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When, where and how? Focus on neuronal calcium dysfunctions in Alzheimer's Disease.
Full text linkAlzheimer’s disease (AD), since its characterization as a precise form of dementia with its own pathological
hallmarks, has captured scientists’ attention because of its complexity. The last 30 years have been
filled with discoveries regarding the elusive aetiology of this disease and, thanks to advances in molecular
biology and live imaging techniques, we now know that an important role is played by calcium (Ca2+).
Ca2+, as ubiquitous second messenger, regulates a vast variety of cellular processes, from neuronal excitation
and communication, to muscle fibre contraction and hormone secretion, with its action spanning
a temporal scale that goes from microseconds to hours. It is therefore very challenging to conceive a
single hypothesis that can integrate the numerous findings on this issue with those coming from the
classical fields of AD research such as amyloid-beta (A) and tau pathology. In this contribution, we will
focus our attention on the Ca2+ hypothesis of AD, dissecting it, as much as possible, in its subcellular
localization, where the Ca2+ signal meets its specificity. We will also follow the temporal evolution of the
Ca2+ hypothesis, providing some of the most updated discoveries. Whenever possible, we will link the
findings regarding Ca2+ dysfunction to the other players involved in AD pathogenesis, hoping to provide
a crossover body of evidence, useful to amplify the knowledge that will lead towards the discovery of an
effective therapy
Delayed activation of the store-operated calcium current induced by calreticulin overexpression in RBL-1 cells
No full textCalreticulin (CRT) is a high-capacity, low-affinity Ca2+-binding protein located in the lumen of the endoplasmic reticulum (ER) of all eukaryotic cells investigated so far. Its high level of conservation among different species suggests that it serves functions fundamental to cell survival. The role originally proposed for CRT, i.e., the main Ca2+ buffer of the ER, has been obscured or even casted by its implication in processes as diverse as gene expression, protein folding, and cell adhesion. In this work we seek the role of CRT in Ca2+ storing and signaling by evaluating its effects on the kinetics and amplitude of the store-operated Ca2+ current (ICRAC). We show that, in the rat basophilic leukemia cell line RBL-1, overexpression of CRT, but not of its mutant lacking the high-capacity Ca2+-binding domain, markedly retards the ICRAC development, however, only when store depletion is slower than the rate of current activation. On the contrary, when store depletion is rapid and complete, overexpression of CRT has no effect. The present results are compatible with a major Ca2+-buffering role of CRT within the ER but exclude a direct, or indirect, role of this protein on the mechanism of ICRAC activation
Receptor-mediated calcium influx in PC12 cells : ATP and bradykinin activate two indipendent pathways
No full textIn the neurosecretory cell line PC12 the cytosolic free Ca2+ concentration, [Ca2+]i, and membrane potential were affected by both external ATP and the nonapeptide bradykinin, BK. The latter caused a rapid and large release of Ca2+ from intracellular stores (Ca2+ redistribution) and, in the presence of external Ca2+, a long lasting, but moderate Ca2+ influx, which was insensitive to dihydropyridine blockers. On the contrary, ATP evoked a [Ca2+]i rise which rapidly inactivated. At least three different mechanisms accounted for the ATP-induced increase in [Ca2+]i: less than 20% of the total response was due to intracellular Ca2+ redistribution, consistent with a small increase in inositol 1,4,5-trisphosphate level; the rest (over 80%) was equally accounted for by ATP-activated cation channels and voltage-gated Ca2+ channels. ATP and BK (the latter after K+ channel blockade) caused plasma membrane depolarization. With both agonists the inward current was carried by both Na+ and Ca2+, although the BK-activated current appeared to be more selective for Ca2+. Channels triggered by ATP and BK differed not only in their cation selectivity, but also in modulation by both [Ca2+]i and drugs such as the phorbol ester phorbol 12-myristate 13-acetate and the new antagonist of ligand-activated Ca2+ influx, SK&F 96365
Stimulation of single L-type calcium channels in rat pituitary GH3 cells by thyrotropin-releasing hormone
No full textHormonal stimulation of voltage-dependent Ca2+ channels in pituitary cells is thought to contribute to the sustained phase of Ca2+ entry and secretion induced by secretion stimulating hormones and has been suggested as a mechanism for refilling the Ca2+ stores. Using the cell-attached patch-clamp technique, we studied the stimulation of single Ca2+ channels by thyrotropin-releasing hormone (TRH) in rat GH3 cells. We show that TRH applied from the bath switched the activity of single L-type Ca2+ channels from a gating mode with very low open probability (po) to a gating mode with slightly smaller conductance but 10 times higher po. Interconversions between these two gating modes were also observed under basal conditions, where the equilibrium was shifted towards the low po mode. TRH applied from the pipette had no effect, indicating the involvement of a cytosolic compound in the stimulatory pathway. We show that TRH does not potentiate all the L-type Ca2+ channels in a given membrane patch and report evidence for co-expression of two functionally different L-type Ca2+ channels. Our results uncover the biophysical mechanism of hormonal stimulation of voltage-dependent Ca2+ channels in GH3 cells and are consistent with differential modulation of different subtypes of dihydropyridine-sensitive Ca2+ channels
Paradoxical Ca2+ Rises Induced by Low External Ca2+ in Hippocampal Neurones
No full textConfocal Ca2+ imaging of rat hippocampal slices shows a paradoxical effect of acute reductions of the [Ca2+]o. Upon slice perfusion with low-Ca2+ media, a prompt intracellular Ca2+ rise selectively occurs in neurones. This response is observed only in slices challenged with agonists of group I metabotropic glutamate or M1 muscarinic receptors. In contrast, the intracellular Ca2+ level of non-stimulated neurones is insensitive to reductions of [Ca2+]o. The phenomenon is observed in 20-25 % of cultured cortical neurones. Evidence is provided demonstrating that: (1) this paradoxical response is not due to a non-specific decrease in divalent cation concentration but it is selectively activated by a reduction in [Ca2+]o, being maximal with [Ca2+]o between 0.25 and 0.5 mM; (2) upon maximal stimulation, 70-90 % of CA1-CA3 pyramidal neurones sense a reduction in [Ca2+]o; a weaker response is observed in neurones from the neocortex, whereas neurones from the dentate gyrus and granule cells from the cerebellum fail to respond; (3) conditions that elicit paradoxical Ca2+ responses cause depolarisation and increase the firing rate of hippocampal neurones; (4) paradoxical Ca2+ rises depend, primarily, on Ca2+ influx through L-type voltage-operated Ca2+ channels and to a lesser extent on release from intracellular Ca2+ stores. Inhibition of phospholipase C or protein kinase C failed to suppress the neuronal response, whereas a selective inhibitor of the Src-family of tyrosine kinases abolishes the paradoxical neuronal Ca2+ rise. A model is presented to explain how this response is elicited by contemporaneous reduction of the [Ca2+]o and metabotropic receptor stimulation; implications for the pathophysiology of the CNS are also discussed
Ca2+ dysregulation mediated by presenilins in Familial Alzheimer's Disease: causing or modulating factor?
No full textCa2+, one of the major intracellular messengers, plays essential roles in neuronal development, synaptic transmission and plasticity, as well as in the regulation of metabolic pathways. A perturbed Ca2+ homeostasis has been demonstrated in Alzheimer’s Disease (AD), one of the most devastating neurological disorder of the elderly. Although the majority of AD cases are sporadic, a small fraction is inherited in a dominant pattern (Familial AD, FAD). Of the three genes involved in the pathogenesis of FAD, two code for the ubiquitously expressed proteins presenilin (PS) 1 and 2. Mutations in PSs have variably been correlated to alterations of Ca2+ signalling and different molecular targets have been identified, suggesting a physiological role for these proteins in multiple intracellular Ca2+ pathways.
According to the renewed “Ca2+ overload” hypothesis for FAD pathogenesis, PS mutations increase the Ca2+ content of the endoplasmic reticulum (ER), thus sensitizing neurons to excitotoxicity and neuronal degeneration. The latter process is closely linked to exaggerated ER Ca2+ release that, in turn, causes abnormal mitochondrial Ca2+ uptake and cell death. New evidence from different groups has however shown exceptions to this scenario: in fact, in addition to an increased, also a reduced, or even an unchanged, ER Ca2+ content has been described in cells over-expressing wild type or FAD mutant PSs. Altogether, these findings suggest that Ca2+ dysregulation in FAD is variable in nature, depending on the type of mutation and the cell model under investigation, thus appearing as a modulator rather than a causing event in AD pathogenesis
Dynamic properties of an inositol 1,4,5-trisphpsphate-and thapsigargin-insensitive calcium pool in mammalian cell lines
Full text linkThe functional characteristics of a nonacidic, inositol 1,4,5-trisphosphate- and thapsigargin-insensitive Ca2+ pool have been characterized in mammalian cells derived from the rat pituitary gland (GH3, GC, and GH3B6), the adrenal tissue (PC12), and mast cells (RBL-1). This Ca2+ pool is released into the cytoplasm by the Ca2+ ionophores ionomycin or A23187 after the discharge of the inositol 1,4,5-trisphosphate-sensitive store with an agonist coupled to phospholipase C activation and/or thapsigargin. The amount of Ca2+ trapped within this pool increased significantly after a prolonged elevation of intracellular Ca2+ concentration elicited by activation of Ca2+ influx. This pool was affected neither by caffeine-ryanodine nor by mitochondrial uncouplers. Probing mitochondrial Ca2+ with recombinant aequorin confirmed that this pool did not coincide with mitochondria, whereas its homogeneous distribution across the cytosol, as revealed by confocal microscopy, and its insensitivity to brefeldin A make localization within the Golgi complex unlikely. A proton gradient as the driving mechanism for Ca2+ uptake was excluded since ionomycin is inefficient in releasing Ca2+ from acidic pools and Ca2+ accumulation/release in/from this store was unaffected by monensin or NH4Cl, drugs known to collapse organelle acidic pH gradients. Ca2+ sequestration inside this pool, thus, may occur through a low-affinity, high-capacity Ca2+-ATPase system, which is, however, distinct from classical endosarcoplasmic reticulum Ca2+-ATPases. The cytological nature and functional role of this Ca2+ storage compartment are discussed
Presenilin-2 modulation of ER-mitochondria interactions. FAD mutations, mechanisms and pathological consequences
No full textPresenilin (PS) mutations are the main cause of Familial Alzheimer's Disease (FAD) and have been demonstrated to cause an imbalance of intracellular Ca(2+) homeostasis. Though PS1 and 2 are generally considered to behave similarly in terms of their effects on Ca(2+) handling, we have recently described a novel function, which is unique to PS2, i.e., the modulation of ER-mitochondria juxtaposition. Accordingly, PS2, but not PS1, affects the Ca(2+) cross-talk between these organelles, a key feature in determining cell fate. In particular, PS2 overexpression, and more drastically that of FAD-linked PS2 mutants, strongly increases the interaction between ER and mitochondria, thus facilitating mitochondrial Ca(2+) uptake. The likely mechanisms behind this phenomenon and its potential effects in cell physiology and pathology are discussed
A beta 42 oligomers selectively disrupt neuronal calcium release
No full textAccumulation of amyloid-beta (A beta) peptides correlates with aging and progression of Alzheimer's disease (AD). A beta peptides, which cause early synaptic dysfunctions, spine loss, and memory deficits, also disturb intracellular Ca2+ homeostasis. By cytosolic and endoplasmic reticulum Ca2+ measurements, we here define the short-term effects of synthetic A beta 42 on neuronal Ca2+ dynamics. When applied acutely at submicromolar concentration, as either oligomers or monomers, A beta 42 did not cause Ca2+ release or Ca2+ influx. Similarly, 1-hour treatment with A beta 42 modified neither the resting cytosolic Ca2+ level nor the long-lasting Ca2+ influx caused by KCl-induced depolarization. In contrast, A beta 42 oligomers, but not monomers, significantly altered Ca2+ release from stores with opposite effects on inositol 1,4,5-trisphosphate (IP3)-and caffeine-induced Ca2+ mobilization without alteration of the total store Ca2+ content. Ca2+ dysregulation by A beta 42 oligomers involves metabotropic glutamate receptor 5 and requires network activity and the intact exo-endocytotic machinery, being prevented by tetrodotoxin and tetanus toxin. These findings support the idea that Ca2+ store dysfunction is directly involved in A beta 42 neurotoxicity and represents a potential therapeutic target in AD-like dementia. (C) 2015 Elsevier Inc. All rights reserved
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