Amentoflavone Promotes Cellular Uptake and Degradation of Amyloid-Beta in Neuronal Cells.

Deposition of fibrillar forms of amyloid ß-protein (Aß) is commonly found in patients with Alzheimer's disease (AD) associated with cognitive decline. Impaired clearance of Aß species is thought to be a major cause of late-onset sporadic AD. Aß secreted into the extracellular milieu can be cleared from the brain through multiple pathways, including cellular uptake in neuronal and non-neuronal cells. Recent studies have showed that the naturally-occurring polyphenol amentoflavone (AMF) exerts anti-amyloidogenic effects. However, its effects on metabolism and cellular clearance of Aß remain to be tested. In the present study, we demonstrated that AMF significantly increased the cellular uptake of both Aß1-40 and Aß1-42, but not inverted Aß42-1 in mouse neuronal N2a cells. Though AMF promoted internalization of cytotoxic Aß1-42, it significantly reduced cell death in our assay condition. Our data further revealed that the internalized Aß is translocated to lysosomes and undergoes enzymatic degradation. The saturable kinetic of Aß uptake and our pharmacologic experiments showed the involvement of receptor-mediated endocytosis, in part, through the class A scavenger receptors as a possible mechanism of action of AMF. Taken together, our findings indicate that AMF can lower the levels of extracellular Aß by increasing their cellular uptake and clearance, suggesting the therapeutic potential of AMF for the treatment of AD.

Sirtuin 1 Mediates Protection Against Delayed Cerebral Ischemia in Subarachnoid Hemorrhage in Response to Hypoxic Postconditioning.

Background Many therapies designed to prevent delayed cerebral ischemia (DCI) and improve neurological outcome in aneurysmal subarachnoid hemorrhage (SAH) have failed, likely because of targeting only one element of what has proven to be a multifactorial disease. We previously demonstrated that initiating hypoxic conditioning before SAH (hypoxic preconditioning) provides powerful protection against DCI. Here, we expanded upon these findings to determine whether hypoxic conditioning delivered at clinically relevant time points after SAH (hypoxic postconditioning) provides similarly robust DCI protection. Methods and Results In this study, we found that hypoxic postconditioning (8% O2 for 2 hours) initiated 3 hours after SAH provides strong protection against cerebral vasospasm, microvessel thrombi, and neurological deficits. By pharmacologic and genetic inhibition of SIRT1 (sirtuin 1) using EX527 and global Sirt1-/- mice, respectively, we demonstrated that this multifaceted DCI protection is SIRT1 mediated. Moreover, genetic overexpression of SIRT1 using Sirt1-Tg mice, mimicked the DCI protection afforded by hypoxic postconditioning. Finally, we found that post-SAH administration of resveratrol attenuated cerebral vasospasm, microvessel thrombi, and neurological deficits, and did so in a SIRT1-dependent fashion. Conclusions The present study indicates that hypoxic postconditioning provides powerful DCI protection when initiated at clinically relevant time points, and that pharmacologic augmentation of SIRT1 activity after SAH can mimic this beneficial effect. We conclude that conditioning-based therapies administered after SAH hold translational promise for patients with SAH and warrant further investigation.

Biflavonoid-Induced Disruption of Hydrogen Bonds Leads to Amyloid-ß Disaggregation.

Deposition of amyloid ß (Aß) fibrils in the brain is a key pathologic hallmark of Alzheimer's disease. A class of polyphenolic biflavonoids is known to have anti-amyloidogenic effects by inhibiting aggregation of Aß and promoting disaggregation of Aß fibrils. In the present study, we further sought to investigate the structural basis of the Aß disaggregating activity of biflavonoids and their interactions at the atomic level. A thioflavin T (ThT) fluorescence assay revealed that amentoflavone-type biflavonoids promote disaggregation of Aß fibrils with varying potency due to specific structural differences. The computational analysis herein provides the first atomistic details for the mechanism of Aß disaggregation by biflavonoids. Molecular docking analysis showed that biflavonoids preferentially bind to the aromatic-rich, partially ordered N-termini of Aß fibril via the π-π interactions. Moreover, docking scores correlate well with the ThT EC50 values. Molecular dynamic simulations revealed that biflavonoids decrease the content of ß-sheet in Aß fibril in a structure-dependent manner. Hydrogen bond analysis further supported that the substitution of hydroxyl groups capable of hydrogen bond formation at two positions on the biflavonoid scaffold leads to significantly disaggregation of Aß fibrils. Taken together, our data indicate that biflavonoids promote disaggregation of Aß fibrils due to their ability to disrupt the fibril structure, suggesting biflavonoids as a lead class of compounds to develop a therapeutic agent for Alzheimer's disease.

SIRT1 mediates hypoxic preconditioning induced attenuation of neurovascular dysfunction following subarachnoid hemorrhage.

BACKGROUND AND PURPOSE: Vasospasm and delayed cerebral ischemia (DCI) contribute significantly to the morbidity/mortality associated with aneurysmal subarachnoid hemorrhage (SAH). While considerable research effort has focused on preventing or reversing vasospasm, SAH-induced brain injury occurs in response to a multitude of concomitantly acting pathophysiologic mechanisms. In this regard, the pleiotropic epigenetic responses to conditioning-based therapeutics may provide an ideal SAH therapeutic strategy. We previously documented the ability of hypoxic preconditioning (PC) to attenuate vasospasm and neurological deficits after SAH, in a manner that depends on the activity of endothelial nitric oxide synthase. The present study was undertaken to elucidate whether the NAD-dependent protein deacetylase sirtuin isoform SIRT1 is an upstream mediator of hypoxic PC-induced protection, and to assess the efficacy of the SIRT1-activating polyphenol Resveratrol as a pharmacologic preconditioning therapy. METHODS: Wild-type C57BL/6J mice were utilized in the study and subjected to normoxia or hypoxic PC. Surgical procedures included induction of SAH via endovascular perforation or sham surgery. Multiple endpoints were assessed including cerebral vasospasm, neurobehavioral deficits, SIRT1 expression via quantitative real-time PCR for mRNA, and western blot for protein quantification. Pharmacological agents utilized in the study include EX-527 (SIRT1 inhibitor), and Resveratrol (SIRT1 activator). RESULTS: Hypoxic PC leads to rapid and sustained increase in cerebral SIRT1 mRNA and protein expression. SIRT1 inhibition blocks the protective effects of hypoxic PC on vasospasm and neurological deficits. Resveratrol pretreatment dose-dependently abrogates vasospasm and attenuates neurological deficits following SAH - beneficial effects that were similarly blocked by pharmacologic inhibition of SIRT1. CONCLUSION: SIRT1 mediates hypoxic preconditioning-induced protection against neurovascular dysfunction after SAH. Resveratrol mimics this neurovascular protection, at least in part, via SIRT1. Activation of SIRT1 is a promising, novel, pleiotropic therapeutic strategy to combat DCI after SAH.

Amentoflavone: A Bifunctional Metal Chelator that Controls the Formation of Neurotoxic Soluble Aß42 Oligomers.

Alzheimer's disease (AD) is the most common neurodegenerative disorder, yet the cause and progression of this disorder are not completely understood. While the main hallmark of AD is the deposition of amyloid plaques consisting of the ß-amyloid (Aß) peptide, transition metal ions are also known to play a significant role in disease pathology by expediting the formation of neurotoxic soluble ß-amyloid (Aß) oligomers, reactive oxygen species (ROS), and oxidative stress. Thus, bifunctional metal chelators that can control these deleterious properties are highly desirable. Herein, we show that amentoflavone (AMF), a natural biflavonoid compound, exhibits good metal-chelating properties, especially for chelating Cu2+ with very high affinity (pCu7.4 = 10.44). In addition, AMF binds to Aß fibrils with a high affinity (Ki = 287 ± 20 nM), as revealed by a competition thioflavin T (ThT) assay, and specifically labels the amyloid plaques ex vivo in the brain sections of transgenic AD mice, as confirmed via immunostaining with an Aß antibody. The effect of AMF on Aß42 aggregation and disaggregation of Aß42 fibrils was also investigated and revealed that AMF can control the formation of neurotoxic soluble Aß42 oligomers, both in the absence and presence of metal ions, as confirmed via cell toxicity studies. Furthermore, an ascorbate consumption assay shows that AMF exhibits potent antioxidant properties and can chelate Cu2+ and significantly diminish the Cu2+-ascorbate redox cycling and reactive oxygen species (ROS) formation. Overall, these studies strongly suggest that AMF acts as a bifunctional chelator that can interact with various Aß aggregates and reduce their neurotoxicity and can also bind Cu2+ and mediate its deleterious redox properties. Thus AMF has the potential to be a lead compound for further therapeutic agent development for AD.

Monocyte chemotactic protein-1-induced protein 1 contributes to neuronal injury following hypoxic-ischemia in the neonatal mouse brain.

Neuroinflammation has been implicated in neurodegenerative diseases and acute brain injuries such as stroke. Monocyte chemotactic protein-1-induced protein-1 (Mcpip1) is a multifunction protein known to have pro-apoptotic or anti-apoptotic actions depending on the nature of experimental settings. However, its role in brain damage after asphyxia in the developing brain has not been studied. We, therefore, explored the role of Mcpip1 in brain injury after hypoxic-ischemia in neonatal mice. At postnatal day 7, Mcpip1-deficient and wild type mice underwent a carotid artery ligation and exposure to hypoxia (8% oxygen). After hypoxic-ischemic insult, we determined the time-course of apoptotic cell death and the expression levels of genes encoding proinflammatory factors. The impact of Mcpip1 on long-term brain damage was assessed 1 week post-hypoxic-ischemia by cresyl violet staining. We found caspase-3 activity was significantly increased in the ipsilateral brain tissues within 12-24 h after hypoxic-ischemia. There was a marked increase in the levels of mRNA transcripts encoding Mcpip1, TNFα, and CCL2 in the ipsilateral brain tissues 6-48 h after hypoxic-ischemia. We found hypoxic-ischemia-induced caspase-3 activity and the levels of the proinflammatory genes were attenuated in Mcpip1-knockout mice compared to wild type mice. Histological assessment revealed that hypoxic-ischemia-induced brain tissue loss was significantly attenuated in the hippocampus of Mcpip1-knockout mice than that of wild type mice (9.0 ± 5.6% vs. 33.9 ± 11.0%, P < 0.05). Our data suggest that Mcpip1 contributes to acute and delayed brain damage, in part, via regulation of neuroinflammation after hypoxic-ischemic insult in the developing mouse brain.

Polyphenolic Biflavonoids Inhibit Amyloid-Beta Fibrillation and Disaggregate Preformed Amyloid-Beta Fibrils.

Alzheimer's disease (AD) is a devastating neurodegenerative disease and a major cause of dementia in elderly individuals worldwide. Increased deposition of insoluble amyloid ß (Aß) fibrils in the brain is thought be a key neuropathological hallmark of AD. Many recent studies show that natural products such as polyphenolic flavonoids inhibit the formation of insoluble Aß fibrils and/or destabilize ß-sheet-rich Aß fibrils to form non-cytotoxic aggregates. In the present study, we explored the structure-activity relationship of naturally-occurring biflavonoids on Aß amyloidogenesis utilizing an in vitro thioflavin T assay with Aß1-42 peptide which is prone to aggregate more rapidly to fibrils than Aß1-40 peptide. Among the biflavonoids we tested, we found amentoflavone revealed the most potent effects on inhibiting Aß1-42 fibrillization (IC50: 0.26 µM), as well as on disassembling preformed Aß1-42 fibrils (EC50: 0.59 µM). Our structure-activity relationship study suggests that the hydroxyl groups of biflavonoid compounds play an essential role in their molecular interaction with the dynamic process of Aß1-42 fibrillization. Our atomic force microscopic imaging analysis demonstrates that amentoflavone directly disrupts the fibrillar structure of preformed Aß1-42 fibrils, resulting in conversion of those fibrils to amorphous Aß1-42 aggregates. These results indicate that amentoflavone affords the most potent anti-amyloidogenic effects on both inhibition of Aß1-42 fibrillization and disaggregation of preformed mature Aß1-42 fibrils.

Evaluation of 64Cu-Based Radiopharmaceuticals that Target Aß Peptide Aggregates as Diagnostic Tools for Alzheimer's Disease.

Positron emission tomography (PET) imaging agents that detect amyloid plaques containing amyloid beta (Aß) peptide aggregates in the brain of Alzheimer's disease (AD) patients have been successfully developed and recently approved by the FDA for clinical use. However, the short half-lives of the currently used radionuclides 11C (20.4 min) and 18F (109.8 min) may limit the widespread use of these imaging agents. Therefore, we have begun to evaluate novel AD diagnostic agents that can be radiolabeled with 64Cu, a radionuclide with a half-life of 12.7 h, ideal for PET imaging. Described herein are a series of bifunctional chelators (BFCs), L1-L5, that were designed to tightly bind 64Cu and shown to interact with Aß aggregates both in vitro and in transgenic AD mouse brain sections. Importantly, biodistribution studies show that these compounds exhibit promising brain uptake and rapid clearance in wild-type mice, and initial microPET imaging studies of transgenic AD mice suggest that these compounds could serve as lead compounds for the development of improved diagnostic agents for AD.

A novel fluorescent imaging technique for assessment of cerebral vasospasm after experimental subarachnoid hemorrhage.

Various techniques have been developed to study changes in the cerebral vasculature in numerous neuropathological processes including subarachnoid hemorrhage (SAH). One of the most widely employed techniques uses India ink-gelatin casting, which presents numerous challenges due to its high viscosity, rapid solidification, and its impact on immunohistochemical analysis. To overcome these limitations, we developed a novel technique for assessing cerebral vasospasm using cerebrovascular perfusion with ROX, SE (5-Carboxy-X-Rhodamine, Succinimidyl Ester), a fluorescent labeling dye. We found that ROX SE perfusion achieves excellent delineation of the cerebral vasculature, was qualitatively and quantitatively superior to India ink-gelatin casting for the assessment of cerebral vasospasm, permits outstanding immunohistochemical examination of non-vasospasm components of secondary brain injury, and is a more efficient and cost-effective experimental technique. ROX SE perfusion is therefore a novel and highly useful technique for studying cerebrovascular pathology following experimental SAH.

Heparan sulfate proteoglycans mediate Aß-induced oxidative stress and hypercontractility in cultured vascular smooth muscle cells.

BACKGROUND: Substantial evidence suggests that amyloid-ß (Aß) species induce oxidative stress and cerebrovascular (CV) dysfunction in Alzheimer's disease (AD), potentially contributing to the progressive dementia of this disease. The upstream molecular pathways governing this process, however, are poorly understood. In this report, we examine the role of heparan sulfate proteoglycans (HSPG) in Aß-induced vascular smooth muscle cell (VSMC) dysfunction in vitro. RESULTS: Our results demonstrate that pharmacological depletion of HSPG (by enzymatic degradation with active, but not heat-inactivated, heparinase) in primary human cerebral and transformed rat VSMC mitigates Aß(1-40⁻) and Aß(1-42⁻)induced oxidative stress. This inhibitory effect is specific for HSPG depletion and does not occur with pharmacological depletion of other glycosaminoglycan (GAG) family members. We also found that Aß(1-40) (but not Aß(1-42)) causes a hypercontractile phenotype in transformed rat cerebral VSMC that likely results from a HSPG-mediated augmentation in intracellular Ca(2+) activity, as both Aß(1-40⁻)induced VSMC hypercontractility and increased Ca(2+) influx are inhibited by pharmacological HSPG depletion. Moreover, chelation of extracellular Ca(2+) with ethylene glycol tetraacetic acid (EGTA) does not prevent the production of Aß(1-40⁻) or Aß(1-42⁻)mediated reactive oxygen species (ROS), suggesting that Aß-induced ROS and VSMC hypercontractility occur through different molecular pathways. CONCLUSIONS: Taken together, our data indicate that HSPG are critical mediators of Aß-induced oxidative stress and Aß(1-40⁻)induced VSMC dysfunction.

Passive immunotherapy targeting amyloid-ß reduces cerebral amyloid angiopathy and improves vascular reactivity.

Prominent cerebral amyloid angiopathy is often observed in the brains of elderly individuals and is almost universally found in patients with Alzheimer's disease. Cerebral amyloid angiopathy is characterized by accumulation of the shorter amyloid-ß isoform(s) (predominantly amyloid-ß40) in the walls of leptomeningeal and cortical arterioles and is likely a contributory factor to vascular dysfunction leading to stroke and dementia in the elderly. We used transgenic mice with prominent cerebral amyloid angiopathy to investigate the ability of ponezumab, an anti-amyloid-ß40 selective antibody, to attenuate amyloid-ß accrual in cerebral vessels and to acutely restore vascular reactivity. Chronic administration of ponezumab to transgenic mice led to a significant reduction in amyloid and amyloid-ß accumulation both in leptomeningeal and brain vessels when measured by intravital multiphoton imaging and immunohistochemistry. By enriching for cerebral vascular elements, we also measured a significant reduction in the levels of soluble amyloid-ß biochemically. We hypothesized that the reduction in vascular amyloid-ß40 after ponezumab administration may reflect the ability of ponezumab to mobilize an interstitial fluid pool of amyloid-ß40 in brain. Acutely, ponezumab triggered a significant and transient increase in interstitial fluid amyloid-ß40 levels in old plaque-bearing transgenic mice but not in young animals. We also measured a beneficial effect on vascular reactivity following acute administration of ponezumab, even in vessels where there was a severe cerebral amyloid angiopathy burden. Taken together, the beneficial effects ponezumab administration has on reducing the rate of cerebral amyloid angiopathy deposition and restoring cerebral vascular health favours a mechanism that involves rapid removal and/or neutralization of amyloid-ß species that may otherwise be detrimental to normal vessel function.

A Phase I proof-of-concept and safety trial of sildenafil to treat cerebral vasospasm following subarachnoid hemorrhage.

OBJECTIVE: Studies show that phosphodiesterase-V (PDE-V) inhibition reduces cerebral vasospasm (CVS) and improves outcomes after experimental subarachnoid hemorrhage (SAH). This study was performed to investigate the safety and effect of sildenafil (an FDA-approved PDE-V inhibitor) on angiographic CVS in SAH patients. METHODS: A2-phase, prospective, nonrandomized, human trial was implemented. Subarachnoid hemorrhage patients underwent angiography on Day 7 to assess for CVS. Those with CVS were given 10 mg of intravenous sildenafil in the first phase of the study and 30 mg in the second phase. In both, angiography was repeated 30 minutes after infusion. Safety was assessed by monitoring neurological examination findings and vital signs and for the development of adverse reactions. For angiographic assessment, in a blinded fashion, pre- and post-sildenafil images were graded as "improvement" or "no improvement" in CVS. Unblinded measurements were made between pre- and post-sildenafil angiograms. RESULTS: Twelve patients received sildenafil; 5 patients received 10 mg and 7 received 30 mg. There were no adverse reactions. There was no adverse effect on heart rate or intracranial pressure. Sildenafil resulted in a transient decline in mean arterial pressure, an average of 17% with a return to baseline in an average of 18 minutes. Eight patients (67%) were found to have a positive angiographic response to sildenafil, 3 (60%) in the low-dose group and 5 (71%) in the high-dose group. The largest degree of vessel dilation was an average of 0.8 mm (range 0-2.1 mm). This corresponded to an average percentage increase in vessel diameter of 62% (range 0%-200%). CONCLUSIONS: The results from this Phase I safety and proof-of-concept trial assessing the use of intravenous sildenafil in patients with CVS show that sildenafil is safe and well tolerated in the setting of SAH. Furthermore, the angiographic data suggest that sildenafil has a positive impact on human CVS.

HIF-1α Mediates Isoflurane-Induced Vascular Protection in Subarachnoid Hemorrhage.

OBJECTIVE: Outcome after aneurysmal subarachnoid hemorrhage (SAH) depends critically on delayed cerebral ischemia (DCI) - a process driven primarily by vascular events including cerebral vasospasm, microvessel thrombosis, and microvascular dysfunction. This study sought to determine the impact of postconditioning - the phenomenon whereby endogenous protection against severe injury is enhanced by subsequent exposure to a mild stressor - on SAH-induced DCI. METHODS: Adult male C57BL/6 mice were subjected to sham, SAH, or SAH plus isoflurane postconditioning. Neurological outcome was assessed daily via sensorimotor scoring. Contributors to DCI including cerebral vasospasm, microvessel thrombosis, and microvascular dysfunction were measured 3 days later. Isoflurane-induced changes in hypoxia-inducible factor 1alpha (HIF-1α)-dependent genes were assessed via quantitative polymerase chain reaction. HIF-1α was inhibited pharmacologically via 2-methoxyestradiol (2ME2) or genetically via endothelial cell HIF-1α-null mice (EC-HIF-1α-null). All experiments were performed in a randomized and blinded fashion. RESULTS: Isoflurane postconditioning initiated at clinically relevant time points after SAH significantly reduced cerebral vasospasm, microvessel thrombosis, microvascular dysfunction, and neurological deficits in wild-type (WT) mice. Isoflurane modulated HIF-1α-dependent genes - changes that were abolished in 2ME2-treated WT mice and EC-HIF-1α-null mice. Isoflurane-induced DCI protection was attenuated in 2ME2-treated WT mice and EC-HIF-1α-null mice. INTERPRETATION: Isoflurane postconditioning provides strong HIF-1α-mediated macro- and microvascular protection in SAH, leading to improved neurological outcome. These results implicate cerebral vessels as a key target for the brain protection afforded by isoflurane postconditioning, and HIF-1α as a critical mediator of this vascular protection. They also identify isoflurane postconditioning as a promising novel therapeutic for SAH.

Development of an ex vivo model for the study of cerebrovascular function utilizing isolated mouse olfactory artery.

OBJECTIVE: Cerebral vessels, such as intracerebral perforating arterioles isolated from rat brain, have been widely used as an ex vivo model to study the cerebrovascular function associated with cerebrovascular disorders and the therapeutic effects of various pharmacological agents. These perforating arterioles, however, have demonstrated differences in the vascular architecture and reactivity compared with a larger leptomeningeal artery which has been commonly implicated in cerebrovascular disease. In this study, therefore, we developed the method for studying cerebrovascular function utilizing the olfactory artery isolated from the mouse brain. METHODS: The olfactory artery (OA) was isolated from the C57/BL6 wild-type mouse brain. After removing connective tissues, one side of the isolated vessel segment (approximately -500 µm in length) was cannulated and the opposite end of the vessel was completely sealed while being viewed with an inverted microscope. After verifying the absence of pressure leakage, we examined the vascular reactivity to various vasoactive agents under the fixed intravascular pressure (60 mm Hg). RESULTS: We found that the isolated mouse OAs were able to constrict in response to vasoconstrictors, including KCl, phenylephrine, endothelin-1, and prostaglandin PGH2. Moreover, this isolated vessel demonstrated vasodilation in a dose-dependent manner when vasodilatory agents, acetylcholine and bradykinin, were applied. CONCLUSION: Our findings suggest that the isolated olfactory artery would provide as a useful ex vivo model to study the molecular and cellular mechanisms of vascular function underlying cerebrovascular disorders and the direct effects of such disease-modifying pathways on cerebrovascular function utilizing pharmacological agents and genetically modified mouse models.

Contribution of reactive oxygen species to cerebral amyloid angiopathy, vasomotor dysfunction, and microhemorrhage in aged Tg2576 mice.

Cerebral amyloid angiopathy (CAA) is characterized by deposition of amyloid ß peptide (Aß) within walls of cerebral arteries and is an important cause of intracerebral hemorrhage, ischemic stroke, and cognitive dysfunction in elderly patients with and without Alzheimer's Disease (AD). NADPH oxidase-derived oxidative stress plays a key role in soluble Aß-induced vessel dysfunction, but the mechanisms by which insoluble Aß in the form of CAA causes cerebrovascular (CV) dysfunction are not clear. Here, we demonstrate evidence that reactive oxygen species (ROS) and, in particular, NADPH oxidase-derived ROS are a key mediator of CAA-induced CV deficits. First, the NADPH oxidase inhibitor, apocynin, and the nonspecific ROS scavenger, tempol, are shown to reduce oxidative stress and improve CV reactivity in aged Tg2576 mice. Second, the observed improvement in CV function is attributed both to a reduction in CAA formation and a decrease in CAA-induced vasomotor impairment. Third, anti-ROS therapy attenuates CAA-related microhemorrhage. A potential mechanism by which ROS contribute to CAA pathogenesis is also identified because apocynin substantially reduces expression levels of ApoE-a factor known to promote CAA formation. In total, these data indicate that ROS are a key contributor to CAA formation, CAA-induced vessel dysfunction, and CAA-related microhemorrhage. Thus, ROS and, in particular, NADPH oxidase-derived ROS are a promising therapeutic target for patients with CAA and AD.

Crystal structure of bis-(4-allyl-2-meth-oxy-phen-yl) terephthalate.

The asymmetric unit of the title compound, C28H26O6, contains one half-mol-ecule, with the complete molecule generated by a crystallographic inversion center. The central terephthalate and meth-oxy-benzene rings are approximately perpendicular, making a dihedral angle of 80.31 (5)°. No specific directional contacts are noted in the crystal packing. The terminal vinyl group is disordered over two orientations with an occupancy ratio of 0.796 (4):0.204 (4).

Cerebral amyloid angiopathy increases susceptibility to infarction after focal cerebral ischemia in Tg2576 mice.

BACKGROUND AND PURPOSE: We and others have shown that soluble amyloid ß-peptide (Aß) and cerebral amyloid angiopathy (CAA) cause significant cerebrovascular dysfunction in mutant amyloid precursor protein (APP) mice, and that these deficits are greater in aged APP mice having CAA compared with young APP mice lacking CAA. Amyloid ß-peptide in young APP mice also increases infarction after focal cerebral ischemia, but the impact of CAA on ischemic brain injury is unknown. METHODS: To determine this, we assessed cerebrovascular reactivity, cerebral blood flow (CBF), and extent of infarction and neurological deficits after transient middle cerebral artery occlusion in aged APP mice having extensive CAA versus young APP mice lacking CAA (and aged-matched littermate controls). RESULTS: We found that aged APP mice have more severe cerebrovascular dysfunction that is CAA dependent, have greater CBF compromise during and immediately after middle cerebral artery occlusion, and develop larger infarctions after middle cerebral artery occlusion. CONCLUSIONS: These data indicate CAA induces a more severe form of cerebrovascular dysfunction than amyloid ß-peptide alone, leading to intra- and postischemic CBF deficits that ultimately exacerbate cerebral infarction. Our results shed mechanistic light on human studies identifying CAA as an independent risk factor for ischemic brain injury.

TrkB agonist antibody pretreatment enhances neuronal survival and long-term sensory motor function following hypoxic ischemic injury in neonatal rats.

Perinatal hypoxic ischemia (H-I) causes brain damage and long-term neurological impairments, leading to motor dysfunctions and cerebral palsy. Many studies have demonstrated that the TrkB-ERK1/2 signaling pathway plays a key role in mediating the protective effect of brain-derived neurotrophic factor (BDNF) following perinatal H-I brain injury in experimental animals. In the present study, we explored the neuroprotective effects of the TrkB-specific agonist monoclonal antibody 29D7 on H-I brain injury in neonatal rats. First, we found that intracerebroventricular (icv) administration of 29D7 in normal P7 rats markedly increased the levels of phosphorylated ERK1/2 and phosphorylated AKT in neurons up to 24 h. Second, P7 rats received icv administration of 29D7 and subjected to H-I injury induced by unilateral carotid artery ligation and exposure to hypoxia (8% oxygen). We found that 29D7, to a similar extent to BDNF, significantly inhibited activation of caspase-3, a biochemical hallmark of apoptosis, following H-I injury. Third, we found that this 29D7-mediated neuroprotective action persisted at least up to 5 weeks post-H-I injury as assessed by brain tissue loss, implicating long-term neurotrophic effects rather than an acute delay of cell death. Moreover, the long-term neuroprotective effect of 29D7 was tightly correlated with sensorimotor functional recovery as assessed by a tape-removal test, while 29D7 did not significantly improve rotarod performance. Taken together, these findings demonstrate that pretreatment with the TrkB-selective agonist 29D7 significantly increases neuronal survival and behavioral recovery following neonatal hypoxic-ischemic brain injury.

2-(2,5-Dimeth-oxy-phen-yl)-N-[2-(4-hy-droxy-phen-yl)eth-yl]acetamide.

In the title compound, C(18)H(21)NO(4), the dihedral angles between the acetamide group and the meth-oxy- and hy-droxy-substitured benzene rings are 80.81 (5) and 8.19 (12)°, respectively. The benzene rings are twisted with respect to each other, making a dihedral angle of 72.89 (5)°. In the crystal, N-H⋯O and O-H⋯O hydrogen bonds link the mol-ecules into a three-dimensional network.

Taraxerone enhances alcohol oxidation via increases of alcohol dehyderogenase (ADH) and acetaldehyde dehydrogenase (ALDH) activities and gene expressions.

The present study, taraxerone (d-friedoolean-14-en-3-one) was isolated from Sedum sarmentosum with purity 96.383%, and its enhancing effects on alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) activities were determined: EC(50) values were 512.42 ± 3.12 and 500.16 ± 3.23 µM for ADH and ALDH, respectively. In order to obtain more information on taraxerone related with the alcohol metabolism, 40% ethanol (5 mL/kg body weight) with 0.5-1mM of taraxerone were administered to mice. The plasma alcohol and acetaldehyde concentrations of taraxerone-treated groups were significantly lowered than those of the control group (p<0.01): approximately 20-67% and 7-57% lowered for plasma alcohol and acetaldehyde, respectively. Compare to the control group, the ADH and ALDH expressions in the liver tissues were abruptly increased in the taraxerone-treated groups after ethanol exposure. In addition, taraxerone prevented catalase, superoxide dismutase, and reduced glutathione concentrations from the decrease induced by ethanol administration with the concentration dependent manner.