Production Of Amyloid Peptides Research Articles

Toward Understanding the Molecular Role of SNX27/Retromer in Human Health and Disease.

Aberrations in membrane trafficking pathways have profound effects in cellular dynamics of cellular sorting processes and can drive severe physiological outcomes. Sorting nexin 27 (SNX27) is a metazoan-specific sorting nexin protein from the PX-FERM domain family and is required for endosomal recycling of many important transmembrane receptors. Multiple studies have shown SNX27-mediated recycling requires association with retromer, one of the best-known regulators of endosomal trafficking. SNX27/retromer downregulation is strongly linked to Down’s Syndrome (DS) via glutamate receptor dysfunction and to Alzheimer’s Disease (AD) through increased intracellular production of amyloid peptides from amyloid precursor protein (APP) breakdown. SNX27 is further linked to addiction via its role in potassium channel trafficking, and its over-expression is linked to tumorigenesis, cancer progression, and metastasis. Thus, the correct sorting of multiple receptors by SNX27/retromer is vital for normal cellular function to prevent human diseases. The role of SNX27 in regulating cargo recycling from endosomes to the cell surface is firmly established, but how SNX27 assembles with retromer to generate tubulovesicular carriers remains elusive. Whether SNX27/retromer may be a putative therapeutic target to prevent neurodegenerative disease is now an emerging area of study. This review will provide an update on our molecular understanding of endosomal trafficking events mediated by the SNX27/retromer complex on endosomes.

Glycosaminoglycans from Litopenaeus vannamei Inhibit the Alzheimer's Disease β Secretase, BACE1.

Only palliative therapeutic options exist for the treatment of Alzheimer’s Disease; no new successful drug candidates have been developed in over 15 years. The widely used clinical anticoagulant heparin has been reported to exert beneficial effects through multiple pathophysiological pathways involved in the aetiology of Alzheimer’s Disease, for example, amyloid peptide production and clearance, tau phosphorylation, inflammation and oxidative stress. Despite the therapeutic potential of heparin as a multi-target drug for Alzheimer’s disease, the repurposing of pharmaceutical heparin is proscribed owing to the potent anticoagulant activity of this drug. Here, a heterogenous non-anticoagulant glycosaminoglycan extract, obtained from the shrimp Litopenaeus vannamei, was found to inhibit the key neuronal β-secretase, BACE1, displaying a more favorable therapeutic ratio compared to pharmaceutical heparin when anticoagulant activity is considered.

P.1.005 - Mutating the cholesterol binding site on amyloid precursor protein alters the processing and production of Aβ-amyloid peptides

Levels of cholesterol are elevated in the brains of Alzheimer’s disease (AD) patients [1,2]. While the precise pathophysiological consequences remain unclear, an increase in cholesterol at the plasma membrane promotes endocytosis of the amyloid precursor protein (APP) thereby leading to increased Aβ production [3]. An interaction between the Aβ fragment C99 (resulting from cleavage of APP by β-secretase), and cholesterol was recently proposed [4]. In the present work, we studied the nature of this putative cholesterol binding site (CBS) on APP by analyzing APP mutants within the CBS (located between 691-710 of APP770). We measured the influence of mutants on production of amyloid peptides, cleavage by secretases and the subcellular localization of APP mutants in HEK293T cells. A variety of mutants of the CBS of APP751-mCherry (APP isoform which contain 751 residues fused to the fluorescent protein mCherry) were constructed using site-directed mutagenesis: E693K, S697A, K699A, K699E, G700A, G704A, V710A, S697A/K699A and G700A/G704A (numbering according to APP770 sequence). The amounts of secreted Aβ40 and Aβ42 peptides were measured by ELISA (Meso Scale Discovery) and short peptides (28 to 42aa) by ELISA from IBL (Immuno-Biological Laboratories) in the culture medium of HEK293T cells transfected with various mutants. We studied the activity of secretases by analyzing the levels of C99 and AICD (APP Intracellular Domain) using western blot. In addition, we analyzed the subcellular localization of mutant APP-mCherry by confocal microscopy, using immunocytochemistry with anti-Early Endosome Antigen 1 antibodies. Levels of secreted Aβ40 and Aβ42 were significantly decreased in HEK293T cells transfected with all mutants (except for S697A) as compared to HEK293T cells transfected with APPwt (p In conclusion, these results suggest that the CBS is a key player in the processing of APP and generation of neurotoxic fragments like Aβ42. Further, a specific interaction between cholesterol and the APP CBS controls the size as well as amount of Aβ peptides. We hypothesize that CBS mutants are positioned more deeply in the membrane thus being processed differently leading to the formation of shorter Aβ peptides. These data demonstrate the functional importance of an interaction between cholesterol and APP, provide insights into the pathology of AD, and suggest potential routes to novel therapies.

NGP 555, a γ-secretase modulator, lowers the amyloid biomarker, Aβ42, in cerebrospinal fluid while preventing Alzheimer's disease cognitive decline in rodents

IntroductionAlzheimer's disease (AD) is defined by the progressive accumulation of amyloid plaques and neurofibrillary tangles in the brain which precedes cognitive decline by years. MethodsUsing amyloid biomarkers, chemical modeling, mouse behavioral models, and drug development techniques, we investigate the properties of NGP 555, a clinical-stage γ-secretase modulator. ResultsNGP 555 shifts amyloid peptide production to the smaller, nonaggregating forms of amyloid. Our preclinical studies show beneficial effects on amyloid biomarkers, pathology, and cognition. NGP 555 has successfully completed chemistry, pharmacology, toxicity, metabolism, and safety studies. DiscussionAbundant data support Aβ42 as a target for prophylactic or early-stage intervention therapies in AD. The γ-secretase modulator, NGP 555 is being actively developed in human clinical trials for the prevention of Alzheimer's disease with the overall aim to achieve an appropriate balance of potency/efficacy on reducing the toxic forms of amyloid versus safety.

Presenilin 1 and Presenilin 2 Target γ-Secretase Complexes to Distinct Cellular Compartments

γ-Secretase complexes achieve the production of amyloid peptides playing a key role in Alzheimer disease. These proteases have many substrates involved in important physiological functions. They are composed of two constant subunits, nicastrin and PEN2, and two variable ones, presenilin (PS1 or PS2) and APH1 (APH1aL, APH1aS, or APH1b). Whether the composition of a given γ-secretase complex determines a specific cellular targeting remains unsolved. Here we combined a bidirectional inducible promoter and 2A peptide technology to generate constructs for the temporary, stoichiometric co-expression of six different combinations of the four γ-secretase subunits including EGFP-tagged nicastrin. These plasmids allow for the formation of functional γ-secretase complexes displaying specific activities and maturations. We show that PS1-containing γ-secretase complexes were targeted to the plasma membrane, whereas PS2-containing ones were addressed to the trans-Golgi network, to recycling endosomes, and, depending on the APH1-variant, to late endocytic compartments. Overall, these novel constructs unravel a presenilin-dependent subcellular targeting of γ-secretase complexes. These tools should prove useful to determine whether the cellular distribution of γ-secretase complexes contributes to substrate selectivity and to delineate regulations of their trafficking.

Cytokine profile in patients with malignant tumors of liver in postoperative period

Cytokine profile in patients with malignant tumors of liver in postoperative period

Cholesterol in brain disease: sometimes determinant and frequently implicated.

Cholesterol is essential for neuronal physiology, both during development and in the adult life: as a major component of cell membranes and precursor of steroid hormones, it contributes to the regulation of ion permeability, cell shape, cell-cell interaction, and transmembrane signaling. Consistently, hereditary diseases with mutations in cholesterol-related genes result in impaired brain function during early life. In addition, defects in brain cholesterol metabolism may contribute to neurological syndromes, such as Alzheimer's disease (AD), Huntington's disease (HD), and Parkinson's disease (PD), and even to the cognitive deficits typical of the old age. In these cases, brain cholesterol defects may be secondary to disease-causing elements and contribute to the functional deficits by altering synaptic functions. In the first part of this review, we will describe hereditary and non-hereditary causes of cholesterol dyshomeostasis and the relationship to brain diseases. In the second part, we will focus on the mechanisms by which perturbation of cholesterol metabolism can affect synaptic function.

Role of cholesterol metabolism in the pathogenesis of Alzheimer's disease.

Cholesterol has been shown to stimulate the cleavage of amyloid precursor protein (APP) into amyloid peptides involved in Alzheimer's disease. However, high level of peripheral cholesterol as a risk factor for Alzheimer's disease is still debated. This current review provides an update of the recent literature on cholesterol and APP metabolisms in the brain. First, a new relationship between neuronal APP and cholesterol has been shown in which this protein controls cholesterol turnover required for neuronal activity. Second, oxysterols are able to stimulate the synthesis of ATP-binding cassette transporters involved in the exchange of amyloid peptides between the blood and the brain. Third, changes in APP targeting to lipid rafts and/or their composition in cholesterol regulate amyloid peptide production. These recent findings open new areas of investigations to control the neuronal activity and to decrease the amyloid peptide levels in brain, opening on new preventive and therapeutic strategies for Alzheimer's disease.

Beta-amyloidolysis and glutathione in Alzheimer's disease

In this review, we hypothesized the importance of the interaction between the brain glutathione (GSH) system, the proteolytic tissue plasminogen activator (t-PA)/plasminogen/ plasmin system, regulated by plasminogen activator inhibitor (PAI-1), and neuroserpin in the pathogenesis of Alzheimer’s disease. The histopathological characteristic hallmark that gives personality to the diagnosis of Alzheimer’s disease is the accumulation of neurofibroid tangles located intracellularly in the brain, such as the protein tau and extracellular senile plaques made primarily of amyloidal substance. These formations of complex etiology are intimately related to GSH, brain protective antioxidants, and the proteolytic system, in which t-PA plays a key role. There is scientific evidence that suggests a relationship between aging, a number of neurodegenerative disorders, and the excessive production of reactive oxygen species and accompanying decreased brain proteolysis. The plasminogen system in the brain is an essential proteolytic mechanism that effectively degrades amyloid peptides (“beta-amyloidolysis”) through action of the plasmin, and this physiologic process may be considered to be a means of prevention of neurodegenerative disorders. In parallel to the decrease in GSH levels seen in aging, there is also a decrease in plasmin brain activity and a progressive decrease of t-PA activity, caused by a decrease in the expression of the t-PA together with an increase of the PAI-1 levels, which rise to an increment in the production of amyloid peptides and a lesser clearance of them. Better knowledge of the GSH mechanism and cerebral proteolysis will allow us to hypothesize about therapeutic practices.

Physical evidence supporting a ribosomal shunting mechanism of translation initiation for BACE1 mRNA.

In Alzheimer disease, elevated levels of the BACE1 enzyme are correlated with increased production of amyloid peptides and disease pathology. The increase in BACE1 levels is post-transcriptional and may involve altered translation efficiency. Earlier studies have indicated that translation of BACE1 mRNA is cap-dependent. As ribosomal subunits move from the cap-structure to the initiation codon, they fail to recognize several AUG codons in the 5′ leader. In this study, we looked for physical evidence of the mechanism underlying ribosomal scanning or shunting along the BACE1 5′ leader by investigating structural stability in the 5′ leaders of endogenous mRNAs in vivo. To perform this analysis, we probed RNAs using lead(II) acetate, a cell-permeable chemical that induces cleavage of unpaired nucleotides having conformational flexibility. The data revealed that the ≈440-nt 5′ leader was generally resistant to cleavage except for a region upstream of the initiation codon. Cleavage continued into the coding region, consistent with destabilization of secondary structures by translating ribosomes. Evidence that a large segment of the BACE1 5′ leader was not cleaved indicates that this region is structurally stable and suggests that it is not scanned. The data support a mechanism of translation initiation in which ribosomal subunits bypass (shunt) part of the BACE1 5′ leader to reach the initiation codon. We suggest that a nucleotide bias in the 5′ leader may predispose the initiation codon to be more accessible than other AUG codons in the 5′ leader, leading to an increase in its relative utilization.

Cross-Linking of Cell Surface Amyloid Precursor Protein Leads to Increased -Amyloid Peptide Production in Hippocampal Neurons: Implications for Alzheimer's Disease

The accumulation of the β-amyloid peptide (Aβ) in Alzheimer's disease (AD) is thought to play a causative role in triggering synaptic dysfunction in neurons, leading to their eventual demise through apoptosis. Aβ is produced and secreted upon sequential cleavage of the amyloid precursor protein (APP) by β-secretases and γ-secretases. However, while Aβ levels have been shown to be increased in the brains of AD patients, little is known about how the cleavage of APP and the subsequent generation of Aβ is influenced, or whether the cleavage process changes over time. It has been proposed that Aβ can bind APP and promote amyloidogenic processing of APP, further enhancing Aβ production. Proof of this idea has remained elusive because a clear mechanism has not been identified, and the promiscuous nature of Aβ binding complicates the task of demonstrating the idea. To work around these problems, we used an antibody-mediated approach to bind and cross-link cell-surface APP in cultured rat primary hippocampal neurons. Here we show that cross-linking of APP is sufficient to raise the levels of Aβ in viable neurons with a concomitant increase in the levels of the β-secretase BACE1. This appears to occur as a result of a sorting defect that stems from the caspase-3-mediated inactivation of a key sorting adaptor protein, namely GGA3, which prevents the lysosomal degradation of BACE1. Together, our data suggest the occurrence of a positive pathogenic feedback loop involving Aβ and APP in affected neurons possibly allowing Aβ to spread to nearby healthy neurons.

Physiological functions of the amyloid precursor protein secretases ADAM10, BACE1, and Presenilin

Alzheimer's disease causing mutations in the amyloid precursor protein (APP) or in the Presenilin 1 (PS1) or Presenilin 2 (PS2) genes increase the production of amyloid peptides (Aβ) that precipitate in amyloid plaques. Since amyloid plaques are also a prominent feature of sporadic Alzheimer's disease (AD), abnormal proteolysis of APP and the generation of amyloid beta (Aβ) are key events in the pathogenesis of AD. The proteases (secretases) that cleave APP are therefore important therapeutic targets, both for the rare familial forms but likely also for the sporadic forms of AD. The identification and understanding of the (neuro)biological functions of the α-, β-, and presenilin/γ-secretase (complexes) is important for the development of drugs and the delineation of their associated side effects. The potential impact of this type of research exceeds the AD field since the function of these secretases are also linked to cellular pathways like ectodomain shedding of growth factors and regulated intramembrane proteolysis of receptors in developmental biology, tissue homeostasis, and tumorigenesis. The generation of mice deficient in presenilin 1, presenilin 2, the α-secretase ADAM10, and the β-secretases BACE1 and BACE2 were instrumental for the elucidation of the physiological functions of these proteases. Using these mouse models understanding how these secretases regulate amyloid peptide formation and how they exert their diverse biological functions could be significantly increased. This review attempts to summarize selected aspects of the current view of the multiple roles such proteases play in health and disease.

Neurotransmitters and Energy Metabolites in Amyloid-Bearing APPSWE×PSEN1dE9 Mouse Brain

Alzheimer's disease is characterized by amyloid peptide formation and deposition, neurofibrillary tangles, synaptic loss and central cholinergic dysfunction, dysfunction of energy metabolism, and dementia; however, the interactions between these hallmarks remain poorly defined. We studied a well characterized mouse model of amyloid deposition, the doubly transgenic APP(SWE)xPSEN1dE9 mouse. At 10 to 14 months of age, these mice had high levels of amyloid peptides (6.6 microg/g wet weight) and widespread amyloid plaques. Extracellular levels of acetylcholine (ACh) were determined by microdialysis in the hippocampus and were comparable with nontransgenic mice from the same colony. In the open field, both mouse strains responded with a 3-fold increase of hippocampal ACh release. Exploratory behavior of the transgenic mice appeared normal. Infusion of scopolamine evoked 5- to 6-fold increases of ACh levels in both mouse strains. High-affinity choline uptake and cholinesterase activities were identical in both mouse lines. Extracellular levels of glucose and glycerol were similar in control and transgenic mice, whereas lactate levels were slightly (p = 0.06) and glutamate levels significantly (p = 0.02) lower in transgenic mice. Exploration caused increases of glucose and lactate, whereas infusion of scopolamine (1 microM) increased glucose but not lactate. Glutamate levels were increased by scopolamine, whereas glycerol remained constant under all the conditions. We conclude that amyloid peptide production and plaque deposition causes minor changes in cholinergic function and energy metabolites in transgenic mice in vivo. Amyloid peptide formation and/or deposition may not be sufficient for long-term cholinergic or metabolic dysfunction.

GSK‐3: A key player in neurodegeneration and memory

Abnormalities in molecular signalling have been implicated in neurodegeneration. It is emerging that glycogen synthase kinase-3 (GSK-3) is a key signalling molecule that induces neurodegeneration and deficits in memory formation related to Alzheimer's disease (AD). Early stages of AD are associated with deficits in memory formation before neuronal cell death is detectable. Recent studies in rodents have suggested that these impairments in memory formation might result from increased GSK-3 signalling, because enhanced GSK-3 activity impairs hippocampal memory formation. GSK-3 activity blocks synaptic long-term potentiation and induces long-term depression. Furthermore, increased GSK-3 signalling is likely to be a key contributor to the formation of the pathological hallmarks in AD, neurofibrillary tangles (NFTs) and amyloid plaques. Recent studies with mouse models have indicated that GSK-3, but not cyclin-dependent kinase 5, is critical for hyperphosphorylation of the cytoskeletal protein tau, which is the prerequisite for NFT formation in AD. Furthermore, increased GSK-3 signalling in AD mice causes abnormal processing of the amyloid precursor protein so that amyloid peptide production augments and neurotoxicity is induced. Taken together, the current evidences suggest that increased GSK-3 signalling may be responsible for the deficits in memory formation in early stages of AD and neurodegeneration in later stages of the disease.

Expression of BRI2 mRNA and protein in normal human brain and familial British dementia: its relevance to the pathogenesis of disease

Two different disease-specific mutations in the BRI2 gene, situated on chromosome 13, have been identified as giving rise to familial British dementia (FBD) and familial Danish dementia (FDD). Each mutation results in extension of the open reading frame generating the disease-specific precursor proteins which are cleaved by furin-like proteolysis releasing the amyloidogenic C-terminal peptides ABri and ADan in FBD and FDD, respectively. To understand the mechanism of the formation of amyloid lesions in FBD, we studied the origin of the precursor proteins and furin in the human brain. We used control brains, cases of sporadic Alzheimer's disease (AD), variant AD with cotton wool plaques and FBD to study BRI2 mRNA expression using in situ hybridization. Furin and BRI2 protein expression was investigated using Western blotting and immunohistochemistry. BRI2 mRNA and BRI2 protein are widely expressed primarily by neurones and glia and are deposited in the amyloid lesions in FBD. They were, however, not expressed by cerebrovascular components. Furin expression showed a similar pattern except that it was also present in cerebrovascular smooth muscle cells. These findings suggest that neurones and glia and are a major source of BRI2 protein and that in FBD, the mutated precursor protein may undergo furin cleavage within neurones to produce the amyloid peptide ABri. The failure to demonstrate BRI2 in blood vessels under the conditions tested suggests that vascular amyloid peptide production does not contribute significantly to cerebral amyloid angiopathy (CAA) in FBD and FDD, lending indirect support to the drainage hypothesis of CAA.

Molecular and Biochemical Features in Alzheimers Disease

The purpose of this review is to discuss the pathophysiological pathways involved in pathogenesis of Alzheimer's disease pointing out current and future pharmacological targets. Alzheimer's disease is one of the most important neurodegenerative disorders in the developed world together with Parkinson's disease. Although this disease was described almost a century ago, the molecular mechanisms that lead to the development of the neuronal pathology are not clear at the moment. Furthermore, although enormous efforts have been done, an efficient treatment for the disease does not exist yet because the mechanism of neuronal cell death is unknown. In the present work we discuss, in depth, the potential mechanisms involved in apoptosis and neuronal death in Alzheimer's disease. The biology, structure and physiological properties of beta-amyloid peptide and related proteases (secretases) are discussed, as well as existing therapeutics and future strategies for the treatment of Alzheimer's disease. Inhibition of production of amyloid peptides by secretase inhibitors has been suggested as one of the most rational and specific therapeutic approaches. Inhibition of apoptosis mediated by oxidative stress generation and mitochondrial alteration, or blockade of NMDA receptors could constitute suitable therapeutic strategies for Alzheimer's disease. Finally, a multiple therapy with antioxidants, cell cycle inhibitors and other drugs modulating APP processing could be, in the future, a suitable strategy in order to delay Alzheimer's disease progression.

Neuronal membrane cholesterol loss enhances amyloid peptide generation

Recent experimental and clinical retrospective studies support the view that reduction of brain cholesterol protects against Alzheimer's disease (AD). However, genetic and pharmacological evidence indicates that low brain cholesterol leads to neurodegeneration. This apparent contradiction prompted us to analyze the role of neuronal cholesterol in amyloid peptide generation in experimental systems that closely resemble physiological and pathological situations. We show that, in the hippocampus of control human and transgenic mice, only a small pool of endogenous APP and its β-secretase, BACE 1, are found in the same membrane environment. Much higher levels of BACE 1–APP colocalization is found in hippocampal membranes from AD patients or in rodent hippocampal neurons with a moderate reduction of membrane cholesterol. Their increased colocalization is associated with elevated production of amyloid peptide. These results suggest that loss of neuronal membrane cholesterol contributes to excessive amyloidogenesis in AD and pave the way for the identification of the cause of cholesterol loss and for the development of specific therapeutic strategies.

Ion channel regulation by chronic hypoxia in models of acute oxygen sensing

Several potentially life-threatening cardiovascular and respiratory disorders result in prolonged deprivation of oxygen, which in turn results in significant cellular adaptation, or remodelling. An important component of this functional adaptation arises as a direct consequence of altered ion channel expression by chronic hypoxia. In this review, we discuss current understanding of this hypoxic remodelling process, with particular reference to regulation of L-type Ca 2+ channels and high-conductance, Ca 2+-sensitive K + (BK) channels. In systems where this remodelling occurs, changes in functional expression of these particular channels evokes marked alteration in, or responses to, Ca 2+-dependent events. Evidence to date indicates that channel expression can be modulated at the transcriptional level but, additionally, that crucial post-transcriptional events are also regulated by chronic hypoxia. Importantly, such remodelling is, in some cases, strongly associated with production of amyloid peptides of Alzheimer’s disease, implicating chronic hypoxia as a causative factor in the progression of specific pathology. Moreover, subtle changes in functional expression of BK channels implicates chronic hypoxia as an important regulator of cell excitability.

Central cholinergic functions in human amyloid precursor protein knock-in/presenilin-1 transgenic mice

Alzheimer's disease is characterized by amyloid peptide formation and deposition, neurofibrillary tangles, central cholinergic dysfunction, and dementia; however, the relationship between these parameters is not well understood. We studied the effect of amyloid peptide formation and deposition on central cholinergic function in knock-in mice carrying the human amyloid precursor protein (APP) gene with the Swedish/London double mutation (APP-SL mice) which were crossbred with transgenic mice overexpressing normal (PS1wt) or mutated (M146L; PS1mut) human presenilin-1. APP-SL×PS1mut mice had increased levels of Aβ peptides at 10 months of age and amyloid plaques at 14 months of age while APP-SL×PS1wt mice did not have increased peptide levels and did not develop amyloid plaques. We used microdialysis in 15–27 months old mice to compare hippocampal acetylcholine (ACh) levels in the two mouse lines and found that extracellular ACh levels were slightly but significantly reduced in the APP-SL×PS1mut mice (−26%; P=0.044). Exploratory activity in the open field increased hippocampal ACh release by two-fold in both mouse lines; total and relative increases were not significantly different for the two strains under study. Similarly, infusion of scopolamine (1 μM) increased hippocampal ACh release to a similar extent (3–5-fold) in both groups. High-affinity choline uptake, a measure of the ACh turnover rate, was identical in both mouse lines. Neurons expressing choline acetyltransferase were increased in the septum of APP-SL×PS1mut mice (+26%; P=0.046). We conclude that amyloid peptide production causes a small decrease of extracellular ACh levels. The deposition of amyloid plaques, however, does not impair stimulated ACh release and proceeds without major changes of central cholinergic function.

Capacitive Calcium Entry Is Directly Attenuated by Mutant Presenilin-1, Independent of the Expression of the Amyloid Precursor Protein

Mutant presenilin-1 (PS1) increases amyloid peptide production, attenuates capacitative calcium entry (CCE), and augments calcium release from the endoplasmatic reticulum (ER). Here we measured the intracellular free Ca(2+) concentration in hippocampal neurons from six different combinations of transgenic and gene-ablated mice to demonstrate that mutant PS1 attenuated CCE directly, independent of the expression of the amyloid precursor protein (APP). On the other hand, increased Ca(2+) release from the ER in mutant PS1 neurons, as induced by thapsigargin, was clearly dependent on the presence of APP and its processing by PS1, i.e. on the generation of the amyloid peptides and the APP C99 fragments. This observation was corroborated by the thapsigargin-induced increase in cytosolic [Ca(2+)](i) in PS1 deficient neurons, which accumulate C99 fragments due to deficient gamma-secretase activity. Moreover, co-expression of mutant APP[V717I] in PS1-deficient neurons further increased the apparent size of the ER calcium stores in parallel with increasing levels of the APP processing products. We conclude that mutant PS1 deregulates neuronal calcium homeostasis by two different actions: (i) direct attenuation of CCE at the cell-surface independent of APP; and (ii) indirect increase of ER-calcium stores via processing of APP and generation of amyloid peptides and C99 fragments.