Dissertations / Theses on the topic 'Intracellular calcium homeostasis'

Spatiotemporal increase of the intracellular Ca2+ is the most universal way to regulate the function of a eukaryotic cell. Owing to a host of actin-binding proteins and enzymes whose activities are modulated by the local concentration of Ca2+, free Ca2+ in cytosol serves as a pivotal second messenger in a variety of cell functions. The rise and fall of intracellular Ca2+ wave has been best illustrated in eggs at fertilization. However, the molecular mechanism by which intracellular Ca2+ is increased in the fertilized egg is largely unknown despite the discoveries of the distinct Ca2+-mobilizing second messengers in the past 30 years. In this thesis, I have used the starfish oocytes to study how Ca2+ signaling can be modulated by the actin cytoskeleton, which is known to be dynamically remodelled during meiotic maturation and fertilization of the egg. The principal issues of my experimental work are: (i) to establish the role of actin-binding proteins and PIP2 in the regulation of the Ca2+ signaling; (ii) to study the effect of the Ca2+ -store depletion on Ca2+ signaling and on the structure and function of the actin cytoskeleton, and (iii) to study the role of the actin-cytoskeleton in establishing the block to polyspermy. Microinjected into starfish eggs, actin-binding protein gelsolin, function- blocking antibody to depactin, and the PIP2-sequestering fusion protein that indirectly alters the actin cytoskeleton, all changed a certain aspect of Ca2+ signaling. Depletion of the Ca2+ store with ionomycin in turn drastically changed the cortical structure and the actin cytoskeleton of the eggs, eventually leading to a deleterious effect on egg activation and early development. Finally, the alteration of the actin cytoskeleton led to failure to establish a fast and slow block to polyspermy. Taken together, this study indicated that the actin cytoskeleton is an important factor that optimizes the Ca2+ response at egg activation and guides monospermic fertilization.

The effect of estrogen on the vasculature is mediated in part by influences on NO bioavailability. Nitric oxide (NO) is a potent vasodilator which is synthesized in endothelial cell by endothelial nitric oxide synthase (eNOS) catalyzed conversion of L-arginine to L-citrulline. Although estrogen has been shown to increase eNOS expression and/or activity, the mechanism of estrogen-mediated increased eNOS activity in endothelial cells remains elusive. The Ca 2+ /calmodulin complex is known to aid in eNOS activation by dissociating eNOS from the membrane bound protein, caveolin-1. We investigated the role of estrogen on the Ca 2+ homeostasis of the human endothelial cell line, EA.hy926, using thapsigargin (TG), a sarco(endo)plasmic reticulum Ca 2+ -ATPase, or ATP, a purinergic receptor agonist, to evoke increased intracellular calcium concentration ([Ca 2+ ] i ). [Ca 2+ ] i in Fura 2-AM-loaded EA.hy926 cell populations were measured by fluorescence spectrophotometry. Treatment of cells with 17 β-estradiol (E 2 , 1 μM, 24 hours) showed an increased agonist-evoked [Ca 2+ ] i increase due to both higher Ca 2+ release and Ca 2+ influx, which accompanied an increased eNOS protein expression. Both increased [Ca 2+ ] i and eNOS expression were attenuated with the nonselective estrogen receptor (ER) inhibitor, ICI 182,780. We further analyzed the role of ER in E 2 - mediated effects using ERα-knockdown cells. ERα-knockdown was achieved by transfecting the cells with ERα-specific siRNA. E 2 did not influence agonist-evoked [Ca 2+ ] i increase in the ERα-knockdown cells, indicating that the E 2 -mediated effects were ERα-dependent. In the vasculature, both the genomic and nongenomic effects of estrogen are mediated via ERα. In the current study, the effect of E 2 on agonist-evoked [Ca 2+ ] i increase was only observed in chronically treated (1 μM, 24 hours) cells and not acutely treated (1 μM, 5 minutes) cells, suggesting a genomic action of E 2 . The genomic action was verified by treating cells with E 2 in the presence of actinomycin D, a transcription inhibitor. Actinomycin D attenuated the effect of E 2 on agonist-evoked [Ca 2+ ] i increase. The present work revealed a transcription-dependent and ERα-mediated modulation of Ca 2+ homeostasis in human endothelial cells treated with estrogen for the long-term. This data suggests a novel mechanism by which estrogen-mediated NO release may occur in endothelial cells.

Familial Alzheimer's disease (FAD) -linked mutations in presenilin 1 and 2 (PS1, PS2) have been causally implicated in neurodegeneration and eventually neuronal cell death by amyloid toxicity and perturbation of cellular Ca2+ homeostasis. The mechanism governing this latter phenomenon remains unclear. In the cytosol, upon stimulation, both exaggerated and reduced Ca2+ release have been reported in different cell lines and neurons expressing PS1 and PS2 mutants. Despite the contradicting data yet available, it is undisputable that FAD-linked PS mutants cause imbalances of cellular Ca2+ homeostasis. Recent independent reports have strongly suggested that the FAD-linked PS1 and PS2 mutants interact directly or indirectly with both the ER Ca2+ uptake and release machinery by modulating SERCA pump, IP3R- and RyR channel activity. In the study described here, we took advantage of two available lines of transgenic (tg) mice expressing a mutant PS2 alone or together with a mutant amyloid precursor protein (APP) - both linked to FAD - in order to investigate its effects on Ca2+ homeostasis in a physiological environment more relevant to the pathology under study. We particularly focused our attention on Ca2+ dysregulation in cortical neurons from tg mice either single homozygous for human PS2-N141I or double homozygous for human PS2-N141I and human APP swedish mutations. Ca2+ measurements were carried by the fura-2/AM technique. This study highlights the role of PS2-N141I in modulating Ca2+ homeostasis of cortical neurons. We have demonstrated that, irrespective of the presence of mutant APP, modest expression of PS2-N141I altered the Ca2+ dynamics of intracellular stores. The total Ca2+ content of intracellular stores is partially depleted, as demonstrated by reduced Ca2+ release upon ionomycin stimulation. Consequently, the tg neurons have reduced Ca2+ release in response to IP3-generating agonists. However, the PS2 mutant does not affect the protein expression levels for both SERCA pump and IP3Rs. Our results suggest that the PS2-N141I FAD mutant causes a functional defect in ER Ca2+ entry/exit pathways. We also show that both tg neurons express very low levels of the mutant protein but show Ca2+ dysregulation, similar quantitatively and qualitatively to that previously reported in cell lines upon transient over-expression of the same mutant protein. Conversely, measurements of Ca2+ release via RyRs revealed a novel and unexpected finding in PS2 mutant tg mice i.e. increased Ca2+ release in response to caffeine. In addition, RyR2 protein expression level was elevated in tg neurons. Upregulation of RyRs function and protein levels could save as an adaptation phenomenon to compensate the reduction in Ca2+ release via IP3Rs or a direct effect of mutant PS2 on RyR or a secondary effect. Furthermore, PS2-N141I causes alteration in neuronal Ca2+ excitability. The tg neurons had significantly elevated number of picrotoxin-evoked synchronous Ca2+ oscillations, which required extracellular Ca2+ influx but not Ca2+ release from intracellular stores. Interestingly, while Ca2+ dysregulation appeared to be similar qualitatively and quantitatively in both single and double tg mouse models, the total amount of brain Ab42 and Ab40 peptides (ELISA) as well as their ratios were strikingly different between the two tg lines. These results strongly suggest that in tg mice the expression of mutant APP and/or Ab levels have no primary effect on the store Ca2+ content at this early stage and provide evidence that the quite similar effects on Ca2+ dynamics observed in both tg mice are due to the mutant PS2. Finally, results presented in this work suggest that although Ca2+ dysregulation is an early event in FAD, it does not affect neuronal response and vulnerability to cytotoxic stimuli at this early stage. The second part of this study focused on the role of Ab42 oligomers in cellular Ca2+ dynamics. Synthetic Ab42 oligomers reduced Ca2+ release in response to IP3 generating agonists in wt neurons but did not affect the total Ca2+ content as monitored by ionomycin. Conversely, Ab42 oligomers increased the Ca2+ release induced by caffeine. It is likely that Ab42 oligomers exert their effect on the activation pathway from IP3 generating agonists to IP3Rs. However, the mechanisms through which Ab42 deranges intracellular Ca2+ homeostasis require further investigation. Nonetheless, it is conceivable that in addition to Ab42 oligomers, also intracellular Ca2+ stores could become likely therapeutic targets in FAD and AD in general
E stato dimostrato che mutazioni in presenilina 1 e 2 (PS1, PS2) legate alle forme familiari della malattia di Alzheimer (FAD) sono implicate nella neurodegenerazione e in ultima istanza nella morte cellulare per effetto della tossicità del peptide amiloide e della perturbazione dell'omeostasi del Ca2+ cellulare. Il meccanismo alla base di quest'ultimo fenomeno non è ancora stato chiarito. Si è visto che in linee cellulari e in neuroni esprimenti mutazioni in PS1 e PS2, il rilascio di Ca2+ nel citosol in seguito a stimolazione cellulare può essere sia aumentato che ridotto. Nonostante i dati contraddittori attualmente disponibili, è innegabile che i mutanti di PS legati a FAD provocano uno squilibrio nell'omeostasi del Ca2+ cellulare. Recentemente studi indipendenti hanno dimostrato che i mutanti FAD in PS1 o PS2 interagiscono sia con i meccanismi di rilascio che di accumulo di Ca2+ nel reticolo endoplasmatico (RE), modulando sia l'attività  della pompa SERCA che i canali di tipo IP3R e RyR. In questo studio abbiamo impiegato due linee già  disponibili di topi transgenici (tg) esprimenti una PS2 mutata, da sola o assieme alla Proteina Precursore dell'Amiloide (APP) (entrambe con mutazioni legate a FAD) al fine di investigarne gli effetti sull'omeostasi del Ca2+ in una condizione fisiologica più rilevante per la patologia oggetto dello studio. Abbiamo focalizzato in particolare la nostra attenzione sull'alterazione del Ca2+ in neuroni corticali ottenuti da topi tg omozigoti per il solo mutante PS2-N141I o doppi omozigoti per le proteine mutate PS2-N141I e APPswe. Le misure di Ca2+ sono state effettuate con la tecnica del Fura-2/AM. Questo studio mette in evidenza il ruolo della PS2-N141I nel modulare l'omeostasi del Ca2+ in neuroni corticali murini. Abbiamo dimostrato che, indipendentemente dalla presenza del mutante di APP, l'espressione della PS2-N141I, in quantità  moderata, inficia le dinamiche del Ca2+ dei depositi intracellulari. In particolare,i depositi del Ca2+ sono parzialmente svuotati, come dimostrato dal ridotto rilascio di Ca2+ in seguito a stimolazione con ionomicina. Conseguentemente i neuroni tg hanno un ridotto rilascio di Ca2+in risposta ad agonisti legati alla generazione di IP3. Occorre notare che la PS2 mutata non altera i livelli di espressione delle proteine SERCA e IP3R. I nostri risultati suggeriscono che il mutante FAD PS2-N141I causa un difetto funzionale nelle vie di entrata e di uscita del Ca2+ dal RE. Abbiamo inoltre dimostrato che i neuroni di entrambe le linee tg esprimono bassi livelli di proteina mutante ma mostrano un'alterazione del segnale Ca2+ qualitativamente e quantitativamente simile a quella precedentemente riportata per le linee cellulari sovra-esprimenti la stessa proteina. Al contrario, misure del rilascio di Ca2+ via RyR hanno rivelato un effetto inatteso, ovvero un aumentato rilascio di Ca2+ in risposta a caffeina nei neuroni tg. Inoltre, i livelli proteici di RyR2 sono aumentati nei neuroni tg. Un aumento della funzionalità  e dei livelli proteici di RyR2 potrebbero essere un fenomeno adattativo per compensare la riduzione del rilascio di Ca2+ via IP3R oppure essere un effetto diretto dei mutanti PS2 su RyR2 o ancora un effetto secondario. Infine abbiamo dimostrato che la PS2-N141I causa un'alterazione nell'eccitabilità  neuronale. I neuroni tg hanno un numero significativamente più elevato di oscillazioni di Ca2+ sincronizzate, evocate da picrotossina, che dipendono dall' ingresso di Ca2+ extracellulare e non dal rilascio di Ca2+ dai depositi. E interessante notare che, mentre l'alterazione del Ca2+ è qualitativamente e quantitativamente simile nei singoli e doppi tg, sia il livello totale di peptidi Ab42 e Ab40 nel tessuto cerebrale (misurati mediante ELISA) che il loro rapporto sono notevolmente diversi tra le due linee tg. Questi risultati suggeriscono che nei topi tg l'espressione di APP mutata o i livelli di Ab non hanno alcun effetto primario sul contenuto dei depositi di Ca2+ in questo stadio precoce, e suggeriscono che gli effetti sulle dinamiche del segnale Ca2+, così simili nelle due linee tg, siano dovuti alla PS2 mutata. Infine, i risultati qui presentati suggeriscono che nonostante l'alterazione del segnale Ca2+ sia un evento precoce nei neuroni a questo stadio, non altera la vulnerabilità  neuronale a stimoli citotossici che agiscono atrraverso i depositi del Ca2+ . La seconda parte di questo studio si è concentrata sul ruolo degli oligomeri di Ab42 sulle dinamiche del Ca2+ cellulare. Nei neuroni di topi wt, oligomeri di Ab42 sintetico riducono il rilascio di Ca2+ in risposta ad agonisti legati alla produzione di IP3, ma non riducono il contenuto totale di Ca2+ dei depositi misurato mediante applicazione di ionomicina. D'altra parte, gli oligomeri di Ab42 aumentano il rilascio di Ca2+ indotto da caffeina. E' probabile che gli oligomeri di Ab42 agiscano sulla via attivata dagli agonisti legati alla produzione di IP3 e sull' IP3R. I meccanismi attraverso cui Ab42 altera l'omeostasi intracellulare del Ca2+ richiedono tuttavia ulteriori indagini. In conclusione, oltre agli oligomeri di Ab42 anche i depositi intracellulari di Ca2+ possono diventare un importante bersaglio terapeutico per intervenire sulla patologia di Alzheimer familiare ma anche sulla forma sporadica

I investigated the effects of Aβ₄₂ on the Ca²⁺ signalling capacity of human neuroblastoma SH-SY5Y cells and primary hippocampal cultures. I developed an in vitro model system of dissociated hippocampal neurons and glial cells in order to reflect as closely as possible the mature hippocampus. Extensive characterisation of the culture revealed that functional neuronal networks were established by day in vitro 11, as demonstrated by the occurrence of spontaneous oscillations in both membrane potential and intracellular Ca²⁺ levels. Neurons exhibited functional ionotropic and metabotropic signalling systems which, in turn, rendered them sensitive to cell death induced by excitotoxic stimuli. Samples of synthetic Aβ₄₂ were prepared according to two published protocols. One protocol produced Aβ₄₂ samples which exhibited highly dynamic aggregation kinetics, the other produced homogeneous Aβ₄₂ oligomers which were stable in their conformational state for up to 24 hours. While both Aβ₄₂ preparations impaired cell viability following 24-hour treatment, only Aβ₄₂ oligomers elicited robust Ca²⁺ responses following their extracellular application to cells. Aβ₄₂ oligomers elicited distinct but different effects on the Ca²⁺ signalling capacity of SH-SY5Y cells and primary hippocampal neurons, respectively. in SH-SY5Y cells, Aβ₄₂ oligomers acted to deplete the content of the intracellular ER Ca²⁺ store, in part through InsP₃ receptors and in part through an as yet unidentified leak pathway. In primary hippocampal neurons, the application of Aβ₄₂ oligomers resulted in a sustained and elevated increase in intracellular Ca²⁺ concentrations. It is postulated that these early cellular events, although distinct, will ultimately converge on a common pathway resulting in dysregulation of Ca²⁺ homeostasis and cell death. Thus, the findings of this thesis support the mounting body of evidence implicating the role of Ca²⁺ dysregulation in mediating the neurotoxic effects of Aβ₄₂ oligomers.

Neurons are specialized cells that communicate via electrical and chemical signaling. It is well-known that homeostatic mechanisms exist to potentiate neuronal output when activity falls. Likewise, while neurons rely on excitable states to function, these same excitable states must be kept in check for stable function. However, the identity of molecular factors and pathways regulating these pathways remain elusive. Chapter 2 of this thesis reports the findings from an RNA interference- and electrophysiology-based screen to identify factors necessary for the long-term maintenance of homeostatic synaptic potentiation. Data is reported to resolve a long-standing question as to the role of presynaptic Cav2-type channels in homeostatic synaptic potentiation at the Drosophila NMJ. It is shown that reduction in Cav2 channel expression and resultant activity is not sufficient to occlude homeostatic potentiation. Thus, the homeostatic block of a amino-acid substituted Cav2-type calcium channel (cacS) channel is presumed to be due to loss of a specific signaling or binding activity, but not due to overall diminishment in channel function. It is also reported that both Drosophila homologs of phospholipase Cβ (PLCβ) and its putative activator Gαq were found to be necessary for a scaling up of neurotransmitter release upon genetic ablation of glutamate receptors. These factors are canonically involved in the activation of intracellular calcium stores through the inositol trisphosphate receptor (IP3R) and the closely related ryanodine receptor (RyR). Likewise, the Drosophila homolog of Cysteine String Protein (Csp) is identified as important for long-term homeostatic potentiation. CSP has also been reported to be involved in regulation of intracellular calcium. PLCβ, Gαq, and CSP are also known to regulate Cav2-type channels directly, and this possibility, as well as others, are discussed as mechanisms underlying their roles in homeostatic potentiation. Chapter 3 of this thesis reports the extended findings from expression of a gain-of-function Cav2-type channel. The Cav2.1 channel in humans is known to cause a dominant, heritable form of migraine called familial hemiplegic migraine (FHM). Two amino-acid substitutions causative for migraine were cloned into their analogous residues of the Drosophila Cav2 homolog. Expression of these migraine-modeled channels gave rise to several forms of hyperexcitability. Hyperexcitability defects included abnormal evoked waveforms, generation of spontaneous action potential-like events, and multi-quantal release. It is shown that these forms of hyperexcitability can be mitigated through targeted down-regulation of the PLCβ-IP3R-RyR intracellular signaling pathway. Chapter 4 presents an extended discussion as to the roles for presynaptic calcium channels, PLCβ, and CSP in homeostatic synaptic potentiation, and the mechanism underlying hyperexcitability downstream of gain-of-function Cav2-type channels. The proposed model aims to bridge the involvement of the PLCβ pathway in both homeostatic potentiation and neuronal excitability. Last, the implications for these findings on human disease conditions are elucidated.

Parathyroid hormone (PTH) secretion maintains free-ionised extracellular calcium (Ca2+o) homeostasis under the control of the calcium-sensing receptor (CaR). In humans and dogs, blood acidosis and alkalosis is associated with increased or suppressed PTH secretion respectively. Furthermore, large (1.0 pH unit) changes in extracellular pH (pHo) alter Ca2+o sensitivity of the CaR in CaR-transfected HEK-293 cells (CaR-HEK). Indeed, it has been found in this laboratory that even pathophysiological acidosis (pH 7.2) renders CaR less sensitive to Ca2+o while pathophysiological alkalosis (pH 7.6) increases its Ca2+o sensitivity, both in CaR-HEK and parathyroid cells. If true in vivo, then CaR’s pHo sensitivity might represent a mechanistic link between metabolic acidosis and hyperparathyroidism in ageing and renal disease. However, in acidosis one might speculate that the additional H+ could displace Ca2+ bound to plasma albumin, thus increasing free-Ca2+ concentration and so compensating for the decreased CaR responsiveness. Therefore, I first demonstrated that a physiologically-relevant concentration of albumin (5% w/v) failed to overcome the inhibitory effect of pH 7.2 or stimulatory effect of pH 7.6 on CaR-induced intracellular Ca2+ (Ca2+i) mobilisation. Determining the molecular basis of CaR pHo sensitivity would help explain cationic activation of CaR and permit the generation of experimental CaR models that specifically lack pHo sensitivity. With extracellular histidine and free cysteine residues the most likely candidates for pHo sensing (given their sidechains’ pK values), all 17 such CaR residues were mutated to non-ionisable residues. However, none of the resulting CaR mutants exhibited significantly decreased CaR pHo sensitivity. Even co-mutation of the two residues whose individual mutation appeared to elicit modest reductions (CaRH429V and CaRH495V) failed to exhibit any change in CaR pHo sensitivity. I conclude therefore, that neither extracellular histidine nor free cysteine residues account for CaR pHo sensitivity. Next, it is known that cytosolic cAMP drives PTH secretion in vivo and that cAMP potentiates Ca2+o-induced Ca2+i mobilisation in CaR-HEK cells. Given the physiological importance of tightly controlled PTH secretion and Ca2+o homeostasis, here I investigated the influence of cAMP on CaR signalling in CaR-HEK cells. Agents that increase cytosolic cAMP levels such as forskolin and isoproterenol potentiated Ca2+o-induced Ca2+i mobilisation and lowered the Ca2+o threshold for Ca2+i mobilisation. Indeed, forskolin lowered the EC50 for Ca2+o on CaR (2.3 ± 0.1 vs. 3.0 ± 0.1 mM control, P<0.001). Forskolin also potentiated CaR-induced ERK phosphorylation; however protein kinase A activation appeared uninvolved in any of these effects. Pertussis toxin, used to block CaR-induced suppression of cAMP accumulation, also lowered the Ca2+o threshold for Ca2+i mobilisation though appeared to do so by increasing efficacy (Emax). Furthermore, mutation of the CaR’s two putative PKA consensus sequences (CaRS899 and CaRS900) to a non-phosphorylatable residue (alanine) failed to alter the potency of Ca2+o for CaR or attenuate the forskolin response. In contrast, phosphomimetic mutation of CaRS899 (to aspartate) did increase CaR sensitivity to Ca2+o. Together this suggests that PKA-mediated CaRS899 phosphorylation could potentiate CaR activity but that this does not occur following Ca2+o treatment in CaR-HEK cells. Together, these data show that cAMP regulates the Ca2+o threshold for Ca2+i mobilisation, thus helping to explain differential efficacy between CaR downstream signals. If true in vivo, this could help explain how multiple physiological signal inputs may be integrated in parathyroid cells.

Presenilin-1 (PS1) mutations caused by the PSEN1 gene mutations are the major cause of early onset familial Alzheimer’s disease (EOFAD). Two Chinesespecific EOFAD related PS1 mutations, V97L and A136G, have been found. Studies suggested that V97L mutation lead to the overexpression of Aβ42 and tau hyperphosphorylation, which are the major hallmarks of Alzheimer’s disease (AD), while properties of A136G were unclear. Since calcium dysregulation was suggested to play an important role in AD, the research project investigated if V97L and A136G mutations also lead to altered endoplasmic reticulum (ER) 〖Ca〗^(2+) regulation. SH-SY5Y cells transduced with retrovirus carrying V97L mutant or A136 mutant PSEN1 were used as the experiment models. In Western blotting, while the PS1 expression level was unaffected in V97L mutant, the expression level was significantly lower in A136G mutant. In carbachol (CCh) perfusion experiment, V97L mutant was found to exaggerate ER 〖Ca〗^(2+) release when stimulated by higher concentration (30, 100 and 300 μM) CCh, while A136G mutant exaggerated ER Ca2+ release when stimulated by 30 μM and 300 μM CCh, but not 100 μM CCh. In 5% fetal bovine serum (FBS) perfusion experiment, both V97L and A136G mutants were found to sensitize 〖Ca〗^(2+) oscillation, which the sensitization effect of V97L was 3 folds of A136G. The results suggested that V97L mutation exaggerates ER 〖Ca〗^(2+) release, possibly via interaction with IP3R. However the results of A136G were inconclusive and contradicting, therefore further investigation is needed.
published_or_final_version
Physiology
Master
Master of Medical Sciences

Calcium (Ca2+) is one of the major intracellular messengers that impacts nearly every aspect of cell life. In particular, it plays essential roles in neuronal development, synaptic transmission and plasticity, as well as in the regulation of metabolic pathways and cell fate decisions. The first goal of my work was to study more in details the Golgi apparatus (GA), as an intracellular Ca2+ store. The Golgi complex may store up to 5% of the total cellular Ca2+ at higher concentrations and contains several different luminal Ca2+-binding proteins and Ca2+-release channels. The use of a specific Cameleon Ca2+ sensor targeted to the trans-Golgi, allowed us to directly demonstrate the functional GA heterogeneity by showing the distinct behaviour of this sub-compartment: it takes up Ca2+ almost exclusively via SPCA1 (and not by SERCA); it does not release Ca2+ in response to IP3 generation, but rather accumulates the cation as a consequence of the cytoplasmic Ca2+ rise. Using a novel GFP-based Ca2+ probe selectively localized to the medial-Golgi (mGo-D1cpV), we have demonstrated that this sub-compartment behaves in a unique way, releasing Ca2+ upon cell stimulation not only with IP3-generating agonists, but also through Ryanodin receptors (RyRs) and relying, for its Ca2+ uptake, on both SERCA and SPCA1 pumps. The GA represents, thus, an intracellular Ca2+ store with an high complexity level. In a space of a few microns and despite its continuous inter-mixing dynamics, the Ca2+ toolkit undergoes major changes and distinct, separate functional sub-regions, with different Ca2+ features, contribute to its Ca2+ handling. The second aim of my work was to study, at the single cell level by employing specifically targeted cameleon Ca2+ probes, the effect of Familial Alzheimer'€™s Disease (FAD) presenilin 1 and 2 (PS1, PS2) mutants on Ca2+ homeostasis in different sub-cellular compartments: the endoplasmic reticulum (ER), the medial and trans-Golgi. Ca2+ dyshomeostasis has been demonstrated in AD, in particular in the rare genetically inherited FAD cases (mostly due to mutations in PS1 and PS2 genes). In our lab has been recently shown, by employing an ER-targeted aequorin Ca2+ sensor at the cell population level, that FAD-PS2 mutants reduce the Ca2+ content of the major intracellular Ca2+ store, the ER, mostly by inhibiting SERCA activity. In order to deeper investigate the effect of the expression of FAD-PS mutants on ER Ca2+ handling in single cell FRET-experiments, we created a novel ER-targeted cameleon Ca2+ probe (ER-D4) with optimized Ca2+ affinity and dynamic range. In addition, the new cameleon Ca2+ probe targeted to the medial-Golgi, above described, was also used for a similar purpose. Ca2+ handling by the distinct intracellular compartments, at single cell level, was evaluated in SHSY5Y cell line over-expressing PS2 wild type (wt), FAD-PS2-T122R, PS1 wt or FAD-PS1-A246E and also in human fibroblasts from a FAD-PS2-N141I and a FAD-PS1-A246E patient (and corresponding controls from individuals of the same age and sex). Ca2+ concentration ([Ca2+]) of the ER and the medial-Golgi, in resting cells, is strongly reduced by PS2, but not PS1, mutants expression, while that of the trans-Golgi compartment results unaffected by both mutants. The Ca2+ uptake rate in these stores is reduced by PS2 mutants expression and, only within the medial-Golgi, also the Ca2+ leak rate is potentiated. These findings strength our previous data showing the inhibitory effect of FAD-PS2 on SERCA activity. They are also consistent with the fact that, within the Golgi, only the medial-compartment is endowed with IP3 receptors and SERCA pumps, whereas the trans-Golgi is devoid of both. In conclusion, the data highlight the molecular heterogeneity of different intracellular Ca2+ stores (and in distinct sub-compartments of the same organelle) and provide new insights for the comprehension of the mechanisms through which PSs influences cellular Ca2+ homeostasis and FAD pathogenesis. In the future, these cameleon probes will be applied to study Ca2+ alterations induced by FAD-PS2 mutations and, eventually, in vivo by means of viral vectors.
Il Calcio (Ca2+) è uno dei principali messaggeri intracellulari coinvolto quasi in tutti gli aspetti della vita cellulare. In particolare, il Ca2+ gioca un ruolo essenziale nello sviluppo neuronale, plasticità e trasmissione sinaptica, come anche nella regolazione delle vie metaboliche e dell’apoptosi. Il primo obbiettivo del mio lavoro è stato studiare in dettaglio l’apparato di Golgi (AG) come riserva intracellulare di Ca2+. Il complesso del Golgi può contenere più del 5% della totalità del Ca2+ cellulare a concentrazioni significativamente maggiori che in altre regioni cellulari. L’apparato del Golgi contiene tipicamente anche una moltitudine di proteine leganti Ca2+ e canali per il Ca2+. L’uso di una sonda cameleon specificamente indirizzata al trans-Golgi, ci ha permesso di dimostrare direttamente l'eterogeneità funzionale dell’AG mettendo in luce le caratteristiche distinte di questo subcompartimento : l’accumulo di Ca2+ avviene esclusivamente tramite SPCA1 (e non tramite SERCA); inoltre, in risposta a generazione di IP3 non c’è rilascio di Ca2+, ma un suo piccolo accumulo come conseguenza dell’aumento di Ca2+ citosolico. Usando una nuova sonda per il Ca2+ basata sulle GFP selettivamente indirizzata nel cis/medial-Golgi, (mGo-D1cpv), abbiamo dimostrato che il medial-Golgi si comporta in una maniera unica, rilasciandolo Ca2+ dopo stimolazione non solo di agonisti liberanti IP3, ma anche attraverso il recettore rianodinico (RyR) e basandosi, per l’accumulo di Ca2+, sia sulla SERCA che su SPCA1. In conclusione, l’AG rappresenta uno store intracellulare di Ca2+ con un alto grado di complessità . In uno spazio di pochi micron e nonostante il suo continuo intermescolamento dei vari subcompartimenti, il set di proteine che regolano l’omeostasi Ca2+ cambia in maniera consistente, separando sub-regioni funzionali, con differenti proteine regolanti il Ca2+, contribuendo alla sua omeostasi. Il secondo obbiettivo del mio lavoro è stato studiare in dettaglio, utilizzando sonde cameleon specificamente indirizzate, l’effetto di mutazioni familiari del morbo di Alzheimer (FAD) nei geni delle preseniline (PSs) 1 e 2. Nel nostro laboratorio è stato recentemente visto che la mutazione FAD PS2 riduce il contenuto di Ca2+ del principale store cellulare: il reticolo endoplasmatico (RE), principalmente inibendo l'attività della SERCA, come dimostrato a livello di popolazione cellulare utilizzando la sonda Ca2+ equorina indirizzata al RE. Per studiare l’effetto dell’espressione delle mutazioni FAD sull’omeostasi del Ca2+ nel RE in esperimenti FRET su singola cellula, abbiamo creato la nuova sonda cameleon indirizzata al RE (ERD4) con ottimizzate affinità al Ca2+ e range dinamico. L’omeostasi del Ca2+ nei diversi subcompartimenti, a livello di singola cellula, sono stati studiati nella linea cellulare SH-SY5Y sovra-esprimendo PS2 wild type (wt), PS2-T122R, PS1 wt opppure PS1-A246E, e anche in fibroblasti umani da pazienti FAD-PS2-N141I e FAD-PS1-A246E (con i rispettivi fibroblasti di controllo da controlli sani della stessa età e sesso). La concentrazione di Ca2+ ([Ca2+]) di RE e medial-Golgi, in cellule a riposo, è fortemente ridotta dalle PS2 mutate o wt (quest’ultima solo in sovra-espressione), ma non dalla PS1 mutata. Nel trans-Golgi, invece, la [Ca2+] in cellule a riposo risulta non essere alterata in nessun caso. Inoltre, il tasso di accumulo di Ca2+ in questi stores viene ridotto dalle PS2 mutate o wt (quest’ultima solo in sovra-espressione) e, solo nel medial-Golgi, il tasso di fuoriuscita del Ca2+ è potenziata. Questi risultati rafforzano la nostra ipotesi di un effetto inibitorio da parte della PS2 mutata sull'attività della SERCA. Tali risultati sono anche consistenti con il fatto che, all’interno del Golgi, solo il compartimento cis/medial ha il recettore IP3 e la pompa SERCA, mentre il trans-Golgi è mancante di entrambi. In conclusione, i nostri dati fanno luce sull'eterogeneità molecolare di diversi store intracellulari per il Ca2+ (e in distinti subcompartimenti dello stesso organello) e aggiungono nuovi dettagli per comprendere i meccanismi attraverso cui le PSs influenzano l’omeostasi del Ca2+ e la patogenesi delle FAD. In prospettiva futura, queste sonde cameleon saranno utilizzate per studiare alterazioni sull’omeostasi del Ca2+ indotte da mutazioni FAD-PS2 e, eventualmente, in vivo utilizzando vettori virali.

Nel primo progetto abbiamo indagato il coinvolgimento dell’autofagia nella patogenesi delle Malformazioni Cavernose Cerebrali (CCM). CCM è una grave malattia cerebrovascolare che colpisce circa lo 0.3-0.5% della popolazione ed è caratterizzata da capillari dilatati e fragili che predispongono a convulsioni, deficit neurologici e fatali emorragie intracerebrali. CCM è una malattia genetica che può insorgere in modo sporadico o essere ereditata con modalità di trasmissione autosomica dominante. La causa è stata identificata in mutazioni che determinano la perdita di tre geni, KRIT1 (CCM1), CCM2 (MGC4607) e PDCD10 (CCM3), che si verificano nelle forme sia sporadiche sia familiari. L'autofagia è un processo di degradazione che preserva l'omeostasi intracellulare e svolge funzioni essenziali di controllo qualità. Infatti, diversi studi hanno identificato un’associazione tra disregolazione dell’autofagia e diverse patologie umane. Nel nostro lavoro mostriamo che la perdita del gene KRIT1 sopprime l'autofagia, portando ad un aberrante accumulo dell'adattatore autofagico p62/SQSTM1, difetti nei sistemi di controllo qualità e aumento dello stress intracellulare. La perdita di funzione della proteina KRIT1 attiva il pathway mTOR-ULK1, il principale regolatore dell'autofagia, e il trattamento con inibitori di mTOR reverte alcuni dei fenotipi molecolari e cellulari associati alle CCM. La riduzione nei livelli di autofagia è evidente anche in cellule endoteliali umane in cui è stato silenziato CCM2, in cellule e in tessuti di topo CCM3 knockout endotelio-specifico, e in lesioni CCM umane. Inoltre, la deregolazione dell'autofagia è altamente correlata alla transizione endoteliale-mesenchimale, evento cruciale che contribuisce alla progressione delle CCM. Complessivamente, i nostri dati indicano un ruolo chiave per la deregolazione dell’autofagia nella patogenesi delle CCM, fornendo così nuove possibilità per lo sviluppo di strategie farmacologiche per prevenire o contrastare gli esiti clinici avversi conseguenti alle lesioni. [da: Marchi et al. (2015) Defective autophagy is a key feature of cerebral cavernous malformations. EMBO Mol Med 7(11):1403-17]. Il secondo progetto è focalizzato sullo studio del ruolo del proto-oncogene c-Src nella modulazione del segnale Ca2+ intracellulare. c-Src è una tirosin chinasi di tipo non recettoriale e svolge un ruolo chiave in diverse vie di segnalazione coinvolte in eventi cellulari fondamentali, quali crescita cellulare, sopravvivenza, adesione, migrazione e invasione. È nota la correlazione diretta tra deregolazione o sovraespressione di c-Src e diversi tumori umani. Il calcio (Ca2+) è un secondo messaggero intracellulare altamente versatile che agisce in funzioni cellulari cruciali. Durante la carcinogenesi e la progressione del tumore, il segnale Ca2+ viene rimodellato in modo tale da supportare la maggior parte delle caratteristiche tipiche del cancro. In questo progetto abbiamo identificato una funzione chiave per c-Src nella modulazione dell'omeostasi del Ca2+ intracellulare. c-Src riduce il rilascio di Ca2+ dal reticolo endoplasmatico (ER) in seguito a stimolazione con agonista e altera i livelli intracellulari di Ca2+. Tale regolazione del segnale Ca2+ mediata da c-Src è strettamente dipendente dalla sua attività catalitica e dalla sua localizzazione. I nostri risultati hanno stabilito che c-Src controlla i livelli di Ca2+ intracellulare attraverso la fosforilazione di uno specifico target. Questa fosforilazione diretta media l'effetto regolatorio di c-Src sul rilascio di Ca2+ dal ER, influenzando quindi il segnale Ca2+ negli altri compartimenti. Nel complesso il nostro lavoro ha identificato un nuovo ruolo per c-Src nel controllo delle dinamiche del Ca2+ e contribuisce a chiarire in modo più dettagliato come c-Src agisca sia in un contesto fisiologico sia patologico.
In the first project we investigated the involvement of the autophagy in the pathogenesis of the Cerebral Cavernous Malformation (CCM). Cerebral cavernous malformation is a major cerebrovascular disease affecting approximately 0.3-0.5% of the population and is characterized by enlarged and leaky capillaries that predispose to seizures, focal neurological deficits, and fatal intracerebral hemorrhages. Cerebral cavernous malformation is a genetic disease that may arise sporadically or be inherited as an autosomal dominant condition with incomplete penetrance and variable expressivity. Causative loss-of-function mutations have been identified in three genes, KRIT1 (CCM1), CCM2 (MGC4607), and PDCD10 (CCM3), which occur in both sporadic and familial forms. Autophagy is a bulk degradation process that maintains intracellular homeostasis and that plays essential quality control functions within the cell. Indeed, several studies have identified the association between dysregulated autophagy and different human diseases. Here, we show that the ablation of the KRIT1 gene strongly suppresses autophagy, leading to the aberrant accumulation of the autophagy adaptor p62/SQSTM1, defective quality control systems, and increased intracellular stress. KRIT1 loss-of-function activates the mTOR-ULK1 pathway, which is a master regulator of autophagy, and treatment with mTOR inhibitors rescues some of the molecular and cellular phenotypes associated with CCM. Insufficient autophagy is also evident in CCM2-silenced human endothelial cells and in both cells and tissues from an endothelial-specific CCM3-knockout mouse model, as well as in human CCM lesions. Furthermore, defective autophagy is highly correlated to endothelial-to-mesenchymal transition, a crucial event that contributes to CCM progression. Taken together, our data point to a key role for defective autophagy in CCM disease pathogenesis, thus providing a novel framework for the development of new pharmacological strategies to prevent or reverse adverse clinical outcomes of CCM lesions. [From: Marchi et al. (2015) Defective autophagy is a key feature of cerebral cavernous malformations. EMBO Mol Med 7(11):1403-17]. The second project was focused on the investigation of a potential role for the proto-oncogene c-Src in the modulation of intracellular Ca2+ signalling. c-Src is a non-receptor tyrosine kinase that plays a pivotal role in several signalling pathways involved in fundamental cellular events, including cell growth, survival, cell adhesion, migration and invasion. It is well established the link existing between c-Src deregulation or overexpression and several human cancers. Calcium (Ca2+) is a highly versatile intracellular second messenger that acts in crucial cellular functions. During carcinogenesis and tumour progression, Ca2+ signalling is significantly remodelled in a way that sustains most of typical cancer hallmarks, as uncontrolled proliferation and evasion of programmed cell death. We identified a key function for c-Src in the modulation of intracellular Ca2+ homeostasis. c-Src downregulates the Ca2+ release from the endoplasmic reticulum (ER) upon agonist stimulation and significantly alters the intracellular Ca2+ levels. c-Src regulation of Ca2+ signalling is strictly dependent on its catalytic activity and subcellular localization. Our results established that c-Src controls Ca2+ handling through phosphorylation of a specific molecular target. This direct phosphorylation mediates the c-Src effect on Ca2+ release from the intracellular store, thus affecting Ca2+ homeostasis at the other intracellular compartments. Overall, our work identified a new role for c-Src in the control of the intracellular Ca2+ dynamics and contribute to deeply understand how c-Src acts in the context of its physiological and pathological functions.

Des études récentes montrent que l'homéostasie calcique intracellulaire, ainsi que l'expression et l'activité de canaux ioniques jouent un rôle essentiel dans le contrôle de la prolifération cellulaire aussi bien dans un contexte physiologique que dans certains cancers. Cependant, aucune approche proposant les canaux ioniques comme cible thérapeutique n'est actuellement envisagée dans le cadre des traitements des cancers de la prostate. Dans ce travail nous avons mis en évidence l'expression, la fonctionnalité et l'implication des canaux potassiques calcium-activés (IKca1) dans la prolifération des lignées cellulaires cancéreuses de la prostate humaine. Ces études ont également montré que l'activation du canal lKca1 favorise l'entrée de calcium via un canal calcique de la famille des TRP, le canal TRPV6, impliqué dans l'entrée passive de calcium dans les cellules cancéreuses prostatiques. De plus, par des études de co-immunoprécipitations, nous avons montré que le canal IKca1 et le canal calcique TRPV6 sont associés formant ainsi un complexe moléculaire fonctionnel permettant l'entrée de calcium et la prolifération des cellules cancéreuses prostatiques humaines. Par ailleurs, une suppression du canal IKcat induit la différenciation neuroendocrine des cellules cancéreuses prostatiques humaines. Ceci suggère un rôle essentiel joué par le canal IKca1 pour favoriser la croissance ou induire la différenciation cellulaire. Nos études ont également mis en évidence une surexpression de l'ARNm et de la protéine d'IKca1 dans les tissus prostatiques atteints d'un cancer de la prostate humaine. Ces données permettraient de proposer ces canaux ioniques comme marqueurs de cancer et/ou comme cibles thérapeutiques potentielles dans le traitement des cancers de la prostate humaine
Recent studies show that the intracellular calcium homeostasis, as well as expression and the activity of ionic channels play an essential role in the control of cell proliferation as weIl in a physiological context as in certain cancers. However, no approaches offering ionic channels as therapeutic target is nowadays envisaged as part of the treatments of the cancers of the prostate. ln the present work, we showed the expression, functionality and involvement of calcium-activated potassium channels (IKCa1) in the proliferation of the human prostate cancer cells. These studies also showed that the activation of the IKca1 channel favours the calcium entry via a member of the TRP family of calcium channels, TRPV6, in the prostate cancer cells. Besides, by studies of co-immunoprécipitations, we showed that the IKca1 potassium channel and the TRPV6 calcium channel form a functional molecular complex allowing the calcium entry and the proliferation of the prostate cancer cells. Moreover, a down-regulation of the 1Kca1 channel leads to the neuroendocrine differentiation of the human prostate cancer cells. This suggests an essential role played by the 1Kca1 channel to favour growth or lead to cell differentiation. Our immunohistotluorescence studies also showed an overexpression of IKca1 mRNA and protein in human prostate cancer compared to non-tumor tissues. These data would allow to propose these ion channels as markers and/or as potential therapeutic targets in the treatment of the human prostate cancers

博士
中山醫學大學
微生物免疫研究所
100
Background: Type 2 transglutaminase (TG2) is a multifunctional protein, which catalyzes Ca2+-dependent protein modifications, acts as a G protein in transmembrane signaling and cell surface adhesion mediator. It not only has a pro-apoptotic function but also presences in the mitochondria and participates in the molecular events of apoptosis. Modulating calcium signaling from the endoplasmic reticulum (ER) to mitochondria can be critical in the induction of mitochondrial dependent cell death pathway; however, the mechanism of action of TG2 is unknown. Accordingly, we investigate the effect of TG2 in calcium signaling pathway which are known to regulate apoptosis. Materials and methods: Jurkat cells were transfected with human wild type TG2 (wtTG2) and TG2C277S which is the transamidation activity mutant by a tetracycline-induced system. Mouse embryonic fibroblasts deficient for TG2 (TG2 KO MEF) were used for calcium experiment. Apoptotsis was determined by DNA fragmentation and flow cytometry. The levels of mitochondria, cytosolic and ER calcium were determined in cells staining with Rhod-2-AM, Fura-2 and Mag-Fura 2, respectively. TMB-8 and ryanodine were used to determine TG2 participation of the inositol trisphosphate receptor and ryanodine-sensitive receptor mediated calcium release from the ER, respectively. Two-dimensional gel electrophoresis (2-DE) was used to analyze proteins on the TG2 overexpressing cells. Results: Overexpression of wtTG2 induced Jurkat cells apoptosis. TG2 was found to be colocalized with calcium in mitochondria. Overexpression of wtTG2 in Jurkat cell has more mitochondrial calcium accumulation. Mitochondrial calcium uptake and ER calcium release were enhanced immediately in wtTG2 cells evoked by thapsigargin (tg). Using TG2 KO MEF cells evoked by tg, mitochondrial calcium uptake and ER calcium release were decreased. Treatment of cells with TMB-8 or Ryanodine resulted in suppressed the elevation of mitochondrial calcium uptake in cells evoked by tg. After 2-DE analysis, RAP1GDS1 became a candidate of intracellular calcium signaling regulated protein. RAP1GDS1 depletion suppressed TG2 modulating mitochondrial calcium uptake and ER calcium release in Tet-on wtTG2 cells evoked by tg. Conclusions: The transamidation activity of TG2 increase mitochondrial calcium uptake mediated calcium release from ER, which contribute to cell death. RAP1GDS1 is the substrate of TG2 and influences TG2 modulating intracellular calcium homeostasis.

Calbindin-D28k (CaBP) is a 28 kD calcium-binding protein found in specific neuronal populations in the mammalian brain. The hypothesized Ca²⁺-buffering action of CaBP is the basis of suggestions that this protein may serve to protect neurons against cell death mediated by large or prolonged increases in intracellular free Ca²⁺ concentration. However, to date, there is little direct evidence to support this hypothesis. To address this question directly, we have examined Ca²⁺-buffering by CaBP in stably transfected HEK 293 and HeLa cell lines. A variety of methods were employed to induce calcium transients, including transfection of NMD A receptors followed by activation with glutamate. In all experiments there was evidence of CaBP-mediated Ca²⁺-buffering. Moreover, when NMDAR transfected cells were exposed to excitotoxic concentrations of glutamate, cells expressing CaBP exhibited enhanced survival over controls. CaBP was unable to prevent acute necrotic cell death but significantly protected cells from delayed, presumably apoptotic cell death. To examine the potential influence of CaBP upon intracellular Ca²⁺-oscillations, stably-transfected HeLa cells were treated with histamine, while measuring intracellular Ca²⁺. The observation that CaBP flattened the profile of component Ca²⁺ peaks, coupled with data from HEK cell lines, provides unequivocal evidence that CaBP can act to buffer increases in intracellular Ca²⁺. Utilizing a novel method for resolving intracellular Ca²⁺ waves, it was found that transfection with CaBP, or loading with artificial Ca²⁺ buffers, attenuated the velocity of Ca²⁺ waves. The scope of attenuation appeared to be a function of the buffer binding kinetics. The rate of Ca²⁺-binding by CaBP was apparently too slow to influence Ca²⁺- interaction between the closely situated IP₃ receptors in initiation sites, where the faster on rate buffer BAPTA exerted a significant effect. However, both CaBP and BAPTA had significant effects upon events which were more distal to the source of Ca²⁺ release, including effects between the more sparsely distributed IP₃ receptors involved in the propagation of Ca²⁺ waves, and global changes in Ca²⁺. In view of the fact that Ca²⁺ waves and oscillations have been shown to modulate neuron development and gene expression, it is possible that the effects of CaBP may include influencing these processes.

碩士
國立臺灣大學
分子與細胞生物學研究所
101
Previously we reported an intronic microRNA (miR), miR-In300 or miR-3906, which locates at the first intron of zebrafish myf5 gene, suppresses the transcription of myf5 through silencing dickkopf-related protein 3 (dkk3r/dkk3a) during early development such as 16 hpf when myf5 is highly transcribed. However, when myf5 mRNAs are gradually reduced to undetectable level in mature somites at late developmental stage, miR-3906 is still predominant. We had been performed LAMP assay and in vitro/in vivo luciferase reporter assay, and found that miR-3906 enabled to silence reporter gene fused by homer-1b-3’UTR. The line of these evidences give a clue that (1) miR-3906 may have its own promoter which can transcribe miR-3906 at late stage; and (2) miR-3906 plays an unknown function through regulating homer-1b in muscle development at late stage since Dkk3a is not detected anymore in muscle at that stage. Firstly, we constructed plasmids in which the luciferase gene was driven by various lengths of upstream segment of miR-3906. After luciferase activity assay was performed in embryos, we found that a motif located at +303/+402 was able to activate luciferase activity, suggesting that +303/+402 cis-element possesses a promoter activity when myf5 mRNA does not exist. Secondly, using WISH, q-PCR and western blotting, we confirmed that miR-3906 negatively modulates the expression levels of homer-1b mRNA and Homer-1b protein. Thirdly, we found that the expression levels of fast muscle-specific gene fmhc4 and calcium-sensitive gene atp2a1 were increased, but slow muscle-specific gene smhc1 was unchanged, in the miR-3906-MO-injected and homer-1b-mRNA-injected embryos. The downregulated expressions of fmhc4 and atp2a1 induced by excessive miR-3906 or absent homer-1b could be rescued by adding exogenous homer-1b mRNA. Fourthly, we observed that the sarcomeric actin arrangement and Z disk were disorganized in the fast muscle of miR-3906-MO-injected and homer-1b-mRNA-injected embryos. They also exhibited abnormal swimming abilities. Furthermore, we found that the [Ca2+]i in the fast muscle cells of either knockdown of miR-3906 embryos or overexpression of homer-1b embryos was increased 82.9-97.3% higher than that of control embryos. In contrast, the [Ca2+]i of the excessive miR-3906 and absent homer-1b embryos was decreased 22-29.6% lower than that of control group. Taken together, we concluded that miR-3906, which is transcribed from its own promoter at late developmental stage, controls [Ca2+]i homeostasis in fast muscle through fine tuning homer-1b expression during differentiation in order to maintain normal muscle development during embryogenesis.

Neutrophils playa key role in the systemic inflammatory response which may lead to serious tissue injury and multiple organ dysfunction. In this setting, activated neutrophils, largely in response to tumour necrosis factor-alpha (TNF-α), secrete reactive oxidants, granule proteases and bioactive lipids, as well as pro-inflammatory cytokines, emphasising the importance of these cells as targets for anti-inflammatory therapies. There are, however, only a few currently available agents that directly modulate neutrophil pro-inflammatory responses in clinical practice, with corticosteroids being relatively ineffective against these cells. Although, the anti-inflammatory potential of cAMP-elevating agents has been recognised, the exact molecular/biochemical mechanisms which underlie the anti-inflammatory actions of epinephrine and related β-agonists with neutrophils, have not been established. Epinephrine treatment of neutrophils resulted in increased intracellular cAMP and dose-related inhibition of both superoxide production and elastase release, which was potentiated by the type 4 phosphodiesterase inhibitor, rolipram, further supporting a cAMP-mediated effect. Although epinephrine did not affect the release of Ca2+ from neutrophil intracellular stores, the rate of clearance of cytosolic Ca2+ was accelerated by this agent. In the setting of decreased efflux and a reduction in store-operated influx of Ca2+, these effects of epinephrine are compatible with enhancement of the cAMP-dependent Ca2+ sequestering/resequestering endo-membrane Ca2+-ATPase. Epinephrine therefore down-regulates the pro-inflammatory activation of neutrophils by cAMP-mediated enhancement of the clearance of cytosolic Ca2+. Comparison of the effects of 4 selective (fenoterol, formoterol, salbutamol and salmeterol) and 3 non-selective (epinephrine, norepinephrine and isoproterenol) β-adrenoreceptor agonists, on the pro-inflammatory activities of human neutrophils, demonstrated that the agents tested clearly differ with respect to anti-inflammatory potential. Epinephrine, isoproterenol, fenoterol and formoterol significantly increased intracellular concentrations of cAMP in neutrophils, an activity which was paralleled by inhibition of the production of reactive oxidants and release of elastase from FMLP-activated cells. Salbutamol and salmeterol on the other hand, did not cause significant suppression of the pro-inflammatory activities of these cells. The effect of norepinephrine was intermediate between these two groups. The inhibitory effects of βagonists are mediated via β2-adrenergic receptors on the neutrophil membrane. The relationship between activation of NAOPH oxidase, alterations in membrane potential and triggering of Ca2+ fluxes in human phagocytes has been investigated using neutrophils from 4 subjects with chronic granulomatous disease (CGO). Activation of CGO neutrophils was accompanied by a prolonged increase in cytosolic Ca2+, occurring in the setting of trivial membrane depolarisation and accelerated influx of Ca2+. This was associated with hyperactivity of the cells with excessive elastase release, which was attenuated by the type 4 phosphodiesterase inhibitor, rolipram. These findings support the involvement of NAOPH oxidase in regulating membrane potential and Ca2+ influx in activated neutrophils, and may explain the disordered inflammatory responses, and granuloma formation, which are characteristic of CGO. Store-operated influx of Ca2+ into activated neutrophils is stringently regulated, presumably to prevent hyperactivation of the cells. The major contributors to this physiologic, anti-inflammatory process are NAOPH oxidase which, by its membrane depolarising actions excludes extracellular Ca2+, and the plasma membrane and endomembrane Ca2+-ATPases, which mediate clearance of store-derived cation. Subsequent influx of the cation, through store-operated Ca2+ channels is controlled by the relatively slow, restraining, membrane repolarising action of the Na+/Ca2+ exchanger, enabling efficient diversion of incoming cation into stores.
Thesis (DPhil (Immunology))--University of Pretoria, 2005.
Immunology
unrestricted