Mitochondrial dysfunction and role in spreading depolarization and seizure.

The effect of pathological phenomena such as epileptic seizures and spreading depolarization (SD) on mitochondria and the potential feedback of mitochondrial dysfunction into the dynamics of those phenomena are complex and difficult to study experimentally due to the simultaneous changes in many variables governing neuronal behavior. By combining a model that accounts for a wide range of neuronal behaviors including seizures, normoxic SD, and hypoxic SD (HSD), together with a detailed model of mitochondrial function and intracellular Ca2+ dynamics, we investigate mitochondrial dysfunction and its potential role in recovery of the neuron from seizures, HSD, and SD. Our results demonstrate that HSD leads to the collapse of mitochondrial membrane potential and cellular ATP levels that recover only when normal oxygen supply is restored. Mitochondrial organic phosphate and pH gradients determine the strength of the depolarization block during HSD and SD, how quickly the cell enters the depolarization block when the oxygen supply is disrupted or potassium in the bath solution is raised beyond the physiological value, and how fast the cell recovers from SD and HSD when normal potassium concentration and oxygen supply are restored. Although not as dramatic as phosphate and pH gradients, mitochondrial Ca2+ uptake has a similar effect on neuronal behavior during these conditions.

Analyzing and Quantifying the Gain-of-Function Enhancement of IP3 Receptor Gating by Familial Alzheimer's Disease-Causing Mutants in Presenilins.

Familial Alzheimer's disease (FAD)-causing mutant presenilins (PS) interact with inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) Ca(2+) release channels resulting in enhanced IP3R channel gating in an amyloid beta (Aß) production-independent manner. This gain-of-function enhancement of IP3R activity is considered to be the main reason behind the upregulation of intracellular Ca(2+) signaling in the presence of optimal and suboptimal stimuli and spontaneous Ca(2+) signals observed in cells expressing mutant PS. In this paper, we employed computational modeling of single IP3R channel activity records obtained under optimal Ca(2+) and multiple IP3 concentrations to gain deeper insights into the enhancement of IP3R function. We found that in addition to the high occupancy of the high-activity (H) mode and the low occupancy of the low-activity (L) mode, IP3R in FAD-causing mutant PS-expressing cells exhibits significantly longer mean life-time for the H mode and shorter life-time for the L mode, leading to shorter mean close-time and hence high open probability of the channel in comparison to IP3R in cells expressing wild-type PS. The model is then used to extrapolate the behavior of the channel to a wide range of IP3 and Ca(2+) concentrations and quantify the sensitivity of IP3R to its two ligands. We show that the gain-of-function enhancement is sensitive to both IP3 and Ca(2+) and that very small amount of IP3 is required to stimulate IP3R channels in the presence of FAD-causing mutant PS to the same level of activity as channels in control cells stimulated by significantly higher IP3 concentrations. We further demonstrate with simulations that the relatively longer time spent by IP3R in the H mode leads to the observed higher frequency of local Ca(2+) signals, which can account for the more frequent global Ca(2+) signals observed, while the enhanced activity of the channel at extremely low ligand concentrations will lead to spontaneous Ca(2+) signals in cells expressing FAD-causing mutant PS.

Data-driven modeling of mitochondrial dysfunction in Alzheimer's disease.

Intracellular accumulation of oligomeric forms of ß amyloid (Aß) are now believed to play a key role in the earliest phase of Alzheimer's disease (AD) as their rise correlates well with the early symptoms of the disease. Extensive evidence points to impaired neuronal Ca2+ homeostasis as a direct consequence of the intracellular Aß oligomers. However, little is known about the downstream effects of the resulting Ca2+ rise on the many intracellular Ca2+-dependent pathways. Here we use multiscale modeling in conjunction with patch-clamp electrophysiology of single inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) and fluorescence imaging of whole-cell Ca2+ response, induced by exogenously applied intracellular Aß42 oligomers to show that Aß42 inflicts cytotoxicity by impairing mitochondrial function. Driven by patch-clamp experiments, we first model the kinetics of IP3R, which is then extended to build a model for the whole-cell Ca2+ signals. The whole-cell model is then fitted to fluorescence signals to quantify the overall Ca2+ release from the endoplasmic reticulum by intracellular Aß42 oligomers through G-protein-mediated stimulation of IP3 production. The estimated IP3 concentration as a function of intracellular Aß42 content together with the whole-cell model allows us to show that Aß42 oligomers impair mitochondrial function through pathological Ca2+ uptake and the resulting reduced mitochondrial inner membrane potential, leading to an overall lower ATP and increased production of reactive oxygen species and H2O2. We further show that mitochondrial function can be restored by the addition of Ca2+ buffer EGTA, in accordance with the observed abrogation of Aß42 cytotoxicity by EGTA in our live cells experiments.

Analyzing optical imaging of Ca2+ signals via TIRF microscopy: The limits on resolution due to chemical rates and depth of the channels.

High resolution total internal reflection (TIRF) microscopy (TIRFM) together with detailed computational modeling provides a powerful approach towards the understanding of a wide range of Ca2+ signals mediated by the ubiquitous inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) channel. Exploiting this fruitful collaboration further requires close agreement between the models and observations. However, elementary Ca2+ release events, puffs, imaged through TIRFM do not show the rapid single-channel openings and closings during and between puffs as are present in simulated puffs using data-driven single channel models. TIRFM also shows a rapid equilibration of 10ms after a channel opens or closes which is not achievable in simulation using standard Ca2+ diffusion coefficients and reaction rates between indicator dye and Ca2+. Furthermore, TIRFM imaging cannot decipher the depth of the channel with respect to the microscope, which will affect the change in fluorescence that the microscope detects, thereby affecting its sensitivity to fast single-channel activity. Using the widely used Ca2+ diffusion coefficients and reaction rates, our simulations show equilibration rates that are eight times slower than TIRFM imaging. We show that to get equilibrium rates consistent with observed values, the diffusion coefficients and reaction rates have to be significantly higher than the values reported in the literature, and predict the channel depth to be 200-250nm. Finally, we show that with the addition of noise, short events due to 1-2ms opening and closing of channels that are observed in computational models can be missed in TIRFM.

The gain-of-function enhancement of IP3-receptor channel gating by familial Alzheimer's disease-linked presenilin mutants increases the open probability of mitochondrial permeability transition pore.

Mutants in presenilins (PS1 or PS2) are the major cause of familial Alzheimer's disease (FAD). They affect intracellular Ca(2+) homeostasis by increasing the open probability (Po) of inositol 1,4,5-trisposphate (IP3) receptor (IP3R) Ca(2+) release channel located on the endoplasmic reticulum (ER) leading to exaggerated Ca(2+) release into a cytoplasmic microdomain formed by neighboring cluster of a few IP3R channels and mitochondrial Ca(2+) uniporter (MCU). Ca(2+) concentration in the microdomain ( [Formula: see text] ) depends on the distance between the cluster and MCU (r); the number of IP3R in the cluster releasing Ca(2+) to the cytoplasm ( [Formula: see text] ), and Po of IP3R. Using experimental whole-cell IP3R-mediated cytosolic Ca(2+) data, in conjunction with a computational model of cell bioenergetics, a data-driven Markov chain model for IP3R gating, and a model for the dynamics of the mitochondrial permeability transition pore (PTP), we explore differences in mitochondrial Ca(2+) uptake in cells expressing wild type (PS1-WT) and FAD-causing mutant (PS1-M146L) PS. We find that increased mitochondrial [Formula: see text] due to the gain-of-function enhancement of IP3R channels in the cells expressing PS1-M146L leads to the opening of PTP in high conductance state (PTPh), where the latency of opening is inversely correlated with r and proportional to [Formula: see text] . Furthermore, we observe diminished inner mitochondrial membrane potential (ΔΨm), [NADH], [Formula: see text] , and [ATP] when PTP opens. Additionally, we explore how parameters such as the pH gradient, inorganic phosphate concentration, and the rate of the Na(+)/Ca(2+)-exchanger affect the latency of PTP to open in PTPh.

Impaired mitochondrial function due to familial Alzheimer's disease-causing presenilins mutants via Ca(2+) disruptions.

Mutants in presenilins (PS1 or PS2) is the major cause of familial Alzheimer's disease (FAD). FAD causing PS mutants affect intracellular Ca(2+) homeostasis by enhancing the gating of inositol trisphosphate (IP3) receptor (IP3R) Ca(2+) release channel on the endoplasmic reticulum, leading to exaggerated Ca(2+) release into the cytoplasm. Using experimental IP3R-mediated Ca(2+) release data, in conjunction with a computational model of cell bioenergetics, we explore how the differences in mitochondrial Ca(2+) uptake in control cells and cells expressing FAD-causing PS mutants affect key variables such as ATP, reactive oxygen species (ROS), NADH, and mitochondrial Ca(2+). We find that as a result of exaggerated cytosolic Ca(2+) in FAD-causing mutant PS-expressing cells, the rate of oxygen consumption increases dramatically and overcomes the Ca(2+) dependent enzymes that stimulate NADH production. This leads to decreased rates in proton pumping due to diminished membrane potential along with less ATP and enhanced ROS production. These results show that through Ca(2+) signaling disruption, mutant PS leads to mitochondrial dysfunction and potentially to cell death.