Enhanced ryanodine receptor recruitment contributes to Ca2+ disruptions in young, adult, and aged Alzheimer's disease mice

Abstract

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) and several AD mouse models (PS1KI, 3xTg-AD, and APPSweTauP301L) at young (6 week), adult (6 months), and old (18 months) ages. At all ages, the PS1KI and 3xTg-AD mice displayed exaggerated endoplasmic reticulum (ER) Ca2+ signals relative to NonTg mice. The PS1 mutation was the predominant "calciopathic" factor, because responses in 3xTg-AD mice were similar to PS1KI mice, and APPSweTauP301L 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 exaggerated signals in 3xTg-AD and PS1KI mice resulted primarily from enhanced RyR-Ca2+ release and were associated with increased 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 discrete "calciumopathy," not merely an acceleration of normal aging.

Figure 1.

Imaging IP₃- and spike-evoked Ca²⁺ signals in NonTg and Tg neurons. A, Fura-2 fluorescence in a neuron from a 6-week-old NonTg mouse. The left panel shows resting fluorescence (F₀) levels. The middle panels show pseudocolored images of Ca²⁺ signals evoked after photolysis of caged IP₃ (20 and 100 ms flash durations, as indicated). Each panel is an average of ∼10 sequential video frames around the time of maximal signal; different colors correspond to fluorescence ratio changes as indicated by the bar. Superimposed traces to the right show increasing somatic fluorescence ratio signals evoked by flashes of 10, 20, 30, 50, and 100 ms. Ca²⁺ signals were measured from the soma (excluding the nucleus). B–D, Corresponding Ca²⁺ images and traces obtained, respectively, in a representative 6-week-old APPTau neuron, a 6-week-old PS1_KI neuron, and a 6-week-old 3xTg-AD neuron. E–H, Ca²⁺ images (average of 15 frames) captured during a train of action potentials and time courses of somatic Ca²⁺ signal during these action potential trains, from the corresponding neurons in A–D.

Figure 2.

ER Ca²⁺ signaling remains elevated throughout the lifetime of PS1 mutant-expressing mice. Panels show identical data grouped by age (A–D) and by transgene (E–H). A, IP₃-evoked Ca²⁺ signals in cortical neurons from NonTg (•), PS1_KI (▴), 3xTg-AD (▪), and APPTau (★) mice at 6 weeks of age. Data points show mean fluorescence ratio changes evoked 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-year-old mice. D, Summary histograms, showing maximal IP₃-evoked Ca²⁺ responses (pooled 100 and 200 ms flash data) for each of the transgenic groups at 6 weeks, 6 months, and 1.5 years of age. Asterisks indicate significant differences (*p < 0.05) from the NonTg controls within each age group. E–G, IP₃- and spike-evoked Ca²⁺ signals grouped by transgene (respectively, in neurons from NonTg, PS1_KI, and 3xTg-AD mice). In each panel, data points show mean responses from 6-week-old (□), 6-month-old (⊠), and 1.5-year-old (▪) mice. H, Summary histograms, showing maximal IP₃-evoked Ca²⁺ responses (pooled 100 and 200 ms flash data) for each age within the different transgenic groups. Asterisks indicate significant differences (∗p < 0.05) from the response at 6 weeks within each transgenic group. Error bars represent SEM.

Figure 3.

Amplitude of the spike-evoked AHP in NonTG, PS1_KI, and 3xTg-AD mice at different ages. A, Representative spike train and subsequent AHP. Resting potential was −62 mV; action potential peaks are clipped. The gray area indicates regions of traces shown in B. B, Representative AHPs recorded in 3xTg-AD neurons at 6 weeks, 6 months, and 1.5 years of age. C, Bar graphs plot mean AHP amplitudes in NonTg and AD-Tg mice at 6 weeks (right), 6 months (middle), and 1.5 years (left) of age. Numbers of cells in each group are indicated within parentheses. Asterisks indicate a significant increase in AHP amplitude relative to the 6 week measurement within each transgene group (NonTg, F_(2,37) = 3.7, p < 0.05; PS1, F_(2,29) = 3.3, p ≤ 0.05; 3xTg-AD, F_(2,31) = 3.8, p < 0.05). Error bars represent SEM.

Figure 4.

Amplitude of the IP₃-evoked hyperpolarization is increased by expression of PS1 mutation but does not increase with age. A, The inset shows a representative IP₃-evoked hyperpolarization evoked by a 100 ms photolysis flash (arrows) in a neuron from a 6-week-old mouse. Resting potential was −62 mV in all groups. Bar graphs show mean IP₃-evoked hyperpolarizations for each transgenic groups at 6 weeks. The asterisk indicates significant increases in IP₃-evoked hyperpolarizations with respect to NonTg controls at this age (∗p < 0.05). B, C, Corresponding hyperpolarizing responses and mean data from 6-month-old and 1.5-year-old mice, respectively. Error bars represent SEM.

Figure 5.

Expression levels of brain RyR protein at different ages and across transgenic groups. In each panel, histograms show mean (±1 SEM; n = 3 replicates) RyR immunoblot density relative to β-actin expression for NonTg (left), PS1_KI (middle), and 3xTg-AD (right) brains. Asterisks mark significant (∗p < 0.05) differences from NonTg controls. Representative immunoblots of RyR protein (top) and representative β-actin levels (bottom) are shown at the right of each age tested. Data are shown from brains from 6-week-old (A), 6-month-old (B), and 1.5-year-old (C) mice.

Figure 6.

Caffeine-evoked Ca²⁺ liberation through RyR is potentiated in PS1_KI and 3xTg-AD neurons. A, Superimposed traces at the left show representative Ca²⁺ signals evoked in the soma of NonTg (gray) and 3xTg-AD (black) neurons by bath application of 20 mm caffeine. Rates of rise were measured from the maximal slope of sigmoid curves fitted to the data (dashed curves). The bar graph at the right plots mean rates of rise of caffeine-evoked fluorescent signals in the soma of NonTg (gray bar; n = 10) and Tg (black bar; n = 9) neurons. B, IP₃-evoked Ca²⁺ signals are reduced by the RyR blocker dantrolene. Traces show Ca²⁺ responses evoked by a 50 ms flash in control conditions (black) and in the presence of bath-applied dantrolene (gray) in representative NonTg (left) and 3xTg-AD (right) neurons. C, Mean percentage reductions in amplitudes of IP₃-evoked Ca²⁺ responses (50 ms flash duration) and spike-evoked Ca²⁺ signals resulting from application of dantrolene (10 μm) in NonTg (n = 6), PS1_KI (n = 6), and 3xTg-AD (n = 7) neurons. D, Effect of dantrolene on the dose–response relationship of IP₃-evoked Ca²⁺ signals. Points show measurements from NonTg neurons (n = 12; squares) and pooled measurements from 3xTg-AD and PS1_KI neurons (Tg, n = 25; circles) before (filled symbols) and after (open symbols) applying dantrolene. Data at the right show respective spike-evoked Ca²⁺ signals. E, Filled symbols show mean amplitudes of IP₃- and spike-evoked Ca²⁺ signals measured from pooled Tg neurons (n = 12) with ryanodine (30 μm) included in the pipette solution). For comparison, open symbols reproduce the measurements in C from Tg neurons exposed to dantrolene. Error bars represent SEM.

Figure 7.

IP₃-evoked membrane hyperpolarizations are strongly suppressed by dantrolene. A, Traces show changes in membrane potential in representative neurons from NonTg (left) and 3xTg-AD mice (right) after photolysis flashes of 100 ms duration in control conditions (top) and after adding 10 μm dantrolene (bottom). B, Relationships between photolysis flash duration and magnitude of the IP₃-evoked hyperpolarization. The main graph shows data from NonTg (n = 17; black squares) and Tg (n = 31; gray circles) neurons before (filled symbols) and during (open symbols) dantrolene application. The inset shows mean data comparing effects of ryanodine in Tg neurons (n = 16; closed circles) with dantrolene in Tg neurons (open squares; same data as in the main graph). C, IP₃-mediated reduction in spiking frequency is suppressed by dantrolene. The top trace shows spikes evoked by periodic injections of depolarizing current. A photolysis flash (100 ms) was delivered at the arrow to photorelease IP₃, resulting in a reduced spiking frequency for several seconds. The bottom trace was obtained using the same protocol in the same neuron while continually superfusing dantrolene (10 μm). Error bars represent SEM.

Figure 8.

The relationship between the IP₃-evoked Ca²⁺ signal and membrane hyperpolarization is steeper in Tg than in NonTg neurons. A, Scatter plot showing the relationship between IP₃-evoked Ca²⁺ signal in the soma and the magnitude of the accompanying membrane hyperpolarization in neurons from NonTg mice (n = 14; open symbols; gray line) and Tg mice (n = 23; closed symbols; black line). Points show means ±1 SEM obtained after binning over selected ranges of fluorescence amplitudes. B, Corresponding data for measurements in the proximal dendrites. C, Corresponding data in dantrolene (10 μm) obtained after pooling data from soma and dendrites. D, Dantrolene strongly reduces the slope of the relationship between IP₃-evoked membrane hyperpolarization and Ca²⁺ fluorescence signal in Tg neurons but has negligible effect in NonTg neurons. Slope data were derived from the plots in A–C. E, Bar graphs show the amplitudes of IP₃-evoked Ca²⁺ signals (left) and membrane hyperpolarizations (right) in Tg neurons as percentages of those in NonTg neurons (dashed line). Data are shown for PS1_KI (gray) and 3xTg-AD neurons (black) before (solid bars; n = 55 and 81, respectively) and after (striped bars; n = 20 and 31) adding dantrolene.

Figure 9.

Schematic time line illustrating changes in AD-linked pathology during aging of NonTg, PS1_KI, and 3xTg-AD mice. Data on cognitive abilities are from studies by Verbitsky et al. (2004), Billings et al. (2005), and Janus et al. (2000), and data on appearance of plaques and tangles are from the studies by Oddo et al. (2003a,b).