Imbalance in Fatty-Acid-Chain Length of Gangliosides Triggers Alzheimer Amyloid Deposition in the Precuneus

Amyloid deposition, a crucial event of Alzheimer’s disease (AD), emerges in distinct brain regions. A key question is what triggers the assembly of the monomeric amyloid ß-protein (Aß) into fibrils in the regions. On the basis of our previous findings that gangliosides facilitate the initiation of Aß assembly at presynaptic neuritic terminals, we investigated how lipids, including gangliosides, cholesterol and sphingomyelin, extracted from synaptic plasma membranes (SPMs) isolated from autopsy brains were involved in the Aß assembly. We focused on two regions of the cerebral cortex; precuneus and calcarine cortex, one of the most vulnerable and one of the most resistant regions to amyloid deposition, respectively. Here, we show that lipids extracted from SPMs isolated from the amyloid-bearing precuneus, but neither the amyloid-free precuneus nor the calcarine cortex, markedly accelerate the Aß assembly in vitro. Through liquid chromatography-mass spectrometry of the lipids, we identified an increase in the ratio of the level of GD1b-ganglioside containing C20:0 fatty acid to that containing C18:0 as a cause of the enhanced Aß assembly in the precuneus. Our results suggest that the local glycolipid environment play a critical role in the initiation of Alzheimer amyloid deposition.     

Pioglitazone Improves Reversal Learning and Exerts Mixed Cerebrovascular Effects in a Mouse Model of Alzheimer’s Disease with Combined Amyloid-β and Cerebrovascular Pathology

Animal models of Alzheimer’s disease (AD) are invaluable in dissecting the pathogenic mechanisms and assessing the efficacy of potential new therapies. Here, we used the peroxisome proliferator-activated receptor gamma agonist pioglitazone in an attempt to rescue the pathogenic phenotype in adult (12 months) and aged (>18 months) bitransgenic A/T mice that overexpress a mutated human amyloid precursor protein (APPSwe,Ind) and a constitutively active form of transforming growth factor-β1 (TGF-β1). A/T mice recapitulate the AD-related cognitive deficits, amyloid beta (Aβ) and cerebrovascular pathologies, as well as the altered metabolic and vascular coupling responses to increased neuronal activity. Pioglitazone normalized neurometabolic and neurovascular coupling responses to sensory stimulation, and reduced cortical astroglial and hippocampal microglial activation in both age groups. Spatial learning and memory deficits in the Morris water maze were not rescued by pioglitazone, but reversal learning was improved in the adult cohort notwithstanding a progressing Aβ pathology. While pioglitazone preserved the constitutive nitric oxide synthesis in the vessel wall, it unexpectedly failed to restore cerebrovascular reactivity in A/T mice and even exacerbated the dilatory deficits. These data demonstrate pioglitazone’s efficacy on selective AD hallmarks in a complex AD mouse model of comorbid amyloidosis and cerebrovascular pathology. They further suggest a potential benefit of pioglitazone in managing neuroinflammation, cerebral perfusion and glucose metabolism in AD patients devoid of cerebrovascular pathology.     

Mitotic Spindle Defects and Chromosome Mis-Segregation Induced by LDL/Cholesterol—Implications for Niemann-Pick C1, Alzheimer’s Disease,and Atherosclerosis

Elevated low-density lipoprotein (LDL)-cholesterol is a risk factor for both Alzheimer’s disease (AD) and Atherosclerosis (CVD), suggesting a common lipid-sensitive step in their pathogenesis. Previous results show that AD and CVD also share a cell cycle defect: chromosome instability and up to 30% aneuploidy–in neurons and other cells in AD and in smooth muscle cells in atherosclerotic plaques in CVD. Indeed, specific degeneration of aneuploid neurons accounts for 90% of neuronal loss in AD brain, indicating that aneuploidy underlies AD neurodegeneration. Cell/mouse models of AD develop similar aneuploidy through amyloid-beta (Aß) inhibition of specific microtubule motors and consequent disruption of mitotic spindles. Here we tested the hypothesis that, like upregulated Aß, elevated LDL/cholesterol and altered intracellular cholesterol homeostasis also causes chromosomal instability. Specifically we found that: 1) high dietary cholesterol induces aneuploidy in mice, satisfying the hypothesis’ first prediction, 2) Niemann-Pick C1 patients accumulate aneuploid fibroblasts, neurons, and glia, demonstrating a similar aneugenic effect of intracellular cholesterol accumulation in humans 3) oxidized LDL, LDL, and cholesterol, but not high-density lipoprotein (HDL), induce chromosome mis-segregation and aneuploidy in cultured cells, including neuronal precursors, indicating that LDL/cholesterol directly affects the cell cycle, 4) LDL-induced aneuploidy requires the LDL receptor, but not Aß, showing that LDL works differently than Aß, with the same end result, 5) cholesterol treatment disrupts the structure of the mitotic spindle, providing a cell biological mechanism for its aneugenic activity, and 6) ethanol or calcium chelation attenuates lipoprotein-induced chromosome mis-segregation, providing molecular insights into cholesterol’s aneugenic mechanism, specifically through its rigidifying effect on the cell membrane, and potentially explaining why ethanol consumption reduces the risk of developing atherosclerosis or AD. These results suggest a novel, cell cycle mechanism by which aberrant cholesterol homeostasis promotes neurodegeneration and atherosclerosis by disrupting chromosome segregation and potentially other aspects of microtubule physiology.     

The Proton-Pump Inhibitor Lansoprazole Enhances Amyloid Beta Production

A key event in the pathogenesis of Alzheimer’s disease (AD) is the accumulation of amyloid-β (Aβ) species in the brain, derived from the sequential cleavage of the amyloid precursor protein (APP) by β- and γ-secretases. Based on a systems biology study to repurpose drugs for AD, we explore the effect of lansoprazole, and other proton-pump inhibitors (PPIs), on Aβ production in AD cellular and animal models. We found that lansoprazole enhances Aβ37, Aβ40 and Aβ42 production and lowers Aβ38 levels on amyloid cell models. Interestingly, acute lansoprazole treatment in wild type and AD transgenic mice promoted higher Aβ40 levels in brain, indicating that lansoprazole may also exacerbate Aβ production in vivo. Overall, our data presents for the first time that PPIs can affect amyloid metabolism, both in vitro and in vivo.     

Intravenous Delivery of Targeted Liposomes to Amyloid-β Pathology in APP/PSEN1 Transgenic Mice

Extracellular amyloid-β (Aβ) plaques and intracellular neurofibrillary tangles constitute the major neuropathological hallmarks of Alzheimer’s disease (AD). It is now apparent that parenchymal Aβ plaque deposition precedes behavioral signs of disease by several years. The development of agents that can target these plaques may be useful as diagnostic or therapeutic tools. In this study, we synthesized an Aβ-targeted lipid conjugate, incorporated it in stealth liposomal nanoparticles and tested their ability to bind amyloid plaque deposits in an AD mouse model. The results show that the particles maintain binding profiles to synthetic Aβ aggregates comparable to the free ligand, and selectively bind Aβ plaque deposits in brain tissue sections of an AD mouse model (APP/PSEN1 transgenic mice) with high efficiency. When administered intravenously, these long circulating nanoparticles appear to cross the blood-brain barrier and bind to Aβ plaque deposits, labeling parenchymal amyloid deposits and vascular amyloid characteristic of cerebral amyloid angiopathy.     

Humanized Tau Mice with Regionalized Amyloid Exhibit Behavioral Deficits but No Pathological Interaction

Alzheimer’s disease (AD) researchers have struggled for decades to draw a causal link between extracellular Aβ aggregation and intraneuronal accumulation of microtubule-associated protein tau. The amyloid cascade hypothesis posits that Aβ deposition promotes tau hyperphosphorylation, tangle formation, cell loss, vascular damage, and dementia. While the genetics of familial AD and the pathological staging of sporadic disease support this sequence of events, attempts to examine the molecular mechanism in transgenic animal models have largely relied on models of other inherited tauopathies as the basis for testing the interaction with Aβ. In an effort to more accurately model the relationship between Aβ and wild-type tau in AD, we intercrossed mice that overproduce human Aβ with a tau substitution model in which all 6 isoforms of the human protein are expressed in animals lacking murine tau. We selected an amyloid model in which pathology was biased towards the entorhinal region so that we could further examine whether the anticipated changes in tau phosphorylation occurred at the site of Aβ deposition or in synaptically connected regions. We found that Aβ and tau had independent effects on locomotion, learning, and memory, but found no behavioral evidence for an interaction between the two transgenes. Moreover, we saw no indication of amyloid-induced changes in the phosphorylation or aggregation of human tau either within the entorhinal area or elsewhere. These findings suggest that robust amyloid pathology within the medial temporal lobe has little effect on the metabolism of wild type human tau in this model.     

Functional Impairment of Microglia Coincides with Beta-Amyloid Deposition in Mice with Alzheimer-Like Pathology

Microglial cells closely interact with senile plaques in Alzheimer’s disease and acquire the morphological appearance of an activated phenotype. The significance of this microglial phenotype and the impact of microglia for disease progression have remained controversial. To uncover and characterize putative changes in the functionality of microglia during Alzheimer’s disease, we directly assessed microglial behavior in two mouse models of Alzheimer’s disease. Using in vivo two-photon microscopy and acute brain slice preparations, we found that important microglial functions - directed process motility and phagocytic activity - were strongly impaired in mice with Alzheimer’s disease-like pathology compared to age-matched non-transgenic animals. Notably, impairment of microglial function temporally and spatially correlated with Aβ plaque deposition, and phagocytic capacity of microglia could be restored by interventionally decreasing amyloid burden by Aβ vaccination. These data suggest that major microglial functions progressively decline in Alzheimer’s disease with the appearance of Aβ plaques, and that this functional impairment is reversible by lowering Aβ burden, e.g. by means of Aβ vaccination.     

Alzheimer’s disease-associated β-amyloid does not protect against herpes simplex virus 1 infection in the mouse brain

Alzheimer’s disease (AD) is a devastating fatal neurodegenerative disease. An alternative to the amyloid cascade hypothesis is that a viral infection is key to the etiology of late-onset AD, with β-amyloid (Aβ) peptides playing a protective role. In the current study, young 5XFAD mice that overexpress mutant human amyloid precursor protein with the Swedish, Florida, and London familial AD mutations were infected with one of two strains of herpes simplex virus 1 (HSV-1), 17syn+ and McKrae, at three different doses. Contrary to previous work, 5XFAD genotype failed to protect mice against HSV-1 infection. The region- and cell-specific tropisms of HSV-1 were not affected by the 5XFAD genotype, indicating that host–pathogen interactions were not altered. Seven- to ten-month-old 5XFAD animals in which extracellular Aβ aggregates were abundant showed slightly better survival rate relative to their wild-type (WT) littermates, although the difference was not statistically significant. In these 5XFAD mice, HSV-1 replication centers were partially excluded from the brain areas with high densities of Aβ aggregates. Aβ aggregates were free of HSV-1 viral particles, and the limited viral invasion to areas with a high density of Aβ aggregates was attributed to phagocytic activity of reactive microglia. In the oldest mice (12–15 months old), the survival rate did not differ between 5XFAD and WT littermates. While the current study questions the antiviral role of Aβ, it neither supports nor refutes the viral etiology hypothesis of late-onset AD.     

Prophylaxis and Treatment of Alzheimer's Disease by Delivery of an Adeno-Associated Virus Encoding a Monoclonal Antibody Targeting the Amyloid Beta Protein

We previously reported on a monoclonal antibody (mAb) that targeted amyloid beta (Aß) protein. Repeated injection of that mAb reduced the accumulation of Aß protein in the brain of human Aß transgenic mice (Tg2576). In the present study, cDNA encoding the heavy and light chains of this mAb were subcloned into an adeno-associated virus type 1 (AAV) vector with a 2A/furin adapter. A single intramuscular injection of 3.0×10¹⁰ viral genome of these AAV vectors into C57BL/6 mice generated serum anti-Aß Ab levels up to 0.3 mg/ml. Anti-Aß Ab levels in excess of 0.1 mg/ml were maintained for up to 64 weeks. The effect of AAV administration on Aß levels in vivo was examined. A significant decrease in Aß levels in the brain of Tg2576 mice treated at 5 months (prophylactic) or 10 months (therapeutic) of age was observed. These results support the use of AAV vector encoding anti-Aß Ab for the prevention and treatment of Alzheimer''s disease.     

Microglial PD‐1 stimulation by astrocytic PD‐L1 suppresses neuroinflammation and Alzheimer’s disease pathology

Chronic neuroinflammation is a pathogenic component of Alzheimer’s disease (AD) that may limit the ability of the brain to clear amyloid deposits and cellular debris. Tight control of the immune system is therefore key to sustain the ability of the brain to repair itself during homeostasis and disease. The immune‐cell checkpoint receptor/ligand pair PD‐1/PD‐L1, known for their inhibitory immune function, is expressed also in the brain. Here, we report upregulated expression of PD‐L1 and PD‐1 in astrocytes and microglia, respectively, surrounding amyloid plaques in AD patients and in the APP/PS1 AD mouse model. We observed juxtamembrane shedding of PD‐L1 from astrocytes, which may mediate ectodomain signaling to PD‐1‐expressing microglia. Deletion of microglial PD‐1 evoked an inflammatory response and compromised amyloid‐β peptide (Aβ) uptake. APP/PS1 mice deficient for PD‐1 exhibited increased deposition of Aβ, reduced microglial Aβ uptake, and decreased expression of the Aβ receptor CD36 on microglia. Therefore, ineffective immune regulation by the PD‐1/PD‐L1 axis contributes to Aβ plaque deposition during chronic neuroinflammation in AD.     

Endothelial expression of human amyloid precursor protein leads to amyloid β in the blood and induces cerebral amyloid angiopathy in knock-in mice

The deposition of amyloid β (Aβ) in blood vessels of the brain, known as cerebral amyloid angiopathy (CAA), is observed in most patients with Alzheimer’s disease (AD). Compared with the pathology of CAA in humans, the pathology in most mouse models of AD is not as evident, making it difficult to examine the contribution of CAA to the pathogenesis of AD. On the basis of biochemical analyses that showed blood levels of soluble amyloid precursor protein (APP) in rats and mice were markedly lower than those measured in human samples, we hypothesized that endothelial APP expression would be markedly lower in rodents and subsequently generated mice that specifically express human WT APP (APP770) in endothelial cells (ECs). The resulting EC-APP770⁺ mice exhibited increased levels of serum Aβ and soluble APP, indicating that endothelial APP makes a critical contribution to blood Aβ levels. Even though aged EC-APP770⁺ mice did not exhibit Aβ deposition in the cortical blood vessels, crossing these animals with APP knock-in mice (App^NL-F/NL-F) led to an expanded CAA pathology, as evidenced by increased amounts of amyloid accumulated in the cortical blood vessels. These results highlight an overlooked interplay between neuronal and endothelial APP in brain vascular Aβ deposition. We propose that these EC-APP770⁺:App^NL-F/NL-F mice may be useful to study the basic molecular mechanisms behind the possible breakdown of the blood–brain barrier upon administration of anti-Aβ antibodies.     

Sodium Chloride Increases Aβ Levels by Suppressing Aβ Clearance in Cultured Cells

Recent studies suggest that high-salt diet is associated with cognitive decline in human and mouse. The fact that genetic factors account for less than 50% cases of sporadic Alzheimer’s disease (AD) highlights the important contribution of environmental factors, such as high-salt diet, in AD pathogenesis. However, whether and how high-salt diet fits the “amyloid cascade” hypothesis remains unexplored. Here, we show sodium chloride (NaCl) could increase Aβ levels in the medium of HEK293 cells overexpressing amyloid precursor protein (APP) or C99 fragment. NaCl treatment dose not affect APP level, gamma secretase level or activity. Instead, NaCl treatment suppresses the capacity of cells to clear Aβ and reduces Apolipoprotein E (ApoE) level. Finally, NaCl treated THP-1 or BV2 cells are inefficient in clearing Aβ when co-cultured with rat primary neurons. Our study suggests that high-salt diet may increase AD risk by directly modulating Aβ levels.     

Tailoring the Antibody Response to Aggregated A? Using Novel Alzheimer-Vaccines

Recent evidence suggests Alzheimer-Disease (AD) to be driven by aggregated Aß. Capitalizing on the mechanism of molecular mimicry and applying several selection layers, we screened peptide libraries for moieties inducing antibodies selectively reacting with Aß-aggregates. The technology identified a pool of peptide candidates; two, AFFITOPES AD01 and AD02, were assessed as vaccination antigens and compared to Aβ1-6, the targeted epitope. When conjugated to Keyhole Limpet Hemocyanin (KLH) and adjuvanted with aluminum, all three peptides induced Aß-targeting antibodies (Abs). In contrast to Aß1-6, AD01- or AD02-induced Abs were characterized by selectivity for aggregated forms of Aß and absence of reactivity with related molecules such as Amyloid Precursor Protein (APP)/ secreted APP-alpha (sAPPa). Administration of AFFITOPE-vaccines to APP-transgenic mice was found to reduce their cerebral amyloid burden, the associated neuropathological alterations and to improve their cognitive functions. Thus, the AFFITOME-technology delivers vaccines capable of inducing a distinct Ab response. Their features may be beneficial to AD-patients, a hypothesis currently tested within a phase-II-study.     

Advances on the Understanding of the Origins of Synaptic Pathology in AD

Although Alzheimer’s disease (AD) was first discovered a century ago, we are still facing a lack of definitive diagnosis during the patient’s lifetime and are unable to prescribe a curative treatment. However, the past 10 years have seen a “revamping” of the main hypothesis about AD pathogenesis and the hope to foresee possible treatment. AD is no longer considered an irreversible disease. A major refinement of the classic β-amyloid cascade describing amyloid fibrils as neurotoxins has been made to integrate the key scientific evidences demonstrating that the first pathological event occurring in AD early stages affects synaptic function and maintenance. A concept fully compatible with synapse loss being the best pathological correlate of AD rather than other described neuropathological hallmarks (amyloid plaques, neurofibrillary tangles or neuronal death). The notion that synaptic alterations might be reverted, thus offering a potential curability, was confirmed by immunotherapy experiments targeting β-amyloid protein in transgenic AD mice in which cognitive functions were improved despite no reduction in the amyloid plaques burden. The updated amyloid cascade now integrates the synapse failure triggered by soluble Aβ-oligomers. Still no consensus has been reached on the most toxic Aβ conformations, neither on their site of production nor on their extra- versus intra-cellular actions. Evidence shows that soluble Aβ oligomers or ADDLs bind selectively to neurons at their synaptic loci, and trigger major changes in synapse composition and morphology, which ultimately leads to dendritic spine loss. However, the exact mechanism is not yet fully understood but is suspected to involve some membrane receptor(s).     

MicroRNA-149 is downregulated in Alzheimer’s disease and inhibits β-amyloid accumulation and ameliorates neuronal viability through targeting BACE1

Beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) plays a critical role in Alzheimer’s disease (AD) pathogenesis. This study aimed to investigate the relationship between microRNA-149 (miR-149) and BACE1, and evaluate the clinical significance and biological function of miR-149 in AD progression. Bioinformatics analysis and a luciferase reporter assay were used to confirm the interaction between miR-149 and BACE1. Expression of miR-149 and BACE1 was estimated using quantitative real-time PCR. The clinical significance of miR-149 in AD diagnosis and severity determination was evaluated using ROC analysis. The effect of miR-149 on Aβ accumulation and neuronal viability was analyzed in Aβ-treated SH-SY5Y cells. miR-149 was found directly binding the 3’-UTR of BACE1 and was negatively correlated with BACE1 in AD patients and cell model. Serum miR-149 expression was downregulated in AD patients and served as a potential diagnostic biomarker. The overexpression of miR-149 in Aβ-treated SH-SY5Y cells resulted in inhibited Aβ accumulation and enhanced neuronal viability. This study demonstrated that serum miR-149 is decreased in AD patients and serves as a candidate diagnostic biomarker, and that the overexpression of miR-149 may suppress Aβ accumulation and promote neuronal viability by targeting BACE1 in AD model cells. Keywords: MicroRNA-149, Alzheimer’s disease, BACE1, diagnosis, APP     

The Preventive and Therapeutic Effects of Intravenous Human Adipose-Derived Stem Cells in Alzheimer’s Disease Mice

Alzheimer’s disease (AD) is characterized by the accumulation of amyloid plaques and neurofibrillary tangles accompanied by cognitive dysfunction. The aim of the present study was to elucidate preventive and therapeutic potential of stem cells for AD. Among stem cells, autologous human adipose-derived stem cells (hASCs) elicit no immune rejection responses, tumorigenesis, or ethical problems. We found that intravenously transplanted hASCs passed through the BBB and migrated into the brain. The learning, memory and pathology in an AD mouse model (Tg2576) mice greatly improved for at least 4 months after intravenous injection of hASC. The number of amyloid plaques and Aβ levels decreased significantly in the brains of hASC-injected Tg mice compared to those of Tg-sham mice. Here, we first report that intravenously or intracerebrally transplanted hASCs significantly rescues memory deficit and neuropathology, in the brains of Tg mice by up-regulating IL-10 and VEGF and be a possible use for the prevention and treatment of AD.     

Traditional Chinese Nootropic Medicine Radix Polygalae and Its Active Constituent Onjisaponin B Reduce β-Amyloid Production and Improve Cognitive Impairments

Decline of cognitive function is the hallmark of Alzheimer’s disease (AD), regardless of the pathological mechanism. Traditional Chinese medicine has been used to combat cognitive impairments and has been shown to improve learning and memory. Radix Polygalae (RAPO) is a typical and widely used herbal medicine. In this study, we aimed to follow the β-amyloid (Aβ) reduction activity to identify active constituent(s) of RAPO. We found that Onjisaponin B of RAPO functioned as RAPO to suppress Aβ production without direct inhibition of β-site amyloid precursor protein cleaving enzyme 1 (BACE1) and γ-secretase activities. Our mechanistic study showed that Onjisaponin B promoted the degradation of amyloid precursor protein (APP). Further, oral administration of Onjisaponin B ameliorated Aβ pathology and behavioral defects in APP/PS1 mice. Taken together, our results indicate that Onjisaponin B is effective against AD, providing a new therapeutic agent for further drug discovery.     

Triptolide treatment reduces Alzheimer’s disease (AD)-like pathology through inhibition of BACE1 in a transgenic mouse model of AD

The complex pathogenesis of Alzheimer’s disease (AD) involves multiple contributing factors, including amyloid β (Aβ) peptide accumulation, inflammation and oxidative stress. Effective therapeutic strategies for AD are still urgently needed. Triptolide is the major active compound extracted from Tripterygium wilfordii Hook.f., a traditional Chinese medicinal herb that is commonly used to treat inflammatory diseases. The 5-month-old 5XFAD mice, which carry five familial AD mutations in the β-amyloid precursor protein (APP) and presenilin-1 (PS1) genes, were treated with triptolide for 8 weeks. We observed enhanced spatial learning performances, and attenuated Aβ production and deposition in the brain. Triptolide also inhibited the processing of amyloidogenic APP, as well as the expression of βAPP-cleaving enzyme-1 (BACE1) both in vivo and in vitro. In addition, triptolide exerted anti-inflammatory and anti-oxidative effects on the transgenic mouse brain. Triptolide therefore confers protection against the effects of AD in our mouse model and is emerging as a promising therapeutic candidate drug for AD.KEY WORDS: Alzheimer’s disease, Amyloid β, 5XFAD mice, BACE1, Inflammation, Triptolide     

Liquid–liquid phase separation of amyloid-β oligomers modulates amyloid fibrils formation

Soluble amyloid-β oligomers (AβOs) are proposed to instigate and mediate the pathology of Alzheimer’s disease, but the mechanisms involved are not clear. In this study, we reported that AβOs can undergo liquid–liquid phase separation (LLPS) to form liquid-like droplets in vitro. We determined that AβOs exhibited an α-helix conformation in a membrane-mimicking environment of SDS. Importantly, SDS is capable of reconfiguring the assembly of different AβOs to induce their LLPS. Moreover, we found that the droplet formation of AβOs was promoted by strong hydrated anions and weak hydrated cations, suggesting that hydrophobic interactions play a key role in mediating phase separation of AβOs. Finally, we observed that LLPS of AβOs can further promote Aβ to form amyloid fibrils, which can be modulated by (−)-epigallocatechin gallate. Our study highlights amyloid oligomers as an important entity involved in protein liquid-to-solid phase transition and reveals the regulatory role of LLPS underlying amyloid protein aggregation, which may be relevant to the pathological process of Alzheimer’s disease.     

A Structure-Toxicity Study of A?42 Reveals a New Anti-Parallel Aggregation Pathway

Amyloid beta (Aβ) peptides produced by APP cleavage are central to the pathology of Alzheimer’s disease. Despite widespread interest in this issue, the relationship between the auto-assembly and toxicity of these peptides remains controversial. One intriguing feature stems from their capacity to form anti-parallel ß-sheet oligomeric intermediates that can be converted into a parallel topology to allow the formation of protofibrillar and fibrillar Aβ. Here, we present a novel approach to determining the molecular aspects of Aß assembly that is responsible for its in vivo toxicity. We selected Aß mutants with varying intracellular toxicities. In vitro, only toxic Aß (including wild-type Aß₄₂) formed urea-resistant oligomers. These oligomers were able to assemble into fibrils that are rich in anti-parallel ß-sheet structures. Our results support the existence of a new pathway that depends on the folding capacity of Aß .