Neuronal Ca2?signaling through inositol triphosphate receptors (IP3R) and ryanodine receptors (RyRs) must be tightly regulated to
maintain cell viability, both acutely and over a lifetime. Exaggerated intracellular Ca2?levels have been associated with expression of
Alzheimer’s disease (AD) mutations in young mice, but little is known of Ca2?dysregulations during normal and pathological aging
processes. Here, we used electrophysiological recordings with two-photon imaging to study Ca2?signaling in nontransgenic (NonTg)
mice were not different from controls. In addition, we uncovered powerful signaling interactions and differences between IP3R- and
RyR-mediated Ca2?components in NonTg and AD mice. In NonTg mice, RyR contributed modestly to IP3-evoked Ca2?, whereas the
RyR expression across all ages. Moreover, IP3-evoked membrane hyperpolarizations in AD mice were even greater than expected from
exaggerated Ca2?signals, suggesting increased coupling efficiency between cytosolic [Ca2?] and K?channel regulation. We conclude
that lifelong ER Ca2?disruptions in AD are related to a modulation of RyR signaling associated with PS1 mutations and represent a
Neuronal Ca2?signaling is tightly controlled to ensure proper
al., 1998, 2000). Two major sources contribute to cytosolic Ca2?
channels and an internal reservoir in the ER liberated through
of Ca2?-induced Ca2?release (CICR), which enables interac-
tions between these pathways (Finch et al., 1991; Friel and Tsien,
1992; Fagni et al., 2000).
Growing evidence implicates Ca2?signaling disruptions in
the etiology of neurological diseases (Mattson et al., 2000;
LaFerla, 2002; Stutzmann, 2005). In particular, presenilin (PS)
in cell-based models (Guo et al., 1996; Leissring et al., 1999) and
used mutant PS1 knock-in mice, which despite their failure to
develop the hallmark ?-amyloid (A?) plaques and neurofibril-
lary tangles, nonetheless, have provided valuable insights into
the 3xTg-AD mice [expressing mutant PS1, amyloid ? protein
precursor (APP), and tau], which develop both plaques and tan-
gles in an age- and region-dependent manner (Oddo et al.,
2003a,b), enable us to compare neuronal Ca2?signaling in con-
trol, PS1KI, and 3xTg-AD mice at varying ages to determine the
age progression and respective contributions of the different AD
mutations. Our results indicate that the PS1 mutation is the pre-
dominant determinant underlying the exaggerated IP3-evoked
Ca2?signals at all ages.
Most studies of AD Ca2?disruptions focused on responses
or indirectly by agonist application (Guo et al., 1996; Etcheberri-
garay et al., 1998), and did not explicitly address the role of RyR.
expression levels are increased in cultured neurons expressing
mutant PS1 (Chan et al., 2000; Smith et al., 2005b), the RyR
PS1 (Popescu et al., 2004), and the RyR agonist caffeine evokes
els (Smith et al., 2005b). Moreover, we observed elevated RyR
Correspondence should be addressed to Grace E. Stutzmann, Rosalind Franklin University of Medicine
and Science, The Chicago Medical School, 3333 Green Bay Road, North Chicago, IL 60064. E-mail:
5180 • TheJournalofNeuroscience,May10,2006 • 26(19):5180–5189
levels throughout the lifetime of the PS1KIand 3xTg-AD mice,
paralleling the pattern of enhanced IP3-evoked ER Ca2?release.
We thus explored RyR function in both normal neuronal
physiology and during Ca2?signaling disruptions associated
with AD. We show that RyR activation contributes modestly to
Ca2?signals in NonTg mice but accounts for most of the exag-
and old ages. Moreover, in all groups, IP3-dependent membrane
hyperpolarizations are regulated primarily through RyR, and the
transgenic mice show hyperpolarizing responses even greater
than expected from the enhanced Ca2?signals.
Transgenic mice and slice preparation. The derivation and characteriza-
tion of the PS1KIand 3xTg-AD mice have been described previously
(Guo et al., 1999; Oddo et al., 2003a). NonTg mice were of the same
other homozygous 129/B6 mice to minimize strain differences and are
considered excitotoxic-resistant strains, thereby reducing confounding
differences. Brain slices were prepared as described previously (Stutz-
sity of California Irvine Institutional Animal Care and Use Committee.
Whole-cell patch-clamp recording was performed using an infrared/dif-
CSF (in mM: 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 10 D-glucose, 25
NaHCO3, 2 CaCl2, 1.2 MgSO4, pH 7.3–7.4) bubbled with 95% O2/5%
CO2at room temperature (22–24°C). Patch pipettes (4–5 M?) were
filled with intracellular solution containing the following (in mM): 135
K-methlysulfonate, 10 HEPES, 10 Na-phosphocreatine, 2 MgCl2, 4
potentials were recorded in current-clamp mode acquired at 1 kHz with
an Axopatch 1C amplifier and analyzed using pClamp 8.1 (Molecular
Devices, Union City, CA). Depolarizing current injections (0.1–0.2 nA)
for 500 ms were used to evoke trains of five to seven action potentials.
Ca2?imaging and flash photolysis. Imaging was performed using a
custom-made video-rate two-photon microscope, as described previ-
2005). In brief, excitation was provided by trains (80 MHz) of ?100 fs
pulses at 780 nm from a Ti:sapphire laser (Tsunami; Spectra-Physics,
Mountain View, CA). The laser beam was scanned at 30 fps and focused
through a 40? water-immersion objective (numerical aperture, 0.8).
Emitted fluorescence light was detected by a wide-field photomultiplier
to derive a video signal that was captured and analyzed by the Meta-
of fura-2 fluorescence are expressed as inverse pseudoratios so that in-
was accomplished by flashes of UV light (340–400 nm) derived from a
100 W Hg arc lamp coupled to an electronically controlled shutter
(Uniblitz). The irradiance at the specimen was ?50 mW/mm2, focused
as a uniform circle (radius, ?50 ?m) centered on the imaging field.
Stimulus strength was regulated by the flash duration. On the basis of
previous calibration (Parker and Ivorra, 1992), a flash of 10 ms duration
would photolyse ?4% of the total caged IP3.
Immunoblot analysis. Whole-brain homogenates were prepared from
4- to 6-week-old (NonTg), PS1M146Vknock-in (PS1KI), and triple trans-
genic (3xTg-AD) mice. Detailed Western blot methodology has been
described previously (Smith et al., 2005b). Protein extracts were moni-
tored by quantitative immunoblotting. Three to four samples were ana-
lyzed for each group, and all samples were normalized to ?-actin levels.
Antibody sources and dilutions were as follows: anti-calsenilin, 1:100
(Zymed Laboratories, San Francisco, CA); anti-calbindin, 1:5000
(Sigma, St. Louis, MO); anti-SERCA 2b, 1:45,000 and anti-RyR, 1:1000
from Sigma (anti-rabbit, 1:20,000; anti-mouse, 1:50,000).
Data analysis. Data are presented as mean ? 1 SEM, where n is the
number of neurons examined. Student’s t tests were performed as a
pared, and a one-way ANOVA was used to compare the samples. Schef-
fe ´’s post hoc analysis was subsequently performed on ANOVA results to
Two lines of transgenic mice expressing human AD-linked mu-
and a nontransgenic control (NonTg) on the same background
strain were used to examine the relationship between aging and
AD mutations on neuronal Ca2?signaling and the respective
contributions of IP3R and RyR to the Ca2?signals. Three age
points were selected (6 weeks, 6 months ? 1 month, and 1.5
years ? 3 months) based, respectively, on previous studies of
early A? pathology, and extensive subsequent plaque and tangle
by backcrossing the 3xTg-AD mice to NonTg mice, it was possi-
ble to replace the mutant PS1 allele with the mouse wild-type
version, essentially generating double transgenic mouse express-
ing mutant APP and tau (APPTau mice); these mice were evalu-
ated in this study at the 6-week time point to assess the relative
contribution of the PS1 mutation to the overall Ca2?dysregula-
tion. Lack of older mice precluded age-dependent comparisons.
Pyramidal neurons from the prefrontal cortex were chosen for
Rakic, 1995). Passive membrane properties of membrane poten-
group are listed in Table 1. No significant differences were ob-
different ages ( p ? 0.05).
Neurons were loaded with caged IP3and fura-2 by dialysis
through the patch pipette, and flashes of UV light of varying
durations were applied to photorelease IP3. The resulting libera-
tion of Ca2?from the ER was monitored by imaging fura-2 flu-
orescence in the soma (excluding the nucleus) and by measuring
changes in membrane potential resulting from activation of
Ca2?-dependent K?channels (Sah, 1996; Stutzmann et al.,
2003). In addition, depolarizing current pulses were applied to
evoke action potentials and accompanying entry of Ca2?
through voltage-gated Ca2?channels (VGCCs).
Stutzmannetal.•Ca2?inNormalAgingandAlzheimer’sDiseaseJ.Neurosci.,May10,2006 • 26(19):5180–5189 • 5181
Ca2?signals in representative neurons
PS1KI, and 3xTg-AD mice are illustrated
in Figure 1, demonstrating some of the
key similarities and differences among
controls (Fig. 1A) and the APPTau neu-
rons (Fig. 1B), photoreleased IP3consis-
tently evoked larger responses in the
PS1KI(Fig. 1C) and 3xTg-AD (Fig. 1D)
neurons. In contrast, Ca2?signals dur-
ing action potential trains were similar
across all three groups (Fig. 1E–H).
We quantified measurements of somatic
amounts of photoreleased IP3(linearly
proportional to photolysis flash duration)
in neurons from the different transgenic
animals at different ages. For clarity, we
present these data arranged in two ways:
grouped according to age (Fig. 2A–D, left
column) and according to transgene (Fig.
2E–H, right column). The graphs also in-
clude measurements of Ca2?signals
evoked in the soma by trains of action po-
ages, the data from 6-week-old mice
(Fig. 2A) confirm our previous findings
neurons showed greatly (twofold to
threefold) exaggerated IP3-evoked Ca2?
signals at all flash durations compared
with NonTg controls. Most importantly,
we now show that mean Ca2?signals in
the 3xTg-AD neurons were essentially
indistinguishable from those in the
PS1KIneurons, whereas signals in the
APPTau neurons were no greater than in
controls (Fig. 2D). At this young age, we
thus conclude that the AD-linked mutation
that mutant forms of APP and tau do not
affect Ca2?signals when coexpressed either
Neurons from 6-month-old NonTg
mice (Fig. 2B) showed no appreciable differences in IP3-
evoked Ca2?signals compared with 6-week-old NonTg mice.
In contrast, signals in both the PS1KIand 3xTg-AD neurons
were uniformly smaller than at 6 weeks, so that the exaggera-
tion of Ca2?signals by these mutants was appreciably dimin-
ished at this age. A significant difference remained, however,
flash durations (Fig. 2D).
In the 1.5-year-old animals, the IP3-evoked Ca2?signals in
NonTg neurons were reduced (to ?60%) versus the 6 week
and 6 month age groups (Fig. 2C,D). Maximal signals in the
PS1KIanimals were also reduced ?34% compared with the 6
week age group (Fig. 2C,D), although the 3xTg-AD neurons
showed amplitudes that had recovered close to that in the 6
week group (Fig. 2C,D). Thus, expression of mutant PS1KI
again resulted in a large exaggeration of Ca2?signals in 1.5-
year-old mice, although this effect was muted at 6 months.
Summarizing next the results from the perspective of dif-
ferences among the transgenes (Fig. 2E–H), the principal
finding is that IP3-evoked Ca2?signals in PS1KIand 3xTg-AD
5182 • J.Neurosci.,May10,2006 • 26(19):5180–5189 Stutzmannetal.•Ca2?inNormalAgingandAlzheimer’sDisease
neurons were exaggerated compared with NonTg controls
across all ages examined, but this exaggeration was most
marked in 6-week-old animals, reduced appreciably at 6
months, and recovered substantially at 1.5 years of age. To test
for statistical significance for age- and transgene-related
changes in IP3-evoked Ca2?signals and possible interactions
between these factors, we performed a two-way ANOVA on
pooled responses evoked by strong (100 and 200 ms) photol-
ysis flashes. There was no overall effect of age as a variable
when the different transgenic groups were collapsed together
( p ? 0.05); however, there was a significant main effect of
transgene (F(2,104)? 8.05; p ? 0.05) with the PS1KIand
3xTg-AD mice having significantly larger Ca2?signals than
the NonTg mice. There was no significant interaction between
age and transgene.
Ca2?signals evoked by spike trains showed no appreciable
C,E–G). Thus, it appears that the age- and transgene-related
changes in IP3-evoked Ca2?signals are specific to ER Ca2?sig-
naling and do not reflect, for example, changes in cytosolic Ca2?
fluorescence measurements were not different in older mice re-
gardless of transgene expression ( p ? 0.05; data not shown).
Decay rates of the spike and IP3-evoked Ca2?transients were
from the cytosol. The time constants of exponential fits to the
0.05; data not shown).
Cytosolic Ca2?signals play a major role in neuronal function by
activating K?channels that modulate membrane excitability
(Sah, 1996). To explore age- and transgene-related changes in
Ca2?-dependent K?channel activation, we first measured the
afterhyperpolarization (AHP) after trains of six to nine action
potentials generated by a depolarizing current pulse (0.2 nA; 500
ms). The peak AHP occurred ?200 ms after the termination of
the pulse (Fig. 3A), approximately corresponding to the time
course of the medium-IAHPresulting from Ca2?entry through
voltage-gated plasma membrane Ca2?channels (Sah, 1996).
Within each age group, the presence or absence of AD-related
alleles had no significant effect on the AHP amplitude (Fig. 3B)
( p ? 0.05). However, as the mice aged, the AHP amplitude in-
both control and transgenic animals.
A different pattern emerged for membrane potential hyper-
C). Across all age groups, PS1KIand 3xTg-AD transgenic mice
had consistently larger IP3-evoked membrane hyperpolariza-
tions compared with the NonTg controls. However, within each
transgenic group, the amplitude of the IP3-evoked hyperpolar-
ization changed little with age.
Western blots were prepared to examine whether the enhanced
ER-Ca2?release in PS1KIand 3xTg-AD neurons may be at-
tributed to increased expression levels of Ca2?signaling-
related proteins. We found no significant differences in IP3R,
SERCA-2b, calsenilin, calbindin-D, or calreticulin levels be-
tween age-matched NonTg and AD-transgenic brains ( p ?
0.05; data not shown). However, RyR levels were significantly
increased in the PS1KIand 3xTg-AD brains relative to NonTg
controls at the 6 week group (Fig. 5A) and 1.5 year group (Fig.
5C); at 6 months, the differences were less pronounced, and at
this age, only the 3xTg-AD brains showed significantly ele-
vated RyR levels (Fig. 5B).
We obtained an independent and direct measure of Ca2?flux
through RyR channels by bath applying the RyR agonist caffeine
6A, left). The rate of rise of Ca2?in the soma of both PS1KIand
3xTg-AD neurons was appreciably greater than in NonTg con-
trols but was not significantly different between the two AD
IP3-evoked Ca2?signals in cortical neurons from NonTg (F), PS1KI(Œ), 3xTg-AD (f), and
by photolysis flashes of increasing durations and (at right) signals evoked by trains of action
potentials. B, Corresponding data from 6-month-old mice. C, Corresponding data from 1.5-
in neurons from NonTg, PS1KI, and 3xTg-AD mice). In each panel, data points show mean
ER Ca2?signaling remains elevated throughout the lifetime of PS1 mutant-
Stutzmannetal.•Ca2?inNormalAgingandAlzheimer’sDiseaseJ.Neurosci.,May10,2006 • 26(19):5180–5189 • 5183
these AD Tg groups (n ? 9), the caffeine-
evoked Ca2?rise was 2.8 times faster than
To ascertain the relative contribution
compared somatic signals evoked by pho-
toreleased IP3 before and after bath-
applying 10 ?M dantrolene to block RyR
(Xu et al., 1998). In NonTg neurons, dan-
trolene caused a modest (20 ? 7%; n ? 6)
reduction in signals evoked by 50 ms
flashes, and action potential-evoked Ca2?
signals were reduced by 15 ? 5%. In
marked contrast, dantrolene substantially
PS1KIneurons (by 59 ? 11%; n ? 6; p ?
0.01) and in 3xTg-AD neurons (by 71 ?
9%; n ? 7; p ? 0.01). However, similar to
NonTg neurons, dantrolene produced
only modest (15–20%) reductions of the
The effects of dantrolene on the dose–
in PS1KIand 3xTg-AD neurons were not
significantly different from one another
( p ? 0.05), and we therefore combined
these data (Tg) for analysis. Dantrolene
strongly suppressed Ca2?signals in the
range of flash durations tested (Fig. 6D,
circles), whereas the reduction in NonTg
6D, squares). Importantly, there were no
Tg and NonTg groups in the Ca2?signals
remaining in the presence of dantrolene,
suggesting that Ca2?flux through the
IP3R channels themselves is not appreciably enhanced by the
AD-linked mutations, but rather that larger responses in the Tg
neurons arise principally from greater CICR through RyR. In
contrast to the IP3-evoked Ca2?signals, spike-evoked Ca2?sig-
nals were reduced to a similar extent in both the NonTg and Tg
activation of Ca2?-dependent K?channels (Sah, 1996; Stutz-
mann et al., 2003), and this hyperpolarization is enhanced in
PS1KImice (Stutzmann et al., 2004). Here, we sought to deter-
mine whether the K?channel regulation primarily involves
Ca2?liberated through the IP3R channels themselves or is con-
sequent to CICR through RyR channels.
Representative membrane potential responses to photore-
7A and were appreciably smaller and of lower sensitivity in the
NonTg cells. These differences did not arise through alterations
in initial resting membrane potential (set to ?60 mV by current
injection) or input resistance (Table 1). Strikingly, all responses
were substantially abolished by dantrolene (Fig. 7A, bottom
Mean data for NonTg and Tg neurons in control and dantrolene
conditions are plotted in Figure 7B (main graph). Hyperpolariz-
ing responses in both NonTg and Tg neurons increased with
increasing photorelease of IP3but, for a given flash duration, the
responses in Tg neurons were nearly three times as large (3.04-
polarizations remained with the strongest flashes and were not
significantly different between NonTg and Tg neurons ( p ?
0.05). We were also able to replicate the effects of dantrolene by
preincubating slices in ryanodine (30 ?M) to block RyR (Fig. 7B,
IP3-evoked changes in membrane conductance strongly reg-
ulate spiking patterns, and photorelease of IP3caused a long-
lasting reduction in the numbers of action potentials evoked by
was abolished by dantrolene (Fig. 7C, bottom trace). Moreover,
similar results were obtained using ryanodine (n ? 8; data not
shown). Dantrolene did not appear to affect other membrane
5184 • J.Neurosci.,May10,2006 • 26(19):5180–5189 Stutzmannetal.•Ca2?inNormalAgingandAlzheimer’sDisease
rhyperpolarization), suggesting that that the IP3modulation of
spike frequency specifically involves RyR.
The greater IP3-evoked membrane hyperpolarization seen in
consequence of the enhanced ER Ca2?release. However, this
appears not to be the sole mechanism, because scatter graphs
plotting the relationship between IP3-evoked hyperpolarization
nals (F0/?F) revealed markedly different slopes between NonTg
and Tg neurons for both soma (Fig. 8A) and dendrite (Fig. 8B).
That is to say, a given cytosolic Ca2?signal was associated with a
larger membrane hyperpolarization in Tg neurons, suggesting
between cytosolic Ca2?signals and activation of membrane K?
conductance, as well as enhancing the Ca2?signals.
To explore the mechanism underlying this effect, we con-
structed a similar scatter plot of hyperpolarization versus Ca2?
signal amplitude after adding dantrolene to block RyR (Fig. 8C).
and dendrite to obtain sufficient data points. Regression lines
showed a slope for NonTg neurons that was not appreciably dif-
ferent from that in control conditions without dantrolene,
whereas in Tg neurons, the slope was dramatically reduced as a
result of blocking RyR. Our findings are further summarized in
Figure 8, D and E. Key points are as follows: (1) the slope of the
relationship between membrane hyperpolarization (??V) and
Ca2?(F0/?F) was steeper (5.9) in Tg than in NonTg neurons
(3.15); (2) the slope in Tg neurons was greatly reduced by dan-
trolene but was almost unchanged in NonTg neurons; (3) the
amplitudes of IP3-evoked Ca2?signals (measured from the
soma, averaged across all flash durations) in Tg neurons were
approximately double that in NonTg neurons, whereas mem-
brane hyperpolarizations were more than three times larger; and
(4) in the presence of dantrolene, there was no significant differ-
ence between Ca2?signals in Tg and NonTg neurons (Fig. 8E,
striped bars). Thus, RyRs are critically involved in mediating the
hyperpolarizing response evoked by IP3. Moreover, AD-linked
mutations appear to result in greater hyperpolarizing responses
of enhanced coupling efficiency between RyR and Ca2?-
Long-term elevations of cytosolic [Ca2?] leave neurons vulner-
able to metabolic stressors, consistent with the Ca2?hypothesis
of AD (Khachaturian, 1994; Toescu and Verkhratsky, 2003). We
examined Ca2?liberation from the ER and Ca2?influx through
voltage-gated channels, and in each case measured cytosolic
polarization following trains of action potentials nearly doubled
with age. Given that membrane potential and input resistance
were similar between groups (Table 1), this suggests an age-
dependent increase in K?channel activation underlying the in-
creased AHP. It seems unlikely that the enhanced K?current
results from increased K?channel density, because IP3-evoked
hyperpolarizations did not increase with age. An alternative
mechanism, as proposed in hippocampal neurons, is that the
density of VGCCs coupled to K?currents increases with age
(Landfield and Pitler, 1984; Landfield, 1996; Thibault and Land-
field, 1996). Although we did not find any concomitant increase
possible that the fluorescence signals do not reflect the Ca2?
entry that activates the AHP; for example, the K?channels may
be located predominantly in the dendrites (Sah and Bekkers,
1996), whereas our measurements were confined to the soma.
The results with PS1KImice replicate our previous findings
tiation of IP3-evoked Ca2?signals associated with PS mutations
we now demonstrate that PS1 is solely involved in causing the
mice showed Ca2?signals that were similar to the PS1KImice,
density relative to ?-actin expression for NonTg (left), PS1KI(middle), and 3xTg-AD (right)
Expression levels of brain RyR protein at different ages and across transgenic
Stutzmannetal.•Ca2?inNormalAgingandAlzheimer’sDisease J.Neurosci.,May10,2006 • 26(19):5180–5189 • 5185
whereas mice expressing mutant APP and
Tau displayed signals no greater than
NonTg controls. Thus, it appears that the
presence of APP and Tau mutations does
not disrupt neuronal Ca2?signals. More-
over, the appearance of overt AD histopa-
thology, such as intracellular A? and tan-
gle deposition between 6 and 18 months,
failed to exacerbate the exaggeration of
The mechanisms by which PS1 muta-
tions exaggerate ER Ca2?signals remain
unresolved (Smith et al., 2005a). In agree-
ment with results from cultured embry-
onic neurons expressing PS1 mutations
(Chan et al., 2000; Smith et al., 2005b), we
pression in the PS1KIand 3xTg-AD neu-
rons in the intact adult brain and further
show this upregulation persists into old
allel the age-dependent pattern of Ca2?
signaling alterations. These findings also
support our conclusion that the exaggera-
tion of IP3-evoked Ca2?signals arises in
induced Ca2?release through RyR and
not simply because of increased Ca2?flux
through the IP3R.
AD pathology may well involve alter-
ations in Ca2?homeostasis beyond those
measured here. An important caveat is
from selected “healthy” neurons to facili-
tate whole-cell patching and may thus
have excluded cells displaying more pro-
The functional roles of neuronal Ca2?
stores are becoming increasingly recog-
nized and include modulation of mem-
brane excitability (Davies et al., 1996;
Stutzmann et al., 2003), synaptic activity
strom and Naranjo, 2001). However, to
gain a better understanding of intracellu-
thology, we attempted to parse the IP3R
and RyR components to identify interac-
tions between these channels, as well as
compartmentalize specific functions as-
cribed to each.
Here, we show that IP3-evoked Ca2?
primarily through IP3receptors them-
RyR. The relative weights of these two
components change dramatically in AD
transgenic mice. Presenilin mutations are
control conditions (black) and in the presence of bath-applied dantrolene (gray) in representative NonTg (left) and 3xTg-AD (right)
5186 • J.Neurosci.,May10,2006 • 26(19):5180–5189Stutzmannetal.•Ca2?inNormalAgingandAlzheimer’sDisease
known to exaggerate ER Ca2?signaling in many cell types, but
this has implicitly been assumed to arise from increased flux
through IP3R channels (Guo et al., 1996; Leissring et al., 2000;
Stutzmann et al., 2004). Instead, our results demonstrate that
Ca2?flux through RyR accounts for the great majority of the
exaggerated IP3-evoked Ca2?response in AD transgenic mice.
Consistent with this, neurons from AD transgenic mice showed
larger Ca2?signals in response to caffeine and enhanced expres-
was not different from the NonTg.
The RyR-mediated component of the intracellular Ca2?sig-
nals almost certainly arises because CICR through RyR is trig-
gered by, and amplifies, Ca2?liberated through IP3R. Increased
RyR expression provides a likely explanation for the exaggerated
IP3-evoked Ca2?signals. Moreover, CICR may be further en-
hanced by PS1 mutations to enhance Ca2?filling of ER stores
(Leissring et al., 2000; Mattson et al., 2000), because elevated
lumenal [Ca2?] is known to increase the sensitivity of RyR to
both cytosolic Ca2?and caffeine (Shmigol et al., 1996; Koizumi
et al., 1999). Although increased store filling might also be ex-
pected to result in greater Ca2?flux through IP3R, as has been
observed in Xenopus oocytes, which lack RyR (Leissring et al.,
1999, 2001), our present results may be
ficiently to sensitize RyR, while causing
only a modest increase in Ca2?flux
through IP3R. Questions remain, how-
ever, as to why the Ca2?signals evoked by
action potentials show relatively little
is no appreciable enhancement of these
signals in the AD transgenic mouse mod-
nels in the plasma membrane are located
channels and are thus relatively ineffective
in inducing CICR.
The mechanisms by which PS muta-
gerated ER Ca2?release are presently un-
clear. One explanation draws on evidence
showing that the PS mutations result in
altered ?-secretase activity, which is re-
sponsible for the proteolysis of APP
(LaFerla, 2002). APP proteolysis generates
several fragments, including the APP-
intracellular domain fragment (AICD),
which has been shown to regulate IP3-
mediated Ca2?signaling by possible tran-
scriptional mechanisms (Cao and Sudhof,
2001; Leissring et al., 2002). Although the
target proteins are not known, altered
AICD transcriptional activity may influ-
ence expression or function of the RyR.
This mechanism appears to require mu-
tant PS, because the APPTau mice, which
would be expected to express increased
AICD levels, exhibit normal Ca2?signal-
ing at 6 weeks of age.
Previously, we found that hyperpolarizing responses to IP3are
show a similar exaggeration in 3xTg-AD neurons. We further
demonstrate that in 3xTg-AD, PSKI, and NonTg mice, these
membrane responses are mediated primarily by RyR rather than
through IP3R. In particular, blocking RyR greatly reduced IP3-
evoked hyperpolarizations in both NonTg and Tg neurons, re-
sulting in almost identical membrane responses to a given flash
simply be a direct consequence of the greater Ca2?signal. How-
ever, this does not appear to be the sole explanation, because
membrane responses accompanying Ca2?signals of a given size
were approximately twice as large in Tg versus NonTg neurons;
in other words, the Tg neurons showed a greater “coupling effi-
ciency” between cytosolic Ca2?and activation of Ca2?-
dependent K?current. This may result if RyRs are closer to the
Ca2?-dependent K?channels than are the IP3R. On this basis,
the disproportionate hyperpolarization in Tg neurons arises be-
cause most of their exaggerated Ca2?signal arises through RyR;
whereas after blocking RyR, both Tg and NonTg neurons show
comparably small hyperpolarizations that are driven by the re-
maining IP3R-mediated Ca2?liberation.
The relationship between the IP3-evoked Ca2?signal and membrane hyperpolarization is steeper in Tg than in
Stutzmannetal.•Ca2?inNormalAgingandAlzheimer’sDiseaseJ.Neurosci.,May10,2006 • 26(19):5180–5189 • 5187
The age progression of various AD markers and related Ca2?-
dependent functions is shown schematically in Figure 9. This
highlights the persistent exaggeration of RyR expression levels,
ER Ca2?release, and IP3-evoked hyperpolarization in the PS1KI
and 3xTg-AD mice as contrasted with the later appearance of
plaques, tangles, and abrupt cognitive decline (as measured by
spatial learning tasks) in the 3xTg-AD mice.
Our results reveal important new aspects of Ca2?signaling
evoked neuronal Ca2?signals is principally linked to mutations
in PS and is largely independent of expression of A? plaques or
neurofibrillar tangles; these exaggerated signals are manifest
throughout life and do not represent an acceleration of a normal
aging process and arise principally through enhanced Ca2?flux
through RyR, not IP3R. Although the familial AD-linked muta-
tions account for a minority of total AD cases, the progression of
neuronal pathology is thought to be the same as in sporadic AD,
and therefore, understanding consequences of pathogenic cellu-
lar signaling can generate insight into mechanisms underlying
AD pathology in all cases. Although additional studies are
needed, these findings further strengthen the growing consensus
that a calciumopathy may be partly responsible for neuronal de-
generation in AD (LaFerla, 2002; Smith et al., 2005a).
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