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Alzheimer’s disease (AD) is a progressive and irreversible neurodegenerative disorder. Familial AD (FAD) mutations in presenilins have been linked to calcium (Ca2+) signaling abnormalities. To explain these results we previously proposed that presenilins function as endoplasmic reticulum (ER) passive Ca2+ leak channels. To directly investigate the role of presenilins in neuronal ER Ca2+ homeostasis we here performed a series of Ca2+ imaging experiments with primary neuronal cultures from conditional presenilin double-knockout mice (PScDKO mice; PS1dTAG/dTAG, PS2−/−) and from a triple transgenic AD mice (3xTg mice; KI-PS1M146V, Thy1-APPKM670/671NL, Thy1-tauP301L). Obtained results provided further support to the hypothesis that presenilins function as ER Ca2+ leak channels in neurons. Interestingly, we discovered that presenilins play a major role in ER Ca2+ leak function in hippocampal but not in striatal neurons. We further discovered that in hippocampal neurons loss of presenilin-mediated ER Ca2+ leak function was compensated by an increase in expression and function of ryanodine receptors (RyanR). Long-term feeding of RyanR inhibitor dantrolene to APPPS1 mice (Thy1-APPKM670/671NL, Thy1-PS1L166P) resulted in an increased amyloid load, loss of synaptic markers and neuronal atrophy in hippocampal and cortical regions. These results indicate that disruption of ER Ca2+ leak function of presenilins may play an important role in AD pathogenesis.
Alzheimer’s disease (AD) is the most common form of age-related dementia in human beings over the age of 60 years. Most cases of AD are idiopathic, but the small fractions of AD cases are familial and characterized by an earlier onset and genetic inheritance. Mutations in presenilin-1 (PS1) and presenilin-2 (PS2) account for about 40% of all known familial AD (FAD) cases in which a genetic cause has been identified (Tandon and Fraser, 2002). Presenilins are 50 kDa proteins that contain nine transmembrane domains (Laudon et al., 2005; Spasic et al., 2006) and reside in the endoplasmic reticulum (ER) membrane (Annaert et al., 1999). Presenilins undergo endoproteolytic cleavage to amino-terminal (NTF) and carboxy-terminal (CTF) fragments. The complex of cleaved presenilins with nicastrin, aph-1, and pen-2 subunits moves from ER to plasma membrane, where it functions as a γ-secretase, which cleaves the amyloid precursor protein (APP) and releases the amyloid β-peptide (Aβ), the principal constituent of the amyloid plaques in the brains of AD patients. Consistent with the role of presenilins as catalytic subunits of a γ-secretase (De Strooper et al., 1998; Wolfe et al., 1999), FAD mutations in presenilins affect APP processing.
In addition to changes in APP processing, many FAD mutations in presenilins result in deranged Ca2+ signaling and growing evidence indicate that neuronal Ca2+ signaling disruptions may play an early and important role in AD pathogenesis (Bezprozvanny and Mattson, 2008). The connection between presenilins and Ca2+ signaling was initially uncovered when it was reported that fibroblasts from FAD patients release supranormal amounts of Ca2+ in response to InsP3 (Ito et al., 1994). Similar results were obtained in experiments with cells expressing FAD mutant presenilins (Leissring et al., 1999a; Leissring et al., 1999b) and in cortical and hippocampal neurons from presenilin FAD mutant knock-in mice (Guo et al., 1999; Chan et al., 2000; Schneider et al., 2001; Stutzmann et al., 2004; Smith et al., 2005; Stutzmann et al., 2006; Chakroborty et al., 2009). To explain these results it has been suggested that mutant presenilins affect store-operated Ca2+ influx (Leissring et al., 2000; Yoo et al., 2000; Herms et al., 2003; Akbari et al., 2004), increase activity and/or expression of intracellular Ca2+ release channels such as ryanodine receptors (RyanR) (Chan et al., 2000; Smith et al., 2005; Stutzmann et al., 2006; Hayrapetyan et al., 2008; Rybalchenko et al., 2008) and inositol-1,4,5-trisphosphate receptors (InsP3R) (Cai et al., 2006; Cheung et al., 2008)or modulate function of smooth endoplasmic reticulum Ca2+-ATPase (SERCA) pump (Green et al., 2008). Based on results obtained in bilayer reconstitution electrophysiological experiments and Ca2+ imaging experiments with mouse embryonic fibroblasts (MEFs) from PS DKO mice we previously suggested that presenilins function as passive ER Ca2+ leak channels which control steady-state ER Ca2+ levels (Tu et al., 2006). We also found that many FAD mutations in presenilins result in loss of ER Ca2+ leak function, leading to ER Ca2+ overload and supranormal Ca2+ release from the ER (Tu et al., 2006; Nelson et al., 2007; Nelson et al., 2010). The ER Ca2+ leak function of presenilins was independent of their γ-secretase function as it could be rescued by PS1-D257A mutant which lacks γ-secretase activity and it was maintained in Aph1 triple-knock out MEF cells which lack γ-secretase but express full length presenilins (Tu et al., 2006).
To directly investigate the role of presenilins in neuronal ER Ca2+ homeostasis we here performed a series of Ca2+ imaging experiments with primary neuronal cultures from conditional presenilin double-knockout mice (PScDKO mice; PS1dTAG/dTAG, PS2−/−) and from a triple transgenic AD mice (3xTg mice; KI-PS1M146V, Thy1-APPKM670/671NL, Thy1-tauP301L) (Oddo et al., 2003). Obtained results supported the hypothesis that presenilins function as ER Ca2+ leak channels in neurons. We further discovered that a loss of ER Ca2+ leak function of presenilins in hippocampal neurons was compensated by increased expression and activity of ryanodine receptors (RyanR). Furthemore, long-term feeding of a double transgenic AD mice (APPPS1 mice; Thy1-APPKM670/671NL, Thy1-PS1L166P) (Radde et al., 2006) with RyanR inhibitor dantrolene resulted in increased amyloid load, loss of synaptic markers and neuronal atrophy in hippocampal and cortical regions of these mice. Our results provide new evidence that disruption of ER Ca2+ leak function of presenilins and resulting neuronal Ca2+ dyshomeostasis play an important role in AD pathogenesis. Our results obtained with the three different presenilin genetic mouse models (knockin, conditional knockout and transgenic) also provide a beginning of common foundation for the currently contradictory view on the role that presenilins play in neuronal Ca2+ homeostasis.
The PScDKO mice (PS1dTAG/dTAG, PS2−/−) were generated by crossing PS1dTAG/dTAG mice with PS2−/− mice (Herreman et al., 1999). PS1dTAG/dTAG mice were generated by knockin strategy. The first exon of PSEN1 gene was surrounded by loxP sites (Supplementary Fig 1A). In addition, a double tag encoding for calmodulin binding protein (CBP) followed by 3xFlag epitope tag was inserted immediately after the ATG start codon of PSEN1 (Supplementary Fig 1A). The generation and characterization of 3xTg mice (KI-PS1M146V, Thy1-APPKM670/671NL, Thy1-tauP301L) has been previously described (Oddo et al., 2003). The wild type (WT) mice of the same mixed background strain (C7BL/6;129X1/SvJ;129S1/Sv) was used as a control for experiments with 3xTg mice. 3xTg mice and control WT mice were kindly provided by Frank LaFerla (UC Irvine). The generation and characterization of APPPS1 mice (Thy1-APPKM670/671NL, Thy1-PS1L166P) has been previously described (Radde et al., 2006). The APPPS1 mice were kindly provided by Mathias Jucker (University of Tubingen). The three transgenic mouse lines and WT mice were housed in a pathogen-free facility in a temperature-controlled room at 22–24 °C with a 12 hr light:dark cycle and were fed a standard laboratory chow diet and double-distilled water ad libitum. All procedures involving mice were approved by Institutional Animal Care and Use Committee (IACUC) of UT Southwestern Medical Center at Dallas, in accord with the NIH Guidelines for the Care and Use of Experimental Animals.
Anti-CTF-PS1 mAb (MAB5232) and anti-actin mAb (MAB1501) were from Chemicon, anti-FLAG mAb (F3165) was from Sigma, anti-RyanR mAb (MA3-925) was from ABR, polyclonal antibody for InsP3R1 (T443) was previously described (Kaznacheyeva et al., 1998), polyclonal antibody for SERCA2b was provided by Dr. Frank Wuytack (KU Leuven), monoclonal antibody for PSD95 (MA1-045) was purchased from Thermo; NeuN mAb (MAB377) was from Millipore, DARPP-32 mAb (#2306) was from Cell Signaling, anti-Aβ 6E10 mAb (SIG-39300) was from Covance, Alexa Fluor-488 or Fluor-594 anti-rabbit and anti-mouse secondary antibodies (A21121, A11012 and A11008) were from Invitrogen, HRP-conjugated anti-rabbit and anti-mouse secondary antibodies (115-035-146 and 111-035-144) were from Jackson ImmunoResearch, biotinylated anti-mouse IgG reagent was from Vector Laboratories (M.O.M. kit, BMK-2202). For Western blotting, analysis proteins were resolved on 6% (for RyanR and InsP3R1), 10% (for actin and SERCA2B) or 12% (for PS1) SDS-PAGE gels, transferred to nitrocellulose paper, and probed with the corresponding primary and secondary antibodies.
Shuttle plasmid constructs encoding NLS-GFP-Cre and NLS-GFP were provided by Thomas Sudhof (Stanford University). Lenti-Cre and Lenti-GFP viruses were generated by co-transfection of shuttle vectors with HIV-1 packaging vector 8.9 and VSVG envelope glycoprotein plasmids into the packaging cell line HEK293T as we previously described (Tang et al., 2009). The generated viral stocks were aliquoted into 1 ml tubes, immediately frozen in liquid nitrogen and stored at −80°C. Prior to use the aliquots of generated lentiviruses were thawed and warmed up in the 37°C incubator. Each batch of generated Lenti-Cre and Lenti-GFP lentiviruses was tested in pilot cortical neuronal culture infection experiments and the titer with minimal toxicity and maximum infection efficiency (>95%) was used in all experiments.
The hippocampal and striatal cultures of PScDKO, 3xTg and WT mice were established from P0-P1 pups and maintained in culture as we previously described (Tang et al., 2005; Zhang et al., 2006). Lenti-CRE and Lenti-GFP viruses were added to PScDKO cultures at DIV4. Wild type PS1 and mutant PS1 constructs (L166P, ΔE9, M146V, D257A, D385A) were amplified by PCR using constructs in pcDNA3 (Tu et al., 2006; Nelson et al., 2007) and cloned into lentiviral shuttle vector. An amino-terminal HA tag was added to all lentiviral PS1 constructs by PCR. Ctrl-shRNAi (SHC002) and RyR-shRNAi (SHCLNG_NM_009109, TRCN0000103010) lentivirus shuttle constructs were obtained from Sigma. Lenti-PS1 and Lenti-shRNAi viruses were generated by following the same procedures as for Lenti-Cre and Lenti-GFP viruses. The Lenti-PS1 rescue viruses were added to Lenti-Cre-infected PScDKO hippocampal neuronal cultures at DIV6. Lenti-shRNAi viruses were added to WT and 3xTg hippocampal neuronal cultures at DIV6.
Fura-2 Ca2+ imaging experiments with DIV12 or DIV13 hippocampal and striatal cultures were performed as previously described (Tang et al., 2005; Tu et al., 2006). Briefly, the cells were maintained in artificial cerebrospinal fluid(aCSF) (140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 10 mM Hepes, pH 7.3). Fura-2 340/380 ratio images were collected every 2 sec for the duration of the experiment using a DeltaRAM-X illuminator, an IC-300 camera, and IMAGEMASTER PRO software (all from PTI). The region of interest (ROI) used in the image analysis corresponded to the entire cell soma. For caffeine experiments, 25 mM caffeine in aCSF was applied. For ionomycin (IO) experiments, the cells were first washed with the Ca2+-free aCSF (omitted CaCl2 from aCSF and supplemented with 100 μM EGTA) for 30 sec or 2 min as indicated, followed by addition of 5 μM IO. For KCl refill experiments, the neurons were first challenged with 15 mM KCl for 1 min in aCSF, then perfused with Ca2+-free aCSF for 30 sec or 2 min as indicated, followed by addition of 5 μM IO. In dantrolene experiments 50 nM of dantrolene was added to the culture medium at DIV7, DIV9, and DIV11, and Ca2+ imaging experiments were performed at DIV13. All Ca2+ imaging experiments were done in room temperature. In caffeine experiments maximal amplitude (peak) response was determined from Fura-2 340/380 ratios. The size of the ER Ca2+ pool was calculated by integrating an area under the IO-induced Fura-2 Ca2+ response curve as we previously described (Tu et al., 2006). IO30 and IO120 correspond to the size of IO-releasable Ca2+ pool after 30 sec and 120 sec incubation in Ca2+-free aCSF, respectively. There was no significant difference in basal Fura-2 ratio values in different groups of neurons prior to addtion of ionomycin. The only exception were neurons infected with PS1-ΔE9 virus which yielded higher basal Ca2+ levels and increased Fura-2 ratios, presumably because this mutant acts as “superleaky” channel in the ER membrane (Tu et al., 2006).
D1ER expression plasmid was kindly provided by Dr Roger Tsien (UCSD) (Palmer et al., 2004). D1ER is a FRET-based cameleon Ca2+ indicator composed of enhanced cyan fluorescent protein (ECFP) and citrin fluorescent protein separated by a linker encoding calmodulin (CaM) and CaM-binding M13 peptide sequence (Palmer et al., 2004; McCombs and Palmer, 2008). Calreticulin (CRT) targeting sequence (MLLPVLLLGLLGAAAD) was added to amino-terminus and ER retension sequence (KDEL) was added to the carboxy-terminus of D1ER protein to facilitate ER targeting and retension.
Wild type (WT) and PS double-knockout (DKO) MEF cells were previously described (Herreman et al., 2000; Tu et al., 2006). WT and DKO MEF cells were transfected with D1ER plasmid using Lipofectamin 2000. DIV7 WT and 3xTg primary hippocampal neuronal cultures were transfected with D1ER plasmid using polyethylenimine (PEIs) method. Imaging experiments with transfected MEF cells and hippocampal neurons were performed 48 hours after D1ER transfection using Deltavision RT wide-field epifluorescence deconvolution microscope (Applied Precision) equipped with a Photometrics CoolSNAPHQ monochromatic digital camera (Roper Scientific) controlled by the SoftWorx image acquisition software package (Applied Precision). The filter sets used for FRET imaging experiments were CFPx 436/10 and YFPx 492/18 for excitation, CFPm 465/30 and YFPm 535/30 for emission. The series of three images: CFPx/CFPm (CFP), YFPx/YFPm (YFP) and CFPx/YFPm (FRET) were acquired every 25sec. After collection of intial data points (100 sec), the cells were washed with Ca2+-free aCSF and transferred to Ca2+-free aCSF containing 2 μM of SERCA pump inhibitor thapsigargin for 600 sec to deplete the ER Ca2+ stores. Following thapsigarin treatment, the cells were exposed to 5 μM ionomycin (IO). At conclusion of each experiment the FRET/CFP ratio signals were calibrated in the presence 5 μM ionomycin using a series of calibration buffers (125 mM KCl, 25 mM NaCl, 10 mM HEPES, 0.8 mM EGTA, pH 7.3) with free Ca2+ clamped to defined concentrations (1 μM, 10 μM, 50 μM, 100 μM, 200 μM, 400 μM, 800 μM, 5 mM). The free Ca2+ concentration in the calibration buffers was calculated by WEBMAXC STANDARD program. Calibration and analysis of the data was performed using Image J software. The ER Ca2+ concentration was calculated from FRET/CFP ratio data for each cell using corresponding empirical calibration curves for the same type of cells.
Dantrolene was delivered to APPPS1 and WT mice by using an approach that we used previously for dantrolene evaluation in SCA3 and SCA2 mice (Chen et al., 2008; Liu et al., 2009). Briefly, six APPPS1 hemizygous mice and six WT mice were fed with 100 μg of dantrolene suspended in 50 μl of PBS with 2% corn flour resulting in the final dose of 5 mg/kg. Thecontrol group of six APPPS1 hemizygous mice and six WT mice were fed with 2% cornflour in PBS. All mice were fed orally twice a week from 2 to 8 months of age. At conclusion of dantrolene feeding all mice were terminally anesthetized and perfused transcardially with 30 ml of ice-cold PBS followed by 50 ml of fixative (4% paraformaldehyde in 0.1 M PBS, pH 7.4) as we previously described (Chen et al., 2008; Liu et al., 2009; Tang et al., 2009). All brains were removed from skull, postfixed overnight at 4°C in 4% paraformaldehyde and equilibrated in 20–30% (w/v) sucrose in PBS. The brains were sliced to 30 μm thick coronal sections using SM2000R sliding microtome (Leica).
For beta-amyloid plaque staining, the 30 μm coronal sections from APPPS1 mice spaced throughout the forebrain were stained with 6E10 anti-Aβ mAb (1:1000 dilution) followed by staining with Alexa Flour-488 secondary anti-mouse IgG (1:2000 dilution). The quantitative analysis of amyloid load was performed blindly using Isocyte laser scanner system (Molecular Devices). The average area of the amyloid plaques (AREA), the average intensity of fluorescent signal in the plaques (IINT-1), and the number of plaques was calculated automatically for each slice by using proprietary image analysis software (Molecular Devices). Twenty coronal sections from each of the twelve mice were quantified for the analysis and the data were averaged within dantrolne-fed and control groups.
The immunostaining experiments were performed as we previously described (Chen et al., 2008; Liu et al., 2009; Tang et al., 2009). The hippocampal sections were stained with PSD95 mAb and Alexa Fluor-488 anti-rabbit IgG. The hippocampal nuclei were stained with propidium iodide (PI). The striatal sections were stained with DARPP-32 mAb and with Alexa Fluor-594 anti-mouse IgG. The images from hippocampal and striatal sections were collected using Zeiss Axoivert 100M confocal microscope. The striatal sections were also stained with anti-NeuN mAb and biotinylated anti-mouse IgG. Signal was amplified with an ABC Elite kit (Vector Laboratories) and detected with diaminobenzidine (Vector Laboratories). The Bielschowsky silver staining was performed on four sections for each APPPS1 mice and WT mice by following published procedures (Schwab et al., 2004). Briefly, the sections were closed and mounted to slides. The slides were placed in 0.1% silver nitrate solution, containing citric acid in a 65°C water bath for seven minutes. The slides were rinsed in 2 changes of ddH2O, followed by rinse in 95% ethanol, and then rinsed with 2 changes of absolute ethanol. All slides were immersed in filtered 2.5% gum mastic ethanol solution for 5 min, then put into developing solution (10 ml 2.5% gum mastic ethanol solution, 30 ml 1.7% Hydroquinone, add 0.9 ml of 1% silver nitrate) in a 65°C water bath until dark brown color developed (approximately 12 minutes). The slides were rinsed in water, dehydrated and mounted. The pictures for NeuN and silver staining were taken using Nikon Eclipse 80i microscope.
For quantification of PSD95 density confocal data were collected using 40x objective at Zeiss 100M microscope. The collected data were exported in tiff file format using Zeiss confocal software. The tiff file containing PSD95 staining signal (green channel) was imported into ImageJ. The region of interest (ROI) 200 × 200 pixels was randomly selected in Imagej and the software automatically calculated the number of PSD95 puncta and area of PSD95-positive pixels in the selected ROI. For quantificaton of neurites density the images of the hippocampal slides stained with Bielschowsky silver staining method were taken using 40x objective using Nikon Eclipse 80i microsope and exported as tiff files using Stereo Investigator software (MicroBrightField Inc). The tiff files were imported into ImageJ for analysis and 200 × 200 pixels ROI was selected as described above. The smallest cell body in the ROI was manually chosen and all objects with larger diameter than the selected cell body were excluded from the analysis to filter out signals from neuronal cell bodies and amyloid plaques. The remaining pixels correspond to the area covered by neurites. The area covered by neurites was automatically calculated for each ROI by MetaXpress. For PSD95 and silver staining quantification 4 ROI were randomly chosen for each analyzed section. Six CA1 hippocampal sections were analyzed from each mice, resulting in 24 ROI for each mice. The data from all 24 ROI were averaged for each mice. The obtained results from each of the 6 mice in vehicle- and dantrolene-treated groups were averaged and presented as mean ± S.E. (n = 6).
Statistical comparisons of results obtained in experiments were performed by one-way Anova test or Student’s t test. The p values are indicated in the text and figure legends as appropriate. The differences between control and experimental groups were determined to be non-significant (n.s.) in cases where p > 0.05.
Previous Ca2+ imaging studies indicated that ryanodine receptor (RyanR)-mediated Ca2+ release is enhanced in cortical and hippocampal neurons from 3xTg mice (KI-PS1M146V, Thy1-APPKM670/671NL, Thy1-tauP301L) and KI-PS1M146V mice (Guo et al., 1999; Chan et al., 2000; Smith et al., 2005; Stutzmann et al., 2006; Chakroborty et al., 2009). Consistent with these findings, we discovered that application of RyanR agonist caffeine resulted in much greater Ca2+ responses in postnatal hippocampal neuronal cultures from 3xTg mice than from the wild type (WT) mice (Fig 1A). On average, the peak amplitude of caffeine-induced Ca2+ responses was 4-fold higher in 3xTg hippocampal neurons than in wild type neurons (Fig 1C). By RT-PCR analysis we established that APPKM670/671NL and tauP301L transgenes (driven by Thy1 promoter) are not expressed in these cultures prior to DIV18 (data not shown), and therefore observed differences in Ca2+ signals in DIV13 hippocampal neurons (Fig 1A) are primarily due to PS1-M146V mutation. This is consistent with the previous comparison of Ca2+ signals in cortical neurons from KI-PS1M146V and 3xTg mice (Stutzmann et al., 2004; Smith et al., 2005; Stutzmann et al., 2006). An increase in the RyanR expression levels has been proposed to be responsible for the enhanced caffeine response in 3xTg and KI-PS1M146V neurons (Chan et al., 2000; Smith et al., 2005; Stutzmann et al., 2006; Chakroborty et al., 2009). Western blot analysis of lysates prepared from DIV13 hippocampal cultures revealed 1.8-fold increase in RyanR expression levels in 3xTg cultures when compared to WT cultures (Fig 1D, Supplementary Fig 2). The levels of InsP3R1 were also increased in 3xTg cultures, although the difference with WT has not reach statistically significant levels (Fig 1D, Supplementary Fig 2). The SERCA2b Ca2+ pump was expressed at similar levels in 3xTg and WT neurons (Fig 1D, Supplementary Fig 2). Selective increase in neuronal RyanR expression levels is in agreement with previous studies with 3xTg and KI-PS1M146V mice (Chan et al., 2000; Smith et al., 2005; Stutzmann et al., 2006; Chakroborty et al., 2009).
In the previous studies with mouse embryonic fibroblasts (MEF) we discovered that presenilins function as endoplasmic reticulum (ER) Ca2+ leak channels, and that disruption of ER Ca2+ leak function of presenilins results in increased ER Ca2+ levels (Tu et al., 2006; Nelson et al., 2007; Nelson et al., 2010). To determine if presenilins function as ER Ca2+ leak channels in neurons, we established primary neuronal cultures from PScDKO mice (PS1dTAG/dTAG; PS2−/−). In control rescue experiments with DKO MEF cells we confirmed that the mouse dTAG-PS1 construct retains ER Ca2+ leak channel activity (data not shown). To generate PS double knockout (DKO) neurons, the PScDKO neurons were infected with lentiviruses encoding nuclear-targeted GFP-Cre fusion protein (NLS-Cre). In parallel control experiments PScDKO cultures were infected with lentiviruses encoding nuclear-targeted GFP protein (NLS-GFP). The absence of PS2 in these cultures was confirmed by Western blotting of lysates with anti-PS2 mAb (data not shown). To determine time-course of PS1 disappearance in cortical PScDKO cultures infected with Lenti-Cre, the lysates for Western blotting experiments were prepared at variable time points following infection. From this analysis we determined that the levels of full-length dTAG-PS1 are reduced below detection limit by DIV12 in these cultures (Supplementary Fig 1B). In contrast, levels of dTAG-PS1 remained unchanged in PScDKO cultures infected with control Lenti-GFP virus (Supplementary Fig 1B). Thus, we concluded that infection of PScDKO neurons with Lenti-Cre virus yielded PS DKO neurons. In Ca2+ imaging experiments we discovered that application of caffeine induced significantly greater Ca2+ responses in PScDKO hippocampal neurons infected with Lenti-Cre viruses than in neurons infected with Lenti-GFP viruses (Figs 1B, 1C). On average the peak amplitude of caffeine-induced Ca2+ response was 2-fold larger in Cre-infected PScDKO hippocampal neurons than in GFP-infected PScDKO neurons (Fig 1C). Western blot analysis revealed a trend towards increase in RyanR expression in Cre-infected PScDKO cultures, although the difference with GFP-infected cultures has not reached the level of statistical significance (Fig 1D, Supplementary Fig 2). The levels of InsP3R1 and SERCA2b were similar in both Cre-infected and GFP-infected PScDKO cultures (Fig 1D, Supplementary Fig 2).
We reasoned that enhanced caffeine responses in PS DKO cultures may partially be due to increased levels of RyanR expression (Fig 1D) but may also be due to loss of ER Ca2+ leak function of presenilins and overloaded ER Ca2+ stores. To determine an importance of ER Ca2+ leak function of presenilins, we performed a series of rescue experiments. In these experiments PScDKO neurons were co-infected with Lenti-Cre viruses and lentiviruses encoding HA-tagged human PS1 rescue constructs. As a result, endogenous mouse dTAG-PS1 protein in these neurons is deleted and replaced with overexpressed human HA-PS1 protein. We found that infection with wild type Lenti-PS1 viruses reduced caffeine responses in PS DKO neurons to wild type levels (Fig 1B, 1C). In contrast, infection with PS1-L166P FAD construct which lost Ca2+ leak channel function (Nelson et al., 2007) did not reduce caffeine responses in PS DKO neurons (Fig 1B, 1C). Expression of PS1-L166P instead resulted in a further increase in the size of caffeine-induced Ca2+ response (Fig 1B, 1C), most likely due to dominant negative effects of this mutant on ER Ca2+ leak activity of remaining endogenous PS1. Infection with PS1-D257A construct which lost γ-secretase function but not Ca2+ leak channel function (Tu et al., 2006) rescued caffeine responses in PS DKO neurons (Fig 1B, 1C). Infection with PS1-D385A construct that lost both γ-secretase and Ca2+ leak channel functions (Omar Nelson and Ilya Bezprozvanny, in preparation) did not reduce the size of the caffeine response and actually increased it, similar to PS1-L166P FAD mutant (Fig 1C). Infection with PS1-ΔE9 FAD construct, which encodes a gain of function ER Ca2+ leak mutant (Tu et al., 2006) rescued the size of the caffeine response in PS DKO neurons (Fig 1B, 1C). Based on these results we concluded that loss of ER Ca2+ leak function and not γ-secretase function of presenilins resulted in increased caffeine responses in PS DKO neurons.
The increased responses to caffeine observed in 3xTg and PS DKO hippocampal neurons (Figs 1A, 1B, 1C) could be due to increased levels of RyanR expression (Fig 1D, Supplementary Fig 2) or due to increased levels of ER Ca2+. To evaluate the ER Ca2+ content more directly, we measured the size of ionomycin (IO)-sensitive Ca2+ pool in these neurons. Ionomycin is a Ca2+ ionophore that facilitates facilitates transport of Ca2+ across membranes. Addition of ionomycin induces exit of all ER-accumulated Ca2+ into the cytosol. The size of IO-sensitive Ca2+ pool can be estimated by integrating a signal reported by cytosolic Fura-2 following exposure to ionomycin. In the previous studies we used a similar approach to evaluate ER Ca2+ content in wild type and PS DKO MEF cells (Tu et al., 2006; Nelson et al., 2007). In experiments with hippocampal neurons the cultures were transferred from aCSF (containing 2 mM Ca2+) to Ca2+-free media for 30 sec and then challenged with 5 μM ionomycin. An increase in the cytosolic Fura-2 signal observed immediately after ionomycin addition was integrated and designated as IO30. We found that application of ionomycin resulted in significantly greater responses in 3xTg hippocampal cultures than in WT cultures (Fig 2A). On average, the size of IO30 Ca2+ pool was 2.7-fold greater in 3xTg neurons than in WT neurons (Fig 2C).
We found the IO-induced responses were also greater in PScDKO neurons infected with Lenti-Cre viruses than in neurons infected with Lenti-GFP viruses (Fig 2B). On average, the size of IO30 Ca2+ pool was 1.7-fold greater in Cre-infected neurons (Fig 2C). Co-infection with wild type PS1 virus or with the virus expressing PS1-D257A “γ-secretase mutant” rescued IO-induced responses back to the levels observed in GFP-infected cultures (Figs 2B, 2C). In contrast, co-infection with “loss of ER Ca2+ leak” function FAD mutant PS1-L166P did not rescue IO-responses and instead resulted in enhanced responses (Fig 2B). On average, the size of IO30 Ca2+ pool was 3-fold greater in PScDKO neurons co-infected with Cre and PS1-L166P viruses than in PScDKO neurons infected with GFP viruses (Fig 2C). Similar results were observed in experiments with PS1-D385A mutant which lost both γ-secretase and ER Ca2+ leak functions (Fig 2C). As discussed above, it is likely that PS1-L166P and PS1-D385A mutants exert dominant negative effect on ER Ca2+ leak activity of endogenous PS1 still remaining in Cre-infected PScDKO neurons. Co-infection of Lenti-Cre with the “gain of Ca2+ leak function” PS1-ΔE9 FAD mutant reduced the size of IO responses to the levels even below levels observed in GFP-infected PScDKO neurons, consistent with “superleaky” ER membrane (Figs 2B, 2C). Based on these results we concluded that loss of ER Ca2+ leak function and not γ-secretase function of presenilins resulted in the increased size of IO-sensitive Ca2+ pool in PS DKO neurons.
Ionomycin is a Ca2+ ionophore that in our experiments facilitates Ca2+ transport across all intracellular membranes and not only across ER membranes. Thus, some of the Ca2+ in IO-sensitive Ca2+ pool may come from other intracellular compartments, such as Goldgi apparatus or mitochondria. To confirm the results obtained with ionomycin, we took an advantage of recently developed ER-targeted genetic Ca2+ indicator D1ER (Palmer et al., 2004; McCombs and Palmer, 2008). D1ER is a FRET-based cameleon Ca2+ indicator composed of ECFP and citrin fluorescent proteins separated by a linker encoding calmodulin (CaM) and CaM-binding M13 peptide sequence (Fig 3A). Upon Ca2+ binding to CaM, CaM binds to M13 target sequence and brings ECFP and citrin proteins in close proximity, which facilitates FRET between these proteins (Fig 3A). Calreticulin (CRT) targeting sequence was added to amino-terminus and ER retension sequence was added to carboxy-terminus of D1ER protein to facilitate ER targeting and retension (Fig 3A) (Palmer et al., 2004). To validate utility of D1ER indicator for our experiments, we transiently transfected D1ER plasmid to wild type and PS DKO MEF cells. Consistent with previous results obtained using Mag-Fura-2 ER Ca2+ indicator (Tu et al., 2006), we discovered that ER Ca2+ levels reported by D1ER are several fold higher in PS DKO MEF cells when compared to wild type cells (data not shown). In experiments with neurons D1ER expression plasmid was transiently transfected to WT and 3xTg hippocampal neuronal cultures. Proper targeting of D1ER construct to neuronal ER compartment was confirmed in immunostaining experiments with polyclonal anti-calreticulin (CRT) antibodies (Fig 3B). To minimize effects of cell-to-cell variability in D1ER expression levels, we normalized FRET signal from D1ER indicator to CFP signal observed for the same cell. When cultured in aCSF media containing 2 mM Ca2+, 3xTg neurons had significantly higher FRET/CFP ratio than WT neurons, consistent with elevated ER Ca2+ concentration (Fig 3C). Following transfer to Ca2+-free aCSF media and addition of 2 μM of SERCA pump inhibitor thapsigargin, FRET/CFP ratios were reduced in both WT and 3xTg neurons, indicating store-depletion due to ER Ca2+ leak activity. In WT cells FRET/CFP ratios were reduced to basal levels within 75 sec after addition of thapsigarin (Fig 3C). In contrast, it took at least 250 sec for FRET/CFP ratios reduction to a new basal state in 3xTg neurons, consistent with impaired ER Ca2+ leak function (Fig 3C). Also, basal FRET/CFP ratios following thapsigargin application were significantly higher in 3xTg neurons than in WT neurons (Fig 3C). Addition of 5 μM ionomycin had no further effect on FRET/CFP in WT neurons, but caused further reduction of FRET/CFP ratios in 3xTg neurons to the same levels as observed in WT neurons (Fig 3C). From these experiments we concluded that in 3xTg neurons significant portion of Ca2+ is accumulated in thapsigargin-insensitive ER pool. This pool was not observed in our experiments with WT neurons, were all ER Ca2+ could be released by thapsigarin (Fig 3C). Thus, appearance of this new pool is likely to be a result of compensation to impaired ER Ca2+ leak function in 3xTg neurons. Further studies will be required to identify nature of this ER subcompartment. Another possibility that the rate of ER Ca2+ leak was so much reduced in 3xTg neurons that 600 sec incubation with thapsigargin was insufficient to deplete the stores completely. In order to convert FRET/CFP ratios to absolute ER Ca2+ values, we concluded each experiment with a calibration procedure using a series of extracellular solutions with defined Ca2+ concentrations. As a result of this procedure, calibration D1ER curves were constructed for WT and 3xTg neurons (Supplementary Fig 3). Using these calibration curves, we estimated that in the basal the ER Ca2+ levels were equal to 207 ± 47 μM (n = 100) in WT hippocampal neurons and 623 ± 108 μM (n = 51) in 3xTg neurons (p < 0.001). The 3-fold increase in ER Ca2+ levels in 3xTg neurons reported by D1ER indicator is in near perfect agreement with 2.5-fold increase in the size of IO-sensitive Ca2+ pool that we measured for the same neurons using Fura-2 (Figs 2A, 2C). Thus, in the future experiments we focused on IO-sensitive Ca2+ pool measurements due to a relative simplicity of experimental procedures involved in these measurements.
The filling state of ER Ca2+ pool regulates store-operated Ca2+ entry (SOC) pathway (Venkatachalam et al., 2002). To investigate effects of presenilin deletions and mutations on activaton of neuronal SOC pathway, we transferred Fura-2 loaded hippocampal neurons from aCSF media to Ca2+-free media for 2 min and then returned neurons to aCSF media. The ER Ca2+ pool is depleted during incubation in Ca2+-free media, and “Ca2+ re-addition” results in activation of SOC-mediated Ca2+ entry. We found that PScDKO neurons infected with Lenti-GFP respond to “Ca2+ re-addition” much stronger than PScDKO neurons infected with Lenti-Cre (Fig 4A). We also discovered that “Ca2+ re-addition” results in much stronger responses in WT hippocampal neurons than in 3xTg neurons (Fig 4B). On average, the size of the peak Ca2+ response was reduced 2-fold in Cre-infected PScDKO neurons and in 3xTg neurons when compared to control groups (Fig 4C). Our results (Fig 4) are in agreement with the previous studies of SOC in PS-FAD mutant cells (Leissring et al., 2000; Yoo et al., 2000; Herms et al., 2003; Akbari et al., 2004), but our conclusion is that presenilins affect SOC in hippocampal neurons indirectly, by controlling the filling state of ER Ca2+ stores. Similar hypothesis has been previously proposed based on the analysis of SOC in KI-PS1M146V fibroblasts (Leissring et al., 2000).
The results obtained from IO30 measurements (Fig 2) are consistent with the hypothesis that the ER Ca2+ leak pathway is impaired in 3xTg and PS DKO hippocampal neurons. To measure the rate of passive ER Ca2+ leak in these neurons more directly, we compared the sizes of IO-induced Ca2+ responses following 30 sec (IO30) and 120 sec (IO120) incubation in Ca2+-free media. We used the IO120/IO30 ratio as a quantitative measure of passive Ca2+ loss from the ER within 90 sec time period due to endogenous ER Ca2+ leak pathway activity. Because the size of IO30 pool is very small in WT neurons and in PScDKO neurons infected with Lenti-GFP (Fig 2), in these experiments we refilled the stores with Ca2+ by 1 min exposure to 15 mM KCl in aCSF. Resulting depolarization induces opening of voltage-gated Ca2+ channels and rapid Ca2+ influx into the hippocampal neurons, which leads to filling of the ER Ca2+ stores. We did not see much difference in KCl-induced Ca2+ responses between different groups of cells (data not shown). Immediately after KCl depolarization the neurons were transferred to Ca2+-free media for 30 sec and challenged with ionomycin. Application of ionomycin in these conditions resulted in similar responses in PScDKO hippocampal neurons infected with Lenti-GFP and Lenti-Cre viruses (Fig 5A), indicating that the stores were completely full in both cultures. When length of incubation in Ca2+-free media was extended to 120 sec, ionomycin induced significantly smaller responses in PScDKO neurons infected with Lenti-GFP than in neurons infected with Lenti-Cre (Fig 5A). On average, IO120/IO30 ratio was equal to 0.35 for GFP-infected PScDKO neurons and 0.93 for Cre-infected PScDKO neurons (Fig 5B). We interpreted these results as indication that PScDKO neurons infected with Lenti-GFP lost 65% of accumulated ER Ca2+ following 90 sec incubation in Ca2+-free media. In contrast, PScDKO neurons infected with Lenti-Cre only lost 7% of accumulated ER Ca2+ during the same time period. Thus, the ER membrane in GFP-infected PScDKO neurons is much more leaky for Ca2+ than the ER membrane in Cre-infected PScDKO hippocampal neurons. This conclusion did not depend on using the KCl refill protocol, as the IO120/IO30 ratio was equal to 0.8 for PScDKO neurons infected with Lenti-Cre in the absence of KCl refill step (Fig 5B). The IO120/IO30 ratio could not be measured for PScDKO neurons infected with Lenti-GFP in the absence of KCl refill as IO30 pool was too small for these neurons (Fig 2). Co-infection of Cre with the wild type PS1 virus resulted in partial rescue of the leak function, yielding IO120/IO30 ratio of 0.5 following KCl refill (Fig 5B). In contrast, co-infection of Cre virus with the mutant PS1-M146V virus did not rescue the leak function, yielding IO120/IO30 ratio of 1.15 following KCl refill and 0.95 in the absence of KCl refill (Fig 5B). A similar paradigm was applied to compare ER Ca2+ leak in WT and 3xTg neurons. We found that IO120/IO30 ratio was equal to 0.25 in WT neurons following KCl refill protocol and 0.79 in 3xTg neurons (Fig 5B). Thus, within 90 sec incubation in Ca2+-free media WT neurons lost 75% of accumulated Ca2+ and 3xTg neurons only lost 21%. The IO120/IO30 ratio could not be measured in WT neurons without KCl refill as the size of IO30 Ca2+ pool is too small in these neurons (Fig 2). From these results we concluded that ER Ca2+ leak function is impaired in 3xTg and PS DKO neurons. We further concluded that ER Ca2+ leak function in PS DKO neurons can be rescued by expression of wild type PS1 but not PS1-M146V mutant.
AD preferentially affects hippocampal and cortical neurons, whereas other neuronal populations are not affected (Gomez-Isla et al., 2008). The reasons for the selective vulnerability of hippocampal and cortical neurons in AD are not well understood. Although the role of ER Ca2+ leak function of presenilins in AD pathogenesis is not yet clear, we reasoned that it may be of interest to evaluate ER Ca2+ leak function of presenilins in vulnerable and resistant neuronal populations. In experiments described above we demonstrated that ER Ca2+ stores in hippocampal neurons are very leaky and that FAD mutations or genetic deletion of presenilins dramatically impair ER Ca2+ leak function in these neurons (Figs 2 and and5).5). For the next series of experiments we focused on striatal medium spiny neurons (MSN), which are selectively affected in Huntington’s disease (Vonsattel and DiFiglia, 1998) but largely spared in AD (Gomez-Isla et al., 2008). The MSN PScDKO neuronal cultures were established and infected with Lenti-GFP and Lenti-Cre viruses as described above for hippocampal neurons. Similar to procedures used for hippocampal neurons, MSN neurons were transferred form aCSF media to Ca2+-free media for 30 sec or 120 sec and challenged with ionomycin. In contrast to hippocampal neurons we found that PScDKO MSN neurons infected with Lenti-GFP respond strongly to ionomycin after 30 sec and 120 sec incubation in Ca2+ free media (Fig 6A). On average, the IO120/IO30 ratio was equal to 0.75 for PScDKO MSN neurons infected wih Lenti-GFP (Fig 6C). The PScDKO MSN neurons infected with Lenti-Cre virus also responded strongly to ionomycin after 30 sec and 120 sec incubation in Ca2+-free media (Fig 6A). The average IO120/IO30 ratio for these neurons was equal to 1.2 (Fig 6C). Similar to PScDKO MSN neurons, we found that both WT and 3xTg MSN neurons responded strongly to ionomycin after 30 sec and 120 sec in Ca2+-free media (Fig 6B). On average, IO120/IO30 ratio was equal to 0.5 for WT neurons and 1.05 for 3xTg neurons (Fig 6C). From these results we concluded that presenilins play a role as ER Ca2+ leak channels in MSN neurons, but the ER Ca2+ leak activity is much less potent in MSN neurons than in hippocampal neurons.
To explain the differences between MSN and hippocampal neurons, we evaluated expression of full-length PS1 in lysates prepared from hippocampal and MSN PScDKO neurons infected with Lenti-GFP and Lenti-Cre viruses. Actin was used as a loading control in these experiments (Fig 6D). The monoclonal anti-FLAG anibodies were used in these experiments for PS1 detection by taking an advantage of dTAG epitope inserted into PS1 amino terminal locus. Consistent with the previous studies (Annaert et al., 1999), we detected both full-length and cleaved forms of PS1 in lysates prepared from GFP-infected PScDKO hippocampal and MSN neurons (Fig 6D). As expected, in both types of neurons cleaved PS1 form (PS1-NTF and PS1-CTF) was significantly more abundant than the holoprotein form (Fig 5D). Infection with Lent-Cre resulted in complete loss of holoprotein and PS1-CTF forms and major reduction in the PS1-NTF form in both neuronal populations (Fig 6D). As we previously reported, only holoprotein presenilins support ER Ca2+ leak activity (Tu et al., 2006). It is of interest that the PS1 holoprotein was less abundant in MSN neuronal lysates than in hippocampal neuronal lysates (Fig 6D). Thus, it is possible that reduced ER Ca2+ leak activity in MSN neurons is related to comparatively lower levels of PS1 holoprotein expressed in these neurons.
Western blot analysis revealed significant increase in RyanR expression levels in 3xTg hippocampal neurons (Fig 1D, Supplementary Fig 2). There was also a trend towards increase in RyanR expression levels in PS DKO hippocampal neurons (Fig 1D, Supplementary Fig 2). The effect on RyanR expression was specific, as expression of InsP3R1 in the same cultures was affected much less than expression of RyanR and expression of SERCA2b was not affected (Fig 1D, Supplementary Fig 2). We hypothesized that increase in RyanR expression may be a compensatory response to impaired ER Ca2+ leak function in these neurons. To test this hypothesis, we cultured WT and 3xTg hippocampal neurons for 6 days (DIV7 – DIV13) in the presence of 50 nM dantrolene, a membrane-permeable RyanR inhibitor (Krause et al., 2004). Following exposure to dantrolene neurons were moved from aCSF to Ca2+-free media for 30 sec and stimulated with ionomycin. We found that incubation with dantrolene had only minor effect on IO-induced responses in WT neurons (Fig 7A). Although some upward trend was observed, on average the size of IO30 Ca2+ pool was not significantly different between control and dantrolene-exposed groups of WT hippocampal neurons (Fig 7B). In contrast, incubation with dantrolene dramatically enhanced IO-induced Ca2+ responses in 3xTg cells (Fig 7A). On average, the IO30 Ca2+ pool size was 2-fold larger in 3xTg hippocampal neurons exposed to dantrolene than in control group of 3xTg neurons and 8-fold larger than in control group of WT neurons (Fig 7B). Consistent with the previous findings (Figs 2A, 2C), the IO30 Ca2+ pool size was 4-fold larger in control group of 3xTg neurons than in control group of WT neurons (Fig 7B).
Dantrolene is relatively specific for RyanR (Krause et al., 2004), but also inhibits other targets such as channels involved in store-operated Ca2+ entry (Zhao et al., 2006). To test the role of RyanR more specifically, we infected WT and 3xTg neuronal cultures with lentiviruses encoding shRNAi against RyanR (RyR-shRNAi) or control shRNAi (Ctrl-shRNAi). RyR-shRNAi is directed against mouse RyanR1 sequence, but in control qPCR experiments we discovered that infection with RyR-shRNAi lentiviruses also reduced expression of RyanR2 and RyanR3 in hippocampal neurons (data not shown). An efficient knockdown of RyanR in 3xTg hippocampal neurons infected with RyR-shRNAi was confirmed in control Western blotting experiments with pan-RyanR antibodies (Supplementary Fig 4). We found that IO-induced Ca2+ responses were increased in WT hippocampal neurons infected with RyR-shRNAi when compared to neurons infected with Ctrl-shRNAi (Fig 7C). On average, the size of IO30 Ca2+ pool was 5-fold higher in WT neurons infected with RyR-shRNAi, comparable in size to IO30 Ca2+ pool in 3xTg neurons (Fig 7D). The 3xTg neurons infected with RyR-shRNAi responded to ionomycin much stronger than 3xTg neurons infected with Ctrl-shRNAi (Fig 7C). On average, the IO30 Ca2+ pool size was 3-fold larger in 3xTg neurons infected with RyR-shRNAi than in 3xTg neurons infected with Ctrl-shRNAi and more than 15-fold larger than in WT neurons infected with Ctrl-shRNAi (Fig 7D). From these results we concluded that RyanR play a role in ER Ca2+ leak pathway in WT hippocampal neurons, but this role becomes more critical in 3xTg hippocampal neurons which lost ER Ca2+ leak function of presenilins due to PS1-M146V mutation.
The results obtained with 3xTg hippocampal neurons pointed to a close relationship between presenilin-mediated and RyanR-mediated Ca2+ leak pathways (Fig 7). To understand the importance of this relationship in vivo, we set out to study the effect of dantrolene in an AD mouse model. For these studies we focused on APPPS1 double transgenic mice (Thy1-APPKM670/671NL, Thy1-PS1L166P) because of the early and robust amyloid accumulation in these mice (Radde et al., 2006; Serneels et al., 2009). Dantrolene was delivered to APPPS1 mice and WT control mice by using an approach that we usedpreviously for dantrolene trials in SCA3 and SCA2 mice (Chen et al., 2008; Liu et al., 2009). In these experiments six APPPS1 hemizygous female mice and six WT female mice were fed with dantrolene resuspended in PBS with 2% corn flour. The control group of six APPPS1 hemizygous female mice and six WT female mice were fed with 2% cornflour in PBS. Littermate mice were selected for four groups to reduce variability. All mice were fed orally twice a week from 2 to 8 months of age and then sacrificed and processed for histological analysis.
To quantify amyloid load in these mice, the coronal slices from both groups of APPPS1 mice were stained with anti-Aβ monoclonal antibody followed by staining with fluorescently-labeled anti-mouse IgG. The stained slices were imaged by using micron resolution laser scanning system. Visual inspection of obtained results indicated that amyloid plaques are more abundant and fluorescent in dantrolene-fed group of APPPS1 mice than in control group (Fig 8A). To quantify these results we estimated the average number of plaques present in a slice and the average plaque fluorescent signal intensity. From this analysis we concluded that there was a 2-fold increase in average plaque number in APPPS1 mice fed with dantrolene when compared to control group of APPPS1 mice (Fig 8B). There was also 2-fold increase in average size and fluorescent intensity of amyloid plaques in dantrolene-fed APPPS1 mice when compared to control group (Fig 8B).
Amyloid load increase in dantrolene-fed APPPS1 mice (Figs 8A, 8B) suggested that neuropathological processes may also be accelerated in these mice. To test this hypothesis we stained hippocampal slices from vehicle-fed and dantrolne-fed APPPS1 mice with antibodies against the excitatory synaptic marker PSD95. Loss of synaptic connections is one of the earliest events in human AD (Selkoe, 2002; Gomez-Isla et al., 2008) and we reasoned that earliest stages of the pathology may be detected by reduction in PSD95 staining. In control experiments we also performed PSD95 staining of hippocampal slices from vehicle-fed and dantrolene-fed WT mice. We discovered that feeding dantrolene resulted in reduction in PSD95 staining density in both WT and APPPS1 mice, but that reduction in APPPS1 mice was more severe (Fig 9A). To compare these results quantitatively, we normalized PSD95-positive area in hippocampal slices from dantrolene-fed mice to the mean PSD95-positive area in hippocampal slices from vehicle-treated mice from the same group. We found that feeding of dantrolene reduced PSD95-positive area to 73% ± 4% (n = 6) of vehicle control in WT mice to 51% ± 4% (n = 6) of vehicle control in APPPS1 mice (p < 0.01). On average, a number of the PSD95-positive puncta per 100 μm2 was reduced from 55 ± 3 (n = 6) in vehicle-treated APPPs1 mice to 21 ± 2 (n = 6) in dantrolene-fed APPPS1 mice. The number of neuronal cell bodies visualized by propidium iodide (PI) staining was slightly reduced in CA1 region of dantrolene-fed APPPS1 mice (Fig 9A). Thus, we concluded that feeding of dantrolene accelerated synaptic loss more significantly than neuronal loss at this early stage of the APPPS1 mice pathology in the hippocampus. The density of amyloid plaques in dantrolene-fed APPPS1 mice was increased in cortical and hippocampal regions and in basal ganglia (Fig 8A). Thus, we investigated if striatal neurons are also affected in these mice. By using neuronal nuclear marker NeuN and cytosolic MSN marker DARPP-32 we did not observe significant differences between control and dantrolene-fed group of APPPS1 mice (Figs 9B and 9C), suggesting that striatal neurons are not effected in these mice.
To further extend pathological characterization of these mice we performed silver staining of hippocampal, cortical and striatal regions from vehicle-fed and dantrolene-fed WT and APPPS1 mice. By using Bielschowsky staining method, we observed significant reduction in neuronal fiber density and abundance of dystrophic neurites in hippocampal and cortical regions of dantrolene-fed APPPS1 mice (Fig 10). Quantitative analysis of silver staining data revealed that the neurites occupied 42% ± 3% (n = 6) of surface area in hippocampal sections from control group and only 31% ± 2% (n = 6) in dantrolene-fed group. We also observed significant thinning of CA1 cell body layer in the hippocampus and appearance of many spaces without cell bodies in the hippocampal and cortical neuropil of dantrolene-fed mice (Fig 10). All of these changes were consistent with on-going neuronal atrophy and loss in hippocampal and cortical regions of dantrolene-fed APPPS1 mice. Consistent with the immunostaining data (Fig 9C, 9D), silver staining showed that striatal MSN neurons were not affected in dantrolene-fed APPPS1 mice but that the cortical projection fibers were undergoing dystrophic changes (Fig 10). The observed effects were specific for APPPS1 mice, as no significant abnormalities were observed in hippocampal, cortical or striatal regions of dantrolene-fed WT mice (Fig 10).
In the previous studies with MEF cells from PS DKO mice we discovered that presenilins control ER Ca2+ levels by acting as passive ER Ca2+ leak channels (Tu et al., 2006). We further demonstrated that many FAD mutations in presenilins result in loss of ER Ca2+ leak function, leading to ER Ca2+ overload and supranormal Ca2+ release from the ER (Tu et al., 2006; Nelson et al., 2007; Nelson et al., 2010). Our hypothesis has been challenged based on experiments with PS-transfected Xenopus oocytes and chicken B-cells DT40 cell line (Cheung et al., 2008). As an alternative explanation Cheung at al proposed that FAD mutant presenilins affect Ca2+ signaling by modulating channel openings of InsP3R1 (Cheung et al., 2008). Results obtained in the present study strongly support our hypothesis that presenilins in fact function as ER Ca2+ leak channels. In experiments with hippocampal neuronal cultures from 3xTg and PScDKO mice we demonstrated that PS1-M146V mutation or genetic deletion of presenilins resulted in enhanced caffeine-induced Ca2+ release from the ER (Fig 1), increase in the size of ionomycin-sensitive Ca2+ pool (Fig 2), reduced store-operated Ca2+ entry (Fig 3) and reduction in passive ER Ca2+ leak rate (Fig 4). The defects in Ca2+ signaling observed in PS DKO hippocampal neurons could be rescued by expression of wild type PS1 or PS1-D257A mutant deficient in γ-secretase activity but not by PS1-L166P or PS1-M146V FAD mutants deficient in ER Ca2+ leak channel activity or PS1-D385A mutant deficient in both γ-secretase and ER Ca2+ leak activities (Figs 1, ,2,2, ,5).5). From these results we concluded that presenilins function as ER Ca2+ leak channels in hippocampal neurons, a function independent from their γ-secretase activity (Fig 11A). Our results provide a coherent explanation for numerous previous reports of abnormal Ca2+ signaling observed in hippocampal and cortical neurons expressing PS1 or PS2 FAD mutants (Begley et al., 1999; Guo et al., 1999; Chan et al., 2000; Yoo et al., 2000; Schneider et al., 2001; Herms et al., 2003; Stutzmann et al., 2004; Smith et al., 2005; Stutzmann et al., 2006; Chakroborty et al., 2009). The main findings reported in these studies, such as enhanced ER Ca2+ release via InsP3R and RyanR and impaired SOC Ca2+ entry, can be accounted for by the loss of ER Ca2+ leak function of presenilins and overloaded ER Ca2+ stores. The same mechanism may also contribute to abnormal presynaptic ER Ca2+ signaling and impaired Ca2+-dependent synaptic plasticity in PS DKO hippocampal neurons (Zhang et al., 2009).
Interestingly, the ER membrane of striatal medium spiny neurons (MSN) is much less leaky for Ca2+ than ER membrane of hippocampal neurons and presenilins appear to play a lesser role in controlling ER Ca2+ homeostasis in MSN than in hippocampal neurons (Fig 6). Full-length PS1 is present in relatively lower amounts in MSN neurons when compared to hippocampal neurons (Fig 6D), providing a possible biochemical explanation to these findings. In contrast to hippocampal and cortical neurons, striatal MSN are not affected in AD (Gomez-Isla et al., 2008), and future studies will be needed to further investigate whether a correlation exist between the potency of ER Ca2+ leak pathway and selective vulnerability of different neuronal populations in AD.
An increase in RyanR expression levels has been previously reported for 3xTg and KI-PS1M146V hippocampal and cortical neurons (Chan et al., 2000; Smith et al., 2005; Stutzmann et al., 2006; Chakroborty et al., 2009). We have also observed significant increase in RyanR expression levels in 3xTg hippocampal neuronal cultures (Fig 1D, Supplementary Fig 2). The increase in RyanR expression levels has been previously proposed to be responsible for enhanced caffeine-induced Ca2+ responses in 3xTg and KI-PS1M146V neurons (Chan et al., 2000; Smith et al., 2005; Stutzmann et al., 2006; Chakroborty et al., 2009). We would like to revise this view and suggest that the increase in RyanR levels plays a compensatory role for the loss of presenilin leak function. Our experiments with RyR-shRNAi indicated that in wild type hippocampal neurons RyanR contribute to the ER Ca2+ leak function (Fig 11A). In 3xTg hippocampal neurons the PS1-M146V mutation impairs presenilin-mediated ER Ca2+ leak and the role of RyanR becomes more critical. Most likely increase in RyanR expression levels is an attempt by 3xTg hippocampal neurons to boost the RyanR-mediated Ca2+ leak pathway which would then conceivably compensate for the loss of presenilin-mediated ER Ca2+ leak more efficiently (Fig 11B). The sensitivity of RyanR to cytosolic Ca2+ is known to be modulated by ER Ca2+ levels. When ER Ca2+ levels become too high even basal cytosolic Ca2+ levels become sufficient to result in RyanR opening. This is so-called “store-overload induced Ca2+ release” mechanism (SOICR) which has been well described in cardiac cells (Jiang et al., 2004; Jones et al., 2008). We postulate that similar SOICR mechanism supported by RyanR is also functional in neurons, in particular in condition of impaired ER Ca2+ leak such as due to PS mutations.
If RyanR-mediated Ca2+ leak is inhibited in this situation (by dantrolene or by RyR-shRNAi knockdown of RyanR expression), then ER Ca2+ stores become very full, with total ER Ca2+ content 10–15 fold higher than in wild type cells (Fig 11C). Resulting Ca2+ dyshomeostasis likely to affects other neuronal functions, such as for example synaptic transmission and plasticity (Chakroborty et al., 2009). Further studies will be required to understand the mechanism involved in upregulation of RyanR resulting from PS1 FAD mutations. Three-fold increase in RyanR2 mRNA has been discovered in samples extracted from adult hippocampus of 3xTg mice (Chakroborty et al., 2009), and 4-fold increase in RyanR3 mRNA was reported for samples from KI-PS1M146V adult mice hippocampus (Chan et al., 2000). We have also observed 2–3 fold increase in RyanR2 and RyanR3 mRNA in qPCR experiments with 3xTg hippocampal neuronal cultures (data not shown). These results suggest that impaired ER Ca2+ leak function and increased ER Ca2+ levels are linked with an increase in transcription of genes encoding RyanR2 and RyanR3 via “ER Ca2+ homeostasis” negative feedback mechanism. The molecular underpinnings of this feedback mechanism need to be clarified in further studies.
Our results indicate that RyanR may play a protective role in the context of AD. The Ca2+ homeostasis model that we propose (Fig 11) predicts that blocking RyanR in AD could have a disastrous effect on the pathology. We tested this hypothesis by long-term feeding of dantrolene to APPPS1 mice. We discovered that long-term feeding of dantrolene to APPPS1 mice results in 3-fold increase in amyloid load (Figs 8A, 8B), loss of excitatory markers of synaptic transmission in hippocampus (Fig 9A), neuronal atrophy and loss in hippocampus and cortex (Fig 10). The results with dantrolene are somewhat paradoxical, as high (1–10 μM) concentrations of dantrolene reduced glutamate-induced excitotoxicity in KI-PS1M146V mouse hippocampal neurons (Guo et al., 1999). We obtained similar results in studies with 3xTg hippocampal neuronal cultures (data not shown). Moreover, we previously reported that long-term feeding of dantrolene to SCA3 and SCA2 mouse models slowed progression of motor phenotype and alleviated age-dependent neuronal loss in these mice (Chen et al., 2008; Liu et al., 2009). These results suggest that while in general dantrolene acts as anti-excitotoxic agent, it specifically accelerated pathology in APPPS1 mice. These results agree with our hypothesis that the loss of ER Ca2+ leak function of presenilins in APPPS1 mice (due to PS1-L166P mutation) is compensated by increased RyanR-mediated Ca2+ flux from the ER. Blocking RyanR by dantrolene in this situation leads to further increase in neuronal ER Ca2+ levels and accelerated pathology (Fig 11C).
Interestingly, the potentially protective role of RyanR has been proposed previously based on studies of APP transgenic mice (TgCRND8 mice; PrP-APPKM670/671NL). These investigators discovered that the levels of RyanR3 mRNA and protein are significantly increased in the cortex of adult TgCRND8 mice and in TgCRND8 cortical neuronal cultures (Supnet et al., 2006). Moreover, direct exposure of wild type cortical neuronal cultures to Aβ42 resulted in 3.5-fold increase in RyanR3 mRNA levels (Supnet et al., 2010). These effects were specific for RyanR3, as exposure to Aβ42 did not affect expression levels of RyanR2, InsP3R1 or SERCA2b (Supnet et al., 2006). Formation of amyloid plaques and accumulation of extracellular Aβ42 causes destablization of neuronal Ca2+ signals and increase in neuronal excitability (Busche et al., 2008; Kuchibhotla et al., 2008), and Supnet at al. argued that upregulation of RyanR3 could be a compensatory response aimed to protect against increased cortical neurons excitability and abnormal Ca2+ signals induced by Aβ42 exposure. Consistent with this prediction the same authors recently discovered that knockdown of RyanR3 by RNAi accelerated neuronal death of TgCRND8 cultured cortical neurons but not wild type cultured cortical neurons (Supnet et al., 2010) Thus, upregulation of RyanR may have protective effects in both mutant presenilin and mutant APP models of AD, as we recently discussed (Supnet and Bezprozvanny, 2010).
Our main goal was to define the role played by presenilins in neuronal ER Ca2+ leak pathway. In addition to this specific issue, some of our results have implications for the more general understanding of existing connections between neuronal Ca2+ signaling, amyloid accumulation and neuronal pathology in AD. A few previous studies addressed potential connection between Ca2+ signaling and amyloid production (Green et al., 2007). Some of these studies concluded that increased cytosolic Ca2+ signals enhance production of Aβ42 and increase Aβ42:Aβ40 ratio (Querfurth and Selkoe, 1994; Pierrot et al., 2004; Green et al., 2007). Other studies indicated that increased Ca2+ signals reduce production of Aβ42 and reduce Aβ42:Aβ40 ratio (Dreses-Werringloer et al., 2008). Most of these studies have been performed in non-neuronal systems. In our experiments we observed 2–3 fold increases in amyloid load in dantrolene-fed APPPS1 mice (Figs 8A, 8B). Although exact mechanism responsible for these effects still needs to be clarified, these results suggest that abnormal ER Ca2+ signaling is able to drive amyloid accumulation in the brain.
Cortical and hippocampal neuronal atrophy is typical of human AD (Gomez-Isla et al., 1996; Price et al., 2001; Gomez-Isla et al., 2008) and correlates much better with cognitive decline than the amyloid load (Giannakopoulos et al., 2003; Gomez-Isla et al., 2008). However, neuronal atrophy has been very difficult to reproduce in APP and presenilin transgenic mouse modes (Takeuchi et al., 2000; Schwab et al., 2004; Gomez-Isla et al., 2008). Interestingly, we found that feeding of dantrolene to APPPS1 mice resulted in significant loss of hippocampal excitatory synaptic markers (Fig 9A) and in neuronal atrophy in hippocampal and cortical regions (Fig 10). Despite comparable increases in amyloid load in the striatal region (Fig 8A), striatal MSN neurons were not significantly affected by dantrolene feeding to APPPS1 mice (Figs 9B, 9C, ,10).10). As discussed above, the ER Ca2+ leak pathway is less potent in striatal MSN neurons than in hippocampal neurons, which may explain why striatal neurons are less affected when ER Ca2+ leak pathway is impaired by combined effects of PS1-L166P mutation and dantrolene feeding in APPPS1 mice.
In conclusion, here we report analysis of the role played by presenilins in neuronal Ca2+ signaling. We conclude that presenilins function as ER Ca2+ leak channels in hippocampal neurons and that the loss of ER Ca2+ leak function of presenilins was partially compensated by an increase in RyanR expression. We further found that if both PS-mediated and RyanR-mediated ER Ca2+ leak pathways are impaired, then ER Ca2+ levels in hippocampal neurons increase dramatically. Simultaneous block of both PS-mediated and RyanR-mediated ER Ca2+ leak pathways in vivo resulted in an increase in amyloid accumulation, loss of synaptic markers in hippocampus and neuronal atrophy in hippocampus and cortex. Our results further support a close connection between neuronal Ca2+ signaling, amyloid and AD pathogenesis (Bezprozvanny and Mattson, 2008).
We are grateful to Frank LaFerla for providing 3xTg mice and to Mathias Jucker for providing the APPPS1 mice, Frank Wuytack for providing the SERCA2b antibody, to Marek Michalek for providing CRT antibodies, to Thomas Sudhof for providing the PSD95 antibodies and Lenti-Cre constructs, to Roger Tsien for providing D1ER plasmid, to Omar Nelson and Huarui Liu for help with making reagents, to Leah Benson for administrative assistance. IB is a holder of Carla Cocke Francis Professorship in Alzheimer’s Research and this work was supported by Alzheimer’s Association, Alzheimer’s Disease Drug Discovery Foundation, McKnight Brain Disorders Award and NIH grant R01AG030746 (IB), a Methusalem grant from the Flemish government and the KULeuven, the Fund for Scientific Research (Flanders, Belgium), the Federal Office for Scientific Affairs, (IUAP P6/43, Belgium) and MEMOSAD (F2-2007-200611) of the European Union (BDS).