Ian F. Smith

Essential Regulation of Cell Bioenergetics by Constitutive InsP3 Receptor Ca2+ Transfer to Mitochondria

Mechanisms that regulate cellular metabolism are a fundamental requirement of all cells. Most eukaryotic cells rely on aerobic mitochondrial metabolism to generate ATP. Nevertheless, regulation of mitochondrial activity is incompletely understood. Here we identified an unexpected and essential role for constitutive InsP(3)R-mediated Ca(2+) release in maintaining cellular bioenergetics. Macroautophagy provides eukaryotes with an adaptive response to nutrient deprivation that prolongs survival. Constitutive InsP(3)R Ca(2+) signaling is required for macroautophagy suppression in cells in nutrient-replete media. In its absence, cells become metabolically compromised due to diminished mitochondrial Ca(2+) uptake. Mitochondrial uptake of InsP(3)R-released Ca(2+) is fundamentally required to provide optimal bioenergetics by providing sufficient reducing equivalents to support oxidative phosphorylation. Absence of this Ca(2+) transfer results in enhanced phosphorylation of pyruvate dehydrogenase and activation of AMPK, which activates prosurvival macroautophagy. Thus, constitutive InsP(3)R Ca(2+) release to mitochondria is an essential cellular process that is required for efficient mitochondrial respiration and maintenance of normal cell bioenergetics.

Enhanced Ryanodine Receptor Recruitment Contributes to Ca2+ Disruptions in Young, Adult, and Aged Alzheimer's Disease Mice

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 Alzheimers 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.

SERCA pump activity is physiologically regulated by presenilin and regulates amyloid β production

In addition to disrupting the regulated intramembraneous proteolysis of key substrates, mutations in the presenilins also alter calcium homeostasis, but the mechanism linking presenilins and calcium regulation is unresolved. At rest, cytosolic Ca2+ is maintained at low levels by pumping Ca2+ into stores in the endoplasmic reticulum (ER) via the sarco ER Ca2+-ATPase (SERCA) pumps. We show that SERCA activity is diminished in fibroblasts lacking both PS1 and PS2 genes, despite elevated SERCA2b steady-state levels, and we show that presenilins and SERCA physically interact. Enhancing presenilin levels in Xenopus laevis oocytes accelerates clearance of cytosolic Ca2+, whereas higher levels of SERCA2b phenocopy PS1 overexpression, accelerating Ca2+ clearance and exaggerating inositol 1,4,5-trisphosphate–mediated Ca2+ liberation. The critical role that SERCA2b plays in the pathogenesis of Alzheimers disease is underscored by our findings that modulating SERCA activity alters amyloid β production. Our results point to a physiological role for the presenilins in Ca2+ signaling via regulation of the SERCA pump.

Enhanced caffeine-induced Ca2+ release in the 3xTg-AD mouse model of Alzheimer's disease

Alzheimers disease (AD) is the most prevalent form of dementia among the elderly and is a complex disorder that involves altered proteolysis, oxidative stress and disruption of ion homeostasis. Animal models have proven useful in studying the impact of mutant AD‐related genes on other cellular signaling pathways, such as Ca2+ signaling. Along these lines, disturbances of intracellular Ca2+ ([Ca2+]i) homeostasis are an early event in the pathogenesis of AD. Here, we have employed microfluorimetric measurements of [Ca2+]i to investigate disturbances in Ca2+ homeostasis in primary cortical neurons from a triple transgenic mouse model of Alzheimers disease (3xTg‐AD). Application of caffeine to mutant presenilin‐1 knock‐in neurons (PS1KI) and 3xTg‐AD neurons evoked a peak rise of [Ca2+]i that was significantly greater than those observed in non‐transgenic neurons, although all groups had similar decay rates of their Ca2+ transient. This finding suggests that Ca2+ stores are greater in both PS1KI and 3xTg‐AD neurons as calculated by the integral of the caffeine‐induced Ca2+ transient signal. Western blot analysis failed to identify changes in the levels of several Ca2+ binding proteins (SERCA‐2B, calbindin, calsenilin and calreticulin) implicated in the pathogenesis of AD. However, ryanodine receptor expression in both PS1KI and 3xTg‐AD cortex was significantly increased. Our results suggest that the enhanced Ca2+ response to caffeine observed in both PS1KI and 3xTg‐AD neurons may not be attributable to an alteration of endoplasmic reticulum store size, but to the increased steady‐state levels of the ryanodine receptor.

Imaging the quantal substructure of single IP3R channel activity during Ca2+ puffs in intact mammalian cells

The spatiotemporal patterning of Ca2+ signals regulates numerous cellular functions, and is determined by the functional properties and spatial clustering of inositol trisphosphate receptor (IP3R) Ca2+ release channels in the endoplasmic reticulum membrane. However, studies at the single-channel level have been hampered because IP3Rs are inaccessible to patch-clamp recording in intact cells, and because excised organelle and bilayer reconstitution systems disrupt the Ca2+-induced Ca2+ release (CICR) process that mediates channel-channel coordination. We introduce here the use of total internal reflection fluorescence microscopy to image single-channel Ca2+ flux through individual and clustered IP3Rs in intact mammalian cells. This enables a quantal dissection of the local calcium puffs that constitute building blocks of cellular Ca2+ signals, revealing stochastic recruitment of, on average, approximately 6 active IP3Rs clustered within <500 nm. Channel openings are rapidly (≈10 ms) recruited by opening of an initial trigger channel, and a similarly rapid inhibitory process terminates puffs despite local [Ca2+] elevation that would otherwise sustain Ca2+-induced Ca2+ release indefinitely. Minimally invasive, nano-scale Ca2+ imaging provides a powerful tool for the functional study of intracellular Ca2+ release channels while maintaining the native architecture and dynamic interactions essential for discrete and selective cell signaling.

Enhanced ryanodine-mediated calcium release in mutant PS1-expressing Alzheimer's mouse models.

Abstract:  Intracellular Ca2+ signaling involves Ca2+ liberation through both inositol triphosphate and ryanodine receptors (IP3R and RyR). However, little is known of the functional interactions between these Ca2+ sources in either neuronal physiology, or during Ca2+ disruptions associated with Alzheimers disease (AD). By the use of whole‐cell recordings and 2‐photon Ca2+ imaging in cortical slices we distinguished between IP3R‐ and RyR‐mediated Ca2+ components in nontransgenic (non‐Tg) and AD mouse models and demonstrate powerful signaling interactions between these channels. Ca2+‐induced Ca2+ release (CICR) through RyR contributed modestly to Ca2+ signals evoked by photoreleased IP3 in cortical neurons from non‐Tg mice. In contrast, the exaggerated signals in 3×Tg‐AD and PS1KI mice resulted primarily from enhanced CICR through RyR, rather than through IP3R, and were associated with increased RyR expression levels. Moreover, membrane hyperpolarizations evoked by IP3 in neurons from AD mouse models were even greater than expected simply from the exaggerated Ca2+ signals, pointing to an increased coupling efficiency between cytosolic [Ca2+] and K+ channel regulation. Our results highlight the critical roles of RyR‐mediated Ca2+ signaling in both neuronal physiology and pathophysiology, and point to presenilin‐linked disruptions in RyR signaling as an important genetic factor in AD.

Localization of puff sites adjacent to the plasma membrane; functional and spatial characterization of Ca2+ signaling in SH-SY5Y cells utilizing membrane-permeant caged IP3

The Xenopus oocyte has been a favored model system in which to study spatio-temporal mechanisms of intracellular Ca2+ dynamics, in large part because this giant cell facilitates intracellular injections of Ca2+ indicator dyes, buffers and caged compounds. However, the recent commercial availability of membrane-permeant ester forms of caged IP3 (ci-IP3) and EGTA, now allows for facile loading of these compounds into smaller mammalian cells, permitting control of [IP3]i and cytosolic Ca2+ buffering. Here, we establish the human neuroblastoma SH-SY5Y cell line as an advantageous experimental system for imaging Ca2+ signaling, and characterize IP3-mediated Ca2+ signaling mechanisms in these cells. Flash photo-release of increasing amounts of i-IP3 evokes Ca2+ puffs that transition to waves, but intracellular loading of EGTA decouples release sites, allowing discrete puffs to be studied over a wide range of [IP3]. Puff activity persists for minutes following a single photo-release, pointing to a slow rate of i-IP3 turnover in these cells and suggesting that repetitive Ca2+ spikes with periods of 20-30s are not driven by oscillations in [IP3]. Puff amplitudes are independent of [IP3], whereas their frequencies increase with increasing photo-release. Puff sites in SH-SY5Y cells are not preferentially localized near the nucleus, but instead are concentrated close to the plasma membrane where they can be visualized by total internal reflection microscopy, offering the potential for unprecedented spatio-temporal resolution of Ca2+ puff kinetics.

Ca 2+ Puffs Originate from Preestablished Stable Clusters of Inositol Trisphosphate Receptors

Localized calcium signals called Ca2+ puffs arise at preestablished clusters of IP3Rs. No Assembly Required The second messenger inositol trisphosphate (IP3) acts on IP3 receptors (IP3Rs) in the endoplasmic reticulum (ER) membrane to elicit Ca2+ release from intracellular stores, enabling the generation of intracellular Ca2+ signals from extracellular messengers. The regulation of these Ca2+ signals depends on the clustered organization of IP3R. Using optical techniques capable of recording single-channel IP3R activity in intact mammalian cells, Smith et al. explored the dynamics of IP3-dependent Ca2+ signals and concluded that local Ca2+ signals (known as puffs) arise from preassembled clusters of IP3R rather than depending on rapid and dynamic clustering in response to the IP3 signal itself. Intracellular calcium ion (Ca2+) signaling crucially depends on the clustered organization of inositol trisphosphate receptors (IP3Rs) in the endoplasmic reticulum (ER) membrane. These ligand-gated ion channels liberate Ca2+ to generate local signals known as Ca2+ puffs. We tested the hypothesis that IP3 itself elicits rapid clustering of IP3Rs by using flash photolysis of caged IP3 in conjunction with high-resolution Ca2+ imaging to monitor the activity and localization of individual IP3Rs within intact mammalian cells. Our results indicate that Ca2+ puffs arising with latencies as short as 100 to 200 ms after photorelease of IP3 already involve at least four IP3R channels, and that this number does not subsequently grow. Moreover, single active IP3Rs show limited mobility, and stochastic simulations suggest that aggregation of IP3Rs at puff sites by a diffusional trapping mechanism would require many seconds. We thus conclude that puff sites represent preestablished, stable clusters of IP3Rs and that functional IP3Rs are not readily diffusible within the ER membrane.

Active Generation and Propagation of Ca2+ Signals within Tunneling Membrane Nanotubes

A new mechanism of cell-cell communication was recently proposed after the discovery of tunneling nanotubes (TNTs) between cells. TNTs are membrane protrusions with lengths of tens of microns and diameters of a few hundred nanometers that permit the exchange of membrane and cytoplasmic constituents between neighboring cells. TNTs have been reported to mediate intercellular Ca(2+) signaling; however, our simulations indicate that passive diffusion of Ca(2+) ions alone would be inadequate for efficient transmission between cells. Instead, we observed spontaneous and inositol trisphosphate (IP(3))-evoked Ca(2+) signals within TNTs between cultured mammalian cells, which sometimes remained localized and in other instances propagated as saltatory waves to evoke Ca(2+) signals in a connected cell. Consistent with this, immunostaining showed the presence of both endoplasmic reticulum and IP(3) receptors along the TNT. We propose that IP(3) receptors may actively propagate intercellular Ca(2+) signals along TNTs via Ca(2+)-induced Ca(2+) release, acting as amplification sites to overcome the limitations of passive diffusion in a chemical analog of electrical transmission of action potentials.

Role of Calcium in the Pathogenesis of Alzheimer’s Disease and Transgenic Models

Alzheimers disease (AD) is a progressive neurodegenerative disorder of the elderly that is characterized by memory loss. Neuropathologically, the AD brain is marked by an increased AP burden, hyperphosphorylated tau aggregates, synaptic loss, and inflammatory responses. Disturbances in calcium homeostasis are also one of the earliest molecular changes that occur in AD patients, alongside alterations in calcium-dependent enzymes in the post-mortem brain. The sum of these studies suggests that calcium dyshomeostasis is an integral part of the pathology, either influencing AP production, mediating its effects or both. Increasing evidence from in vitro studies demonstrates that the AP peptide could modulate a number of ion channels increasing calcium influx, including voltage-gated calcium and potassium channels, the NMDA receptor, the nicotinic receptor, as well as forming its own calcium-conducting pores. In vivo evidence has shown that A3 impairs both LTP and cognition, whereas all of these ion channels cluster at the synapse and underlie synaptic transmission and hence cognition. Here we consider the evidence that AP causes cognitive deficits through altering calcium homeostasis at the synapse, thus impairing synaptic transmission and LTP. Furthermore, this disruption appearr to occur without overt or extensive neuronal loss, as it is observed in transgenic mouse models of AD, but may contribute to the synaptic loss, which is an early event that correlates best with cognitive decline.