Calcium-dependent Processes Research Articles

A comparative study of the effects of Pr3+ and La3+ ions on calcium dependent processes in frog cardiac muscle and rat heart mitochondria

The inotropic effect of Pr3+ and La3+ ions on the heart muscle of frog Rana ridibunda, as well as the influence of the ions on respiration, swelling, and the potential (ΔΨmito) on the inner membrane of Ca2+- loaded rat heart mitochondria, energized by glutamate and malate or succinate in the presence of rotenone were studied. It was found that 2 mM Pr3+ in Ringer’s solution reduces the force of spontaneous contractions and those induced by electrical stimulation in the heart; it had a negative chronotropic effect, decreasing the frequency of spontaneous contractions. Pr3+ and La3+ prevented a decrease in the 2,4-dinitrophenol (DNP)- uncoupled respiration of energized rat heart mitochondria, swelling of these organelles in salt media, and a reduction in ΔΨmito on the inner mitochondrial membrane that were induced by Ca2+ ions. Retardation by Pr3+ and La3+ ions of these calcium-induced effects may suggest that in the inner mitochondrial membrane these metals inhibit the opening of the mitochondrial permeability transition pore caused by Ca2+ overload of mitochondria. The data we obtained are important for a better understanding of the mechanisms of the damaging action of rare-earth elements on Ca2+-dependent processes in the vertebrate myocardium.

Neuronal Calcium Sensor 1 Has Two Variants with Distinct Calcium Binding Characteristics.

Neuronal calcium sensor-1 (NCS-1 Var1) is a calcium-binding protein expressed in most tissues. We examined a poorly characterized variant of NCS-1 (Var2), identified only in humans where the N-terminal 22 amino acid residues of native NCS-1(MGKSNSKLKPEVVEELTRKTY) were replaced with 4 different residues (MATI). Because alterations in the level of expression of NCS-1 Var1 and the expression of NCS-1 variants have been correlated with several neurological diseases, the relative expression and functional role of NCS-1 Var2 was examined. We found that NCS-1 Var2 mRNA levels are not found in mouse tissues and are expressed at levels ~1000-fold lower than NCS-1 Var1 in three different human cell lines (SHSY5Y, HEK293, MB231). Protein expression of both variants was only identified in cell lines overexpressing exogenous NCS-1 Var2. The calcium binding affinity is ~100 times weaker in purified NCS-1 Var2 than NCS-1 Var1. Because truncation of NCS-1 Var1 has been linked to functional changes in neurons, we determined whether the differing properties of the NCS-1 variants could potentially contribute to the altered cell function. In contrast to previous reports showing that overexpression of NCS-1 Var1 increases calcium-dependent processes, functional differences in cells overexpressing NCS-1 Var2 were undetectable in assays for cell growth, cell death and drug (paclitaxel) potency. Our results suggest that NCS-1 Var1 is the primary functional version of NCS-1.

Experimental setup for studying dynamics of the calcium interaction in cells

A calcium cell signaling system is one of the first, which were formed in the course of evolution of systems. The understanding of calcium binding–uncaging dynamics is crucial in studies of corresponding intracellular processes. By now, a great number of calcium-dependent processes have been investigated. However, works that fully consider these processes are absent. This is specified in many respects by the instrumental abilities. In this work, requirements for the experimental setup intended for comprehensive studies of calcium interaction dynamics are briefly formulated, its block diagram is described, and the results of test experiments are presented.

Discriminating Lipid- from Protein-Calcium Binding To Understand the Interaction between Recoverin and Phosphatidylglycerol Model Membranes.

Recoverin is a protein involved in the phototransduction cascade by regulating the activity of rhodopsin kinase through a calcium-dependent binding process at the surface of rod outer segment disk membranes. Understanding how calcium modulates these interactions and how it interacts with anionic lipid membranes is necessary to gain insights into the function of recoverin. In this work, infrared spectroscopy allowed us to show that the availability of calcium to recoverin is modulated by the presence of complexes involving phosphatidylglycerol (PG), which in turn regulates its interactions with this negatively charged lipid. Calcium can indeed be sequestered into strongly bound complexes with PG and is thus sparingly available to recoverin. The thermal stability of recoverin then decreases, which results in weakened interactions with PG. By contrast, when calcium is fully available to recoverin, the protein is thermally stable, indicating that it binds two calcium ions, which results in favorable interactions with negatively charged lipids. Consequently, the protein induces an increase in the chain-melting phase transition temperature of PG, which is indicative of an enhanced lipid chain packing resulting from the peripheral location of the protein. The secondary structure of recoverin is not affected by its interactions with anionic membrane lipids. Similar results have been obtained with saturated and unsaturated anionic lipids. This work shows that the recruitment of recoverin at the surface of anionic lipid membranes is dependent on the availability of calcium.

Comparative study of Y3+ effect on calcium-dependent processes in frog cardiac muscle and mitochondria of rat cardiomyocytes

Inotropic effects of yttrium acetate (Y³⁺) on contractions of myocardium preparations of the frog Ra- na ridibunda as well as on respiration and the inner membrane potential (Δψmito) of isolated rat heart mi- tochondria were studied. It was found that 2 mM yttrium in Ringer solution significantly reduced the am- plitude of myocardium contractions evoked by electric stimulation and increased the half-relaxation time (n = 5). In experiments with Ca²⁺, Y³⁺ decreased Ca²⁺-dependent oxygen consumption rate of rat heart mitochondria, energized by glutamate and malate, impeded the reduction in respiration of these mito- chondria in state 3 or uncoupled by 2,4-dinitrophenol, and inhibited Ca²⁺-induced decrease in their inner membrane potential. These data are important to better understand the mechanisms of Y³⁺ effects on myocardial calcium-dependent processes. Possible mechanisms of negative inotropic effect of Y³⁺ on the myocardium and its influence on the Ca²⁺-dependent processes in rat mitochondria are discussed.

Altered expression of stromal interaction molecule (STIM)-calcium release-activated calcium channel protein (ORAI) and inositol 1,4,5-trisphosphate receptors (IP3Rs) in cancer: will they become a new battlefield for oncotherapy?

The stromal interaction molecule (STIM)-calcium release-activated calcium channel protein (ORAI) and inositol 1,4,5-trisphosphate receptors (IP3Rs) play pivotal roles in the modulation of Ca2+-regulated pathways from gene transcription to cell apoptosis by driving calcium-dependent signaling processes. Increasing evidence has implicated the dysregulation of STIM–ORAI and IP3Rs in tumorigenesis and tumor progression. By controlling the activities, structure, and/or expression levels of these Ca2+-transporting proteins, malignant cancer cells can hijack them to drive essential biological functions for tumor development. However, the molecular mechanisms underlying the participation of STIM–ORAI and IP3Rs in the biological behavior of cancer remain elusive. In this review, we summarize recent advances regarding STIM–ORAI and IP3Rs and discuss how they promote cell proliferation, apoptosis evasion, and cell migration through temporal and spatial rearrangements in certain types of malignant cells. An understanding of the essential roles of STIM–ORAI and IP3Rs may provide new pharmacologic targets that achieve a better therapeutic effect by inhibiting their actions in key intracellular signaling pathways.

Immature human dendritic cells enhance their migration through KCa3.1 channel activation

Migration capacity is essential for dendritic cells (DCs) to present antigen to T cells for the induction of immune response. The DC migration is supposed to be a calcium-dependent process, while not fully understood. Here, we report a role of the KCa3.1/IK1/SK4 channels in the migration capacity of both immature (iDC) and mature (mDC) human CD14+-derived DCs. KCa3.1 channels were shown to control the membrane potential of human DC and the Ca2+ entry, which is directly related to migration capacities. The expression of migration marker such as CCR5 and CCR7 was modified in both types of DCs by TRAM-34 (100nM). But, only the migration of iDC was decreased by use of both TRAM-34 and KCa3.1 siRNA. Confocal analyses showed a close localization of CCR5 with KCa3.1 in the steady state of iDC. Finally, the implication of KCa3.1 seems to be limited to the migration capacities as T cell activation of DCs appeared unchanged. Altogether, these results demonstrated that KCa3.1 channels have a pro-migratory effect on iDC migration. Our findings suggest that KCa3.1 in human iDC play a major role in their migration and constitute an attractive target for the cell therapy optimization.

Considering calcium-binding proteins in invertebrates: multi-functional proteins that shape neuronal growth.

Calcium is a critical second messenger molecule in all cells and is vital in neurons for synaptic transmission. Given this importance, calcium ions are tightly controlled by a host of molecular players including ion channels, sensors, and buffering proteins. Calcium can act directly by binding to signaling molecules or calcium's effects can be indirect, for example by altering nuclear histones which can lead to changes in gene transcription (Rishal and Fainzilber, 2014). All of these mechanisms come into play in developing axons as calcium is required for both axon pathfinding and branching. Furthermore, after neuronal injury, waves of calcium originating at the site of axon segmentation and propagating to the nucleus have long been known to be required for regeneration (Rishal and Fainzilber, 2014). These changes in intracellular calcium concentrations [Ca2+]i must be properly controlled or else new growth cones may fail to form and degeneration of the neuron may occur. While many of the molecular players involved in these calcium-dependent processes have been identified, calcium buffering proteins have often been undervalued for their role in regulating axon growth either during normal development or in the event of injury.

Analysis of Perforin Assembly by Quartz Crystal Microbalance Reveals a Role for Cholesterol and Calcium-independent Membrane Binding

Perforin is an essential component in the cytotoxic lymphocyte-mediated cell death pathway. The traditional view holds that perforin monomers assemble into pores in the target cell membrane via a calcium-dependent process and facilitate translocation of cytotoxic proteases into the cytoplasm to induce apoptosis. Although many studies have examined the structure and role of perforin, the mechanics of pore assembly and granzyme delivery remain unclear. Here we have employed quartz crystal microbalance with dissipation monitoring (QCM-D) to investigate binding and assembly of perforin on lipid membranes, and show that perforin monomers bind to the membrane in a cooperative manner. We also found that cholesterol influences perforin binding and activity on intact cells and model membranes. Finally, contrary to current thinking, perforin efficiently binds membranes in the absence of calcium. When calcium is added to perforin already on the membrane, the QCM-D response changes significantly, indicating that perforin becomes membranolytic only after calcium binding.

The ionic mechanism of the Purkinje cell dendritic spikes generation and propagation: a model exploration

Due to the lack of sodium channel expression in the Purkinje cell (PC) dendrite, the passively propagated sodium spikes can hardly be detected in the dendrite. However, the substantial expression of T type calcium channels (ICaT) and P type calcium channels (ICaP) can cause local regenerative spikes to occur in the dendrite after receiving a parallel fiber (PF) stimulus [1]. In this study, with the previously published PC dendrite model [2] from our group as the starting point, we have built a new PC dendrite model incorporating the low threshold A-type K+ current (IKA) [3], high threshold activated K+ current (IKV3) and hyperpolarization-activated cation current (Ih). In our model, in agreement with experimental observations [1], the EPSP amplitude increases nonlinearly with the strength of the PF synaptic input until it reaches the spike threshold. During this range, we find that ICaT amplifies the EPSP caused by PF synaptic input to boost the EPSP close to the spike threshold. Around the spike threshold there is an obvious spike delay due to the interactive role of IKA and ICaT. Above the spike threshold, high threshold activated ICaP takes over and depolarizes the dendrite rapidly, finally leading to the generation of dendritic spikes. The amplitude of dendritic spikes doesn't increase further with the stimulation intensity and is mainly determined by the struggle among ICaP, IKV3 and high conductance calcium activated K+ currents (IBK). Injecting a negative current can eliminate the PF input evoked dendritic spikes by lowering the membrane potential which implies less activation of the ICaT and subsequent less amplification of the EPSP. We also find that the simulated dendritic spikes can't propagate to the whole dendrite due to the regulating role of mainly IBK and IKV3. Surprisingly by partially blocking IKV3 and IBK parallel fiber evoked dendritic spikes can propagate to the whole dendrite because of the broadening of its width. The calcium influx by dendritic spikes is vital to a lot of calcium dependent processes like synaptic plasticity. Understanding the mechanism of generation of the local dendritic spikes evoked by PF stimulus and how their localized nature works will help us better explore how information is processed in the PC dendrite.

Pharmacological characterization of the involvement of protein kinase C in oscillatory and non-oscillatory calcium increases in astrocytes

Evidence increasingly shows that astrocytes play a pivotal role in brain physiology and pathology via calcium dependent processes, thus the characterization of the calcium dynamics in astrocytes is of growing importance. We have previously reported that the epidermal growth factor and basic fibroblast growth factor up-regulate the oscillation of the calcium releases that are induced by stimuli, including glutamate in cultured astrocytes. This calcium oscillation is assumed to involve protein kinase C (PKC), which is activated together with the calcium releases as a consequence of inositol phospholipid hydrolysis. In the present study, this issue has been investigated pharmacologically by using astrocytes cultured with and without the growth factors. The pharmacological activation of PKC largely reduced the glutamate-induced oscillatory and non-oscillatory calcium increases. Meanwhile, PKC inhibitors increased the total amounts of both calcium increases without affecting the peak amplitudes and converted the calcium oscillations to non-oscillatory sustained calcium increases by abolishing the falling phases of the repetitive calcium increases. Furthermore, the pharmacological effects were consistent between both glutamate- and histamine-induced calcium oscillations. These results suggest that PKC up-regulates the removal of cytosolic calcium in astrocytes, and this up-regulation is essential for calcium oscillation in astrocytes cultured with growth factors.

Purinergic signalling in the enteric nervous system (An overview of current perspectives).

Purinergic Signalling in the Enteric Nervous System involves the regulated release of ATP (or a structurally-related nucleotide) which activates an extensive suite of membrane-inserted receptors (P2X and P2Y subtypes) on a variety of cell types in the gastrointestinal tract. P2X receptors are gated ion-channels permeable to sodium, potassium and calcium. They depolarise cells, act as a pathway for calcium influx to activate calcium-dependent processes and initiate gene transcription, interact at a molecular level as a form of self-regulation with lipids within the cell wall (e.g. PIP2) and cross-react with other membrane-inserted receptors to regulate their activity (e.g. nAChRs). P2Y receptors are metabotropic receptors that couple to G-proteins. They may release calcium ions from intracellular stores to activate calcium-dependent processes, but also may activate calcium-independent signalling pathways and influence gene transcription. Originally ATP was a candidate only for NANC neurotransmission, for inhibitory motoneurons supplying the muscularis externa of the gastrointestinal tract and bringing about the fast IJP. Purinergic signalling later included neuron-neuron signalling in the ENS, via the production of either fast or slow EPSPs. Later still, purinergic signalling included the neuro-epithelial synapse-for efferent signalling to epithelia cells participating in secretion and absorption, and afferent signalling for chemoreception and mechanoreception at the surface of the mucosa. Many aspects of purinergic signalling have since been addressed in a series of highly-focussed and authoritative reviews. In this overview however, the current focus is on key aspects of purinergic signalling where there remains uncertainty and ambiguity, with the view to stimulating further research in these areas.

Role of domain calcium in purinergic P2X2 receptor channel desensitization.

Activation of P2X2 receptor channels (P2X2Rs) is characterized by a rapid current growth accompanied by a decay of current during sustained ATP application, a phenomenon known as receptor desensitization. Using rat, mouse, and human receptors, we show here that two processes contribute to receptor desensitization: bath calcium-independent desensitization and calcium-dependent desensitization. Calcium-independent desensitization is minor and comparable during repetitive agonist application in cells expressing the full size of the receptor but is pronounced in cells expressing shorter versions of receptors, indicating a role of the COOH terminus in control of receptor desensitization. Calcium-dependent desensitization is substantial during initial agonist application and progressively increases during repetitive agonist application in bath ATP and calcium concentration-dependent manners. Experiments with substitution of bath Na(+) with N-methyl-d-glucamine (NMDG(+)), a large organic cation, indicate that receptor pore dilation is a calcium-independent process in contrast to receptor desensitization. A decrease in the driving force for calcium by changing the holding potential from -60 to +120 mV further indicates that calcium influx through the channel pores at least partially accounts for receptor desensitization. Experiments with various receptor chimeras also indicate that the transmembrane and/or intracellular domains of P2X2R are required for development of calcium-dependent desensitization and that a decrease in the amplitude of current slows receptor desensitization. Simultaneous calcium and current recording shows development of calcium-dependent desensitization without an increase in global intracellular calcium concentrations. Combined with experiments with clamping intrapipette concentrations of calcium at various levels, these experiments indicate that domain calcium is sufficient to establish calcium-dependent receptor desensitization in experiments with whole-cell recordings.

Mercury-induced neurotoxicity and neuroprotective effects of berberine.

Mercury-induced neurotoxicity and neuroprotective effects of berberine.

Actin dynamics and the evolution of the memory trace

The goal of this essay is to link the regulation of actin dynamics to the idea that the synaptic changes that support long-term potentiation and memory evolve in temporally overlapping stages—generation, stabilization, and consolidation. Different cellular/molecular processes operate at each stage to change the spine cytoarchitecture and, in doing so, alter its function. Calcium-dependent processes that degrade the actin cytoskeleton network promote a rapid insertion of AMPA receptors into the post synaptic density, which increases a spine׳s capacity to express a potentiated response to glutamate. Other post-translation events then begin to stabilize and expand the actin cytoskeleton by increasing the filament actin content of the spine and reorganizing it to be resistant to depolymerizing events. Disrupting actin polymerization during this stabilization period is a terminal event—the actin cytoskeleton shrinks and potentiated synapses de-potentiate and memories are lost. Late-arriving, new proteins may consolidate changes in the actin cytoskeleton. However, to do so requires a stabilized actin cytoskeleton. The now enlarged spine has properties that enable it to capture other newly transcribed mRNAs or their protein products and thus enable the synaptic changes that support LTP and memory to be consolidated and maintained.This article is part of a Special Issue entitled SI: Brain and Memory.

Riboflavin and vitamin E increase brain calcium and antioxidants, and microsomal calcium-ATP-ase values in rat headache models induced by glyceryl trinitrate.

The essential use of riboflavin is the prevention of migraine headaches, although its effect on migraines is considered to be associated with the increased mitochondrial energy metabolism. Oxidative stress is also important in migraine pathophysiology. Vitamin E is a strong antioxidant in nature and its analgesic effect is not completely clear in migraines. The current study aimed to investigate the effects of glyceryl trinitrate (GTN)-sourced exogen nitric oxide (NO), in particular, and also riboflavin and/or vitamin E on involved in the headache model induced via GTN-sourced exogen NO on oxidative stress, total brain calcium levels, and microsomal membrane Ca(2+)-ATPase levels. GTN infusion is a reliable method to provoke migraine-like headaches in experimental animals and humans. GTN resulted in a significant increase in brain cortex and microsomal lipid peroxidation levels although brain calcium, vitamin A, vitamin C, and vitamin E, and brain microsomal-reduced glutathione (GSH), glutathione peroxidase (GSH-Px), and plasma-membrane Ca(2+)-ATPase values decreased through GTN. The lipid peroxidation, GSH, vitamin A, β-carotene, vitamin C, and vitamin E, and calcium concentrations, GSH-Px, and the Ca(2+)-ATPase activities were increased both by riboflavin and vitamin E treatments. Brain calcium and vitamin A concentrations increased through riboflavin only. In conclusion, riboflavin and vitamin E had a protective effect on the GTN-induced brain injury by inhibiting free radical production, regulation of calcium-dependent processes, and supporting the antioxidant redox system. However, the effects of vitamin E on the values seem more important than in riboflavin.

Calcium restores the macrophage response to nontypeable haemophilus influenzae in chronic obstructive pulmonary disease.

Alveolar macrophages in chronic obstructive pulmonary disease (COPD) have demonstrated impaired bacterial phagocytosis and disordered cytokine secretion, which are calcium-dependent processes. We determined how calcium moderates the macrophage response to nontypeable Haemophilus influenzae (NTHI). We hypothesized that augmenting extracellular calcium during bacterial challenge would restore bacterial phagocytosis and cytokine secretion in monocyte-derived macrophages (MDMs) from subjects with COPD. We further determined whether restoration of pattern recognition and scavenger receptors correlated with the calcium-induced improvements in macrophage function. Monocytes were purified from whole blood from healthy control subjects (n = 20) and patients with moderate to severe COPD (n = 35), and cultured in suspension with granulocyte macrophage colony-stimulating factor to generate MDMs. The MDMs were incubated with fluorescently labeled NTHI with and without calcium lactate and calcium channel inhibitors. Phagocytosis efficiency was evaluated by flow cytometry. Supernatants were assayed for cytokines using bead array technology. Cell surface receptor expression was assayed by multicolor flow cytometry. Extracellular calcium significantly improved phagocytosis and cytokine secretion (IL-8, TNF-α, and macrophage inflammatory protein [MIP]-1α, and -1β) in COPD MDMs. NTHI challenge led to statistically significant reductions in CD16 (FcγRIII), and extracellular calcium up-regulated both CD16 and macrophage receptor with collagenous structure (MARCO). Specific calcium channel inhibitors abrogated calcium-mediated MARCO up-regulation and cytokine secretion. Extracellular calcium improved phagocytosis, restored innate cytokine secretion, and increased cell surface expression of bacterial recognition receptors, CD16 and MARCO. These observations support the therapeutic use of calcium to improve macrophage function in COPD to decrease exacerbations and chronic bacterial infection.

Contact-induced mitochondrial polarization supports HIV-1 virological synapse formation.

ABSTRACTRapid HIV-1 spread between CD4 T lymphocytes occurs at retrovirus-induced immune cell contacts called virological synapses (VS). VS are associated with striking T cell polarization and localized virus budding at the site of contact that facilitates cell-cell spread. In addition to this, spatial clustering of organelles, including mitochondria, to the contact zone has been previously shown. However, whether cell-cell contact specifically induces dynamic T cell remodeling during VS formation and what regulates this process remain unclear. Here, we report that contact between an HIV-1-infected T cell and an uninfected target T cell specifically triggers polarization of mitochondria concomitant with recruitment of the major HIV-1 structural protein Gag to the site of cell-cell contact. Using fixed and live-cell imaging, we show that mitochondrial and Gag polarization in HIV-1-infected T cells occurs within minutes of contact with target T cells, requires the formation of stable cell-cell contacts, and is an active, calcium-dependent process. We also find that perturbation of mitochondrial polarization impairs cell-cell spread of HIV-1 at the VS. Taken together, these data suggest that HIV-1-infected T cells are able to sense and respond to contact with susceptible target cells and undergo dynamic cytoplasmic remodeling to create a synaptic environment that supports efficient HIV-1 VS formation between CD4 T lymphocytes.IMPORTANCE HIV-1 remains one of the major global health challenges of modern times. The capacity of HIV-1 to cause disease depends on the virus's ability to spread between immune cells, most notably CD4 T lymphocytes. Cell-cell transmission is the most efficient way of HIV-1 spread and occurs at the virological synapse (VS). The VS forms at the site of contact between an infected cell and an uninfected cell and is characterized by polarized assembly and budding of virions and clustering of cellular organelles, including mitochondria. Here, we show that cell-cell contact induces rapid recruitment of mitochondria to the contact site and that this supports efficient VS formation and consequently cell-cell spread. Additionally, we observed that cell-cell contact induces a mitochondrion-dependent increase in intracellular calcium, indicative of cellular signaling. Taken together, our data suggest that VS formation is a regulated process and thus a potential target to block HIV-1 cell-cell spread.

High extracellular magnesium inhibits mineralized matrix deposition and modulates intracellular calcium signaling in human bone marrow-derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) have the potential to differentiate into several cell types and provide an attractive source of autologous cells for regenerative medicine. However, their cellular biology is not fully understood. Similar to Ca2+, extracellular Mg2+ plays an important role in the functions of the skeletal system. Here, we examined the effects of extracellular Mg2+ on the deposition of calcium phosphate matrix and Ca2+ signaling with or without ATP stimulation in human bone marrow-derived mesenchymal stem cells (hBMSCs). We found that high extracellular Mg2+ concentration ([Mg2+]e) inhibited extracellular matrix mineralization in hBMSCs in vitro. hBMSCs also produced a dose-dependent decrease in the frequency of calcium oscillations during [Mg2+]e elevation with a slight suppression on oscillation amplitude. In addition, spontaneous ATP release was inhibited under high [Mg2+]e levels and exogenous ATP addition stimulated oscillation reappear. Taken together, our results indicate that high [Mg2+]e modulates calcium oscillations via suppression of spontaneous ATP release and inactivates purinergic receptors, resulting in decreased extracellular mineralized matrix deposition in hBMSCs. Therefore, the high magnesium environment created by the rapid corrosion of Mg alloys may result in the dysfunction of calcium-dependent physiology processes and be disadvantageous to hBMSCs physiology.

Calpain activation and CaMKIV reduction in spinal cords from hSOD1G93A mouse model.

Amyotrophic Lateral Sclerosis (ALS), a severe neurodegenerative disease, affects the upper and lower motor neurons in the brain and spinal cord. In some studies, ALS disease progression has been associated with an increase in calcium-dependent degeneration processes. Motoneurons are specifically vulnerable to sustained membrane depolarization and excessive elevation of intracellular calcium concentration. The present study analyzed intracellular events in embryonic motoneurons and adult spinal cords of the hSOD1G93A ALS mouse model. We observed activation of calpain, a calcium-dependent cysteine protease that degrades a variety of substrates, and a reduction in calcium-calmodulin dependent protein kinase type IV (CaMKIV) levels in protein extracts from spinal cords obtained at several time-points of hSOD1G93A mice disease progression. However, in cultured embryonic motoneurons these differences between controls and hSOD1G93A mutants are not evident. Our results support the hypothesis that age-dependent changes in calcium homeostasis and resulting events, e.g., calpain activation and CaMKIV processing, are involved in ALS pathogenesis.