Abeta(31-35) peptide induce apoptosis in PC 12 cells: contrast with Abeta(25-35) peptide and examination of underlying mechanisms

The toxic behaviour of the two shorter sequences of the native Abeta amyloid peptide required for cytotoxicity i.e., Abeta(31-35) and Abeta(25-35) peptides, was studied. We have shown that Abeta(31-35) peptide induces neurotoxicity in undifferentiated PC 12 cell via an apoptotic cell death pathway, including caspase activation and DNA fragmentation. Abeta(25-35) peptide, like the shorter amyloid peptide has the ability to induce neurotoxicity, as evaluated by the MTS reduction assay and by adherent cell count, but the Abeta(25-35) peptide-induced neurotoxicity is not associated with any biochemical features of apoptosis. The differences observed between the neurotoxic properties of Abeta(31-35) and Abeta(25-35) peptides might result on their different ability to be internalised within the neuronal cells. Furthermore, this study reveals that the redox state of methionine residue, C-terminal in Abeta(31-35) and Abeta(25-35) peptides affect in a different way the toxic behaviour of these two short amyloid fragments. Taken together our results suggest that Abeta(31-35) peptide induces cell death by apoptosis, unlike the Abeta(25-35) peptide and that role played by methionine-35 in Abeta induced neurotoxicity might be related to the Abeta aggregation state.     

The spirostenol (22R, 25R)-20alpha-spirost-5-en-3beta-yl hexanoate blocks mitochondrial uptake of Abeta in neuronal cells and prevents Abeta-induced impairment of mitochondrial function

Abeta(1-42) has been shown to uncouple the mitochondrial respiratory chain and promote the opening of the membrane permeability transition (MPT) pore, leading to cell death. We have previously reported that the spirostenol derivative (22R, 25R)-20alpha-spirost-5-en-3beta-yl hexanoate (SP-233) protects neuronal cells against Abeta(1-42) toxicity by binding to and inactivating the peptide. Picomolar concentrations of Abeta(1-42) decreased the mitochondrial respiratory coefficient in mitochondria isolated from the rat forebrain, and this decrease was partially reversed by SP-233. SP-233 abolished the uncoupling of oxidative phosphorylation induced by carbonyl cyanide 3-chlorophenylhydrazone on isolated mitochondria. These results are consistent with a direct effect of SP-233 on the MPT. Moreover, SP-233 displayed a neuroprotective effect on SK-N-AS human neuroblastoma cells treated with the MPT promoter, phenylarsine oxide. Treatment of SK-N-AS cells with Abeta(1-42) resulted in an accumulation of the peptide in the mitochondrial matrix; SP-233 completely scavenged Abeta(1-42) from the matrix. In addition, SP-233 protected the cells against mitochondrial toxins targeting complexes IV and V of the respiratory chain. These results indicate that Abeta(1-42) and SP-233 exert direct effects on mitochondrial function and SP-233 protects neuronal cells against Abeta-induced toxicity by targeting Abeta directly.     

Neurotoxic Abeta peptides increase oxidative stress in vivo through NMDA-receptor and nitric-oxide-synthase mechanisms, and inhibit complex IV activity and induce a mitochondrial permeability transition in vitro

Beta amyloid (Abeta) peptides accumulate in Alzheimer's disease and are neurotoxic possibly through the production of oxygen free radicals. Using brain microdialysis we characterized the ability of Abeta to increase oxygen radical production in vivo. The 1-40 Abeta fragment increased 2,3-dehydroxybenzoic acid efflux more than the 1-28 fragment, in a manner dependent on nitric oxide synthase and NMDA receptor channels. We then examined the effects of Abeta peptides on mitochondrial function in vitro. Induction of the mitochondrial permeability transition in isolated rat liver mitochondria by Abeta(25-35) and Abeta(35-25) exhibited dose dependency and required calcium and phosphate. Cyclosporin A prevented the transition as did ruthenium red, chlorpromazine, or N-ethylmaleimide. ADP and magnesium delayed the onset of mitochondrial permeability transition. Electron microscopy confirmed the presence of Abeta aggregates and swollen mitochondria and preservation of mitochondrial structure by inhibitors of mitochondrial permeability transition. Cytochrome c oxidase (COX) activity was selectively inhibited by Abeta(25-35) but not by Abeta(35-25). Neurotoxic Abeta peptide can increase oxidative stress in vivo through mechanisms involving NMDA receptors and nitric oxide sythase. Increased intracellular Abeta levels can further exacerbate the genetically driven complex IV defect in sporadic Alzheimer's disease and may precipitate mitochondrial permeability transition opening. In combination, our results provide potential mechanisms to support the feed-forward hypothesis of Abeta neurotoxicity.     

Enhanced G-protein activation by a mixture of Abeta(25-35), Abeta(1-40/42) and zinc

Beta-amyloid peptides (Abetas) bind to several G-protein coupled receptor proteins and stimulate GTPase activity in neurons. In this study we determined the effects of Abeta(1-42), Abeta(1-40), Abeta(25-35) and their mixtures on [(35)S]GTP binding in rat brain cortical membranes in the absence and presence of zinc. We found that the Abetas alone induced a concentration-dependent activation of G-proteins (IC50 approximately 10(-6) m), while aggregated Abeta fibrils only affected GTP binding at concentrations above 10(-5) m. Mixing Abeta(25-35) with Abeta(1-42) or Abeta(1-40) induced a several-fold increase in GTP-binding. This potentiation followed a bell shaped curve with a maximum at 50 : 50 ratios. No potentiating effect could be seen by mixing Abeta(1-40) and Abeta(1-42) or highly aggregated Abetas. Zinc had no effect on Abeta(1-40/42) but strongly potentiated the Abeta(25-35) or the mixed peptides-induced GTP-binding. Changes in secondary structure accompanied the mixed peptides or the peptide/zinc complexes induced potentiation, revealing that structural alterations are behind the increased biological action. These concentration dependent potentiating effects of zinc and the peptide mixtures could be physiologically important at brain regions where peptide fragments and/or zinc are present at elevated concentrations.     

Catechin and epicatechin from Smilacis chinae rhizome protect cultured rat cortical neurons against amyloid beta protein (25-35)-induced neurotoxicity through inhibition of cytosolic calcium elevation

We previously reported that the Smilacis chinae rhizome inhibits amyloid beta protein (25-35) (Abeta (25-35))-induced neurotoxicity in cultured rat cortical neurons. Here, we isolated catechin and epicatechin from S. chinae rhizome and also studied their neuroprotective effects on Abeta (25-35)-induced neurotoxicity in cultured rat cortical neurons. Catechin and epicatechin inhibited 10 microM Abeta (25-35)-induced neuronal cell death at a concentration of 10 microM, which was measured by a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay and Hoechst 33342 staining. Catechin and epicatechin inhibited 10 microM Abeta (25-35)-induced elevation of cytosolic calcium concentration ([Ca2+]c), which was measured by a fluorescent dye, Fluo-4 AM. Catechin and epicatechin also inhibited glutamate release into medium induced by 10 microM Abeta (25-35), which was measured by HPLC, generation of reactive oxygen species (ROS) and activation of caspase-3. These results suggest that catechin and epicatechin prevent Abeta (25-35)-induced neuronal cell damage by interfering with the increase of [Ca2+]c, and then by inhibiting glutamate release, generation of ROS and caspase-3 activity. Furthermore, these effects of catechin and epicatechin may be associated with the neuroprotective effect of the S. chinae rhizome.     

Interaction between Alzheimer's Abeta(25-35) peptide and phospholipid bilayers: the role of cholesterol

There is mounting evidence that the lipid matrix of neuronal cell membranes plays an important role in the accumulation of beta-amyloid peptides into senile plaques, one of the hallmarks of Alzheimer's disease (AD). With the aim to clarify the molecular basis of the interaction between amyloid peptides and cellular membranes, we investigated the interaction between a cytotoxic fragment of Abeta(1-42), i.e., Abeta(25-35), and phospholipid bilayer membranes. These systems were studied by Electron Paramagnetic Resonance (EPR) spectroscopy, using phospholipids spin-labeled on the acyl chain. The effect of inclusion of charged phospholipids or/and cholesterol in the bilayer composition was considered in relation to the peptide/membrane interaction. The results show that Abeta(25-35) inserts in bilayers formed by the zwitterionic phospholipid dilauroyl phosphatidylcholine (DLPC), positioning between the outer part of the hydrophobic core and the external hydrophilic layer. This process is not significantly influenced by the inclusion of the anionic phospholipid phosphatidylglycerol (DLPG) in the bilayer, indicating the peptide insertion to be driven by hydrophobic rather than electrostatic interactions. Cholesterol plays a fundamental role in regulating the peptide/membrane association, inducing a membrane transition from a fluid-disordered to a fluid-ordered phase. At low cholesterol content, in the fluid-disordered phase, the insertion of the peptide in the membrane causes a displacement of cholesterol towards the more external part of the membrane. The crowding of cholesterol enhances its rigidifying effect on this region of the bilayer. Finally, the cholesterol-rich fluid-ordered membrane looses the ability to include Abeta(25-35).     

Abeta(25-35) and Abeta(1-40) act on different calcium channels in CA1 hippocampal neurons

The acute effects of beta-amyloid (25-35) and (1-40) on high voltage activated calcium channels were compared in CA1 pyramidal cells of adult mouse hippocampal slices using the whole-cell patch-clamp recording. Bath application of oligomeric beta-amyloid (25-35) reversibly increased the barium current (I(Ba)) to 1.61 (normalized amplitude), while oligomeric beta-amyloid (1-40) reversibly enhanced the I(Ba) to 1.74. Reverse-sequence beta-amyloid [(35-25) and (40-1)] had no effect. The effect of beta-amyloid (25-35) was blocked by nifedipine, a selective antagonist of L-type calcium channels. In contrast, the effect of beta-amyloid (1-40) was not blocked by nifedipine and I(Ba) was enhanced to 4.96. It is concluded that these oligomeric peptides may act through different types of calcium channels and/or receptors. The toxicity of Abeta(25-35) implicates a potentiation of L-type calcium channels while the one of Abeta(1-40) is related to an increase of non-L-type calcium channels, which may involve an increase in transmitter release.     

杏仁核注射Aβ 25-35后大鼠脑内细胞周期蛋白、tau蛋白和Bax蛋白的异常表达

近年来研究发现,阿尔茨海默病(Alzheimer′s disease,AD)病人脑内神经元细胞周期相关蛋白的异常表达与AD相关病理改变存在关联。为探讨β-淀粉样蛋白(β—amyloid,Aβ)的毒性作用能否导致成年脑神经元表达细胞周期相关蛋白,以及细胞周期相关蛋白表达与神经损伤之间的关系,我们运用免疫组化、积分光密度分析等方法对Aβ25-35多肽片段单侧杏仁核注射的大鼠脑进行了研究。结果显示,Aβ25-35注射的大鼠脑内除了有与神经纤维缠结相关的磷酸化tau蛋白和凋亡相关蛋白Bax蛋白水平增加外,术后7d细胞周期相关蛋白cyclin A和cyclin B1蛋白在神经元内异常表达,但术后21d时cyclin A的表达有所降低,而cyclin B1在脑内神经元中已检测不到;免疫荧光双标结果显示Aβ25-35注射后7d的大鼠脑内有较多的cyclin B1和Bax、cyclin B1和磷酸化tau蛋白共存的神经元,而Bax与磷酸化tau蛋白阳性信号很少共存在同一细胞上。以上结果提示,Aβ可导致成年脑神经元表达细胞周期相关蛋白,这些神经元可能会通过与Bax相关的凋亡途径死亡,或首先导致与AD神经纤维缠结相关的tau蛋白磷酸化。     

Membrane interactions and the effect of metal ions of the amyloidogenic fragment Abeta(25-35) in comparison to Abeta(1-42)

Abeta(1-42) peptide, found as aggregated species in Alzheimer's disease brain, is linked to the onset of Alzheimer's disease. Many reports have linked metals to inducing Abeta aggregation and amyloid plaque formation. Abeta(25-35), a fragment from the C-terminal end of Abeta(1-42), lacks the metal coordinating sites found in the full-length peptide and is neurotoxic to cortical cortex cell cultures. We report solid-state NMR studies of Abeta(25-35) in model lipid membrane systems of anionic phospholipids and cholesterol, and compare structural changes to those of Abeta(1-42). When added after vesicle formation, Abeta(25-35) was found to interact with the lipid headgroups and slightly perturb the lipid acyl-chain region; when Abeta(25-35) was included during vesicle formation, it inserted deeper into the bilayer. While Abeta(25-35) retained the same beta-sheet structure irrespective of the mode of addition, the longer Abeta(1-42) appeared to have an increase in beta-sheet structure at the C-terminus when added to phospholipid liposomes after vesicle formation. Since the Abeta(25-35) fragment is also neurotoxic, the full-length peptide may have more than one pathway for toxicity.     

Methionine 35 oxidation reduces toxic effects of the amyloid beta-protein fragment (31-35) on human red blood cell

Amyloid beta-peptide, the central constituent of senile plaques in Alzheimer's disease brain, has been shown to be a source of free radical oxidative stress that may lead to neurodegeneration. In particular, it is well known that oxidation of methionine 35, is strongly related to the pathogenesis of Alzheimer's disease, since it represents the residue in the beta-amyloid peptide most susceptible to oxidation "in vivo". In this study, the fragment 31-35 of the beta-amyloid peptide, which has a single methionine at residue 35, was used to investigate the influence of the oxidation state of methionine-35 on the beta-amyloid peptide (31-35) mediated cytotoxic effects. Because no extensive studies have yet addressed whether amyloid beta peptides-mediated toxic effects can occur in the absence of mitochondria, human red blood cells were used as cell model. Exposure of intact red blood cells to beta-amyloid peptide (31-35) induced a marked stimulation (approximately 45%) of the pentose phosphate pathway and a significant inhibition of the red cell enzyme catalase, compared with the results observed in control red blood cells. In contrast, exposure of red blood cells to the beta-amyloid peptide (31-35)-Met35OX i.e. in which the sulfur of methionine is oxidised to sulfoxide, induced a slight activation of PPP (approximately 19%), and an inhibition of catalase activity lower with respect to the results observed in beta-amyloid peptide (31-35)-treated red blood cells. Since the activities of red cell phosphofructokinase, glucose-6-phosphate dehydrogenase, glutathione peroxidase, glutathione reductase and the functionality of hemoglobin were not modified within the red cell following to beta-amyloid peptides exposure, it is likely that beta-amyloid (31-35)-catalase interaction may represent a selective toxic event. Together, these results support the hypothesis that Abeta peptide and the oxidative state of Met-35 may be involved in the mechanisms responsible of neurodegeneration in Alzheimer's disease.     

Role of fibrillar Abeta25-35 in the inflammation induced rat model with respect to oxidative vulnerability

The major pathological ramification of Alzheimer's disease (AD) is accumulation of beta-Amyloid (Abeta) peptides in the brain. An emerging therapeutic approach for AD is elimination of excessive Ass peptides and preventing its re-accumulation. Immunization is the most effective strategy in removing preexisting cerebral Abetas and improving the cognitive capacity as shown in transgenic mice model of AD. However, active immunization is associated with adverse effect such as encephalitis with perivascular inflammation and hemorrhage. Details about the mechanistic aspects of propagation of these toxic effects are matter of intense enquiry as this knowledge is essential for the understanding of the AD pathophysiology. The present work aimed to study the oxidative vulnerability in the plasma, liver and brain of the inflammation-induced rats subjected to Ass immunization. Induction of inflammation was performed by subcutaneous injection of 0.5?ml of 2% silver nitrate. Our present result shows that the proinflammatory cytokines such as IL1alpha and TNFalpha are increased significantly in the inflammation-induced, Abeta1-42, Abeta25-35 treated groups and inflammation with Abeta25-35 treated group when compared to control, complete Freund's adjuvant and Abeta35-25 treated groups. These increased proinflammatory cytokines concurrently releases significant amount of free radicals in the astrocytes of respected groups. The present result shows that nitric oxide (NO) level was significantly higher (P<0.001) in plasma, liver and brain of the rat subjected to inflammation, Abeta1-42, Abeta25-35 and inflammation with Abeta25-35 injected groups when compared to control. The increased level of lipid peroxides (LPO) (P<0.001) and decreased antioxidant status (P<0.05) were observed in the plasma, liver and brain of inflammation-induced group when compared to control. Our result shows that significant oxidative vulnerability was observed in the inflammation with Ass treated rats when compared to other groups. Based on our results, we suggest that immunization of AD patients with Ass should be done with caution as the increase in Ass could trigger the brain inflammation in uncontrollable level.     

Effects of amyloid-beta peptides on hydrogen peroxide-metabolizing enzymes in rat brain in vivo

Amyloid-beta (Abeta) peptides are components of senile plaques initiating degeneration of brain neurons in Alzheimer's disease. They increase reactive oxygen species generation that may exceed the defensive capacity of cells. To test the hypothesis, this study investigated the in vivo effects of Abeta peptides on mitochondrial and non-mitochondrial enzymic sources of reactive oxygen species and antioxidant enzymes in rat brain. Continuous intracerebroventricular infusion of both Abeta(25-35) and Abeta(1-40) for up to 14 days stimulated the hydrogen peroxide (H2O2) generation in isolated neocortex mitochondria. Infusion of Abeta(1-40) led to an increase in Mn-superoxide dismutase activity and a decrease in activities of catalase and glutathione peroxidase in mitochondria, to elevation of activities of Cu,Zn-superoxide dismutase and aldehyde oxidase, forwarded the conversion of xanthine dehydrogenase to xanthine oxidase and corresponding increase in the rate of H2O2 formation in the cytosol. Thus, Abeta peptides increase H2O2-formation and H2O2-forming enzyme activities and inhibit H2O2-consuming enzyme activities in mitochondria and cytosol in vivo. These studies suggest that disbalance between H2O2-generating and H2O2-metabolizing enzyme activities can contribute to oxidative stress underlying neurodegeneration and neuronal death in Alzheimer's disease.     

Synthesis and biological properties of beta-turned Abeta(31-35) constrained analogues

A series of constrained pentapeptide analogues of the fragment Abeta(31-35) has been prepared using solid phase synthesis protocols. The results of conformational studies and surface plasmon resonance (SPR) experiments seem to indicate that the affinity of these constrained analogues for immobilized Abeta(25-35) peptide could be related to their ability to adopt a Leu34N-Ile31O beta-turn-like folded conformation.     

The correlation between neurotoxicity, aggregative ability and secondary structure studied by sequence truncated Abeta peptides

Aggregated beta-amyloid (Abeta) peptides are neurotoxic and cause neuronal death both in vitro and in vivo. Although the formation of a beta-sheet structure is usual required to form aggregates, the relationship between neurotoxicity and the Abeta sequence remains unclear. To explore the correlation between Abeta sequence, secondary structure, aggregative ability, and neurotoxicity, we utilized both full-length and fragment-truncated Abeta peptides. Using a combination of spectroscopic and cellular techniques, we demonstrated that neurotoxicity and aggregative ability are correlated while the relationship between these characteristics and secondary structure is not significant. The hydrophobic C-terminus, particularly the amino acids of 17-21, 25-35, and 41-42, is the main region responsible for neurotoxicity and aggregation. Deleting residues 17-21, 25-35 or 41-42 significantly reduced the toxicity. On the other hand, truncation of the peptides at either residues 22-24 or residues 36-40 had little effect on toxicity and aggregative ability. While the N-terminal residues 1-16 may not play a major role in neurotoxicity and aggregation, a lack of N-terminal fragment Abeta peptide, (e.g. Abeta17-35), does not display the neurotoxicity of either full-length or 17-21, 25-35 truncated Abeta peptides.     

Single chain Fv antibodies against the 25-35 Abeta fragment inhibit aggregation and toxicity of Abeta42

Alzheimer's disease (AD) is characterized by the deposition of amyloid-beta (Abeta) protein in the brain. Immunization studies have demonstrated that anti-Abeta antibodies reduce Abeta deposition and improve clinical symptoms seen in AD. However, conventional antibody-based therapies risk an inflammatory response that can result in meningoencephalitis and cerebral hemorrhage. Here we report on the development of human-based single chain variable domain antibody fragments (scFvs) directed against the Abeta 25-35 region as potential therapeutics for AD that do not risk an inflammatory response. The 25-35 region of Abeta represents a promising therapeutic target since it promotes aggregation and is highly toxic. Two scFvs with differing affinities for Abeta were studied, and both inhibited aggregation of Abeta42 as determined by thioflavin T binding assay and atomic force microscopy analysis and blocked Abeta-induced toxicity toward human neuroblastoma SH-SY5Y cells as determined by MTT and LDH release assays. These results provide additional evidence that scFvs against Abeta provide an attractive alternative to more conventional antibody-based therapeutics for controlling aggregation and toxicity of Abeta.     

beta-Sheet structured beta-amyloid(1-40) perturbs phosphatidylcholine model membranes

The disruption of intracellular calcium homeostasis plays a central role in the pathology of Alzheimer's disease, which is also characterized by accumulation of the amyloid-beta peptides Abeta40 and Abeta42. These amphipathic peptides may become associated with neuronal membranes and affect their barrier function, resulting in the loss of calcium homeostasis. This suggestion has been extensively investigated by exposing protein-free model membranes, either vesicles or planar bilayers, to soluble Abeta. Primarily unstructured Abeta has been shown to undergo a membrane-induced conformational change to either primarily beta-structure or helical structure, depending, among other factors, on the model membrane composition. Association of Abeta renders lipid bilayers permeable to ions but there is dispute whether this is due to the formation of discrete transmembrane ion channels of Abeta peptides, or to a non-specific perturbation of bilayer integrity by lipid head group-associated Abeta. Here, we have attempted incorporation of Abeta in the hydrophobic core of zwitterionic bilayers, the most simple model membrane system, by preparing proteoliposomes by hydration of a mixed film of Abeta peptides and phosphatidylcholine (PC) lipids. Despite the use of a solvent mixture in which Abeta40 and Abeta42 are almost entirely helical, the Abeta analogs were beta-structured in the resulting vesicle dispersions. When Abeta40-containing vesicles were fused into a zwitterionic planar bilayer, the typical irregular "single channel-like" conductance of Abeta was observed. The maximum conductance increased with additional vesicle fusion, while still exhibiting single channel-like behavior. Supported bilayers formed from Abeta40/PC vesicles did not exhibit any channel-like topological features, but the bilayer destabilized in time. Abeta40 was present primarily as beta-sheets in supported multilayers formed from the same vesicles. The combined observations argue for a non-specific perturbation of zwitterionic bilayers by surface association of small amphipathic Abeta40 assemblies.     

Secreted Abeta does not mediate neurotoxicity by antibody-stimulated amyloid precursor protein

Antibodies against APP, a precursor of Abeta deposited in Alzheimer's disease brain, have been shown to cause neuronal death. Therefore, it is important to determine whether Abeta mediates antibody-induced neurotoxicity. When primary neurons were treated with anti-APP antibodies, Abeta40 and Abeta42 in the cultured media were undetectable by an assay capable of detecting 100 nM Abeta peptides. However, exogenously treated Abeta1-42 or Abeta1-43 required >3 microM to exert neurotoxicity, and 25 microM Abeta1-40 was not neurotoxic. Glutathione-ethyl-ester inhibited neuronal death by anti-APP antibody, but not death by Abeta1-42, whereas serum attenuated toxicity by Abeta1-42, but not by anti-APP antibody. Using immortalized neuronal cells, we specified the domain responsible for toxicity to be cytoplasmic His(657)-Lys(676), but not the Abeta1-42 region, of APP. This indicates that neuronal cell death by anti-APP antibody is not mediated by secreted Abeta.     

Genistein ameliorates beta-amyloid peptide (25-35)-induced hippocampal neuronal apoptosis

beta-Amyloid protein (Abeta), a major component of senile plaques of Alzheimer's disease (AD) brain, causes elevation of the intracellular free Ca2+ level and the production of robust free radicals, both of which contribute greatly to the AD-associated cascade including severe neuronal loss in the hippocampus. Genistein, the most active molecule of soy isoflavones, protects diverse kinds of cells from damage caused by a variety of toxic stimuli. In the present study, we investigated the neuroprotective effect of genistein against Abeta25-35-induced apoptosis in cultured hippocampal neurons, as well as the underlying mechanism. Abeta25-35-induced apoptosis, characterized by decreased cell viability, neuronal DNA condensation, and fragmentation, is associated with an increase in intracellular free Ca2+ level, the accumulation of reactive oxygen species (ROS), and the activation of caspase-3. All these phenotypes induced by Abeta25-35 are reversed by genistein. Our results further show that at the nanomolar (100 nM) level, genistein protects neurons from Abeta25-35-induced damage largely via the estrogen receptor-mediated pathway, and at the micromolar (40 microM) level, the neuroprotective effect of genistein is mediated mainly by its antioxidative properties. Our data suggest that genistein attenuates neuronal apoptosis induced by Abeta25-35 via various mechanisms.     

Salvianolic acid B, an antioxidant from Salvia miltiorrhiza, prevents Abeta(25-35)-induced reduction in BPRP in PC12 cells

Several lines of evidence support that beta-amyloid (Abeta)-induced neurotoxicity is mediated through the generation of reactive oxygen species (ROS) and elevation of intracellular calcium. Salvianolic acid B (Sal B), the major and most active anti-oxidant from Salvia miltiorrhiza, protects diverse kinds of cells from damage caused by a variety of toxic stimuli. In the present study, we investigated the effects of Sal B against beta-amyloid peptide 25-35 (Abeta(25-35))-induced neurotoxicity, focused mainly on the neurotoxic effects of Abeta(25-35) and the neuroprotective effects of Sal B on the expression of brain-pancreas relative protein (BPRP), which is a new protein and mainly expressed in brain and pancreas. Following exposure of PC12 cells to 20 microM Abeta(25-35), a marked reduction in the expression of BPRP was observed, accompanied with decreased cell viability and increased cell apoptosis, as well as increased ROS production and calcium influx. Treatment of the PC12 cells with Sal B significantly reversed the expression of BPRP and cell viability while it decreased ROS production and intracellular calcium. These data indicate that Abeta(25-35) decreases the expression of BPRP via enhanced formation of intracellular ROS and increased intracellular calcium, and that Sal B, as an anti-oxidant, protects against Abeta(25-35)-induced reduction in expression of BPRP through its effects on suppressing the production of ROS, calcium flux, and apoptosis. However, the role(s) of BPRP in AD and the definite mechanisms by which Sal B protects against Abeta(25-35)-induced reduction in the expression of BPRP require further study.     

Lipid-induced conformational transition of the amyloid core fragment Abeta(28-35) and its A30G and A30I mutants

The interaction of the beta-amyloid peptide (Abeta) with neuronal membranes could play a key role in the pathogenesis of Alzheimer's disease. Recent studies have focused on the interactions of Abeta oligomers to explain the neuronal toxicity accompanying Alzheimer's disease. In our study, we have investigated the role of lipid interactions with soluble Abeta(28-35) (wild-type) and its mutants A30G and A30I in their aggregation and conformational preferences. CD and Trp fluorescence spectroscopic studies indicated that, immediately on dissolution, these peptides adopted a random coil structure. Upon addition of negatively charged 1,2-dipalmitoyl-syn-glycero-3-phospho-rac-(glycerol) sodium salt (PG) lipid, the wild-type and A30I mutant underwent reorganization into a predominant beta-sheet structure. However, no conformational changes were observed in the A30G mutant on interaction with PG. In contrast, the presence of zwitterionic 1,2-dipalmitoyl-syn-glycero-3-phosphatidylcholine (PC) lipid had no effect on the conformation of these three peptides. These observations were also confirmed with atomic force microscopy and the thioflavin-T assay. In the presence of PG vesicles, both the wild-type and A30I mutant formed fibrillar structures within 2 days of incubation in NaCl/P(i), but not in their absence. Again, no oligomerization was observed with PC vesicles. The Trp studies also revealed that both ends of the three peptides are not buried deep in the vesicle membrane. Furthermore, fluorescence spectroscopy using the environment-sensitive probe 1,6-diphenyl-1,3,5-hexatriene showed an increase in the membrane fluidity upon exposure of the vesicles to the peptides. The latter effect may result from the lipid head group interactions with the peptides. Fluorescence resonance energy transfer experiments revealed that these peptides undergo a random coil-to-sheet conversion in solution on aging and that this process is accelerated by negatively charged lipid vesicles. These results indicate that aggregation depends on hydrophobicity and propensity to form beta-sheets of the amyloid peptide, and thus offer new insights into the mechanism of amyloid neurodegenerative disease.