Aβ oligomers, and their extracellular accumulation, although the underpinning molecular machinery remains elusive. We propose a novel model for Alzheimer's Aβ accumulation based on Ca2+-dependent exosome release from astrocytes. Moreover, we exploit our model to assess how temperature dependence of exosome release could interact with Aβ neurotoxicity. We predict that voltage-gated Ca2+ channels (VGCCs) along with the transient-receptor potential M8 (TRPM8) channel are crucial molecular components in Alzheimer's progression.Exosomes are nano-sized extracellular vesicles that perform a variety of biological functions linked to the pathogenesis of various neurodegenerative disorders. In Alzheimer's disease (AD), for examples, exosomes are responsible for the release of  Protein misfolding, oligomerization, and aggregation are responsible for the initiation of pathological disorders in the brain   Jain, . Specifi+) Jain,  have sho+) Jain, , a questAβ can substantially affect intracellular Ca2+ homeostasis both in neurons and in astrocytes  astrocytic exosome exocytosis mediated by Aβ in AD, and (iii) temperature dependence of neuron models including TRPM8 currents. In section 3, we present numerical simulations based on the developed neuron-glial model. Finally, we discuss our results and outline future directions in section 4.This study is organized as follows. In section 2, we describe our model  when the channels are opened (CL|opened) and closed (CL|closed), low-voltage activated T-type Ca2+ channels, as well as the characterization of Ca2+ below the plasma membrane (Cm) in the bulk cytosol (Cc), and in the endoplasmic reticulum (Cr). In this fashion, exosomal release can be expressed as a function of L-type Ca2+ microdomain concentrations and plasma membrane Ca2+ concentrations, respectively, are as follows:The calcium-mediated exosomal release is restricted to active zones that contain VGCCs that control ć et al. . To linkCL|closed = Cm  CL; (ii) Cm; (iii) Cc and (iv) Cr. The equations for compartment-specific Ca2+ concentrations  are described by λd, λm, λc, and λr, respectively, kPMCA is the rate of Ca2+ adenosine triphosphatase (ATPase) at the plasma membrane level, pleak is the rate of the leak current from the r to the cytosol, and kSERCA is the amount of Ca2+ sequestration into the r by the sarco/endoplasmic Ca2+ ATPase pump. The gating variables in the steady-state are defined in Table 2:In terms of coefficients of the equations, x =  and where Ca2+-mediated exocytosis by neurons is regulated by intracellular Ca2+, where electrical activity pattern determines the exocytosis Ca2+ threshold  channels, voltage-gated sodium (Na+) channels, a leak current, and an induced control signal/current (Iind) as Veletić et al. , with the hypothesis that a quantized amount of IP3 molecules is released after glutamate levels rise due to pre-synaptic spiking activity , as a function of a generic control signal vind applied to depolarize the astrocyte, is given as follows  by setting vm = Vm + vind in all corresponding equations, where Vm = −70mV gives the resting astrocytic membrane potential. The Ca2+ concentrations in single microdomains surrounding high-voltage activate N-type Ca2+ channels when the channels are opened and closed, and the plasma membrane (Cm) lead to the following equations  with CN|closed = Cm, where iCN = gCN(Vm + vind − VC)/NN and gCN is the membrane conductance of N-type Ca2+ channels. In modeling exosomal release from astrocytes, by manipulating the Nadkarni-Jung model that is further based on the Li-Rinzel model ·Cc, d5, d6), hP,∞ = Q, Cc, d6) adopted from Veletić et al. . Since internal Ca2+ stores are also responsive to Ca2+, an increase in Ca2+ concentration deploys sufficient Ca2+ release. This biological process is known as Calcium-Induced Calcium Release (CICR) which shows the first term of Equation (12). Additional Ca2+ flow from the endoplasmic reticulum into the cytosol usually arises (leakage flow), while Ca2+ based ATPase pumps (SERCA) work in the opposite direction to uptake Ca2+ (second term of Equation 12) back into the stores for potential use (pump flow). The balance between passive leakage from the endoplasmic reticulum and SERCA uptake regulates Ca2+ concentration at rest. Sneyd and Li and Rinzel have identified analytically the Ca2+ dynamics and release/uptake processes triggered by IP3 , respectively, and the collective exosomal release rate in astrocytes is defined as follows . The distribution of Ca2+ astrocyte signaling plays an important role in AD. We have modified our previously elaborated model of Ca2+-mediated exosomal dynamics in neural cells to study spontaneous Ca2+oscillations in astrocytes in order to investigate the impact of Aβ on intracellular Ca2+ dynamics during AD. By activating the L-type VGCCs and metabolic glutamate receptors, or by increasing ryanodine receptor sensitivity and Ca2+leakage, Aβ will increase the resting concentration of intracellular Ca2+and adjust the regime of Ca2+ oscillations. The primary target of Aβ neurotoxicity is thought to be astrocytes  and vm is the membrane potential as given in Equation (9), VC is the constant Nernst potential for calcium and other relevant parameters are given in where l to reflect a fixed amount of Aβ concentration present in the environment to investigate the effects of Aβ. In addition, we use AVL to monitor the strength of Aβ effects on the L-type VGCC current pathway, resulting in a total Ca2+current as follows:We use parameter where the corresponding flux is defined asIP3 catalyzed by phospholipase (PLC) is enhanced by cytoplasmic Ca2+ in this model. The IP3 receptors (IP3R) are mediated by cytoplasmic Ca2+ and IP3, inducing Ca2+ flow out of the endoplasmic reticulum by CICR. Importantly, CICR from the endoplasmic reticulum is perhaps the most well-studied Ca2+ signaling pathway in astrocytes , ryanodine receptor (RyR) channels, and membrane leak Jin . The leak flux due to the concentration gradient is indicated by the “leak” from the endoplasmic reticulum (Jr). The other Ca2+fluxes JCICR, JSERCA, JRyR, Jr, Jin, and Jpm are defined as follows:The concrete formula for each type of calcium current is given in detail in Jin, JPLC, and JRyR, we also use Ain, ARyR, Am to monitor the intensity of these different effects, as well as l to represent the impact of Aβ. The essential parameters used in our analysis are listed in Ca2+concentrations in the cytosol, endoplasmic reticulum, and the IP3 concentrations in the IPc cell (Pc) are defined as follows:In Cc = 0.1μM, Pc = 0.1μM, Cr = 1.5μM at t = 0 motivated and validated by Gao et al. . However, the bulk cytosol, endoplasmic reticulum, and Pc concentrations are the same as defined in Equations (27–29). Therefore, the relative exosomal release incorporating Aβ would be the same as defined in Equation (16) by using the concrete formula for each type of calcium current is given in Ca2+ levels can be thought of as a form of Ca2+ excitability  is the Heaviside function . Therefore, the modified membrane potential for the neuron-astrocyte network model is defined as follows  for the cold sensing TRPM8 channel in contrast to the prior Hodgkin-Huxley model, prompted by McGahan and Keener  will be used. Moreover, we will develop a biologically driven model of a Ca2+-mediated exosomal release in astrocytes mediated by Aβ of a specific cold thermosensor with the existence of TRPM8 channels. Therefore, we couple the developed neuronal model with TRPM8 channels for analyzing the temperature threshold dependence on cold-sensing neurons, utilizing (Equations 35–37).where ΔCa2+ mediated exocytosis on the membrane potential with a focus on microdomain Ca2+ concentrations around high-voltage activated L-type and low-voltage activated T-type Ca2+ channels. Also, a definition of Ca2+ below the plasma membrane, in the bulk cytosol, and in the endoplasmic reticulum using the Ca2+-mediated exosomal dynamics neuronal model. Moreover, the mechanism of Ca2+-dependant exosome release in response to square pulses of DC input currents of different amplitudes, both in neurons and astrocytes on the Ca2+-mediated exosomal dynamics is also investigated. The numerical results provided in this section are obtained by using the parameter values collected from Veletić et al.  is shown in 2. As evident from IP3 development rate is linearly proportional to the external stimuli vind adopted from Veletić et al.  and is shown in vind = 10 − 30 mV , L-type Ca2+ concentrations, and sub-membrane Ca2+ concentrations (for weak depolarization). Astrocytes, unlike neurons, are electrically silent and incapable of generating action potentials  by integrating different types of VGCCs  act to harm astrocytes by raising the frequency of Ca2+ oscillations, lowering the membrane threshold for Ca2+ oscillations, and enhancing the stable state concentration of Ca2+. Furthermore, by increasing Jin, Aβ expands the membrane potential spectrum and raises resting Ca2+ concentrations to a low steady-state. The clustering of mGluR triggered by Aβ causes the oscillating range to change to a lower potential, as demonstrated in Gao et al.  by applying an external stimulus presented in Aβ. The effects of different values of Aβ on calcium concentrations for Cc and Pc without applying the induced/control signals stimulus is presented in Aβ alters the membrane potential which in turn can enhance the regime of Ca2+ oscillations and increase the stable state concentration of Ca2+. The Ca2+ oscillations demonstrate that astrocytes have ionic excitability mediated by Aβ, making them possible targets for Aβ neurotoxicity. A pathological rise in Aβ may cause functional and structural abnormalities in glial cells, including Ca2+ dysregulation. This calcium/gliotransmission alteration might route a key component in the pathophysiology of AD. The calcium hypothesis of AD proposes that activation of the amyloidogenic pathway retrofits neuronal Ca2+ signaling, affecting normal Ca2+ homeostasis and the processes involved in learning and memory. Our results show that the presence of Aβ aggregates raises cytosolic Ca2+ levels. Exaggerated Pc concentrations evoked Ca2+ release with the influence of Aβ raises the amplitude of a Ca2+-activated hyperpolarizing current, which suppresses membrane excitability. Furthermore, as AD progresses, increasing the threshold for spike activation may have an effect on coincidence detection and local circuit activity. Additionally, the effect of the control signal presented in 2 results in a corresponding increase in the concentration of Pc and Cc. Cytosolic buffering determines the presence of a control signal across the plasma membrane in response to an increase in free cytosolic Ca2+ concentration, the rate of removal from the cytosol by sequestration into organelles (primarily r), and cell extrusion over the plasma membrane. Furthermore, external stimuli leverage subsequent Ca2+ influx by inactivating voltage- or receptor-operated channels in the bulk cytosol, activating K channels that lower membrane excitability, or influencing Ca2+ release from intracellular depots. The spatial distribution of Ca2+ signals is greatly influenced by cytosolic Ca2+ buffering. One of the major pathways for Ca2+ release from the endoplasmic reticulum is through IP3 receptors. The diffusion of IP3 concentration in the IPc cell can stimulate Ca2+ release from the endoplasmic reticulum in response to the activation of the firing of action potentials by external stimuli. Increased concentrations of Pc pathways may then encourage the transition to a low-threshold activated L-type Ca2+ current, causing additional disturbance of intracellular Ca2+ homeostasis which is a key phenomenon for AD . This means that astrocyte secretion will generate new synaptic connections for different values of Aβ, thus, increasing complexity of the neural network. Hence, increasing the values of Aβ would lead to a significant increase in the spiking sequence of exosomal release from astrocytes, while the effect on the concentrations of exosomal release rate is quite negligible. In addition, the effect of the control signal presented in Ca2+-dependent exosomal release from astrocytes has been presented in Aβ. Aβ is involved in the model. The external stimulus enhances the influence of activity-dependent Aβ and the spiking sequences are significantly larger for the release of exosomes from astrocytes. The spiking sequences are also significantly reduced without the influence of activity-dependent Aβ as depicted in Furthermore, the Aβ enhances exosomal release from the astrocytes. Astrocytes, unlike neurons, are electrically quiet and cannot trigger action potentials. Because Aβ triggers astrocytes and neurophysiological properties of selected neurons by shifting from a high-threshold to a low-threshold triggered L-type Ca2+ current, this hints that a variety of pathways, including chemical processes involving Aβ, cause an increase in astrocyte intracellular Ca2+ levels. It could be an underlying mechanism for the early metabolic and noncognitive symptoms of AD caused by hypothalamic dysfunction.Our findings suggest that Pc is triggered by glutamate elevations in the astrocyte domain, which activates intracellular Ca2+ dynamics. The amplitude, frequency, and propagation of intracellular Ca2+ oscillations produced in astrocytes are regulated by the intrinsic properties of both neuronal inputs and astrocytes. Astrocytes can distinguish between numerous forms of neuronal inputs and incorporate concomitant inputs in response to calcium elevations. Calcium dynamics are regulated by the interaction of CICR, which is a nonlinear amplification mechanism dependent on calcium channels opening to calcium stores, such as the endoplasmic reticulum. The action of active transporters causes a reverse flow (SERCA pumps). Signals impinging on the cell from an outside environment directly regulate the level of Pc , modulating pre-synaptic and post-synaptic neural activities. When a neuron fires, small quantities of neurotransmitters (glutamate) are released into the synaptic cleft. The release of intracellular Pc is triggered when neurotransmitters bind to glutamate receptors on astrocytes. The action potentials generated by the neuron injected with a constant current Iind, trigger an increase of the internal Ca2+ concentration of the astrocyte. This event feedbacks an inward current to the neuron (Iastro).In this section, we will discuss the dynamics of neural activity in the presence of exosomal release from astrocytes by analyzing it with a model involving an astrocyte coupled to a single neuron, as developed in section 2.3. The biological processes involving astrocytes take place in close proximity to the synapses of neurons. They are sensitive to neuronal activity sensors that react to glutamate synaptic release with oscillations in intracellular calcium concentration. The concentration of Ca2+ concentrations in the presence of exosomal release from the astrocyte, when the neuron is injected with the current Iind = 20 μA/cm2, are presented in Ca2+ level in the astrocyte is not sufficient to trigger a feedback response in the neurons (see Equation 32). The increase of the production rate of Pc amplifies the Ca2+ response in the astrocyte and so leads to the generation of membrane potentials within a well-defined time window. The neural dynamics of membrane potential (vm given in Equation 32) and Cc are altered by the surrounding activity, i.e., the astrocyte feedback. To illustrate the impact of the astrocytic feedback on neural excitability, we study the neural activity dynamics generated by our model both with and without Aβ. Numerical simulations of dynamical regimes in which neuronal firing is sustained indefinitely revealed that cytosolic Ca2+ concentration and membrane potential fluctuate rapidly under the control of Aβ while spiking sequences are greatly decreased when Aβ is not present  and without the temperature effects have been presented in The excitability and response characteristics of a neuron might change depending on the temperature of the surroundings. The effects of the induced pulse of 20 μA/cmCa2+ concentrations in the closed and open channels of L-type, plasma membrane, bulk cytosol, and endoplasmic reticulum are significantly overestimated if the effect of temperature is neglected and the spiking sequences are also significantly reduced. Indeed, incorporating temperature will result in a corresponding decrease in the concentration of Cr, Cm, Cc, and CL concentrations, and the spiking sequences are also significantly reduced. The findings underpin Huxley's theory that thermally induced block is caused by increased activation of voltage-dependent potassium ion channels in response to depolarization, particularly at higher temperatures  with increased temperature which enhances neural activity. Thus, the exosomal release rate of the targeted neuron is significantly affected by the changes in temperature.The effects of the temperature on the microdomain calcium concentrations have been presented in gm8 nontrivial . We begin by using (Equation 36) of TRPM8 channels at different physiological voltages in response to temperature. Ca2+ concentrations at different temperature levels (see Equation 35). From Ca2+ concentrations decreases with rising temperatures. Additionally, as the temperature rises, the average amplitude of the TRPM8 current grows. We should recognize that since Vm8 = 0 , which leads to a rise in action potential frequencies, yet, when the temperature is increased, certain nerve cells exhibit an increase in frequency.Examining the behavior of the established model as 2 on the cytosolic calcium and the IP3 concentrations in the IPc cells in regards to astrocytic exosome exocytosis mediated by Aβ in AD (see section 2.3), with temperature (T = 25°C) and without the temperature effects have been presented in Cc and IP3 was markedly accelerated by increasing temperature. At T = 25 oC, Aβ accumulation and hyperexcitability. The effect of temperature has been examined on cytosolic Ca2+ concentrations and the IP3 concentrations mediated by Aβ. Importantly, Ca2+ wave propagation is thought to be a reaction/diffusion system requiring several cycles of Ca2+ release from IP3 clusters and diffusion to nearby clusters to trigger CICR. The study found that the concentrations of Ca2+ and IP3 in the IPc cells decrease monotonically with temperature which disturbs the brain dynamics and could lead to the pathophysiology of AD.The effects of the induced pulse of 20 μA/cmCa2+-mediated astrocytic exosome exocytosis mediated by Aβ (l = 0.4) in AD. The TRPM8 part of the model is added by setting gm8 to nonzero. The open possibility of TRPM8 channels, am8, in response to temperature at various physiological voltages is first added. All other parameter values are set to Hodgkin-Huxley norms and gm8 = 3. As the fixed temperature level is raised, a few distinct features emerge that include the amplitudes of the action potentials increase, resulting in an increase in the maximum voltage reached. As a result, the current model emphasizes the importance of TRPM8 channels in determining temperature-dependent activation and inactivation thresholds. Furthermore, our findings shed light on what happens at the temperature levels at which these neurons shut down, as well as the role sodium and leak currents can play. It has been demonstrated by using both TRPM8 and the stimulus of induced control signal triggered the calcium concentrations of Cc and Pc defined in . From Cc and Pc declines. It can be seen from T = 5 to 15°C would result in a corresponding decrease in the concentration of Cc and Pc in the presence of gm8 = 3. However, TRPM8 channels were shown to be expressed in the endoplasmic reticulum where their modulation by activators and/or inhibitors was demonstrated to be crucial for intracellular Ca2+ signaling. The rise in inward flux is primarily responsible for the shift in the TRPM8 current amplitude, in fact, it is worth noting that from T = 5 to 15°C there is an increase in outward current. These findings imply that TRPM8 channels confer temperature sensitivity to the endoplasmic reticulum, which permits Ca2+ release by facilitating Ca2+ efflux into the cytosol and therefore contributing to CICR viaIP3 and ryanodine receptors. Although the IP3 evoked Ca2+ signals were qualitatively comparable at 5–15 Ca2+, this difference in temperature should take into account the temperature sensitivity of IP3-mediated signal amplitudes. The transition temperature was 25°C in all cases, which might indicate a phase change in the lipids of the cytoplasmic membrane. Our findings demonstrate that using this temperature range (from T = 5 to 15°C) significantly increases the amplitude and lowers the frequency of global IP3-mediated Ca2+ signals, which is consistent with findings from a variety of different cell types. For instance, fast cooling elicits strong oscillatory Ca2+-activated leak currents when the IP3 pathway is active and has been shown to enhance the amplitudes of IP3-mediated Ca2+ signals in several cultured glial cells, including Schwann cells and olfactory ensheathing cells, as well as astrocytes. As a result, the temperature sensitivity of the cytosolic Ca2+ concentration underpinning global IP3-mediated signals appears to be a common occurrence, which must be taken into account when extending data obtained at room temperature to body temperature.Moreover, a study has been conducted to quantify the effects of TRPM8 channels in the modeling of Ca2+ compartments and astrocytes in exosomal release regulation. We implemented a novel model for accurately quantifying the Ca2+-mediated astrocytic exosome exocytosis mediated by amyloid-beta in AD. In addition, a comparative analysis has been conducted to quantify the effect of temperature and cold-sensitive neurons on the Ca2+-dependent exosomal release mediated by VGCCs and amyloid-beta in AD. Moreover, we calculated the astrocytic current as a function of both the pre-synaptic neuron current and the astrocyte (Li-Rinzel model). This work describes the synapse and astrocyte couplings in a computationally efficient model. It is possible to simulate real-time spiking artificial neuron-glia networks using the model proposed in this study, revealing the mechanism that appears to be a necessary part of the regulation of spiking activities. We showed that this model can be used to simulate the neuron astrocyte interaction. The results obtained with the developed model suggest that cell depolarization in neurons is directly related to the exosomal release which is proportional to the applied stimulation. The novelty of the present research is in the development of the Ca2+-mediated exosomal dynamics model of neurons accounting for the temperature effects with emphasis on TRPM8-mediated modulations of membrane potential. Further, it has been observed that Ca2+ concentrations in the respective compartments, and thus the overall Ca2+-mediated exosomal dynamics are significantly affected by the changes in temperature and TRPM8 channels. The findings show that TRPM8 and VGCCs play an important role in determining temperature-dependent activation and inactivation at numerous threshold levels. Thermal effects caused by cold detecting neurons cause Ca2+ to be released from the endoplasmic reticulum of primary spiral ganglion neurons. The activation of TRPM8 channels causes Ca2+ release, which is amplified by CICR. TRPM8 channel that colocalizes with the endoplasmic reticulum, is immunostained in the neurons. Indeed, the original and modified Hodgkin/Huxley models have a high degree of qualitative agreement, and the findings of this study are a significant move toward a better understanding of a novel modality for altering neural activity. The developed neuronal model provides an important step not only for our better understanding of the exosomal dynamics in neurons and astrocytes but also paves the way for the generation of new models aiming at optimizing and designing exosome-based drug delivery systems for the treatment of brain pathologies and neurodegenerative disorders such as AD.We presented an integrated mathematical model for therapeutic exosomal release modulated by an externally applied stimulus. The proposed model combines cell activation, intercellular signaling, and exocytosis and allows to shed light on the relative roles of different subcellular via unknown mechanisms , emphasizing its importance. To further substantiate our findings presented on this study, a variety of future investigations into the astrocyte sources and cytotoxic mechanisms of complement proteins in astrocyte-derived exosomes will be needed. However, the definitive etiological relationships between the neuronal accumulation of primary neurotoxic proteins such as amyloid-beta, tau, and reductions in synaptic proteins that contribute to early synaptic dysfunction are now being discovered (Goetzl et al., Ca2+ signaling decrease neuronal interactions and enhance both acute and chronic degenerative diseases of the nervous system. In the present study, we found that due to the biophysical properties of voltage-gated and ligand-activated ion channels and receptors, Ca2+ fluxes through the neuronal membrane are strictly time-constrained. The neural activity could be enhanced by Ca2+-dependent receptors and channels, constantly rearranged as they are embedded in the crowded dynamic environment of biological membranes, allowing for temporary interaction and the creation of transient signals. In a highly dynamic environment, efficient Ca2+-mediated signal transduction necessitates mechanisms that support the very precise spatiotemporal alignment of the Ca2+ source and Ca2+-dependent exosomal exocytosis (De Pittà, Ca2+ signaling are a promising avenue for pharmaceutical intervention in nervous system neurodegenerative diseases, such as AD. Moreover, several intracellular Ca2+ signaling regulators found on the plasma membrane and intracellular organelles have been implicated in many of these pathophysiological processes (Valori et al., Ca2+ channels in synapse formation and function in the healthy central nervous system. Importantly, the previous studies were focused on the effect of temperature and TRPM8 channels on Ca2+-dependent exosome-release, where the temperature has been linked to dementia and may play a role in clinical phenotypes, particularly in the frontotemporal lobar degeneration continuum, but the cause of these symptoms has yet to be determined (Fletcher et al., Ca2+ compartments linked to the integration of experimental mice model data of AD and on the development of a new stochastic model based on the ideas highlighted in this study.Our model supports the view that astrocytes normally serve as neuronal signaling events, but in AD, they transform into astrocyte-derived exosomes, which can disrupt neurons The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.HS: methods and materials, data curation, formal analysis, investigation, writing, and original draft preparation. SS and RM: designed research. SS: methodology, formal analysis, writing, review and editing, and supervision. RM: conceptualization, supervision, and reviews. All authors approved the final submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Mitochondria and ER are established storage organelles controlling Ca2+ dynamics in neurons. Mitochondria also generate a majority of energy used within postsynaptic spines to support the downstream events associated with neuronal stimulus. Recently, high resolution microscopy has unveiled direct contact sites between the ER and the mitochondria (MERCs), which directly channel Ca2+ release from the ER into the mitochondrial membrane. In this study, we develop a computational 3D reaction-diffusion model to investigate the role of MERCs in regulating Ca2+ and ATP dynamics. This spatiotemporal model accounts for Ca2+ oscillations initiated by glutamate stimulus of metabotropic and ionotropic glutamate receptors and Ca2+ changes in four different compartments: cytosol, ER, mitochondria, and the MERC microdomain. Our simulations predict that the organization of these organelles and inter-organellar contact sites play a key role in modulating Ca2+ and ATP dynamics.Spatiotemporal compartmentation of calcium dynamics is critical for neuronal function, particularly in postsynaptic spines. This exquisite level of Ca2+ dynamics in synaptic signaling. Overall, our model predicts that a combination of MERC linkage and mitochondria size is necessary for optimal ATP production in the cytosol.We further show that the crosstalk between geometry (mitochondria and MERC) and metabolic parameters  influences the neuronal energy state. Our findings shed light on the importance of organelle interactions in predicting Ca In excitatory neurons during synaptic transmission, glutamate released from the presynaptic bouton leads to a localized increase in calcium at the postsynaptic dendrite that is critical for the induction of synaptic plasticity3. At the postsynaptic site, small bulbous protrusions called dendritic spines act as biochemical computation units that regulate the duration and spread of postsynaptic calcium fluxes produced by glutamatergic neurotransmission4. The temporal dynamics of calcium, and the coupling to downstream signaling pathways in dendritic spines depend on many factors, including the nature of stimulus as well as the positioning of the calcium storage organelles, the endoplasmic reticulum (ER), and the mitochondria6. A continuous tubular network of ER spreads throughout the dendrites and extends into the spines either as a simple smooth tubule or a spine apparatus9.Compartmentalized calcium handling in postsynaptic structures underlies synaptic communication and controls synaptic plasticity, which is the bidirectional modulation of synaptic strength that is thought to underlie learning and memory formation12. The intimate contact between mitochondria and ER along dendrites allows for a functional inter-organellar coupling and plays a central role in the regulation of the postsynaptic calcium dynamics  production22. While the exact mechanism for the formation of these sites is unclear, there is evidence that binding proteins link via calcium channels on the surface of the respective organelle23. The role of organelles and MERC in calcium dynamics of dendritic spines is not yet fully understood.Physical contact sites between mitochondria and ER  were first observed by electron microscopy, then confirmed by dimerization-dependent fluorescent proteins26. Bertram et al.27, Han et al.28, Wacquier et al.29, and others have created models addressing the role of calcium in mitochondria. Bertram et al.27 simplified a mathematical model for mitochondrial calcium dynamics in pancreatic beta cells originally developed by Magnus et al.30 to explain experimentally derived results31 in which calcium increases NADH under low glucose and reduces NADH in high glucose. Han et al.28 explicitly modeled the role MERC in a pancreatic beta-cell in a compartmental model for the case of a healthy and diabetic cells and predicted a connection between obesity, MERCs, and calcium dynamics. Qi et al. similarly modeled mitochondrial-ER calcium flux into the mitochondria as a function of distance between IP3 receptor (IP3R) and Mitochondrial Calcium Uniporter (MCU) to predict an optimal ER-to-mitochondria distance for the regulation of mitochondrial calcium32. However, these models are not specific to neurons and the unique circumstances of a dendritic spine. Previously, we and others have shown that spatial modeling of signaling pathways can provide insight into how cell shape and organelle organization can affect the spatiotemporal dynamics of second messengers and signaling cascades38. Although spatial models for neuronal calcium signaling exist40, these do not focus on the metabolic consequences of calcium dynamics and do not incorporate mitochondria.Computational modeling has played a pivotal role in providing insight into calcium dynamics in neurons41, mitochondria27, and neuronal calcium signaling models39 to generate a spatial model in dendrites incorporating mitochondria and MERC in calcium signaling and ATP generation , in response to a glutamate stimulus, in the spine head, spine shaft, and the mitochondria? How does the presence or absence of MERCs affect the dynamics of these second messengers and alter the energy landscape in these locations? And finally, how do different geometric features such as mitochondrial length and MERC surface area ratio affect Ca2+ handling and energy generation in spines? To answer these questions, we developed a spatiotemporal model of Ca2+ dynamics in a portion of a dendrite with spatially resolved ER and mitochondria in idealized geometries. We found that the MERC leads to a significant increase in mitochondrial calcium, and subsequently ATP production. Additionally, increasing the surface area of the MERC, increases the calcium influx into the mitochondria, providing insight into how the different extents of MERC can give rise to different calcium states. Finally, we predict metabolic parameters, such as cytosolic ATP hydrolysis, nucleotide transport from mitochondria to the cytoplasm, and rate of ATP synthesis to be key deciding factors in the delicate balance of calcium signaling and energy homeostasis in the postsynaptic spine.In this study, we investigated the role of the ER, mitochondria, and MERC in modulating the spatiotemporal dynamics of calcium and localized ATP production in postsynaptic spines using computational modeling. We combined key cascades and parameters of glutamatergic receptor39, in this study we simplify the complex spine morphology to idealized geometries to focus on the role of mitochondria in neuronal calcium dynamics. While the interconnected ER tubules are widely distributed throughout the cytoplasm, we approximate the ER within the dendritic shaft as long cylinders, keeping ER to cytoplasmic volume ratios constant. The mitochondrion was modeled as a wide cylinder in the center of these four ER. Although dendritic mitochondria have a wide range of sizes from sub micron to to more than 10 μm with complex inner membrane and outer membrane morphologies42, we approximate a relatively small mitochondria of 0.6 μm localized at the base of a dendritic spine. The sizes of the compartments in the model are given in Supplementary Table 45. The details of the model development and numerical methods are given in the supplementary material , the cytosol, one mitochondria, the ER, and a mitochondria ER Contact region (MERC) Fig. a, b. In We conducted simulations with a 1 Hz stimulus of glutamate for 5 s and ran the simulations until an approximate steady state is reached  and one with MERC consisting of 10% of the total surface area  and about half that in the dendritic shaft (2.5 μM) proximal to the stimulated spine. In contrast, the neighboring spine has a peak Ca2+ concentration dynamics in both the stimulated and neighboring spines.When we look at the simulations without MERC Fig. b, we not2+ from the base of the stimulated spine  location and PIP2 hydrolysis from the plasma membrane. The neighboring spine receives IP3 only through diffusion and therefore has a lower concentration and no observable gradients of IP3. When there is no MERC, IP3 concentrations are similar to the case with MERC because IP3 is an early response to mGluR and not affected at these times by Ca2+.Comparing the scenario with and without MERC, we note that the MERC compartment essentially allows for an increased and rapid early release of Ca2+ dynamics in the ER and mitochondria in the same simulation condition as Fig. 3R), rapidly release calcium into the cytosol46. Sacroplasmic/endoplasmic reticulum Ca2+-ATPases (SERCAs) can result in high uptake of Ca2+ into the ER ( > 300 μM47) through the consumption of ATP. Ca2+ is then released from the ER through IP3R and ryanodine receptors (RyRs)48. Ca2+ regulation and mitochondrial ATP production are crucial for synaptic function and neuroplasticity49; mitochondria in synaptic terminals aid neurotransmission by producing ATP, buffering calcium, and local protein translation50. The metabolic cost of synaptic plasticity has been estimated to be between 3.4 and 9.4 × 105 ATP molecules per min and the cost of postsynaptic current is 8.4 × 106 ATP molecules per min  at 10s, 30s, and 60s for mitochondrial and cytosolic ATP to obtain the total number of molecules of ATP available in the system.Next, we investigated the effect of MERCs on Ca2+ dynamics in organelles, consistent with other hypotheses in the literature53. The ER Ca2+ also changes with more decline in the presence MERC  are also altered in the presence of MERCs  is also known to vary19. Do these physical variables have an impact on the production of ATP in the mitochondria? To address this question, we conducted two sets of simulations: (i) vary mitochondrial length while maintaining MERC area fraction, and (ii) vary MERC area fraction in mitochondria of 3 sizes  are required to balance ATP production and supply in dendrites. ANT also drives ADP transport between the cytosol and mitochondria, which is a necessary substrate for ATP production. For effective ATP availability in the dendrites our model predicts two requirements—production of ATP in the mitochondria and availability of ATP in the cytosol. Production of ATP in the mitochondria depends on MERC area fraction and mitochondrial size. Availability of ATP in the cytosol depends on the flux of ATP from the mitochondria through the ANT and the consumption of ATP through a lumped hydrolysis rate.2+, we varied the area fraction of MERCs with mitochondria of different sizes . These lengths are supported by observations that Ca-CaMKII activity can decrease mitochondrial length over a range of synaptic activity levels56. Particularly, we sought to find if increasing MERC area fraction could confer an advantage to smaller mitochondria, compensating for volumetric disadvantage with increased calcium. Experimental evidence indicates that MERCs range from 2 to 20% of mitochondria surface area depending on the metabolic state and type of cell57. We conducted simulations in small (0.3 μm), medium (0.6 μm), and large mitochondria (1.2 μm), with a range of MERC area fractions ranging from 2.5 to 30%  influenced by mitochondrial size and MERC area fraction? We conducted a series of simulations to map the corresponding phase spaces that hold the answer to this question. For all simulations, we varied mitochondrial size from 0.3 to 1.2 μm and MERC area fraction from 0 (no contact) to 0.2. Then, we calculated the AUC for mitochondrial ATP and the AUC for cytosolic ATP as our scalar readout at 10s 56. While our spatial model does not cover the entire dendrite , we note that our model predicts that dendritic energy availability is directly proportional to mitochondrial length. Divakaruni et al. report that mitochondrial length is linearly proportional to dendritic length56; thus the scaling we find here suggests a common size principle for dendritic energy availability. Separately, Hirayabashi and colleagues reported that PDZ Domain-Containing Protein 8 (PDZD8) is required for MERC formation and that the MERC area fraction ranges from 0.02 to 0.16 in HeLa cells, and 0.04 in neurons58.Recently, the mitochondrial dynamics, including fission2+ transfer and impairs activity-dependent cytoplasmic Ca2+ dynamics in neurons15. In addition to having multiple contact sites, the ER forms an elaborate network around mitochondria in neuronal processes66. MERC distribution and ER topology in dendrites are also implicated in the regulation of mitochondrial dynamics and the formation of a spatially stable mitochondrial pool near active dendritic spines to fuel synaptic plasticity67. We note that increasing MERC-to-mitochondria surface area ratios influences ATP production dynamics as well as total ATP level. Although MERCs can have a substantial impact on mitochondrial ATP generation, in contrast to cases without MERC, the MERC area fraction does not show a linear dependence on ATP production as was observed for mitochondrial size.Perturbation of MERCs halts ER-mitochondria Ca2+ dependent increase in oxidative phosphorylation68. We also show that biochemical parameters associated with neuronal metabolism such as ATP production, nucleotide transport, and ATP hydrolysis also play an important role in the cytosolic energy landscape by allowing for a graded behavior. As demonstrated in our model, the spatiotemporal compartmentalization of Ca2+ flux and ATP source via MERCs provides a unique balance between local energy metabolism and synaptic activity. This balance is further regulated in cells by Ca2+-dependent mitochondrial fission to complete the feedback loop of signaling and mitochondrial size regulation, and will require a deeper understanding of the underlying coupled mechanochemical processes69. The subprocesses of synaptic plasticity, including actin remodeling, protein synthesis, receptor trafficking, and phosphorylation require a surge of ATP production62. In our work, MERC’s are able to transiently raise ATP generation at the cost of lower overall energy production.Neuronal activity, while consuming ATP, also stimulates ATP production via a Ca2+ and ATP dynamics72. Second, we note that we assume that Ca2+ and ATP are present in large enough amounts to justify a deterministic approach. We recognize that a stochastic approach would likely give rise to insights in limit cases of few molecules or in noisy environments40. Finally, we note that a more complete Ca2+ influx model would account for voltage-gated calcium channel dynamics as well74. This is a focus of technical development and ongoing effort in our group.We note that our model has made some simplifying assumptions to keep the simulations tractable. First, all our geometries are simplified, allowing us to construct a spatial model of multiple organelles. We note that current efforts include using realistic geometries of spines and organelles derived from microscopy to investigate how these play a role in Ca75, biophysical mechanisms of organelle transport and tethering, and metabolic pathways, giving a glimpse into the complex regulation of synaptic plasticity by functional inter-organellar coupling.This work sets the stage for future investigation on the coupling between biochemical signalingIn this section, we discuss the details of the model development, assumptions, parameters, and numerical methods.We built a model with five compartments: the postsynaptic density (PSD), the cytosol, one mitochondria, the ER, and a mitochondria ER Contact region (MERC) Fig. . In our 33, in this study we simplify complex spine morphology in idealized geometries to focus on the role of mitochondria in neuronal calcium dynamics. While the ER is distributed throughout the cytoplasm, we approximate the ER within the dendritic shaft as long cylinders, keeping volumetric ER to cytoplasm ratios constant. The sizes of these compartments are given in Supplementary Table 24. While we acknowledge that these simplifications do not capture geometric complexity of a dendrite45, they allow us to explore the role of spatial organization of the spine in a computational framework.Although the morphology of a spine has been shown to govern the magnitude and stability of calcium transients in dendrites in previous studies76 to emulate signaling in an established spine. Mathematically, we approximate glutamate stimulus as discrete pulses in a well-mixed system within COMSOL. Since we are not modeling synaptic vesicles or presynaptic neuron signaling, we apply a series of delta functions temporally spaced according to the desired pulse train (i.e. 1 Hz stimulus has delta functions spaced 1 seconds apart). Glutamate then decays with a time constant of 1.2 ms77. Key contributing factors to this decay are rapid reabsorption by the signaling neurons and astrocytic cells as well as diffusion78.While there are a multitude of signaling frequencies and patterns, in this work we focus on a simple periodic stimulus, 1 Hz for 5 s79. The calcium dynamics in our model are based on work done by refs. 80, and parameters were adapted and suitably modified to reflect neuronal calcium metabolism.Postsynaptic area and Receptor Dynamics: This compartment includes membrane-bound molecules on the postsynaptic area: NMDA receptor (NMDAR), free mGluRs (R2), glutamate-bound mGluRs (DIM), phosphorylated mGluRs (DIMp), and second messenger Diacylglycerol (DAG). These molecules localize to the PSD area of spine heads. Calcium enters the spine head through NMDAR. The NMDAR dynamics are described in a multistate model81. NMDAR-induced calcium influx is modeled as:INMDA is the current associated with calcium influx. Mathematical descriptions of all terms are expanded in Supplementary Table qα to separate from Gqβγ and activate PLCβ enzyme. PLCβ enzyme cleaves PIP2 (a membrane phospholipid) and generates DAG and IP3. While DAG remains on the membrane and activates Protein kinase C (PKC), which then may phosphorylate mGluR; phosphatases dephosphorylate mGluR. This phosphorylation can inhibit the dimerization of mGluR. IP3 diffuses through the cytosol and initiates Ca2+ release from internal stores by binding to receptors (IP3R) on the ER membrane. IP3 in the cytosol is degraded to IP2 and IP4. The degradation is assumed to recycle phospholipids such that IP3 production is not rate limited by PIP2 availability. Ca2+ can also be released from ER by Ryanodine receptors and conversely can be actively transported back into the ER by Sarcoplasmic/endoplasmic reticulum calcium-ATPase (SERCA) on the ER membrane. Equations associated with the mGluR cascade are included in Supplementary Table  mGluRs R, glutama mGluRs R, glutamaCytosolic Calcium Dynamics: Ca2+ transients that accumulate due to influx at the PSD are able to diffuse throughout the cytosol. While we do not explicitly model calcium buffers, buffering capacity is included through boundary conditions between compartments. Cytosolic Ca2+ dynamics in 3D is defined as:2 is Laplacian operator in 3D. The boundary condition for Ca2+ influx through NMDAR in postsynaptic area and Ca2+ efflux from cytosol is defined as,mp is a geometric factor for fluxes on the boundary and is defined as the ratio of the cytosol volume to the postsynaptic surface area. JNMDA is defined as the flux as a result of the NMDA receptor activity. Jeff is defined as the activity as a result of plasma membrane calcium ATPase. The boundary fluxes on ER for cytosolic Ca2+ are defined as:c is the buffering capacity of cytosol and α is ER to cytosol volume ratio. 2+ flux from the ER into the cytosol through the IP3 receptor, JRYR is the Ca2+ flux from the ER into the cytosol through the Ryanodine receptor, JSERCA is the Ca2+ flux from the cytosol into the ER through the SERCA ATPase pumps, and me is a geometric factor for fluxes on the boundary and is defined as the ratio of the cytosolic volume to the ER surface area. JRYR are defined with respect to ER volume and JSERCA is defined with respect to the cytosol volume. The mitochondrial boundary fluxes for cytosolic Ca2+ are defined as:β is mitochondria to cytosol volume ratio. JMCU is Ca2+ flux from the cytosol into the mitochondrion through the MCU channel, JNCX is Ca2+ flux from the mitochondrion into the cytosol through Na+/Ca2+ exchanger, and JmPTP is Ca2+ bidirectional flux of the mitochondrial Permeability Transition Pore. All of the fluxes within Eq. 5 are defined with respect to mitochondrion volume.Calcium buffering: Calcium buffering is explicitly included as a volumetric reaction between free calcium and buffer terms, denoted as JBuff.bm, bc, ber and bMERC.Mitochondrial Calcium Dynamics: The spatiotemporal dynamics of mitochondrial Ca2+ is given bym is the buffering capacity of Ca2+ in the mitochondrion. We do not consider the distinction between the outer and inner mitochondrial membrane. Voltage-dependent anion channels transport calcium highly efficiently during neuronal activation. Thus, we may assume, as done in refs. 28, that the calcium concentrations in the cytosol and outer mitochondrial calcium approach a rapid equilibrium. The inner mitochondrial membrane calcium fluxes are dependent solely on the cytosolic calcium. The same applies to fluxes regarding the mitochondria ER contact microdomain, which is represented in this model as a separate compartment.ER Calcium Dynamics: The only variable in ER compartment is Ca2+ in the ER is defined as:e is the buffering capacity of ER. While the endoplasmic reticulum is a sprawling network of interconnected membranes containing a wide range of proteins and ribosomes, we simplify this network into a series of tubes that span the length of the model dendritic shaft.Mito-ER Contacts: The ER is known to closely contact the mitochondria and form junctions known as mitocondria-ER Contacts53. This close proximity, which can be as small as 10 nm and is filled with actin82, calcium binding proteins20, and pathways that inhibit calcium reuptake53. Therefore we model the calcium dynamics in these regions as a separate microdomain from the cytosol. In order to activate IP3R on the ER surface facing this microdomain, IP3 must also be present. Thus there are 2 main components within this microdomain, Ca2+ and IP3. We do not model the molecular composition of the MERC microdomain that control the influx and efflux of Ca2+. Rather, we use the diffusive fluxes at the MERC boundaries as lumped parameters to reflect the different protein distributions across the MERC boundaries. We model the geometry of this microdomain as a shell that surrounds the circumference of the mitochondria and connects to the endoplasmic reticulum. This shape was chosen to reflect EM images of MERCs and to efficiently model the portion of ER in contact with the mitochondria. The calcium concentration within this compartment is given by:After glutamate binds to receptors on the post-synaptic density (PSD), there is a large calcium influx from the extracellular space and endoplasmic reticulum. This signaling initiates at the dendritic spine and propagates to the dendritic shaft, where the mitochondria are primarily located. We assume that the extracellular calcium does not deplete and does not alter the flux of synaptic calcium signaling. Extracellular calcium concentrations are typically several orders of magnitude higher than intracellular calcium27. Our metabolic model focuses on NAD and NADH within the mitochondria, and ATP and ADP in both the mitochondria and cytosol. To avoid modeling the complex spatial patterns of the inner mitochondrial membrane, we assume that ADP conversion into ATP is a volumetric reaction within the mitochondria. Redox and mitochondrial potential are likewise modeled as volumetric terms within the mitochondria.JHYD.In this model, we focus on the impact on calcium on mitochondrial ATP production, as done in2+ concentrations and other variables concentrations in all compartments are high enough to be modeled through a deterministic approach. Simulations were conducted using the commercially available finite-element software COMSOL Multiphysics 5.483. In order to solve our system of partial differential equations, we used time-dependent general partial differential equations and general boundary partial differential equations modules83. Starting with a coarse and unstructured mesh, we decreased the mesh size until we obtained the same results when using the maximum mesh size. COMSOL was allowed to optimize the element sizes through the "physics-controlled mesh" option. The linear system was solved directly by using the PARADISO solver on a Linux-based compute cluster. Newton’s method (nonlinear method) was used to linearize the system. Time integration was performed using a backward differentiation formula (BDF) with both adaptive order and adaptive step sizes. All COMSOL source files will be available on Rangamani lab Github page upon publication.The CaSupplementary Information


Aim: Reperfusion after myocardial ischemia causes cellular injury, in part due to changes in mitochondrial Ca2+ handling, oxidative stress, and myocyte energetics. We have previously shown that the 18-kDa translocator protein of the outer mitochondrial membrane (TSPO) can modulate Ca2+ handling. Here, we aim to evaluate the role of the TSPO in ischemia/reperfusion (I/R) injury.Methods: Rabbit ventricular myocytes underwent simulated acute ischemia (20 min) and reperfusion  in the absence and presence of 50 μM PK11195, a TSPO inhibitor. Cell death was measured by lactate dehydrogenase (LDH) assay, while changes in mitochondrial Ca2+, membrane potential (ΔΨm), and reactive oxygen species (ROS) generation were monitored using confocal microscopy in combination with fluorescent indicators. Substrate utilization was measured with Biolog mitochondrial plates.Results: Cell death was increased by ~200% following I/R compared to control untreated ventricular myocytes. Incubation with 50 μM PK11195 during both ischemia and reperfusion did not reduce cell death but increased mitochondrial Ca2+ uptake and ROS generation. However, application of 50 μM PK11195 only at the onset and during reperfusion effectively protected against cell death. The large-scale oscillations in ΔΨm observed after ~1 h of reperfusion were significantly delayed by 1 μM cyclosporin A and almost completely prevented by 50 μM PK11195 applied during 3 h of reperfusion. After an initial increase, mitochondrial Ca2+, measured with Myticam, rapidly declined during 3 h of reperfusion after the initial transient increase. This decline was prevented by application of PK11195 at the onset and during reperfusion. PK11195 prevented a significant increase in succinate utilization following I/R and succinate-induced forward-mode ROS generation. Treatment with PK11195 was also associated with a significant increase in glutamate and a decrease in leucine utilization.Conclusion: PK11195 administered specifically at the moment of reperfusion limited ROS-induced ROS release and cell death, likely in part, by a shift from succinate to glutamate utilization. These data demonstrate a unique mechanism to limit cardiac injury after I/R. Acute myocardial infarction and ischemic heart disease remain the leading cause of death worldwide . The mosWe and others have shown that the 18-kDa translocator protein (TSPO), previously known as the peripheral benzodiazepine receptor (PBR), likely plays a key role in cardiac injury during heart failure and myocardial infarction . TSPO isInterventions to modulate TSPO expression and function in the heart have had mixed results. We recently demonstrated that genetic cardiospecific ablation of TSPO significantly delayed HF development and improved cardiac bioenergetics in a murine model of pressure overload . In cont2+ handling and lower ROS production. Furthermore, PK11195 strongly inhibited ROS-induced ROS release (RIRR) and cell death when applied at reperfusion. This effect of PK11195 was comparable and even stronger compared to the known mPTP desensitizer, cyclosporin A (CsA), suggesting inhibition of mPTP by PK11195. Given the fact that some studies reported the inhibitory effect of PK11195 on the c subunit of the mitochondrial ATP synthase complex .2+-free minimal essential medium (MEM). The right atrium was excised and the aorta opened to visualize the left coronary ostium, which was then cannulated using a 5-F Judkins right catheter . Perfusion of the left ventricle and left atrium was established before removal of the right ventricular free wall and application of a purse-string suture to secure the catheter in place. Then, the heart was perfused with MEM solution containing 20 μmol/l Ca2+ and 22.5 μg/mg Liberase TH at 37°C until tissue was digested. The left ventricle was cut from the heart and minced, filtered, and washed in a MEM solution containing 50 μmol/l Ca2+ and 10 mg/ml bovine serum albumin. Isolated cells were kept in MEM solution with 50 μmol/l Ca2+ at room temperature (22–24°C) until used for experimentation or placed in a short-term cell culture (see below).For cardiomyocyte isolation, rabbits were subjected to general anesthesia induced with 2 mg/kg propofol followed by 2–5% isoflurane in 100% oxygen . After a2, 2 CaCl2, and 10 Hepes, pH 6.4 for 20 min , inhibition of glycolysis (glucose replaced with 20 mM deoxyglucose), and inhibition of mitochondrial respiration  using glucose-free modified Tyrode solution containing (in mM) 20 2-deoxyglucose, 2 NaCN, 135 NaCl, 4 KCl, 1 MgClr 20 min . Reperfu2+ concentration ([Ca2+]m) were measured in freshly isolated ventricular myocytes using the Ca2+-sensitive fluorescent probe X-Rhod-1/AM  was monitored in intact cardiomyocytes using the potentiometric probe tetramethylrhodamine methyl ester . PI is a cell impermeable fluorescent dye which binds to double-stranded DNA. Therefore, PI will only stain DNA in cells where the plasma membrane has been damaged. About 500 nM propidium iodide was added in the end of reperfusion.Cell death measurements were performed by counting cells which shrunk at the end of experiments and had nuclei positively stained with propidium iodide  for 30 min at 37°C (0), and expressed as ΔF/F0 or as rate of fluorescence increase normalized to basal level. Changes in MitoSox Red fluorescence were recorded every 1 min.Reactive oxygen species generation was measured in freshly isolated myocytes loaded with 0.5 μM MitoSox Red . For this assay, freshly isolated cardiomyocytes were counted, resuspended at 1 × 106 cells/ml density and split into three groups. One group was kept in Tyrode solution, and the two other groups were exposed to simulated ischemia for 20 min. After 20 min, control or ischemia solutions were removed, and cells from the three groups were resuspended in Biolog mitochondrial assay solution (MAS) containing 30 μg/ml saponin and the Redox Dye MC  in the absence or presence of 50 μM PK11195. Next, 30 μl of cells was added into the wells preloaded with different cytosolic and mitochondrial metabolites. Each substrate follows a different route, using different transporters to enter the mitochondria and different dehydrogenases to produce NAD(P)H or FADH2. The electrons travel from the beginning (complex I or II) to the distal portion of the electron transport chain where a tetrazolium redox dye acts as a terminal electron acceptor that turns purple upon reduction. Absorbance at 590 nm was measured using BioTek Synergy Mx multiplate reader. The initial rate of absorbance increase was measured in each experimental group.Mitochondrial substrate utilization was assayed by measuring the rates of electron flow into and through the electron transport chain from metabolic substrates that produce NAD(P)H or FADHCell death was measured by the lactate dehydrogenase (LDH) release into the extracellular medium after 20 min of simulated ischemia and 15 min, 1 h, and 3 h of reperfusion . The LDHt-tests or two-way analysis of variance with Tukey’s post-hoc, using GraphPad Prism version 8.0  and p < 0.05 was considered statistically significant. All data are reported as mean +/−SEM.All data were analyzed using unpaired n = 12) and remained at the same level after 1 h (219 ± 14%) and 3 h (224 ± 14%) of reperfusion (black bars). Cell treatment with 50 μM PK11195 during both ischemia and reperfusion did not prevent cell death on reperfusion, and even exacerbated cell death during ischemia itself . However, 50 μM PK11195 exposure only at the onset of reperfusion effectively prevented cell death at 15 min , 1 h , and 3 h  reperfusion time (blue bars). Time control performed in the absence of ischemia demonstrated that 50 μM PK11195 by itself did not induce cell death (green bars). Therefore, these data suggest that 50 μM PK11195 was amplifying some signaling pathways leading to cell death during ischemia, possibly Ca2+ or ROS or both.To evaluate the effect of PK11195 on cellular death during I/R, freshly isolated rabbit ventricular myocytes were exposed to 20 min of simulated ischemia followed by 15 min, 1 h, and 3 h reperfusion time as shown in 2+ uptake under conditions of heart failure (2+ uptake during I/R. To monitor [Ca2+]m changes during our long protocol for ischemia-reperfusion (more than 3 h), the mitochondrially targeted Ca2+-sensitive fluorescent protein, Mitycam , such that increases in [Ca2+]m are an upward deflection . [Ca2+]m then progressively declined to levels below baseline, reaching normalized levels of −0.196 ± 0.003, n = 10. The initial decline might reflect a recovery of mitochondrial Ca2+ overload, but the further fall below baseline might also reflect some loss of mitochondrial integrity . Upon reperfusion, [Ca2+]m rose transiently, as expected, but then continuously declined during 3 h of reperfusion , but still resulted in major progressive [Ca2+]m decline during the reperfusion  paralleling that seen without drug. However, when 50 μM PK11195 was introduced only during the reperfusion phase, it prevented the progressive [Ca2+]m decline and stabilized [Ca2+]m slightly above the baseline value . These data suggest that PK11195 administration during the reperfusion phase protected against the rapid [Ca2+]m decline, possibly by limiting mPTP opening or promoting MCU-dependent mitochondrial Ca2+ uptake with the subsequent improvement in mitochondrial energetics.Treatment of cardiomyocytes with 50 μM PK11195 during both ischemia and reperfusion, significantly increased mitochondrial m increased during ischemia  to cytochrome c where a tetrazolium redox dye acts as a terminal electron acceptor that turns purple upon reduction.Previous studies have suggested that an increased accumulation of succinate during I/R drives reperfusion injury . Here, wBefore loading into the plates, cells were exposed to simulated ischemia or control Tyrode solution for 20 min, and then the medium was changed to Biolog MAS to simulate reperfusion in the absence or presence of 50 μM PK11195. MAS contained a redox dye and low concentration of digitonin required for plasma membrane permeabilization in order to allow pre-loaded cytosolic and mitochondrial substrates and intermediates to enter the cells. Next, cardiomyocytes were loaded into the 96-well Biolog MitoPlates to examine the major changes in glycolytic metabolites , mitochoHowever, the most significant change during I/R was an increase in the utilization of glycogen and glucose-1-phosphate, which is the product of glycogenolysis, indicating increased reliance on endogenous glucose sources  in simulated ischemia and at the beginning of reperfusion. There was also increased utilization of D-gluconate-6-phosphate (an intermediate in pentose phosphate pathway) and D,L-α-glycerol-phosphate . In contrast, treatment with 50 μM PK11195 on reperfusion reversed the changes observed with simulated I/R, suggesting decreased reliance of the heart on the internal energy sources such as glycogen in order to sustain anaerobic glycolysis.via anaplerosis.Next, using TCA cycle metabolites pre-loaded into the assay plate, we determined that there was a significant decrease in utilization of early TCA cycle intermediates such as citrate, cis-aconitate, isocitrate, and α-ketoglutarate with I/R ,B. DespiThe Biolog assay also allowed us to examine utilization of several amino acids. We found reduced utilization of L-glutamine (−43%) and L-glutamate (−60%), which are precursors for the TCA cycle anaplerotic entry point at the level of α-ketoglutarate, following 20 min of simulated ischemia compared to control cells. Cardiomyocytes treatment with PK11195 during simulated reperfusion increased utilization of both L-glutamine (+54%) and L-glutamate (+32%) even above the levels observed in control cells . Since αTo summarize, our cellular model of simulated ischemia and reperfusion revealed changes in cardiac glucose, amino acids, and fatty acid metabolism similar to that reported in human  and animvia inhibition of succinate utilization. The exact mechanism will require further investigation.In order to test whether succinate utilization during reperfusion was the main source of ROS, we performed experiments where control rabbit myocytes were exposed to a high concentration of cell-permeable succinate, 10 mM of dimethyl succinate. Cells were loaded with superoxide-sensitive fluorescent indicator MitoSox Red to monitor superoxide generation in mitochondria. All cells were electrically-field stimulated at 0.5 Hz to maintain excitation-contraction coupling. p < 0.05). However, when mitochondrial complex I was blocked with 1 μM Rotenone, the maximal superoxide generation was observed in ventricular myocytes, which was blocked by inhibiting complex III of the mitochondrial respiratory chain with AntA. These data suggest that PK11195 did not act as a superoxide scavenger, and its effects were specific for succinate availability and utilization at mitochondrial complex II.Interestingly, we did not observe any significant effect of PK11195 on superoxide generation when mitochondrial complex I activity was stimulated by 5 mM glutamate in combination with 5 mM pyruvate. The rate of mitochondrial superoxide generation was much lower  compared2+]m during reperfusion coupled with limited succinate oxidation. Together, these data suggest a protective role of PK11195 during reperfusion following an ischemic episode.This study has elucidated key actions of PK11195, which is reported to bind to TSPO and change its conformation and activity , on mito2+ uptake and bioenergetics. Our earlier study demonstrated that the endogenous TSPO ligand hemin (protoporphyrin IX) dose-dependently (2.5–10 μM) reduced Ca2+ uptake in isolated heart mitochondria, possibly by modulating the open probability of the VDAC  observed in the closed states  than that in the open state. In agreement with that,  induced a maximal mitochondrial Ca2+ uptake and mPTP activation which was not affected by the VDAC inhibitor G3139. These observations led to the conclusion that VDAC could also be a potential target of PK11195 when used at a higher concentration. However, it could be also explained by the fact that 50 μM PK11195 induced the highest mitochondrial Ca2+ uptake in brain mitochondria. The Ca2+ sensitivity of cardiac mitochondria for mPTP activation could be different compared to brain mitochondria.Transporter protein expression responds to injury, increasing almost 3-fold in experimental heart failure in mice . This inthe VDAC . When ththe VDAC . At conc’s group  demonstrth that,  showed t2+-induced mPTP opening in permeabilized cardiomyocytes or calcium retention capacity in isolated cardiac mitochondria from cardiospecific TSPO KO and corresponding WT control mice. This, however, cannot exclude that TSPO and its modulators could affect mPTP activity indirectly. For example, several studies demonstrated that TSPO ligands 4'-chlorodiazepam and TRO40403 delayed mPTP opening following I/R due to decrease in mitochondrial cholesterol accumulation  subunit  myocytes  by the protein kinase A (PKA), which is recruited to the mitochondria by TSPO in complex with the Golgi Acyl-CoA binding domain containing 3 (ACBD3) protein , and metabolite  transport into and out of mitochondria .In the current experiments, succinate-induced ROS generation was only partially affected by complex I inhibition with rotenone (−40%) and almost completely abolished by the complex III inhibitor AntA −60%; . This invia glycogenolysis was required to maintain cardiac energetics during ischemia and was associated with better functional recovery on reperfusion, but only in the hearts from fasted rats. Interestingly, the increased glycogen levels in fed rats perfused with insulin to increase glucose uptake resulted in worse functional recovery compared control untreated rats.We also demonstrated that PK11195 diminished a significant reliance on glycogen utilization, which possibly contributed to succinate formation during ischemia as suggested by 4  parmination ; or (3) in vivo and large animal studies is required to account for the extent and substrate utilization changes in the working heart. A single concentration of PK11195 was employed based on prior studies , it did not provide any protection. These data emphasize the differences in PK11195 effects in terms of cell survival during I/R with careful attention to the timing of administration – the protective effect was observed only when the drug was administered at the onset and during reperfusion.In summary, using a model of simulated I/R, we demonstrated that PK11195 administration only at reperfusion limited cell death, prevented loss of [CaThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.All animal handling and laboratory procedures were performed in accordance with the approved protocols of the Institutional Animal Care and Use Committee of the University of California, Davis conforming to the Guide for the Care and Use of Laboratory Animals published by the United States National Institute of Health .ED designed and supervised this study and wrote the manuscript. LS, BH, PT, and ED performed the experiments and analyzed the data. SS and DB edited the manuscript and provided the financial support. There are no competing financial interests. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Extensive structural and functional studies have advanced the knowledge of the molecular mechanism by which Orai channels are activated. However, the gating mechanism that allows Ca2+ permeation through Orai channels is less well explained. Here, we reviewed and summarized the existing structural studies of Orai channels. We detailed the structural features of Orai channels, described structural comparisons of their closed and open states, and finally proposed a “push–pull” model of Ca2+ permeation.Orai channels belong to the calcium release-activated calcium (CRAC) channel family. Orai channels are responsible for the influx of extracellular Ca Therefore, maintaining Ca2+ homeostasis inside the ER is required for proper Ca2+ signaling [2+ entry (SOCE) is one of the major pathways of extracellular Ca2+ influx [2+ depletion from the ER, calcium release-activated calcium (CRAC) channels are activated and open to allow Ca2+ influx, resulting in an increase in cytosolic Ca2+ concentrations to generate sustained Ca2+ signals [2+ into cells through CRAC channels plays a major role in many cell types [Calcium ions  and stromal interaction molecules (STIMs) [2+ concentration and then regulate the opening of Orais [The SOCE process requires two components: Ca (STIMs) ,9. Orais (STIMs) ,11,12. Sof Orais ,14.2+ permeation across Orai channels. The role of counterions in the flow of cations was emphasized.Here, we reviewed and summarized what is known about Orai dynamics and functions. We described the molecular features and gating of Orai channels. Furthermore, we discussed the molecular mechanism that allows Ca2+ store via Ca2+ release through IP3 receptors. Subsequently, ER-localized STIMs oligomerize and accumulate at ER–PM junctions. Oligomeric STIMs contact the negatively charged cytosolic face of the PM and directly interact with PM-localized Orai channels through their STIM1–Orai-activating region (SOAR) domains [Three Orai genes (Orai1–3) and two STIM genes (STIM1 and STIM2) have been identified in mammals ,6. Orai1 domains ,17,18,192+ channels, activated CRAC channels show more than 1000-fold higher selectivity for Ca2+ ions than for Na+ or K+ ions [2+ concentration gradient across the PM  drive Ca2+ influx, Orais have extremely low Ca2+ conductance and thus 100-times slower ion permeation than most Ca2+ channels [2+ concentration to approximately 200 fS in the absence of divalent cations [2+ selectivity and slow permeation rate, Orai channels can produce sustained elevations in cytosolic Ca2+ concentration and prevent Ca2+ overload in cells.There are several notable features of activated Orai channels. The current through Orai channels is inwardly rectifying and voltage independent for tens of seconds. Similar to voltage-gated Ca K+ ions . Althougchannels . When ex cations ,23,24. BSTIM-activated Orai channels are essential for immune functions by activating immune response genes in T cells ,25,26. L2+ permeation across unique Ca2+ Orai channels. The first Orai structural information came from the crystal structure of the Drosophila melanogaster Orai (dOrai) at a resolution of 3.35 Å (PDB code: 4HKR) [Structural studies are good approaches to elucidate the molecular mechanism underlying Cae: 4HKR) . The dOr2+ permeation [2+ permeation. The V174A mutant dOrai channel is constitutively activated [2+ permeation  [EM) [The H206A mutation of the dOrai channel (dOrai-H206A), which was shown to generate an activated channel with high selectivity for Ca06Axtal)  and lateal) [EM) . Structual) [EM) .Our group reported the structure of another constitutively active dOrai mutant, P288L (dOrai-P288L), by both X-ray crystallography at a resolution of 4.5 Å and cryo-electron microscopy at an overall resolution of 5.7 Å . Compare2+ influx without attenuating the association of STIM1 with hOrai1 [2+ influx [Within each protomer, the conformational transduction pathway starts at the peripheral TM4 helix and continues along the middle TM3 helix to the basic section of the N-terminal region of the TM1 helix inset in f. Mutatih hOrai1 . Further+ influx ,40. Thes+ influx .2+ influx in the closed state. However, contradicting this proposal, mutations, such as R83A-K87A and R77A-K78A in the constitutively mutant hOrai1-P245L [2+. These results indicated that the basic region near the cytosolic face of the pore may play some unexpected but important roles in Ca2+ permeation.In the open state dOrai-P288L structure and the closed state dOrai structure, an anion binding pocket was observed around the basic section ,39. The i1-P245L , were in2+ channels. Understanding the molecular details underlying how Ca2+ permeates through Orai pores will definitely advance the knowledge of ion conduction and chemical screening against related diseases. Both membrane potential and a 20,000-fold Ca2+ concentration gradient across the PM produce a substantial driving force for Ca2+ permeation through other Ca2+-conducting channels, but not through Orai channels. Ca2+ permeating through the pores of Orai channels must pass the selection filter, the unusual hydrophobic gate and the rarely seen positively charged blocker, as shown by structural investigation [2+ permeation; in this model, the pore helix is rotated to move the hydrophobic gate away from the central pore axis upon channel activation [2+ permeation [2+ permeation [Orai channels are unique among all kinds of Catigation . Yamashitivation . Later, rmeation . Althougrmeation ,46.2+ permeation model”, which fits well with published functional results [2+ permeation model”, to a shorter name, “push–pull model” (2+ gradient drives Ca2+ from the extracellular space into the pore. Inside the pore, the front Ca2+ will be pushed along the pore axis by the back Ca2+ due to cation–cation repulsion. Additionally, the positively charged blocker undergoes dilation and exposes positive charges toward the cytosol to aggregate anions. Thus, the Ca2+ inside the pore will be pulled by the anions located at the cytosolic end of the pore. The combination of the push force and the pull force presumably facilitates Ca2+ permeation through the hydrophobic gate.Recently, our group proposed an “anion-assisted Ca results . Here, wl model” . Upon th2+ channels, have provided an extensive understanding of channel assembly, ion selection, gating mechanisms and Ca2+ permeation. However, to date, only dOrai structure information has been obtained, and structural information on human Orai channels is lacking. Will the mechanism by which hOrai channels assemble be similar to that by which dOrai channels assemble? In earlier studies, Orai channels were proposed to be tetramers [Structural studies of Orais, very unique Caetramers ,48 or peetramers . Until tetramers , Orai chetramers ,51. Ther