Intraneuronal amyloid-beta1-42 production triggered by sustained increase of cytosolic calcium concentration induces neuronal death

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the presence in the brain of senile plaques which contain an amyloid core made of beta-amyloid peptide (Abeta). Abeta is produced by the cleavage of the amyloid precursor protein (APP). Since impairment of neuronal calcium signalling has been causally implicated in ageing and AD, we have investigated the influence of an influx of extracellular calcium on the metabolism of human APP in rat cortical neurones. We report that a high cytosolic calcium concentration, induced by neuronal depolarization, inhibits the alpha-secretase cleavage of APP and triggers the accumulation of intraneuronal C-terminal fragments produced by the beta-cleavage of the protein (CTFbeta). Increase in cytosolic calcium concentration specifically induces the production of large amounts of intraneuronal Abeta1-42, which is inhibited by nimodipine, a specific antagonist of l-type calcium channels. Moreover, calcium release from endoplasmic reticulum is not sufficient to induce the production of intraneuronal Abeta, which requires influx of extracellular calcium mediated by the capacitative calcium entry mechanism. Therefore, a sustained high concentration of cytosolic calcium is needed to induce the production of intraneuronal Abeta1-42 from human APP. Our results show that this accumulation of intraneuronal Abeta1-42 induces neuronal death, which is prevented by a functional gamma-secretase inhibitor.     

Caspase-6 role in apoptosis of human neurons, amyloidogenesis, and Alzheimer's disease.

Neuronal cell death, neurofibrillary tangles, and amyloid beta peptide (Abeta) deposition depict Alzheimer's disease (AD) pathology, but neuronal loss correlates best with dementia. We have shown that increased production of Abeta is a consequence of neuronal apoptosis, suggesting that apoptosis activates proteases involved in amyloid precursor protein (APP) processing. Here, we investigate key effectors of cell death, caspases, in human neuronal apoptosis and APP processing. We find that caspase-6 is activated and responsible for neuronal apoptosis by serum deprivation. Caspase-6 activity precedes the time of commitment to neuronal apoptosis by 10 h, indicating possible activity without subsequent apoptosis. Inhibition of caspase-6 activity prevents serum deprivation-mediated increase of Abeta. Caspase-6 directly cleaves APP at the C terminus and generates a C-terminal fragment of 3 kDa (Capp3) and an Abeta-containing 6.5-kDa fragment, Capp6.5, that increases in serum-deprived neurons. A pulse-chase experiment reveals a precursor-product relationship between Capp6.5, intracellular Abeta, and secreted Abeta, indicating a potential alternate amyloidogenic pathway. Caspase-6 proenzyme is present in adult human brain tissue, and the p10 active caspase-6 fragment is detected in AD brain tissue. These results indicate a possible alternate pathway for APP amyloidogenic processing in human neurons and a potential implication for this pathway in the neuronal demise of AD.     

Caspase cleavage of the amyloid precursor protein modulates amyloid beta-protein toxicity

The amyloid beta-protein precursor (APP) is proteolytically cleaved to generate the amyloid beta-protein (Abeta), the principal constituent of senile plaques found in Alzheimer's disease (AD). In addition, Abeta in its oligomeric and fibrillar forms have been hypothesized to induce neuronal toxicity. We and others have previously shown that APP can be cleaved by caspases at the C-terminus to generate a potentially cytotoxic peptide termed C31. Furthermore, this cleavage event and caspase activation were increased in the brains of AD, but not control, cases. In this study, we show that in cultured cells, Abeta induces caspase cleavage of APP in the C-terminus and that the subsequent generation of C31 contributes to the apoptotic cell death associated with Abeta. Interestingly, both Abeta toxicity and C31 pathway are dependent on the presence of APP. Both APP-dependent Abeta toxicity and C31-induced apoptotic cell death involve apical or initiator caspases-8 and -9. Our results suggest that Abeta-mediated toxicity initiates a cascade of events that includes caspase activation and APP cleavage. These findings link C31 generation and its potential cell death activity to Abeta cytotoxicity, the leading mechanism proposed for neuronal death in AD.     

Intracellular amyloid-beta 1-42, but not extracellular soluble amyloid-beta peptides, induces neuronal apoptosis

Alzheimer disease (AD), the most frequent cause of dementia, is characterized by an important neuronal loss. A typical histological hallmark of AD is the extracellular deposition of beta-amyloid peptide (A beta), which is produced by the cleavage of the amyloid precursor protein (APP). Most of the gene mutations that segregate with the inherited forms of AD result in increasing the ratio of A beta 42/A beta 40 production. A beta 42 also accumulates in neurons of AD patients. Altogether, these data strongly suggest that the neuronal production of A beta 42 is a critical event in AD, but the intraneuronal A beta 42 toxicity has never been demonstrated. Here, we report that the long term expression of human APP in rat cortical neurons induces apoptosis. Although APP processing leads to production of extracellular A beta 1-40 and soluble APP, these extracellular derivatives do not induce neuronal death. On the contrary, neurons undergo apoptosis as soon as they accumulate intracellular A beta 1-42 following the expression of full-length APP or a C-terminal deleted APP isoform. The inhibition of intraneuronal A beta 1-42 production by a functional gamma-secretase inhibitor increases neuronal survival. Therefore, the accumulation of intraneuronal A beta 1-42 is the key event in the neurodegenerative process that we observed.     

Molecular mechanisms for neuronal cell death by Alzheimer's amyloid precursor protein-relevant insults

To develop a therapeutic intervention for Alzheimer's disease (AD), it is necessary to clarify the mechanisms underlying the pathogenesis of AD, in which senile plaques, neurofibrillary tangles and neuronal loss in the cerebrum are the central abnormalities. A number of studies have focused on the major component of the senile plaques, which is amyloid-beta (Abeta) and its precursor protein APP, and have investigated the roles of these molecules in the onset, progression and inhibition of AD. For multiple reasons, however, their roles in AD, especially in neuronal death, remain elusive and a unified concept for their roles has not yet been established. Recently, it has been found that APP functions normally as a neuronal surface transmembrane protein. In this article, we review the molecular mechanisms of neuronal cell death by these APP-relevant insults and discuss the functions of APP in regard to intracellular signal transducers, including c-Jun N-terminal kinase. We also revise the roles of Abeta in neuronal death and survival.     

Triple-transgenic model of Alzheimer's disease with plaques and tangles: intracellular Abeta and synaptic dysfunction

The neuropathological correlates of Alzheimer's disease (AD) include amyloid-beta (Abeta) plaques and neurofibrillary tangles. To study the interaction between Abeta and tau and their effect on synaptic function, we derived a triple-transgenic model (3xTg-AD) harboring PS1(M146V), APP(Swe), and tau(P301L) transgenes. Rather than crossing independent lines, we microinjected two transgenes into single-cell embryos from homozygous PS1(M146V) knockin mice, generating mice with the same genetic background. 3xTg-AD mice progressively develop plaques and tangles. Synaptic dysfunction, including LTP deficits, manifests in an age-related manner, but before plaque and tangle pathology. Deficits in long-term synaptic plasticity correlate with the accumulation of intraneuronal Abeta. These studies suggest a novel pathogenic role for intraneuronal Abeta with regards to synaptic plasticity. The recapitulation of salient features of AD in these mice clarifies the relationships between Abeta, synaptic dysfunction, and tangles and provides a valuable model for evaluating potential AD therapeutics as the impact on both lesions can be assessed.     

Downregulation of myosin II-B by siRNA alters the subcellular localization of the amyloid precursor protein and increases amyloid-beta deposition in N2a cells

The Alzheimer's disease (AD) brain pathology is characterized by extracellular deposits of amyloid-beta (Abeta) peptides and intraneuronal fibrillar structures. These pathological features may be functionally linked, but the mechanism by which Abeta accumulation relates to neuronal degeneration is still poorly understood. Abeta peptides are fragments cleaved from the amyloid precursor protein (APP), a transmembrane protein ubiquitously expressed in the nervous system. Although the proteolytic processing of APP has been implicated in AD, the physiological function of APP and the subcellular site of APP cleavages remain unknown. The overall structure of the protein and its fast anterograde transport along the axon support the idea that APP functions as a vesicular receptor for cytoskeletal motor proteins. In the current study, we test the hypothesis that myosin II, important contributor to the cytoskeleton of neuronal cells, may influence the trafficking and/or the processing of APP. Our results demonstrate that downregulation of myosin II-B, the major myosin isoform in neurons, is able to increase Abeta deposition, concomitantly altering the subcellular localization of APP. These new insights might be important for the understanding of the function of APP and provide a novel conceptual framework in which to analyze its pathological role.     

Alternative, non-secretase processing of Alzheimer's beta-amyloid precursor protein during apoptosis by caspase-6 and -8.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder. Although the pathogenesis of AD is unknown, it is widely accepted that AD is caused by extracellular accumulation of a neurotoxic peptide, known as Abeta. Mutations in the beta-amyloid precursor protein (APP), from which Abeta arises by proteolysis, are associated with some forms of familial AD (FAD) and result in increased Abeta production. Two other FAD genes, presenilin-1 and -2, have also been shown to regulate Abeta production; however, studies examining the biological role of these FAD genes suggest an alternative theory for the pathogenesis of AD. In fact, all three genes have been shown to regulate programmed cell death, hinting at the possibility that dysregulation of apoptosis plays a primary role in causing neuronal loss in AD. In an attempt to reconcile these two hypotheses, we investigated APP processing during apoptosis and found that APP is processed by the cell death proteases caspase-6 and -8. APP is cleaved by caspases in the intracellular portion of the protein, in a site distinct from those processed by secretases. Moreover, it represents a general effect of apoptosis, because it occurs during cell death induced by several stimuli both in T cells and in neuronal cells.     

HH Domain of Alzheimer’s Disease Aβ Provides Structural Basis for Neuronal Binding in PC12 and Mouse Cortical/Hippocampal Neurons

A key question in understanding AD is whether extracellular Aβ deposition of parenchymal amyloid plaques or intraneuronal Aβ accumulation initiates the AD process. Amyloid precursor protein (APP) is endocytosed from the cell surface into endosomes where it is cleaved to produce soluble Aβ which is then released into the brain interstitial fluid. Intraneuronal Aβ accumulation is hypothesized to predominate from the neuronal uptake of this soluble extracellular Aβ rather than from ER/Golgi processing of APP. We demonstrate that substitution of the two adjacent histidine residues of Aβ40 results in a significant decrease in its binding with PC12 cells and mouse cortical/hippocampal neurons. These substitutions also result in a dramatic enhancement of both thioflavin-T positive fibril formation and binding to preformed Aβ fibrils while maintaining its plaque-binding ability in AD transgenic mice. Hence, alteration of the histidine domain of Aβ prevented neuronal binding and drove Aβ to enhanced fibril formation and subsequent amyloid plaque deposition - a potential mechanism for removing toxic species of Aβ. Substitution or even masking of these Aβ histidine residues might provide a new therapeutic direction for minimizing neuronal uptake and subsequent neuronal degeneration and maximizing targeting to amyloid plaques.     

Proteases and lipoprotein receptors in Alzheimer's disease

Alzheimer's disease (AD) is the leading cause of senile dementia, and is a complex disorder. The pathological hallmarks of AD were discovered by Dr. Alois Alzheimer in 1907, and include deposits of amyloid or senile plaques and neurofibrillar tangles. Plaques are composed of a peptide, termed the Abeta peptide, that is derived by proteolytic processing of the amyloid precursor protein (APP), while neurofibrillar tangles result from a hyperphosphorylation of the tau protein. Mechanisms associated with the formation of plaques and neurofibrillar tangles and their respective contributions to the disease process have been intensely investigated. Proteolytic processing of APP that results in the generation of the Abeta peptide is now well understood and is influenced by several proteins. Recent evidence suggests that the Abeta levels are carefully regulated, and several proteases play an important role in removing the Abeta peptide. Finally, it is becoming apparent that several members of the LDL receptor family play important roles in the brain, and may modulate the course of AD.     

Molecular and cellular mechanisms for Alzheimer's disease: understanding APP metabolism

Alzheimer's disease (AD) is the most common neurodegenerative disease associated with aging. One important pathologic feature of AD is the formation of extracellular senile plaques in the brain, whose major components are small peptides called beta-amyloid (Abeta) that are derived from beta-amyloid precursor protein (APP) through sequential cleavages by beta-secretase and gamma-secretase. Because of the critical role of Abeta in the pathogenesis of AD, unraveling the cellular and molecular events underlying APP/Abeta metabolism has been and remains, of paramount importance to AD research. In this article we will focus on the regulation of APP metabolism leading to Abeta generation. We will review current knowledge of the secretases (alpha-, beta-, and gamma-secretases) involved in APP processing and various molecular and cellular mechanisms underlying intracellular trafficking of APP, which is a highly regulated process and whose disturbance has direct impacts on the production of Abeta.     

Molecular dissection of the interaction between amyloid precursor protein and its neuronal trafficking receptor SorLA/LR11

SorLA/LR11 is a sorting receptor that regulates the intracellular transport and processing of the amyloid precursor protein (APP) in neurons. SorLA/LR11-mediated binding results in sequestration of APP in the Golgi and in protection from processing into the amyloid-beta peptide (Abeta), the principal component of senile plaques in Alzheimer's disease (AD). To gain insight into the molecular mechanisms governing sorLA and APP interaction, we have dissected the respective protein interacting domains. Using a fluorescence resonance energy transfer (FRET) based assay of protein proximity, we identified binding sites in the extracellular regions of both proteins. Fine mapping by surface plasmon resonance analysis and analytical ultracentrifugation of recombinant APP and sorLA fragments further narrowed down the binding domains to the cluster of complement-type repeats in sorLA that forms a 1:1 stoichiometric complex with the carbohydrate-linked domain of APP. These data shed new light on the molecular determinants of neuronal APP trafficking and processing and on possible targets for intervention with senile plaque formation in patients with AD.     

RAC1 inhibition targets amyloid precursor protein processing by gamma-secretase and decreases Abeta production in vitro and in vivo

beta-Amyloid peptides (Abeta) that form the senile plaques of Alzheimer disease consist mainly of 40- and 42-amino acid (Abeta 40 and Abeta 42) peptides generated from the cleavage of the amyloid precursor protein (APP). Generation of Abeta involves beta-secretase and gamma-secretase activities and is regulated by membrane trafficking of the proteins involved in Abeta production. Here we describe a new small molecule, EHT 1864, which blocks the Rac1 signaling pathways. In vitro, EHT 1864 blocks Abeta 40 and Abeta 42 production but does not impact sAPPalpha levels and does not inhibit beta-secretase. Rather, EHT 1864 modulates APP processing at the level of gamma-secretase to prevent Abeta 40 and Abeta 42 generation. This effect does not result from a direct inhibition of the gamma-secretase activity and is specific for APP cleavage, since EHT 1864 does not affect Notch cleavage. In vivo, EHT 1864 significantly reduces Abeta 40 and Abeta 42 levels in guinea pig brains at a threshold that is compatible with delaying plaque accumulation and/or clearing the existing plaque in brain. EHT 1864 is the first derivative of a new chemical series that consists of candidates for inhibiting Abeta formation in the brain of AD patients. Our findings represent the first pharmacological validation of Rac1 signaling as a target for developing novel therapies for Alzheimer disease.     

Partial reduction of BACE1 has dramatic effects on Alzheimer plaque and synaptic pathology in APP Transgenic Mice

The aspartyl protease beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) initiates processing of amyloid precursor protein (APP) into amyloid beta (Abeta) peptide, the major component of Alzheimer disease (AD) plaques. To determine the role that BACE1 plays in the development of Abeta-driven AD-like pathology, we have crossed PDAPP mice, a transgenic mouse model of AD overexpressing human mutated APP, onto mice with either a homozygous or heterozygous BACE1 gene knockout. Analysis of PDAPP/BACE(-/-) mice demonstrated that BACE1 is absolutely required for both Abeta generation and the development of age-associated plaque pathology. Furthermore, synaptic deficits, a neurodegenerative pathology characteristic of AD, were also reversed in the bigenic mice. To determine the extent of BACE1 reduction required to significantly inhibit pathology, PDAPP mice having a heterozygous BACE1 gene knock-out were evaluated for Abeta generation and for the development of pathology. Although the 50% reduction in BACE1 enzyme levels caused only a 12% decrease in Abeta levels in young mice, it nonetheless resulted in a dramatic reduction in Abeta plaques, neuritic burden, and synaptic deficits in older mice. Quantitative analyses indicate that brain Abeta levels in young APP transgenic mice are not the sole determinant for the changes in plaque pathology mediated by reduced BACE1. These observations demonstrate that partial reductions of BACE1 enzyme activity and concomitant Abeta levels lead to dramatic inhibition of Abeta-driven AD-like pathology, making BACE1 an excellent target for therapeutic intervention in AD.     

Correlation between beta-amyloid peptide production and human APP-induced neuronal death

The production of amyloid peptide (Abeta) from its precursor (APP) plays a key role in Alzheimer's disease (AD). However, the link between Abeta production and neuronal death remains elusive. We studied the biological effects associated with human APP expression and metabolism in rat cortical neurons. Human APP expressed in neurons is processed to produce Abeta and soluble APP. Moreover, human APP expression triggers neuronal death. Pepstatin A, an inhibitor of aspartyl proteases that reduces Abeta production, protects neurons from APP-induced neurotoxicity. This suggests that Abeta production is likely to be the critical event in the neurodegenerative process of AD.     

Review on the APP/PS1KI mouse model: intraneuronal Abeta accumulation triggers axonopathy, neuron loss and working memory impairment

Accumulating evidence points to an important role of intraneuronal Abeta as a trigger of the pathological cascade of events leading to neurodegeneration and eventually to Alzheimer's disease (AD) with its typical clinical symptoms, like memory impairment and change in personality. As a new concept, intraneuronal accumulation of Abeta instead of extracellular Abeta deposition has been introduced to be the disease-triggering event in AD. The present review compiles current knowledge on the amyloid precursor protein (APP)/PS1KI mouse model with early and massive intraneuronal Abeta42 accumulation: (1) The APP/PS1KI mouse model exhibits early robust brain and spinal cord axonal degeneration and hippocampal CA1 neuron loss. (2) At the same time-point, a dramatic, age-dependent reduced ability to perform working memory and motor tasks is observed. (3) The APP/PS1KI mice are smaller and show development of a thoracolumbar kyphosis, together with an incremental loss of body weight. (4) Onset of the observed behavioral alterations correlates well with robust axonal degeneration in brain and spinal cord and with abundant hippocampal CA1 neuron loss.     

Regulation of Alzheimer beta-amyloid precursor trafficking and metabolism

Alzheimer's disease (AD) is characterized by the intracranial accumulation of the 4 kDa amyloid-beta peptide (Abeta), following proteolysis of a approximately 700-amino acid, integral membrane precursor, the Alzheimer amyloid precursor protein (APP). The best evidence causally linking APP to AD has been provided by the discovery of mutations within the APP coding sequence that segregate with disease phenotypes in autosomal dominant forms of familial AD (FAD). Though FAD is rare ( < 10% of all AD), the hallmark features (amyloid plaques, neurofibrillary tangles, synaptic and neuronal loss, neurotransmitter deficits and dementia) are indistinguishable when FAD is compared with typical, common, 'non-familial', or sporadic, AD (SAD). Studies of some clinically relevant mutant APP molecules from FAD families have yielded evidence that APP mutations can lead to the enhanced generation or aggregability of Abeta, consistent with a pathogenic role in AD. Other genetic loci for FAD have been discovered which are distinct from the immediate regulatory and coding regions of the APP gene, indicating that defects in molecules other than APP can also specify cerebral amyloidogenesis and FAD. To date, all APP and non-APP FAD mutations can be demonstrated to have the common feature of promoting amyloidogenesis of Abeta. Epidemiological studies indicate that postmenopausal women on estrogen replacement therapy (ERT) have their relative risk of developing SAD diminished by about one third as compared with age-matched women not receiving ERT [M.X. Tang, D. Jacobs, Y. Stern, K. Marder, P. Schofield, B. Gurland, H. Andrews, R. Mayeux, Effect of estrogen during menopause on risk and age at onset of Alzheimer's disease, Lancet 348 (2000) 429432]. Because of the key role of cerebral Abeta accumulation in initiating AD pathology, it is most attractive that estradiol might modulate SAD risk or age-at-onset by inhibiting Abeta accumulation. A possible mechanistic basis for such a scenario is reviewed here.     

Detection of Neuritic Plaques in Alzheimer's Disease Mouse Model

Alzheimer''s disease (AD) is the most common neurodegenerative disorder leading to dementia. Neuritic plaque formation is one of the pathological hallmarks of Alzheimer''s disease. The central component of neuritic plaques is a small filamentous protein called amyloid β protein (Aβ)¹, which is derived from sequential proteolytic cleavage of the beta-amyloid precursor protein (APP) by β-secretase and γ-secretase. The amyloid hypothesis entails that Aγ-containing plaques as the underlying toxic mechanism in AD pathology². The postmortem analysis of the presence of neuritic plaque confirms the diagnosis of AD. To further our understanding of Aγ neurobiology in AD pathogenesis, various mouse strains expressing AD-related mutations in the human APP genes were generated. Depending on the severity of the disease, these mice will develop neuritic plaques at different ages. These mice serve as invaluable tools for studying the pathogenesis and drug development that could affect the APP processing pathway and neuritic plaque formation. In this protocol, we employ an immunohistochemical method for specific detection of neuritic plaques in AD model mice. We will specifically discuss the preparation from extracting the half brain, paraformaldehyde fixation, cryosectioning, and two methods to detect neurotic plaques in AD transgenic mice: immunohistochemical detection using the ABC and DAB method and fluorescent detection using thiofalvin S staining method.     

Estrogen-induced cell signalling in a cellular model of Alzheimer's disease

Alzheimer's disease (AD) is characterised by deposition of a 4 kDa amyloid-beta peptide (Abeta) into senile plaques of the affected brain. Abeta is a proteolytic product of the membrane protein, amyloid precursor protein (APP). An alternative cleavage pathway involves alpha-secretase activity and results in secretion of a 100 kDa non-amyloidogenic APP (sAPPalpha) and therefore a potential reduction in Abeta secretion. We have shown that estrogen induces alpha-cleavage and therefore results in the secretion of sAPPalpha. This secretion is signalled via MAP-kinase and PI-3 kinase signal-transduction pathways. These pathways also have the potential to inhibit the activation of glycogen synthase kinase 3beta (GSK), a protein involved in cell death. Therefore, the aim of this work was to further elucidate the estrogen-mediated signaling pathways involved in APP processing, with particular emphasis on GSK activity. By stimulating rat hypothalamic neuronal GT1-7 cells with estradiol, we found that estrogen decreases the activation state of GSK via the MAP kinase pathway. Moreover, the inhibition of GSK activity by LiCl causes enhanced sAPPalpha secretion in a pattern similar to that seen in response to estrogen, suggesting a pivotal role for this deactivation in APP processing. Further, inactivation of GSK by estrogen can be confirmed in an in vivo model. Elucidation of the signaling pathways involved in APP processing may help to understand the pathology of AD and may also prove beneficial in developing therapeutic strategies to combat AD.