APP-transfected Cells Research Articles

A Class I HDAC Inhibitor Rescues Synaptic Damage and Neuron Loss in APP-Transfected Cells and APP/PS1 Mice through the GRIP1/AMPA Pathway.

As a neurodegenerative disease, Alzheimer’s disease (AD) seriously affects the health of older people. Changes in synapses occur first over the course of the disease, perhaps even before the formation of Aβ plaques. Histone deacetylase (HDAC) mediates the damage of Aβ oligomers to dendritic spines. Therefore, we examined the relationship between HDAC activity and synaptic defects using an HDAC inhibitor (HDACI), BG45, in the human neuroblastoma SH-SY5Y cell line with stable overexpression of Swedish mutant APP (APPsw) and in APP/PS1 transgenic mice during this study. The cells were treated with 15 μM BG45 and the APP/PS1 mice were treated with 30 mg/kg BG45. We detected the levels of synapse-related proteins, HDACs, tau phosphorylation, and amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors using Western blotting and immunohistochemistry. We also measured the expression of cytoskeletal proteins in the cell model. The mRNA levels of the glutamate ion receptor alginate subunit 2 (GRIK2), sodium voltage-gated channel beta subunit (SCN3B), synaptophysin (SYP), Grm2 (the gene encoding glutamate receptor subunit 2 (GluR2)), Grid2IP, glutamate receptor interacting protein 1 (GRIP1), and GRIP2 were detected to explore the effects of the HDACI on regulating the expression of synaptic proteins and AMPA receptors. According to our studies, the expressions of HDAC1, HDAC2, and HDAC3 were increased, which were accompanied by the downregulation of the synapse-related proteins SYP, postsynaptic dendritic protein (PSD-95), and spinophilin as early as 24 h after transfection with the APPsw gene. BG45 upregulated the expression of synapse-related proteins and repaired cytoskeletal damage. In vivo, BG45 alleviated the apoptosis-mediated loss of hippocampal neurons, upregulated synapse-related proteins, reduced Aβ deposition and phosphorylation of tau, and increased the levels of the synapse-related genes GRIK2, SCN3B, SYP, Grm2, and Grid2IP. BG45 increased the expression of the AMPA receptor subunits GluA1, GluA2, and GluA3 on APPsw-transfected cells and increased GRIP1 and GRIP2 expression and AMPA receptor phosphorylation in vivo. Based on these results, HDACs are involved in the early process of synaptic defects in AD models, and BG45 may rescue synaptic damage and the loss of hippocampal neurons by specifically inhibiting HDAC1, HDAC2, and HDAC3, thereby modulating AMPA receptor transduction, increasing synapse-related gene expression, and finally enhancing the function of excitatory synapses. BG45 may be considered a potential drug for the treatment of early AD in further studies.

Mutations in the β-amyloid precursor protein in familial Alzheimer’s disease increase Aβ oligomer production in cellular models

Soluble oligomers of amyloid-β (Aβ) peptides (AβOs) contribute to neurotoxicity in Alzheimer’s disease (AD). However, it currently remains unknown whether an increase in AβOs is the common phenotype in cellular and animal models. Furthermore, it has not yet been established whether experimental studies conducted using models overexpressing mutant genes of the amyloid precursor protein (APP) are suitable for investigating the underlying molecular mechanism of AD. We herein employed the Flp-In™ T-REx™-293 (T-REx 293) cellular system transfected with a single copy of wild-type, Swedish-, Dutch-, or London-type APP, and quantified the levels of Aβ monomers (Aβ1-40 and Aβ1-42) and AβOs using an enzyme-linked immunosorbent assay (ELISA). The levels of extracellular AβOs were significantly higher in Dutch- and London-type APP-transfected cells than in wild-type APP-transfected cells. Increased levels were also observed in Swedish-type APP-transfected cells. On the other hand, intracellular levels of AβOs were unaltered among wild-type and mutant APP-transfected cells. Intracellular levels of Aβ monomers were undetectable, and no common abnormality was observed in their extracellular levels or ratios (Aβ1-42/Aβ1-40) among the cells examined. We herein demonstrated that increased levels of extracellular AβOs are the common phenotype in cellular models harboring different types of APP mutations. Our results suggest that extracellular AβOs play a key role in the pathogenesis of AD.

Allopregnanolone and its analog BR 297 rescue neuronal cells from oxidative stress-induced death through bioenergetic improvement

Allopregnanolone (AP) is supposed to exert beneficial actions including anxiolysis, analgesia, neurogenesis and neuroprotection. However, although mitochondrial dysfunctions are evidenced in neurodegenerative diseases, AP actions against neurodegeneration-induced mitochondrial deficits have never been investigated. Also, the therapeutic exploitation of AP is limited by its difficulty to pass the liver and its rapid clearance after sulfation or glucuronidation of its 3-hydroxyl group. Therefore, the characterization of novel potent neuroprotective analogs of AP may be of great interest. Thus, we synthesized a set of AP analogs (ANS) and investigated their ability to counteract APP-overexpression-evoked bioenergetic deficits and to protect against oxidative stress-induced death of control and APP-transfected SH-SY5Y cells known as a reliable cellular model of Alzheimer's disease (AD). Especially, we examined whether ANS were more efficient than AP to reduce mitochondrial dysfunctions or bioenergetic decrease leading to neuronal cell death.Our results showed that the ANS BR 297 exhibits notable advantages over AP with regards to both protection of mitochondrial functions and reduction of oxidative stress. Indeed, under physiological conditions, BR 297 does not promote cell proliferation but efficiently ameliorates the bioenergetics by increasing cellular ATP level and mitochondrial respiration. Under oxidative stress situations, BR 297 treatment, which decreases ROS levels, improves mitochondrial respiration and cell survival, appears more potent than AP to protect control and APP-transfected cells against H2O2-induced death.Our findings lend further support to the neuroprotective effects of BR 297 emphasizing this analog as a promising therapeutic tool to counteract age- and AD-related bioenergetic deficits.

Sphingosine Kinases/Sphingosine-1-Phosphate and Death Signalling in APP-Transfected Cells

It has been postulated that disturbances in the sphingolipid metabolism play a key role in the pathogenesis of Alzheimer’s disease (AD). An alteration in sphingosine kinases 1, 2 (SphK1/2) and sphingosine-1-phosphate (S1P) was recently reported in AD. However, the effect of AD-related amyloid beta (Aβ) peptides on SphK1/2 and the role of S1P in Aβ toxicity have not been fully elucidated. In this study the relationship between the Aβ concentration and SphK1/2 expression/activity was analysed in PC12 cells transfected with the Aβ precursor protein, wild-type (APPwt) or bearing a double Swedish mutation (APPsw). The role of SphK(s)/S1P in cell survival and death was also investigated. Our results indicated that endogenously liberated Aβ significantly decreases expression and activity of SphK1/2. The SphK(s) inhibitor (SKI II, 10 μM) decreased the viability of APPwt, APPsw as well as empty vector-transfected PC12 control cells. Our data demonstrated that expression of S1P receptor-1 (S1P1) was significantly reduced in APP-transfected cells. The effect of S1P applied exogenously was cell type-dependent. In control and APPwt cells S1P reduced the effect of the SphK1 inhibitor on death signalling. Conversely, it decreased the survival of APPsw cells and had no protective effect on cells treated with SKI II. Using the S1P1 agonist (SEW2871, 5 μM) and antagonist (W123, 20 μM), we demonstrated that the cytoprotective effect of S1P was receptor-independent. Summarising, we showed that Aβ peptides evoke down-regulation of gene expression and activity for SphK(s) and S1P1. Inhibition of SphK(s) significantly decreased cell survival. The effect of exogenous S1P depended on the concentration of Aβ peptides.

Alterations of Cyclin dependent kinase 5 expression and phosphorylation in Amyloid precursor protein (APP)-transfected PC12 cells

The aim of the present study was to analyse the alterations of Cyclin dependent kinase 5 (Cdk5) expression and phosphorylation in PC12 cells overexpressing Amyloid precursor protein (APP). Our results demonstrated enhanced cell death and increased levels of mRNA for the Cdk5 gene in APP-transfected cells. Significantly decreased phosphorylation of Cdk5 at Tyr15 was observed in APPsw cells, which is responsible for a reduction in Cdk5 activity. Cdk5-dependent phosphorylation of Glycogen synthase kinase-3β (Gsk-3β) at Ser9 was also decreased, which can lead to the increase of Gsk-3β activity and hyperphosphorylation of MAP tau. Our results demonstrate for the first time, a deregulation of Cdk5 phosphorylation in APP-transfected cells.

The role of low-density receptor-related protein 1 (LRP1) as a competitive substrate of the amyloid precursor protein (APP) for BACE1

Cleavage of APP by BACE1 is the first proteolytic step in the production of amyloid-beta (Aβ), which accumulates in senile plaques in Alzheimer's disease. Through its interaction with APP, the low-density receptor-related protein 1 (LRP1) enhances APP internalization. Recently, BACE1 has been shown to interact with and cleave the light chain (lc) of LRP1. Since LRP1 is known to compete with APP for cleavage by gamma-secretase, we tested the hypothesis that LRP1 also acts as a competitive substrate for β-secretase. We found that the increase in secreted APP (sAPP) mediated by over-expression of BACE1 in APP-transfected cells could be decreased by simultaneous LRP1 over-expression. Analysis by multi-spot ELISA revealed that this is due to a decrease in sAPPβ, but not sAPPα. Interaction between APP and BACE1, as measured by immunoprecipitation and fluorescence lifetime assays, was impaired by LRP1 over-expression. We also demonstrate that APP over-expression leads to decreased LRP1 association with and cleavage by BACE1. In conclusion, our data suggest that – in addition to its role in APP trafficking – LRP1 affects APP processing by competing for cleavage by BACE1.

How do soluble oligomers of amyloid β-protein impair hippocampal synaptic plasticity?

How do soluble oligomers of amyloid β-protein impair hippocampal synaptic plasticity?

The cytoplasmic adaptor protein X11α and extracellular matrix protein Reelin regulate ApoE receptor 2 trafficking and cell movement

The goal of this study was to determine the effect of X11alpha on ApoE receptor 2 (ApoEr2) trafficking and the functional significance of this interaction on cell movement in MCF 10A epithelial cells. We found that X11alpha increased surface levels of ApoEr2 by 64% compared to vector control, as determined by surface protein biotinylation. To examine the functional significance of this effect, we tested whether ApoEr2 played a novel role in cell movement in a wound-healing assay. We found that overexpression of ApoEr2 in MCF 10A cells increased cell migration velocity by 87% (P<0.01, n=4) compared to GFP control. Cotransfection of X11alpha had an additive effect on average velocity compared to ApoEr2 alone (13%; P<0.05, n=4). In addition, we tested whether ApoEr2 ligands altered the effect of ApoEr2 on cell movement. We found that treatment with concentrated medium containing the extracellular matrix protein Reelin, but not control medium, further increased the velocity of ApoEr2- but not APP-transfected cells (20%; P<0.001, n=4). Similarly, Reelin treatment increased cell velocity in the presence of ApoEr2 and X11alpha (10%; P<0.05, n=4). In the present study, we are the first to demonstrate that ApoEr2 regulates cell movement, and both X11alpha and Reelin enhance this effect.

Amyloid-beta peptide degradation in cell cultures by mycoplasma contaminants

BackgroundCell cultures have become an indispensable tool in Alzheimer's disease research for studying amyloid-β (Aβ) metabolism. It is estimated that up to 35% of cell cultures in current use are infected with various mycoplasma species. In contrast with common bacterial and fungal infections, contaminations of cell cultures with mycoplasmas represent a challenging issue in terms of detectability and prevention. Mycoplasmas are the smallest and simplest self-replicating bacteria and the consequences of an infection for the host cells are variable, ranging from no apparent effect to induction of apoptosis.FindingsHere we present evidence that mycoplasmas from a cell culture contamination are able to efficiently and rapidly degrade extracellular Aβ. As a result, we observed no accumulation of Aβ in the conditioned medium of mycoplasma-positive cells stably transfected with the amyloid-β precursor protein (APP). Importantly, eradication of the mycoplasma contaminant – identified as M. hyorhinis – by treatments with a quinolone-based antibiotic, restored extracellular Aβ accumulation in the APP-transfected cells.ConclusionThese data show that mycoplasmas degrade Aβ and thus may represent a significant source of variability when comparing extracellular Aβ levels in different cell lines. On the basis of these results, we recommend assessment of mycoplasma contaminations prior to extracellular Aβ level measurements in cultured cells.

Effect of amyloid peptides on serum withdrawal-induced cell differentiation and cell viability

Abnormal deposition of amyloid-beta(A beta) peptides and formation of neuritic plaques are recognized as pathological processes in Alzheimer's disease (AD) brain. By using amyloid precursor protein (APP) transfected cells, this study aims to investigate the effect of overproduction of A beta on cell differentiation and cell viability. It was shown that after serum withdrawal, untransfected cell (N2a/Wt) and vector transfected cells (N2a/vector) extended long and branched cell processes, whereas no neurites was induced in wild type APP (N2a/APP695) and Swedish mutant APP (N2a/APPswe) transfected N2a cells. After differentiation by serum withdrawal, the localization of APP/A beta and neurofilament was extended to neurites, whereas those of APP-transfected cells were still restricted within the cell body. Levels of both APP and A beta were significantly higher in N2a/APP695 and N2a/APPswe than in N2a/Wt, as determined by Western blot and Sandwich ELISA, respectively. To further investigate the effect of A beta on the inhibition of cell differentiation, we added exogenously the similar level or about 10-times of the A beta level produced by N2a/APP695 and N2a/APPswe to the culture medium and co-cultured with N2a/Wt for 12 h, and we found that the inhibition of serum withdrawal-induced differentiation observed in N2a/APP695 and N2a/APPswe could not be reproduced by exogenous administration of A beta into N2a/Wt. We also observed that neither endogenous production nor exogenous addition of A beta 1-40 or A beta 1- 42, even to hundreds fold of the physiological concentration, affected obviously the cell viability. These results suggest that the overproduction of A beta could not arrest cell differentiation induced by serum deprivation and that, at least to a certain degree and in a limited time period, is not toxic to cell viability.

Difluoro ketone peptidomimetics suggest a large S1 pocket for Alzheimer's gamma-secretase: implications for inhibitor design.

The final step in the generation of the amyloid-beta protein (Abeta), implicated in the etiology of Alzheimer's disease, is proteolysis within the transmembrane region of the amyloid precursor protein (APP) by gamma-secretase. Although considered an important target for therapeutic design, gamma-secretase has been neither well-characterized nor definitively identified. Previous studies in our laboratory using substrate-based difluoro ketone and difluoro alcohol transition-state analogue inhibitors suggest that gamma-secretase is an aspartyl protease with loose sequence specificity. To further characterize the active site of gamma-secretase, we prepared a series of difluoro ketone peptide analogues with varying steric bulkiness in the P1 position and tested the ability of these compounds to inhibit Abeta production in APP-transfected cells. Incorporation of bulky, aliphatic P1 side chains, such as sec-butyl or cyclohexylmethyl, led to increased gamma-secretase inhibitory potency, suggesting a large S1 pocket to accommodate these substituents and providing further evidence for loose sequence specificity. The cyclohexylmethyl P1 substituent allowed N-terminal truncation to a low-molecular-weight compound (<600 Da) that effectively blocked Abeta production (IC(50) approximately 5 microM). This finding suggests that optimal S1 binding may allow the development of potent inhibitors with ideal pharmaceutical properties. Moreover, a difluoro alcohol analogue with a cyclohexylmethyl P1 substituent was equipotent with its difluoro ketone counterpart, providing strong evidence that gamma-secretase is an aspartyl protease. All new analogues inhibited total Abeta and Abeta(42) production with the same rank order of potency and increased Abeta(42) production at low concentrations, providing further evidence for distinct gamma-secretases that are nevertheless closely similar with respect to active site topology and mechanism.

Alternative, Non-secretase Processing of Alzheimer's β-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.

γ-Secretase Cleavage Is Distinct from Endoplasmic Reticulum Degradation of the Transmembrane Domain of the Amyloid Precursor Protein

One of the critical cleavage events that generates Alzheimer's amyloid Abeta peptide occurs within the transmembrane domain (TMD) of the amyloid precursor protein (APP) and is carried out by a poorly understood enzyme activity known as gamma-secretase. To investigate this processing, a probe molecule, H26-57C, was constructed containing the TMD of APP flanked immediately on each side by unique epitope tags. H26-57C-transfected cells secrete a approximately 2.9-kDa fragment, indicating that the lumenal and cytosolic domains of APP are not required for gamma-secretase processing. Pulse-chase experiments indicate that the probe turns over with a half-life of 8 min. No degradation intermediates are detected during the chase period, indicating that TMD turnover is a highly processive mechanism. The protease inhibitors, ALLN and MG132, cause a dramatic (50-fold) increase in the steady-state amount of the probe. All of the inhibitors that prevent degradation of the probe in the rough endoplasmic reticulum increase the amount of the approximately 2.9-kDa fragment that is secreted into the media and also causes a similar increase the secretion of 4 kDa Abeta from APP-transfected cells. These results indicate that the system responsible for the degradation of the probe in the rough endoplasmic reticulum and the intramembrane cleavage by gamma-secretase that produces soluble, secreted Abeta are distinct and opposing processes.

Particulate forms of APP in the extracellular milieu of cultured cells

The principle externalized forms of amyloid precursor protein (APP) are soluble and well-characterized, but some evidence has suggested the additional presence of externalized APP in a nonsoluble form. To further assess this possibility, the current study has applied high resolution microscopy protocols in addition to immunoprecipitation to characterize externalized APP in three commonly used cell culture models (SH-SY5Y human neuroblastoma cells, fetal rat brain cells, and HEK 293 human embryonic kidney cells). Confocal immunofluorescence microscopy, using an antiserum against the c-terminal domain of APP, showed typical cell-associated APP, but hot spots of APP also were evident in cell-free areas, apparently associated with the culture substrata. These hot-spots were examined for evidence of cellular deterioration by whole mount transmission electron microscopy. Neither cell debris nor disrupted cells were present. Instead, the hot spots of substratum-bound APP comprised discrete microparticles, ∼50–100 nm across. These microparticles also could be found near cells and in some cases were attached to cell surface fibrils. Substratum-bound APP also could be found clustered within the extracellular matrix made by primary cell cultures. Occurrence of APP in extracellular microparticles was verified by centrifugation-immunoprecipitation analysis of media conditioned by APP-transfected cells. Radiolabeling data showed that particulate APP was from metabolically active cells. Metabolic labeling of particle-associated APP, as well as the absence of cellular debris near the APP-containing particles, suggests that the occurrence of nonsoluble APP in the extracellular milieu derives from a physiologically active process.