Withanoside IV and its active metabolite, sominone, attenuate Abeta(25-35)-induced neurodegeneration

At the present, medication of dementia is limited to symptomatic treatments such as the use of cholinesterase inhibitors. To cure dementia completely, that is regaining neuronal function, reconstruction of neuronal networks is necessary. Therefore, we have been exploring antidementia drugs based on reconstructing neuronal networks in the damaged brain and found that withanoside IV (a constituent of Ashwagandha; the root of Withania somnifera) induced neurite outgrowth in cultured rat cortical neurons. Oral administration of withanoside IV (10 micromol/kg/day) significantly improved memory deficits in Abeta(25-35)-injected (25 nmol, i.c.v.) mice and prevented loss of axons, dendrites, and synapses. Sominone, an aglycone of withanoside IV, was identified as the main metabolite after oral administration of withanoside IV. Sominone (1 microM) induced axonal and dendritic regeneration and synaptic reconstruction significantly in cultured rat cortical neurons damaged by 10 microM Abeta(25-35). These data suggest that orally administrated withanoside IV may ameliorate neuronal dysfunction in Alzheimer's disease and that the active principle after metabolism is sominone.     

A natural scavenger of peroxynitrites, rosmarinic acid, protects against impairment of memory induced by Abeta(25-35)

Peroxynitrite (ONOO⁻)-mediated damage is regarded to be responsible for the cognitive dysfunction induced by amyloid beta protein (Aβ) in Alzheimer's disease (AD). In the present study, we examined the protective effects of rosmarinic acid (RA), a natural scavenger of ONOO⁻, on the memory impairment in a mouse model induced by acute i.c.v. injection of Aβ_25–35. Mice daily received i.p. several doses of RA after the injection of Aβ_25–35. RA prevented the memory impairments induced by Aβ_25–35 in the Y maze test and novel object recognition task. RA, at the effective lowest dose (0.25 mg/kg), prevented Aβ_25–35-induced nitration of proteins, an indirect indicator of ONOO⁻ damage, in the hippocampus. At this dose, RA also prevented nitration of proteins and impairment of recognition memory induced by ONOO⁻-i.c.v.-injection. Co-injection of the non-memory-impairing dose of ONOO⁻ with Aβ_25–35 blocked the protective effects of RA (0.25 mg/kg). These results demonstrated that the memory protective effects of RA in the neurotoxicity of Aβ_25–35 is due to its scavenging of ONOO⁻, and that daily consumption of RA may protect against memory impairments observed in AD.     

LRP-mediated clearance of Abeta is inhibited by KPI-containing isoforms of APP

The pathogenesis of Alzheimer's disease (AD) involves the abnormal accumulation and deposition of beta-amyloid in cerebral blood vessels and in the brain parenchyma. Critical in modulating beta-amyloid deposition in brain is the flux of Abeta across the blood brain barrier. The low-density lipoprotein receptor-related protein (LRP), is a large endocytic receptor that mediates the efflux of Abeta out of brain and into the periphery. The first step in the LRP-mediated clearance of Abeta involves the formation of a complex between Abeta and the LRP ligands apolipoprotein E (apoE) or alpha(2)-macroglobulin (alpha(2)M). The Abeta/chaperone complexes then bind to LRP via binding sites on apoE or alpha(2)M. The efflux of Abeta/chaperone complexes out of the neuropil and into the periphery may be attenuated by LRP-ligands that compete with apoE or alpha(2)M for LRP binding. LRP is also the cell surface receptor for Kunitz Protease Inhibitor (KPI) containing isoforms of Abeta's parent protein, the amyloid protein precursor (APP). Protein and mRNA levels of KPI-containing APP isoforms (APP-KPI) are elevated in AD brain and are associated with increased Abeta production. In this study we show that soluble non-amyloidogenic APP-KPI can also inhibit the uptake of Abeta/alpha(2)M in a cell culture model of LRP mediated Abeta clearance. Clearance of Abeta/apoE complexes was not inhibited by APP-KPI. Our findings are consistent with studies showing that apoE and alpha(2)M have discrete binding sites on LRP. Most significantly, our data suggests that the elevated levels of APP-KPI in AD brain may attenuate the clearance of Abeta, the proteins own amyloidogenic catabolic product.     

Neuroprotection by D-securinine against neurotoxicity induced by beta-amyloid (25-35)

The effects of (+) securinine on behavior and morphological changes after intracerebral ventricle injection of beta-amyloid (25-35) (Abeta(25-35)) in rats were investigated. A single high dose of Abeta(25-35) could impair the spatial cognitive function. The latency of locating the platform was longer in the model group than in the sham-operated group. While chronic administration of D-securinine (40 mg kg(-1)) could significantly shorten the latency. After Morris water maze, the activity of choline acetyltransferase (ChAT) and acetylcholinesterase (AchE) were detected. The results showed that D-securinine could decrease the AchE activity significantly and have no effect on ChAT. Meanwhile, immunohistochemistry analysis revealed that D-securinine could reduce the glial inflammatory responses induced by beta-amyloid protein. These results suggest that the effect of D-securinine in improving the cognitive deficits and neurodegeneration in betaAP(25-35)-treated rats may involve direct and indirect actions on targets. The GABA receptor plays a key role in these therapeutic effects and may be helpful in the treatment of Alzheimer's disease.     

Abeta(25-35) and its C- and/or N-blocked derivatives: copper driven structural features and neurotoxicity

The toxic properties of beta-amyloid protein, Abeta(1-42), the major component of senile plaques in Alzheimer's disease, depend on nucleation-dependent oligomerization and aggregation. In addition, Abeta(1-42) toxicity is favored by the presence of trace metals, which affect the secondary structure of the peptide. A peptide comprising 11 residues within Abeta(1-42) [Abeta(25-35)] aggregates and retains the neurotoxic activity of Abeta(1-42). We have used both Abeta(25-35) and its C-amidated or N-acetylated/C-amidated derivatives to investigate the role of copper(II) in modulating the conformation and aggregation state as well as the neurotoxic properties of amyloid peptides. Electrospray ionization mass spectrometry (ESI-MS) and electron paramagnetic resonance (EPR) measurements were performed to verify the formation of copper(II)/Abeta(25-35) complexes and to determine the coordination mode, respectively. Abeta(25-35) and its derivatives were analyzed by circular dichroism spectroscopy to assess their secondary structure, subjected to thioflavine-T (Th-T) binding assay to reveal beta-sheet structured aggregates formation, and imaged by scanning force microscopy. Toxicity was assessed on mature cultures of rat cortical neurons. We found that beta-sheet-structured species of Abeta(25-35) were neurotoxic, whereas the random-coil-structured derivatives were devoid of effect. Interestingly, copper promoted the random-coil/beta-sheet transition of Abeta(25-35), with ensuing peptide toxicity, but it induced the toxicity of the N-acetylated/C-amidated derivative without affecting peptide folding. Moreover, copper did not influence either the folding or the activity of the C-amidated Abeta(25-35), suggesting that blockade of the C-terminus of Abeta peptides might be sufficient to prevent Abeta toxicity.     

The hydrophobic environment of Met35 of Alzheimer's Abeta(1-42) is important for the neurotoxic and oxidative properties of the peptide

In Alzheimer's disease (AD) brain increased lipid peroxidation is found. Amyloid β-peptide [Aβ(1–42)] induces oxidative stress (including lipid peroxidation) and neurotoxicity, and the single methionine residue (Met35), is important for these properties. In the current study, we tested the hypothesis that removal of Met35 from lipid bilayer would abrogate the oxidative stress and neurotoxic properties of Aβ(1–42), i.e. we tested the hypothesis and found that lipid peroxidation initiated by oxidation of the Met35 is an early event in Aβ(1–42) neurotoxicity. Substitution of negatively charged aspartic acid for glycine residue 37 is not predicted to bring the Met35 residue out of the hydrophobic lipid bilayer. In this study, we showed that G37D substitution in Aβ(1–42) completely abolishes neurotoxic and oxidative processes associated with the parent peptide. This is demonstrated by the lack of cell toxicity and protein oxidation in contrast to the treatment with native Aβ(1–42). Additionally, the G37D peptide does not display the aggregation properties that are associated with native Aβ as seen in the thioflavin T (ThT) assay and fibril morphology. The results presented in this work are thus consistent with the notion of the importance of methionine 35 of Aβ(1–42) in the lipid-initiated oxidative cascade and subsequent neurotoxicity in AD brain.     

Effects of Abeta25-35 on neurogenesis in the adult mouse subventricular zone and dentate gyrus

It has been demonstrated that neuorgenesis driven by neural precursor cells persists well into the adult period. This study was to observe the effects of Amyloid-beta (25-35) peptide (Abeta(25-35)) on neurogenesis in the subventricular zone and dentate gyrus of adult mouse brain. Aggregated Abeta(25-35)(1 mg/ml, 3 microl) was injected into the lateral ventricle of adult mouse. Animals were transcardially perfused with 4% paraformaldehyde in PBS, respectively at 5, 10, 20, 30 days after the Abeta(25-35) injection. All the animals were injected with BrdU (50 mg/kg, i. p) to label the neural precursor cells 24 h before the each perfusion. NeuN immunofluorescence and BrdU immunohistology were performed. It was found that Abeta(25-35) could injure the mature neurons and decrease the number of NeuN positive neurons. It also showed that Abeta(25-35) inhibited neurogenesis and significantly decreased the number of BrdU positive cells in the dentate gyrus of hippocampus, but it had no obvious effects on neurogenesis in the subventricular zone. The present results indicated that Abeta(25-35) could impair neurogenesis in the hippocampus of adult mouse brain.     

Autoimmune responses to amyloid structures of Abeta(25-35) peptide and human lysozyme in the serum of patients with progressive Alzheimer's disease

We have found an increased level of serum antibodies to the prefibrillar structures of both Abeta(25-35) peptide and human lysozyme in Alzheimer's disease (AD) patients compared to age-matched controls, indicating that autoimmunity is implicated in AD. In the serum of AD patients with a long-term duration (>15 years) the titer of serum antibodies to aggregates of Abeta(25-35) peptide increased by approximately 5-fold, whilst the antibody titer to lysozyme protofilaments decreased by approximately 8-fold compared to patients with AD duration of <5 years. The content of immunoglobulins of the A, G and M types declined, particularly in AD duration of >15 years. An increase in the concentration of immune complexes and higher lysozyme activity was detected in the serum of all patients and this was suggestive of an inflammatory reaction. We propose that the autoimmune response to different amyloid structures in AD can be viewed as a clearance pathway targeting amyloid development. Autoimmune response can be exploited as a marker of ongoing protein aggregation and hence be used as a diagnostic feature of AD.     

S14G-Humanin ameliorates Abeta25-35-induced behavioral deficits by reducing neuroinflammatory responses and apoptosis in mice

Cerebral amyloid-beta protein (Abeta) deposition and associated neuroinflammation and apoptosis are increasingly recognized as an important component leading to cognitive impairment in Alzheimer's disease (AD). Humanin (HN) and its derivative, S14G-HN (HNG), are best known for their ability to suppress neuronal death induced by AD-related insults in vitro. Furthermore, limited in vivo studies show that HNG can ameliorate memory impairment induced by intracerebroventricular injection of anti-cholinergic drugs or Abeta25-35. However, the mechanism underlying the in vivo effect remains unclear. In this study, we sought to determine the effects of HNG on neuroinflammatory responses and apoptosis associated with behavioral deficits induced by Abeta25-35 in vivo. Our results indicate that intracerebroventricular injection of aggregated Abeta25-35 induced impairment of learning and memory, markedly elevated numbers of reactive astrocytes, activated microglia, and apoptotic cells, as well as remarkable increased levels of IL-6 and TNFalpha. Moreover, intraperitoneal HNG treatment ameliorated behavioral deficits, and reduced neuroinflammatory responses and apoptotic cells in the brain. Cumulatively, these finding demonstrate for the first time that HNG may have the potential for attenuating Abeta-induced cognitive deficits by reducing inflammatory responses and apoptosis in vivo, which may add to the novel evidence for anti-inflammatory and antiapoptosis properties of HNG in AD treatment.     

Neuroprotective effects of extracellular glutamate are absent in hippocampal organotypic cultures treated with the amyloid peptide Abeta(25-35).

Hippocampal cells are particularly vulnerable in Alzheimer's disease but the cause of cell death is unknown. Amyloid toxicity has been implicated in hippocampal cell death, but its specific mechanisms are poorly understood. We used confocal microscopy to examine the effects of the amyloid peptide fragment 25-35 (Abeta(25-35)) on cell death in organotypic hippocampal slice cultures. Addition of glutamate to the culture medium significantly improved nerve cell survival in cultures subjected to consecutive medium exchanges. This effect was lost if cultures were treated with the amyloid peptide fragment Abeta(25-35) but not the inactive peptide 35-25. These data suggest that one of the mechanisms responsible for amyloid toxicity may be inhibition of the survival promoting effects of extracellular glutamate.     

Implanted cannula-mediated repetitive administration of Abeta25-35 into the mouse cerebral ventricle effectively impairs spatial working memory

Amyloid beta (Abeta) is closely related to the onset of Alzheimer's disease (AD). To construct AD animal models, a bolus administration of a large dose of toxic Abeta into the cerebral ventricles of rodents has been performed in earlier studies. In parallel, a continuous infusion system via an osmotic pump into the cerebral ventricle has been developed to make a rat AD model. In this study, we developed a mouse AD model by repetitive administration of Abeta25-35 via a cannula implanted into the cerebral ventricle. Using this administration system, we reproducibly constructed a mouse with impaired spatial working memory. In accordance with the occurrence of the abnormal mouse behavior, we found that the number of choline acetyltransferase (ChAT)-positive neurons was reduced in paraventricular regions of brains of Abeta25-35-administered mice in a dose-dependent manner. Considering that the repetitive administration of a small dose of toxic Abeta via an implanted cannula leads to a brain status more resembling that of the AD patients than a bolus injection of a large dose of Abeta, and therapeutic as well as toxic agents are able to be repeatedly and reliably administered via an implanted cannula, we concluded that the implanted cannula-bearing AD mouse model is useful for development of new AD therapy.     

Aβ_(25-35)诱导PC12细胞损伤的蛋白质组学研究

目的 识别AD细胞模型中蛋白质组改变,在蛋白质水平上揭示Aβ的作用机制.方法 利用双向差异凝胶电泳技术(2D-DIGE)和质谱(MS)技术.探索20μmol/L浓度Aβ_(25-35)肽段作用48h后PC12细胞蛋白质组的改变.结果 2D-DIGE图像上出现约2000个蛋白点.与对照组比较,Aβ_(25-35)作用下共有29个蛋白的表达有显著差异,其中25个蛋白表达量上调及4个蛋白表达量下调超过30%.质谱鉴定出7个蛋白点,分为3类:(1)具有分子伴侣活性的蛋白:葡萄糖调节蛋白75(glucose-regulated protein75,GRP75)、热休克同源蛋白71(heat shock cognate 71 kDa protein,HSC71)、calreticulin表达量均上调.(2)细胞骨架蛋白:β-微管蛋白(tubulin beta chain 15,TBETA-15)和低分子量神经细丝蛋白(neurofilament light polypeptide,NF-L)表达量亦上调.(3)与能量代谢有关的酶:肌酸激酶B(creatine kinase-B,CKB)和醛缩酶A(aldolaseA)蛋白表达量均下调.结论 本实验首次将DIGE和MS方法应用于Aβ_(25-35)神经毒性作用机制研究,在蛋白质组水平上揭示了Aβ_(25-35)毒性作用的早期机制.     

The effect of fibrinogen degradation products and some lysine analogues on the dissociation of plasmin(ogen) - fibrin complexes

Fibrin carries sites which bind plasmin and preactivated plasminogen (mol.wt. 84,000) allowing the formation of stable complexes. Fibrinogen degradation product E interferes with this complex formation suggesting that the plasmin(ogen) binding site is present in this fragment. The complexes are also dissociated by the anti-fibrinolytic agents 6-aminohexanoic acid and aminomethyl cyclohexanoic acid which both compete for the fibrin binding site of plasmin and plasminogen.     

The Survivin-mediated radioresistant phenotype of glioblastomas is regulated by RhoA and inhibited by the green tea polyphenol (-)-epigallocatechin-3-gallate

INTRODUCTION: Glioblastoma multiforme's (GBM) aggressiveness is potentiated in radioresistant tumor cells. The combination of radiotherapy and chemotherapy has been envisioned as a therapeutic approach for GBM. The goal of this study is to determine if epigallocatechin-3-gallate (EGCg), a green tea-derived anti-cancer molecule, can modulate GBMs' response to ionizing radiation (IR) and whether this involves mediators of intracellular signaling and inhibitors of apoptosis proteins. MATERIAL AND METHODS: U-87 human GBM cells were cultured and transfected with cDNAs encoding for Survivin, RhoA or Caveolin-1. Mock and transfected cells were irradiated at sublethal single doses. Cell proliferation was analyzed by nuclear cell counting. Apoptosis was detected using a fluorometric caspase-3 assay. Analysis of protein expression was accomplished by Western immunoblotting. RESULTS: IR (10 Gy) reduced control U-87 cell proliferation by 40% through a caspase-independent mechanism. The overexpression of Survivin induced a cytoprotective effect against IR, while the overexpression of RhoA conferred a cytosensitizing effect upon IR. Control U-87 cells pretreated with EGCg exhibited a dose-dependent decrease in their proliferation rate. The growth inhibitory effect of EGCg was not antagonized by overexpressed Survivin. However, Survivin -transfected cells pretreated with EGCg became sensitive to IR, and their RhoA expression was downregulated. A potential therapeutic effect of EGCg targeting the prosurvival intracellular pathways of cancer cells is suggested to act synergistically with IR. CONCLUSION: The radioresistance of GBM is possibly mediated by a mechanism dependent on Survivin in conjunction with RhoA. The combination of natural anti-cancerous molecules such as EGCg with radiotherapy could improve the efficacy of IR treatments.     

Plasma Abeta(1-40) and Abeta(1-42) and the risk of dementia: a prospective case-cohort study

BACKGROUND: Amyloid beta peptides (Abeta) are important components of plaques in Alzheimer's disease. Plasma concentrations of Abeta(1-40) and Abeta(1-42) rise with age and are increased in people with mutations that cause early-onset Alzheimer's disease. However, Abeta(1-42) concentrations may decrease early in the dementia process. We postulated that concentrations of Abeta(1-40) and Abeta(1-42) in plasma are associated with risk of dementia. METHODS: We did a case-cohort study embedded in the prospective, population-based Rotterdam Study. Of 6713 participants at risk for dementia, a random sample of 1756 people was drawn. During follow-up (mean 8.6 years), 392 incident dementia cases were identified. We investigated the association between plasma Abeta concentrations and risk of dementia and its subtypes using Cox proportional hazard models. FINDINGS: High concentrations of Abeta(1-40) but not Abeta(1-42) at baseline were associated with an increased risk of dementia. Compared with the first quartile of Abeta(1-40), age and sex-adjusted hazard ratios for dementia for the second, third, and fourth quartiles were 1.07 (95% CI 0.72-1.58), 1.16 (0.78-1.70), and 1.46 (1.01-2.12). People with an increased Abeta(1-42)/Abeta(1-40) ratio had a reduced risk of dementia. Compared with the first quartile of the Abeta(1-42)/Abeta(1-40) ratio, hazard ratios for the second, third, and fourth quartiles were 0.74 (0.53-1.02), 0.62 (0.44-0.88), and 0.47 (0.33-0.67). Associations were similar for Alzheimer's disease and vascular dementia. INTERPRETATION: High plasma concentrations of Abeta(1-40), especially when combined with low concentrations of Abeta(1-42), indicate an increased risk of dementia. A potential role of plasma Abeta concentrations as a marker of incipient dementia warrants further investigation.     

Interaction of amyloid beta protein (25-35) with tachykinin receptors.

Amyloid beta protein (25-35) failed to significantly interact with tachykinin NK1 (rat forebrain, guinea-pig ileum) NK2 (rabbit pulmonary artery, hamster trachea) or NK-3 (guinea-pig cortex) receptors, as determined by radioligand binding and functional assays. A weak interaction (Ki 14.8 microM) was detected with NK2 receptors in rat small intestine. It appears unlikely that direct interaction with tachykinin receptors may account for the reported ability of amyloid beta protein (25-35) to affect neuronal survival.     

The trophic effect of beta-amyloid 25-35 peptide is not mediated by NK1 or bombesin receptors.

beta-Amyloid peptide and spantide have previously been described to have trophic effects on hippocampal neurones in vitro. We report here that bombesin and [Pro9]-substance P also show a neurotrophic effect on cultured hippocampal neurones. The neurotrophic effect of spantide or a beta-amyloid fragment containing amino acids 25 to 35 was not blocked by addition of the NK1 receptor agonist, substance P or the nonpeptide NK1 antagonist, RP 67580. For the bombesin-related peptides, the antagonist [Tyr4-DPhe12]-bombesin also provokes a trophic response, but the agonist alytesin and the antagonist [Leu13-psi-(CH2NH) Leu14]-bombesin have no effect on neurite growth. These results suggest that the observed trophic responses are unlikely to be mediated by a classical NK1 or bombesin receptor.     

Scutellaria baicalensis stem-leaf total flavonoid reduces neuronal apoptosis induced by amyloid beta-peptide (25-35)

Scutellaria baicalensis stem-leaf total flavonoid might attenuate learning/memory impairment and neuronal loss in rats induced by amyloid beta-peptide. This study aimed to explore the effects of Scutellaria baicalensis stem-leaf total flavonoid on amyloid beta-peptide-induced neuronal apoptosis and the expression of apoptosis-related proteins in the rat hippocampus. Male Wistar rats were given intragastric administration of Scutellaria baicalensis stem-leaf total flavonoid, 50 or 100 mg/kg, once per day. On day 8 after administration, 10 μg amyloid beta-peptide (25-35) was injected into the bilateral hippocampus of rats to induce neuronal apoptosis. On day 20, hippocampal tissue was harvested and probed with the terminal deoxyribonucleotidyl transferase-mediated biotin-16-dUTP nick-end labeling assay. Scutellaria baicalensis stem-leaf total flavonoid at 50 and 100 mg/kg reduced neuronal apoptosis induced by amyloid beta-peptide (25-35 in the rat hippocampus. Immunohistochemistry and western blot assay revealed that expression of the pro-apoptotic protein Bax, cytochrome c and caspase-3 was significantly diminished by 50 and 100 mg/kg Scutellaria baicalensis stem-leaf total flavonoid, while expression of the anti-apoptotic protein Bcl-2 was increased. Moreover, 100 mg/kg Scutellaria baicalensis stem-leaf total flavonoid had a more dramatic effect than the lower dosage. These experimental findings indicate that Scutellaria baicalensis stem-leaf total flavonoid dose-dependently attenuates neuronal apoptosis induced by amyloid beta-peptide in the hippocampus, and it might mediate this by regulating the expression of Bax, cytochrome c, caspase-3 and Bcl-2.