A major neuropathological hallmark of Alzheimer’s disease (AD) is the deposition of aggregated β amyloid (Aβ) peptide in the senile plaques. Aβ is a peptide of 38–43 amino acids and its accumulation and aggregation plays a key role early in the disease. A large fraction of β amyloid is N-terminally truncated rendering a glutamine that can subsequently be cyclized into pyroglutamate (pE). This makes the peptide more resistant to proteases, more prone to aggregation and increases its neurotoxicity. The enzyme glutaminyl cyclase (QC) catalyzes this conversion of glutamine to pE. In brains of AD patients, the expression of QC is increased in the earliest stages of pathology, which may be an important event in the pathogenesis. In this study we aimed to investigate the regulatory mechanism underlying the upregulation of QC expression in AD. Using differentiated SK-N-SH as a neuronal cell model, we found that neither the presence of Aβ peptides nor the unfolded protein response, two early events in AD, leads to increased QC levels. In contrast, we demonstrated increased QC mRNA levels and enzyme activity in response to another pathogenic factor in AD, perturbed intracellular Ca2+ homeostasis. The QC promoter contains a putative binding site for the Ca2+ dependent transcription factors c-fos and c-jun. C-fos and c-jun are induced by the same Ca2+-related stimuli as QC and their upregulation precedes QC expression. We show that in the human brain QC is predominantly expressed by neurons. Interestingly, the Ca2+- dependent regulation of both c-fos and QC is not observed in non-neuronal cells. Our results indicate that perturbed Ca2+ homeostasis results in upregulation of QC selectively in neuronal cells via Ca2+- dependent transcription factors. This suggests that disruption of Ca2+ homeostasis may contribute to the formation of the neurotoxic pE Aβ peptides in Alzheimer’s disease.
Citation: De Kimpe L, Bennis A, Zwart R, van Haastert ES, Hoozemans JJM, Scheper W (2012) Disturbed Ca2+ Homeostasis Increases Glutaminyl Cyclase Expression; Connecting Two Early Pathogenic Events in Alzheimer’s Disease In Vitro. PLoS ONE 7(9): e44674. https://doi.org/10.1371/journal.pone.0044674
Editor: Koichi M. Iijima, Thomas Jefferson University, United States of Amercia
Received: May 10, 2012; Accepted: August 10, 2012; Published: September 7, 2012
Copyright: © De Kimpe et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by the European Commission as part of the 6th Framework Program (EDAR, contract # 37670) and the 7th Framework program (NAD, grant agreement # 212043), Internationale Stichting Alzheimer Onderzoek Nederland (ISAO #07506) and the Netherlands Organisation for Scientific Research (NWO; Meervoud Grant #836.05.060). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
The formation of Aβ peptides from the amyloid precursor protein (APP) is a crucial event in Alzheimer’s disease (AD). Proteolytic processing of APP results in different forms of the Aβ protein with different characteristics . C-terminal cleavage of the Aβ sequence by γ-secretase leads to the formation of Aβ1-x varying in length from 38–43 amino acids. In addition, N-terminal truncations have been identified that expose a glutamic acid at position 3 or 11. This glutamate residue can be cyclized into a pyroglutamate (pE) N-terminus which leads to the formation of Aβ3(pE)-x and Aβ11(pE)-x in a reaction catalyzed by glutaminyl cyclases (QCs). The pE residue stabilizes the peptide by protection against degradation by aminopeptidases  and lysosomal proteases . In addition, the conversion of the glutamatic acid into a pE residue results in a loss of charge and the consequent increased hydrophobicity leads to an increase in aggregation propensity , . It has been suggested that pE Aβ provides a seed for the Aβ aggregation process . The change in physicochemical properties is accompanied by increased neurotoxicity of pE Aβ peptides compared to unmodified Aβ species –.
QC is abundantly expressed in the cortex and hippocampus and its expression correlates with the appearance of pE Aβ , . In a transgenic AD mouse model, overexpression of human QC results in an increase in Aβ3(pE)-42 peptides, plaque formation as well as memory impairments . Moreover, the formation of the neurotoxic pE Aβ peptides can be prevented by inhibition of QC in vitro and in vivo , . Oral application of a QC inhibitor diminishes plaque formation and improves performance in spatial learning tests and context memory in two transgenic mouse models of AD . QC expression is upregulated at the mRNA and protein level in the AD brain , which may be an important pathogenic factor triggered early in the development of AD pathogenesis.
Accumulation of Aβ is an early pathogenic event in AD. In particular, oligomeric assemblies of Aβ interfere with cellular physiology. AD is a multifactorial disease, and additional dysfunction that is not directly related to Aβ may converge with the toxic effects of Aβ. For example, our lab has previously shown that the unfolded protein response (UPR) is activated early in AD , . The UPR is a stress response of the endoplasmic reticulum (ER) that is activated in response to disturbed ER homeostasis. Several homeostatic and biosynthetic pathways are located in the ER: it is a site for Ca2+ homeostasis, redox balance, lipid synthesis and the synthesis and folding of membrane bound and secretory proteins. We showed that increased production of Aβ , as well as the presence of early oligomeric Aβ aggregates  sensitize cells for UPR induction and ER stress toxicity, indicating an interaction between these two early events in AD pathology. Recently our group demonstrated that the accumulation of pE Aβ aggregates in human cortex is highly dependent on the presence of AD pathology, but also on age . Also changes in Ca2+ homeostasis that have been suggested to be involved early in AD pathology are highly age related –. The above illustrates that multiple factors can affect AD pathogenesis. In this study we investigated whether these key early pathogenic events -Aβ, UPR activation and perturbed Ca2+ homeostasis - affect QC expression.
Upregulation of QC mRNA is a very Early Event in AD Pathology
Elaborating on the previously reported QC mRNA expression data in the neocortex of AD patients , we determined the expression of QC mRNA in the hippocampus/entorhinal cortex of a cohort of age-matched patients with varying stages of AD pathology. We observe a significant increase in mRNA levels of QC in the patients with the earliest stages of pathology (Fig. 1). Levels are the highest in low Braak scores for tau pathology (1–3) with many amyloid deposits in the hippocampus. In patients with more advanced tau pathology the QC levels are lower, in accordance with the observations in the cortex in the previous study. This indicates that QC is upregulated in the earliest stages of AD pathology.
Expression of QC mRNA was determined in a cohort of patients with varying stages of AD pathology (see materials and methods for details). CON and AD refer to the clinical diagnosis, BS is Braak score for tau pathology, PL is the plaqueload in the hippocampus/entorhinal cortex. Shown is a box-plot of results of the pathological groups as indicated in hippocampus/entorhinal cortex. The expression levels were normalized to eEF2α mRNA. Kruskall-Wallis test was used to evaluate differences between groups followed by the Mann-Whitney U test, to test differences between pairs of groups.
QC Expression is not Upregulated by Aβ or the UPR
Because QC is increased very early in AD, we investigated if early events occurring in AD might be involved in the regulation. Therefore differentiated SK-N-SH were treated with increasing concentrations of either oligomeric or fibrillar Aβ1–42 for 24 h and subsequently the expression level of QC mRNA was measured by semi-quantitative real-time PCR analysis (Fig. 2A). Treatment with Aβ did not increase the QC mRNA level - fibrillar Aβ1–42 at 2 µM even slightly reduced the QC level - indicating that this is not a signal to increase QC expression.
A. Differentiated SK-N-SH cells were treated with 1 µM and 2 µM oligomeric (O) and fibrillary (F) Aβ1–42 for 24 h. Shown is the average + SD of normalized QC mRNA levels of triplicates from a representative experiment B. Differentiated SK-N-SH cells were treated with different UPR inducers, TM (0.2 µg/µl and 0.5 µg/µl), TG (1 µM and 2 µM) and DD (20 mM) for 16 h. Shown is the average + SD of normalized QC mRNA levels of n = 9 from 3 independent experiments. Normalized BiP mRNA levels are shown as positive control for UPR induction (n = 6 from two independent experiments). The expression levels were normalized to eEF2α mRNA, the levels in untreated cells are set to 1. Asterisks indicate a significant difference compared with control (*p≤0.01, **p≤0.001, ***p≤0.0001).
Previously, our group demonstrated that UPR activation, which is known to affect the expression of several genes, is an early event in Alzheimer’s disease brain. Therefore we used different chemical compounds (tunicamycin [TM], thapsigargin [TG] and 2-deoxy-d-glucose [DG]), to induce the UPR in the differentiated SK-N-SH (Fig. 2B). All treatments increase the levels of the UPR-responsive mRNA BiP, confirming that the UPR is activated. In contrast, the QC mRNA expression level was not increased by TM or DG, but only after treatment with TG indicating that activation of the UPR per se does not regulate QC gene expression.
Disturbed Ca2+ Homeostasis Increases QC Expression and Enzyme Activity
To further explore the TG induced gene expression of QC, differentiated SK-N-SH cells were treated with increasing concentrations of TG. As shown in Figure 3A a dose-dependent increase in QC mRNA expression levels was observed. The TG induced expression of QC indicates Ca2+-dependent gene regulation since TG, a specific and irreversible sarco-endoplasmic reticulum ATPase (SERCA) pump inhibitor, blocks the re-uptake of cytosolic Ca2+ into the ER and as a consequence depletes the ER Ca2+ and increases the cytosolic Ca2+ as has been shown by fura-2 Ca2+ imaging –.
A. Differentiated SK-N-SH cells were treated with different concentrations TG as indicated. Shown are the average + SD of normalized QC mRNA levels of n = 6 from two independent experiments. The expression levels were normalized to eEF2α mRNA, the expression levels in untreated cells are set to 1. B. Differentiated SK-N-SH cells were treated with 1 µM TG for 16 h. The enzymatic activity of QC activity was determined in protein lysates as described in materials and methods, activity in untreated cells is set to 1. Shown is average +SEM of n = 15 from 5 independent experiments. Asterisks indicate a significant difference compared to control (*p≤0.001, **p≤0.0001).
To investigate if TG also increased QC activity, we used an enzymatic activity assay. The activity of the enzyme is determined by the ability of QC to convert a glutaminyl residue to a pyroglutamyl residue on a fluorogenic substrate. Differentiated SK-N-SH cells were treated with 1 µM TG for 16 h. The fluorescence, indicative of the activity of QC, was measured and the data show that treatment with TG induces the fluorescence which is in line with the mRNA expression data (Fig. 3B).
ER Ca2+ Depletion Increases QC Levels
Conceivably two TG-dependent events could regulate QC mRNA levels (i) TG-mediated ER Ca2+ pool depletion and/or (ii) TG-initiated rise in cytosolic Ca2+. To investigate which of these two mechanism might be responsible for the TG initiated increase in QC mRNA levels, differentiated SK-N-SH were pretreated (1 h) with the membrane permeable Ca2+ chelator BAPTA-AM (5 µM) before treatment with TG (16 h) (Fig. 4). Once BAPTA-AM enters the cytosol, it is cleaved to membrane-impermeable BAPTA and significantly reduces and maintains a low cytoplasmic Ca2+ concentration even upon subsequent stimulation with TG , . When BAPTA-AM was added separately no increase in QC mRNA expression level was observed. However, the combination of BAPTA and TG strongly potentiated the TG induced QC mRNA expression level (Fig. 4), indicating that it is probably not the rise in the cytosolic Ca2+ but rather the depletion of the ER Ca2+ that is responsible for the TG induced QC gene expression.
Differentiated SK-N-SH cells were treated with 1 µM TG for 16 h with or without pre-incubation with 5 µM BAPTA-AM (B) for 1 h. Shown are the average + SD of normalized QC mRNA levels of n = 9 from 3 independent experiments. The expression levels were normalized to eEF2α mRNA, the expression levels in untreated cells are set to 1. Asterisks indicate a significant difference compared to control (*p≤0.0001).
Induction of c-fos and c-jun Precedes Increased QC Expression
To further analyze the effect of TG on QC expression we determined the expression of QC at different time points (Fig. 5A). The earliest timepoint where a slight increase is observed is 5 h, but the levels are further increased at 16h. This relatively slow effect indicates an indirect mechanism, and can be explained if the effect is mediated via the upregulation of a Ca2+ -dependent transcription factor.
Differentiated SK-N-SH cells were treated with 1 µM TG for the times indicated. Shown is the average + SD of normalized QC (A), c-fos (B) and c-jun (C) mRNA levels of triplicates from a representative experiment of three. The expression levels were normalized to eEF2α mRNA, the expression levels in untreated cells are set to 1. Asterisks indicate a significant difference compared to control (*p<0.05; **p≤0.001; ***p≤0.0001).
We used Tfsearch (www.cbrc.jp/research/db/TFSEARCH.html) to identify possible Ca2+ -dependent transcription factor binding sites in the proximal promoter region of the QC gene. The program identified a putative binding site for heterodimers of the transcription factors encoded by the immediate early genes (IEGs) c-fos and c-jun. Disturbed ER Ca2+ homeostasis in neurons leads to the rapid activation of c-fos and c-jun . Therefore we investigated the effect of TG on c-fos and c-jun expression. We found that both IEGs are rapidly induced by TG, c-fos faster than c-jun, but both are high at 5 h after treatment, whereas after 16 h, both are back to their normal levels (Fig. 5B,C). Like QC, the expression of c-fos by TG is also potentiated by treatment with BAPTA-AM. Again, this effect is observed at 5 h for c-fos, and at 16 h for QC (Fig. 6). These data demonstrate that c-fos and c-jun expression precedes QC expression and suggest that the upregulation of QC may involve activation of the QC promoter via c-fos and c-jun.
Differentiated SK-N-SH cells were treated with 1 µM TG for 5 h and 16 h with or without pre-incubation with 5 µM BAPTA-AM (B) for 1 h. Shown are the average + SD of triplicates of normalized QC (A) and c-fos (B) mRNA levels from a representative experiment of three. The expression levels were normalized to eEF2α mRNA, the expression levels in untreated cells are set to 1. Asterisks indicate a significant difference compared to control (*p<0.05, **p≤0.02 ***p≤0.001).
Ca2+ Induced QC and c-fos mRNA Expression is Neuronal Cell Type Specific
Since Aβ is known to be produced by different cell types in the brain, we investigated which cells express QC. We employed in situ hybridization using LNA probes, to detect the QC mRNA in human temporal cortex and hippocampus/entorhinal cortex. In sequential slides, probes recognizing the QC mRNA and the neuron specific microRNA hsa-mir-134 were used. This demonstrated that QC is predominantly expressed in neurons in different brain areas (Fig. 7A).
A. In situ hybridizations were performed on sequential sections of temporal cortex and hippocampus/entorhinal cortex of a patient with early AD pathology (I,B) as described in materials and methods. Shown are incubations without probe, using a probe directed against the QC mRNA or a probe recognizing the neuron-specific microRNA hsa-mir-134. Scale bar: 100 µM. B. Hek293 and C. Hela cells were treated with 1 µM TG for 5 h and 16 h. Shown is the average + SD of normalized QC and c-fos mRNA levels of n = 6 from 2 experiments (B) and n = 12 from 4 experiments (C). The expression levels were normalized to eEF2α mRNA, the expression levels in untreated cells are set to 1. Asterisks indicate a significant difference compared to control (*p<0.05, ***p≤0.0001).
To analyze whether the Ca2+-dependent regulation of QC is cell-type specific, we treated the non-neuronal HEK293 and Hela cell lines with TG. QC is not upregulated by TG treatment in either cell line (Fig. 7B,C). At 5h, the QC expression is even significantly reduced, but at 16 h there is no effect of TG observed. Interestingly, also the induction of c-fos by TG appears to be cell type specific. In the Hela cells the response is much lower than in the SK-N-SH cells and in 293 cells TG does not elicit any effect on the c-fos levels (Fig. 7B,C). These data suggest that the Ca2+-dependent regulation of QC expression is specific for neuronal cells.
The expression of QC, the enzyme that catalyzes the conversion of Aβ to the more toxic pE Aβ, is upregulated in AD cortex. Here we demonstrate that also in hippocampus/entorhinal cortex the upregulation of QC occurs at the earliest stages of AD pathology. In this study we investigated whether early pathogenic events that occur in AD brain could be directly involved in this regulation. We demonstrated that QC mRNA expression was not increased by oligomeric or fibrillar Aβ aggregates. Also induction of the UPR, using TM and 2DG was without effect. Only when the UPR was induced by the SERCA pump inhibitor TG the QC mRNA level was increased, indicating the involvement of Ca2+. We tried three different commercially available QC antibodies to directly determine the QC protein levels, but none worked in our hands. Instead we demonstrated that the QC enzyme activity is increased by TG. Aβ has been shown before to affect Ca2+ homeostasis , but apparently in a different manner or to a lesser extent than the Ca2+ perturbing stimuli that induce QC expression. Ca2+ signalling plays an important role in the regulation of many processes in cells. In neurons, Ca2+ acts as a second messenger and co-factor for many processes, e.g. neurotransmitter release and long-term potentiation, the cellular mechanism underlying learning and memory . Since Ca2+ is involved in such a broad variety of processes, the Ca2+ signals need to be regulated precisely and this involves regulated changes in Ca2+ concentration in the different cellular compartments. Under resting conditions the cytosolic Ca2+ concentration is kept low by an array of buffers, pumps and transport mechanisms, creating an electro-chemical gradient across the plasma membrane and the intracellular Ca2+ stores, including the ER. Ca2+ is predominantly released from the ER through activation of inositol-1,4,5-triphosphate receptors (IP3Rs) and ryanodine receptors (RyRs), whereas the SERCA pumps refill the store after depletion . Inhibition of SERCA leads to an increase in cytosolic Ca2+ concentration and ER Ca2+ depletion. Our data show that chelation of intracellular Ca2+ using BAPTA-AM potentiates the QC induction by TG, but does not have an effect by itself. Combined use of TG and BAPTA-AM results in faster and increased release of Ca2+ from the ER. This indicates that depletion of ER Ca2+ stores is the main trigger to increase the expression of QC. To further define the mechanism we analysed the effect of SERCA inhibition in combinations with pharmacological inhibition of the IP3Rs and RyRs. Unfortunately these experiments were not informative, probably by artefacts generated by the use of combinations of different pharmacological inhibitors. In addition, it has been shown before that the ER Ca2+ leak is not affected by inhibition of the IP3Rs or of the RyRs , probably because of the presence of other Ca2+ leak mediators .
Activation of immediate early gene expression is a key event in stress-induced neuronal cell injury . Analysis of the QC promoter showed the presence of a potential binding site for the heterodimer of the transcription factors c-fos and c-jun, both IEGs. We found that c-fos and c-jun mRNA are induced by the same stimuli as QC mRNA. It was reported before that c-fos is induced in neurons by treatment with TG . Similar to what we report in this study for QC, this increase is caused by Ca2+ depletion of the ER, rather than increased cytosolic Ca2+ levels. The induction of the IEGs is much faster and precedes the upregulation of QC. This is compatible with a model where the upregulation of QC by ER Ca2+ depletion is mediated by c-fos and c-jun, although this requires further study. Interestingly, both c-fos and c-jun are upregulated in AD hippocampus .
Using in situ hybridization we demonstrated that QC is predominantly expressed in neurons in the human brain, both in temporal cortex as well as in the earlier affected area in AD, the hippocampus/entorhinal cortex. The regulation of QC expression by TG appears to be specific for neuronal cells, because it is not observed in non-neuronal cells. Also c-fos is less responsive in non-neuronal cells, which strengthens the hypothesis that IEG induction is required for Ca2+ -regulated QC expression. BiP is upregulated in response to TG treatment in these cells, indicating that the treatment has resulted in depletion of ER Ca2+ levels. It is possible that non-neuronal cells have more resources to deal with disturbances in Ca2+ homeostasis, or that neuronal cells are more dependent on proper Ca2+ homeostasis for their function. It has been shown before that a different isoform of QC, isoQC, is involved in monocyte infiltration via modification of cytokines, and therefore it has a major role in cell types involved in the immune response . Our data suggest that QC performs a specific function in neurons. The formation of an N-terminal pE residue is an important event in the processing of numerous bioactive neuropeptides, hormones and cytokines during their maturation in the secretory pathway , . These peptides need the conformational change to bind to their receptors and/or to protect the N terminus from degradation. It is possible that QC is upregulated under stress conditions in neurons to stabilise specific peptides and potentiate their function. Whether the modification of Aβ is part of this or whether this is a pathological side-effect requires further investigation.
Perturbed Ca2+ homeostasis has been found in both sporadic and in familial (F)AD , . The majority of cases of FAD originate from a mutant PS gene. In addition to effects on Aβ formation, PS-FAD mutations lead to perturbed neuronal ER Ca2+ signalling , . It was found that PS can function as low-conductance passive ER Ca2+ leak channel and that the FAD mutations disrupt this Ca2+ leak function . This results in higher steady-state intraluminal Ca2+ levels and lower cytosolic Ca2+ homeostasis levels and leads to exaggerated release of Ca2+ from the ER, both in in vitro and in vivo , . In this respect it is interesting that the intraneuronal levels of pE Aβ are higher in PS1 FAD patients than in sporadic AD patients . In sporadic AD, aging is the major risk factor for developing the neurodegenerative disease. The Ca2+ hypothesis of aging emerged two decades ago  and is supported by studies that show age-related alterations in specific Ca2+ homeostasis -regulating systems in neurons , . In the aged brain there is an increased Ca2+ release from the ER stores through both the RyR and the IP3 receptors . Interestingly, our group recently demonstrated that the accumulation of intracellular pE aggregates in human brain is also highly dependent on age . The data presented here suggest that perturbed ER Ca2+ homeostasis facilitates the production of pE modified Aβ and may thereby contribute to the amyloid pathology in AD.
Materials and Methods
Cell Culture and Treatment
SK-N-SH (European Collection of Cell Cultures #86012802, Salisbury, UK), 293 and Hela cells were cultured in Dulbecco’s modified Eagle’s medium with GlutaMAX (Gibco BRL, Carlsbad, CA, USA) supplemented with 10% fetal calf serum (FCS, Gibco BRL), 100 µg/ml streptomycin and 100 U/ml penicillin. SK-N-SH cells were differentiated in culture medium supplemented with all trans-retinoic acid (Sigma, St Louis, MO, USA) at a final concentration of 10 µM for 5 days. Differentiated cells were treated with Aβ1–42 oligomers or fibrils, TG, TM, DG (all from Sigma) and BAPTA-AM (Calbiochem, Darmstadt, Germany) at the indicated concentrations and time points.
Preparation of Different Aβ Species
Synthetic Aβ1–42 was purchased from Anaspec (San Jose, CA, USA). Aβ1–42 was dissolved in hexafluorisopropanol (HFIP) at a final concentration of 1 mg/ml, aliquoted and dried under vacuum. The resulting peptide film was stored at −80°C until further use. To obtain low molecular weight Aβ, the peptide film was dissolved in DMSO (5 mM), and sonicated in a bath sonicator for 10 minutes. Aβ was subsequently diluted in PBS to a final concentration of 50 µM. To enrich for oligomers, this mixture was incubated at 4°C for 24 h. High order aggregates were removed by centrifugation (10 min, 14 000 rpm) at 4°C. To obtain fibrillar Aβ, the peptide film was dissolved in DMSO (5 mM). Then 10 mM HCl was added to bring the peptide to 100 µM concentration and incubated for 24h at 37°C. The final concentration of the oligomeric and the fibrillar Aβ 1–42 preparations was determined using Bradford protein assay and the β-sheet content was determined by a Thioflavin T assay.
Post-mortem Brain Tissue
Post-mortem brain material was obtained from the Netherlands Brain Bank (Amsterdam, The Netherlands). All donors or their next of kin provided written informed consent for brain autopsy and use of tissue and medical records for research purposes. The parameters of all tissue samples used in this study are listed in Table 1.
RNA Isolation and Real-time Quantitative PCR
Total RNA was isolated from differentiated SK-N-SH cells, untreated or treated, and post-mortem brain tissue with the RNAeasy minikit (Qiagen, Netherlands). cDNA synthesis was performed using Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA) and oligo-dT primers. Real-time quantitative PCR (qPCR) was performed using the Light Cycler 480 system (Roche Applied Science, USA). Reaction volumes of 5 µl contained cDNA, 0.1 µM Universal Probe Library probe (Roche Applied Science, USA), 0.4 µM forward primer, 0.4 µM reverse primer and 2.5 µl 2x LightCycler 480 Probes Master (Roche Applied Science, USA). After denaturation for 10 min at 95°C, amplification was performed using 35 cycles of denaturation (95°C for 10 s), followed by annealing (58°C for 15 s), and elongation (72°C for 15 s). Results were analysed using the LightCycler 480 software (Roche Applied Science, USA) version 1.5. Expression levels were normalized using eEF2α. Primers and probes used in this study are listed in Table 2.
Cells were washed with PBS and lysed in PBS+1%Triton. Subsequently the supernatant was collected after centrifugation 12000×g at 4°C for 10 minutes. The protein concentration was determined by the Bradford Assay (Bio-Rad, Laboratories, Veenendaal, The Netherlands). 100 µl lysate (0.5 mg/ml) was mixed with 100 µl of 200 µM Q-AMC (Bachem, The Netherlands) in assay buffer (25 mM HEPES, pH 7.0) and incubated in dark tubes at room temperature for 20 minutes. The reaction was stopped at 100°C for 5 minutes and the incubations were cooled on ice for 3 minutes. Subsequently 200 µl of rhPGPEP-1 (0.1 µg/ml, 6278-CY, R&D systems, Europe) was added to each vial and the mixture was incubated at room temperature for 10 minutes. Fluorescence was measured using a FLUOstar Omega (BMG LABTECH GmbH, Ortenberg, Germany).
In situ Hybridization
In situ hybridization was essentially performed as described before . For human QC a 5′ fluorescein labeled 19mer antisense oligonucleotide was used, containing Locked Nucleic Acid (LNA) and 2′OME RNA moieties (FAM – AuuTucTucAuuGucAucC, capitals indicate LNA, lower case indicates 2′OME RNA). An antisense oligo targeting the neuron-specific hsa-miR-134 was used (FAM - CucTggTcaAccAguCacA) to detect neurons and as negative control an oligo that does not detect a signal in human brain (FAM - TaaCccTaaGgcAauTccT). The oligonucleotides were synthesized by Ribotask ApS, Odense, Denmark. The hybridizations were done on 5 µM sections of paraffin embedded temporal cortex material obtained from the Netherland Brain Bank. In brief, sections were deparaffinized, treated with proteinase K (20 µg/ml) for 5 min. Hybridizations were done at 60°C for 90 min in hybridization mix (50% (vol/vol) deionized formamide, 600 mM NaCl, 10 mM HEPES buffer, pH 7.5, 1 mM EDTA, 5×Denhardt’s reagent and 200 µg/ml denatured herring sperm DNA). The oligonucleotide concentration in the hybridization mix was 1 µM. The QC incubations were also performed at lower concentration (0.25 µM) with similar result. After hybridization the tissue sections were washed consecutively for 5 min with 2×SSC, 0.5×SSC and 0.2×SSC at 60°C. The hybridization signal was detected using a rabbit polyclonal α-fluorescein/Oregon green antibody (Molecular Probes) and a horse radish peroxidase (HRP) labeled goat α-rabbit polyclonal antibody as secondary antibody. Signal was detected with standard 3-amino-9-ethylcarbazole staining (AEC) and hematoxylin was used as a nuclear counterstain.
Conceived and designed the experiments: LDK WS. Performed the experiments: LDK AB RZ EvS. Analyzed the data: LDK AB RZ EvS JJMH WS. Wrote the paper: LDK WS.
- 1. De Kimpe L, Scheper W (2010) From alpha to omega with Aβ: targeting the multiple molecular appearances of the pathogenic peptide in Alzheimer’s disease. Curr Med Chem 17: 198–212.
- 2. Schilling S, Wasternack C, Demuth HU (2008) Glutaminyl cyclases from animals and plants: a case of functionally convergent protein evolution. Biol Chem 389: 983–991.
- 3. De Kimpe L., Van Haastert ES, Kaminari A, Zwart R, Rutjes H, et al.. (2012) Intracellular accumulation of aggregated pyroglutamate amyloid beta: convergence of aging and Aβ pathology at the lysosome. AGE 10.1007/s11357-012-9403-0 [doi].
- 4. He W, Barrow CJ (1999) The Aβ 3-pyroglutamyl and 11-pyroglutamyl peptides found in senile plaque have greater beta-sheet forming and aggregation propensities in vitro than full-length Aβ. Biochemistry 38: 10871–10877.
- 5. Schlenzig D, Manhart S, Cinar Y, Kleinschmidt M, Hause G, et al. (2009) Pyroglutamate formation influences solubility and amyloidogenicity of amyloid peptides. Biochemistry 48: 7072–7078.
- 6. Schilling S, Lauber T, Schaupp M, Manhart S, Scheel E, et al. (2006) On the seeding and oligomerization of pGlu-amyloid peptides (in vitro). Biochemistry 45: 12393–9.
- 7. Saido TC, Iwatsubo T, Mann DM, Shimada H, Ihara Y, et al. (1995) Dominant and differential deposition of distinct beta-amyloid peptide species, A beta N3(pE), in senile plaques. Neuron 14: 457–466.
- 8. Mori H, Takio K, Ogawara M, Selkoe DJ (1992) Mass spectrometry of purified amyloid beta protein in Alzheimer’s disease. J Biol Chem 267: 17082–17086.
- 9. Schilling S, Hoffmann T, Manhart S, Hoffmann M, Demuth HU (2004) Glutaminyl cyclases unfold glutamyl cyclase activity under mild acid conditions. FEBS Lett 563: 191–196.
- 10. Cynis H, Scheel E, Saido TC, Schilling S, Demuth HU (2008) Amyloidogenic processing of amyloid precursor protein: evidence of a pivotal role of glutaminyl cyclase in generation of pyroglutamate-modified amyloid-beta. Biochemistry 47: 7405–7413.
- 11. Jawhar S, Wirths O, Schilling S, Graubner S, Demuth HU, et al. (2011) Overexpression of glutaminyl cyclase, the enzyme responsible for pyroglutamate Aβ formation, induces behavioral deficits, and glutaminyl cyclase knock-out rescues the behavioral phenotype in 5XFAD mice. J Biol Chem 286: 4454–4460.
- 12. Schilling S, Zeitschel U, Hoffmann T, Heiser U, Francke M, et al. (2008) Glutaminyl cyclase inhibition attenuates pyroglutamate Abeta and Alzheimer’s disease-like pathology. Nat Med 14: 1106–1111.
- 13. Hoozemans JJ, Veerhuis R, Van Haastert ES, Rozemuller JM, Baas F, et al. (2005) The unfolded protein response is activated in Alzheimer’s disease. Acta Neuropathol (Berl) 110: 165–172.
- 14. Hoozemans JJM, Van Haastert ES, Nijholt DAT, Rozemuller AJM, Eikelenboom P, et al. (2009) The Unfolded Protein Response is activated in pretangle neurons in Alzheimer’s disease hippocampus. Am J Pathol 174: 1241–1251.
- 15. Chafekar SM, Zwart R, Veerhuis R, Vanderstichele H, Baas F, et al. (2008) Increased Abeta1–42 production sensitizes neuroblastoma cells for ER stress toxicity. Curr Alzheimer Res 5: 469–74.
- 16. Chafekar SM, Hoozemans JJ, Zwart R, Eikelenboom P, Baas F, et al. (2007) Abeta1–42 induces mild endoplasmic reticulum stress in an aggregation state dependent manner. Antioxid Redox Signal 9: 2245–54.
- 17. Emilsson L, Saetre P, Jazin E (2006) Alzheimer’s disease: mRNA expression profiles of multiple patients show alterations of genes involved with calcium signaling. Neurobiol Dis 21: 618–625.
- 18. Khachaturian ZS (1987) Hypothesis on the regulation of cytosol calcium concentration and the aging brain. Neurobiol Aging 8: 345–346.
- 19. Mattson MP, Magnus T (2006) Ageing and neuronal vulnerability. Nat Rev Neurosci 7: 278–294.
- 20. Murchison D, Griffith WH (2007) Calcium buffering systems and calcium signaling in aged rat basal forebrain neurons. Aging Cell 6: 297–305.
- 21. Chung KC, Sung JY, Ahn W, Rhim H, Oh TH, et al. (2001) Intracellular calcium mobilization induces immediate early gene pip92 via Src and mitogen-activated protein kinase in immortalized hippocampal cells. J Biol Chem 276: 2132–2138.
- 22. Gruszczynska-Biegala J, Pomorski P, Wisniewska MB, Kuznicki J (2011) Differential roles for STIM1 and STIM2 in store-operated calcium entry in rat neurons. PLoS One 6: e19285. 10.1371/journal.pone.0019285 [doi].
- 23. Moore WC, Hargrove HM, Salama AI, Patel J (1991) Calcium mobilization and entry by thapsigargin in neuronal tumor cell line, SK-N-SH. Neuroreport 2: 124–126.
- 24. Weber JT, Rzigalinski BA, Ellis EF (2001) Traumatic injury of cortical neurons causes changes in intracellular calcium stores and capacitative calcium influx. J Biol Chem 276: 1800–1807.
- 25. Muallem S, Khademazad M, Sachs G (1990) The route of Ca2+ entry during reloading of the intracellular Ca2+ pool in pancreatic acini. J Biol Chem 265: 2011–2016.
- 26. Preston SF, Berlin RD (1992) An intracellular calcium store regulates protein synthesis in HeLa cells, but it is not the hormone-sensitive store. Cell Calcium 13: 303–312.
- 27. Gissel C, Doutheil J, Paschen W (1997) Temporal analysis of changes in neuronal c-fos mRNA levels induced by depletion of endoplasmic reticulum calcium stores: effect of clamping cytoplasmic calcium activity at resting levels. J Neurochem 69: 2538–2545.
- 28. Demuro A, Parker I, Stutzmann GE (2010) Calcium signaling and amyloid toxicity in Alzheimer disease. J Biol Chem 285: 12463–12468.
- 29. Berridge MJ, Bootman MD, Lipp P (1998) Calcium-a life and death signal. Nature 395: 645–648.
- 30. Stutzmann GE, Mattson MP (2011) Endoplasmic reticulum Ca(2+) handling in excitable cells in health and disease. Pharmacol Rev 63: 700–727.
- 31. Hofer AM, Curci S, Machen TE, Schulz I (1996) ATP regulates calcium leak from agonist-sensitive internal calcium stores. FASEB J 10: 302–308.
- 32. Camello C, Lomax R, Petersen OH, Tepikin AV (2002) Calcium leak from intracellular stores–the enigma of calcium signalling. Cell Calcium 32: 355–361.
- 33. Herrera DG, Robertson HA (1996) Activation of c-fos in the brain. Prog Neurobiol 50: 83–107.
- 34. Sajan FD, Martiniuk F, Marcus DL, Frey WH, Hite R, et al. (2007) Apoptotic gene expression in Alzheimer’s disease hippocampal tissue. Am J Alzheimers Dis Other Demen 22: 319–328.
- 35. Cynis H, Hoffmann T, Friedrich D, Kehlen A, Gans K, et al. (2011) The isoenzyme of glutaminyl cyclase is an important regulator of monocyte infiltration under inflammatory conditions. EMBO Mol Med 3: 545–558.
- 36. Hinke SA, Pospisilik JA, Demuth HU, Mannhart S, Kuhn-Wache K, et al. (2000) Dipeptidyl peptidase IV (DPIV/CD26) degradation of glucagon. Characterization of glucagon degradation products and DPIV-resistant analogs. J Biol Chem 275: 3827–3834.
- 37. Van CE, Proost P, Van A, I, Struyf S, Polfliet M, et al. (1998) Functional comparison of two human monocyte chemotactic protein-2 isoforms, role of the amino-terminal pyroglutamic acid and processing by CD26/dipeptidyl peptidase IV. Biochemistry 37: 12672–12680.
- 38. Toescu EC, Vreugdenhil M (2010) Calcium and normal brain ageing. Cell Calcium 47: 158–164.
- 39. Supnet C, Bezprozvanny I (2011) Presenilins function in ER calcium leak and Alzheimer’s disease pathogenesis. Cell Calcium 50: 303–309.
- 40. Supnet C, Bezprozvanny I (2010) The dysregulation of intracellular calcium in Alzheimer disease. Cell Calcium 47: 183–189.
- 41. Demuro A, Parker I, Stutzmann GE (2010) Calcium signaling and amyloid toxicity in Alzheimer disease. J Biol Chem 285: 12463–12468.
- 42. Tu H, Nelson O, Bezprozvanny A, Wang Z, Lee SF, et al. (2006) Presenilins form ER Ca2+ leak channels, a function disrupted by familial Alzheimer’s disease-linked mutations. Cell 126: 981–993.
- 43. Bezprozvanny I, Mattson MP (2008) Neuronal calcium mishandling and the pathogenesis of Alzheimer’s disease. Trends Neurosci 31: 454–463.
- 44. Smith IF, Green KN, LaFerla FM (2005) Calcium dysregulation in Alzheimer’s disease: recent advances gained from genetically modified animals. Cell Calcium 38: 427–437.
- 45. Wirths O, Erck C, Martens H, Harmeier A, Geumann C, et al. (2010) Identification of low molecular weight pyroglutamate Aβ oligomers in Alzheimer disease: a novel tool for therapy and diagnosis. J Biol Chem 285: 41517–41524.
- 46. Thibault O, Gant JC, Landfield PW (2007) Expansion of the calcium hypothesis of brain aging and Alzheimer’s disease: minding the store. Aging Cell 6: 307–317.
- 47. Budde BS, Namavar Y, Barth PG, Poll-The BT, Nurnberg G, et al. (2008) tRNA splicing endonuclease mutations cause pontocerebellar hypoplasia. Nat Genet 40: 1113–1118.