Amyloid-β-(1-42) increases ryanodine receptor-3 expression and function in neurons of TgCRND8 mice

Disruption of intracellular calcium homeostasis precedes the neurodegeneration that occurs in Alzheimer disease (AD). Of the many neuronal calcium-regulating proteins, we focused on endoplasmic reticulum (ER)-resident ryanodine receptors (RyRs) because they are increased in the hippocampus of mice expressing mutant presenilin-1 and are associated with neurotoxicity. Others have observed that ryanodine binding is elevated in human post-mortem hippocampal regions suggesting that RyR(s) are involved in AD pathogenesis. Here we report that extracellular amyloid-(cid:1) (A (cid:1) )-(1–42) specifically increased RyR-3, but not RyR-1 or RyR-2, gene expression in cortical neurons from C57Bl6 mediates disruption of Ca 2 i homeostasis in Here we that high levels of A (cid:1) selectively elevated RyR-3 and protein levels in vitro and in vivo . Functionally, increased RyR-3 facilitated significant increases in Ca 2 (cid:1) i levels following ryanodine or glutamate treatment. These observations highlight a possible role for RyR-3 in the AD pathology.

Disruption of intracellular calcium homeostasis precedes the neurodegeneration that occurs in Alzheimer disease (AD). Of the many neuronal calcium-regulating proteins, we focused on endoplasmic reticulum (ER)-resident ryanodine receptors (RyRs) because they are increased in the hippocampus of mice expressing mutant presenilin-1 and are associated with neurotoxicity. Others have observed that ryanodine binding is elevated in human postmortem hippocampal regions suggesting that RyR(s) are involved in AD pathogenesis. Here we report that extracellular amyloid-␤(A␤)-(1-42) specifically increased RyR-3, but not RyR-1 or RyR-2, gene expression in cortical neurons from C57Bl6 mice. Furthermore, endogenously produced A␤-(1-42) increased RyR-3 mRNA and protein in cortical neurons from transgenic (Tg)CRND8 mice, a mouse model of AD. Increased RyR-3 mRNA and protein was also observed in brain tissue from 4-to 4.5month-old Tg animals compared with non-Tg littermate controls. In experiments performed in nominal extracellular calcium, neurons from Tg mice had significant increases in intracellular calcium following ryanodine or glutamate treatment compared with littermate controls, which was abolished by treatment with small interfering RNA directed to RyR-3, indicating that the higher levels of calcium originated from RyR-3-regulated stores. Taken together, these observations suggest that A␤-(1-42)-mediated changes in intracellular calcium homeostasis is regulated in part through a direct increase of RyR-3 expression and function.
Alzheimer disease (AD) 3 is a neurodegenerative disorder characterized by neuritic plaques composed of aggregated extracellular amyloid-␤ (A␤), intracellular neurofibrillary tangles, and containing selective neuron and synapse loss leading to irreversible cognitive decline and memory impairment (1). Although the cause of AD is still unknown, it is believed that accumulation of A␤ in the brain is an initial pathogenic event (2). A␤-(1-42) is a fibrillogenic peptide resulting from the sequential endoproteolytic cleavage of amyloid precursor protein by ␤-and ␥-secretase, and its overproduction and deposition are associated with early-onset (or familial) and sporadic AD (3). Consequently, there is an extensive effort to determine the molecular and cellular actions of A␤ on neurons and glial cells.
Alterations in intracellular calcium (Ca 2ϩ i ) homeostasis constitute an early signaling event that initiates the manifestation of AD (4). Ca 2ϩ i levels are maintained by voltage-, ligand-gated, or store-operated Ca 2ϩ channels on the plasmalemma and by endoplasmic reticulum (ER)-resident channels (5). Several studies have indicated that disruption of ER-regulated Ca 2ϩ i homeostasis is associated with neurotoxicity in AD (6). The ER is an important dynamic Ca 2ϩ source and sink, it regulates neurotransmitter release, synaptic plasticity, gene expression, and signal transduction to the nucleus (7). Ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP 3 Rs) are the two major Ca 2ϩ release channels found on the ER (8) and are activated through Ca 2ϩ -induced Ca 2ϩ release and IP 3 -induced Ca 2ϩ release, respectively (9).
Our study focuses on the role of RyRs in the regulation of Ca 2ϩ i homeostasis because others have observed that hippocampal brain specimens from AD patients with early cognitive decline have increased ryanodine binding, suggestive of increased RyR levels (1). We observed that RyR expression and function were increased in neurons that expressed mutant PS-1 (10). Furthermore, high levels of RyRs mediated dysregulation of Ca 2ϩ i in cortical neurons from other mouse models of AD (11)(12)(13). In brain, RyRs are concentrated in postsynaptic neurons, astrocytes and oligodendrocytes, where they regulate Ca 2ϩ i levels, membrane potential, and second messenger systems. Three isoforms of RyRs have been identified as follows: type-1, which is found in skeletal muscle; type-2, which is concentrated in the heart; and type-3, which is concentrated in hippocampus (14), cerebral cortex, and corpus striatum (15). All isoforms exist in brain, where RyR-2 is the most abundant, but previous studies give no indication as to which isoform is * This work was supported in part by the Manitoba Health Research Council, an industry award from the Alzheimer Society of Canada, Canadian Institute for Health Research, and AstraZeneca. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1  important in the dysregulation of Ca 2ϩ i homeostasis in AD neurons.
The way A␤ regulates the expression and function of the machinery that mediates disruption of Ca 2ϩ i homeostasis in AD remains unclear. Here we report that high levels of A␤-(1-42) selectively elevated RyR-3 mRNA and protein levels in vitro and in vivo. Functionally, increased RyR-3 facilitated significant increases in Ca 2ϩ i levels following ryanodine or glutamate treatment. These observations highlight a possible role for RyR-3 in the AD pathology.

EXPERIMENTAL PROCEDURES
Animals and Neuronal Cultures-APP695 (KM670/ 671NLϩV717F) transgenic (Tg) CRND8 mice were a gift from Dr. David Westaway (University of Toronto) and have been described previously to exhibit A␤ plaques and cognitive defects by 3 months of age (16). Timed pregnant C57Bl6 mice were obtained from the animal housing facility at the University of Manitoba. Cerebral cortices were dissected from embryonic day 16 C57Bl6 or CRND8 (non-Tg or Tg) fetuses and used for primary culture of cortical neurons as described previously (10). CRND8 fetuses were genotyped for the APP695 double mutation by real time PCR (Platinum TaqDNA polymerase; Invitrogen) before cell dissociation and tissue pooling. The sequences of primers used for double mutant APP695 were as follows: 5Ј TGT CCA AGA TGC AGC AGA ACG GCT ACG AAAA and 3Ј AGA AAT GAA GAA ACG CCA AGC GCC GTG ACT. PCR products were generated by 30 cycles of 94/68/ 72°C. PCR product levels were determined during each cycle by SYBR-Green I (1/10,000 dilution; Molecular Probes, Eugene, OR) using iCycler iQ real time PCR detection system (Bio-Rad). Non-Tg and TgCRND8 cortical tissues were pooled separately and seeded at 8 ϫ 10 5 cells per well onto poly-D-lysine (Sigma)coated 6-well culture plates, 6.5 ϫ 10 5 cells per 40-mm glass coverslips for Ca 2ϩ imaging experiments, or 8 ϫ 10 5 cells per 25-mm glass coverslips for immunofluorescence assay. CRND8 cortical neurons were maintained as above. Experiments were performed on C57Bl6 primary cortical neurons following 8 days of in vitro culture. Experiments conducted on non-Tg or TgCRND8 cultures were completed following 5 days of in vitro culture to ensure high viability of Tg neurons. Cultures were ϳ80% MAP-2 (Chemicon, Sunnyville, CA)-positive neurons and 20% were glial fibrillary acidic protein (Abcam, Cambridge, MA)-positive astrocytes by immunofluorescence assay (data not shown). Reverse Transcription-PCR-Total RNA was purified from 8 ϫ 10 5 neurons or a small cortical section of the right hemisphere of the brain (ϳ10 mg) from non-Tg and TgCRND8 mice using the GenElute Total Mammalian RNA isolation kit (Sigma). RT-PCR amplification of RyR-1 and RyR-3 mRNA was conducted as outlined previously (10). PCR amplification of RyR-2 mRNA was conducted using primers 5Ј GAA TCA GTG AGT TAC TGG GCA TGG and 3Ј CTG GTC TCT GAG TTC TCC AAA AGC, yielding a product of 650 bp. RyR-2 PCR products were generated by 35 cycles of 94/53/72°C. IP 3 R-1 levels were determined using 5Ј TCA TGG AAA GCA GAC ACG AT and 3Ј CCG CTC TGT GGT GTA ATA TA yielding a PCR product of 404 bp, generated by 30 cycles of 94/54/72°C. SERCA-2b levels were determined using 5Ј GGG AGT GGG GCA GTG GCA GC and 3Ј CGT CTC TCT GGG CTG AGG GG yielding a PCR product of 350 bp, generated by 30 cycles of 94/62/72°C. ␤-Actin levels were determined using 5Ј ATG GAT GAC GAT ATC GCT GC and 3Ј CGT ACA TGG CTG GGG TGT TG primers that yielded a PCR product of 399 bp, generated by 25 cycles of 94/53/72°C. Alignment analysis of each primer was conducted to ensure that they were gene-and isoform-specific. Densitometric analysis of RT-PCR products was performed using NIH Image software, version 1.60. RyR pixel density levels were normalized to ␤-actin levels and are depicted as fold changes, where the untreated condition is considered 1-fold.
Western Blot Analysis-Non-Tg and TgCRND8 brains were harvested from 4-to 4.5-month-old mice (16 -18 weeks) sacrificed by intraperitoneal injection of 50 mg/kg pentobarbital (Bimeda-MTC Animal Health Inc., Cambridge, Ontario, Canada) followed by decapitation. Whole brains were dissected on ice into cerebellar, cortical, hippocampal, and basal regions (the remainder of tissue after the removal of the cortex, hippocampus, and brain stem). Tissue was immediately homogenized on ice with 400 l of lysis buffer containing 25 mM Tris, 50 mM HEPES, pH 7.4, 137 mM NaCl, 1% CHAPS, 2.5 mM dithiothreitol (Fisher) and protease inhibitor mixture (Roche Applied Science). Samples were incubated on ice for 1 h and then centrifuged at 950 ϫ g to pellet insoluble proteins. 125 go f supernatant was loaded onto a 3-8% Tris acetate NuPAGE gradient gel (Invitrogen) and transferred under reducing conditions onto 0.4 m polyvinylidene difluoride membranes (Fisher) on ice for 24 h at 20 V. Membranes were blocked with 5% nonfat milk in TBS for1hatroom temperature and incubated in blocking buffer with 1/1000 dilution of RyR-1, -2, or -3 antibodies overnight at 4°C (isoform-specific RyR rabbit polyclonal antibodies were a gift from Dr. V. Sorrentino, University of Siena, Italy). The same membranes were probed for actin (Santa Cruz Biotechnology, Santa Cruz, CA). RyR pixel density levels were normalized to actin levels and are depicted as fold changes, where the untreated condition is considered 1-fold.
Small Interfering RNA (siRNA) Design and Delivery-siRNAs were generated using the Dicer siRNA generation kit (Genelantis, San Diego) and were based on the rabbit RyR-3 mRNA sequence (GenBank TM accession number X68650). The region of the RyR-3 mRNA chosen showed 88% alignment with mouse RyR-3, 9.6% with mouse RyR-2, and 0% with mouse RyR-1 after BLAST analysis. Primers used to amplify RyR-3 from plasmid for diced siRNA generation were specific to mouse and rabbit RyR-3 as determined by alignment analysis. Tg neurons seeded at 8 ϫ 10 5 per well in 6-well plates were transfected with 200 ng of siRyR-3 or nonspecific control siRNA (Qiagen, Mississauga, Ontario, Canada) using 3 l of GeneSilencer siRNA transfection reagent (Genelantis) per ml of conditioned media for 24 h. Gene silencing was confirmed 72 h post-transfection by RT-PCR and Western blot analysis as described previously. Transfection efficiency was determined using 1 g of AlexaFluor 546-labeled nonspecific siRNA (Qiagen) and calculated as the number of nuclei localized with siRNA Ϭ total number of nuclei ϫ 100%.
Measurement of Free Intracellular Calcium-Free intracellular Ca 2ϩ (Ca 2ϩ i ) levels were quantified using fluorescence ratio microscopy as described elsewhere (10), using the Ca 2ϩ indicator dye Fura-2 (Molecular Probes). Fura-2 bound to Ca 2ϩ , an indication of free Ca 2ϩ levels, and was excited at 340 nm, and unbound Fura-2 was excited at 380 nm. Ca 2ϩ i levels are depicted as a ratio of 340/380 nm emission at 510 nm. Briefly, 6 ϫ 10 5 cells were loaded for 30 min with 1 g/ml of acetoxymethyl ester (AM) Fura-2 in Locke's buffer. Cells were imaged on an inverted Zeiss microscope using a CoolSnapHQ (Roper Scientific Inc., Trenton, NJ) camera with a 40ϫ oil objective lens. Changes in Ca 2ϩ i levels were determined using Stallion Imaging software (Downingtown, PA). L-(ϩ)-glutamic acid (EM Science, Gibbstown, NJ) was converted to 1 M stock of glutamate by addition of 10 N NaOH (Fisher) to pH 7.2. Ryanodine (Sigma) was prepared as a stock solution of 50 mM in 100% Me 2 SO (Sigma). Treatments were delivered to neurons by perfusion. Experiments were performed in nominal extracellular Ca 2ϩ (Locke's buffer without CaCl 2 ). Each experiment was repeated at least three separate times, 15-30 cells imaged at each time. Of the cells imaged, data were pooled from cells that responded to both glutamate and ryanodine.
Statistical Analysis-Densitometric analysis was performed using NIH Scion Image software, version 1.60. Data were ana-lyzed using GraphPad Prism, version 3.02 (San Diego, CA). One-way ANOVA with Student-Newman-Keuls post-test was performed to determine the difference between means after treatment. Two-way ANOVA with Bonferroni post-test was used to determine the difference between genotypes and treatments and the possible interactions of each. Differences were considered significant at p Ͻ 0.05.

A␤-induced RyR-3 mRNA Expression in Primary Cortical
Neurons-To test the hypothesis that A␤ directly increased RyR mRNA levels, C57Bl6 cortical neurons were treated with A␤-(1-42) peptide (1000 nM) for 18 h and analyzed for RyR mRNA levels (Fig. 1A). As determined by RT-PCR, C57Bl6 primary cortical cells incubated with 1000 nM A␤-(1-42)  (Fig. 1A). Furthermore, identical treatments did not alter RyR-2, IP 3 R-1, or SERCA-2b mRNA levels (not shown). To rule out the involvement of rapid increases in Ca 2ϩ i in the up-regulation of RyR gene expression, we treated C57Bl6 primary cortical neurons with an ionophore, ionomycin, for 30 min. The treatment was removed and replaced with conditioned media for 18 h at 37°C. Consistent with the observations made in a previous study (18), we did not see an induction of RyR gene expression after treatment with ionomycin (n ϭ 2). Cell viability was unaltered by a 30-min treatment with ionomycin as assessed 18 h after treatment by trypan blue exclusion assay (data not shown).
RyR-3 Transcription and Protein Levels Are Increased in TgCRND8 Mice-Because A␤ increased RyR gene expression in primary cortical neurons, we hypothesized that RyR-3 expression is elevated in TgCRND8 mice, a mouse model of Alzheimer disease that produces high levels of A␤ and expresses early cognitive decline (16). Examination of cerebrum (forebrain) from the right hemisphere of 4 -4.5-monthold TgCRND8 animals showed that RyR-3 mRNA levels were ϳ2.6 Ϯ 0.43-fold S.E. higher (n ϭ 16, p Ͻ 0.01, two-way ANOVA, Bonferroni post-test) compared with their non-Tg littermates (Fig. 1B). There was no change in RyR-1 and RyR-2 ( Fig. 1B)orinIP 3 R-1 and SERCA-2b mRNA levels (not shown). To determine where in TgCRND8 brain the increases in RyR were located, lysates of different brain regions dissected from 4to 4.5-month-old CRND8 mice were analyzed for RyR protein by Western blot analysis using isoform-specific antibodies (17). Similar to mRNA levels, RyR-3 levels were increased ϳ2.7 Ϯ 0.42-fold S.E. (n ϭ 14, p Ͻ 0.001, two-way ANOVA, Bonferroni post-test) in the cortex of Tg mice as compared with non-Tg, whereas RyR-1 and RyR-2 levels were unchanged (Fig. 1C). RyR-3 levels were increased 1.9 Ϯ 0.26-fold S.E. in the hippocampus as well (n ϭ 14, p Ͻ 0.01, two-way ANOVA, Bonferroni post-test) but were unchanged in the basal and cerebellar regions (Fig. 1D). Our statistical tests reveal that these differences in RyR-3 expression are attributed to the genotype (p Ͻ 0.0001) and specific to the brain region (p ϭ 0.0011).
Targeting RyR-3 by Gene Silencing Techniques-It was reported recently that RyR levels are increased in other models of AD, and as a result neurons from transgenic mice have elevated responses to 20 -25 mM caffeine (12,13). Given our observations showing increased RyR-3 levels in TgCRND8 neurons, we wanted to determine whether RyR-3 was involved in the elevated response. Because of a lack of pharmacological tools to selectively block RyR-3, we opted for a molecular approach to target RyR-3 and depress its expression in Tg neurons prior to Ca 2ϩ imaging studies. Diced siRNAs selective for RyR-3 were generated to span a 613-bp section of the mRNA and knock down RyR-3 levels. 200 ng of siRyR-3 was transfected into Tg neurons with GeneSilencer reagent (GSR) for 24 h. At 72 h post-transfection, RyR-3 levels were significantly decreased by 2.9 Ϯ 0.47-fold S.E. compared with untreated (n ϭ 4, p Ͻ 0.001, two-way ANOVA, Bonferroni post-test) and were comparable with non-Tg neurons (Fig. 3A), whereas RyR-1 and RyR-2 levels did not change (fold changes not shown). Tg neurons treated with GSR, siRyR-3 alone, nonspecific siRNA alone (200 ng), and siRNA with GSR did not show significant changes in RyR-1, -2 (not shown), or -3 (Fig.  3A) mRNA levels. To determine the transfection efficiency of Calcium Released from ER Stores Is Increased in TgCRND8 Neurons Expressing Elevated Levels of RyR-3-To determine some of the functional consequences of increased levels of RyR-3 in TgCRND8 mice, we measured Ca 2ϩ i levels of neurons after treatments of glutamate and ryanodine. Because of our interest in the contribution of ER Ca 2ϩ stores to the overall Ca 2ϩ i response, experiments were conducted in the presence of nominal extracellular Ca 2ϩ to minimize the contribution of Ca 2ϩ influx. Increased Ca 2ϩ i because of glutamate is closely associated with neurotoxicity (19 -23) and with alterations in AD neurophysiology (24); therefore, we sought to determine whether Tg neurons had altered Ca 2ϩ i responses because of increased RyR-3. TgCRND8 neurons at in vitro day 5 demonstrated an elevated Ca 2ϩ i response to 250 M glutamate treatment as compared with non-Tg littermate controls (Fig.  4A), with a peak response of 0.67 Ϯ 0.01 S.E. Fura-2 fluorescence ratio 340/380 nM (n ϭ 15, p Ͻ 0.001, oneway ANOVA, Student-Newman-Keuls post-test) for Tg neurons was in contrast to 0.55 Ϯ 0.03 S.E. 340/ 380 nM for non-Tg (n ϭ 18) (Fig.  4B). To determine the contribution of RyR-3 to the observed increases in Ca 2ϩ i levels, Tg neurons were treated with siRyR-3 for 72 h prior to Ca 2ϩ imaging experiments. Knockdown of RyR-3 levels resulted in a decreased Ca 2ϩ response of Tg neurons to glutamate (Fig. 4A), with a peak response of 0.52 Ϯ 0.02 S.E. ratio 340/380 nM (Fig. 4B) that was significantly lower than non-Tg (0.55 Ϯ 0.06 S.E., n ϭ 15, p Ͻ 0.05, one-way ANOVA, Student-Newman-Keuls post-test).
To determine the direct contribution of RyR-gated Ca 2ϩ stores to Ca 2ϩ i levels, we performed experiments using an agonistic concentration (100 nM) of ryanodine (Ry) in the presence of nominal extracellular Ca 2ϩ . After treatment of glutamate, ER stores become charged with Ca 2ϩ and are then available for Ca 2ϩ liberation (25). TgCRND8 neurons had increased Ca 2ϩ i levels after 100 nM Ry treatment (Fig. 4A), with a peak response of 0.50 Ϯ 0.01 S.E. ratio 340/380 nM (n ϭ 15, p Ͻ 0.001, one-way ANOVA, Student-Newman-Keuls post-test) as compared with littermate controls (Fig. 4C). siRyR-3 treatment of Tg neurons prior to the experiment reduced the Ca 2ϩ i response to Ry (Fig.  4A) to peak levels that were not significantly different from littermate controls (Fig. 4C).
To determine the role of ER Ca 2ϩ load, we calculated the area under the curve (AUC) of our Fura-2 traces (Fig. 4, D and E). AUC calculations, or the ratio of 340/380 nM multiplied by time and depicted in arbitrary units, represent the total amount of Ca 2ϩ being liberated from the ER after glutamate or Ry treatment. In both instances, Tg neurons had higher levels of ER Ca 2ϩ released after agonist treatment (n ϭ 15, p Ͻ 0.01, p Ͻ Neurons cultured for 4 days were treated with 1000 nM A␤ peptides as before (Fig. 1A). Cells were collected on in vitro day 5. Non-Tg (N) are littermate controls, and Tg (T) neurons are heterozygous for the APP695 double mutation. Values are the mean Ϯ S.E. of determinations made in samples from three cultures. Tg values were compared with littermate controls (non-Tg) at all treatments (ns indicates no statistical difference; ***, p Ͻ 0.001), and A␤-treated non-Tg values were compared with untreated (u) non-Tg (###,pϽ 0.001, two-way ANOVA with Bonferroni post-test). B, representative agarose gel showing fold changes of RyR-3 RT-PCR products from 4-day-old cultures treated with 1.3 g/ml A␤-(1-42) antibody or 1.3 g/ml gp-116 antibody (nonspecific monoclonal antibody control) for 18 h at 37°C before analysis (cells collected at in vitro day 5). Values are the mean Ϯ S.E. of determinations made in samples from three cultures. Tg values were compared with littermate controls (non-Tg) at all treatments (ns indicates no statistical difference; ***,pϽ 0.001) and anti-A␤treated Tg values were compared with untreated Tg (###,pϽ 0.001, two-way ANOVA with Bonferroni posttest). C, immunofluorescence assay for RyR-1, RyR-2, and RyR-3 protein in non-Tg and Tg primary cortical neurons is representative of three separate cultures. Non-Tg neurons are treated with 1000 nM A␤-(1-42) peptide as before (Fig. 1A). Cells were imaged at 400ϫ magnification.

DISCUSSION
Several studies now clearly implicate a role for RyRs in the development and progression of Alzheimer disease (10 -13). Findings here have implicated RyR-3 as a key player in altering Ca 2ϩ homeostasis of neurons in response to A␤. We have come to this conclusion based on the following evidence. First, our in vitro studies show that A␤- , regardless of the source of peptide (endogenously produced or synthetic), increased levels of RyR-3. As a direct consequence, Tg neurons had exaggerated Ca 2ϩ i responses to ryanodine and to glutamate, which can trigger CICR from RyR stores (4, 26 -29). Second, after knockdown of RyR-3 levels by siRNA, Ca 2ϩ imaging experiments confirmed that the RyR-3 pools represented the majority of the glutamate-induced increase in Ca 2ϩ in Tg neurons. Finally, RyR-3 levels were increased in vivo in the cortex and hippocampus, areas of the brain affected in AD, and suggest an important role for RyR-3 in disease progression.
It is well documented that alterations in Ca 2ϩ homeostasis occur early in AD, before profound neuronal damage and cell death (4,30). These changes in Ca 2ϩ i levels appear to correlate more closely with cognitive decline compared with the formation of A␤-containing senile plaques, the pathological hallmark of AD (31). It is interesting to note that TgCRND8 mice begin to display cognitive defects in spatial learning tasks at 19 -22 weeks of age, where their ability to acquire new information is impaired (32). Of relevance to our study, we observed an increase in RyR-3 levels and Ca 2ϩ disruption within this time frame.
The impact of dysregulated Ca 2ϩ homeostasis as a result of increased RyR-3 in the in AD is still unclear. In fact, the exact function of RyR-3 in brain has yet to be determined, although their relative abundance in neurons of the hippocampus suggests a role for RyR-3 in hippocampal Ca 2ϩ regulation (14). The generation of RyR-3 knock-out mice has given some insight into its function. Two independent groups have generated RyR-3 knock-out mice, and each group performed electrophysiological and behavioral studies to determine the role of RyR-3 in synaptic plasticity and hippocampus-dependent learning (33,34). Both groups demonstrated evidence of increased long term potentiation (LTP), a physiological corre- late for learning and memory, after particular types of stimulation, namely short or weak tetanus, suggesting a role for RyR-3 in the depression of LTP and long term depression (LTD). However, after subjecting the RyR-3-deficient mice to the Morris water maze to assess spatial learning competencies, the two groups came to contradictory conclusions. Although Futatsugi et al. (34) showed that mutant mice had enhanced spatial learning, which would be expected if RyR-3 mediated the depression of LTP, Balschun et al. (33) observed that RyR-3 knock outs had significant deficiencies in their ability to acquire new information compared with mice with wild-type RyR. Interestingly, it has been shown that RyR-mediated Ca 2ϩ release from postsynaptic CA1 (35,36) or pre-synaptic CA3 (35,37) neurons is required for the induction of LTD. Of the three different RyR isoforms, RyR-3 would be the likely candidate for the fine-tuning of these synaptic changes. RyR-3 is less sensitive to Ca 2ϩ in comparison to RyR-1 and RyR-2 and therefore requires higher cytosolic Ca 2ϩ levels to be activated and deactivated (38). Taken together, these observations suggest that RyR-3 may contribute to synaptic plasticity by modulating the balance between LTP and LTD, and the up-regulation of RyR-3 could be a response to an increase in neuronal excitability.
Such a mechanism could be at play in AD, and the up-regulation of RyR-3 might be an effort mounted by neurons to protect against the increased excitability brought about by the pathology of AD, specifically A␤ accumulation. Other aspects of Ca 2ϩ i signaling in AD neurons are also enhanced, in particular N-methyl-D-aspartic acid receptor (39) and IP 3 R-mediated signaling (40), leading to increased excitotoxicity. In fact, Stutzmann et al. (12) showed that RyR Ca 2ϩ stores were directly responsible for the exaggerated hyperpolarization responses of PS1 mutant neurons to IP 3 and therefore impacted membrane excitability. Furthermore, RyR-3 mRNA was transiently increased in rat brain after kainic acid-induced status elipticus (41), implicating a role in modulating excitability. Higher levels of RyRs could be considered advantageous in protecting against putative excitotoxicity in AD, but the implications of chronic up-regulation of RyR-3, which could occur as long as A␤ is being produced, might lead to the opposite effect. As the balance of excitation and depression are skewed, the resulting effect could be an increase to overall Ca 2ϩ i levels, oxidative stress, and activation of pathways leading to toxicity and neurodegeneration (10,11,41). Further studies are required to resolve why RyR-3 is being up-regulated so significantly and specifically in TgCRND8 mice.
The results of our study describe a mechanism by which A␤ contributes to the dysregulation of intraneuronal Ca 2ϩ homeostasis by up-regulating the expression and function of a specific ER Ca 2ϩ release channel, RyR-3. There is much evidence to suggest that RyR Ca 2ϩ pools play an important role in the neuronal dysfunction initiated by A␤ deposition in AD (10 -13). Our findings further focus on RyRs and implicate RyR-3 as the primary ER channel that facilitates Ca 2ϩ disruption in AD. This information could be significant in understanding the neurobiology of AD pathology. RyR(s) may play a role in normal aging as well as contribute to AD pathology. In a report by Lu et al. (42), RyR-1 mRNA was increased significantly in aged human cortical tissue from individuals that were not demented, which suggests that increased RyRs may have a protective or positive role to play in neuronal function. Furthermore, it has been shown recently that RyR-3 mRNA and protein levels are increased in the superior cervical ganglia of aged rats and may serve to protect neurons from oxidative stress by modulating and increasing the protein expression of neuronal nitric-oxide synthase (43). A clearer picture of how RyRs are involved in balancing normal and pathological aging is required before they can be considered a feasible therapeutic target.