Calcium signaling in the nucleus

Cytosolic Ca(2+) is a versatile secondary messenger that regulates a wide range of cellular activities. In the past decade, evidence has accumulated that free Ca(2+) within the nucleus also plays an important messenger function. Here we review the mechanisms and effects of Ca(2+) signals within the nucleus. In particular, evidence is reviewed that the nucleus contains the machinery necessary for production of inositol 1,4,5-trisphosphate and for inositol 1,4,5-trisphosphate receptor-mediated Ca(2+) release. The role of Ca(2+) signals within the nucleus is discussed including regulation of such critical cell functions as gene expression, activation of kinases, and permeability of nuclear pores.     

Nicotinic acid adenine dinucleotide phosphate-induced Ca(2+) release. Interactions among distinct Ca(2+) mobilizing mechanisms in starfish oocytes

An intracellular mechanism activated by nicotinic acid adenine dinucleotide phosphate (NAADP(+)) contributes to intracellular Ca(2+) release alongside inositol 1,4,5-trisphosphate (Ins-P(3)) and ryanodine receptors. The NAADP(+)-sensitive mechanism has been shown to be operative in sea urchin eggs, ascidian eggs, and pancreatic acinar cells. Furthermore, most mammalian cell types can synthesize NAADP(+), with nicotinic acid and NADP(+) as precursors. In this contribution, NAADP(+)-induced Ca(2+) release has been investigated in starfish oocytes. Uncaging of injected NAADP(+) induced Ca(2+) mobilization in both immature oocytes and in oocytes matured by the hormone 1-methyladenine (1-MA). The role of extracellular Ca(2+) in NAADP(+)-induced Ca(2+) mobilization, which was minor in immature oocytes, was instead essential in mature oocytes. Thus, the NAADP(+)-sensitive Ca(2+) pool, which is known to be distinct from those sensitive to inositol 1,4,5-trisphosphate or cyclic ADPribose, apparently migrated closer to (or became part of) the plasma membrane during the maturation process. Inhibition of both Ins-P(3) and ryanodine receptors, but not of either alone, substantially inhibited NAADP(+)-induced Ca(2+) mobilization in both immature and mature oocytes. The data also suggest that NAADP(+)-induced Ca(2+) mobilization acted as a trigger for Ca(2+) release via Ins-P(3) and ryanodine receptors.     

Inositol trisphosphate isomers, but not inositol 1,3,4,5-tetrakisphosphate, induce calcium influx in Xenopus laevis oocytes

To investigate the mechanisms by which inositol phosphates regulate cytosolic free Ca2+ concentration ([Ca2+]c), we injected Xenopus oocytes with inositol phosphates and measured Ca2+-activated Cl- currents as an assay of [Ca2+]c. Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) injection (0.1-10.0 pmol) induced an initial transient Cl- current (I1) followed by a second more prolonged Cl- current (I2). Both currents were Ca2+-dependent, but the source of Ca2+ was different. Release of intracellular Ca2+ stores produced I1, whereas influx of extracellular Ca2+ produced I2; Ca2+-free bathing media and inorganic calcium channel blockers (Mn2+, Co2+) did not alter I1 but completely and reversibly inhibited I2. Injection of the Ins(1,4,5)P3 metabolite, inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) (0.2-10.0 pmol) generated a Ca2+-dependent Cl- current with superimposed current oscillations that resulted from release of intracellular Ca2+, not Ca2+ influx. Injection of the Ins(1,3,4,5)P4 metabolite, inositol 1,3,4-trisphosphate (10.0 pmol), or the synthetic inositol trisphosphate isomer, inositol 2,4,5-trisphosphate (1.0-10.0 pmol), mimicked the effect of Ins(1,4,5)P3, stimulating an I1 resulting from release of intracellular Ca2+ and an I2 resulting from influx of extracellular Ca2+. The results indicate that several inositol trisphosphate isomers stimulate both release of intracellular Ca2+ and influx of extracellular Ca2+. Ins(1,3,4,5)P4 also stimulated release of intracellular Ca2+, but it was neither sufficient nor required for Ca2+ influx.     

Regenerative release of calcium from functionally discrete subcellular stores by inositol trisphosphate.

Fluorescence imaging was used to determine the spatial and temporal patterns of subcellular calcium (Ca2+) liberation induced in Xenopus oocytes by photorelease of inositol 1,4,5-trisphosphate (InsP3) from a caged precursor. Increasing levels of InsP3 evoked Ca2+ release that began in a graded manner but, at varying threshold levels of InsP3, localized sites then showed transient and asynchronous 'puffs' of Ca2+ release. With higher levels of InsP3, Ca2+ from adjacent sites formed a focus for initiation of a propagating Ca2+ wave. The results show that InsP3-sensitive Ca2+ stores are arranged as distinct and functionally independent units, and that Ca2+ is released in both graded and regenerative fashions.     

Signaling mechanism for receptor-activated canonical transient receptor potential 3 (TRPC3) channels

Canonical transient receptor potential 3 (TRPC3) is a receptor-activated, calcium permeant, non-selective cation channel. TRPC3 has been shown to interact physically with the N-terminal domain of the inositol 1,4,5-trisphosphate receptor, consistent with a "conformational coupling" mechanism for its activation. Here we show that low concentrations of agonists that fail to produce levels of inositol 1,4,5-trisphosphate sufficient to induce Ca(2+) release from intracellular stores substantially activate TRPC3. By several experimental approaches, we demonstrate that neither inositol 1,4,5-trisphosphate nor G proteins are required for TRPC3 activation. However, diacylglycerols were sufficient to activate TRPC3 in a protein kinase C-independent manner. Surface receptor agonists and exogenously applied diacylglycerols were not additive in activating TRPC3. In addition, inhibition of metabolism of diacylglycerol slowed the reversal of receptor-dependent TRPC3 activation. We conclude that receptor-mediated activation of phospholipase C in intact cells activates TRPC3 via diacylglycerol production, independently of G proteins, protein kinase C, or inositol 1,4,5-trisphosphate.     

Amyloid induced suicidal erythrocyte death.

Amyloid peptides are known to induce apoptosis in a wide variety of cells. Erythrocytes may similarly undergo suicidal death or eryptosis, which is characterized by scrambling of the cell membrane with subsequent exposure of phosphatidylserine (PS) at the cell surface. Eryptosis is triggered by increase of cytosolic Ca(2+) activity and by activation of acid sphingomyelinase with subsequent formation of ceramide. Triggers of eryptosis include energy depletion and isosmotic cell shrinkage (replacement of extracellular Cl(-) by impermeable gluconate for 24 h). The present study explored whether amyloid peptide Abeta (1-42) could trigger eryptosis and to possibly identify underlying mechanisms. Erythrocytes from healthy volunteers were exposed to amyloid and PS-exposure (annexin V binding), cell volume (forward scatter), cytosolic Ca(2+) activity (Fluo3 fluorescence) and ceramide formation (anti-ceramide antibody) were determined by FACS analysis. Exposure of erythrocytes to the amyloid peptide Abeta (1-42) (> or = 0.5 microM) for 24 h significantly triggered annexin V binding, an effect mimicked to a lesser extent by the amyloid peptide Abeta (1-40) (1 microM). Abeta (1-42) (> or = 1.0 microM) further significantly decreased forward scatter of erythrocytes. The effect of Abeta (1-42) (> or = 0.5 microM) on erythrocyte annexin V binding was paralleled by formation of ceramide but not by significant increase of cytosolic Ca(2+) activity. The presence of Abeta (1-42) further significantly enhanced the eryptosis following Cl(-) depletion but not of glucose depletion for 24 hours. The present observations disclose a novel action of Abeta (1-42), which may well contribute to the pathophysiological effects of amyloid peptides, such as vascular complications in Alzheimer's disease.     

Reassessment of the Ca2+ sensing property of a type I metabotropic glutamate receptor by simultaneous measurement of inositol 1,4,5-trisphosphate and Ca2+ in single cells

Transient transfection of Chinese hamster ovary or baby hamster kidney cells expressing the Group I metabotropic glutamate receptor mGlu1alpha with green fluorescent protein-tagged pleckstrin homology domain of phospholipase Cdelta1 allows real-time detection of inositol 1,4,5-trisphosphate. Loading with Fura-2 enables simultaneous measurement of intracellular Ca(2+) within the same cell. Using this technique we have studied the extracellular calcium sensing property of the mGlu1alpha receptor. Quisqualate, in extracellular medium containing 1.3 mm Ca(2+), increased inositol 1,4,5-trisphosphate in all cells. This followed a typical peak and plateau pattern and was paralleled by concurrent increases in intracellular Ca(2+) concentration. Under nominally Ca(2+)-free conditions similar initial peaks in inositol 1,4,5-trisphosphate and Ca(2+) concentration occurred with little change in either agonist potency or efficacy. However, sustained inositol 1,4,5-trisphosphate production was substantially reduced and the plateau in Ca(2+) concentration absent. Depletion of intracellular Ca(2+) stores using thapsigargin abolished quisqualate-induced increases in intracellular Ca(2+) and markedly reduced inositol 1,4,5-trisphosphate production. These data suggest that the mGlu1alpha receptor is not a calcium-sensing receptor because the initial response to agonist is not sensitive to extracellular Ca(2+) concentration. However, prolonged activation of phospholipase C requires extracellular Ca(2+), while the initial burst of activity is highly dependent on Ca(2+) mobilization from intracellular stores.     

Inducible nitric-oxide synthase attenuates vasopressin-dependent Ca2+ signaling in rat hepatocytes

Increases in both Ca(2+) and nitric oxide levels are vital for a variety of cellular processes; however, the interaction between these two crucial messengers is not fully understood. Here, we demonstrate that expression of inducible nitric-oxide synthase in hepatocytes, in response to inflammatory mediators, dramatically attenuates Ca(2+) signaling by the inositol 1,4,5-trisphosphate-forming hormone, vasopressin. The inhibitory effects of induction were reversed by nitric oxide inhibitors and mimicked by prolonged cyclic GMP elevation. Induction was without effect on Ca(2+) signals in response to AlF(4)(-) or inositol 1,4,5-trisphosphate, indicating that phospholipase C activation and release of Ca(2+) from inositol 1,4,5-trisphosphate-sensitive Ca(2+) stores were not targets for nitric oxide inhibition. Vasopressin receptor levels, however, were dramatically reduced in induced cultures. Our data provide a possible mechanism for hepatocyte dysfunction during chronic inflammation.     

Calreticulin modulates capacitative Ca2+ influx by controlling the extent of inositol 1,4,5-trisphosphate-induced Ca2+ store depletion

Calreticulin (CRT) is a highly conserved Ca(2+)-binding protein that resides in the lumen of the endoplasmic reticulum (ER). We overexpressed CRT in Xenopus oocytes to determine how it could modulate inositol 1,4,5-trisphosphate (InsP(3))-induced Ca(2+) influx. Under conditions where it did not affect the spatially complex elevations in free cytosolic Ca(2+) concentration ([Ca(2+)](i)) due to InsP(3)-induced Ca(2+) release, overexpressed CRT decreased by 46% the Ca(2+)-gated Cl(-) current due to Ca(2+) influx. Deletion mutants revealed that CRT requires its high capacity Ca(2+)-binding domain to reduce the elevations of [Ca(2+)](i) due to Ca(2+) influx. This functional domain was also required for CRT to attenuate the InsP(3)-induced decline in the free Ca(2+) concentration within the ER lumen ([Ca(2+)](ER)), as monitored with a "chameleon" indicator. Our data suggest that by buffering [Ca(2+)](ER) near resting levels, CRT may prevent InsP(3) from depleting the intracellular stores sufficiently to activate Ca(2+) influx.     

Cell side-specific sensitivities of intracellular Ca2+ stores for inositol 1,4,5-trisphosphate, cyclic ADP-ribose, and nicotinic acid adenine dinucleotide phosphate in permeabilized pancreatic acinar cells from mouse.

In pancreatic acinar cells hormonal stimulation leads to a cytosolic Ca(2+) wave that starts in the apical cell pole and subsequently propagates toward the basal cell side. We used permeabilized pancreatic acinar cells from mouse and the mag-fura-2 technique, which allows direct monitoring of changes in [Ca(2+)] of intracellular stores. We show here that Ca(2+) can be released from stores in all cellular regions by inositol 1,4,5-trisphosphate. Stores at the apical cell pole showed a higher affinity to inositol 1,4,5-trisphosphate (EC(50) = 89 nm) than those at the basolateral side (EC(50) = 256 nm). In contrast, cADP-ribose, a modifier of Ca(2+)-induced Ca(2+) release, and nicotinic acid adenine dinucleotide phosphate (NAADP) were able to release Ca(2+) exclusively from intracellular stores located at the basolateral cell side. Our data agree with observations that upon stimulation Ca(2+) is released initially at the apical cell side and that this is caused by high affinity inositol 1,4,5-trisphosphate receptors. Moreover, our findings allow the conclusion that in Ca(2+) wave propagation from the apical to the basolateral cell side observed in pancreatic acinar cells Ca(2+)-induced Ca(2+) release, modulated by cADP-ribose and/or NAADP, might be involved.     

Inositol 1,4,5-trisphosphate receptor contains multiple cavities and L-shaped ligand-binding domains

Calcium concentrations are strictly regulated in all biological cells, and one of the key molecules responsible for this regulation is the inositol 1,4,5-trisphosphate receptor, which was known to form a homotetrameric Ca(2+) channel in the endoplasmic reticulum. The receptor is involved in neuronal transmission via Ca(2+) signaling and for many other functions that relate to morphological and physiological processes in living organisms. We analysed the three-dimensional structure of the ligand-free form of the receptor based on a single-particle technique using an originally developed electron microscope equipped with a helium-cooled specimen stage and an automatic particle picking system. We propose a model that explains the complex mechanism for the regulation of Ca(2+) release by co-agonists, Ca(2+), inositol 1,4,5-trisphosphate based on the structure of multiple internal cavities and a porous balloon-shaped cytoplasmic domain containing a prominent L-shaped density which was assigned by the X-ray structure of the inositol 1,4,5-trisphosphate binding domain.     

Inositol tetrakisphosphates as second messengers induce Ca(++)-dependent chloride currents in Xenopus laevis oocytes.

Microinjection of inositol 1,3,4,5-tetrakisphosphate or inositol 1,4,5-trisphosphate induced distinct chloride membrane currents in defolliculated Xenopus laevis oocytes. To decide whether these Cl(-)-currents were due to the injected compounds or their metabolic products, [3H]Ins(1,3,4,5)P4 or [3H]Ins(1,4,5)P3 were injected into oocytes and their metabolites were analyzed by HPLC. Our results indicate that Ins(1,3,4,5)P4 itself or its metabolite Ins(1,3,4,6)P4 is able to induce Cl(-)-membrane currents, most likely by increasing the cytosolic Ca(++)-concentration.     

The overexpression of presenilin2 and Alzheimer's-disease-linked presenilin2 variants influences TRPC6-enhanced Ca2+ entry into HEK293 cells

Mutations in the presenilin (PS) genes are linked to the development of early-onset Alzheimer's disease by a gain-of-function mechanism that alters proteolytic processing of the amyloid precursor protein (APP). Recent work indicates that Alzheimer's-disease-linked mutations in presenilin1 and presenilin2 attenuate calcium entry and augment calcium release from the endoplasmic reticulum (ER) in different cell types. However, the regulatory mechanisms underlying the altered profile of Ca(2+) signaling are unknown. The present study investigated the influence of two familial Alzheimer's-disease-linked presenilin2 variants (N141I and M239V) and a loss-of-function presenilin2 mutant (D263A) on the activity of the transient receptor potential canonical (TRPC)6 Ca(2+) entry channel. We show that transient coexpression of Alzheimer's-disease-linked presenilin2 mutants and TRPC6 in human embryonic kidney (HEK) 293T cells abolished agonist-induced TRPC6 activation without affecting agonist-induced endogenous Ca(2+) entry. The inhibitory effect of presenilin2 and the Alzheimer's-disease-linked presenilin2 variants was not due to an increase in amyloid beta-peptides in the medium. Despite the strong negative effect of the presenilin2 and Alzheimer's-disease-linked presenilin2 variants on agonist-induced TRPC6 activation, conformational coupling between inositol 1,4,5-trisphosphate receptor type 3 (IP(3)R3) and TRPC6 was unaffected. In cells coexpressing presenilin2 or the FAD-linked presenilin2 variants, Ca(2+) entry through TRPC6 could still be induced by direct activation of TRPC6 with 1-oleoyl-2-acetyl-sn-glycerol (OAG). Furthermore, transient coexpression of a loss-of-function PS2 mutant and TRPC6 in HEK293T cells enhanced angiotensin II (AngII)- and OAG-induced Ca(2+) entry. These results clearly indicate that presenilin2 influences TRPC6-mediated Ca(2+) entry into HEK293 cells.     

SERCA pump activity is physiologically regulated by presenilin and regulates amyloid beta production

In addition to disrupting the regulated intramembraneous proteolysis of key substrates, mutations in the presenilins also alter calcium homeostasis, but the mechanism linking presenilins and calcium regulation is unresolved. At rest, cytosolic Ca(2+) is maintained at low levels by pumping Ca(2+) into stores in the endoplasmic reticulum (ER) via the sarco ER Ca(2+)-ATPase (SERCA) pumps. We show that SERCA activity is diminished in fibroblasts lacking both PS1 and PS2 genes, despite elevated SERCA2b steady-state levels, and we show that presenilins and SERCA physically interact. Enhancing presenilin levels in Xenopus laevis oocytes accelerates clearance of cytosolic Ca(2+), whereas higher levels of SERCA2b phenocopy PS1 overexpression, accelerating Ca(2+) clearance and exaggerating inositol 1,4,5-trisphosphate-mediated Ca(2+) liberation. The critical role that SERCA2b plays in the pathogenesis of Alzheimer's disease is underscored by our findings that modulating SERCA activity alters amyloid beta production. Our results point to a physiological role for the presenilins in Ca(2+) signaling via regulation of the SERCA pump.     

Humoral immune response to fibrillar beta-amyloid peptide

The beta-amyloid peptide (Abeta) is a normal product of the proteolytic processing of its precursor (beta-APP). Normally, it elicits a very low humoral immune response; however, the aggregation of monomeric Abeta to form fibrillar Abeta amyloid creates a neo-epitope, to which antibodies are generated. Rabbits were injected with fibrillar human Abeta(1-42), and the resultant antibodies were purified and their binding properties characterized. The antibodies bound to an epitope in the first eight residues of Abeta and required a free amino terminus. Additional residues did not affect the affinity of the epitope as long as the peptide was unaggregated; the antibody bound Abeta residues 1-8, 1-11, 1-16, 1-28, 1-40, and 1-42 with similar affinities. In contrast, the antibodies bound approximately 1000-fold more tightly to fibrillar Abeta(1-42). Their enhanced affinity did not result from their bivalent nature: monovalent Fab fragments exhibited a similar affinity for the fibrils. Nor did it result from the particulate nature of the epitope: monomeric Abeta(1-16) immobilized on agarose and soluble Abeta(1-16) exhibited similar affinities for the antifibrillar antibodies. In addition, antibodies raised to four nonfibrillar peptides corresponding to internal Abeta sequences did not exhibit enhanced affinity for fibrillar Abeta(1-42). Antibodies directed to the C-terminus of Abeta bound poorly to fibrillar Abeta(1-42), which is consistent with models where the carboxyl terminus is buried in the interior of the fibril and the amino terminus is on the surface. When used as an immunohistochemical probe, the antifibrillar Abeta(1-42) IgG exhibited enhanced affinity for amyloid deposits in the cerebrovasculature. We hypothesize either that the antibodies recognize a specific conformation of the eight amino-terminal residues of Abeta, which is at least 1000-fold more favored in the fibril than in monomeric peptides, or that affinity maturation of the antibodies produces an additional binding site for the amino-terminal residues of an adjacent Abeta monomer. In vivo this specificity would direct the antibody primarily to fibrillar vascular amyloid deposits even in the presence of a large excess of monomeric Abeta or its precursor. This observation may explain the vascular meningeal inflammation that developed in Alzheimer's disease patients immunized with fibrillar Abeta. Passive immunization with an antibody directed to an epitope hidden in fibrillar Abeta and in the transmembrane region of APP might be a better choice in the search for an intervention to remove Abeta monomers without provoking an inflammatory response.     

Roles of Ca2+ on the inositol 1,4,5-trisphosphate-induced release of Ca2+ from saponin-permeabilized single cells of the porcine coronary artery

The release of Ca2+ from the intracellular store site, as induced by inositol 1,4,5-trisphosphate, was studied in relation to free Ca2+ concentrations or amounts of stored Ca2+ in smooth muscle cells. The maximal Ca2+ release induced by inositol 1,4,5-trisphosphate was observed when the amount of Ca2+ in the store site was about 50% of the maximal capacity of the Ca2+ storage, and when the extravesicular free Ca2+ concentration was less than 1.5 X 10(-6) M. The Ca2+ release induced by inositol 1,4,5-trisphosphate was accelerated by ATP and 5'-adenylylimidodiphosphate (AMPPNP), but not by ADP and AMP. This inositol 1,4,5-trisphosphate-induced Ca2+ release appeared to be specific for intracellular Ca2+ store sites (mainly sarcoplasmic reticulum), and this Ca2+ release was not apparent in the sarcolemmal fraction.     

Role of voltage-dependent modulation of store Ca2+ release in synchronization of Ca2+ oscillations

Slow waves are rhythmic depolarizations that underlie mechanical activity of many smooth muscles. Slow waves result through rhythmic Ca(2+) release from intracellular Ca(2+) stores through inositol 1,4,5-trisphosphate (IP(3)) sensitive receptors and Ca(2+)-induced Ca(2+) release. Ca(2+) oscillations are transformed into membrane depolarizations by generation of a Ca(2+)-activated inward current. Importantly, the store Ca(2+) oscillations that underlie slow waves are entrained across many cells over large distances. It has been shown that IP(3) receptor-mediated Ca(2+) release is enhanced by membrane depolarization. Previous studies have implicated diffusion of Ca(2+) or the second messenger IP(3) across gap junctions in synchronization of Ca(2+) oscillations. In this study, a novel mechanism of Ca(2+) store entrainment through depolarization-induced IP(3) receptor-mediated Ca(2+) release is investigated. This mechanism is significantly different from chemical coupling-based mechanisms, as membrane potential has a coupling effect over distances several orders of magnitude greater than either diffusion of Ca(2+) or IP(3) through gap junctions. It is shown that electrical coupling acting through voltage-dependent modulation of store Ca(2+) release is able to synchronize oscillations of cells even when cells are widely separated and have different intrinsic frequencies of oscillation.     

Presence of a putative vesicular inositol 1,4,5-trisphosphate-sensitive nucleoplasmic Ca2+ store

The inositol 1,4,5-trisphosphate receptors (IP(3)Rs) are widely localized in both the heterochromatin and euchromatin regions. We found recently the presence of nucleoplasmic complexes that are composed of phospholipids, IP(3)R/Ca(2+) channels, and Ca(2+) storage protein chromogranin B (CGB). Close examination and 3D image reconstruction of these complexes revealed numerous vesicular structures with an average diameter of approximately 50 nm that are primarily interspersed between the heterochromatins. IP(3) rapidly released Ca(2+) from these structures, but other inositol phosphates, inositol 1,4-bisphosphate, inositol 1,3,4-trisphosphate, and inositol 1,3,4,5-tetrakisphosphate, failed to release Ca(2+). Addition of heparin or IP(3)R antibody blocked the IP(3)-induced Ca(2+) releases, indicating the release of Ca(2+) through the IP(3)R/Ca(2+) channels. Given the presence of the IP(3)R/Ca(2+) channels and Ca(2+) storage protein CGB in these vesicular structures, we postulate that these vesicles are the IP(3)-sensitive nucleoplasmic Ca(2+) stores. Abundance of the vesicular Ca(2+) stores between the heterochromatins appeared to imply critical roles these vesicular Ca(2+) stores play in controlling the Ca(2+) concentrations of the chromosomes.     

Mechanism of Ca2+ disruption in Alzheimer's disease by presenilin regulation of InsP3 receptor channel gating

Mutations in presenilins (PS) are the major cause of familial Alzheimer's disease (FAD) and have been associated with calcium (Ca2+) signaling abnormalities. Here, we demonstrate that FAD mutant PS1 (M146L)and PS2 (N141I) interact with the inositol 1,4,5-trisphosphate receptor (InsP3R) Ca2+ release channel and exert profound stimulatory effects on its gating activity in response to saturating and suboptimal levels of InsP3. These interactions result in exaggerated cellular Ca2+ signaling in response to agonist stimulation as well as enhanced low-level Ca2+signaling in unstimulated cells. Parallel studies in InsP3R-expressing and -deficient cells revealed that enhanced Ca2+ release from the endoplasmic reticulum as a result of the specific interaction of PS1-M146L with the InsP3R stimulates amyloid beta processing,an important feature of AD pathology. These observations provide molecular insights into the "Ca2+ dysregulation" hypothesis of AD pathogenesis and suggest novel targets for therapeutic intervention.     

Calcium dyshomeostasis in beta-amyloid and tau-bearing skeletal myotubes

The relative scarcity of inclusion-affected muscle cells or markers of cell death in inclusion body myositis (IBM) is in distinction to the specific and early intracellular deposition of several Alzheimer's Disease (AD)-related proteins. The current study examined the possible correlation between myotube beta-amyloid and/or Tau accumulations and a widespread mishandling of intracellular muscle calcium concentration that could potentially account for the unrelenting weakness in affected patients. Cultured myogenic cells (C(2)C(12)) expressed beta-amyloid-42 (Abeta(42)) and fetal Tau peptides, as human transgenes encoded by herpes simplex virus, either individually or concurrently. Co-expression of Abeta(42) in C(2)C(12) myotubes resulted in hyperphosphorylation of Tau protein that was not observed when Tau was expressed alone. Resting calcium concentration and agonist-induced RyR-mediated Ca(2+) release were examined using calcium-specific microelectrodes and Fluo-4 epifluorescence, respectively. Co-expression of Abeta(42) and Tau cooperatively elevated basal levels of myoplasmic-free calcium, an effect that was accompanied by depolarization of the plasma membrane. Sarcoplasmic reticulum (SR) calcium release, induced by KCl depolarization, was not affected by Abeta(42) or Tau. In contrast, expression of Abeta(42), Tau, or Abeta(42) together with Tau resulted in enhanced sensitivity of ryanodine receptors to activation by caffeine. Notably, expression of beta-amyloid, alone, was sufficient to result in an increased sensitivity to direct activation by caffeine. Current results indicate that amyloid proteins cooperate to raise resting calcium levels and that these effects are associated with a passive SR Ca(2+) leak and Tau hyperphosphorylation in skeletal muscle.