Aβ-mediated Toxicity Research Articles

Aggregation and cytotoxic properties towards cultured cerebrovascular cells of Dutch-mutated Aβ40 (DAβ 1-40) are modulated by sulfate moieties of heparin

Glycosaminoglycans (GAGs), in particular as part of heparan sulfate proteoglycans, are associated with cerebral amyloid angiopathy (CAA). Similarly, GAGs are also associated with the severe CAA found in patients suffering from hereditary cerebral hemorrhage with amyloidosis of the Dutch type (HCHWA-D), where the amyloid β (Aβ) peptide contains the Dutch mutation (DAβ 1-40). This suggests a role for GAGs in vascular Aβ aggregation. It was the aim of this study to investigate the effect of different GAGs (heparin, chondroitin sulfate, heparan sulfate), the macromolecule dextran sulfate and, using desulfated heparins, the role of GAG sulfate moieties on the in vitro aggregation of CAA-associated DAβ 1-40 and on DAβ 1-40-induced toxicity of cultured cerebrovascular cells. We also aimed to study the in vivo distribution of various sulfated heparan sulfate GAG epitopes in CAA. Of all GAGs tested, heparin was the strongest inducer of aggregation of DAβ 1-40 in the different aggregation assays, with both heparin and heparan sulfate reducing Aβ-induced cellular toxicity. Furthermore, (partial) removal of the sulfate moieties of heparin partially abolished the effects of heparin on aggregation and cellular toxicity, suggesting an essential role for the sulfate moieties in heparin. Finally, we demonstrated the in vivo association of sulfated heparan sulfate (HS) GAGs with CAA. We conclude that sulfate moieties within GAGs, like heparin and HS, have an important role in Aβ aggregation in CAA and in Aβ-mediated toxicity of cerebrovascular cells.

Apolipoprotein E protects cultured pericytes and astrocytes from D-Aβ 1–40-mediated cell death

Cerebral amyloid angiopathy (CAA) is a common pathological finding in Alzheimer's disease and hereditary cerebral hemorrhage with amyloidosis of the Dutch type; in this latter condition it is caused by deposition of mutated amyloid β protein (Aβ Glu22Gln; D-Aβ 1–40). Previously, we found a dependence of the Aβ-mediated toxicity and apolipoprotein E (apoE) production by cultured pericytes on apoE genotype. Given their close association with the cerebrovascular wall both astrocytes and pericytes may be involved in CAA development, a process that includes Aβ deposition and clearance and that may be affected by interaction with locally produced apolipoprotein E (apoE). Although astrocytes are regarded as the major source of apolipoprotein E (apoE) in the brain, also pericytes produce apoE. In this study we compared the apoE production capacity, the effects of apoE on D-Aβ 1–40 internalization, D-Aβ 1–40 cell surface accumulation and the vulnerability for D-Aβ 1–40-induced toxicity of either cell type in order to quantify the relative contributions of astrocytes and pericytes in the various processes that contribute to CAA formation. Strikingly, cultured astrocytes produced only 3–10% of the apoE amounts produced by pericytes. Furthermore, pericytes with the apoE ɛ4 allele produced three times less apoE and were more vulnerable to D-Aβ 1–40 treatment than pericytes without an ɛ4 allele. Such relations were not observed with astrocytes in vitro. Both pericytes and astrocytes, however, were protected from Aβ-induced cytotoxicity by high levels of pericyte-derived apoE, but not recombinant apoE. In addition, pericyte-derived apoE dose-dependently decreased both internalization of Aβ and Aβ accumulation at the cell surface in either cell type. The present data suggest that apoE produced by pericytes, rather than astrocyte-produced apoE, modulates Aβ cytotoxicity and Aβ removal near the vasculature in the brain. Furthermore, since apoE production in pericytes is genotype dependent, this may contribute to the apoE genotype-dependent development of CAA in vivo.

Linking Amyloid and Tau Pathology in Alzheimer's Disease: The Role of Membrane Cholesterol in Aβ-Mediated Tau Toxicity: Figure 1.

Alzheimer's disease (AD) is characterized by extracellular deposition of β-amyloid (Aβ) as senile plaques, as well as intracellular accumulation of hyperphosphorylated, aggregated tau as neurofibrillary tangles. Increased production and/or deposition of Aβ is thought to precede tangle formation,

Characterization of microglia derived from bone marrow hematopoietic stem cells: In vitro model for amyloid-beta toxicity and phagocytosis

Bone marrow (BM) derived microglia contribute to reduction of Amyloid-beta (Aβ) burden in Alzheimer's disease as previously demonstrated in vitro and in vivo. However, the nature of BM derived microglia in Aβ phagocytosis and Aβ-induced toxicity and inflammation remain largely unknown. The aim of this study was to generate a model reproducing differentiation of monocyte/microglia from BM hematopoietic stem cells (HSC) and to investigate the phagocytic capacity of BM derived monocyte/microglia towards Aβ and contribution to Aβ-mediated toxicity in vitro. Mouse BM CD117 HSC were isolated by immunomagnetic separation and differentiated into monocyte/microglia lineage. Cell differentiation was determined with flow cytometry. Cells were stimulated with lipopolysaccharide (LPS) and soluble and fibrillar Aβ to determine their inflammatory capacity and effect on cellular signaling and viability, utilizing cytokine ELISA, intracellular calcium imaging and MTT cell viability assay. Cells' capacity for Aβ phagocytosis was determined from hippocampal brain sections prepared from aged APP/PS1 mice in ex vivo assay utilizing Aβ immunostaining and Aβ1-40 and Aβ1-42 ELISA. BM HSC derived monocyte/microglia showed characteristics of monocytes and microglia expressing CD11b, and CD68, marker for lysosomal activity. BM HSC derived monocyte/microglia showed morphological changes and inflammatory cytokine TNFα release in response to inflammatory stimulus LPS, comparable to that of primary microglia. Soluble/oligomeric Aβ interfered with cellular signaling and reduced cell viability. In contrast, BM derived monocyte/microglia presented phagocytic capacity towards Aβ plaques, comparable to BM derived CD11b monocytes. Differentiation of mouse BM HSC into monocyte/microglia lineage represents an in vitro model for BM derived microglia. These cells present phagocytic capacity towards Aβ and could be utilized in further in vitro and in vivo studies for studying the role of BM derived cells in Alzheimer's disease in detail.

P4-328: Interception of ABAD-Aβ interaction protects against Aβ-mediated mitochondrial toxicity in the transgenic animal model of Alzheimer disease

P4-328: Interception of ABAD-Aβ interaction protects against Aβ-mediated mitochondrial toxicity in the transgenic animal model of Alzheimer disease

Apolipoprotein E Genotype Regulates Amyloid-β Cytotoxicity

The epsilon4 allele of apolipoprotein E (ApoE) is a risk factor for Alzheimer's disease (AD), whereas the epsilon2 allele may be relatively protective. Both alleles are risk factors for cerebral amyloid angiopathy (CAA)-related hemorrhages. CAA is associated with degeneration of smooth muscle cells and pericytes. Previously, we described that synthetic amyloid-beta1-40 peptide (Abeta1-40) with the 22Glu--> Gln "Dutch" mutation caused pericyte death in vitro by a mechanism that involves Abeta fibril-like assembly at the cell surface. It is known that ApoE binds to Abeta and may modify its biological activities. In the present study, we evaluated the effect of ApoE on Abeta-mediated toxicity of cerebrovascular cells. We observed that cultured cells with an epsilon4/epsilon4 genotype were more vulnerable to Abeta than cultures with an epsilon3/epsilon3 or epsilon3/epsilon4 genotype. The one cell culture with the epsilon2/epsilon3 genotype was relatively resistant to Abeta compared with other cultures. Furthermore, we observed a dose-dependent protective effect of native ApoE against Abeta-mediated toxicity of cerebrovascular cells and, in addition, ApoE epsilon2/epsilon3 cells secreted more ApoE protein compared with cells with other ApoE genotypes, in particular, compared with epsilon4/epsilon4 cells. Thus, the disparity between ApoE genotype and Abeta-mediated toxicity might be related to differences in the cellular capacity to secrete ApoE. The present data suggest that one mechanism by which ApoE may alter the risk for AD is a genotype-dependent regulation of Abeta cytotoxicity, possibly via variations in its secretion levels, whereby extracellular ApoE may bind to Abeta and thereby modify Abeta-mediated cell death.

Neurotoxicity and oxidative stress in D1M-substituted Alzheimer's Aβ(1-42): relevance to N-terminal methionine chemistry in small model peptides

Small model peptides containing N-terminal methionine are reported to form sulfur-centered-free radicals that are stabilized by the terminal N atom. To test whether a similar chemistry would apply to a disease-relevant longer peptide, Alzheimer's disease (AD)-associated amyloid beta-peptide 1-42 was employed. Methionine at residue 35 of this 42-mer has been shown to be a key amino acid residue involved in amyloid beta-peptide 1-42 [Aβ1-42]-mediated toxicity and therefore, the pathogenesis of AD. Previous studies have shown that mutation of the methionine residue to norleucine abrogates the oxidative stress and neurotoxic properties of Aβ(1-42). In the current study, we examined if the position of methionine at residue 35 is a criterion for toxicity. In doing so, we tested the effects of moving methionine to the N-terminus of the peptide in a synthetic peptide, Aβ(1-42)D1M, in which methionine was substituted for aspartic acid at the N-terminus of the peptide and all subsequent residues from D1 to L34 were shifted one position towards the carboxy-terminus. Aβ(1-42)D1M exhibited oxidative stress and neurotoxicity properties similar to those of the native peptide, Aβ(1-42), all of which are inhibited by the free radical scavenger Vitamin E, suggesting that reactive oxygen species may play a role in the Aβ-mediated toxicity. Additionally, substitution of methionine at the N-terminus by norleucine, Aβ(1-42)D1Nle, completely abrograted the oxidative stress and neurotoxicity associated with the Aβ(1-42)D1M peptide. The results of this study validate the chemistry reported for short peptides with N-terminal methionines in a disease-relevant peptide.

A Cdk5 inhibitory peptide reduces tau hyperphosphorylation and apoptosis in neurons

The extracellular aggregation of amyloid beta (Abeta) peptides and the intracellular hyperphosphorylation of tau at specific epitopes are pathological hallmarks of neurodegenerative diseases such as Alzheimer's disease (AD). Cdk5 phosphorylates tau at AD-specific phospho-epitopes when it associates with p25. p25 is a truncated activator, which is produced from the physiological Cdk5 activator p35 upon exposure to Abeta peptides. We show that neuronal infections with Cdk5 inhibitory peptide (CIP) selectively inhibit p25/Cdk5 activity and suppress the aberrant tau phosphorylation in cortical neurons. Furthermore, Abeta(1-42)-induced apoptosis of these cortical neurons was also reduced by coinfection with CIP. Of particular importance is our finding that CIP did not inhibit endogenous or transfected p35/Cdk5 activity, nor did it inhibit the other cyclin-dependent kinases such as Cdc2, Cdk2, Cdk4 and Cdk6. These results, therefore, provide a strategy to address, and possibly ameliorate, the pathology of neurodegenerative diseases that may be a consequence of aberrant p25 activation of Cdk5, without affecting 'normal' Cdk5 activity.

P75 Neurotrophin Receptor Protects Primary Cultures of Human Neurons against Extracellular Amyloid β Peptide Cytotoxicity

The cytotoxicity of extracellular amyloid beta peptide (Abeta) has been clearly demonstrated in many cell types. In contrast, primary human neurons in culture are resistant to extracellular Abeta-mediated toxicity. Here, we investigate the involvement of p75 neurotrophin receptor (p75NTR) in Abeta-treated human neurons. We find that Abeta1-40 and Abeta1-42, but not the reverse control peptide, Abeta40-1, rapidly increase the levels of p75NTR in a specific and dose-dependent manner. In contrast to observations in cell lines, enhanced expression of p75NTR in human neurons via a herpes simplex virus amplicon vector does not increase the susceptibility of neurons to Abeta. Unexpectedly, inhibition of p75NTR expression with an antisense expression construct or incubation of the cells with an antibody to the extracellular domain of p75NTR sensitizes human neurons to extracellular nonfibrillar or fibrillar Abeta1-42 cytotoxicity. Unlike intracellular Abeta, extracellular Abeta toxicity is independent of p53 and Bax activity. However, Abeta toxicity is inhibited by caspase inhibitors and the glycogen synthase kinase 3beta inhibitor lithium. Neuroprotection against Abeta is phosphatidylinositide 3-kinase dependent but Akt independent. These results are consistent with a neuroprotective role for p75NTR against extracellular Abeta toxicity in human neurons.

Amyloid β peptide induces tau phosphorylation and loss of cholinergic neurons in rat primary septal cultures

The neuropathological features associated with Alzheimer’s disease (AD) brain include the presence of extracellular neuritic plaques composed of amyloid β protein (Aβ), intracellular neurofibrillary tangles containing phosphorylated tau protein and the loss of basal forebrain cholinergic neurons which innervate regions such as the hippocampus and the cortex. Studies of the pathological changes that characterize AD and several other lines of evidence indicate that Aβ accumulation in vivo may initiate phosphorylation of tau protein, which by disrupting neuronal network may trigger the process of neurodegeneration observed in AD brains. However, the underlying cause of degeneration of the basal forebrain cholinergic neurons and their association, if any, to Aβ peptides or phosphorylated tau remains mostly unknown. In the present study, using rat primary septal cultures, we have shown that aggregated Aβ peptides, in a time (18–96 h)- and concentration (0.7–60 μM)-dependent manner, induce toxicity and decrease choline acetyltransferase enzyme activity in cultured neurons. Using immunocytochemistry and immunoblotting, we have also demonstrated that Aβ treatment can significantly increase the phosphorylation of tau protein in septal cultures. At the cellular level, hyperphosphorylated tau is mostly apparent in the somatodendritic compartment of the neurons. Aβ peptide (10 μM), in addition to tau phosphorylation, also activates mitogen-activated protein kinase and glycogen synthase kinase-3β, the two kinases which are known to be involved in the formation of hyperphosphorylated tau in the AD brain. Exposure to specific inhibitors of the mitogen-activated protein kinase (i.e. PD98059) or glycogen synthase kinase-3β (i.e. LiCl) attenuated the hyperphosphorylation of the tau protein in cultured neurons. Given the evidence that tau phosphorylation can induce cell loss by disrupting neuronal cytoskeleton, it is likely that aggregated Aβ peptide triggers degeneration of septal neurons, including those expressing the cholinergic phenotype, by phosphorylation of the tau protein activated by mitogen-activated protein kinase and glycogen synthase kinase-3β. These results, taken together, suggest that cultured septal cholinergic neurons are vulnerable to Aβ-mediated toxicity and tau phosphorylation may play an important role in Aβ-induced neurodegeneration.

The Human DIMINUTO/DWARF1 Homolog Seladin-1 Confers Resistance to Alzheimer's Disease-Associated Neurodegeneration and Oxidative Stress

In Alzheimer's disease (AD) brains, selected populations of neurons degenerate heavily, whereas others are frequently spared from degeneration. To address the cellular basis for this selective vulnerability of neurons in distinct brain regions, we compared gene expression between the severely affected inferior temporal lobes and the mostly unaffected fronto-parietal cortices by using an mRNA differential display. We identified seladin-1, a novel gene, which was downregulated in large pyramidal neurons in vulnerable regions in AD but not control brains. Seladin-1 is a human homolog of the DIMINUTO/DWARF1 gene described in plants and Caenorhabditis elegans. Its sequence shares similarities with flavin-adenin-dinucleotide (FAD)-dependent oxidoreductases. In human control brain, seladin-1 was highly expressed in almost all neurons. In PC12 cell clones that were selected for resistance against AD-associated amyloid-beta peptide (Abeta)-induced toxicity, both mRNA and protein levels of seladin-1 were approximately threefold higher as compared with the non-resistant wild-type cells. Functional expression of seladin-1 in human neuroglioma H4 cells resulted in the inhibition of caspase 3 activation after either Abeta-mediated toxicity or oxidative stress and protected the cells from apoptotic cell death. In apoptotic cells, however, endogenous seladin-1 was cleaved to a 40 kDa derivative in a caspase-dependent manner. These results establish that seladin-1 is an important factor for the protection of cells against Abeta toxicity and oxidative stress, and they suggest that seladin-1 may be involved in the regulation of cell survival and death. Decreased expression of seladin-1 in specific neurons may be a cause for selective vulnerability in AD.

Decreased thioredoxin and increased thioredoxin reductase levels in alzheimer’s disease brain

Increasing evidence supports the role of reactive oxygen species (ROS) in the pathogenesis of Alzheimer’s disease (AD). Both in vivo and in vitro studies demonstrate that thioredoxin (Trx) and thioredoxin reductase (TR), the enzyme responsible for reduction of oxidized Trx, have protective roles against cytotoxicity mediated by the generation of ROS. The present study measured levels of Trx protein and activities of TR in the brain in AD compared with control subjects, and evaluated the possible protective role of TR and Trx against amyloid β-peptide (Aβ) toxicity in neuronal cultures. Analysis of Trx protein levels in 10 AD and 10 control subjects demonstrated a general decrease in all AD brain regions studied, with statistically significant decreases in the amygdala ( p < .05), hippocampus/parahippocampal gyrus ( p < .05), and marginally significant ( p < .10) depletions in the superior and middle temporal gryi. Thioredoxin reductase activity levels were increased in all AD brain regions studied with statistically significant increases occurring in AD amygdala ( p = .01) and cerebellum ( p = .007). To investigate the protective effects of Trx and TR against Aβ-induced toxicity, primary hippocampal cultures were treated with Trx or TR in combination with toxic doses of Aβ. Treatment of cultures with Trx led to a statistically significant concentration-dependent enhancement in cell survival against Aβ-mediated toxicity as did treatment with TR. Together, these data suggest that, although TR is protective against Aβ-mediated toxicity, the increase observed in AD brain offers no protection due to the significant decrease in Trx levels. This decrease in the antioxidant Trx-TR system may contribute to the increased oxidative stress and subsequent neurodegeneration observed in the brain in AD.

The Toxicity of the Alzheimer's β-Amyloid Peptide Correlates with a Distinct Fiber Morphology

In an attempt to elucidate the relationship among aggregation properties, fiber morphology, and cellular toxicity several β-amyloid peptides (Aβ) were prepared according to a standardized procedure. Peptides either carried mutations inside the membrane anchor segment around amino acid position 35 or their carboxy terminus was shortened from 42 to 41, 40, or 39 amino acids. The time-dependent self-assembly of monomeric Aβ into fibers was simultaneously monitored by electron microscopy, circular dichroism spectroscopy, analytical ultracentrifugation, and Aβ-mediated cellular toxicity using the reduction of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to measure cell viability. The transition of Aβ monomers into fibers was analyzed by more than 600 electron micrographs. Distinct morphological changes from seed-like structures to immature and mature fibers were observed. Seeds were of spherical appearance. Immature fibers were typically elongated structures with a rough surface and with varying thickness depending on the Aβ sequence. Mature fibers were characterized by a periodic variation of their thickness along the fiber axis. The proportion of these different structures and the total amount of aggregated Aβ was amino acid sequence-dependent. Wild-type Aβ1–42and its oxidized derivative carrying a methionine sulfoxide residue at position 35 showed the highest rate of fiber formation and exerted toxic activity in the MTT assay at very low nanomolar concentrations. The fibers formed by these two peptides were predominantly of the mature type. In contrast, carboxyl-terminus truncated peptides Aβ1–41, Aβ1–40, and Aβ1–39or most Aβ1–42derivatives mutated around amino acid position 35 showed a reduced aggregation rate, the immature fibers predominated, and the toxicity was orders of magnitude lower. Thus, a correlation can be drawn among the chemical structure, aggregation properties, fiber morphology, and cellular toxicity.