Evidence for the involvement of calbindin D28k in the presenilin 1 model of Alzheimer's disease

Neuroscience 169 (2010) 532–543

EVIDENCE FOR THE INVOLVEMENT OF CALBINDIN D28k IN THE PRESENILIN 1 MODEL OF ALZHEIMER’S DISEASE G. L. ODERO,a K. OIKAWA,a1 K. A. C. GLAZNER,a,b J. SCHAPANSKY,a,b D. GROSSMAN,a J. D. THIESSEN,a,c,d A. MOTNENKO,a N. GE,a M. MARTIN,b,c,d G. W. GLAZNERa,b AND B. C. ALBENSIa,b*

To date, most types of Alzheimer’s disease (AD) occur in sporadic form, with familial AD (FAD) accounting for less than 10% of all cases (Price et al., 1998). Pathological hallmarks of FAD include memory deficits, accumulation of amyloid beta (A␤) plaques, the appearance of neurofibrillary tangles, and the dysregulation of calcium homeostasis, which has been linked to mutations in the presenilin gene that code for presenilin proteins (Green et al., 2008a; Poirier et al., 2007; Stutzmann et al., 2007). Presenilin proteins (PSs) are a family of multi-pass transmembrane proteins where normal presenilins (PS1 and PS2) are highly localized in the endoplasmic reticulum (ER) (Brunkan and Goate, 2005; Jimenez-Escrig et al., 2005; Kobayashi and Chen, 2005; Link, 2005; Marambaud and Robakis, 2005). Data suggest that normal PS proteins function in protein trafficking, receptor turnover, cleavage of various membrane proteins, and may also play a role in synaptic plasticity (Chan et al., 2002; Brunkan and Goate, 2005; Parent et al., 2005). PSs also function as the catalytic core of the gamma secretase complex that is responsible for enzymatic cleavage of the amyloid precursor protein (Steiner, 2008). PS mutations, however, are correlated with FAD, which is caused by mutations in PS1 (chromosome 14) and/or PS2 (chromosome 1) genes (Nishimura et al., 1999; Drouet et al., 2000; Selkoe, 2000; St GeorgeHyslop and Petit, 2005). In addition, PSs have been shown to differentially modulate endoplasmic reticulum calcium efflux through inositol trisphosphate (IP3R) and ryanodine receptors (RyRs) (Stutzmann et al., 2004, 2007; Green et al., 2008b), cytosolic calcium influx to the ER lumen through sarco/endoplasmic reticulum Ca2⫹-ATPase (Green et al., 2008b), and capacitative calcium entry (Akbari et al., 2004). It has also been proposed (Tu et al., 2006) that PS1, itself forms a passive calcium channel which may contribute an increase in cytosolic calcium concentrations. Collectively, these studies indicate that mutant PS1 variants perturb calcium homeostasis. Calbindin D28k may play a role in AD by rescuing neurons from elevations in intracellular calcium (Lazarov et al., 2006; Leissring et al., 2000). For example, studies in patients with AD have shown reductions in calbindin D28k levels (Hof and Morrison, 1991). Calbindin D28K is a calcium-binding protein, whose expression is induced by activation of the transcription factor, nuclear factor kappa B (NF-␬B). Calbindin D28k is also a member of the calmodulin super family, and has been shown to be neuroprotective, (i.e., when expressed at high levels), against apoptosis and necrosis due to its ability to buffer intracellular calcium ions (Hilton et al., 2005; Fan et al., 2007). Studies utilizing calbindin knock out mice have shown impairments

a

Division of Neurodegenerative Disorders, St. Boniface Research Centre, Winnipeg, MB R2H 2A6, Canada

b

Department of Pharmacology and Therapeutics, University of Manitoba, Winnipeg, MB R3E 0W3, Canada

c

Department of Physics and Astronomy, University of Manitoba, Winnipeg, MB R3T 2N2, Canada

d

Department of Physics, University of Winnipeg, Winnipeg, MB R3B 2E9, Canada

Abstract—Pathological hallmarks of Alzheimer’s disease include memory deficits, accumulation of amyloid beta (A␤) plaques, the appearance of neurofibrillary tangles, and dysregulation of calcium homeostasis, which has been linked to mutations in the presenilin gene that code for presenilin (PS) proteins. PSs are a family of multi-pass transmembrane proteins where normal presenilins (PS1 and PS2) are highly localized in the endoplasmic reticulum (ER). Several past studies have explored alterations in long-term potentiation (LTP), a proposed molecular correlate of memory, and in behavioral tests of spatial memory in a variety of PS1 models. These reports suggest that calcium plays a role in these alterations, but mechanistic explanations for changes in LTP and in behavioral tests of memory are still lacking. To test the hypothesis that calcium-related mechanisms, such as changes in calcium buffering, are associated with alterations in LTP and memory, we utilized in vitro experimental paradigms of LTP in hippocampal slices obtained from the PS1-M146V transgenic mouse model of Alzheimer’s disease (AD). We also used the in vivo Morris water maze (MWM), a test for hippocampal dependent spatial memory. In addition, we used cellular assays to explore molecular mechanisms. We confirm that PS1 mutations (M146V) enhance LTP. We also find increases in some parameters of the MWM, and alterations in other parameters, such as path length indicating impairment in cognitive functioning in PS1-M146V mice. In addition, these findings are observed in association with increased calbindin D28K expression in the CA1 hippocampus of PS1-M146V mice. © 2010 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: synaptic plasticity, memory, Alzheimer’s disease, calcium, calbindin D28k, hippocampus. 1 Contributed equally to this work as first author. *Corresponding author. Tel: ⫹1-204-235-3942; fax: ⫹1-204-237-4092. E-mail address: [email protected] (B. C. Albensi). Abbreviations: A␤, amyloid beta; AD, Alzheimer’s disease; EPSPs, excitatory postsynaptic potentials; ER, endoplasmic reticulum; FAD, familial AD; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HFS, high frequency stimulation; IP3R, inositol trisphosphate; LTP, long-term potentiation; MWM, Morris water maze; PCR, polymerase chain reaction; PSs, Presenilin proteins; RyRs, ryanodine receptors.

0306-4522/10 $ - see front matter © 2010 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2010.04.004

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in long-term potentiation (LTP), presumably by adversely affecting the ability of neurons to appropriately mitigate the exaggerated increase in intracellular calcium (Jouvenceau et al., 2002). Interestingly, studies evaluating the effect of calbindin overexpression have also found alterations in LTP and impaired spatial memory, suggesting that there are normative homeostatic ranges in which calbindin function remains optimal (Dumas et al., 2004). However, little work has been done to investigate the role of calbindin D28k in contexts of synaptic plasticity and memory in PS1-M146V models. Recently, Smith et al. (Smith et al., 2005a) measured calbindin D28k expression and found no difference between wild type (WT) and transgenic (Tg) samples using both PS1-M146V and 3xTg mice. However, they used cortical tissue for their samples, which is curious since all studies showing enhanced LTP thus far have been performed in the hippocampus. To date, several studies (Parent et al., 1999, 2005; Barrow et al., 2000; Zaman et al., 2000; Schneider et al., 2001; Pybus et al., 2003) using various PS1 models (egs, ⌬E9, A245E, M124L, M146V), have explored alterations in synaptic plasticity and memory in AD. Some reports suggest calcium plays a role in these alterations, but mechanistic explanations for changes in LTP and in behavioral tests of memory are still lacking. To test the hypothesis that calcium-related mechanisms, such as changes in calcium buffering mediated by calbindin D28k, are associated with alterations in LTP and memory, we utilized several experimental procedures. This included in vitro experimental paradigms of LTP in hippocampal slices obtained from PS1-M146V mice, a transgenic model of AD. LTP is a molecular mechanism suspected to be associated with memory encoding (Bliss and Collingridge, 1993). The PS1M146V model was used for this investigation for several reasons including the fact that prior studies have reported mutant PS1-M146V mice show deficits in calcium homeostasis (Guo et al., 1999a). We also used the in vivo Morris water maze (MWM), a test for hippocampal dependent spatial memory to assess memory in intact mice where the same mice were utilized for both electrophysiology and MWM testing. In addition, we used molecular assays to explore calcium-related mechanisms. We confirm that PS1-M146V mice show enhanced LTP in CA1 hippocampus; previously described in this mutation under different conditions (Barrow et al., 2000; Oddo et al., 2003; Pybus et al., 2003). We also find alteration in several parameters of the MWM, where improvement is seen in some parameters and impairments are see in other parameters, such as path length indicating changes in cognitive functioning. These results are observed in association with the novel finding that calbindin D28K expression is increased in the CA1 hippocampus of PS1-M146V mice.

EXPERIMENTAL PROCEDURES The animal studies presented here were performed under a protocol approved by the University of Manitoba Protocol Management and Review Committee. The number of animals was minimized, but in accordance with appropriate sample size requirements.

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Animal model PS1 strains have been previously described (Duff et al., 1996; Duff, 1997; Guo et al., 1999a), and have been used in several studies of AD (Parent et al., 1999; Zaman et al., 2000; Stutzmann et al., 2004). Our colony was established with PS1-M146V animals obtained from Mattson and colleagues who described the generation of this colony in detail (Guo et al., 1999a). The genetic background of the mice used in prior PS1-M146V studies (Guo et al., 1999a; Oddo et al., 2003; Sun et al., 2005) and our study was the C57BL6/129 hybrid. To date, the PS1-M146V mouse has shown overexpression of PS1 mutant protein, elevations in A␤42, altered mitochondrial activity, and dysregulation of Ca⫹⫹ homeostasis, but no manifestation of amyloid plaques or tangles (Duff et al., 1996; Duff, 1997). A model of this type yields important information related to calcium dysfunction for AD since any results that are gleaned are independent of overt A␤ and tau pathologies. Prior reports using this model claim that there are no behavioral abnormalities and the mice appear healthy at over a year old (Duff et al., 1996; Duff, 1997). Here, mice were tested at both 3 and 6 months of age for the electrophysiological and behavioral experiments and at ⱕ6 months of age for all the cell and molecular experiments (i.e., RNA extraction, polymerase chain reaction (PCR), and Western blot). Other studies using PS1 models have used mice that primarily ranged in age from 1 to 6 months and so we used mice within this range so our results could be compared to prior studies (Guo et al., 1999a; Parent et al., 1999; Barrow et al., 2000; Zaman et al., 2000; Schapansky et al., 2007) and since these studies have shown that intracellular calcium homeostasis is perturbed in mice at these ages.

Morris water maze Hippocampal-dependent spatial learning and memory was assessed in a standard MWM, which consisted of an 81 cm circular pool, filled with tap water (23.5–24.5 °C) and made opaque (white in color) with powdered milk. Unwanted extra-maze cues were blocked with a black curtain and standard visual cues (green triangle, blue circle, red star and yellow arrow) were positioned equidistant above water level. A non-visible escape platform, 7 cm in diameter, was submerged approximately 5 mm below water surface in the center of the designated target quadrant. For the acquisition phase, mice were given up to 90 s to find the hidden platform and remain seated on the platform for 10 s after which the mice were returned to their cage. If unsuccessful at the end of 90 s, the mouse was manually placed on the platform for 30 s then returned to their cage. Each animal was tested for one block/day (one block consisted of four trials) for seven consecutive days. Live video was recorded for each trial and the videos were analyzed for escape latency and search strategies were assigned as previously described (Janus, 2004). Search strategies were assigned for every trial of every block of the acquisition phase. Search strategies consisted of the following: the animal swims directly onto the platform (spatial direct); the animal searches the correct quadrant before finding the platform (focal: correct target quadrant); the animal searches the incorrect quadrant before finding the platform (focal: incorrect quadrant); the animal searches the pool randomly (random); the animal searches the pool at a fixed distance from the pool wall (scanning); the animal searches in big circles with no particular spatial bias (circling); the animal swims in short circles with no spatial bias (chaining); the animal fails to move when placed in the pool (floating); the animal swims along the side of the pool (thigmotaxis). All search strategies could be grouped into larger categories: spatial (spatial, focal: correct target quadrant, focal: incorrect quadrant); non-spatial, systematic (random, scanning); repetitive looping (chaining, circling, thigmotaxis). All animals were given a 30-min inter-trial period. The retention phase began 24 h after the last block of the acquisition phase. For the retention phase, the platform was removed from the pool and each

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mouse was given a maximum of 2 min to search for the position of the missing platform. Each animal was tested for one block/day (one block consisted of four trials) for three consecutive days. Time in the target quadrant and number of times the mouse crossed the location of the missing platform were recorded. Swim speeds and path length were calculated using Matlab (The MathWorks Co., Natick, MA, USA). Statistical analysis was performed using 2-way ANOVA with repeated measures.

Slice preparation and electrophysiology Electrophysiological techniques with brain slices have been described before by Albensi et al. (Albensi et al., 2000; Albensi and Mattson, 2000). Animals tested in the MWM were subsequently used for these electrophysiology experiments. Mice were killed by decapitation (World Precision Instruments, Inc., Sarasota, FL, USA) under anaesthetized conditions with isoflurane. The brain was rapidly removed and placed in ice-cold (4 – 6 °C) artificial cerebrospinal fluid (aCSF), containing low calcium (in mM): NaCl, 124; KCl, 3; KH2PO4, 1.25; MgCl2, 1.4; CaCl2, 1; NaHCO3, 26; glucose, 10. The aCSF was equilibrated with 95% O2/ 5% CO2 (pH 7.4) throughout the dissection. The left hippocampus was dissected free and sectioned (350 ␮m thick) using a McIlwain tissue chopper (TC752, Campden Instr, Lafayette, IN, USA). Slices were collected in an incubation chamber with oxygenated aCSF (95% O2/5% CO2) at room temperature (⬃21 °C). The incubation temperature was gradually increased up to 31–32 °C. After 60 min recovery, slices were then gently transferred to another incubation chamber or the recording chamber using a standard recording buffer, containing (in mM): NaCl, 124; KCl, 3; KH2PO4, 1.25; MgCl2, 1.4; CaCl2, 2; NaHCO3, 26; glucose, 10. The aCSF was equilibrated with 95% O2/5% CO2 (pH 7.4). Tungsten stimulating electrodes were placed on the Schaffer collaterals in the CA1 subregion of hippocampal slices. To evoke orthodromic field excitatory postsynaptic potentials (fEPSPs or EPSPs), monophasic test pulses were delivered to the slice every 30 s (Grass S48 stimulator, Warwick, RI, USA) with a stimulus duration of 0.1 ms. Recording was accomplished with an AxoClamp 2B amplifier (Axon Instr., Foster City, CA, USA) in continuous current clamp bridge mode. EPSPs were obtained using glass microelectrodes (2.3 M⍀) filled with recording buffer, which were generated in a standard recording chamber (RC-27L, Warner Inst., Hamden, CT, USA) at 31–32 °C. EPSP slope responses were recorded from the CA1 dendritic arbor. EPSP slope values were calculated by measuring the rise/run (i.e., 10%–90% of trace). Responses were amplified (gain 50⫻) low-pass filtered at 6 kHz and digitized (20 kHz) (DIGIDATA 1322A, Axon Instr., Foster City, CA, USA). Input– output curves were generated at the beginning of each experiment to determine maximal and halfmaximal responses and voltage settings (max response was also sometimes determined at the end of the experiment). The voltage was then set to evoke an EPSP response that was approximately half-maximal in amplitude. High frequency stimulation (HFS) consisted of three trains, separated by 0.5 s, of 100 Hz for 1 s each and was applied for inducing LTP responses. A control baseline period was established for 15 min before HFS. The response was

followed for approximately 60 min after HFS. Data were acquired with Clampex 9.2, (Axon Instr., Foster City, CA, USA) analyzed initially with Clampfit 9.2 (Axon Instr., Foster City, CA, USA). The magnitude of LTP in a single slice was determined by comparing the mean response from the post-tetanization period (⬃60 min) with the mean response for the baseline period (⬃15 min) prior to tetanization. Paired pulse stimulation was delivered by using paired stimuli (duration of each pulse, 0.1 ms) at an interstimulus interval of 50 ms. For each experiment the mean response value following stimulation was then normalized by comparison with the mean response value obtained during the baseline recording. Calculation of the paired pulse ratio was done as follows: second EPSP response/first EPSP response. Data were statistically analyzed with SPSS (v13, Chicago, IL, USA). Significance was set at P⬍0.05. Results were analyzed with student’s t-test and 2-way ANOVA. EPSP data represented by mean⫾SEM.

Western blot The entire hippocampus was excised from the brains of experimental animals (transgenics and controls) and processed for protein extraction using the Allprep DNA/RNA/protein Mini Kit (Qiagen, Valencia, CA, USA). In a specific number of samples (n⫽4 WT; n⫽6 PS1-M146V), the CA1 subfield was also isolated and processed for protein extraction. These samples were taken from mice that were not used previously in the electrophysiology and MWM tests in order to generate enough protein for testing. RNA was simultaneously extracted and used for the qRT-PCR experiment. To isolate the CA1 subregion, samples were first sliced at 350 ␮m using a McIllwain tissue chopper and then the CA1 was surgically removed using a dissecting microscope for visual guidance. Protein concentration for these samples was then measured using the BCA Protein Assay Kit (Pierce, Rockford, IL, USA). Subsequently, protein extracts were subjected to SDS-PAGE and then incubated with primary polyclonal antibodies for calbindin D28K (1:1000) (Santa Cruz Technology, Santa Cruz, CA, USA), and ␤-actin 1:5000 (Sigma Aldrich, Oakville, ON, Canada), followed by incubation of a secondary antibody (Santa Cruz Technology). Enhanced chemiluminescence was detected from the blot using the ECL Plus™ (GE Healthcare, Baie d’Urfe, QC, Canada).

RNA extraction, reverse transcript PCR (RT-PCR), PCR Samples from the same animals assayed using Western blotting techniques were also used for PCR experiments. Total RNA was extracted from hippocampal tissue (three animals per group) using the RNAeasy Kit (Qiagen), and 200 ng of RNA was used for first-strand cDNA synthesis with random hexamer primers using the iScript cDNA Synthesis Kit (BIORAD, Mississauga, ON, Canada). The PCR amplification was conducted using the following cycle parameters: 94 °C for 2 min (one cycle), 94 °C for 30 s, 60 °C for 30 s, 72 °C for 1 min (30 cycles), 72 °C for 7 min (one cycle) and then held at 4 °C. RyR3, IP3R1 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were amplified with the primers listed in Table 1 and PCR products were confirmed by agarose gel electrophoresis (1% gel, 100V for 25 min and stained

Table 1. Primer pairs for qRT-PCR Gene

Sequence (5=–3=)

Location

RyR3

CAG CAG GAG CAA GTA CGG GAA G (Forward) CTT TGA CAC GAC CCC TCA TGG T (Reverse) CAG CAG GAA ATC AAG GCG ACA G (Forward) GGA AGA AAG CCA AAG AGC CCA C (Reverse) CGT GTT CCT ACC CCC AAT GTG TCC (Forward) GAA GGT GGT GAA GCA GGC ATC TGA G (Reverse)

14,389 14,475 5927 6039 766 843

IP3R1 GAPDH

Amplicon size (bp) 86

Tm (°C)

Primer conc. (uM)

62

1

112

64.1

1

186

69.4

1

G. L. Odero et al. / Neuroscience 169 (2010) 532–543 with Ethidium Bromide (2 mg/mL)). RNA was used as a negative control to assess gDNA contamination. Serial dilutions of purified PCR products were generated to allow quantitation of the specific mRNA of interest.

Quantitative real-time RT-PCR (qRT-PCR) A total volume of 1 ␮l of cDNA was used as the template in each 25 ␮l PCR reaction with iQSYBR Green (BIORAD). Cycling conditions were: 94 °C for 10 min, followed by 40 cycles at 94 °C for 15 s, Tm for primers (see Table 1) for 30 s, and 72 °C for 30 s, 72 °C for 7 min and held at 4 °C. PCR assays were performed using the iCycler Thermal Cycler (BIORAD, Mississauga, ON, Canada). Crossing threshold (Ct) values for each sample were used to calculate the initial quantity of cDNA template by the standard curve method. Data were normalized from each sample by dividing the copy number of target gene cDNA by the copy number of GAPDH cDNA to correct for variability in individual samples. Negative control reactions were also performed without template cDNA.

Statistical analysis Results are presented as mean⫾SEM in all cases except for the PCR and Western blot experiments where data are presented as mean⫾SD. Data were analyzed using Student’s t-test or two-way analysis of variance (ANOVA) with repeated measures using SPSS (version 13.0, Chicago, IL, USA). In all cases, P⬍0.05 was considered statistically significant.

RESULTS Hippocampal-dependent memory To ascertain potential changes in hippocampal dependent spatial memory in PS-M146V animals, MWM experiments were conducted in two different age groups: 3 and 6 months of age, two groups at risk for early cognitive dysfunction. The acquisition phase of the MWM showed no significant difference (P⬎0.05) in escape latency times over 7 days between the PS-M146V strain and the WT controls in either age group (Fig. 1A). With regard to swim speed, the PS1-M146V mice in both age groups showed a significant increase (P⬍0.05) in their swim speed over 7 days relative to the WT control mice (Fig. 1B) during the acquisition phase. The time in the target quadrant, measured during the retention phase, showed no significant difference (P⬎0.05) between WT and PS1-M146V in 6-month old mice over 3 days, however, there was a statistically significant difference between both groups at 3 months of age over 3 days (P⫽0.01) (Fig. 1C). The PS1M146V mice also showed a significantly greater number of passes (P⬍0.05) over the missing target (5.39 times vs. 3.57 times for the WT) for the 3-month group (Fig. 1D) over 3 days. Likewise, the 6-month old PS1-M146V mice also showed more passes over the missing target (4.86 times vs. 2.31 times for the WT controls) over 3 days. An increase in the number of passes over the missing platform is interpreted as increased spatial accuracy or increased spatial reference memory (D’Hooge and De Deyn, 2001; Lipp and Wolfer, 1998). However, both the WT and PS1M146V strains at both ages performed below chance levels (i.e., ⬍30 s) with regard to the mean time in the target quadrant (2 min total). In addition, path length was mea-

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sured during the escape latency phase of the water maze. Representative traces in Fig. 2A show the disparity in the chosen path between groups. While no difference in path length was found between the groups at 3 months of age, a significant difference (P⬍0.05) was found in mean path length over 7 days at 6 months of age (Fig. 2B). Some investigators claim path length is the most appropriate index of cognitive performance in the MWM (Lindner, 1997; Lindner et al., 1997). Since any single measure of the MWM does not fully reflect the complexities of search behavior, we also assessed search strategies to provide a qualitative measure of learning. We found (Fig. 3) that on the first experimental day all groups used predominately non-spatial strategies (random). Thereafter, 3 and 6-month controls showed progressive and consistent use of spatial search strategies (i.e., spatial direct, focal correct, focal incorrect) over 7 days with an incidence from approximately 22% to over 60% with a corresponding decrease in other strategies (i.e., non spatial and repetitive looping) over this time period. Similar to the controls, we found a comparable pattern in the 3-month PS1-M146V group suggesting these mice also adopted spatial strategies on an increasing and reliable basis. However, the 6-month PS1-M146V group differed markedly from the 6 month control group and spatial search strategy use fluctuated unpredictably over the 7 day period. Of note, the 6 month PS1-M146V group used a combination of non-spatial strategies (scanning, random, chaining) in addition to the spatial strategies over the 7 days. It needs to be acknowledged, however, that in spite of the fact that search strategies have been previously used and documented by many investigators, any post hoc classification of strategies into mutually exclusive categories has the potential to introduce bias in the interpretation of MWM results. In addition, there are instances when mice find the hidden platform by chance such as on the first day of the trial when the mice had no previous knowledge of the platform location. Paired-pulse responses To examine possible perturbations in presynaptic function, we evaluated paired pulse responses, a phenomenon previously described (Zucker, 1989) where the EPSP response to a second stimulus is potentiatied in certain cases (e.g., CA1 region and if the second stimulus is less than ⬃200 ms of the first stimulus). We found (Fig. 4) there were no significant differences (P⬎0.05) in paired pulse ratios in the age groups tested (3 and 6 months old) between PS1-M146V mutants and their controls. Long-term potentiation We then evaluated the effect of LTP-inducing HFS. Such HFS protocols have been previously shown to activate NMDA receptors and initiate calcium-dependent processes leading to LTP (Bliss and Collingridge, 1993). For all groups we found that three trains of 100 Hz tetanic stimulation reliably gave rise to LTP of synaptic transmission (Fig. 5). In addition, we found that PS1-M146V mutants showed enhancements in EPSP slope as compared

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Fig. 1. Morris water maze. Mice were subjected to seven consecutive days (blocks) of training in the hidden platform version of the Morris water maze (acquisition) and 3 days without the platform (retention). Each day consisted of four trials per animal with an inter-trial interval of 30 min. (A) During the acquisition phase, time required to find the platform or escape latency was recorded. (B) Custom Matlab software was used to also determine the average swim speed during the acquisition phase. (C) The time spent in the target quadrant (the quadrant that previously held the position of the platform), and the number of passes over the position of the missing platform (D) were recorded during the retention phase of the experiment. * P⬍0.05; ** P⬍0.01. Error bars represent ⫾SEM. 3 mon n⫽8 WT, n⫽7 PS1-M146V; 6 mon n⫽9 WT, n⫽7 PS1-M146V.

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Fig. 2. Representative Search Paths and Path Lengths for WT versus PS1-M146V strains. (A) Search paths were determined using MatLab software during the acquisition phase in the Morris water maze. Paths were derived from animals that had equal escape latency times ⫾2 s, and display the predominant search strategy from the block chosen. (B) Average path lengths for WT versus PS1-M146V were also plotted for 3 and 6 mon old groups. Path lengths for PS1-M146V mice were significantly increased (* P⬍0.05) over 7 days in the 6 mon group (B, right panel). Closed circles denote the position of the hidden platform and open circles represent the position in the pool in which the mouse was placed. Sample size: 3 mon, n⫽8 WT, n⫽7 PS1-M146V; 6 mon, n⫽9 WT, n⫽7 PS1-M146V. Error bars represent ⫹/⫺SEM.

to controls post-tetanization (from time⫽15 min to time⫽75 min) in both the 3 month group (P⬍0.05), and the 6 month group (P⬍0.05). Expression of RyR3 and IP3R1 mRNA It has previously been reported that presenilin 1 mutations lead to increased RyR expression and abnormally elevated Ca2⫹ signals via IP3Rs (Chan et al., 2000; Stutzmann et al., 2004), so we evaluated relative expression of RyRs and IP3Rs mRNA specifically in the CA1 hippocampal subregion of PS1-M146V mutants (where the enhancements in LTP were recorded) to investigate if enhancements in LTP in the PS1-M146V mutants were linked to changes in the mRNA

expression of these receptors. Although all three RyR isoforms are expressed in area CA1 of the hippocampus in adult mice, the RyR3 isoform was chosen for analysis because it is specifically enriched in the CA1 region (Mori et al., 2000), where this isoform has been reported to play an important role in hippocampal synaptic plasticity and hippocampal-dependent learning tasks (Balschun et al., 1999; Kouzu et al., 2000; Shimuta et al., 2001). The IP3R1 isoform of the IP3R was also used since it too has been shown to play a role in synaptic plasticity and the induction of LTP in the CA1 hippocampal subregion (Fujii et al., 2000). Real-time experiments showed there was no significant difference in the expression of RyR3 and IP3R1 mRNA in the CA1

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3 Month Old

A

6 Month Old

Percent Incidence

WT

PS1-M146V

Spatial Focal: Correct TQ Focal: Incorrect Q

Spatial

Scanning Random Floating

Non-spatial, Systematic Other

Chaining Circling Thigmotaxis

Repetitive Looping

Fig. 3. Search strategies used in the Morris water maze were assigned during the acquisition phase. Search strategies for 3 and 6 month old WT control mice show reliable increases in the use of spatial strategies over 7 days. Similar to the controls, we found a comparable pattern in the 3-month PS1-M146V group suggesting these mice also adopted spatial strategies on an increasing basis. However, the 6-month PS1-M146V group differed markedly from the 6 mon control group and spatial search strategy use fluctuated greatly over the 7 day period. Each block above represents a different day. Sample size: 3 mon, n⫽8 WT, n⫽7 PS1-M146V; 6 mon, n⫽9 WT, n⫽7 PS1-M146V. TQ⫹, target quadrant; Q⫹⫹, quadrant.

Paried Pulse Ratio (EPSP2/EPSP1)

hippocampal subregion of the PS1-M146V mutant strain (Fig. 6).

3 2.5

3 Month Old

6 Month Old

Wild type PS1M146V

2 1.5 1 0.5

Expression of calbindin D28k We then investigated the possibility that perturbed calcium buffering in the PS1-M146V mutants may be associated with alterations in LTP and MWM tests. To investigate this possibility, Western blots were performed to assay the expression of calbindin D28k in whole hippocampus and in isolated CA1 subregion (the region where LTP was recorded). Western blot analysis showed no difference in protein expression in total hippocampal extracts for calbindin D28k in the PS1-M146V mutants relative to their controls (Fig. 7A), however, we observed a two-fold increase in calbindin D28k expression in the PS1-M146V mutant strain relative to the control in the CA1 subregion of the hippocampus (Fig. 7B).

0

Fig. 4. Paired pulse analysis. A 50 ms interstimulus interval was used to generate paired pulses. EPSP responses were measured in CA1 hippocampal slices and then plotted as a ratio of EPSP2/EPSP1. No significant differences (P⬎0.05) in paired pulse ratios were found between PS1-M146V mutants and their controls in either group. Three months old: WT 10 slices/five animals; PS1-M146V nine slices/five animals. Six months old: WT eight slices/five animals; PS1-M146V 10 slices/six animals. Error bars represent ⫾ SEM. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.

DISCUSSION The present findings show that PS1-M146V mice exhibit increases in some parameters, but impairments in other parameters in MWM tests and that hippocampal brain slices from mice with PS1-M146V mutations display enhancements in LTP in CA1 hippocampus. The data confirms previous reports that LTP is enhanced in various PS1 models (Barrow et al., 2000; Zaman et al., 2000; Oddo et al., 2003; Pybus et al., 2003; Parent et al., 2005). Our data

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A

3 Month Old

f EPSP Slope (% of Baseline)

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Time (min) Fig. 5. LTP in hippocampal slices (CA1 subfield) from 3-mon (A) and 6-mon old (B) mice. Representative traces above summary graphs are taken immediately before tetanus and post tetanus at approximately at t⫽70 min. Slices from both the 3- and 6-mon old groups showed significant increases (P⬍0.05) in LTP (time approx. 15–75 min) in the PS1-M146V strain (pink triangles) as compared to WT. Slices were stimulated every 30 s. Sample sizes: 3 mon old WT, n⫽10 slices/five animals; PS1-M146V, n⫽9 slices/five animals; 6 mon WT n⫽12 slices/ eight animals, PS1-M146V n⫽12 slices/eight animals. Delivery of three trains of 100 Hz tetanus (separated by 0.5 s, of 100 Hz for 1 s each) is represented with three solid upward arrows at time⫽15 min. Scale bar: 2 mV, 10 ms. Error bars represent ⫹/⫺SEM. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.

are also consistent with reports that MWM performance is selectively affected in models of PS1 mice (Chan et al., 2000; Huang et al., 2003; Janus et al., 2000; Sun et al., 2005). We also find that calbindin D28k expression is elevated locally in CA1 hippocampus. In our study, we found that several aspects of MWM behavior were different in PS1-M146V mice. Overall, stud-

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ies of spatial memory in PS1 mutants have resulted in conflicting reports including: no difference in learning, differences in memory recall, and severe impairments in spatial learning and memory (Chan et al., 2000; Huang et al., 2003; Janus et al., 2000; Sun et al., 2005). Importantly, studies to date have not yet clearly shown if cognitive function is modified in PS1 mutant models. Of note, some studies (D’Hooge and De Deyn, 2001; Poirier et al., 2007) have claimed that more than two thirds of behavioral variability in MWMs is accounted for by two factors that have no direct relation to spatial memory and learning, which are so-called noncognitive factors (egs., thigmotaxis, passivity). Furthermore, studies have demonstrated that mutant mice may also show excessive thigmotaxis (Poirier et al., 2007). In our MWM experiments we found that the PS1-M146V group showed a higher number of passes over the missing platform in both 3 and 6 month old groups and spent more time in the target quadrant (3 month old). However, the WT and Tg strains at both ages performed below chance levels (i.e., ⬍30 s) with regard to the time in the target quadrant, which complicates interpretation. In a recent study by Sun et al., who looked at MWM behaviors in the PS1-M146V strain, they reported no difference on day 2 for time in target quadrant, but did report decreases in the passes over the platform and time in target quadrant at day 6 in PS1 mice 6 months of age (Sun et al., 2005). In addition, we found increases in swim speed in PS1-M146V mice at 3 and 6 months of age, which has been previously shown by others (Janus et al., 2000) using similar strains (PS1M146L). We also found that 6 month old PS1-M146V mice showed significantly increased path length, which is a measure that indicates impaired cognitive functioning. In fact, some investigators have argued that path length (although not a pure measure) might be the most appropriate index of cognitive performance in the MWM (Lindner, 1997; Lindner et al., 1997). In our experiments, the qualitative assessment of search strategies was additionally utilized. Erratic and nonpredictable use of spatial search strategies was observed in the 6 month PS1-M146V group, while a steady increase in spatial search strategies during consecutive trials was observed in the 6 month control group. The observation that control mice reliably and increasing use spatial strategies and that AD transgenic mice do not use spatial strategies reliably have been seen by other investigators (Janus, 2004; Brody and Holtzman, 2006). These data support our observations and suggests potential impairments in the formation of a cognitive map in older PS1M146V mice. To our knowledge, our study is the first to suggest that alterations in search strategies are present during the acquisition phase of the MWM in the PS1M146V strain. Given that path length was increased and the use of spatial search strategies appeared different between groups, we conclude that cognition was modified in the 6 month old PS1-M146V group. However, these results do not point to robust impairments in cognition, but do suggest alteration of cognitive processes that should be further investigated.

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Fig. 6. Quantitative real-time PCR for RyR3 and IP3R1 in CA1 hippocampal samples showed no significant difference (P⬎0.05) between strains. Panel (A) shows relative RNA expression for IP3R1 and (B) shows relative RNA expression for RyR3. Relative quantitation of RNA was determined using the standard curve method. Samples were normalized to GAPDH. Sample size: n⫽5 animals/group and samples were run in triplicate. Error bars represent ⫾ SDs.

Due to the hypothesized role in modulating intracellular calcium, several presenilin 1 mutants (egs., A246E, ⌬E9, M146L, M146V) have been previously studied to determine their role, if any, in altering synaptic plasticity, a calcium-dependent phenomenon. In general, past LTP experiments assessing synaptic plasticity using the PS1 strain have observed enhancements in LTP (Parent et al., 1999; Barrow et al., 2000; Zaman et al., 2000; Pybus et al., 2003). For example, a study by Pybus et al. (Pybus et al., 2003) was conducted in transgenic rats that carried the

Fig. 7. A representative Western blot analysis (A) for calbindin D28k showed no difference in expression for whole hippocampus in PS1M146V versus WT, whereas PS1-M146V samples from isolated CA1 hippocampus showed increased expression. Quantification of calbindin expression in WT (n⫽4) versus PS1-M146V (n⫽6) samples (B) verified that a significant difference in expression levels existed in CA1 hippocampus * P⬍0.05, but not in whole hippocampus (not shown). Animals were ⱕ6 mon old at time of analysis. Student’s t-test was used for the analysis of relative expression of calbindin D28K. Error bars represent ⫾ SD.

M146V mutation, which showed that enhanced LTP was age-related. In this investigation, 6-month old rats demonstrated no difference in LTP, whereas 18-month old animals exhibited increased LTP in CA1 and dentate subfields of the hippocampus. Schneider et al. (2001) found PS1 slices to have normal LTP when induced with a strong stimulus; however, weak stimulation elicited LTP in mutant PS1 slices. Why LTP is enhanced in some contexts and not others is not clear. In this study, we found that LTP was enhanced in both 3- and 6-month age groups in CA1; however we did not test the dentate subfield or animals older than 6 months. In another study by Barrow et al. (Barrow et al., 2000), which used transgenic mice carrying the M146V mutation, LTP was also enhanced. They also found that afterhyperpolarizations were altered in CA3 pyramidal cells, which may be linked to calcium dysregulation. Collectively, these data suggest that PS1 mutations generally enhance LTP, but how this occurs is not yet clear. Some investigators have suggested that calcium release from the ER via IP3Rs or RyRs can enhance LTP (Raymond and Redman, 2002). However, mice lacking the type 3 RyR showed enhanced LTP (in CA1 hippocampus) and improved spatial learning (Futatsugi et al., 1999). In addition, administration of specific IP3R receptor blockers in CA1 hippocampal brain slices also resulted in enhanced LTP (Taufiq et al., 2005). Moreover, some studies have shown that mice harboring PS1 mutations display increased expression of both IP3R and RyR receptors (Chan et al., 2000; Cheung et al., 2008; Rybalchenko et al., 2008). We evaluated both IP3R and RyR mRNA expression in CA1 hippocampus and found no difference between PS1-M146V and control samples and so this issue appears unresolved at this time. We also evaluated in total hippocampus the expression of calbindin D28k. Due to its function as a key calcium regulating protein, calbindin has been thought to play a role in learning and memory and its cellular correlate, LTP (Chan et al., 2000; Chard et al., 1995; Dumas et al., 2004). We found no change in calbindin D28k expression in PS1-

G. L. Odero et al. / Neuroscience 169 (2010) 532–543

M146V versus control tissue in total hippocampus. However, in isolated CA1 hippocampus, we found that calbindin D28k levels were significantly increased in PS1M146V tissue as compared to control. Recently, Smith et al. (Smith et al., 2005b) used Western blots to measure calbindin D28k expression and found no difference between WT and Tg samples using both PS1-M146V and 3xTg mice. However, they used cortical tissue for their samples, which is surprising since all studies showing enhanced LTP to date have been performed in the hippocampus. It would be interesting in future studies to also evaluate calbindin D28k levels in hippocampal slices before and after LTP induction to see if calbindin levels increase following LTP inducing stimuli. Studies utilizing calbindin knock out mice have shown impairments in LTP, presumably caused by unbuffered increases in intracellular calcium (Jouvenceau et al., 2002). Interestingly, studies evaluating the effect of calbindin overexpression (Dumas et al., 2004) have also found alterations in LTP and impaired spatial memory, suggesting that there are normative homeostatic ranges in which calbindin function remains optimal. In any case, inadequate calcium buffering could lead to increased intracellular calcium thus activating apoptotic pathways and excitotoxic responses, as has been previously shown in PS1 mutant cell culture models (Guo et al., 1999b). Of equal importance, however, is how neurons might respond to prolonged increases in intracellular calcium. In other words, it could be argued that increased expression of calbindin D28k is a homeostatic or a compensatory response to the heightened levels of intracellular calcium in this PS1-M146V mutant, which would be seen early on during disease progression. This would agree with other studies that have linked calbindin D28k to neuroprotection (Chui et al., 1999; Goodman et al., 1993; Mattson et al., 1995, 1991; Toyoshima et al., 1996). Our findings show the increased expression of calbindin D28k in the basal state in PS1 mice and taken together with other studies that show decreased expression in samples from human patients with AD, may suggest that calbindin expression varies with age and/or as a consequence of disease progression. Future studies that measure calcium concentrations and calbindin D28k levels over longer time courses could help shed light on potential age-related changes. An alternative explanation is that calbindin D28k is functionally compromised in this strain. Calbindin D28k has been found to contain both high and low affinity binding sites for calcium (Chan et al., 2000; Nagerl et al., 2000). Perhaps, the capacity for calbindin to bind calcium is dysfunctional. Nagerl et al. (Nagerl et al., 2000) provided evidence that calbindin binds calcium in two distinct kinetic patterns: using high affinity sites with a kon similar to calcium chelator EGTA and low affinity sites which binds calcium eight times faster than the high affinity sites. To compensate for the overwhelming increase in calcium, the lower affinity binding sites of calbindin may be favored since it binds calcium at a faster rate, albeit with a much lower affinity. This would result in decreased capacity of calbindin to

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bind optimally to calcium resulting in increases in calcium and concurrent increases in calbindin expression. We are also left with the question how behavioral alterations seen in the MWM may be related to enhanced LTP and increases in calbindin D28k. According to the synaptic plasticity and memory hypothesis put forth by Martin et al. (Martin et al., 2000) synaptic plasticity as measured by LTP occurs during brain activity and is responsible for memory formation. However, from a survey of the literature, it is far from clear exactly which properties of synaptic plasticity and LTP actually correlate with memory and cognition and the present study is no different. However, it is interesting to note that in humans with AD, one observes that these individuals walk incessantly, exhibit difficulty remembering familiar paths or routes, and “getting lost” is a commonly experienced symptom early in the disease. Our observations of increased swim speed and increased path length would seem to be consistent with these observations in humans. One possibility is that abnormal increases in intracellular calcium (presumed to be linked to enhanced LTP) and increased locomotion could be part of a generalized physiological pattern occurring in response to disease progression. A limited number of studies that have measured calcium in PS1 mice show increases in intracellular calcium (Smith et al., 2005b) supporting a link between increased calcium and enhanced LTP in PS1 animals. Our observations of increased calbindin presumably are intimately connected to changes in calcium functioning in this scenario as well, but requires further study. In summary, we find alterations in specific parameters in the MWM and also enhanced LTP in PS1-M146V mutants, which are associated with the increased expression of calbindin D28k in CA1 hippocampus. These results may be linked to calcium dysfunction in FAD and warrant additional investigation. Acknowledgments—Funding for this work was granted in part by, the Manitoba Health Research Council (MHRC), an award from the Scottish Rite Charitable Foundation of Canada, the St. Boniface General Hospital Research Foundation, and the University of Manitoba. BCA is a Research Affiliate at the University of Manitoba’s Centre on Aging. We also thank Dr. Robert Ariano for his help with statistical considerations and Drs. Smyth, Parkinson, and Fernyhough for a critical reading of the manuscript.

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(Accepted 1 April 2010) (Available online 14 April 2010)