Intracellular Calcium Dynamics Research Articles

Advancing Cardiovascular Drug Screening Using Human Pluripotent Stem Cell-Derived Cardiomyocytes.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have emerged as a promising tool for studying cardiac physiology and drug responses. However, their use is largely limited by an immature phenotype and lack of high-throughput analytical methodology. In this study, we developed a high-throughput testing platform utilizing hPSC-CMs to assess the cardiotoxicity and effectiveness of drugs. Following an optimized differentiation and maturation protocol, hPSC-CMs exhibited mature CM morphology, phenotype, and functionality, making them suitable for drug testing applications. We monitored intracellular calcium dynamics using calcium imaging techniques to measure spontaneous calcium oscillations in hPSC-CMs in the presence or absence of test compounds. For the cardiotoxicity test, hPSC-CMs were treated with various compounds, and calcium flux was measured to evaluate their effects on calcium dynamics. We found that cardiotoxic drugs withdrawn due to adverse drug reactions, including encainide, mibefradil, and cetirizine, exhibited toxicity in hPSC-CMs but not in HEK293-hERG cells. Additionally, in the effectiveness test, hPSC-CMs were exposed to ATX-II, a sodium current inducer for mimicking long QT syndrome type 3, followed by exposure to test compounds. The observed changes in calcium dynamics following drug exposure demonstrated the utility of hPSC-CMs as a versatile model system for assessing both cardiotoxicity and drug efficacy. Overall, our findings highlight the potential of hPSC-CMs in advancing drug discovery and development, which offer a physiologically relevant platform for the preclinical screening of novel therapeutics.

A silicon diode–based optoelectronic interface for bidirectional neural modulation

The development of advanced neural modulation techniques is crucial to neuroscience research and neuroengineering applications. Recently, optical-based, nongenetic modulation approaches have been actively investigated to remotely interrogate the nervous system with high precision. Here, we show that a thin-film, silicon (Si)-based diode device is capable to bidirectionally regulate in vitro and in vivo neural activities upon adjusted illumination. When exposed to high-power and short-pulsed light, the Si diode generates photothermal effects, evoking neuron depolarization and enhancing intracellular calcium dynamics. Conversely, low-power and long-pulsed light on the Si diode hyperpolarizes neurons and reduces calcium activities. Furthermore, the Si diode film mounted on the brain of living mice can activate or suppress cortical activities under varied irradiation conditions. The presented material and device strategies reveal an innovated optoelectronic interface for precise neural modulations.

Microscale geometrical modulation of PIEZO1 mediated mechanosensing through cytoskeletal redistribution

The microgeometry of the cellular microenvironment profoundly impacts cellular behaviors, yet the link between it and the ubiquitously expressed mechanosensitive ion channel PIEZO1 remains unclear. Herein, we describe a fluorescent micropipette aspiration assay that allows for simultaneous visualization of intracellular calcium dynamics and cytoskeletal architecture in real-time, under varied micropipette geometries. By integrating elastic shell finite element analysis with fluorescent lifetime imaging microscopy and employing PIEZO1-specific transgenic red blood cells and HEK cell lines, we demonstrate a direct correlation between the microscale geometry of aspiration and PIEZO1-mediated calcium signaling. We reveal that increased micropipette tip angles and physical constrictions lead to a significant reorganization of F-actin, accumulation at the aspirated cell neck, and subsequently amplify the tension stress at the dome of the cell to induce more PIEZO1’s activity. Disruption of the F-actin network or inhibition of its mobility leads to a notable decline in PIEZO1 mediated calcium influx, underscoring its critical role in cellular mechanosensing amidst geometrical constraints.

Exploring the interplay of intrinsic fluctuation and complexity in intracellular calcium dynamics

The concentration of intracellular calcium ion (Ca2＋) exhibits complex oscillations, including bursting and chaos, as observed experimentally. These dynamics are influenced by inherent fluctuations within cells, which serve as crucial determinants in cellular decision-making processes and fate determination. In this study, we systematically explore the interplay between intrinsic fluctuation and the complexity of various dynamic states of intracellular cytosolic Ca2＋. To investigate this interplay, we employ complexity measures such as permutation entropy and statistical complexity. Using a stochastic chemical Langevin equation, we simulate the dynamics of cytosolic Ca2＋. Our findings reveal that permutation entropy and statistical complexity effectively characterize the diverse, dynamic states of cytosolic Ca2＋ and illustrate their interactions with intrinsic fluctuation. Permutation entropy analysis elucidates that the chaotic state is more sensitive to intrinsic fluctuation than the other periodic states. Furthermore, we identify distinct states of cytosolic Ca2＋ occupying specific locations within the theoretical bounds of the complexity–entropy causality plane. These locations indicate varying complexity and information content as intrinsic fluctuation varies. When adjusting the permutation order, the statistical complexity measure for the different periodic and chaotic states exhibits peaks in an intermediate range of intrinsic fluctuation values. Additionally, we identify scale-free or fractal patterns in this intermediate range, which are further corroborated by multifractal detrended fluctuation analysis. These high-complexity states likely correspond to optimal Ca2＋ dynamics with biological significance, revealing rich and complex dynamics shaped by the interplay of intrinsic fluctuation and complexity. Our investigation enhances our understanding of the intricate regulatory mechanisms governing intracellular Ca2＋ dynamics and how intrinsic fluctuation modulates the complexity of the various dynamics that play crucial roles in biological cells.

Angiotensin II-mediated oxidative stress increased the vulnerability of ventricular arrhythmia in cardiac hypertrophy rabbit model, which is suppressed by CaMKII inhibitor

Abstract Objectives Angiotensin system is a major cause of heart failure and arrhythmia. Despite a lack of direct evidence that oxidative stress causes ventricular arrhythmia, reversal of oxidative stress is considered a plausible therapy. This study evaluated the Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitor could suppress arrhythmia in cardiac hypertrophy rabbit model. Methods Angiotensin II (Ang II) or saline was administrated for 2 weeks via osmotic minipumps implanted subcutaneously in the midclavicular region. Hearts were perfused, mapped optically to analyze action potential durations (APD), and restitution kinetics, and tested for VF vulnerability. The intracellular calcium dynamics were measured in cardiomyocyte treated with Ang II (10 ng/ml) for 1 hours. Results In Ang II rabbit groups, 2(30%) rabbits died and had ventricular arrhythmia and hearts had enlarged left ventricle, longer APD90, slower conduction velocity (CV; P<0.01 versus control) and higher levels of transcripts for CaMKII, PLB, RyR2 (P<0.05 versus control). N-acetylcysteine treatment reversed the transcripts for APD90 and CV (P<0.01). Programmed stimulation triggered VF in Ang II (n=5/6) and Ang II + saline (n=4/6), but not in control (n=0/10, P<0.01). Cardiomyocyte induced with Ang II showed increased spontaneous Ca2+ release (Ca2+ Wave Frequency; 1.0±0.0 vs. 5.1±0.5, p=0.001, Ca2+ Amplitude; 1.0±0.0 vs. 1.3±0.1, p=0.001) with control. CaMKII inhibitor reversed the change of Ca2+. Conclusion The Ang II group had an increased incidence of arrhythmia caused by increased phosphorylation of Ca2+ handling proteins. These changes were partially reversed by CaMKII inhibition.

IL-10 and Cdc42 modulate astrocyte-mediated microglia activation in methamphetamine-induced neuroinflammation.

Methamphetamine (Meth) use is known to induce complex neuroinflammatory responses, particularly involving astrocytes and microglia. Building upon our previous research, which demonstrated that Meth stimulates astrocytes to release tumor necrosis factor (TNF) and glutamate, leading to microglial activation, this study investigates the role of the anti-inflammatory cytokine interleukin-10 (IL-10) in this process. Our findings reveal that the presence of recombinant IL-10 (rIL-10) counteracts Meth-induced excessive glutamate release in astrocyte cultures, which significantly reduces microglial activation. This reduction is associated with the modulation of astrocytic intracellular calcium (Ca2+) dynamics, particularly by restricting the release of Ca2+ from the endoplasmic reticulum to the cytoplasm. Furthermore, we identify the small Rho GTPase Cdc42 as a crucial intermediary in the astrocyte-to-microglia communication pathway under Meth exposure. By employing a transgenic mouse model that overexpresses IL-10 (pMT-10), we also demonstrate in vivo that IL-10 prevents Meth-induced neuroinflammation. These findings not only enhance our understanding of Meth-related neuroinflammatory mechanisms, but also suggest IL-10 and Cdc42 as putative therapeutic targets for treating Meth-induced neuroinflammation.

Intracellular Calcium Dynamics in Primary Human Adrenocortical Cells Deciphered with a Novel Pipeline.

The fluctuations of the intracellular Ca2+ concentration ([Ca2+]i) are key physiological signals for cell function under normal conditions and can undergo profound alterations in disease states, as high blood pressure due to endocrine disorders like primary aldosteronism (PA). However, when assessing such fluctuations several parameters in the Ca2+ signal dynamics need to be considered, which renders their assessmentchallenging. Aimto develop an observer-independent custom-made pipeline to analyze Ca2+ dynamics in terms of frequency and peak parameters, as amplitude, full width at half maximum (FWHM) and area under the curve (AUC). We applied a custom-made methodology to aldosterone-producing adenoma (APA) and APA adjacent cells (AAC) and found this pipeline to be suitable for monitoring and processing a wide-range of [Ca2+]i events in these cell types delivering reproducible results. The designed pipeline can provide a useful tool for [Ca2+]i signal analysis that allows comparisons of Ca2+ dynamics not only in PA, but in other cell phenotypes that are relevant for the regulation of blood pressure.

Exploring Ca2+ Dynamics in Myelinating Oligodendrocytes through rAAV-Mediated jGCaMP8s Expression in Developing Spinal Cord Organ Cultures.

Oligodendrocytes, the myelin-producing glial cells of the central nervous system (CNS), crucially contribute to myelination and circuit function. An increasing amount of evidence suggests that intracellular calcium (Ca2+) dynamics in oligodendrocytes mediates activity-dependent and activity-independent myelination. Unraveling how myelinating oligodendrocytes orchestrate and integrate Ca2+ signals, particularly in relation to axonal firing, is crucial for gaining insights into their role in the CNS development and function, both in health and disease. In this framework, we used the recombinant adeno-associated virus/Olig001 capsid variant to express the genetically encoded Ca2+ indicator jGCaMP8s, under the control of the myelin basic protein promoter. In our study, this tool exhibits excellent tropism and selectivity for myelinating and mature oligodendrocytes, and it allows monitoring Ca2+ activity in myelin-forming cells, both in isolated primary cultures and organotypic spinal cord explants. By live imaging of myelin Ca2+ events in oligodendrocytes within organ cultures, we observed a rapid decline in the amplitude and duration of Ca2+ events across different in vitro developmental stages. Active myelin sheath remodeling and growth are modulated at the level of myelin-axon interface through Ca2+ signaling, and, during early myelination in organ cultures, this phase is finely tuned by the firing of axon action potentials. In the later stages of myelination, Ca2+ events in mature oligodendrocytes no longer display such a modulation, underscoring the involvement of complex Ca2+ signaling in CNS myelination.

Nonlinear super-resolution signal processing allows intracellular tracking of calcium dynamics

Objective. Traditional quantification of fluorescence signals, such as ΔF/F , relies on ratiometric measures that necessitate a baseline for comparison, limiting their applicability in dynamic analyses. Our goal here is to develop a baseline-independent method for analyzing fluorescence data that fully exploits temporal dynamics to introduce a novel approach for dynamical super-resolution analysis, including in subcellular resolution. Approach. We introduce ARES (Autoregressive RESiduals), a novel method that leverages the temporal aspect of fluorescence signals. By focusing on the quantification of residuals following linear autoregression, ARES obviates the need for a predefined baseline, enabling a more nuanced analysis of signal dynamics. Main result. We delineate the foundational attributes of ARES, illustrating its capability to enhance both spatial and temporal resolution of calcium fluorescence activity beyond the conventional ratiometric measure ( ΔF/F ). Additionally, we demonstrate ARES’s utility in elucidating intracellular calcium dynamics through the detailed observation of calcium wave propagation within a dendrite. Significance. ARES stands out as a robust and precise tool for the quantification of fluorescence signals, adept at analyzing both spontaneous and evoked calcium dynamics. Its ability to facilitate the subcellular localization of calcium signals and the spatiotemporal tracking of calcium dynamics—where traditional ratiometric measures falter—underscores its potential to revolutionize baseline-independent analyses in the field.

Loss of glycine receptors in the nucleus accumbens and ethanol reward in an Alzheimer´s Disease mouse model

Alterations in cognitive and non-cognitive cerebral functions characterize Alzheimer's disease (AD). Cortical and hippocampal impairments related to extracellular accumulation of Aβ in AD animal models have been extensively investigated. However, recent reports have also implicated intracellular Aβ in limbic regions, such as the nucleus accumbens (nAc). Accumbal neurons express high levels of inhibitory glycine receptors (GlyRs) that are allosterically modulated by ethanol and have a role in controlling its intake. In the present study, we investigated how GlyRs in the 2xTg mice (AD model) affect nAc functions and ethanol intake behavior. Using transgenic and control aged-matched litter mates, we found that the GlyRα2 subunit was significantly decreased in AD mice (6-month-old). We also examined intracellular calcium dynamics using the fluorescent calcium protein reporter GCaMP in slice photometry. We also found that the calcium signal mediated by GlyRs, but not GABAAR, was also reduced in AD neurons. Additionally, ethanol potentiation was significantly decreased in accumbal neurons in the AD mice. Finally, we performed drinking in the dark (DID) experiments and found that 2xTg mice consumed less ethanol on the last day of DID, in agreement with a lower blood ethanol concentration. 2xTg mice also showed lower sucrose consumption, indicating that overall food reward was altered. In conclusion, the data support the role of GlyRs in nAc neuron excitability and a decreased glycinergic activity in the 2xTg mice that might lead to impairment in reward processing at an early stage of the disease.

Machine learning predictions of T cell antigen specificity from intracellular calcium dynamics.

Adoptive T cell therapies rely on the production of T cells with an antigen receptor that directs their specificity toward tumor-specific antigens. Methods for identifying relevant T cell receptor (TCR) sequences, predominantly achieved through the enrichment of antigen-specific T cells, represent a major bottleneck in the production of TCR-engineered cell therapies. Fluctuation of intracellular calcium is a proximal readout of TCR signaling and candidate marker for antigen-specific T cell identification that does not require T cell expansion; however, calcium fluctuations downstream of TCR engagement are highly variable. We propose that machine learning algorithms may allow for T cell classification from complex datasets such as polyclonal T cell signaling events. Using deep learning tools, we demonstrate accurate prediction of TCR-transgenic CD8+ T cell activation based on calcium fluctuations and test the algorithm against T cells bearing a distinct TCR as well as polyclonal T cells. This provides the foundation for an antigen-specific TCR sequence identification pipeline for adoptive T cell therapies.

The bioelectric mechanisms of local calcium dynamics in cancer cell proliferation: an extension of the A549 in silico cell model.

Advances in molecular targeting of ion channels may open up new avenues for therapeutic approaches in cancer based on the cells' bioelectric properties. In addition to in-vitro or in-vivo models, in silico models can provide deeper insight into the complex role of electrophysiology in cancer and reveal the impact of altered ion channel expression and the membrane potential on malignant processes. The A549 in silico model is the first computational cancer whole-cell ion current model that simulates the bioelectric mechanisms of the human non-small cell lung cancer cell line A549 during the different phases of the cell cycle. This work extends the existing model with a detailed mathematical description of the store-operated Ca2+ entry (SOCE) and the complex local intracellular calcium dynamics, which significantly affect the entire electrophysiological properties of the cell and regulate cell cycle progression. The initial model was extended by a multicompartmental approach, addressing the heterogenous calcium profile and dynamics in the ER-PM junction provoked by local calcium entry of store-operated calcium channels (SOCs) and uptake by SERCA pumps. Changes of cytosolic calcium levels due to diffusion from the ER-PM junction, release from the ER by RyR channels and IP3 receptors, as well as corresponding PM channels were simulated and the dynamics evaluated based on calcium imaging data. The model parameters were fitted to available data from two published experimental studies, showing the function of CRAC channels and indirectly of IP3R, RyR and PMCA via changes of the cytosolic calcium levels. The proposed calcium description accurately reproduces the dynamics of calcium imaging data and simulates the SOCE mechanisms. In addition, simulations of the combined A549-SOCE model in distinct phases of the cell cycle demonstrate how Ca2+ - dynamics influence responding channels such as KCa, and consequently modulate the membrane potential accordingly. Local calcium distribution and time evolution in microdomains of the cell significantly impact the overall electrophysiological properties and exert control over cell cycle progression. By providing a more profound description, the extended A549-SOCE model represents an important step on the route towards a valid model for oncological research and in silico supported development of novel therapeutic strategies.

Effects of Remimazolam on Intracellular Calcium Dynamics in Myotubes Derived from Patients with Malignant Hyperthermia and Functional Analysis of Type 1 Ryanodine Receptor Gene Variants.

Remimazolam is a novel general anesthetic and its safety in patients with malignant hyperthermia (MH) is unknown. We used myotubes derived from the skeletal muscle of patients with MH to examine the response to ryanodine receptor 1 (RYR1) agonist and remimazolam in MH-susceptible patients. Patients underwent muscle biopsy for the Ca2+-induced Ca2+ release (CICR) rate test, a diagnostic tool for MH in Japan. Ten patients had myotubes obtained from skeletal muscle cultures, and the genes associated with malignant hyperthermia in these patients were analyzed. The EC50 of caffeine, cresol, and remimazolam to induce intracellular calcium concentration change were compared between myotubes from CICR-negative genetic test patients and myotubes from other patients. Eight of the ten were CICR-positive, five of whom had RYR1 causative gene mutations or variants. Two patients had CICR-negative genetic tests, and as expected had the highest EC50 (the concentration of a drug that gives a half-maximal response) in response to caffeine, 4CmC and remimazolam. Three patients had a positive CICR but no known variants in RYR1 or CACNA1S (voltage-gated calcium channel subunit alpha1S). Myotubes in these patients had significantly lower EC50s for all agents than myotubes in CICR-negative patients. When myotubes from a patient who was CICR-negative and had no gene variant were used as a control, myotubes from CICR-positive patients were more hyper-responsive than controls to all stimulants used. The EC50 for remimazolam was lowest for myotubes from CICR-positive, RYR1-mutant patients, at 206 µM (corresponding to 123 µg/mL). The concentration was more than 80-times higher than the clinical concentration. RYR1 gene variants in R4645Q and W5020G were shown to be causative gene mutations for MH. Intracellular calcium in myotubes from MH patients are elevated at high concentrations of remimazolam but not at clinically used concentrations of remimazolam. Remimazolam appears to be safe to use in patients with MH.

A microfluidic array enabling generation of identical biochemical stimulating signals to trapped biological cells for single-cell dynamics

Microfluidic-based analyses of single-cell dynamics in response to dynamic biochemical signals are emerging as pivotal approaches for investigating the effects of extracellular microenvironmental biochemical factors on cellular structure, function, and behavior. However, current devices often fail to consistently apply identical dynamic biochemical signals to trapped cells. In this study, we introduce a novel radially distributed single-cell trapping microfluidic array, designed to quantitatively and consistently apply identical biochemical stimulating signals to each trapped cell. Numerical simulations were employed to optimize microchannel geometry, enhancing trapping efficiency while minimizing signal distortion. Experimental validation demonstrated the trapping success rate and the single-cell trapping efficiency exceeding 99% and 85%, respectively. The microarray's capability to deliver identical dynamic biochemical stimulating signals, with various waveforms, to each unit was confirmed through fluorescein transport tests. Furthermore, we examined the intracellular calcium dynamics of U-2 OS human osteosarcoma cells in response to dynamic ATP signals, observing both single-peak calcium responses and calcium oscillations, which were modelled by a second-order system with a natural frequency of 1.6 mHz. Overall, our proposed microfluidic array offers a robust and valuable framework for advancing the understanding of single-cell dynamics.

Modeling of olfactory transduction in AWCON neuron via coupled electrical-calcium dynamics.

Amphid wing "C" (AWC) neurons are among the most important and studied neurons of the nematode Caenorhabditis elegans. In this work, we unify the existing electrical and intracellular calcium dynamics descriptions to obtain a biophysically accurate model of olfactory transduction in AWCON neurons. We study the membrane voltage and the intracellular calcium dynamics at different exposure times and odorant concentrations to grasp a complete picture of AWCON functioning. Moreover, we investigate the complex cascade of biochemical processes that allow AWC activation upon odor removal. We analyze the behavior of the different components of the models and, by suppressing them selectively, we extrapolate their contribution to the overall neuron response and study the resilience of the dynamical system. Our results are all in agreement with the available experimental data. Therefore, we provide an accurate mathematical and biophysical model for studying olfactory signal processing in C. elegans.

Insights on the mechanism of flecainide in catecholaminergic polymorphic ventricular tachycardia.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmia syndrome characterized by defective cardiac ryanodine receptor (RyR2) calcium release during times of adrenergic stimulation, resulting in bidirectional or polymorphic ventricular tachycardia. Flecainide is a class 1c anti-arrhythmic drug that has demonstrated therapeutic efficacy in treating CPVT. However, its mechanism of action remains disputed. One group proposes a direct effect of flecainide on RyR2-mediated calcium release, while another proposes an indirect effect via sodium channel blockade and modulation of intracellular calcium dynamics. In light of recent studies, this commentary aims to explore and discuss the evidence base for these potential mechanisms.

Neuronal actin cytoskeleton gain of function in the human brain

While advancements in imaging techniques have led to major strides in deciphering the human brain, successful interventions are elusive and represent some of the most persistent translational gaps in medicine. Human restricted CHRFAM7A has been associated with neuropsychiatric disorders. The physiological role of CHRFAM7A in human brain is explored using multiomics approach on 600 post mortem human brain tissue samples. The emerging pathways and mechanistic hypotheses are tested and validated in an isogenic hiPSC model of CHRFAM7A knock-in medial ganglionic eminence progenitors and neurons. CHRFAM7A is identified as a modulator of intracellular calcium dynamics and an upstream regulator of Rac1. Rac1 activation re-designs the actin cytoskeleton leading to dynamic actin driven remodeling of membrane protrusion and a switch from filopodia to lamellipodia. The reinforced cytoskeleton leads to an advantage to tolerate stiffer mechanical properties of the extracellular environment. CHRFAM7A modifies the actin cytoskeleton to a more dynamic and stiffness resistant state in an α7nAChR dependent manner. CHRFAM7A may facilitate neuronal adaptation to changes in the brain environment in physiological and pathological conditions contributing to risk or recovery. Understanding how CHRFAM7A affects human brain requires human studies in the areas of memory formation and erasure, cognitive reserve, and neuronal plasticity. This work is supported in part by the Community Foundation for Greater Buffalo (Kinga Szigeti). Also, in part by the International Society for Neurochemistry (ISN) and The Company of Biologists (Nicolas Rosas). ROSMAP is supported by NIA grants P30AG10161, P30AG72975, R01AG15819, R01AG17917. U01AG46152, and U01AG61356.

Target identification and validation of the alpha7 nicotinic acetylcholine receptor as a potential therapeutic target in retinal disease.

The role of acetylcholine (ACh) in visual processing in the mammalian retina has been the focus of research for many decades. Pioneering work on the localization of ACh discovered that the neurotransmitter is synthesized and stored in a distinct subpopulation of amacrine (starburst) cells. It has been shown that ACh release is regulated to a low resting "tonic" level, much like what is observed at the neuromuscular junction (NMJ). If there were a dysfunction in the tonic release of ACh, might post-synaptic changes render the targets of ACh [i.e., retinal ganglion cells (RGCs)] vulnerable to disease? During my time at Pharmacia & Upjohn (PNU), selective nicotinic ACh receptor (nAChR) agonists (e.g., PNU-282987) were developed as a possible therapy for central nervous system (CNS) diseases. As RGCs are the main targets of neurodegeneration in glaucoma, could the activation of this target provide neuroprotection? In response to this question, experiments to identify alpha7 nAChRs in the retina (i.e., target ID studies) followed by "proof-of-concept" experiments were conducted. Target ID studies included binding studies with retinal homogenates, [125I]-alpha-bungarotoxin (α-BTX) autoradiography, and fluorescently tagged α-BTX binding in retinal slices. Imaging studies of intracellular calcium dynamics in the retinal slice were conducted. Reverse transcription-polymerase chain reaction (RT-PCR) analysis with alpha7 nAChR knockout mice using the "laser-capture microdissection" technique, in situ hybridization studies, and RT-PCR analysis of the human retina were conducted. Collectively, these experiments confirmed the presence of alpha7 nAChRs on specific cells in the retina. "Proof-of-concept" neuroprotection studies demonstrated that PNU-282987 provided significant protection for RGCs. This protection was dose dependent and was blocked with selective antagonists. More recently, evidence for the generation of new RGCs has been reported with PNU-282987 in rodents. Interestingly, the appearance of new RGCs is more pronounced with eye-drop application than with intravitreal injection. One could postulate that this reflects the neurogenic activation of alpha7 receptors on the retinal pigment epithelium (RPE) (eye drops) vs. a neuroprotective effect on RGCs (injections). In conclusion, there does appear to be a cholinergic retinal "tone" associated with RGCs that could be utilized as a neuroprotective therapy. However, a distinct cholinergic neurogenic mechanism also appears to exist in the outer retina that could possibly be exploited to generate new RGCs lost through various disease processes.

Regulation of specific abnormal calcium signals in the hippocampal CA1 and primary cortex M1 alleviates the progression of temporal lobe epilepsy.

Temporal lobe epilepsy is a multifactorial neurological dysfunction syndrome that is refractory, resistant to antiepileptic drugs, and has a high recurrence rate. The pathogenesis of temporal lobe epilepsy is complex and is not fully understood. Intracellular calcium dynamics have been implicated in temporal lobe epilepsy. However, the effect of fluctuating calcium activity in CA1 pyramidal neurons on temporal lobe epilepsy is unknown, and no longitudinal studies have investigated calcium activity in pyramidal neurons in the hippocampal CA1 and primary motor cortex M1 of freely moving mice. In this study, we used a multi-channel fiber photometry system to continuously record calcium signals in CA1 and M1 during the temporal lobe epilepsy process. We found that calcium signals varied according to the grade of temporal lobe epilepsy episodes. In particular, cortical spreading depression, which has recently been frequently used to represent the continuously and substantially increased calcium signals, was found to correspond to complex and severe behavioral characteristics of temporal lobe epilepsy ranging from grade II to grade V. However, vigorous calcium oscillations and highly synchronized calcium signals in CA1 and M1 were strongly related to convulsive motor seizures. Chemogenetic inhibition of pyramidal neurons in CA1 significantly attenuated the amplitudes of the calcium signals corresponding to grade I episodes. In addition, the latency of cortical spreading depression was prolonged, and the above-mentioned abnormal calcium signals in CA1 and M1 were also significantly reduced. Intriguingly, it was possible to rescue the altered intracellular calcium dynamics. Via simultaneous analysis of calcium signals and epileptic behaviors, we found that the progression of temporal lobe epilepsy was alleviated when specific calcium signals were reduced, and that the end-point behaviors of temporal lobe epilepsy were improved. Our results indicate that the calcium dynamic between CA1 and M1 may reflect specific epileptic behaviors corresponding to different grades. Furthermore, the selective regulation of abnormal calcium signals in CA1 pyramidal neurons appears to effectively alleviate temporal lobe epilepsy, thereby providing a potential molecular mechanism for a new temporal lobe epilepsy diagnosis and treatment strategy.

Unveiling the impact of lysosomal ion channels: balancing ion signaling and disease pathogenesis.

Ion homeostasis, which is regulated by ion channels, is crucial for intracellular signaling. These channels are involved in diverse signaling pathways, including cell proliferation, migration, and intracellular calcium dynamics. Consequently, ion channel dysfunction can lead to various diseases. In addition, these channels are present in the plasma membrane and intracellular organelles. However, our understanding of the function of intracellular organellar ion channels is limited. Recent advancements in electrophysiological techniques have enabled us to record ion channels within intracellular organelles and thus learn more about their functions. Autophagy is a vital process of intracellular protein degradation that facilitates the breakdown of aged, unnecessary, and harmful proteins into their amino acid residues. Lysosomes, which were previously considered protein-degrading garbage boxes, are now recognized as crucial intracellular sensors that play significant roles in normal signaling and disease pathogenesis. Lysosomes participate in various processes, including digestion, recycling, exocytosis, calcium signaling, nutrient sensing, and wound repair, highlighting the importance of ion channels in these signaling pathways. This review focuses on different lysosomal ion channels, including those associated with diseases, and provides insights into their cellular functions. By summarizing the existing knowledge and literature, this review emphasizes the need for further research in this field. Ultimately, this study aims to provide novel perspectives on the regulation of lysosomal ion channels and the significance of ion-associated signaling in intracellular functions to develop innovative therapeutic targets for rare and lysosomal storage diseases.