenotype cytokine, interleukin (IL)-17 also involved in the development of MS. Here, we investigated the direct effect of IL-17 on the survival of OLs in the presence of TNF-α and individually in vitro settings. Our findings suggest that IL-17 alone, however, was not able to affect the survival of OLs, but it exacerbates the TNF-α-induced OL apoptosis as compared to individual TNF-α treatment. This effect of cytokines was ascribed to an inhibition of cell survival mechanisms, co-localization of Bid/Bax proteins in the mitochondrial membrane and caspase 8 activation mediated release of apoptosis inducing factor from mitochondria in treated OLs. In addition, cytokine treatment disturbed the mitochondrial membrane potential in OLs with corresponding increase in the generation of reactive oxygen species, which were attenuated by N-acetyl cysteine treatment. In addition, combining of these cytokines induced cell cycle arrest at G1/S phases in OL-like cells and inhibited the maturation of OL progenitor cells (OPCs) that was attenuated by peroxisome proliferator activated receptor (PPAR)-γ/-β agonists. Collectively, these data provide initial evidence that IL-17 exacerbates TNF-α-induced OL loss and inhibits the differentiation of OPCs suggesting that antioxidant- or PPAR agonist-based therapies have potential to limit CNS demyelination in MS or other related demyelinating disorders.
Tumor necrosis factor; Interleukin-17; oligodendrocyte; apoptosis; multiple sclerosis
Previously, we and others documented that statins including—lovastatin (LOV) promote the differentiation of oligodendrocyte progenitor cells (OPCs) and remyelination in experimental autoimmune encephalomyelitis (EAE), an multiple sclerosis (MS) model. Conversely, some recent studies demonstrated that statins negatively influence oligodendrocyte (OL) differentiation in vitro and remyelination in a cuprizone-CNS demyelinating model. Therefore, herein, we first investigated the cause of impaired differentiation of OLs by statins in vitro settings. Our observations indicated that the depletion of cholesterol was detrimental to LOV treated OPCs under cholesterol/serum-deprived culture conditions similar to that were used in conflicting studies. However, the depletion of geranylgeranyl-pp under normal cholesterol homeostasis conditions enhanced the phenotypic commitment and differentiation of LOV-treated OPCs ascribed to inhibition of RhoA-Rho kinase. Interestingly, this effect of LOV was associated with increased activation and expression of both PPAR-γ and PTEN in OPCs as confirmed by various pharmacological and molecular based approaches. Furthermore, PTEN was involved in an inhibition of OPCs proliferation via PI3K-Akt inhibition and induction of cell cycle arrest at G1 phase, but without affecting their cell survival. These effects of LOV on OPCs in vitro were absent in the CNS of normal rats chronically treated with LOV concentrations used in EAE indicating that PPAR-γ induction in normal brain may be tightly regulated — providing evidences that statins are therapeutically safe for humans. Collectively, these data provide initial evidence that statin-mediated activation of the PPAR-γ — PTEN cascade participates in OL differentiation, thus suggesting new therapeutic-interventions for MS or related CNS-demyelinating diseases.
Lovastatin; EAE/MS; oligodendrocyte progenitors; PPAR-γ/PTEN; RhoA-ROCK; Differentiation; remyelination
Rapid-access carotid endarterectomy (RACE) is an evidence-based treatment for symptomatic carotid stenosis. Our vascular centre aims to provide this service within 48 h of symptoms in appropriate patients. This study audits safety and efficacy of the first year of RACE.
SUBJECTS AND METHODS
A clear trust protocol was publicised for the RACE pathway. A prospective database was established for all carotid endarterectomies (CEAs) performed. Outcomes were compared between elective (ECE) and rapid-access operations.
In 1 year, 96 patients received CE; 20 were performed urgently. There were no significant differences in age or gender between ECE and RACE groups. Twenty-three (30%) of ECE were for asymptomatic stenoses; no other significant differences in surgical indication were seen. Of symptomatic ECE, 43% were for completed stroke versus 55% for RACE. Median delay between diagnosis and surgery was 113 days for elective and 2 days for RACE patients. There was one death following ECE (1.3%) and one stroke after RACE (5%), all not significant. Anaesthetic method did not influence outcome. The main reasons for delaying surgery in RACE patients were optimisation of patient fitness and availability of theatre time.
The RACE pathway dramatically reduces delay without compromising patient safety. In the first year of service, we have treated 50% of suitable patients within 48 h. Further education of patients and colleagues should reduce delay and improve outcomes for symptomatic carotid disease.
Rapid-access carotid endarterectomy; RACE; Carotid endarterectomy
Traumatic brain injury (TBI) induces primary and secondary damage in both the endothelium and the brain parenchyma, collectively termed the neurovascular unit. While neurons die quickly by necrosis, a vicious cycle of secondary injury in endothelial cells exacerbates the initial injury in the neurovascular unit following TBI. In activated endothelial cells, excessive superoxide reacts with nitric oxide (NO) to form peroxynitrite. Peroxynitrite has been implicated in blood brain barrier (BBB) leakage, altered metabolic function, and neurobehavioral impairment. S-nitrosoglutathione (GSNO), a nitrosylation-based signaling molecule, was reported not only to reduce brain levels of peroxynitrite and oxidative metabolites but also to improve neurological function in TBI, stroke, and spinal cord injury. Therefore, we investigated whether GSNO promotes the neurorepair process by reducing the levels of peroxynitrite and the degree of oxidative injury.
TBI was induced by controlled cortical impact (CCI) in adult male rats. GSNO or 3-Morpholino-sydnonimine (SIN-1) (50 μg/kg body weight) was administered orally two hours following CCI. The same dose was repeated daily until endpoints. GSNO-treated (GSNO group) or SIN-1-treated (SIN-1 group) injured animals were compared with vehicle-treated injured animals (TBI group) and vehicle-treated sham-operated animals (Sham group) in terms of peroxynitrite, NO, glutathione (GSH), lipid peroxidation, blood brain barrier (BBB) leakage, edema, inflammation, tissue structure, axon/myelin integrity, and neurotrophic factors.
SIN-1 treatment of TBI increased whereas GSNO treatment decreased peroxynitrite, lipid peroxides/aldehydes, BBB leakage, inflammation and edema in a short-term treatment (4-48 hours). GSNO also reduced brain infarctions and enhanced the levels of NO and GSH. In a long-term treatment (14 days), GSNO protected axonal integrity, maintained myelin levels, promoted synaptic plasticity, and enhanced the expression of neurotrophic factors.
Our findings indicate the participation of peroxynitrite in the pathobiology of TBI. GSNO treatment of TBI not only reduces peroxynitrite but also protects the integrity of the neurovascular unit, indicating that GSNO blunts the deleterious effects of peroxynitrite. A long-term treatment of TBI with the same low dose of GSNO promotes synaptic plasticity and enhances the expression of neurotrophic factors. These results support that GSNO reduces the levels of oxidative metabolites, protects the neurovascular unit, and promotes neurorepair mechanisms in TBI.
Krabbe disease is an inherited lysosomal disorder in which galactosylsphingosine (psychosine) accumulates mainly in the central nervous system. To gain insight into the possible mechanism(s) that may be participating in the inhibition of the postnatal somatic growth described in the animal model of this disease (twitcher mouse, twi), we studied their femora. This study reports that twi femora are smaller than of those of wild type (wt), and present with abnormality of marrow cellularity, bone deposition (osteoblastic function), and osteoclastic activity. Furthermore, lipidomic analysis indicates altered sphingolipid homeostasis, but without significant changes in the levels of sphingolipid-derived intermediates of cell death (ceramide) or the levels of the osteoclast-osteoblast coupling factor (sphingosine-1-phosphate). However, there was significant accumulation of psychosine in the femora of adult twi animals as compared to wt, without induction of tumor necrosis factor-alpha or interleukin-6. Analysis of insulin-like growth factor-1 (IGF-1) plasma levels, a liver secreted hormone known to play a role in bone growth, indicated a drastic reduction in twi animals when compared to wt. To identify the cause of the decrease, we examined the IGF-1 mRNA expression and protein levels in the liver. The results indicated a significant reduction of IGF-1 mRNA as well as protein levels in the liver from twi as compared to wt littermates. Our data suggest that a combination of endogenous (psychosine) and endocrine (IGF-1) factors play a role in the inhibition of postnatal bone growth in twi mice; and further suggest that derangements of liver function may be contributing, at least in part, to this alteration.
Galactosylsphingosine; Insulin-like growth factor-1; Krabbe disease; lysosomal disorders; osteopenia; psychosine; twitcher mice
S-nitrosoglutathione (GSNO) is a physiological nitric oxide molecule which regulates biological activities of target proteins via s-nitrosylation leading to attenuation of chronic inflammation. In this study we evaluated the therapeutic efficacy of GSNO in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Oral administration of GSNO (0.5 or 1.0 mg/kg) reduced disease progression in chronic models (SJL and C57BL/6) of EAE induced with PLP(139–151) or MOG(35–55) peptides, respectively. GSNO attenuated EAE disease by reducing the production of IL17 (from Thi or Th17 cells) and the infiltration of CD4 T cells into the central nervous system without affecting the levels of Th1 (IFNγ) and Th2 (IL4) immune responses. Inhibition of IL17 was observed in T cells under normal as well as Th17 skewed conditions. In vitro studies showed that the phosphorylation of STAT3 and expression of RORγ, key regulators of IL17 signaling, were reduced while phosphorylation of STAT4 or STAT6 and expression of T-bet or GATA3 remained unaffected, suggesting that GSNO preferentially targets Th17 cells. Collectively, GSNO attenuated EAE via modulation of Th17 cells and its effects are independent of Th1 or Th2 cells functions, indicating that it may have therapeutic potential for Th17-mediated autoimmune diseases.
EAE; T cells; autoimmunity; GSNO; IL-17; inflammation; MS; STAT; T-bet; ROR-gamma
Peroxisome, a ubiquitous subcellular organelle, plays an important function in cellular metabolism, and its importance for human health is underscored by the identification of fatal disorders caused by genetic abnormalities. Recent findings indicate that peroxisomal dysfunction is not restricted only to inherited peroxisomal diseases but also to disease processes associated with generation of inflammatory mediators that downregulate cellular peroxisomal homeostasis. Evidence indicates that leukodystrophies (X-linked adrenoleukodystrophy, globoid cell leukodystrophy, periventricular leukomalacia) may share common denominators in the development and progression of the inflammatory process and thus in the dysfunctions of peroxisomes. Dysfunctions of peroxisomes may therefore contribute in part to white matter disease and to the mental and physical disabilities that develop in patients affected by these diseases.
cytokines; inflammation; leukodystrophies; myelin; neuroinflammation; peroxisomal disorders
X-adrenoleukodystrophy (X-ALD) is a complex disease where inactivation of ABCD1 gene results in clinically diverse phenotypes, the fatal disorder of cerebral ALD (cALD) or a milder disorder of adrenomyeloneuropathy (AMN). Loss of ABCD1 function results in defective beta oxidation of very long chain fatty acids (VLCFA) resulting in excessive accumulation of VLCFA, the biochemical “hall mark” of X-ALD. At present, the ABCD1-mediated mechanisms that determine the different phenotype of X-ALD are not well understood. The studies reviewed here suggest for a “three-hit hypothesis” for neuropathology of cALD. An improved understanding of the molecular mechanisms associated with these three phases of cALD disease should facilitate the development of effective pharmacological therapeutics for X-ALD.
axonal degeneration; demyelination; neuroinflammation; oxidative stress; peroxisomes; plasmalogens; VLCFA; X-adrenoleukodystrophy
AMP-activated-protein-kinase (AMPK) is a key sensor and regulator of cellular and whole-body energy metabolism and plays a key role in regulation of lipid metabolism. Since lipid metabolism has been implicated in neuronal amyloid-β (Aβ) homeostasis and onset of Alzheimer’s disease, we investigated the involvement of AMPK in neuronal lipid metabolism and Aβ production. We observed in cultured rat cortical neurons that Aβ production was significantly reduced when the neurons were stimulated with AMPK activator, 5-aminoimidazole-4-carboxamide-1-D-ribofuranoside (AICAR), but increased when AMPKα2 was knocked out, thus indicating the role of AMPK in amyloidogenesis. Although the detailed mechanisms by which AMPK regulates Aβ generation is not well understood, AMPK-mediated alterations in cholesterol and sphingomyelin homeostasis and in turn the altered distribution of Aβ precursor-protein (APP) in cholesterol and sphingomyelin rich membrane lipid rafts participate in Aβ generation. Taken together, this is the first report on the role of AMPK in regulation of neuronal amyloidogenesis.
AMPK; Amyloid-β; Amyloid precursor protein; Cholesterol; Lipid rafts; Sphingomyelin
AMP-activated protein kinase (AMPK) is an energy sensing metabolic switch in mammalian cells. Here, we report our novel finding that AMPK is lost in all immune cells of experimental autoimmune encephalomyelitis (EAE), an inflammatory disease of Central Nervous System (CNS). AMPKα1 is predominantly expressed in T cells and antigen presenting cells (APCs), which are primarily involved in EAE disease progression. AMPK is lost at protein level in spleen macrophages, total T cells and their subsets (CD4, CD8 and regulatory T cells) isolated from EAE afflicted animals compared to control, without affecting its mRNA levels suggesting that the loss of AMPK protein is the result of posttranscriptional modification. To examine its pathological relevance in inflammatory disease, EAE was induced in wild type (+/+) and AMPKα1 null mice (−/−) using MOG35–55 peptide. AMPKα1−/− mice exhibited severe EAE disease with profound infiltration of mononuclear cells compared to wild type mice however, AMPKα2 is not involved in enhancing the severity of the disease. Spleen cells isolated from AMPKα1−/− immunized mice exhibited a significant induction in the production of IFNγ. Our study identifies AMPK as a down regulated target during disease in all immune cells and possibly restoring AMPK may serve as a novel therapeutic target in autoimmune diseases like multiple sclerosis (MS).
AMPK; EAE; Multiple sclerosis; T cells; Macrophage
Maternal microbial infections cause adverse fetal developmental outcomes including embryonic resorption, intrauterine fetal death, and preterm labor. Recent studies demonstrated that oxidative-stress plays an important role in chorioamniotitis pathogenesis. Herein we investigated the effect of N-acetyl cysteine (NAC) on lipopolysaccharide-induced preterm labor and fetal demise in murine model. Lipopolysaccharide exposure at embryonic day 18 demonstrated an increase in the abortion rate and fetal demise in pregnant dams. This was associated with increase in an inflammatory response (cytokines, chemokines and iNOS expression) and infiltration of leukocytes (monocytes and polymorphonuclear cells) in the placenta. There was increased expression of cytosolic and secretary phospholipase A2 with increased secretion of prostaglandin-2 and leukotriene B4 in the placenta, suggestive of increased metabolism of phospholipids. In addition, expression of cycloxygenase-2 and malondialdehyde production (oxidative-stress marker) was increased in the placenta. Conversely, NAC pretreatment abolished these effects of lipopolysaccharide in the placenta. Collectively, these data provide evidence that LPS-induced increased inflammation and metabolism of phospholipids at the feto-maternal interface (placenta) is critical for preterm labor and fetal demise during maternal microbial infections which could be blocked by antioxidant-based therapies.
Preterm labor; placenta; maternal microbial infection; Lipopolysaccharide and N-acetyl-cysteine