Pharmacologically induced calcium oscillations protect neurons from increases in cytosolic calcium after trauma

Increases in cytosolic calcium ([Ca(2+)](i)) following mechanical injury are often considered a major contributing factor to the cellular sequelae in traumatic brain injury (TBI). However, very little is known on how developmental changes may affect the calcium signaling in mechanically injured neurons. One key feature in the developing brain that may directly impact its sensitivity to stretch is the reduced inhibition which results in spontaneous [Ca(2+)](i) oscillations. In this study, we examined the mechanism of stretch-induced [Ca(2+)](i) transients in 18-days in vitro (DIV) neurons exhibiting bicuculline-induced [Ca(2+)](i) oscillations. We used an in vitro model of mechanical trauma to apply a defined uniaxial strain to cultured cortical neurons and used increases in [Ca(2+)](i) as a measure of the neuronal response to the stretch insult. We found that stretch-induced increases in [Ca(2+)](i) in 18-DIV neurons were inhibited by pretreatment with either the NMDA receptor antagonist, APV [D(-)-2-Amino-5-phosphonopentanoic acid], or by depolymerizing the actin cytoskeleton prior to stretch. Blocking synaptic NMDA receptors prior to stretch significantly attenuated most of the [Ca(2+)](i) transient. In comparison, cultures with pharmacologically induced [Ca(2+)](i) oscillations showed a substantially reduced [Ca(2+)](i) peak after stretch. We provide evidence showing that a contributing factor to this mechanical desensitization from induced [Ca(2+)](i) oscillations is the PKC-mediated uncoupling of NMDA receptors (NMDARs) from spectrin, an actin-associated protein, thereby rendering neurons insensitive to stretch. These results provide novel insights into how the [Ca(2+)](i) response to stretch is initiated, and how reduced inhibition - a feature of the developing brain - may affect the sensitivity of the immature brain to trauma.     

Fibulin7 aggravates calcium oxalate-induced acute kidney injury by binding to calcium oxalate crystals

Fibulin7 (Fbln7) is a matricellular protein that is structurally similar to short fibulins but does not possess elastogenic abilities. Fbln7 is localized on the cell surface of the renal tubular epithelium in the adult kidney. We previously reported that Fbln7 binds artificial calcium phosphate particles in vitro, and that heparin counteracts this binding by releasing Fbln7 from the cell surface. Fbln7 gene (Fbln7) deletion in vivo decreased interstitial fibrosis and improved renal function in a high phosphate diet-induced chronic kidney disease mouse model. However, the contribution of Fbln7 during acute injury response remains largely unknown. We hypothesized that Fbln7 serves as an exacerbating factor in acute kidney injury (AKI). We employed three AKI models in vivo and in vitro, including unilateral ureteral obstruction (UUO), cisplatin-induced AKI, and calcium oxalate (CaOx)-induced AKI. Here, we report that Fbln7KO mice were protected from kidney damage in a CaOx-induced AKI model. Using HEK293T cells, we found that Fbln7 overexpression enhanced the CaOx-induced upregulation of EGR1 and LAMB3, and that heparin treatment canceled this effect. Interestingly, the protective function observed in Fbln7KO kidneys was limited to the CaOx-induced AKI model, while Fbln7KO mice were not protected against UUO-induced renal fibrosis or cisplatin-induced renal tubular damage. Taken together, our study indicates that Fbln7 mediates the local deposition of CaOx and damages the renal tubular epithelium. Releasing Fbln7 from the cell surface via heparin/heparin derivatives or Fbln7 inhibitory antibodies may provide a general strategy to mitigate calcium crystal-induced kidney injuries.     

Neuronal calcium signaling via store-operated channels in health and disease

Store-operated calcium entry (SOCE) is the flow of calcium ions (Ca²⁺) into cells in response to the depletion of intracellular Ca²⁺ stores that reside predominantly in the endoplasmic reticulum (ER). The role of SOCE has been relatively well understood for non-excitable cells. It is mediated mostly by the ER Ca²⁺ sensor STIM1 and plasma membrane Ca²⁺ channel Orai1 and serves to sustain Ca²⁺ signaling and refill ER Ca²⁺ stores. In contrast, because of the complexity of Ca²⁺ influx mechanisms that are present in excitable cells, our knowledge about the function of neuronal SOCE (nSOCE) is still nascent. This review summarizes the available data on the molecular components of nSOCE and their relevance to neuronal signaling. We also present evidence of disturbances of nSOCE in neurodegenerative diseases (namely Alzheimer’s disease, Huntington’s disease, and Parkinson’s disease) and traumatic brain injury. The emerging important role of nSOCE in neuronal physiology and pathology makes it a possible clinical target.     

Low serum calcium: a new,important indicator of COVID-19 patients from mild/moderate to severe/critical

Background: Coronavirus disease 2019 (COVID-19) virus is still spreading, finding out the initial hits of viral infection is important to minimize the mild/moderate population, prevent disease aggravation and organs dysfunction.Objective: We investigated COVID-19 patients with different serum calcium levels.Design: We checked the serum calcium level of the patients based on days after symptom onset as well as the severity of COVID-19. We also checked multiorgan injuries and immune cytokines level in their blood.Results: Both mild/moderate and severe critical cases we observed showed low calcium level in the early stage of viral infection, while the severe/critical cases showed significant lower calcium level than mild/moderate cases in the early stage. We also found that low calcium level related to severe/critical multiorgan injuries especially in the mild/moderate population. Proinflammatory cytokine IL-6 also correlated to calcium change in both mild/moderate and severe/critical cases.Conclusions: Our finding indicates that calcium balance is a primal hit of COVID-19 and a biomarker of clinical severity at the beginning of symptom onset. Calcium is closely associated with virus-associated multiple organ injuries and the increase in inflammatory cytokines. Our results provide a new, important indicator of COVID-19 patients from mild/moderate to severe/critical: serum calcium.     

Proteasome inhibitor lactacystin disturbs the intracellular calcium homeostasis of dopamine neurons in ventral mesencephalic cultures

Ubiquitin proteasome system (UPS) impairment has been implicated in the pathology of Parkinson's disease, but the mechanisms underlying the UPS impairment-induced dopamine (DA) neuron degeneration remain obscure. To test whether calcium homeostasis disturbance is involved in the DA neuronal injury resulting from UPS impairment, we treated the primary ventral mesencephalic (VM) cultures with the proteasome inhibitor lactacystin, and observed its effects on the expression of the gene Homer 1a that is related to calcium homeostasis, and the intracellular free calcium ([Ca²⁺]_i) levels as well as the DA neuron survival. We also investigated a possible role of the L-type voltage dependent calcium channels (L-VDCC) in these events. We found that the lactacystin exposure induced the Homer 1a expression, lowered the [Ca²⁺]_i levels, reduced the depolarization-induced calcium entry and DA release in the VM cultures, and caused a significant DA neuron loss. Activation of L-VDCC by potassium chloride or its agonists alleviated the effects of lactacystin on the [Ca²⁺]_i levels and promoted DA neuron survival, whereas L-VDCC antagonists blocked the depolarization-mediated neuroprotective effect, and at high concentrations the L-VDCC antagonists aggravated the lactacystin-induced DA neuronal injury. These results indicate that calcium homeostasis disturbance may be a novel pathological mechanism leading to DA neuronal injury under conditions of proteasome inhibition.     

Role of mitochondrial calcium uniporter‐mediated Ca2+ and iron accumulation in traumatic brain injury

Previous studies have suggested that the cellular Ca²⁺ and iron homeostasis, which can be regulated by mitochondrial calcium uniporter (MCU), is associated with oxidative stress, apoptosis and many neurological diseases. However, little is known about the role of MCU‐mediated Ca²⁺ and iron accumulation in traumatic brain injury (TBI). Under physiological conditions, MCU can be inhibited by ruthenium red (RR) and activated by spermine (Sper). In the present study, we used RR and Sper to reveal the role of MCU in mouse and neuron TBI models. Our results suggested that the Ca²⁺ and iron concentrations were obviously increased after TBI. In addition, TBI models showed a significant generation of reactive oxygen species (ROS), decrease in adenosine triphosphate (ATP), deformation of mitochondria, up‐regulation of deoxyribonucleic acid (DNA) damage and increase in apoptosis. Blockage of MCU by RR prevented Ca²⁺ and iron accumulation, abated the level of oxidative stress, improved the energy supply, stabilized mitochondria, reduced DNA damage and decreased apoptosis both in vivo and in vitro. Interestingly, Sper did not increase cellular Ca²⁺ and iron concentrations, but suppressed the Ca²⁺ and iron accumulation to benefit the mice in vivo. However, Sper had no significant impact on TBI in vitro. Taken together, our data demonstrated for the first time that blockage of MCU‐mediated Ca²⁺ and iron accumulation was essential for TBI. These findings indicated that MCU could be a novel therapeutic target for treating TBI.     

Calcium binding to fluorescent calcium indicators: calcium green, calcium orange and calcium crimson

The recently introduced fluorescent calcium sensitive indicators calcium green, calcium orange and calcium crimson suggest important improvements and advantages to detect small calcium transients at low indicator concentrations. Thermodynamic dissociation constants and dissociation rate constants of calcium green, calcium orange and calcium crimson were measured by use of fluorescence titration and stopped flow fluorescence, respectively. Calcium binding to the indicators conforms to a 1:1 calcium:indicator complex although at high concentrations of calcium the fluorescence properties deviate somewhat from the behaviour predicted by the simple model. Dissociation of the calcium-indicator complex was found to be monoexponential under all conditions examined. The affinity for calcium of the three indicators generally increases with raising temperatures (Kd at 11.5 degrees C and 39.7 degrees C (nM): 261, 180 for calcium green; 527, 323 for calcium orange; 261, 204 for calcium crimson) and pH (Kd at pH 6.42 and 7.40 (nM): 314, 226 for calcium green; 562, 457 for calcium orange; 571, 269 for calcium crimson). The changes of the thermodynamic dissociation constant are mainly caused by changes of the association rate constant. The temperature dependence of calcium binding to the indicators revealed that this process is entropically favoured at ambient temperature.     

Stretch-induced injury in organotypic hippocampal slice cultures reproduces in vivo post-traumatic neurodegeneration: role of glutamate receptors and voltage-dependent calcium channels

The relationship between an initial mechanical event causing brain tissue deformation and delayed neurodegeneration in vivo is complex because of the multiplicity of factors involved. We have used a simplified brain surrogate based on rat hippocampal slices grown on deformable silicone membranes to study stretch-induced traumatic brain injury. Traumatic injury was induced by stretching the culture substrate, and the biological response characterized after 4 days. Morphological abnormalities consistent with traumatic injury in humans were widely observed in injured cultures. Synaptic function was significantly reduced after a severe injury. The N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 attenuated neuronal damage, prevented loss of microtubule-associated protein 2 immunoreactivity and attenuated reduction of synaptic function. In contrast, the NMDA receptor antagonists 3-[(R)-2-carboxypiperazin-4-yl]-propyl-1-phosphonic acid (CPP) and GYKI53655, were neuroprotective in a moderate but not a severe injury paradigm. Nifedipine, an L-type voltage-dependent calcium channel antagonist was protective only after a moderate injury, whereas omega-conotoxin attenuated damage following severe injury. These results indicate that the mechanism of damage following stretch injury is complex and varies depending on the severity of the insult. In conclusion, the pharmacological, morphological and electrophysiological responses of organotypic hippocampal slice cultures to stretch injury were similar to those observed in vivo. Our model provides an alternative to animal testing for understanding the mechanisms of post-traumatic delayed cell death and could be used as a high-content screen to discover neuroprotective compounds before advancing to in vivo models.     

Spinal cord mitochondria display lower calcium retention capacity compared with brain mitochondria without inherent differences in sensitivity to cyclophilin D inhibition

The mitochondrial permeability transition (mPT) is a potential pathogenic mechanism in neurodegeneration. Varying sensitivity to calcium-induced mPT has been demonstrated for regions within the CNS possibly correlating with vulnerability following insults. The spinal cord is selectively vulnerable in e.g. amyotrophic lateral sclerosis and increased mPT sensitivity of mitochondria derived from the spinal cord has previously been demonstrated. In this study, we introduce whole-body hypothermia prior to removal of CNS tissue to minimize the effects of differential tissue extraction prior to isolation of spinal cord and cortical brain mitochondria. Spinal cord mitochondria were able to retain considerably less calcium when administered as continuous infusion, which was not related to a general increased sensitivity of the mPT to calcium, its desensitization to calcium by the cyclophilin D inhibitor cyclosporin-A, or to differences in respiratory parameters. Spinal cord mitochondria maintained a higher concentration of extramitochondrial calcium during infusion than brain mitochondria possibly related to an increased set-point concentration for calcium uptake. A hampered transport and retention capacity of calcium may translate into an increased susceptibility of the spinal cord to neurodegenerative processes involving calcium-mediated damage.     

Calreticulin couples calcium release and calcium influx in integrin-mediated calcium signaling

The engagement of integrin alpha7 in E63 skeletal muscle cells by laminin or anti-alpha7 antibodies triggered transient elevations in the intracellular free Ca(2+) concentration that resulted from both inositol triphosphate-evoked Ca(2+) release from intracellular stores and extracellular Ca(2+) influx through voltage-gated, L-type Ca(2+) channels. The extracellular domain of integrin alpha7 was found to associate with both ectocalreticulin and dihydropyridine receptor on the cell surface. Calreticulin appears to also associate with cytoplasmic domain of integrin alpha7 in a manner highly dependent on the cytosolic Ca(2+) concentration. It appeared that intracellular Ca(2+) release was a prerequisite for Ca(2+) influx and that calreticulin associated with the integrin cytoplasmic domain mediated the coupling of between the Ca(2+) release and Ca(2+) influx. These findings suggest that calreticulin serves as a cytosolic activator of integrin and a signal transducer between integrins and Ca(2+) channels on the cell surface.     

Stretch-activated calcium channels relay fast calcium waves propagated by calcium-induced calcium influx

For nearly 30 years, fast calcium waves have been attributed to a regenerative process propagated by CICR (calcium-induced calcium release) from the endoplasmic reticulum. Here, I propose a model containing a new subclass of fast calcium waves which is propagated by CICI (calcium-induced calcium influx) through the plasma membrane. They are called fast CICI waves. These move at the order of 100 to 1000 microm/s (at 20 degrees C), rather than the order of 3 to 30 microm/s found for CICR. Moreover, in this proposed subclass, the calcium influx which drives calcium waves is relayed by stretch-activated calcium channels. This model is based upon reports from approx. 60 various systems. In seven of these reports, calcium waves were imaged, and, in five of these, evidence was presented that these waves were regenerated by CICI. Much of this model involves waves that move along functioning flagella and cilia. In these systems, waves of local calcium influx are thought to cause waves of local contraction by inducing the sliding of dynein or of kinesin past tubulin microtubules. Other cells which are reported to exhibit waves, which move at speeds in the fast CICI range, include ones from a dozen protozoa, three polychaete worms, three molluscs, a bryozoan, two sea urchins, one arthropod, four insects, Amphioxus, frogs, two fish and a vascular plant (Equisetum), together with numerous healthy, as well as cancerous, mammalian cells, including ones from human. In two of these systems, very gentle local mechanical stimulation is reported to initiate waves. In these non-flagellar systems, the calcium influxes are thought to speed the sliding of actinomyosin filaments past each other. Finally, I propose that this mechanochemical model could be tested by seeing if gentle mechanical stimulation induces waves in more of these systems and, more importantly, by imaging the predicted calcium waves in more of them.     

Subcellular calcium oscillators and calcium influx support agonist-induced calcium waves in cultured astrocytes

We have analysed Ca²⁺ waves induced by norepinephrine in rat cortical astrocytes in primary culture using fluorescent indicators fura-2 or fluo-3. The temporal pattern of the average [Ca²⁺]_i responses were heterogeneous from cell to cell and most cells showed an oscillatory response at concentrations of agonist around EC₅₀ (200 nM). Upon receptor activation, [Ca²⁺]_i signals originated from a single cellular locus and propagated throughout the cell as a wave. Wave propagation was supported by specialized regenerative calcium release loci along the length of the cell. The periods of oscillations, amplitudes, and the rates of [Ca²⁺]_i rise of these subcellular oscillators differ from each other. These intrinsic kinetic properties of the regenerative loci support local waves when stimulation is continued over long periods of time. The presence of local waves at specific, invariant cellular sites and their inherent kinetic properties provide for the unique and reproducible pattern of response seen in a given cell. We hypothesize that these loci are local specializations in the endoplasmic reticulum where the magnitude of the regenerative Ca²⁺ release is higher than other regions of the cell. Removal of extracellular Ca²⁺ or blockade of Ca²⁺ channels by inorganic cations (Cd²⁺ and Ni²⁺) during stimulation of adrenergic receptors alter the sustained plateau component of the [Ca²⁺]_i response. In the absence of Ca²⁺ release, due to store depletion with thapsigargin, agonist occupation alone does not induce Ca²⁺ influx in astrocytes. This finding suggests that, under these conditions, receptor-operated Ca²⁺ entry is not operative. Furthermore, our experiments provide evidence for local Ca²⁺ oscillations in cells which can support both wave propagation as well as spatially discrete Ca²⁺ signalling.     

An heuristic hypothesis of chilling injury in plants: a role for calcium as the primary physiological transducer of injury

Abstract. It is suggested that increased levels of free cytosolic calcium ([Ca²⁺]_cyt) may serve as the primary physiological transducer of chilling injury in plants. Numerous similarities between the effects of [Ca²⁺]_cyt-raising treatments on plants and the effects of chilling temperatures on chilling-sensitive (CS) plants are noted. It is proposed that chilling temperatures may lead to increases in [Ca²⁺]_cyt in CS plant cells by reducing the rate at which they exclude Ca²⁺ from their cytosol and that rapid cooling (coldshock) may cause rapid increases in [Ca²⁺]_cyt due to the activation of voltage-dependent cation channels. Chill-induced increases in [Ca²⁺]_cyt in the cells of CS plants may reflect either an inherent inability of such plants to maintain homeostatic levels of Ca²⁺ at low temperatures or a stress-induced reaction which has evolved to enable such cells to cope more effectively with the short-term hardships imposed by cold. Previous proposals concerning the physiological transduction of chilling injury are also discussed. It is argued that there is little evidence to suggest that the immediate effects of low temperatures on CS cells include either decreases in ATP levels, general increases in the passive permeability of membranes, or increased rates of fermentation.     

Cell proliferation, calcium influx and calcium channels

Both increases in the basal cytosolic calcium concentration ([Ca²⁺]_cyt) and [Ca²⁺]_cyt transients play major roles in cell cycle progression, cell proliferation and division. Calcium transients are observed at various stages of cell cycle and more specifically during late G₁ phase, before and during mitosis. These calcium transients are mainly due to calcium release and reuptake by the endoplasmic reticulum (ER) and are observed over periods of hours in oocytes and mammalian cells. Calcium entry sustains the ER Ca²⁺ load and thereby helps to maintain these calcium transients for such a long period. Calcium influx also controls cell growth and proliferation in several cell types. Various calcium channels are involved in this process and the tight relation between the expression and activity of cyclins and calcium channels also suggests that calcium entry may be needed only at particular stages of the cell cycle. Consistent with this idea, the expression of l-type and T-type calcium channels and SOCE amplitude fluctuate along the cell cycle. But, as calcium influx regulates several other transduction pathways, the presence of a specific connection to trigger activation of proliferation and cell division in mammalian cells will be discussed in this review.     

Capacitative calcium entry: sensing the calcium stores

A long-standing mystery in the cell biology of calcium channel regulation is the nature of the signal linking intracellular calcium stores to plasma membrane capacitative calcium entry channels. An RNAi-based screen of selected Drosophila genes has revealed that a calcium-binding protein, stromal interaction molecule (STIM), plays an essential role in the activation of these channels and may be the long sought sensor of calcium store content.     

Release of calcium from endolysosomes increases calcium influx through N-type calcium channels: Evidence for acidic store-operated calcium entry in neurons

Neurons possess an elaborate system of endolysosomes. Recently, endolysosomes were found to have readily releasable stores of intracellular calcium; however, relatively little is known about how such ‘acidic calcium stores’ affect calcium signaling in neurons. Here we demonstrated in primary cultured neurons that calcium released from acidic calcium stores triggered calcium influx across the plasma membrane, a phenomenon we have termed “acidic store-operated calcium entry (aSOCE)”. aSOCE was functionally distinct from store-operated calcium release and calcium entry involving endoplasmic reticulum. aSOCE appeared to be governed by N-type calcium channels (NTCCs) because aSOCE was attenuated significantly by selectively blocking NTCCs or by siRNA knockdown of NTCCs. Furthermore, we demonstrated that NTCCs co-immunoprecipitated with the lysosome associated membrane protein 1 (LAMP1), and that aSOCE is accompanied by increased cell-surface expression levels of NTCC and LAMP1 proteins. Moreover, we demonstrated that siRNA knockdown of LAMP1 or Rab27a, both of which are key proteins involved in lysosome exocytosis, attenuated significantly aSOCE. Taken together our data suggest that aSOCE occurs in neurons, that aSOCE plays an important role in regulating the levels and actions of intraneuronal calcium, and that aSOCE is regulated at least in part by exocytotic insertion of N-type calcium channels into plasma membranes through LAMP1-dependent lysosome exocytosis.