Protective effect of melatonin on beta-amyloid-induced apoptosis in rat astroglioma C6 cells and its mechanism

Astrocytosis is a common feature of amyloid plaques. The Abeta-astrocyte interaction produces a detrimental effect on neurons, which may contribute to neurodegeneration in Alzheimer disease (AD). The regulation of astrocyte apoptosis is essential to physiological and pathological processes in the CNS. Melatonin is a potent antioxidant and free radical scavenger. Previously, we showed that melatonin alleviated the learning and memory deficits in the APP 695 transgenic mouse model of AD. In this study, the importance of melatonin in the management of Abeta-induced apoptosis in an astrocyte-like cell is discussed. We found that rat astroglioma C6 cells treated with Abeta25-35 or Abeta1-42 undergo apoptosis and that melatonin pretreatment at 10(-5), 10(-6), and 10(-7) M significantly attenuates Abeta25-35- or Abeta1-42-induced apoptosis. The antiapoptotic effects of melatonin were extremely reproducible and corroborated by multiple quantitative methods, including an MTT cell viability assay, Hoechst 33342 nuclei staining, DNA fragmentation analysis, and flow cytometric analysis. In addition, melatonin effectively suppressed Abeta1-42-induced nitric oxide formation, remarkably prevented Abeta1-40-induced intracellular calcium overload, and significantly alleviated Abeta1-40-induced membrane rigidity. Our results demonstrate that, in addition to the beneficial effects of providing direct antioxidant protection to neurons, melatonin may enhance neuroprotection against Abeta-induced neurotoxicity by promoting the survival of glial cells.     

Mixtures of wild-type and a pathogenic (E22G) form of Abeta40 in vitro accumulate protofibrils,including amyloid pores

Although APP mutations associated with inherited forms of Alzheimer's disease (AD) are relatively rare, detailed studies of these mutations may prove critical for gaining important insights into the mechanism(s) and etiology of AD. Here, we present a detailed biophysical characterization of the structural properties of protofibrils formed by the Arctic variant (E22G) of amyloid-beta protein (Abeta40(ARC)) as well as the effect of Abeta40(WT) on the distribution of the protofibrillar species formed by Abeta40(ARC) by characterizing biologically relevant mixtures of both proteins that may mimic the situation in the heterozygous patients. These studies revealed that the Arctic mutation accelerates both Abeta oligomerization and fibrillogenesis in vitro. In addition, Abeta40(ARC) was observed to affect both the morphology and the size distribution of Abeta protofibrils. Electron microscopy examination of the protofibrils formed by Abeta40(ARC) revealed several morphologies, including: (1) relatively compact spherical particles roughly 4-5 nm in diameter; (2) annular pore-like protofibrils; (3) large spherical particles 18-25 nm in diameter; and (4) short filaments with chain-like morphology. Conversion of Abeta40(ARC) protofibrils to fibrils occurred more rapidly than protofibrils formed in mixed solutions of Abeta40(WT)/Abeta40(ARC), suggesting that co-incubation of Abeta40(ARC) with Abeta40(WT) leads to kinetic stabilization of Abeta40(ARC) protofibrils. An increase in the ratio of Abeta(WT)/Abeta(MUT(Arctic)), therefore, may result in the accumulation of potential neurotoxic protofibrils and acceleration of disease progression in familial Alzheimer's disease mutation carriers.     

The neuroprotective role of melatonin against amyloid beta peptide injected mice

Widespread cerebral deposition of a 40-42 amino acid peptide called amyloid beta peptide (A beta) in the form of amyloid fibrils is one of the most prominent neuropathologic features of Alzheimer's disease (AD). The clinical study provides evidence that accumulation of protofibrils due to the Arctic mutation (E22G) causes early AD onset. Melatonin showed beneficial effects in an AD mouse model. Mice were divided into four different groups (n=8 per group): (i) control group, (ii) scrambled A beta-injected group, (iii) A beta protofibril-injected group and (iv) melatonin-treated group. A single dose of (5 microg) A beta protofibril was administered to the A beta protofibril-injected and melatonin-treated groups via intracerebroventricular injections. The results demonstrate that melatonin treatment significantly reduces A beta protofibril-induced reactive oxygen species (ROS) production, intracellular calcium levels and acetylcholinesterase activity in the neocortex and hippocampus regions. Based on these findings it is suggested that melatonin therapy might be a useful treatment for AD patients.     

Protective effects of insulin-like growth factor-I on the somatostatinergic system in the temporal cortex of beta-amyloid-treated rats

Insulin-like growth factor-I (IGF-I) has protective effects against beta-amyloid (Abeta)-induced neuronal cell death. Because alterations of the somatostatinergic system have been described in Alzheimer's disease, we investigated the effects of the Abeta peptide and the possible protective role of IGF-I on the somatostatinergic system of the rat temporal cortex and on cell death and phosphorylated (p)-Akt levels in this area. Abeta25-35 was administered intracerebroventricularly to male rats via an osmotic minipump over 14 days (300 pmol/day). Another group received a subcutaneous IGF-I infusion (50 microg/kg/day), concomitant with Abeta25-35 administration, whereas a third group received IGF-I alone. Abeta25-35 significantly decreased the somatostatin (SRIF)-like immunoreactive content and the SRIF receptor density, as a result of a decrease in the levels of the SRIF receptor subtype 2. The inhibitory effect of SRIF on adenylyl cyclase activity was significantly lower after Abeta25-35 infusion, whereas the levels of the inhibitory G protein subunit Gialpha1, Gialpha2 or Gialpha3 were unaltered. Cell death was increased and p-Akt levels decreased in Abeta25-35-treated animals. IGF-I administration increased immunoreactive IGF-I levels in the temporal cortex and restored all parameters affected by Abeta25-35 to baseline values. These findings suggest that IGF-I prevents the deleterious effect of Abeta25-35 on the somatostatinergic system.     

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.     

Sensitive ELISA detection of amyloid-beta protofibrils in biological samples

Amyloid-beta (Abeta) protofibrils are known intermediates of the in vitro Abeta aggregation process and the protofibrillogenic Arctic mutation (APPE693G) provides clinical support for a pathogenic role of Abeta protofibrils in Alzheimer's disease (AD). To verify their in vivo relevance and to establish a quantitative Abeta protofibril immunoassay, Abeta conformation dependent monoclonal antibodies were generated. One of these antibodies, mAb158 (IgG2a), was used in a sandwich ELISA to specifically detect picomolar concentrations of Abeta protofibrils without interference from Abeta monomers or the amyloid precursor protein (APP). The specificity and biological significance of this ELISA was demonstrated using cell cultures and transgenic mouse models expressing human APP containing the Swedish mutation (APPKN670/671ML), or the Swedish and Arctic mutation in combination. The mAb158 sandwich ELISA analysis revealed presence of Abeta protofibrils in both cell and animal models, proving that Abeta protofibrils are formed not only in vitro, but also in vivo. Furthermore, elevated Abeta protofibril levels in the Arctic-Swedish samples emphasize the usefulness of the Arctic mutation as a model of enhanced protofibril formation. This assay provides a novel tool for investigating the role of Abeta protofibrils in AD and has the potential of becoming an important diagnostic assay.     

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。     

Vaccination with soluble Aβ oligomers generates toxicity-neutralizing antibodies

In recent studies of transgenic models of Alzheimer's disease (AD), it has been reported that antibodies to aged beta amyloid peptide 1-42 (Abeta(1-42)) solutions (mixtures of Abeta monomers, oligomers and amyloid fibrils) cause conspicuous reduction of amyloid plaques and neurological improvement. In some cases, however, neurological improvement has been independent of obvious plaque reduction, and it has been suggested that immunization might neutralize soluble, non-fibrillar forms of Abeta. It is now known that Abeta toxicity resides not only in fibrils, but also in soluble protofibrils and oligomers. The current study has investigated the immune response to low doses of Abeta(1-42) oligomers and the characteristics of the antibodies they induce. Rabbits that were injected with Abeta(1-42) solutions containing only monomers and oligomers produced antibodies that preferentially bound to assembled forms of Abeta in immunoblots and in physiological solutions. The antibodies have proven useful for assays that can detect inhibitors of oligomer formation, for immunofluorescence localization of cell-attached oligomers to receptor-like puncta, and for immunoblots that show the presence of SDS-stable oligomers in Alzheimer's brain tissue. The antibodies, moreover, were found to neutralize the toxicity of soluble oligomers in cell culture. Results support the hypothesis that immunizations of transgenic mice derive therapeutic benefit from the immuno-neutralization of soluble Abeta-derived toxins. Analogous immuno-neutralization of oligomers in humans may be a key in AD vaccines.     

Mercury induces cell cytotoxicity and oxidative stress and increases beta-amyloid secretion and tau phosphorylation in SHSY5Y neuroblastoma cells

Concentrations of heavy metals, including mercury, have been shown to be altered in the brain and body fluids of Alzheimer's disease (AD) patients. To explore potential pathophysiological mechanisms we used an in vitro model system (SHSY5Y neuroblastoma cells) and investigated the effects of inorganic mercury (HgCl2) on oxidative stress, cell cytotoxicity, beta-amyloid production, and tau phosphorylation. We demonstrated that exposure of cells to 50 microg/L (180 nM) HgCl2 for 30 min induces a 30% reduction in cellular glutathione (GSH) levels (n = 13, p<0.001). Preincubation of cells for 30 min with 1 microM melatonin or premixing melatonin and HgCl2 appeared to protect cells from the mercury-induced GSH loss. Similarly, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assays revealed that 50 microg/L HgCl2 for 24 h produced a 50% inhibition of MTT reduction (n = 9, p<0.001). Again, melatonin preincubation protected cells from the deleterious effects of mercury, resulting in MTT reduction equaling control levels. The release of beta-amyloid peptide (Abeta) 1-40 and 1-42 into cell culture supernatants after exposure to HgCl2 was shown to be different: Abeta 1-40 showed maximal (15.3 ng/ml) release after 4 h, whereas Abeta 1-42 showed maximal (9.3 ng/ml) release after 6 h of exposure to mercury compared with untreated controls (n = 9, p<0.001). Preincubation of cells with melatonin resulted in an attenuation of Abeta 1-40 and Abeta 1-42 release. Tau phosphorylation was significantly increased in the presence of mercury (n = 9, p<0.001), whereas melatonin preincubation reduced the phosphorylation to control values. These results indicate that mercury may play a role in pathophysiological mechanisms of AD.     

Demonstration of an anti-oxidative stress mechanism of quetiapine: implications for the treatment of Alzheimer's disease

We have shown that quetiapine, a new antipsychotic drug, protects cultured cells against oxidative stress-related cytotoxicities induced by amyloid beta (Abeta)25-35, and that quetiapine prevents memory impairment and decreases Abeta plaques in the brains of amyloid precursor protein (APP)/presenilin-1 (PS-1) double-mutant mice. The aim of this study was to understand why quetiapine has these protective effects. Because the cytotoxicity of both Abeta(25-35) and Abeta(1-40) requires fibril formation, our first experiments determined the effect of quetiapine on Abeta(25-35) aggregation. Quetiapine inhibited Abeta(25-35) aggregation in cell-free aqueous solutions and blocked the fibrillar aggregation of Abeta(25-35), as observed under an electron microscope. We then investigated why quetiapine inhibits Abeta(25-35) aggregation. During the aggregation of Abeta(25-35), a hydroxyl radical (OH*) was released, which in turn amplified Abeta(25-35) aggregation. Quetiapine blocked OH*-induced Abeta(25-35) aggregation and scavenged the OH* produced in the Fenton system and in the Abeta(25-35) solution, as analyzed using electron paramagnetic resonance spectroscopy. Furthermore, new compounds formed by quetiapine and OH* were observed in MS analysis. Finally, we applied Abeta(25-35) to PC12 cells to observe the effect of quetiapine on living cells. Abeta(25-35) increased levels of intracellular reactive oxygen species and calcium in PC12 cells and caused cell death, but these toxic effects were prevented by quetiapine. These results demonstrate an anti-oxidative stress mechanism of quetiapine, which contributes to its protective effects observed in our previous studies and explains the effectiveness of this drug for Alzheimer's disease patients with psychiatric and behavioral complications.     

A novel tricyclic pyrone compound ameliorates cell death associated with intracellular amyloid-beta oligomeric complexes

The neurotoxicity of amyloid-beta protein (Abeta) is widely regarded as one of the fundamental causes of neurodegeneration in Alzheimer's disease (AD). This toxicity is related to Abeta aggregation into oligomers, protofibrils and fibrils. Recent studies suggest that intracellular Abeta, which causes profound toxicity, could be one of the primary therapeutic targets in AD. So far, no compounds targeting intracellular Abeta have been identified. We have investigated the toxicity induced by intracellular Abeta in a neuroblastoma MC65 line and found that it was closely related to intracellular accumulation of oligomeric complexes of Abeta (Abeta-OCs). We further identified a cell-permeable tricyclic pyrone named CP2 that ameliorates this toxicity and significantly reduces the levels of Abeta-OCs. In aqueous solution, CP2 attenuates Abeta oligomerization and prevents the oligomer-induced death of primary cortical neurons. CP2 analogs represent a new class of promising compounds for the amelioration of Abeta toxicities within both intracellular and extracellular sites.     

Unique physicochemical profile of beta-amyloid peptide variant Abeta1-40E22G protofibrils: conceivable neuropathogen in arctic mutant carriers

A new early-onset form of Alzheimer's disease (AD) was described recently where a point mutation was discovered in codon 693 of the beta-amyloid (Abeta) precursor protein gene, the Arctic mutation. The mutation translates into a single amino acid substitution, glutamic acid-->glycine, in position 22 of the Abeta peptide. The mutation carriers have lower plasma levels of Abeta than normal, while in vitro studies show that Abeta1-40E22G protofibril formation is significantly enhanced. We have explored the nature of the Abeta1-40E22G peptide in more detail, in particular the protofibrils. Using size-exclusion chromatography (SEC) and circular dichroism spectroscopy (CD) kinetic and secondary structural characteristics were compared with other Abeta1-40 peptides and the Abeta12-28 fragment, all having single amino acid substitutions in position 22. We have found that Abeta1-40E22G protofibrils are a group of comparatively stabile beta-sheet-containing oligomers with a heterogeneous size distribution, ranging from >100 kDa to >3000 kDa. Small Abeta1-40E22G protofibrils are generated about 400 times faster than large ones. Salt promotes their formation, which significantly exceeds all the other peptides studied here, including the Dutch mutation Abeta1-40E22Q. Position 22 substitutions had significant effects on aggregation kinetics of Abeta1-40 and in Abeta12-28, although the qualitative aspects of the effects differed between the native peptide and the fragment, as no protofibrils were formed by the fragments. The rank order of protofibril formation of Abeta1-40 and its variants was the same as the rank order of the length of the nucleation/lag phase of the Abeta12-28 fragments, E22V>E22A?E22G>E22Q?E22, and correlated with the degree of hydrophobicity of the position 22 substituent. The molecular mass of peptide monomers and protofibrils were estimated better in SEC studies using linear rather than globular calibration standards. The characteristics of the Abeta1-40E22G suggest an important role for the peptide in the neuropathogenesis in the Arctic form of AD.     

Radioiodinated clioquinol as a biomarker for beta-amyloid: Zn complexes in Alzheimer's disease

Neocortical beta-amyloid (Abeta) aggregates in Alzheimer's disease (AD) are enriched in transition metals that mediate assembly. Clioquinol (CQ) targets metal interaction with Abeta and inhibits amyloid pathology in transgenic mice. Here, we investigated the binding properties of radioiodinated CQ ([(125)I]CQ) to different in vitro and in vivo Alzheimer models. We observed saturable binding of [(125)I]CQ to synthetic Abeta precipitated by Zn(2+) (K(d)=0.45 and 1.40 nm for Abeta(1-42) and Abeta(1-40), respectively), which was fully displaced by free Zn(2+), Cu(2+), the chelator DTPA (diethylene triamine pentaacetic acid) and partially by Congo red. Sucrose density gradient of post-mortem AD brain indicated that [(125)I]CQ concentrated in a fraction enriched for both Abeta and Zn, which was modulated by exogenous addition of Zn(2+) or DTPA. APP transgenic (Tg2576) mice injected with [(125)I]CQ exhibited higher brain retention of tracer compared to non-Tg mice. Autoradiography of brain sections of these animals confirmed selective [(125)I]CQ enrichment in the neocortex. Histologically, both thioflavine-S (ThS)-positive and negative structures were labeled by [(125)I]CQ. A pilot SPECT study of [(123)I]CQ showed limited uptake of the tracer into the brain, which did however, appear to be more rapid in AD patients compared to age-matched controls. These data support metallated Abeta species as the neuropharmacological target of CQ and indicate that this drug class may have potential as in vivo imaging agents for Alzheimer neuropathology.     

Stabilization of the cyclin-dependent kinase 5 activator,p35, by paclitaxel decreases beta-amyloid toxicity in cortical neurons

One hallmark of Alzheimer's disease (AD) is the formation of neurofibrillary tangles, aggregated paired helical filaments composed of hyperphosphorylated tau. Amyloid-beta (Abeta) induces tau hyperphosphorylation, decreases microtubule (MT) stability and induces neuronal death. MT stabilizing agents have been proposed as potential therapeutics that may minimize Abeta toxicity and here we report that paclitaxel (taxol) prevents cell death induced by Abeta peptides, inhibits Abeta-induced activation of cyclin-dependent kinase 5 (cdk5) and decreases tau hyperphosphorylation. Taxol did not inhibit cdk5 directly but significantly blocked Abeta-induced calpain activation and decreased formation of the cdk5 activator, p25, from p35. Taxol specifically inhibited the Abeta-induced activation of the cytosolic cdk5-p25 complex, but not the membrane-associated cdk5-p35 complex. MT-stabilization was necessary for neuroprotection and inhibition of cdk5 but was not sufficient to prevent cell death induced by overexpression of p25. As taxol is not permeable to the blood-brain barrier, we assessed the potential of taxanes to attenuate Abeta toxicity in adult animals using a succinylated taxol analog (TX67) permeable to the blood-brain barrier. TX67, but not taxol, attenuated the magnitude of both basal and Abeta-induced cdk5 activation in acutely dissociated cortical cultures prepared from drug treated adult mice. These results suggest that MT-stabilizing agents may provide a therapeutic approach to decrease Abeta toxicity and neurofibrillary pathology in AD and other tauopathies.     

The oxidative stress metabolite 4-hydroxynonenal promotes Alzheimer protofibril formation

4-Hydroxynonenal (4-HNE), formed as a consequence of oxidative stress, exists at increased concentrations in Alzheimer's disease (AD) patients and is found in amyloid beta peptide (Abeta) plaques associated with AD. Although it remains an open question as to whether oxidative stress is a causative factor or a consequence of AD, we show here that 4-HNE, putatively resulting from the peroxidation of lipids, covalently modifies Abeta, triggering its aggregation. These Abeta modifications result from 1,4 conjugate addition and/or Schiff base formation, they occur at multiple locations on a single Abeta peptide, and they result in covalent cross-linking of Abeta peptides. The consequence of these reactions is that 4-HNE accelerates the formation of Abeta protofibrils while inhibiting the production of straight, mature fibrils. Recent studies implicating Abeta oligomers and protofibrils in the neurotoxic process that ultimately leads to AD suggest that the Abeta aggregates induced by 4-HNE may be important in the pathogenesis of AD. These results provide further incentive to understand the role of oxidative stress and small-molecule Abeta modifications in sporadic AD.     

Beta-amyloid treatment of two complementary P301L tau-expressing Alzheimer's disease models reveals similar deregulated cellular processes

Alzheimer's disease (AD) is characterized by Abeta peptide-containing plaques and tau-containing neurofibrillary tangles (NFTs). Both pathologies have been combined by crossing Abeta plaque-forming APP mutant mice with NFT-forming P301L tau mutant mice or by stereotaxically injecting beta-amyloid peptide 1-42 (Abeta42) into brains of P301L tau mutant mice. In cell culture, Abeta42 induces filamentous tau aggregates. To understand which processes are disrupted by Abeta42 in the presence of tau aggregates, we applied comparative proteomics to Abeta42-treated P301L tau-expressing neuroblastoma cells and the amygdala of P301L tau transgenic mice stereotaxically injected with Abeta42. Remarkably, a significant fraction of proteins altered in both systems belonged to the same functional categories, i.e. stress response and metabolism. We also identified model-specific effects of Abeta42 treatment such as differences in cell signaling proteins in the cellular model and of cytoskeletal and synapse associated proteins in the amygdala. By Western blotting (WB) and immunohistochemistry (IHC), we were able to show that 72% of the tested candidates were altered in human AD brain with a major emphasis on stress-related unfolded protein responsive candidates. These data highlight these processes as potentially important initiators in the Abeta42-mediated pathogenic cascade in AD and further support the role of unfolded proteins in the course of AD.     

Structural differences in Abeta amyloid protofibrils and fibrils mapped by hydrogen exchange--mass spectrometry with on-line proteolytic fragmentation

We report here structural differences between Abeta(1-40) protofibrils and mature amyloid fibrils associated with Alzheimer's disease as determined using hydrogen-deuterium exchange-mass spectrometry (HX-MS) coupled with on-line proteolysis. Specifically, we have identified regions of the Abeta(1-40) peptide containing backbone amide hydrogen atoms that are protected from HX or exposed when this peptide is incorporated into protofibrils or amyloid fibrils formed in phosphate-buffered saline without stirring at 37 degrees C. Study of protofibrils was facilitated by use of the protofibril-stabilizing agent calmidazolium chloride. Our data clearly show that both the C-terminal segment 35-40 and the N-terminal segment 1-19 are highly exposed to HX in both fibrils and protofibrils. In contrast, the internal fragment 20-34 is highly protected from exchange in fibrils but much less so in protofibrils. The data suggest that the beta-sheet elements comprising the amyloid fibril are already present in protofibrils, but that they are expanded into some adjacent residues upon the formation of mature amyloid. The N-terminal approximately ten residues appear to be unstructured in both protofibrils and fibrils. The 20-30 segment of Abeta(1-40) is more ordered in fibrils than in protofibrils, suggesting that, if protofibrils are a mechanistic precursor of fibrils, the transition from protofibril to fibril involves substantial ordering of this region of the Abeta peptide.     

Amyloid-beta protofibrils differ from amyloid-beta aggregates induced in dilute hexafluoroisopropanol in stability and morphology

The brains of Alzheimer's disease (AD) patients contain large numbers of amyloid plaques that are rich in fibrils composed of 40- and 42-residue amyloid-beta (Abeta) peptides. Several lines of evidence indicate that fibrillar Abeta and especially soluble Abeta aggregates are important in the etiology of AD. Recent reports also stress that amyloid aggregates are polymorphic and that a single polypeptide can fold into multiple amyloid conformations. Here we demonstrate that Abeta-(1-40) can form soluble aggregates with predominant beta-structures that differ in stability and morphology. One class of aggregates involved soluble Abeta protofibrils, prepared by vigorous overnight agitation of monomeric Abeta-(1-40) at low ionic strength. Dilution of these aggregation reactions induced disaggregation to monomers as measured by size exclusion chromatography. Protofibril concentrations monitored by thioflavin T fluorescence decreased in at least two kinetic phases, with initial disaggregation (rate constant approximately 1 h(-1)) followed by a much slower secondary phase. Incubation of the reactions without agitation resulted in less disaggregation at slower rates, indicating that the protofibrils became progressively more stable over time. In fact, protofibrils isolated by size exclusion chromatography were completely stable and gave no disaggregation. A second class of soluble Abeta aggregates was generated rapidly (<10 min) in buffered 2% hexafluoroisopropanol (HFIP). These aggregates showed increased thioflavin T fluorescence and were rich in beta-structure by circular dichroism. Electron microscopy and atomic force microscopy revealed initial globular clusters that progressed over several days to soluble fibrous aggregates. When diluted out of HFIP, these aggregates initially were very unstable and disaggregated completely within 2 min. However, their stability increased as they progressed to fibers. Relative to Abeta protofibrils, the HFIP-induced aggregates seeded elongation by Abeta monomer deposition very poorly. The techniques used to distinguish these two classes of soluble Abeta aggregates may be useful in characterizing Abeta aggregates formed in vivo.     

Lipids revert inert Abeta amyloid fibrils to neurotoxic protofibrils that affect learning in mice

Although soluble oligomeric and protofibrillar assemblies of Abeta-amyloid peptide cause synaptotoxicity and potentially contribute to Alzheimer's disease (AD), the role of mature Abeta-fibrils in the amyloid plaques remains controversial. A widely held view in the field suggests that the fibrillization reaction proceeds 'forward' in a near-irreversible manner from the monomeric Abeta peptide through toxic protofibrillar intermediates, which subsequently mature into biologically inert amyloid fibrils that are found in plaques. Here, we show that natural lipids destabilize and rapidly resolubilize mature Abeta amyloid fibers. Interestingly, the equilibrium is not reversed toward monomeric Abeta but rather toward soluble amyloid protofibrils. We characterized these 'backward' Abeta protofibrils generated from mature Abeta fibers and compared them with previously identified 'forward' Abeta protofibrils obtained from the aggregation of fresh Abeta monomers. We find that backward protofibrils are biochemically and biophysically very similar to forward protofibrils: they consist of a wide range of molecular masses, are toxic to primary neurons and cause memory impairment and tau phosphorylation in mouse. In addition, they diffuse rapidly through the brain into areas relevant to AD. Our findings imply that amyloid plaques are potentially major sources of soluble toxic Abeta-aggregates that could readily be activated by exposure to biological lipids.     

Abeta protofibrils possess a stable core structure resistant to hydrogen exchange

Protofibrils are transient structures observed during in vitro formation of mature amyloid fibrils and have been implicated as the toxic species responsible for cell dysfunction and neuronal loss in Alzheimer's disease (AD) and other protein aggregation diseases. To better understand the roles of protofibrils in amyloid assembly and Alzheimer's disease, we characterized secondary structural features of these heterogeneous and metastable assembly intermediates. We chromatographically isolated different size populations of protofibrils from amyloid assembly reactions of Abeta(1-40), both wild type and the Arctic variant associated with early onset familial AD, and exposed them to hydrogen-deuterium exchange analysis monitored by mass spectrometry (HX-MS). We show that HX-MS can distinguish among unstructured monomer, protofibrils, and fibrils by their different protection patterns. We find that about 40% of the backbone amide hydrogens of Abeta protofibrils are highly resistant to exchange with deuterium even after 2 days of incubation in aqueous deuterated buffer, implying a very stable, presumably H-bonded, core structure. This is in contrast to mature amyloid fibrils, whose equally stable structure protects about 60% of the backbone amide hydrogens over the same time frame. We also find a surprising degree of specificity in amyloid assembly, in that wild type Abeta is preferentially excluded from both protofibrils and fibrils grown from an equimolar mixture of wild type and Arctic mutant peptides. These and other data are interpreted and discussed in terms of the role of protofibrils in fibril assembly and in disease.