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.

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.

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.

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.

Resorufin analogs preferentially bind cerebrovascular amyloid: potential use as imaging ligands for cerebral amyloid angiopathy.

BACKGROUND: Cerebral amyloid angiopathy (CAA) is characterized by deposition of fibrillar amyloid ß (Aß) within cerebral vessels. It is commonly seen in the elderly and almost universally present in patients with Alzheimer's Disease (AD). In both patient populations, CAA is an independent risk factor for lobar hemorrhage, ischemic stroke, and dementia. To date, definitive diagnosis of CAA requires obtaining pathological tissues via brain biopsy (which is rarely clinically indicated) or at autopsy. Though amyloid tracers labeled with positron-emitting radioligands such as [11C]PIB have shown promise for non-invasive amyloid imaging in AD patients, to date they have been unable to clarify whether the observed amyloid load represents neuritic plaques versus CAA due in large part to the low resolution of PET imaging and the almost equal affinity of these tracers for both vascular and parenchymal amyloid. Therefore, the development of a precise and specific non-invasive technique for diagnosing CAA in live patients is desired. RESULTS: We found that the phenoxazine derivative resorufin preferentially bound cerebrovascular amyloid deposits over neuritic plaques in the aged Tg2576 transgenic mouse model of AD/CAA, whereas the congophilic amyloid dye methoxy-X34 bound both cerebrovascular amyloid deposits and neuritic plaques. Similarly, resorufin-positive staining was predominantly noted in fibrillar Aß-laden vessels in postmortem AD brain tissues. Fluorescent labeling and multi-photon microscopy further revealed that both resorufin- and methoxy-X34-positive staining is colocalized to the vascular smooth muscle (VSMC) layer of vessel segments that have severe disruption of VSMC arrangement, a characteristic feature of CAA. Resorufin also selectively visualized vascular amyloid deposits in live Tg2576 mice when administered topically, though not systemically. Resorufin derivatives with chemical modification at the 7-OH position of resorufin also displayed a marked preferential binding affinity for CAA, but with enhanced lipid solubility that indicates their use as a non-invasive imaging tracer for CAA is feasible. CONCLUSIONS: To our knowledge, resorufin analogs are the fist class of amyloid dye that can discriminate between cerebrovascular and neuritic forms of amyloid. This unique binding selectivity suggests that this class of dye has great potential as a CAA-specific amyloid tracer that will permit non-invasive detection and quantification of CAA in live patients.

Clinical features and outcome of physiotherapy in early presenting congenital muscular torticollis with severe fibrosis on ultrasonography: a prospective study.

BACKGROUND: It has been reported that ultrasonography (US) can detect the severity of congenital muscular torticollis (CMT), and severe fibrosis of the sternocleidomastoid (SCM) muscle noted on US is irreversible and likely to require surgery. Clinical outcome of CMT depends mainly on the patient's age, which is also associated with the severity of fibrosis as determined by US. However, there has been no well-designed study to elucidate the true relationship among these factors nor a definite consensus on treatment of young infants with severe fibrosis in the SCM compared with well-documented reports that late cases require surgery. PURPOSE: The purpose of the current study was to investigate whether severity of SCM fibrosis on US is correlated with clinical severity and outcome of standardized physiotherapy in early presenting CMT. METHODS: Fifty patients with a palpable neck mass, initial deficit of passive neck rotation (ΔROT) more than 10°, and age less than 3 months were classified into 4 US types according to the severity of fibrosis in the SCM and underwent standardized physiotherapy and regular assessment. Relationship between US types and 2 variables (ΔROT and treatment duration) and success rate of physiotherapy was assessed. RESULTS: None of the cases was classified as type 4. Type 3 showed greatest ΔROT and longest mean treatment duration. Both variables showed a significant linear trend of association with US types by P for trend (P = .003, P < .001, respectively). Treatment was "successful" in 49 patients (98%). CONCLUSION: In young infants with CMT, US can document severity; and an early and adequate physiotherapy is a good treatment option, particularly even in those with severe fibrosis.

Risk factors for intrauterine constraint are associated with ultrasonographically detected severe fibrosis in early congenital muscular torticollis.

BACKGROUND/PURPOSE: The etiology of congenital muscular torticollis (CMT) remains controversial. Ultrasonographically, severe fibrosis involving the entire sternocleidomastoid muscle (SCM; type 3 or 4) fibrosis has been associated with poor clinical outcomes and indicates a chronic state of the condition. The purpose of this study was to test whether or not type 3 or 4 fibrosis detected early after birth is associated with factors related to prolonged intrauterine constraint. METHODS: Sixty-seven patients (age, <3 months) with CMT were classified into 4 different ultrasonographic types according to the severity of SCM fibrosis. The odds ratio for the relationship between probability of type 3 or 4 and factors related to intrauterine constraint were calculated by a multivariate logistic regression model. RESULTS: None were classified as type 4. Twenty-three patients (34%) had a history of breech presentation, and 21 (91.3%) of them were delivered by elective cesarean section without likelihood of birth trauma. Compared with normal pregnancy, breech presentation and oligohydramnios showed a 6.7 or 7.5 times higher probability for type 3 fibrosis, respectively. CONCLUSION: Risk factors for intrauterine constraint appear to be associated with ultrasonographically detected severe fibrosis involving the entire SCM muscle in early presenting CMT.

Endothelial nitric oxide synthase mediates endogenous protection against subarachnoid hemorrhage-induced cerebral vasospasm.

BACKGROUND AND PURPOSE: Vasospasm-induced delayed cerebral ischemia remains a major source of morbidity in patients with aneurysmal subarachnoid hemorrhage (SAH). We hypothesized that activating innate neurovascular protective mechanisms by preconditioning (PC) may represent a novel therapeutic approach against SAH-induced vasospasm and neurological deficits and, secondarily, that the neurovascular protection it provides is mediated by endothelial nitric oxide synthase (eNOS). METHODS: Wild-type mice were subjected to hypoxic PC or normoxia followed 24 hours later by SAH. Neurological function was analyzed daily; vasospasm was assessed on post-surgery Day 2. Nitric oxide availability, eNOS expression, and eNOS activity were also assessed. In a separate experiment, wild-type and eNOS-null mice were subjected to hypoxic PC or normoxia followed by SAH and assessed for vasospasm and neurological deficits. RESULTS: PC nearly completely prevented SAH-induced vasospasm and neurological deficits. It also prevented SAH-induced reduction in nitric oxide availability and increased eNOS activity in mice with and without SAH. PC-induced protection against vasospasm and neurological deficits was lost in wild-type mice treated with the nitric oxide synthase inhibitor N(G)-nitro-l-arginine methyl ester and in eNOS-null mice. CONCLUSIONS: Endogenous protective mechanisms against vasospasm exist, are powerful, and can be induced by PC. eNOS-derived nitric oxide is a critical mediator of PC-induced neurovascular protection. These data provide strong "proof-of-principle" evidence that PC represents a promising new strategy to reduce vasospasm and delayed cerebral ischemia after SAH.

Strain measurement from 3D micro-CT images of a breast-mimicking phantom.

Strain distribution in compressed tissues gives information about elasticity of the tissues. We have measured strain from two sets of 3D micro-CT images of a breast-mimicking phantom; one obtained without compressing the phantom and the other with compressing it. To measure strain, we first calculated compression-induced displacements of high-intensity feature patterns in the image. In measuring displacement of a pixel of interest, we searched the pixel in the compressed-phantom image, whose surrounding resembles the uncompressed-phantom image most closely, using the image correlation technique. From the displacement data, we calculated average strain at a region of interest. With the calculated average strains, we could distinguish the hard inclusion in the phantom which was not distinguishable from the background body of the phantom in the ordinary micro-CT images. The calculated strains account for stiffness of the tissue of interest, one of the important parameters for diagnosing malignant tissues. We present experimental results of the displacement and strain measurement along with FEM analysis results.

N-(3,4-Difluoro-phen-yl)-3,4-dimethoxy-benzene-sulfonamide.

In the title sulfonamide derivative, C(14)H(13)F(2)NO(4)S, the dihedral angle between the benzene rings is 66.05 (9)°. The crystal structure is stabilized by weak inter-molecular N-H⋯O hydrogen bonds involving the amine and meth-oxy groups, which link the mol-ecules into a one-dimensional chain. No significant inter-chain contacts are observed.

Evaluation of paclitaxel rearrangement involving opening of the oxetane ring and migration of acetyl and benzoyl groups.

The stability of drug is a critical factor in quality control, drug efficacy, safety, storage, and production conditions. The rearrangement of paclitaxel, which involves opening of the oxetane ring and migration of acetyl group occurred on heating a powder of purified paclitaxel. Subsequently, the unusual migration of benzoyl groups progressed rapidly in organic solvents. These rearrangement derivatives were isolated carefully. The structures of the intermediate derivative A and the product derivative B were confirmed using (1)H NMR, high performance liquid chromatography (HPLC), and mass spectrometry. We proposed the rearrangement pathway here for the first time. Neither derivative exhibited bioactivity in SKOV3 (ovarian cancer) or MDA-MB-435 (breast cancer) cell culture assays.

Polyphenol amentoflavone affords neuroprotection against neonatal hypoxic-ischemic brain damage via multiple mechanisms.

Flavonoids are naturally occurring polyphenolic compounds that have many biological properties, including antioxidative, anti-inflammatory and neuroprotective effects. Here, we report that amentoflavone significantly reduced cell death induced by staurosporine, etoposide and sodium nitroprusside in neuroblastoma SH-SY5Y cells. In post-natal day 7 rats, hypoxic-ischemic (H-I) brain damage induced by unilateral carotid ligation and hypoxia resulted in distinct features of neuronal cell death including apoptosis and necrosis. In this model, a systemic administration of amentoflavone (30 mg/kg) markedly reduced the H-I-induced brain tissue loss with a wide therapeutic time window up to 6 h after the onset of hypoxia. Amentoflavone blocked the activation of caspase 3, characteristic of apoptosis, and the proteolytic cleavage of its substrates following H-I injury. Amentoflavone also reduced the excitotoxic/necrotic cell death after H-I injury in vivo and after oxygen/glucose deprivation in mouse mixed cultures in vitro. Treatment of mouse microglial cells with amentoflavone resulted in a significant decrease in the lipopolysaccharide-induced production of nitric oxide and induction of inducible nitric oxide synthase and cyclo-oxygenase-2. Furthermore, amentoflavone decreased the inflammatory activation of microglia after H-I injury when assessed by the microglial-specific marker OX-42. These data demonstrate for the first time that amentoflavone strongly protects the neonatal brain from H-I injury by blocking multiple cellular events leading to brain damage.

MKP-1 contributes to oxidative stress-induced apoptosis via inactivation of ERK1/2 in SH-SY5Y cells.

Mitogen-activated protein (MAP) kinase phosphatase-1 (MKP-1) is a dual specificity phosphatase that negatively regulates the MAP kinases. In this study, we found that levels of MKP-1 expression were transiently decreased within 3h, followed by an increase 6-9h after H2O2-induced oxidative stress in human neuroblastoma SH-SY5Y cells. There was a strong negative correlation between MKP-1 expression and ERK1/2 phosphorylation levels. Treatment of cells with a proteasomal inhibitor MG132 decreased the oxidative stress-induced degradation of MKP-1, resulting in dephosphorylation of ERK1/2. MG132 potentiated hydrogen peroxide-induced cell death, which was attenuated by a phosphatase inhibitor sodium orthovanadate. Suppression of MKP-1 expression by transfection with siRNA duplexes specific to MKP-1 transcript resulted in a decrease in oxidative stress-induced cell death. These data therefore suggest that MKP-1, a negative regulator of ERK1/2, plays a proapoptotic role in oxidative stress-induced cell death in a neuronal cell line.

Neuroprotective effects of naturally occurring biflavonoids.

We examined neuroprotective effects of naturally occurring biflavonoids on oxidative stress-induced and amyloid beta peptide-induced cell death in neuronal cells. Among the nine biflavonoids tested, amentoflavone, ginkgetin, and isoginkgetin exhibited strong neuroprotection against cytotoxic insults induced by oxidative stress and amyloid beta, suggesting their therapeutic potential against neurodegenerative diseases, including ischemic stroke and Alzheimer's disease.

Ghrelin stimulates proliferation and differentiation and inhibits apoptosis in osteoblastic MC3T3-E1 cells.

Ghrelin is a 28-amino-acid peptide identified in the stomach as an endogenous ligand of the growth hormone secretagogue receptor (GHS-R) that strongly stimulates the release of growth hormone at the hypothalamus and pituitary level. Although GHS-Rs are expressed in a variety of peripheral tissues, little is known about its effect on bone independent of GH/IGF-1 axis. This study was undertaken to investigate whether ghrelin exerts a direct effect on osteoblasts. We identified mRNA and protein expression of GHS-R in primary osteoblasts as well as a number of osteoblastic cell lines, including MC3T3-E1, ROS 17/2.8, UMR-106, MG63, and SaOS2 cells. Treatment of ghrelin (10(-11) to 10(-7) M) to MC3T3-E1 cells showed dose-dependent stimulation of proliferation, which was abrogated by treatment with [d-Lys]-GHRP-6 (10(-3) M), a selective antagonist of the ghrelin receptor. Ghrelin activated ERK1/2 MAPK and pretreatment with MAPK kinase inhibitors, PD98059 attenuated the ghrelin-induced cell proliferation. Ghrelin also inhibited TNFalpha-induced apoptosis and suppressed caspase-3 activation that occurs in response to TNFalpha as well as during in vitro differentiation process. Moreover, ghrelin treatment enhanced in vitro osteoblast differentiation as evidenced by matrix mineralization, alkaline phosphatase activity, and osteoblast-specific gene expression. These results suggest that ghrelin promotes proliferation and differentiation and inhibits apoptosis of osteoblasts.

Neuroprotective effects of flavones on hydrogen peroxide-induced apoptosis in SH-SY5Y neuroblostoma cells.

Neuroprotective effects of flavones were examined. Luteolin and apigenin exhibited neuroprotection against oxidative stress-induced cell death in SH-SY5Y cells. Free radical scavenging activity and neuroprotection assays revealed that flavones exerted their neuroprotective effects via the direct interaction with the apoptotic caspase pathway independently of their antioxidant activity.

Selective, reversible caspase-3 inhibitor is neuroprotective and reveals distinct pathways of cell death after neonatal hypoxic-ischemic brain injury.

Hypoxia-ischemia (H-I) in the developing brain results in brain injury with prominent features of both apoptosis and necrosis. A peptide-based pan-caspase inhibitor is neuroprotective against neonatal H-I brain injury, suggesting a central role of caspases in brain injury. Because previously studied peptide-based caspase inhibitors are not potent and are only partially selective, the exact contribution of specific caspases and other proteases to injury after H-I is not clear. In this study, we explored the neuroprotective effects of a small, reversible caspase-3 inhibitor M826. M826 selectively and potently inhibited both caspase-3 enzymatic activity and apoptosis in cultured cells in vitro. In a rat model of neonatal H-I, M826 blocked caspase-3 activation and cleavage of its substrates, which begins 6 h and peaks 24 h after H-I. Although M826 significantly reduced DNA fragmentation and brain tissue loss, it did not prevent calpain activation in the cortex. This activation, which is associated with excitotoxic/necrotic cell injury, occurred within 30 min to 2 h after H-I even in the presence of M826. Similar to calpain activation, we found evidence of caspase-2 processing within 30 min to 2 h after H-I that was not affected by M826. Caspase-2 processing appeared to be secondary to calpain-mediated cleavage and was not associated with caspase-2 activation. These data suggest that caspase-3 specifically contributes to delayed cell death and brain injury after neonatal H-I and that calpain activation is associated with and likely a marker for the early component of excitotoxic/necrotic brain injury previously demonstrated in this model.