β-Amyloid Neurotoxicity in Human Cortical Culture Is Not Mediated by Excitotoxins

Abstract: β-Amyloid is a metabolic product of the amyloid precursor protein, which accumulates abnormally in senile plaques in the brains of patients with Alzheimer's disease. The neurotoxicity of 0-amyloid has been observed in cell culture and in vivo, but the mechanism of this effect is unclear. In this report, we describe the direct neurotoxicity of β-amyloid in high-density primary cultures of human fetal cortex. In 36-day-old cortical cultures, β-amyloid neurotoxicity was not inhibited by the broad-spectrum excitatory amino acid receptor antagonist kynurenate or the NMDA receptor antagonist D-2-amino-5-phosphonovaleric acid under conditions that inhibited glutamate and NMDA neurotoxicity. In 8-day-old cortical cultures, neurons were resistant to glutamate and NMDA toxicity but were still susceptible to β-amyloid neurotoxicity, which was unaffected by excitatory amino acid receptor antagonists. Treatment with β-amyloid caused chronic neurodegenera-tive changes, including neuronal clumping and dystrophic neurites, whereas glutamate treatment caused rapid neuronal swelling and neurite fragmentation. These results suggest that β-amyloid is directly neurotoxic to primary human cortical neurons by a mechanism that does not involve excitatory amino acid receptors.     

Membrane Currents Induced in Xenopus Oocytes by the C-Terminal Fragment of the β-Amyloid Precursor Protein

Abstract: There is mounting evidence that at least some of the neurotoxicity associated with Alzheimer's disease (AD) is due to proteolytic fragments of the β-amyloid precursor protein (βAPP). Most research has focused on the amyloid β protein (Aβ), which has been shown to possess ion channel activity. However, the possible role of other cleaved products of the βAPP is less clear. We have investigated the ability of various products of βAPP to induce membrane ion currents by applying them to Xenopus oocytes, a model system used extensively for investigating electrophysiological aspects of cellular, including neuronal, signalling. We focussed on the 105-amino-acid C-terminal fragment (CT₁₀₅) (containing the full sequence Aβ), which has previously been found to be toxic to cells, although little is known about its mode of action. We have found that CT₁₀₅ is exceedingly potent, with a threshold concentration of 100–200 n M , in inducing nonselective ion currents when applied from either outside or inside the oocyte and is more effective than either βAPP or the Aβ fragments, β_25–35 or β_1–40. The ion channel activity of CT₁₀₅ was concentration dependent and blocked by a monoclonal antibody to Aβ. These results suggest the possible involvement of CT₁₀₅ in inducing the neural toxicity characteristic of AD.     

Structure-Activity Analyses of β-Amyloid Peptides: Contributions of the β25–35 Region to Aggregation and Neurotoxicity

Abstract: The neurodegeneration of Alzheimer's disease has been theorized to be mediated, at least in part, by insoluble aggregates of β-amyloid protein that are widely distributed in the form of plaques throughout brain regions affected by the disease. Previous studies by our laboratory and others have demonstrated that the neurotoxicity of β-amyloid in vitro is dependent upon its spontaneous adoption of an aggregated structure. In this study, we report extensive structure-activity analyses of a series of peptides derived from both the proposed active fragment of β-amyloid, β25–35, and the full-length protein, β1–42. We examine the effects of amino acid residue deletions and substitutions on the ability of β-amyloid peptides to both form sedimentable aggregates and induce toxicity in cultured hippocampal neurons. We observe that significant levels of peptide aggregation are always associated with significant β-amyloid-induced neurotoxicity. Further, both N- and C-terminal regions of β25–35 appear to contribute to these processes. In particular, significant disruption of peptide aggregation and toxicity result from alterations in the β33–35 region. In β1–42 peptides, aggregation disruption is evidenced by changes in both electrophoresis profiles and fibril morphology visualized at the light and electron microscope levels. Using circular dichroism analysis in a subset of peptides, we observed classic features of β-sheet secondary structure in aggregating, toxic β-amyloid peptides but not in nonaggregating, nontoxic β-amyloid peptides. Together, these data further define the primary and secondary structures of β-amyloid that are involved in its in vitro assembly into neurotoxic peptide aggregates and may underlie both its pathological deposition and subsequent degenerative effects in Alzheimer's disease.     

β-Amyloid Neurotoxicity In Vitro: Evidence of Oxidative Stress but Not Protection by Antioxidants

Abstract: Recent data from several groups suggest that the primary mechanism of β-amyloid neurotoxicity may be mediated by reactive oxygen species. To evaluate this hypothesis, we first compared the efficacy of antioxidant agents in preventing toxicity caused by oxidative insults (iron, hydrogen peroxide, and tert -butyl hydroperoxide) and β-amyloid peptides in cultured rat hippocampal neurons. Tested antioxidants (propyl gallate, Trolox, probucol, and promethazine) generally provided significant protection against oxidative insults but not β-amyloid peptides. Next, we examined whether β-amyloid causes oxidative stress, by comparing levels of lipid peroxidation after exposure to either iron or β-amyloid. In a cell-free system, iron but not β-amyloid generated lipid peroxidation. In culture, both insults caused rapid increases in lipid peroxidation, with iron inducing higher levels at later time points. Pretreatment with the antioxidant probucol significantly reduced lipid peroxidation caused by both insults but only attenuated iron toxicity, suggesting that lipid peroxidation does not contribute directly to cell death induced by β-amyloid. Finally, we observed that increasing basal levels of oxidative stress by pretreating cultures with subtoxic doses of iron significantly increased neuronal vulnerability to β-amyloid. The ability of β-amyloid to induce oxidative stress and the demonstration that oxidative stress potentiates β-amyloid toxicity support the clinical use of antioxidants for AD. However, these data do not support the theory that the primary mechanism of β-amyloid toxicity involves oxidative pathways, indicating a continued need to identify additional cellular responses to β-amyloid that underlie its neurodegenerative actions.     

An Etiological Role of Amyloidogenic Carboxyl-Terminal Fragments of the β-Amyloid Precursor Protein in Alzheimer's Disease

Abstract: Amyloid β protein (Aβ), 39–43 amino acids long, is the principal constituent of the extracellular amyloid deposits in brain that are characteristic of Alzheimer's disease (AD). Several lines of evidence indicate that Aβ may play an important role in the pathogenesis of AD. However, there are several discrepancies between the production of Aβ and the development of the disease. Thus, Aβ may not be the sole active fragment of β-amyloid precursor protein (βAPP) in the neurotoxicity associated with AD. Consequently, the possible effects of other cleaved products of βAPP need to be explored. The recent concentration on other potentially amyloidogenic products of βAPP has produced interesting candidates, the most promising of which are the amyloidogenic carboxyl-terminal (CT) fragments of βAPP. This review discusses a possible etiological role of CT fragments of βAPP in AD.     

Oxidative Stress Induces Dephosphorylation of τ in Rat Brain Primary Neuronal Cultures

Abstract: Oxidative stress and free radical damage have been implicated in the neurodegenerative changes characteristic of several neurodegenerative diseases, including Alzheimer's disease. There is experimental evidence that the neurotoxicity of β-amyloid is mediated via free radicals, and as the deposition of β-amyloid apparently precedes the formation of paired helical filaments (PHF) in Alzheimer's disease, we have investigated whether subjecting primary neuronal cultures to oxidative stress induces changes in the phosphorylation state of the principal PHF protein τ that resemble those found in PHF-τ. Contrary to causing an increase in τ phosphorylation, treatment of neurones with hydrogen peroxide caused a dephosphorylation of τ and so we conclude that oxidative stress is not the direct cause of τ hyperphosphorylation and hence of PHF formation.     

Secreted Forms of β-Amyloid Precursor Protein Protect Against Ischemic Brain Injury

Abstract: The β-amyloid precursor protein (βAPP) is the source of the amyloid β-peptide that accumulates in the brain in Alzheimer's disease. A major processing pathway for βAPP involves an enzymatic cleavage within the amyloid β-peptide sequence that liberates secreted forms of βAPP (APP^Ss) into the extracellular milieu. We now report that postischemic administration of these APP^Ss intracerebroventricularly protects neurons in the CA1 region of rat hippocampus against ischemic injury. Treatment with APP^S695 or APP^S751 resulted in increased neuronal survival, and the surviving cells were functional as demonstrated by their ability to synthesize protein. These data provide direct evidence for a neuroprotective action of APP^Ss in vivo.     

Dysfunction of Cholinergic and Dopaminergic Neuronal Systems in β-Amyloid Protein-Infused Rats

Abstract: Accumulations of β-amyloid protein are characteristic and diagnostic features of the brain of Alzheimer's disease patients; however, the physiological role of this protein in CNS is unknown. We have previously reported that continuous infusion of β-amyloid protein into rat cerebral ventricle impairs learning ability and decreases choline acetyltransferase activity, a marker enzyme of cholinergic neuron. In this study, the effects of β-amyloid protein infusion on the release of neurotransmitters in cholinergic and dopaminergic neuronal systems were investigated by using an in vivo brain microdialysis method. Nicotine-stimulated release of acetylcholine and dopamine in these animals was significantly lower than that in vehicle-infused rats. Further, dopamine release induced by high-K stimulation was decreased in β-amyloid protein-infused rats compared with vehicle-infused rats. These results suggest that the release of the two transmitters, acetylcholine and dopamine, was decreased by β-amyloid protein and that learning deficits observed in the β-amyloid protein-infused rats are partly due to the impairment of neurotransmitter release. Furthermore, continuous infusion of β-amyloid protein may be a useful method to produce the animal model of Alzheimer's disease.     

Neurotoxicity of a Carboxyl-Terminal Fragment of the Alzheimer's Amyloid Precursor Protein

Abstract: We have previously shown that a recombinant carboxyl-terminal 105-amino-acid fragment (CT105) of the amyloid precursor protein (APP) induced strong non-selective inward currents in Xenopus oocytes. Here we investigated the toxic effect of CT105 peptide on the cultured mammalian cells. The CT105 peptide induced a significant lactate dehydrogenase (LDH) release from cultured rat cortical neurons and PC12 cells in a concentration (from 10 µ M )- and time (from 48 h)-dependent manner. The toxic effect of CT105 was more potent than that of any fragments of amyloid β protein (Aβ). However, CT105 peptide did not affect the viability of U251 human glioblastoma cells. In contrast to CT105, Aβ increased LDH release only slightly even at 50 µ M but significantly inhibited 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction at submicromolar concentrations. Among the various neuroprotective drugs tested, only cholesterol, which alters membrane fluidity, could attenuate the cytotoxicity of CT105 significantly. The CT105 peptide formed multiple self-aggregates on solubilization. Pretreatment with a sublethal concentration of CT105 did not significantly alter the susceptibility of cells to hydrogen peroxide and glutamate. Endogenous CT peptides were found not only in the cell lysates but also in the conditioned medium of PC12 cells. These results imply that CT peptide can directly attack the cell membrane probably by making pores or nonselective ion channels, whereas Aβ impairs the intracellular metabolic pathway first. Thus, it is thought that both CT and Aβ, which are formed during the processing of APP, may participate in the neuronal degeneration in Alzheimer's disease by different mechanisms.     

Thrombin Attenuates Neuronal Cell Death and Modulates Astrocyte Reactivity Induced by β-Amyloid In Vitro

Abstract: β-Amyloid protein has been implicated as a potential causative agent in the neuropathology associated with Alzheimer's disease. This possibility is supported by observations that β-amyloid induces neuronal degeneration and astrocyte reactivity in vitro by as yet undefined mechanism(s). In this report, we present data demonstrating that the pathological effects of β-amyloid on cultured cells are modulated by activation of the thrombin receptor. At concentrations between 50 and 500 n M , thrombin pretreatment significantly attenuates neurotoxicity mediated by fibrillar aggregates of β1–42 and β25–35 peptides. In cultured astrocytes, the stellate morphology induced by β1–42 and β25–35 aggregates can be prevented and reversed by thrombin exposures between 10 p M and 1 µ M . In contrast, thrombin potentiates rather than attenuates the β-amyloid-induced increased expression of basic fibroblast growth factor, suggesting that thrombin differentially modulates the effects of β-amyloid on astrocytes. Thrombin's effects on both neurons and astrocytes are mimicked by thrombin receptor-activating peptide and inhibited by two potent thrombin inhibitors, hirudin and protease nexin-1. These data provide both new insight into the signaling pathways underlying the cellular effects of β-amyloid and additional support for the role of thrombin as an important mediator of neuropathological events.     

Rapid Communication: Ca2+ Channel Blockers Attenuate β-Amyloid Peptide Toxicity to Cortical Neurons in Culture

Abstract: Deposit of β-amyloid protein (Aβ) in Alzheimer's disease brain may contribute to the associated neurodegeneration. We have studied the neurotoxicity of Aβ in primary cultures of murine cortical neurons, with the aim of identifying pharmacologic ways of attenuating the injury. Exposure of cultures to Aβ (25–35 fragment; 3–25 4mU M ) generally triggers slow, concentration-dependent neurodegeneration (over 24–72 h). With submaximal Aβ- (25–35) exposure (10 μ M ), substantial (>40% within 48 h) degeneration often occurs and is markedly attenuated by the presence of the Ca²⁺ channel blockers nimodipine (1–20 μ M ) and Co²⁺ (100 μ M ) during the Aβ exposure. However, Aβ neurotoxicity is not affected by the presence of glutamate receptor antagonists. We suggest that Ca²⁺ influx through voltage-gated Ca²⁺ channels may contribute to Aβ-induced neuronal injury and that nimodipine and Co²⁺, by attenuating such influx, are able to attenuate Aβ neurotoxicity.     

Complement component C1q inhibits beta-amyloid- and serum amyloid P-induced neurotoxicity via caspase- and calpain-independent mechanisms

Alzheimer's disease is a neurodegenerative disorder characterized by neuronal loss, β-amyloid (Aβ) plaques, and neurofibrillary tangles. Complement protein C1q has been found associated with fibrillar Aβ deposits, however the exact contributions of C1q to Alzheimer's disease is still unknown. There is evidence that C1q, as an initiator of the inflammatory complement cascade, may accelerate disease progression. However, neuronal C1q synthesis is induced after injury/infection suggesting that it may be a beneficial response to injury. In this study, we report that C1q enhances the viability of neurons in culture and protects neurons against Aβ- and serum amyloid P (SAP)-induced neurotoxicity. Investigation of potential signaling pathways indicates that caspase and calpain are activated by Aβ, but C1q had no effect on either of these pathways. Interestingly, SAP did not induce caspase and calpain activation, suggesting that C1q neuroprotection is in distinct from caspase and calpain pathways. In contrast to Aβ- and SAP-induced neurotoxicity, neurotoxicity induced by etoposide or FCCP was unaffected by the addition of C1q, indicating pathway selectivity for C1q neuroprotection. These data support a neuroprotective role for C1q which should be further investigated to uncover mechanisms which may be therapeutically targeted to slow neurodegeneration via direct inhibition of neuronal loss.     

Defining pre-synaptic nicotinic receptors regulated by beta amyloid in mouse cortex and hippocampus with receptor null mutants

Disruption of neuronal signaling by soluble β-amyloid has been implicated in deficits in short-term recall in the early stages of Alzheimer's disease. One potential target for β-amyloid is the synapse, with evidence for differential interaction with both pre- and post-synaptic elements. Our previous work revealed an agonist-like action of soluble β-amyloid (pM to nM) on isolated pre-synaptic terminals to increase [Ca²⁺]i, with apparent involvement of pre-synaptic nicotinic receptors. To directly establish the role of nicotinic receptors in pre-synaptic Ca²⁺ regulation, we investigated the pre-synaptic action of β-amyloid on terminals isolated from mice harboring either β2 or α7 nicotinic receptor null mutants (knockouts). Average pre-synaptic responses to β-amyloid in hippocampal terminals of α7 knockout mice were unchanged, whereas responses in hippocampal terminals from β2 knockout mice were strongly attenuated. In contrast, pre-synaptic responses to soluble β-amyloid were strongly attenuated in cortical terminals from α7 knockout mice but were moderately attenuated in cortical terminals from β2 knockout mice. The latter responses, having distinct kinetics, were completely blocked by α-bungarotoxin. The use of receptor null mutants thus permitted direct demonstration of the involvement of specific nicotinic receptors in pre-synaptic Ca²⁺ regulation by soluble β-amyloid, and also indicated differential neuromodulation by β-amyloid of synapses in hippocampus and cortex.     

The Y-Box Binding Protein 1 Suppresses Alzheimer’s Disease Progression in Two Animal Models

The Y-box binding protein 1 (YB-1) is a member of the family of DNA- and RNA binding proteins. It is involved in a wide variety of DNA/RNA-dependent events including cell proliferation and differentiation, stress response, and malignant cell transformation. Previously, YB-1 was detected in neurons of the neocortex and hippocampus, but its precise role in the brain remains undefined. Here we show that subchronic intranasal injections of recombinant YB-1, as well as its fragment YB-1₁₋₂₁₉, suppress impairment of spatial memory in olfactory bulbectomized (OBX) mice with Alzheimer’s type degeneration and improve learning in transgenic 5XFAD mice used as a model of cerebral amyloidosis. YB-1-treated OBX and 5XFAD mice showed a decreased level of brain β-amyloid. In OBX animals, an improved morphological state of neurons was revealed in the neocortex and hippocampus; in 5XFAD mice, a delay in amyloid plaque progression was observed. Intranasally administered YB-1 penetrated into the brain and could enter neurons. In vitro co-incubation of YB-1 with monomeric β-amyloid (1–42) inhibited formation of β-amyloid fibrils, as confirmed by electron microscopy. This suggests that YB-1 interaction with β-amyloid prevents formation of filaments that are responsible for neurotoxicity and neuronal death. Our data are the first evidence for a potential therapeutic benefit of YB-1 for treatment of Alzheimer’s disease.     

Application of Surface-Enhanced Raman Spectroscopy for Detection of Beta Amyloid Using Nanoshells

Currently, no methods exist for the definitive diagnosis of AD premortem. β-amyloid, the primary component of the senile plaques found in patients with this disease, is believed to play a role in its neurotoxicity. We are developing a nanoshell substrate, functionalized with sialic acid residues to mimic neuron cell surfaces, for the surface-enhanced Raman detection of β-amyloid. It is our hope that this sensing mechanism will be able to detect the toxic form of β-amyloid, with structural and concentration information, to aid in the diagnosis of AD and provide insight into the relationship between β-amyloid and disease progression. We have been successfully able to functionalize the nanoshells with the sialic acid residues to allow for the specific binding of β-amyloid to the substrate. We have also shown that a surface-enhanced Raman spectroscopy response using nanoshells is stable and concentration-dependent with detection into the picomolar range.     

Actions of Neurotoxic β-Amyloid on Calcium Homeostasis and Viability of PC12 Cells Are Blocked by Antioxidants but Not by Calcium Channel Antagonists

Abstract: The fragment of β-amyloid comprised of amino acids 25–35 induces a rapid, concentration-dependent increase in cytosolic free calcium levels in suspensions of PC12 neuronal cells. This action of β-amyloid 25–35 is not altered by pretreatment with the calcium channel blockers nifedipine or cobalt, with the depleter of intracellular calcium stores cyclopiazonic acid, or with the phospholipase C inhibitor neomycin. However, the effects of β-amyloid 25–35 on cytosolic free calcium are absent in calcium-free buffer and are blocked by the antioxidant lazaroid U-83836E and by vitamin E. β-Amyloid 25–35 is also neurotoxic and produces a concentration-dependent reduction in the viability of PC12 cells in culture. The neurotoxic action of β-amyloid is blocked by U-83836E and vitamin E but not by nifedipine or cobalt. These data indicate that both the disruption of calcium homeostasis and the reduction of cell viability produced by β-amyloid in PC12 cells are mediated by free radical-based processes.     

β-Amyloid Protein Precursor and τ mRNA Levels Versus β-Amyloid Plaque and Neurofibrillary Tangles in the Aged Human Brain

Abstract: To learn whether or not the levels of β-amyloid protein precursor (APP) and τ mRNAs are related to the formation of β-amyloid and neurofibrillary tangles, we quantified these mRNA levels in three cortical regions of 38 aged human brains, which were examined immunocyto-chemically for β-amyloid and tangles. Marked individual variabilities were noted in APP and τ mRNA levels among elderly individuals. The mean APP mRNA level was slightly reduced in the β-amyloid plaque (++) group, but not in the plaque (+) group, compared to the plaque (−) group. Some brains in the plaque (−) group showed increased APP expression, the extent of which was not seen in the plaque (+)or(++) group. The differences in the mean τ mRNA levels were not statistically significant among the tangle (−), (+), and (++) groups. These results show that β-protein and τ deposition do not accompany increased expression of the APP and τ genes, respectively, and thus suggest that factors other than gene expression may be at work in the progression of β-amyloid and/or tangle formation in the aged human brain.     

Enhancement of β-Amyloid Peptide Aβ(1–40)-Mediated Neurotoxicity by Glutamine Synthetase

Abstract: The β-amyloid peptide (Aβ), a main constituent in both senile and diffuse plaques in Alzheimer's disease brains, was previously shown to be neurotoxic and to be able to interact with several macromolecular components of brain tissue. Previous investigations carried out in our laboratory demonstrated free radical species formation in aqueous solutions of Aβ(1–40) and its C-end fragment, Aβ(25–35). Toxic forms of Aβ rapidly inactivate the oxidation-sensitive cytosolic enzyme glutamine synthetase (GS). In this regard, we suggested and subsequently demonstrated that Aβ radicals can cause an oxidative damage of cell proteins and lipids resulting in disruption of membrane functions, enzyme inactivation, and cell death. Because GS can be a substrate for Aβ-derived oxidizing species, the present study was conducted to determine if GS could protect against Aβ neurotoxicity. In contrast to this initial hypothesis, we here report that GS significantly enhances the neurotoxic effects of Aβ(1–40). The Aβ-mediated inactivation of GS was found to be accompanied by the loss of immunoreactive GS and the significant increase of Aβ(1–40) neurotoxicity.     

S100β Increases Levels of β-Amyloid Precursor Protein and Its Encoding mRNA in Rat Neuronal Cultures

Abstract: S100β has been implicated in the formation of dystrophic neurites, overexpressing β-amyloid precursor protein (βAPP), in the β-amyloid plaques of Alzheimer's disease. We assessed the effects of S100β on cell viability of, neurite outgrowth from, and βAPP expression by neurons in primary cultures from fetal rat cortex. S100β (1–10 ng/ml) enhanced neuronal viability (as assessed by increased mitochondrial activity and decreased lactic acid dehydrogenase release) and promoted neurite outgrowth. Higher levels of S100β (100 ng/ml, but not 1 µg/ml) produced qualitatively similar, but less marked, effects. S100β also induced increased neuronal expression of the microtubule-associated protein MAP2, an effect that is consistent with trophic effects of S100β on neurite outgrowth. S100β (10 and 100 ng/ml) induced graded increases in neuronal expression of βAPP and of βAPP mRNA. These results support our previous suggestion that excessive expression of S100β by activated, plaque-associated astrocytes in Alzheimer's disease contributes to the appearance of dystrophic neurites overexpressing βAPP in diffuse amyloid deposits, and thus to the conversion of these deposits into the diagnostic neuritic β-amyloid plaques.     

Aβ Toxicity in Alzheimer's Disease

Alzheimer's Disease (AD), the most common age-related neurodegenerative disorder, is characterized by progressive cognitive decline, synaptic loss, the formation of extracellular β-amyloid plaques and intracellular neurofibrillary tangles, and neuronal cell death. Despite the massive neuronal loss in the ‘late stage’ of disease, dendritic spine loss represents the best pathological correlate to the cognitive impairment in AD patients. The ‘amyloid hypothesis’ of AD recognizes the Aβ peptide as the principal player in the pathological process. Many lines of evidence point out to the neurotoxicity of Aβ, highlighting the correlation between soluble Aβ oligomer accumulation, rather than insoluble Aβ fibrils and disease progression. Pathological increase of Aβ in AD brains, resulting from an imbalance between its production, aggregation and clearance, might target mitochondrial function promoting a progressive synaptic impairment. The knowledge of the exact mechanisms by which Aβ peptide impairs neuronal function will help us to design new pharmacological tools for preventing AD neurodegeneration.