Mitochondria plays a crucial role in cells by maintaining energy metabolism and directing cell death mechanisms by buffering calcium (Ca2+ )from cytosol. Therefore, the Ca2+ overload of mitochondria due to the upregulated cytosolic Ca2+ , observed in many neurological disorders is hypothesized to be a key pathway leading to mitochondrial dysfunction and cell death. In particular, Ca2+ homeostasis disruptions due to Alzheimer’ s disease (AD)-causing presenilins (PS1/PS2) and oligomeric forms of β-amyloid peptides Aβ commonly found in AD patients are presumed to cause detrimental effects on the mitochondria and its ability to function properly. We begin by showing that Familial Alzheimer’s disease (FAD)-causing PS mutants affect intracellular Ca2+ ([Ca2+]i) homeostasis by enhancing the gating of inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) Ca2+ channels on the endoplasmic reticulum (ER), leading to exaggerated Ca2+ release into the cytoplasm. Using experimental IP3R-mediated Ca2+ release data in conjunction with a computational model of mitochondrial bioenergetics, we explore how the differences in mitochondrial Ca2+ uptake in control cells and cells expressing FAD-causing PS mutants affect key variables such as ATP, reactive oxygen species (ROS), NADH, and mitochondrial Ca2+ ([Ca2+ ]m). We find that as a result of exaggerated [Ca2+]i in FAD-causing mutant PS-expressing cells, the rate of oxygen consumption increases dramatically and overcomes the Ca2+ dependent enzymes that stimulate NADH production. This leads to decreased rates of proton pumping due to diminished membrane potential (Ψm) along with less ATP and enhanced ROS production. These results show that through Ca2+ signaling disruption, mutant PS leads to mitochondrial dysfunction and potentially cell death.
Next, the model for the mitochondria is expanded to include the mitochondrial uniporter (MCU) that senses Ca2+ in the microdomain formed by the close proximity of mitochondria and ER. Ca2+ concentration in the microdomain ([Ca2+] mic) depends on the distance between the cluster of IP3R channels (r) on ER and mitochondria, the number of IP3R in the cluster (nIP3R), and open-probability (Po) of IP3R. Using the same experimental results for Ca2+ release though IP3R due to FAD-causing PS mutants, in conjunction with a computational model of mitochondrial 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 the difference in mitochondrial Ca2+ uptake in cells expressing wild type (PS1-WT) and FAD-causing mutant (PS1-M146L) PS. We find that increased mitochondrial [Ca2+]m due to the gain-of-function enhancement of IP3R channels in the cell 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 nIP3R. Furthermore, we observe diminished inner mitochondrial Ψm, [NADH], [Ca2+]m, and [ATP] when PTP opens. Additionally, we explore how parameters such as the pH gradient, inorganic phosphate concentration, and the rate of the Na+/ Ca2+ -exchanger affect the latency of PTP to open in PTPh.
Intracellular accumulation of oligomeric forms of Aβ are now believed to play a key role in the early phase of 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 Aβ oligomers. To study the effect of intracellular Aβ on Ca2+ signaling and the resulting mitochondrial dysfunction, we employed data-driven modeling in conjunction with total internal reflection fluorescence (TIRF) microscopy (TIRFM). High resolution fluorescence TIRFM together with detailed computational modeling provides a powerful approach towards the understanding of a wide range of Ca2+ signals mediated by the IP3R. Achieving this requires a close agreement between Ca2+ signals from computational models and TIRFM experiments. However, we found that elementary Ca2+ release events, puffs, imaged through TIRFM do not show the rapid single-channel opening and closing during x and between puffs using data-driven single channel models. TIRFM also shows a rapid equilibration of 10 ms 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+. 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. Once a close agreement between experiment and model is achieved, we use multiscale modeling in conjunction with patch-clamp electrophysiology of IP3R and fluorescence imaging of whole-cell Ca2+ response, induced by intracellular Aβ42 oligomers to show that Aβ42 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 ER by intracellular Aβ42 oligomers through G-protein-mediated stimulation of IP3 production. The estimated IP3 concentration as a function of intracellular Aβ42 content together with the whole-cell model allows us to show that Aβ42 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 Aβ42 cytotoxicity by EGTA in our live cells experiments.
Finally, our modeling study was extended to other pathological phenomena such as epileptic seizures and spreading depolarizations (SD) and their effects on mitochondria by incorporating conservation of particles and charge, and accounting for the energy required to restore ionic gradients to the neuron. By examining the dynamics as a function of potassium and oxygen we can account for a wide range of neuronal hyperactivity from seizures, normoxic SD, and hypoxic SD (HSD) in the model. Together with a detailed model of mitochondria xi and Ca2+ -release through the ER, we determine mitochondrial dysfunction and potential recovery mechanisms from HSD. Our results demonstrate that HSD causes detrimental mitochondrial dysfunction that can only be recovered by restoration of oxygen. Once oxygen is replenished to the neuron, organic phosphate and pH gradients along the mitochondria determine how rapid the neuron recovers from HSD.
| Identifer | oai:union.ndltd.org:USF/oai:scholarcommons.usf.edu:etd-8849 |
| Date | 04 April 2018 |
| Creators | Toglia, Patrick |
| Publisher | Scholar Commons |
| Source Sets | University of South Flordia |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Graduate Theses and Dissertations |
| Rights | default |
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