Analyzing the effects of Ca²⁺ dynamics on mitochondrial function in health and disease

Author

Patrick Toglia, University of South FloridaFollow

Graduation Year

2018

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Physics

Major Professor

Ghanim Ullah, Ph.D.

Committee Member

Martin Muschol, Ph.D.

Committee Member

Jianjun Pan, Ph.D.

Committee Member

Sagar Pandit, Ph.D.

Committee Member

Sameer Varma, Ph.D.

Keywords

Ca2+ signaling, Neurodegenerative diseases, Data-Driven Modeling

Abstract

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 (Ψ_m) 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 (n_IP3R), and open-probability (P_o) 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 (PTP_h), where the latency of opening is inversely correlated with r and proportional to n_IP3R. Furthermore, we observe diminished inner mitochondrial Ψ_m, [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 PTP_h.

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.

Scholar Commons Citation

Toglia, Patrick, "Analyzing the effects of Ca²⁺ dynamics on mitochondrial function in health and disease" (2018). USF Tampa Graduate Theses and Dissertations.
https://digitalcommons.usf.edu/etd/7652