Geert Callewaert

Role of endoplasmic reticulum depletion and multidomain proapoptotic BAX and BAK proteins in shaping cell death after hypericin-mediated photodynamic therapy

Both the commitment event and the modality of cell death in photodynamic therapy (PDT) remain poorly defined. We report that PDT with endoplasmic reticulum (ER)‐associating hypericin leads to an immediate loss of SERCA2 protein levels, causing disruption of Ca2+ homeostasis and cell death. Protection of SERCA2 protein rescues ER‐Ca2+ levels and prevents cell death, suggesting that SERCA2 photodestruction with consequent incapability of the ER to maintain intracellular Ca2+ homeostasis is causal to cell killing. Apoptosis is rapidly initiated after ER‐Ca2+ depletion and strictly requires the BAX/BAK gateway at the mitochondria. Bax−/− Bak−/− double‐knockout (DKO) cells are protected from apoptosis but undergo autophagy‐associated cell death as revealed by electron microscopy and biochemical analysis. Autophagy inhibitors, but not caspase antagonists, significantly reduce death of DKO cells, suggesting that sustained autophagy is lethal. Thus, following ER photodamage and consequent disruption of Ca2+ homeostasis, BAX and BAK proteins model PDT‐mediated cell killing, which is executed through apoptosis in their presence or via an autophagic pathway in their absence.

Astrocytes regulate GluR2 expression in motor neurons and their vulnerability to excitotoxicity

Influx of Ca2+ ions through α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors contributes to neuronal damage in stroke, epilepsy, and neurodegenerative disorders such as ALS. The Ca2+ permeability of AMPA receptors is largely determined by the glutamate receptor 2 (GluR2) subunit, receptors lacking GluR2 being permeable to Ca2+ ions. We identified a difference in GluR2 expression in motor neurons from two rat strains, resulting in a difference in vulnerability to AMPA receptor-mediated excitotoxicity both in vitro and in vivo. Astrocytes from the ventral spinal cord were found to mediate this difference in GluR2 expression in motor neurons. The presence of ALS-causing mutant superoxide dismutase 1 in astrocytes abolished their GluR2-regulating capacity and thus affected motor neuron vulnerability to AMPA receptor-mediated excitotoxicity. These results reveal a mechanism through which astrocytes influence neuronal functioning in health and disease.

Inhibition and Rapid Recovery of Ca2+ Current During Ca2+ Release From Sarcoplasmic Reticulum in Guinea Pig Ventricular Myocytes

We have investigated the modulation of the L-type Ca2+ channel by Ca2+ released from the sarcoplasmic reticulum (SR) in single guinea pig ventricular myocytes under whole-cell voltage clamp. [Ca2+]i was monitored by fura 2. By use of impermeant monovalent cations in intracellular and extracellular solutions, the current through Na+ channels, K+ channels, nonspecific cation channels, and the Na+-Ca2+ exchanger was effectively blocked. By altering the amount of Ca2+ loading of the SR, the time course of the Ca2+ current (ICa) could be studied during various amplitudes of Ca2+ release. In the presence of a large Ca2+ release, fast inhibition of ICa occurred, whereas on relaxation of [Ca2+]i, fast recovery was observed. The time course of this transient inhibition of ICa reflected the time course of [Ca2+]i. However, the inhibition seen in the first 50 ms, ie, the time of net Ca2+ release from the SR, exceeded the inhibition observed later during the pulse, suggesting the existence of a higher [Ca2+] near the channel during this time. Transient inhibition of ICa during Ca2+ release was observed to a similar degree at all potentials. It could still be observed in the presence of intracellular ATP-gamma-S and of cAMP. Therefore, we conclude that the modulation of ICa by Ca2+ release from the SR is not related to dephosphorylation. It could be related to a reduction in the driving force and to a direct inhibition of the channel by [Ca2+]i. The observation that the degree of inhibition does not depend on membrane potential suggests that the Ca2+ binding site for this modulation is located outside the pore.(ABSTRACT TRUNCATED AT 250 WORDS)

Shear stress-induced calcium transients in endothelial cells from human umbilical cord veins.

1. Changes of the free cytosolic Ca2+ concentration induced by shear stress were measured in Fura‐2 acetoxymethyl ester‐loaded endothelial cells from human umbilical cord veins. 2. We were able to induce Ca2+ transients in almost every cell by blowing a stream of physiological solution onto a single endothelial cell thereby inducing shear stress between 0 and 50 dyn cm‐2. The Ca2+ response could be graded by varying the shear stress, and reached a half‐maximal value at a shear stress of 30 dyn cm‐2. 3. The shear stress responses critically depended on the extracellular Ca2+ concentration and were absent in a Ca(2+)‐free solution. Repetitive application of short pulses of shear stress induced cumulative effects because of the slow decay of the shear stress Ca2+ responses (time constants 82.3 +/‐ 17.8 s from twenty‐five cells). Application of a depolarizing high potassium solution to reduce the driving force for Ca2+ entry decreased the Ca2+ transients in some of the cells. 4. Application of shear stress in the presence of other divalent cations, such as nickel, cobalt or barium, always produced substantial changes in the ratio of the 390/360 nm fluorescence signal, indicating influx of these cations and subsequent quenching of the Fura‐2 fluorescence. 5. Shear stress responses in the presence of 10 mM Ca2+ were completely blocked by application of 1 mM La3+. 6. Incubation of the cells with the phorbol ester 12‐O‐tetradecanoyl phorbol‐13‐acetate (TPA) did not alter the shear stress response, but completely blocked histamine‐induced Ca2+ transients. 7. Small submaximal shear stress potentiated the Ca2+ transients induced by histamine. 8. We conclude that shear stress‐dependent Ca2+ signals are induced by an influx of calcium that is not modulated via protein kinase C and not activated by membrane depolarization. The influx pathway is also permeable to divalent cations such as Ni2+, Co2+ and Ba2+, but is blocked by La3+.

Caspase-3-induced Truncation of Type 1 Inositol Trisphosphate Receptor Accelerates Apoptotic Cell Death and Induces Inositol Trisphosphate-independent Calcium Release during Apoptosis

Inositol 1,4,5-trisphosphate receptor-deficient (IP3RKO) B-lymphocytes were used to investigate the functional relevance of type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) and its cleavage by caspase-3 in apoptosis. We showed that inositol 1,4,5-trisphosphate receptor-deficient cells were largely resistant to apoptosis induced by both staurosporine (STS) and B-cell receptor (BCR) stimulation. Expression of either the wild-type IP3R1 or an N-terminal deletion mutant (Δ1-225) that lacks inositol 1,4,5-trisphosphate-induced Ca2+ release activity restored sensitivity to apoptosis and the consequent rise in free cytosolic Ca2+ concentration ([Ca2+]i). Expression of caspase-3-non-cleavable mutant receptor, however, dramatically slowed down the rate of apoptosis and prevented both Ca2+ overload and secondary necrosis. Conversely, expression of the “channel-only” domain of IP3R1, a fragment of the receptor generated by caspase-3 cleavage, strongly increased the propensity of the cells to undergo apoptosis. In agreement with these observations, caspase inhibitors impeded apoptosis and the associated rise in [Ca2+]i. Both the staurosporine- and B-cell receptor-induced apoptosis and increase in [Ca2+]i could be induced in nominally Ca2+-free and serum-free culture media, suggesting that the apoptosis-related rise in [Ca2+]i was primarily because of the release from internal stores rather than of influx through the plasma membrane. Altogether, our results suggest that IP3R1 plays a pivotal role in apoptosis and that the increase in [Ca2+]i during apoptosis is mainly the consequence of IP3R1 cleavage by caspase-3. These observations also indicate that expression of a functional IP3R1 per se is not enough to generate the significant levels of cytosolic Ca2+ needed for the rapid execution of apoptosis, but a prior activation of caspase-3 and the resulting truncation of the IP3R1 are required.

Depletion of PINK1 affects mitochondrial metabolism, calcium homeostasis and energy maintenance

Loss-of-function mutations in the gene encoding the mitochondrial PTEN-induced putative kinase 1 (PINK1) are a major cause of early-onset familial Parkinsons disease (PD). Recent studies have highlighted an important function for PINK1 in clearing depolarized mitochondria by mitophagy. However, the role of PINK1 in mitochondrial and cellular functioning in physiological conditions is still incompletely understood. Here, we investigate mitochondrial and cellular calcium (Ca2+) homeostasis in PINK1-knockdown and PINK1-knockout mouse cells, both in basal metabolic conditions and after physiological stimulation, using unbiased automated live single-cell imaging in combination with organelle-specific fluorescent probes. Our data reveal that depletion of PINK1 induces moderate fragmentation of the mitochondrial network, mitochondrial membrane depolarization and increased production of reactive oxygen species. This results in reduced uptake of Ca2+ by mitochondria after physiological stimulation. As a consequence, cells with knockdown or knockout of PINK1 display impaired mitochondrial ATP synthesis, which is exacerbated under conditions of increased ATP demand, thereby affecting cytosolic Ca2+ extrusion. The impairment in energy maintenance was confirmed in the brain of PINK1-knockout mice by in vivo bioluminescence imaging. Our findings demonstrate a key role for PINK1 in the regulation of mitochondrial homeostasis and energy metabolism under physiological conditions.

The AMPA receptor antagonist NBQX prolongs survival in a transgenic mouse model of amyotrophic lateral sclerosis

alpha-Amino-3-hydroxy-5-methylisoxazole propionic acid (AMPA) receptor-mediated excitotoxicity has been implicated in the selective motor neuron loss in amyotrophic lateral sclerosis (ALS). The extent to which excitotoxicity contributes to motor neuron death remains incompletely understood. We therefore tested the potent and selective AMPA/kainate receptor antagonist 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide (NBQX) on motor neurons in culture and in the G93A mouse model for familial ALS. Kainate-induced currents and changes in intracellular Ca(2+) concentration were measured with the perforated patch clamp technique combined with Ca(2+) imaging, motor neuron death was quantified by counting experiments and G93A mice were treated with saline or 8 mg/kg NBQX. NBQX blocked kainate-induced currents and concomitant changes in intracellular Ca(2+), prevented the kainate-induced motor neuron death, and prolonged survival of G93A mice.

[Ca2+]i transients and [Ca2+]i‐dependent chloride current in single Purkinje cells from rabbit heart.

1. Single Purkinje cells, enzymatically isolated from rabbit ventricle, were studied under whole‐cell voltage clamp and internally perfused with the fluorescent Ca2+ indicator, indo‐1 (100 microM). 2. Fast [Ca2+]i transients were elicited by brief depolarizations from a holding voltage of ‐45 mV and by repolarization from very positive potentials. The peak [Ca2+]i‐voltage relation was bell‐shaped with a peak around +10 mV. 3. [Ca2+]i transients were completely blocked by the Ca2+ channel antagonist, nisoldipine (10 microM) and were very small when Ca2+ release from the sarcoplasmic reticulum (SR) was prevented by superfusion of cells by caffeine (1 mM) or ryanodine (10 microM). A fast application of caffeine induced a transient increase in [Ca2+]i. These results suggest [Ca2+]i transients are due to Ca(2+)‐induced Ca2+ release from the SR. 4. Rate of decline of the [Ca2+]i transient was voltage dependent, suggesting contribution of the Na(+)‐Ca2+ exchanger to Ca2+ efflux. At very positive potentials (> +60 mV), Ca2+ influx through the Na(+)‐Ca2+ exchanger could be observed. 5. A transient outward current was observed at potentials positive to +10 mV, but only if depolarizing pulses were accompanied by a [Ca2+]i transient. 6. When the amplitude of the [Ca2+]i transient was changed by (1) changes in [Ca2+]o, (2) changes in frequency of depolarization or (3) conditioning prepulses, the amplitude of the outward current changed in the same direction. This suggests activation of the current is dependent on and graded by [Ca2+]i. 7. The outward current was observed in K(+)‐free solutions, in the presence of Cs+ and TEA+, and was not blocked by 4‐aminopyridine (10 mM). In contrast, DIDS (100 microM) decreased the outward current by 70 +/‐ 20% (mean +/‐ S.D., n = 9), without affecting [Ca2+]i. 8. When external Cl‐ was lowered, the amplitude of the outward current decreased; when internal Cl‐ was replaced by aspartate, it became apparent at more negative potentials. These interventions strongly suggest the current was carried by Cl‐; it can therefore be referred to as a [Ca2+]i‐activated Cl‐ current or ICl(Ca). 9. When ICl(Ca) was maximally activated during a conditioning step, steps to negative potentials revealed inward currents through ICl(Ca) (in symmetrical Cl‐ solutions). The fully activated I‐V relation was linear. 10. ICl(Ca) could be activated at membrane potentials between ‐80 and +80 mV by a fast application of caffeine (10 mM), inducing Ca2+ release from the SR, demonstrating that ICl(Ca) does not require membrane depolarization or Ca2+ influx through the Ca2+ channel for its activation.(ABSTRACT TRUNCATED AT 400 WORDS)

GluR2 deficiency accelerates motor neuron degeneration in a mouse model of amyotrophic lateral sclerosis

AMPA receptor-mediated excitotoxicity has been implicated in the selective degeneration of motor neurons in amyotrophic lateral sclerosis (ALS). Motor neurons in vitro are particularly vulnerable to excessive AMPA receptor stimulation and one of the factors underlying this selective vulnerability is the presence of a large proportion of Ca2+-permeable (i.e. GluR2-lacking) AMPA receptors. However, the precise role of GluR2-lacking AMPA receptors in motor neuron degeneration remains to be defined. We therefore studied the impact of GluR2 deficiency on motor neuron death in vitro and in vivo. Cultured motor neurons from GluR2-deficient embryos displayed an increased Ca2+ influx through AMPA receptors and an increased vulnerability to AMPA receptor-mediated excitotoxicity. We deleted the GluR2 gene in mutant SOD1G93A mice by crossbreeding them with GluR2 knockout mice. GluR2 deficiency clearly accelerated the motor neuron degeneration and shortened the life span of mutant SOD1G93A mice. These findings indicate that GluR2 plays a pivotal role in the vulnerability of motor neurons in vitro and in vivo, and that therapies that limit Ca2+ entry through AMPA receptors might be beneficial in ALS patients.

Thimerosal stimulates Ca2+ flux through inositol 1,4,5-trisphosphate receptor type 1, but not type 3, via modulation of an isoform-specific Ca2+-dependent intramolecular interaction

Thiol-reactive agents such as thimerosal have been shown to modulate the Ca2+-flux properties of IP3 (inositol 1,4,5-trisphosphate) receptor (IP3R) via an as yet unidentified mechanism [Parys, Missiaen, De Smedt, Droogmans and Casteels (1993) Pflügers Arch. 424, 516-522; Kaplin, Ferris, Voglmaier and Snyder (1994) J. Biol. Chem. 269, 28972-28978; Missiaen, Taylor and Berridge (1992) J. Physiol. (Cambridge, U.K.) 455, 623-640; Missiaen, Parys, Sienaert, Maes, Kunzelmann, Takahashi, Tanzawa and De Smedt (1998) J. Biol. Chem. 273, 8983-8986]. In the present study, we show that thimerosal potentiated IICR (IP3-induced Ca2+ release) and IP3-binding activity of IP3R1, expressed in triple IP3R-knockout R23-11 cells derived from DT40 chicken B lymphoma cells, but not of IP3R3 or [D1-225]-IP3R1, which lacks the N-terminal suppressor domain. Using a 45Ca2+-flux technique in permeabilized A7r5 smooth-muscle cells, we have shown that Ca2+ shifted the stimulatory effect of thimerosal on IICR to lower concentrations of thimerosal and thereby increased the extent of Ca2+ release. This suggests that Ca2+ and thimerosal synergetically regulate IP3R1. Glutathione S-transferase pull-down experiments elucidated an interaction between amino acids 1-225 (suppressor domain) and amino acids 226-604 (IP3-binding core) of IP3R1, and this interaction was strengthened by both Ca2+ and thimerosal. In contrast, calmodulin and sCaBP-1 (short Ca2+-binding protein-1), both having binding sites in the 1-225 region, weakened the interaction. This interaction was not found for IP3R3, in agreement with the lack of functional stimulation of this isoform by thimerosal. The interaction between the IP3-binding and transmembrane domains (amino acids 1-604 and 2170-2749 respectively) was not affected by thimerosal and Ca2+, but it was significantly inhibited by IP3 and adenophostin A. Our results demonstrate that thimerosal and Ca2+ induce isoform-specific conformational changes in the N-terminal part of IP3R1, leading to the formation of a highly IP3-sensitive Ca2+-release channel.