Endonuclease VIII-like 1 (NEIL1) promotes short-term spatial memory retention and protects from ischemic stroke-induced brain dysfunction and death in mice.

Recent findings suggest that neurons can efficiently repair oxidatively damaged DNA, and that both DNA damage and repair are enhanced by activation of excitatory glutamate receptors. However, in pathological conditions such as ischemic stroke, excessive DNA damage can trigger the death of neurons. Oxidative DNA damage is mainly repaired by base excision repair (BER), a process initiated by DNA glycosylases that recognize and remove damaged DNA bases. Endonuclease VIII-like 1 (NEIL1) is a DNA glycosylase that recognizes a broad range of oxidative lesions. Here, we show that mice lacking NEIL1 exhibit impaired memory retention in a water maze test, but no abnormalities in tests of motor performance, anxiety, or fear conditioning. NEIL1 deficiency results in increased brain damage and a defective functional outcome in a focal ischemia/reperfusion model of stroke. The incision capacity on a 5-hydroxyuracil-containing bubble substrate was lower in the ipsilateral side of ischemic brains and in the mitochondrial lysates of unstressed old NEIL1-deficient mice. These results indicate that NEIL1 plays an important role in learning and memory and in protection of neurons against ischemic injury.

High-dose intravenous immunoglobulin exerts neuroprotective effect in the rat model of neonatal asphyxia.

BACKGROUND: Neonatal asphyxia is one of the leading causes of death in newborn and permanent neurological disabilities in surviving children. The underlying hypoxic-ischemic (HI) injury triggers an inflammatory response leading to neuronal damage. Here, we tested the hypothesis that high-dose intravenous immunoglobulin (IVIG) could exert immunomodulatory effect in rat pups subjected to HI injury. METHODS: HI injury was induced in 7-d-old pups by ligating the common carotid artery followed by exposure to 8% oxygen for 2 h. Brain infarction was evaluated by imaging stained coronal brain sections. Neurological deficits were assessed in weeks 1 through 4 after HI. Western blotting and immunohistochemistry were used to assess complement fragment deposition in the brain tissue. RESULTS: Treatment with IVIG at 2 g/kg significantly and in a dose-responsive manner reduced brain infarction size as well as mortality and neurological deficits caused by HI. Anatomical and functional improvements in IVIG-treated pups correlated with decreased deposition of C3b complement fragments in the injured brain hemisphere. CONCLUSION: IVIG significantly improved the outcome of HI injury in rat pups and could potentially be used for the treatment of human neonatal asphyxia to target proinflammatory complement fragments.

3,6'-dithiothalidomide improves experimental stroke outcome by suppressing neuroinflammation.

Tumor necrosis factor-α (TNF) plays a prominent role in the brain damage and functional deficits that result from ischemic stroke. It was recently reported that the thalidomide analog 3,6'-dithiothalidomide (3,6'-DT) can selectively inhibit the synthesis of TNF in cultured cells. We therefore tested the therapeutic potential of 3,6'-DT in a mouse model of focal ischemic stroke. Administration of 3,6'-DT immediately prior to a stroke or within 3 hr after the stroke reduced infarct volume, neuronal death, and neurological deficits, whereas thalidomide was effective only when administered prior to stroke. Neuroprotection was accompanied by decreased inflammation; 3,6'-DT-treated mice exhibited reduced expression of TNF, interleukin-1ß, and inducible nitric oxide synthase; reduced numbers of activated microglia/macrophages, astrocytes, and neutrophils; and reduced expression of intercellular adhesion molecule-1 in the ischemic brain tissue. 3,6'-DT treatment attenuated stroke-induced disruption of the blood-brain barrier by a mechanism that appears to involve suppression of matrix metalloproteinase-9 and preservation of occludin. Treatment with 3,6'-DT did not reduce ischemic brain damage in mice lacking TNF receptors, consistent with a critical role for suppression of TNF production and TNF signaling in the therapeutic action of 3,6'-DT. These findings suggest that anti-inflammatory mechanisms underlie the therapeutic actions of 3,6-DT in an animal model of stroke.

Inhibitory alkaloids from Dictamnus dasycarpus root barks on lipopolysaccharide-induced nitric oxide production in BV2 cells.

The methanolic extract of Dictamnus dasycarpus root barks afforded one new glycosidic quinoline alkaloid, 3-[1ß-hydroxy-2-(ß-D-glucopyranosyloxy)-ethyl)-4-methoxy-2(1H)-quinolinone (1), together with nine known compounds, preskimmianine (2), 8-methoxy-N-methylflindersine (3), dictamine (4), γ-fagarine (5), halopine (6), skimmianine (7), dictangustine-A (8), iso-γ-fagarine (9), isomaculosidine (10). The isolated alkaloids significantly inhibited nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated BV2 cells. Among them, compounds 3 and 7 showed the most potent inhibitory activities on LPS-induced NO production.

Tumor necrosis factor-α synthesis inhibitor, 3,6'-dithiothalidomide, reverses behavioral impairments induced by minimal traumatic brain injury in mice.

Mild traumatic brain injury (mTBI) patients do not show clear structural brain defects and, in general, do not require hospitalization, but frequently suffer from long-lasting cognitive, behavioral and emotional difficulties. Although there is no current effective treatment or cure for mTBI, tumor necrosis factor-alpha (TNF-α), a cytokine fundamental in the systemic inflammatory process, represents a potential drug target. TNF-α levels increase after mTBI and may induce or exacerbate secondary damage to brain tissue. The present study evaluated the efficacy of the experimental TNF-α synthesis inhibitor, 3,6'-dithiothalidomide, on recovery of mice from mTBI in a closed head weight-drop model that induces an acute elevation in brain TNF-α and an impairment in cognitive performance, as assessed by the Y-maze, by novel object recognition and by passive avoidance paradigms at 72 h and 7 days after injury. These impairments were fully ameliorated in mice that received a one time administration of 3,6'-dithiothalidomide at either a low (28 mg/kg) or high (56 mg/kg) dose provided either 1 h prior to injury, or at 1 or 12 h post-injury. Together, these results implicate TNF-α as a drug target for mTBI and suggests that 3,6'-dithiothalidomide may act as a neuroprotective drug to minimize impairment.

Pregabalin suppresses calcium-mediated proteolysis and improves stroke outcome.

Pregabalin, a Ca(2+) channel α(2)δ-subunit antagonist with analgesic and antiepileptic activity, reduced neuronal loss and improved functional outcome in a mouse model of focal ischemic stroke. Pregabalin administration (5-10mg/kg, i.p.) 30-90 min after transient middle cerebral artery occlusion/reperfusion reduced infarct volume, neuronal death in the ischemic penumbra and neurological deficits at 24h post-stroke. Pregabalin significantly decreased the amount of Ca(2+)/calpain-mediated α-spectrin proteolysis in the cerebral cortex measured at 6h post-stroke. Together with the extensive clinical experience with pregabalin for other neurological indications, our findings suggest the potential for a therapeutic benefit of pregabalin in stroke patients.

CXCR4-STAT3 Axis Plays a Role in Tumor Cell Infiltration in an Orthotopic Mouse Glioblastoma Model.

Glioblastoma multiforme (GBM) is a fatal malignant tumor that is characterized by diffusive growth of tumor cells into the surrounding brain parenchyma. However, the diffusive nature of GBM and its relationship with the tumor microenvironment (TME) is still unknown. Here, we investigated the interactions of GBM with the surrounding microenvironment in orthotopic xenograft animal models using two human glioma cell lines, U87 and LN229. The GBM cells in our model showed different features on the aspects of cell growth rate during their development, dispersive nature of glioma tumor cells along blood vessels, and invasion into the brain parenchyma. Our results indicated that these differences in the two models are in part due to differences in the expression of CXCR4 and STAT3, both of which play an important role in tumor progression. In addition, the GBM shows considerable accumulation of resident microglia and peripheral macrophages, but polarizes differently into tumor-supporting cells. These results suggest that the intrinsic factors of GBM and their interaction with the TME determine the diffusive nature and probably the responsiveness to non-cancer cells in the TME.

Neurogenin-1 Overexpression Increases the Therapeutic Effects of Mesenchymal Stem Cells through Enhanced Engraftment in an Ischemic Rat Brain.

BACKGROUND AND OBJECTIVES: Stem cell therapy is a promising strategy for treating neurological diseases but its effectiveness is influenced by the route of administration and the characteristics of the stem cells. We determined whether neural induction of mesenchymal stem cells (MSCs) was beneficial when the cells were delivered intra-arterially through the carotid artery. METHODS AND RESULTS: MSCs were neurally induced using a retroviral vector expressing the neurogenic transcription factor neurogenin-1 (Ngn1). The LacZ gene encoding bacterial ß-galactosidase was used as a control. Ischemic stroke was induced by transluminal occlusion of the middle cerebral artery and 3 days later the MSCs were delivered intra-arterially through the internal carotid artery. Magnetic resonance imaging analysis indicated that compared to MSCs expressing LacZ (MSCs/LacZ), MSCs expressing Ngn1 (MSCs/Ngn1) exhibited increased recruitment to the ischemic region and populated this area for a longer duration. Immunohistochemical analysis indicated that compared to MSCs/LacZ, MSCs/Ngn1 more effectively alleviated neurological dysfunction by blocking secondary damage associated with neuronal cell death and brain inflammation. Microarray and real-time PCR analysis indicated that MSCs/Ngn1 exhibited increased expression of chemotactic cytokine receptors, adherence to endothelial cells, and migration ability. CONCLUSIONS: Neural induction with Ngn1 increases the homing ability of MSCs, enhancing their engraftment efficiency in the ischemic rat brain. Intra-arterial delivery of neurally induced MSCs/Ngn1 3 days after ischemic injury blocks neuronal cell death and inflammation, and improves functional recovery. Thus, intra-arterial administration of stem cells with neural properties may be a novel therapy for the treatment of ischemic stroke.

ERK Regulates NeuroD1-mediated Neurite Outgrowth via Proteasomal Degradation.

Neurogenic differentiation 1 (NeuroD1) is a class B basic helix-loop-helix (bHLH) transcription factor and regulates differentiation and survival of neuronal and endocrine cells by means of several protein kinases, including extracellular signal-regulated kinase (ERK). However, the effect of phosphorylation on the functions of NeuroD1 by ERK has sparked controversy based on context-dependent differences across diverse species and cell types. Here, we evidenced that ERK-dependent phosphorylation controlled the stability of NeuroD1 and consequently, regulated proneural activity in neuronal cells. A null mutation at the ERK-dependent phosphorylation site, S274A, increased the half-life of NeuroD1 by blocking its ubiquitin-dependent proteasomal degradation. The S274A mutation did not interfere with either the nuclear translocation of NeuroD1 or its heterodimerization with E47, its ubiquitous partner and class A bHLH transcription factor. However, the S274A mutant increased transactivation of the E-box-mediated gene and neurite outgrowth in F11 neuroblastoma cells, compared to the wild-type NeuroD1. Transcriptome and Gene Ontology enrichment analyses indicated that genes involved in axonogenesis and dendrite development were downregulated in NeuroD1 knockout (KO) mice. Overexpression of the S274A mutant salvaged neurite outgrowth in NeuroD1-deficient mice, whereas neurite outgrowth was minimal with S274D, a phosphomimicking mutant. Our data indicated that a longer protein half-life enhanced the overall activity of NeuroD1 in stimulating downstream genes and neuronal differentiation. We propose that blocking ubiquitin-dependent proteasomal degradation may serve as a strategy to promote neuronal activity by stimulating the expression of neuron-specific genes in differentiating neurons.

Uric acid enhances longevity and endurance and protects the brain against ischemia.

Among mammals, there is a positive correlation between serum uric acid (UA) levels and life span. Humans have high levels of UA because they lack a functional urate oxidase (UOX) enzyme that is present in shorter lived mammals. Here, we show that male and female mice with UOX haploinsufficiency exhibit an age-related elevation of UA levels, and that the life span of female but not male UOX+/- mice is significantly increased compared to wild-type mice. Serum UA levels are elevated in response to treadmill exercise in UOX+/- mice, but not wild-type mice, and the endurance of the UOX+/- mice is significantly greater than wild-type mice. UOX+/- mice exhibit elevated levels of brain-derived neurotrophic factor, reduced brain damage and improved functional outcome in a model of focal ischemic stroke. Levels of oxidative protein nitration and lipid peroxidation are reduced in muscle and brain tissues of UOX+/- mice under conditions of metabolic and oxidative stress (running in the case of muscle and ischemia in the case of the brain), consistent with prior evidence that UA can scavenge peroxynitrite and hydroxyl radical. Our findings reveal roles for UA in life span determination, endurance and adaptive responses to brain injury, and suggest novel approaches for protecting cells against injury and for optimizing physical performance.

Antifibrotic activity of diterpenes from Biota orientalis leaves on hepatic stellate cells.

Antifibrotic effect of twelve diterpenes (1-12) from the 90% methanolic fraction of Biota orientalis leaves was evaluated employing HSC-T6 cells by assessing cell proliferation and morphological change. Among these diterpenes, totarol (8) and isopimara-8(14),15-dien-19-oic acid (9) dramatically reduced cell proliferation in dose-and time-dependent manner. Furthermore, treatment with these compounds resulted in the different pattern of morphological changes of HSC-T6 cells. Taken together, antiproliferative activity of diterpenes from B. orientalis might suggest therapeutic potentials against liver fibrosis.

Spatiotemporal Protein Atlas of Cell Death-Related Molecules in the Rat MCAO Stroke Model.

Ischemic stroke and cerebral infarction triggered by the blockage of blood supply can cause damage to the brain via a complex series of pathological changes. Recently, diverse therapies have emerged as promising candidates for the treatment of stroke. These treatments exert therapeutic effects by acting on diverse target molecules and cells in different time windows from the acute to chronic phases. Here, using immunohistochemistry, we show pathophysiological changes in the brain microenvironment at the hyperacute (within 6 h), acute (1~3 days), subacute (7 days), and chronic (1 month) phases following ischemic injury. Ischemic injury in rats was induced by occluding the middle cerebral artery and was validated by magnetic resonance imaging. The progression of damage to the brain was evaluated by immunohistochemistry for NeuN+ neurons, GFAP+ astrocytes, and Iba1+ microglia, and by the emergence of the cell death-related molecules such as AIF, FAF1, and activated caspase-3. Our data regarding the spatial and temporal information on pathophysiological changes may warrant the investigation of the timing of administration of therapeutic treatments in preclinical studies with an animal model of stroke.

Mitochondrial Protein PGAM5 Regulates Mitophagic Protection against Cell Necroptosis.

Necroptosis as a molecular program, rather than simply incidental cell death, was established by elucidating the roles of receptor interacting protein (RIP) kinases 1 and 3, along with their downstream partner, mixed lineage kinase-like domain protein (MLKL). Previous studies suggested that phosphoglycerate mutase family member 5 (PGAM5), a mitochondrial protein that associates with RIP1/RIP3/MLKL complex, promotes necroptosis. We have generated mice deficient in the pgam5 gene and surprisingly found PGAM5-deficiency exacerbated rather than reduced necroptosis in response to multiple in vitro and in vivo necroptotic stimuli, including ischemic reperfusion injury (I/R) in the heart and brain. Electron microscopy, biochemical, and confocal analysis revealed that PGAM5 is indispensable for the process of PINK1 dependent mitophagy which antagonizes necroptosis. The loss of PGAM5/PINK1 mediated mitophagy causes the accumulation of abnormal mitochondria, leading to the overproduction of reactive oxygen species (ROS) that worsen necroptosis. Our results revise the former proposal that PGAM5 acts downstream of RIP1/RIP3 to mediate necroptosis. Instead, PGAM5 protects cells from necroptosis by independently promoting mitophagy. PGAM5 promotion of mitophagy may represent a therapeutic target for stroke, myocardial infarction and other diseases caused by oxidative damage and necroptosis.

The Anti-apoptotic Effect of Ghrelin on Restraint Stress-Induced Thymus Atrophy in Mice.

Thymic atrophy is a complication that results from exposure to many environmental stressors, disease treatments, and microbial challenges. Such acute stress-associated thymic loss can have a dramatic impact on the host's ability to replenish the necessary naïve T cell output to reconstitute the peripheral T cell numbers and repertoire to respond to new antigenic challenges. We have previously reported that treatment with the orexigenic hormone ghrelin results in an increase in the number and proliferation of thymocytes after dexamethasone challenge, suggesting a role for ghrelin in restraint stress-induced thymic involution and cell apoptosis and its potential use as a thymostimulatory agent. In an effort to understand how ghrelin suppresses thymic T cell apoptosis, we have examined the various signaling pathways induced by receptor-specific ghrelin stimulation using a restraint stress mouse model. In this model, stress-induced apoptosis in thymocytes was effectively blocked by ghrelin. Western blot analysis demonstrated that ghrelin prevents the cleavage of pro-apoptotic proteins such as Bim, Caspase-3, and PARP. In addition, ghrelin stimulation activates the Akt and Mitogen-activated protein kinases (MAPK) signaling pathways in a time/dose-dependent manner. Moreover, we also revealed the involvement of the FoxO3a pathway in the phosphorylation of Akt and ERK1/2. Together, these findings suggest that ghrelin inhibits apoptosis by modulating the stress-induced apoptotic signal pathway in the restraint-induced thymic apoptosis.

ESP-102, a standardized combined extract of Angelica gigas, Saururus chinensis and Schizandra chinensis, significantly improved scopolamine-induced memory impairment in mice.

We assessed the effects of oral treatments of ESP-102, a standardized combined extract of Angelica gigas, Saururus chinensis and Schizandra chinensis, on learning and memory deficit. The cognition-enhancing effect of ESP-102 was investigated in scopolamine-induced (1 mg/kg body weight, s.c.) amnesic mice with both passive avoidance and Morris water maze performance tests. Acute oral treatment (single administration prior to scopolamine treatment) of mice with ESP-102 (doses in the range of 10 to 100 mg/kg body weight) significantly reduced scopolamine-induced memory deficits in the passive avoidance performance test. Another noteworthy result included the fact that prolonged oral daily treatments of mice with much lower amounts of ESP-102 (1 and 10 mg/kg body weight) for ten days reversed scopolamine-induced memory deficits. In the Morris water maze performance test, both acute and prolonged oral treatments with ESP-102 (single administration of 100 mg/kg body weight or prolonged daily administration of 1 and 10 mg/kg body weight for ten days, respectively, significantly ameliorated scopolamine-induced memory deficits as indicated by the formation of long-term and/or short-term spatial memory. In addition, we investigated the effects of ESP-102 on neurotoxicity induced by amyloid-beta peptide (Abeta25-35) or glutamate in primary cultured cortical neurons of rats. Pretreatment of cultures with ESP-102 (0.001, 0.01 and 0.1 mug/ml) significantly protected neurons from neurotoxicity induced by either glutamate or Abeta25-35. These results suggest that ESP-102 may have some protective characteristics against neuronal cell death and cognitive impairments often observed in Alzheimer's disease, stroke, ischemic injury and other neurodegenerative diseases.

Vismodegib, an antagonist of hedgehog signaling, directly alters taste molecular signaling in taste buds.

Vismodegib, a highly selective inhibitor of hedgehog (Hh) pathway, is an approved treatment for basal-cell carcinoma. Patients on treatment with vismodegib often report profound alterations in taste sensation. The cellular mechanisms underlying the alterations have not been studied. Sonic Hh (Shh) signaling is required for cell growth and differentiation. In taste buds, Shh is exclusively expressed in type IV taste cells, which are undifferentiated basal cells and the precursors of the three types of taste sensing cells. Thus, we investigated if vismodegib has an inhibitory effect on taste cell turnover because of its known effects on Hh signaling. We gavaged C57BL/6J male mice daily with either vehicle or 30 mg/kg vismodegib for 15 weeks. The gustatory behavior and immunohistochemical profile of taste cells were examined. Vismodegib-treated mice showed decreased growth rate and behavioral responsivity to sweet and bitter stimuli, compared to vehicle-treated mice. We found that vismodegib-treated mice had significant reductions in taste bud size and numbers of taste cells per taste bud. Additionally, vismodegib treatment resulted in decreased numbers of Ki67- and Shh-expressing cells in taste buds. The numbers of phospholipase Cß2- and α-gustducin-expressing cells, which contain biochemical machinery for sweet and bitter sensing, were reduced in vismodegib-treated mice. Furthermore, vismodegib treatment resulted in reduction in numbers of T1R3, glucagon-like peptide-1, and glucagon-expressing cells, which are known to modulate sweet taste sensitivity. These results suggest that inhibition of Shh signaling by vismodegib treatment directly results in alteration of taste due to local effects in taste buds.

Partial loss of the DNA repair scaffolding protein, Xrcc1, results in increased brain damage and reduced recovery from ischemic stroke in mice.

Oxidative DNA damage is mainly repaired by base excision repair (BER). Previously, our laboratory showed that mice lacking the BER glycosylases 8-oxoguanine glycosylase 1 (Ogg1) or nei endonuclease VIII-like 1 (Neil1) recover more poorly from focal ischemic stroke than wild-type mice. Here, a mouse model was used to investigate whether loss of 1 of the 2 alleles of X-ray repair cross-complementing protein 1 (Xrcc1), which encodes a nonenzymatic scaffold protein required for BER, alters recovery from stroke. Ischemia and reperfusion caused higher brain damage and lower functional recovery in Xrcc1(+/-) mice than in wild-type mice. Additionally, a greater percentage of Xrcc1(+/-) mice died as a result of the stroke. Brain samples from human individuals who died of stroke and individuals who died of non-neurological causes were assayed for various steps of BER. Significant losses of thymine glycol incision, abasic endonuclease incision, and single nucleotide incorporation activities were identified, as well as lower expression of XRCC1 and NEIL1 proteins in stroke brains compared with controls. Together, these results suggest that impaired BER is a risk factor in ischemic brain injury and contributes to its recovery.

Requirement of AMPK activation for neuronal metabolic-enhancing effects of antidepressant paroxetine.

Reduced glucose metabolism has been implicated as a pathophysiology of depressive disorder. Normalization of such impaired neurometabolism has been related to the therapeutic actions of antidepressant medication. However, the molecular mechanism underlying the neurometabolic actions of antidepressants has not been fully understood. Given that AMP-activated protein kinase (AMPK) is a master switch for energy homeostasis, we aimed to determine whether selective serotonin reuptake inhibitor paroxetine enhances energy metabolism by activating AMPK in neuroblastoma cells. We found that paroxetine dose dependently increased mitochondrial biogenesis, which involves the AMPK-peroxisome proliferator-activated receptor-γ coactivator-1α pathway. In addition, paroxetine-induced AMPK activation increases glucose uptake and ATP production. These neurometabolic effects of paroxetine were suppressed by cotreatment with compound C (CC), an AMPK inhibitor. These findings suggest a possibility that modulation of the AMPK pathway might be a previously unrecognized mechanism underlying the neurometabolic action of antidepressants. Further study is warranted to examine the region-specific and time-specific effects of AMPK modulation by antidepressants on mood-related behaviors.

Flavonoids from Spatholobus suberectus.

Two pterocarpans [(6aR,11aR)-maackiain, (6aR,11aR)-medicarpin], one flavanone [(2S)-7-hydroxy-6-methoxy-flavanone], one isoflavan (sativan) and two isoflavones (pseudobaptigenin, genistein) were isolated from the Spatholobus suberectus (Leguminosae). Their chemical structures were determined by comparison of their spectroscopic parameters of CD, EIMS, 1D-NMR and 2D-NMR with those reported in the literatures. All of these compounds are reported for the first time from this plant through the present study.

Energy restriction negates NMDA receptor antagonist efficacy in ischemic stroke.

Preclinical evaluation of drugs for neurological disorders is usually performed on overfed rodents, without consideration of how metabolic state might affect drug efficacy. Using a widely employed mouse model of focal ischemic stroke, we found that that the NMDA receptor antagonist dizocilpine (MK-801) reduces brain damage and improves functional outcome in mice on the usual ad libitum diet, but exhibits little or no therapeutic efficacy in mice maintained on an energy-restricted diet. Thus, NMDA receptor activation plays a central role in the mechanism by which a high dietary energy intake exacerbates ischemic brain injury. These findings suggest that inclusion of subjects with a wide range of energy intakes in clinical trials for stroke may mask a drug benefit in the overfed/obese subpopulation of subjects.