SERCA Pump Research Articles

Identification of novel targets for host-directed therapeutics against intracellular Staphylococcus aureus

During patient colonization, Staphylococcus aureus is able to invade and proliferate within human cells to evade the immune system and last resort drugs such as vancomycin. Hijacking specific host molecular factors and/or pathways is necessary for pathogens to successfully establish an intracellular infection. In this study, we employed an unbiased shRNA screening coupled with ultra-fast sequencing to screen 16,000 human genes during S. aureus infection and we identified several host genes important for this intracellular pathogen. In addition, we interrogated our screening results to find novel host-targeted therapeutics against intracellular S. aureus. We found that silencing the human gene TRAM2 resulted in a significant reduction of intracellular bacterial load while host cell viability was restored, showing its importance during intracellular infection. Furthermore, TRAM2 is an interactive partner of the endoplasmic reticulum SERCA pumps and treatment with the SERCA-inhibitor Thapsigargin halted intracellular MRSA survival. Our results suggest that Thapsigargin could be repurposed to tackle S. aureus host cell infection in combination with conventional antibiotics.

Sarco-Endoplasmic Reticulum Calcium Release Model Based on Changes in the Luminal Calcium Content.

The sarcoplasmic/endoplasmic reticulum (SR/ER) is the main intracellular calcium (Ca2+) pool in muscle and non-muscle eukaryotic cells, respectively. The reticulum accumulates Ca2+ against its electrochemical gradient by the action of sarco/endoplasmic reticulum calcium ATPases (SERCA pumps), and the capacity of this Ca2+ store is increased by the presence of Ca2+ binding proteins in the lumen of the reticulum. A diversity of physical and chemical signals, activate the main Ca2+ release channels, i.e. ryanodine receptors (RyRs) and inositol (1, 4, 5) trisphosphate receptors (IP3Rs), to produce transient elevations of the cytoplasmic calcium concentration ([Ca2+]i) while the reticulum is being depleted of Ca2+. This picture is incomplete because it implies that the elements involved in the Ca2+ release process are acting alone and independently of each other. However, it appears that the Ca2+ released by RyRs and IP3Rs is trapped in luminal Ca2+ binding proteins (Ca2+ lattice), which are associated with these release channels, and the activation of these channels appears to facilitate that the trapped Ca2+ ions become available for release. This situation makes the initial stage of the Ca2+ release process a highly efficient one; accordingly, there is a large increase in the [Ca2+]i with minimal reductions in the bulk of the free luminal SR/ER [Ca2+] ([Ca2+]SR/ER). Additionally, it has been shown that active SERCA pumps are required for attaining this highly efficient Ca2+ release process. All these data indicate that Ca2+ release by the SR/ER is a highly regulated event and not just Ca2+ coming down its electrochemical gradient via the open release channels. One obvious advantage of this sophisticated Ca2+ release process is to avoid depletion of the ER Ca2+ store and accordingly, to prevent the activation of ER stress during each Ca2+ release event.

ALS-Associated SOD1(G93A) Decreases SERCA Pump Levels and Increases Store-Operated Ca2+ Entry in Primary Spinal Cord Astrocytes from a Transgenic Mouse Model.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the selective death of motor neurons (MNs), probably by a combination of cell- and non-cell-autonomous processes. The past decades have brought many important insights into the role of astrocytes in nervous system function and disease, including the implication in ALS pathogenesis possibly through the impairment of Ca2+-dependent astrocyte-MN cross-talk. In this respect, it has been recently proposed that altered astrocytic store-operated Ca2+ entry (SOCE) may underlie aberrant gliotransmitter release and astrocyte-mediated neurotoxicity in ALS. These observations prompted us to a thorough investigation of SOCE in primary astrocytes from the spinal cord of the SOD1(G93A) ALS mouse model in comparison with the SOD1(WT)-expressing controls. To this purpose, we employed, for the first time in the field, genetically-encoded Ca2+ indicators, allowing the direct assessment of Ca2+ fluctuations in different cell domains. We found increased SOCE, associated with decreased expression of the sarco-endoplasmic reticulum Ca2+-ATPase and lower ER resting Ca2+ concentration in SOD1(G93A) astrocytes compared to control cells. Such findings add novel insights into the involvement of astrocytes in ALS MN damage.

Cast immobilization of hindlimb upregulates sarcolipin expression in atrophied skeletal muscles and increases thermogenesis in C57BL/6J mice.

Mechanical unloading impairs cytosolic calcium (Ca2+) homeostasis in skeletal muscles. In this study, we investigated whether sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) itself or one of the regulators of the Ca2+ SERCA pump, sarcolipin (SLN), is altered to deregulate Ca2+ homeostasis in cast immobilized, atrophied muscles. Hindlimb muscles of 8-wk-old male C57BL/6J mice were subjected to bilateral cast immobilization for 2 wk. Two-week-cast immobilization induced both body weight and skeletal muscle loss. Highly phosphorylated Ca2+/calmodulin-dependent protein kinase II in the atrophied muscles suggested that cytosolic Ca2+ concentration was elevated. Extremely high expression levels of SLN mRNA and protein were observed in the atrophied muscles. Upregulation of SLN at the transcriptional level was supported by low RCAN1 expression, which is a negative regulator of SLN. We treated C2C12 cells with dexamethasone to mimic muscle atrophy in vitro and showed a direct relationship between high SLN mRNA expression and low Ca2+ uptake by sarcoplasmic reticulum. Since SLN reportedly plays a role in nonshivering thermogenesis, we performed a cold tolerance test of the whole body. As a result, we found that mice with cast immobilization showed high cold tolerance, suggesting that cast immobilization promoted whole body thermogenesis. Although the activity level was decreased during cast immobilization without change in food intake, adipose tissue weights also decreased significantly after cast immobilization. Concomitantly, we conclude that cast immobilization of hindlimb increased thermogenesis in C57Bl/6J mice, probably via high expression of SLN.

Abstract 717: CGMP Expanded First Trimester Human Umbilical Cord Perivascular Cells (FTM HUCPVC) Induce Significant Angiogenesis, Myocardial Regeneration and Sustained Functional Recovery Exceeding Older MSC Sources in the Rat Myocardial Infarction (MI) Model

Introduction: FTM HUCPVC are a novel source of young, immunoprivileged mesenchymal stromal cells (MSC). FTM HUCPVC demonstrate significant homing, angiogenic and tissue remodelling capacities in vitro . We aimed to explore the therapeutic potential of cGMP compliant FTM HUCPVC for myocardial infarction (MI). Methods: FTM HUCPVC and term HUCPVC were expanded in cGMP compliant xeno-free (5% HPL) multilayer cultures (Corning) and Quantum Bioreactor (Terumo). One week post-MI Fox rnu rats (n=6) were intramyocardially injected with 3 million FTM HUCPVC, term HUCPVC, bone marrow MSC (BMSC) or basal media. Cardiac function (echocardiography, pressure/volume catheter and ECG telemetry), infarct scar (Trichrome), ECM remodelling activity (DQ collagenase substrate) vascular density (IB4) and contractile proteins (SarcA) were quantified 4 weeks and 6 months after cell implantation. Myocardial apoptosis (caspase-3) and macrophage infiltration (eNOS, CD68, CD80) were assessed at 5 days after cell treatment. Results: FTM HUCPVC significantly reduced apoptosis at 5 days and infarct size at 4 weeks (5.3±1.1, p<0.05) compared to other treatment groups. Histological analysis showed that the infarcted region of FTM HUCPVC treated hearts displayed significantly higher: M2 macrophage ratio, perivascular gelatinase activity, capillary density (543.4±118.6 vs. 276.5±42.69; p<0.05), abundance of contractile sarcomeric actinin (48.9±6.1 vs. 19.5±7.0; p<0.05), and Ca 2+ pump SERCA (69.9±5.9 vs. 49.8±3.7) over other experimental groups. FTM HUCPVCs induced significant and superior improvements in all measures of contractile function (ESV, EF, dPdt, tau) relative to cell-free media (p<0.001), BMSC (p<0.001) and term HUCPVC (p<0.001). Improvements in inotropy and lusitropy were maintained to 6 months (δ dPdt max 3.8±6.0; δ dPdt min 3.2±8.78) and resulted in near complete abolishment of premature ventricular contractions (<1/min). Conclusion: FTM HUCPVC treatment after MI induces superior structural and functional recovery compared to older MSC sources; characteristics that were maintained during xeno-free upscaling and for up to 6 months post injection. FTM HUCPVC are a promising option as a novel cell therapy for optimal cardiovascular regeneration.

Sarcolipin overexpression impairs myogenic differentiation in Duchenne muscular dystrophy.

Reduction in the expression of sarcolipin (SLN), an inhibitor of sarco(endo)plasmic reticulum (SR) Ca2+-ATPase (SERCA), ameliorates severe muscular dystrophy in mice. However, the mechanism by which SLN inhibition improves muscle structure remains unclear. Here, we describe the previously unknown function of SLN in muscle differentiation in Duchenne muscular dystrophy (DMD). Overexpression of SLN in C2C12 resulted in decreased SERCA pump activity, reduced SR Ca2+ load, and increased intracellular Ca2+ () concentration. In addition, SLN overexpression resulted in altered expression of myogenic markers and poor myogenic differentiation. In dystrophin-deficient dog myoblasts and myotubes, SLN expression was significantly high and associated with defective cycling. The dystrophic dog myotubes were less branched and associated with decreased autophagy and increased expression of mitochondrial fusion and fission proteins. Reduction in SLN expression restored these changes and enhanced dystrophic dog myoblast fusion during differentiation. In summary, our data suggest that SLN upregulation is an intrinsic secondary change in dystrophin-deficient myoblasts and could account for the mishandling, which subsequently contributes to poor myogenic differentiation. Accordingly, reducing SLN expression can improve the cycling and differentiation of dystrophic myoblasts. These findings provide cellular-level supports for targeting SLN expression as a therapeutic strategy for DMD.

A mathematical model for interdependent calcium and inositol 1,4,5-trisphosphate in cardiac myocyte

Calcium ($${\text {Ca}}^{2+}$$) signaling is the secondary signaling processes which have been one of the most vital intracellular signaling mechanisms. Over recent decades, this signaling process has been studied a lot in various cells to understand its mechanisms and also cure of various health hazards. In this paper, an attempt has been made to propose a model for coupled dynamics of $${\text {Ca}}^{2+}$$ and inositol 1,4,5-trisphosphate ($${\text {IP}}_3$$) in cardiac myocyte for a better understanding of the dependence of $${\text {Ca}}^{2+}$$ signaling on other chemical ions such as $${\text {IP}}_3$$ ions. The parameters such as influx, outflux, diffusion coefficient, SERCA pump, and Leak have been incorporated into the model and the finite differences scheme has been employed for the solution of the problem. The numerical results have been used to study the interdependence of $${\text {Ca}}^{2+}$$ and $${\text {IP}}_3$$ in the cardiac myocyte. It is observed that this interdependence is quite significantly affected by all these parameters except Leak. Also, the relationship between $${\text {Ca}}^{2+}$$ and $${\text {IP}}_3$$ dynamics is found to be non-linear. Such realistic models can be useful to generate the information of these dynamics in cardiac cells which can be useful for developing protocols for diagnosis and treatment of heart diseases like abnormal calcium signaling due to mutation of calsequestrin which results in sudden cardiac death and cardiomyopathy that affects the squeezing of the heart muscle.

341-LB: Chronic ER Calcium Depletion Resulting from Sarcoendoplasmic Reticulum Ca2+ Atpase 2b (SERCA2b) Deficiency Leads to Impaired Proinsulin Processing in the Pancreatic ß Cell

Reductions in pancreatic β cell endoplasmic reticulum (ER) Ca2+ levels are associated with the development of ER stress and impaired proinsulin processing in both type 1 and type 2 diabetes. The SERCA pump transports Ca2+ into the ER lumen in order to maintain a steep Ca2+ gradient between the ER and cytosol. SERCA2 activity and expression are reduced in diabetes, however, the relationship between SERCA2 and proinsulin processing remains incompletely understood. To this end, we developed mice with β cell specific deletion of SERCA2 (βS2KO mice) and S2KO INS1 cells. βS2KO mice exhibited glucose intolerance and reduced insulin secretion in response to glucose without evidence of impaired insulin sensitivity. Islets from βS2KO mice had significantly reduced ER Ca2+ levels and impaired glucose-induced Ca2+ oscillations, while serum proinsulin/insulin (PI/I) ratios and whole pancreas PI/I content were elevated. Immunoblot analysis of βS2KO islets and S2KO INS-1 cells revealed reduced total levels of the proinsulin processing enzymes, PC1/3, PC2 and CPE, and increased levels of proPC2. Restoration of SERCA2b via adenoviral transduction in S2KO INS1 cells was sufficient to restore PC1/3 and PC2 maturation and enzyme activity. Brefeldin A treatment in INS1 cells recapitulated the impairments in PC1/3 and PC2 maturation observed in S2KO INS1 cells, suggesting a disturbance in protein trafficking between the ER and Golgi. Trafficking assays were next performed using a vesicular stomatitis virus G (VSVG) protein construct and revealed a significantly slower rate of VSVG movement from the ER to the Golgi in S2KO INS1 cells. Taken together, these data indicate that loss of SERCA2 activity and impairments in ER Ca2+ lead to reduced proinsulin processing via altered protein trafficking through the β cell secretory pathway. Disclosure H. Iida: None. T. Kono: None. X. Tong: None. I. Lindberg: None. C. Evans-Molina: None.

The sarcoplasmic reticulum and SERCA: a nexus for muscular adaptive thermogenesis.

We are currently facing an "obesity epidemic" worldwide. Promoting inefficient metabolism in muscle represents a potential treatment for obesity and its complications. Sarco(endo)plasmic reticulum (SR) Ca2+-ATPase (SERCA) pumps in muscle are responsible for maintaining low cytosolic Ca2+ concentration through the ATP-dependent pumping of Ca2+ from the cytosol into the SR lumen. SERCA activity has the potential to be a critical regulator of body mass and adiposity given that it is estimated to contribute upwards of 20% of daily energy expenditure. More interestingly, this fraction can be modified physiologically in the face of stressors, such as ambient temperature and diet, through its physical interaction with several regulators known to inhibit Ca2+ uptake and muscle function. In this review, we discuss advances in our understanding of Ca2+-cycling thermogenesis within skeletal muscle, focusing on SERCA and its protein regulators, which were thought previously to only modulate muscular contractility. Novelty ATP consumption by SERCA pumps comprises a large proportion of resting energy expenditure in muscle and is dynamically regulated through interactions with small SERCA regulatory proteins. SERCA efficiency correlates significantly with resting metabolism, such that individuals with a higher resting metabolic rate have less energetically efficient SERCA Ca2+ pumping in muscle (i.e., lower coupling ratio). Futile Ca2+ cycling is a versatile heat generating mechanism utilized by both skeletal muscle and beige fat.

Impaired Activity of Ryanodine Receptors Contributes to Calcium Mishandling in Cardiomyocytes of Metabolic Syndrome Rats.

Metabolic syndrome (MetS) has become a global epidemic. MetS is a serious health problem because of its related cardiovascular complications, which include hypertension and delayed heart rate recovery after exercise. The molecular bases of cardiac dysfunction in MetS are still under scrutiny and may be related to anomalies in the activity and expression of key proteins involved in the cardiac excitation–contraction coupling (ECC). The cardiac Ca2+ channel/ryanodine receptor (RyR2) participates in releasing Ca2+ from internal stores and plays a key role in the modulation of ECC. We examined alterations in expression, phosphorylation status, Ca2+ sensitivity, and in situ function (by measuring Ca2+ sparks and Ca2+ transients) of RyR2; alterations in these characteristics could help to explain the Ca2+ handling disturbances in MetS cardiomyocytes. MetS was induced in rats by adding commercially refined sugar (30% sucrose) to their drinking water for 24 weeks. Cardiomyocytes of MetS rats displayed decreased Ca2+ transient amplitude and cell contractility at all stimulation frequencies. Quiescent MetS cardiomyocytes showed a decrease in Ca2+ spark frequency, amplitude, and spark-mediated Ca2+ leak. The [3H]-ryanodine binding data showed that functionally active RyRs are significantly diminished in MetS heart microsomes; and exhibited rapid Ca2+-induced inactivation. The phosphorylation of corresponding Ser2814 (a preferential target for CaMKII) of the hRyR2 was significantly diminished. RyR2 protein expression and Ser2808 phosphorylation level were both unchanged. Further, we demonstrated that cardiomyocyte Ca2+ mishandling was associated with reduced SERCA pump activity due to decreased Thr17-PLN phosphorylation, suggesting a downregulation of CaMKII in MetS hearts, though the SR Ca2+ load remained unchanged. The reduction in the phosphorylation level of RyR2 at Ser2814 decreases RyR2 availability for activation during ECC. In conclusion, the impaired in situ activity of RyR2 may also account for the poor overall cardiac outcome reported in MetS patients; hence, the SERCA pump and RyR2 are both attractive potential targets for future therapies.

Iron-deficiency anemia reduces cardiac contraction by downregulating RyR2 channels and suppressing SERCA pump activity.

Iron deficiency is present in ~50% of heart failure (HF) patients. Large multicenter trials have shown that treatment of iron deficiency with i.v. iron benefits HF patients, but the underlying mechanisms are not known. To investigate the actions of iron deficiency on the heart, mice were fed an iron-depleted diet, and some received i.v. ferric carboxymaltose (FCM), an iron supplementation used clinically. Iron-deficient animals became anemic and had reduced ventricular ejection fraction measured by magnetic resonance imaging. Ca2+ signaling, a pathway linked to the contractile deficit in failing hearts, was also significantly affected. Ventricular myocytes isolated from iron-deficient animals produced smaller Ca2+ transients from an elevated diastolic baseline but had unchanged sarcoplasmic reticulum (SR) Ca2+ load, trigger L-type Ca2+ current, or cytoplasmic Ca2+ buffering. Reduced fractional release from the SR was due to downregulated RyR2 channels, detected at protein and message levels. The constancy of diastolic SR Ca2+ load is explained by reduced RyR2 permeability in combination with right-shifted SERCA activity due to dephosphorylation of its regulator phospholamban. Supplementing iron levels with FCM restored normal Ca2+ signaling and ejection fraction. Thus, 2 Ca2+-handling proteins previously implicated in HF become functionally impaired in iron-deficiency anemia, but their activity is rescued by i.v. iron supplementation.

Substrate Selectivity of a P4‐ATPase Flippase

Phospholipids, the primary structural components of cell membranes, are distributed unequally across the membrane. The sphingolipids, including sphingomyelin (SM) and glycosphingolipids, are enriched on the exofacial surface of the plasma membrane or the luminal surfaces of internal organelles, while the cytofacial leaflet is enriched in the aminophospholipids, phophatidylserine (PS) and phosphatidylethanolamine (PE). Contributing to maintenance of this asymmetry is a combination of slow passive transbilayer phospholipid movement, trapping by cytofacial proteins and the activity of a number of energy‐dependent transporters. The substrate specificity and directionality of these pumps defines the transbilayer asymmetry of phospholipids. Two transporter superfamilies, ABC transporters and P‐type ATPases, account for these activities and move lipids in either the exofacial (floppase) or cytofacial (flippase) directions, respectively. In previous studies we have characterized the biochemical requirements and substrate specificity for a PS specific flippase activity in human red blood cells. Transport is ATP‐dependent and sensitive to vanadate and sulfhydryl‐modification. This flippase is selective for the sn‐1,2‐isomer of PS. Transport is diminished by modification of the PS headgroup amine (with the exception of mono‐methylation), carboxyl and phosphate groups, while a variety of fatty acyl groups and the D‐serine analog are accommodated. Red blood cells express two members of the P4‐type ATPase subfamily, ATP8A1 and ATP11C, that are likely responsible for this activity. Here, we report on the heterologous expression, purification, and functional assaying of hexahistidine‐tagged constructs of Atp8a1 with and without its cognate accessory subunit, Cdc50a. Purified Atp8a1 is vanadate‐sensitive and dependent upon Cdc50a co‐expression and PS for robust ATPase activity, although lipid specificity is not dependent on the presence of Cdc50a. In addition to reconstituting the flippase activity of Atp8a1 and ATP11c in vitro, our efforts are focused on rational mutagenesis of Atp8a1 to identify the lipid transport path and the determinants of substrate specificity, directed by homology modeling and the lipid‐binding characteristics of the closest members of the P‐type superfamily. X‐ray crystal structures of the SERCA pump in conformation E2 (PDB 2AGV) display a PE glycerol backbone and head group tightly pinched by helices M2 and M4 with PE's acyl chains extending between helices M2 and M6. This PE‐binding site implies a mechanism for how the P4lipid‐binding site and transport pathway evolved from ancestor P‐types designed to move simple cations. With these clues, we aim to identify the PS binding site and deduce the origin of the sn‐1,2‐stereospecificity of Atp8a1.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Probing the effects of nonannular lipid binding on the stability of the calcium pump SERCA

The calcium pump SERCA is a transmembrane protein that is critical for calcium transport in cells. SERCA resides in an environment made up largely by the lipid bilayer, so lipids play a central role on its stability and function. Studies have provided insights into the effects of annular and bulk lipids on SERCA activation, but the role of a nonannular lipid site in the E2 intermediate state remains elusive. Here, we have performed microsecond molecular dynamics simulations to probe the effects of nonannular lipid binding on the stability and structural dynamics of the E2 state of SERCA. We found that the structural integrity and stability of the E2 state is independent of nonannular lipid binding, and that occupancy of a lipid molecule at this site does not modulate destabilization of the E2 state, a step required to initiate the transition toward the competent E1 state. We also found that binding of the nonannular lipid does not induce direct allosteric control of the intrinsic functional dynamics the E2 state. We conclude that nonannular lipid binding is not necessary for the stability of the E2 state, but we speculate that it becomes functionally significant during the E2-to-E1 transition of the pump.

Epigenetic regulation of the human ATP2A3 gene promoter in gastric and colon cancer cell lines.

The knowledge about the role of calcium-regulated pathways in cancer cell growth and differentiation could be useful for the development of new therapeutic approaches to diminish its mortality. The ATP2A genes encode for SERCA pumps, which modulate cytosolic Ca2+ concentration, regulating various cellular processes including cell growth. ATP2A3 gene transcriptional down-regulation has been reported in gastric and colon cancer, but there is still a lack of understanding about the epigenetic processes regulating its transcription. In this work, we report that butyrate, trichostatin A, and 5-azacytidine treatments increase SERCA3 expression, increased apoptosis, and decreased cell viability of the KATO-III gastric carcinoma cell line. We analyzed the methylation profile of the ATP2A3 gene promoter CpG island, finding clones with methylated status through -280 to -135 promoter region, harboring Sp1 and AP-2 binding sites, which could have a role in transcriptional repression. Post-translational modifications of histones show a major role in the ATP2A3 transcriptional regulation, and our results show histones marks linked to transcriptional repression associated with the -262 to -135 region, this repressive context changed to transcriptional permissive through SERCA3 re-expressing conditions. These results suggest that the nucleotide sequence from -280 to -135 position is an ATP2A3 epigenetic regulatory CpG region in KATO-III cells. Analyses of online-databases show a decreased SERCA3 expression in gastric and colon tumors, as well as overall survival results, showed that high SERCA3 expression could serve as a favorable prognostic marker for colon and gastric cancer patients.

Reduced SERCA Function Preferentially Affects Wnt Signaling by Retaining E-Cadherin in the Endoplasmic Reticulum

SUMMARYCalcium homeostasis in the lumen of the endoplasmic reticulum is required for correct processing and trafficking of transmembrane proteins, and defects in protein trafficking can impinge on cell signaling pathways. We show here that mutations in the endoplasmic reticulum calcium pump SERCA disrupt Wingless signaling by sequestering Armadillo/β-catenin away from the signaling pool. Armadillo remains bound to E-cadherin, which is retained in the endoplasmic reticulum when calcium levels there are reduced. Using hypomorphic and null SERCA alleles in combination with the loss of the plasma membrane calcium channel Orai allowed us to define three distinct thresholds of endoplasmic reticulum calcium. Wingless signaling is sensitive to even a small reduction, while Notch and Hippo signaling are disrupted at intermediate levels, and elimination of SERCA function results in apoptosis. These differential and opposing effects on three oncogenic signaling pathways may complicate the use of SERCA inhibitors as cancer therapeutics.

Mechanisms of Supralinear Calcium Integration in Dendrites of Hippocampal CA1 Fast-Spiking Cells.

In fast-spiking (FS), parvalbumin-expressing interneurons of the CA1 hippocampus, activation of the GluA2-lacking Ca2+-permeable AMPA receptors (CP-AMPARs) in basal dendrites is coupled to Ca2+-induced Ca2+-release (CICR), and can result in a supralinear summation of postsynaptic Ca2+-transients (post-CaTs). While this mechanism is important in controlling the direction of long-term plasticity, it is still unknown whether it can operate at all excitatory synapses converging onto FS cells or at a set of synapses receiving a particular input. Using a combination of patch-clamp recordings and two-photon Ca2+ imaging in acute mouse hippocampal slices with computational simulations, here we compared the generation of supralinear post-CaTs between apical and basal dendrites of FS cells. We found that, similar to basal dendrites, apical post-CaTs summated supralinearly and relied mainly on the activation of the CP-AMPARs, with a variable contribution of other Ca2+ sources, such as NMDA receptors, L-type voltage-gated Ca2+-channels and Ca2+ release. In addition, supralinear post-CaTs generated in apical dendrites had a slower decay time and a larger cumulative charge than those in basal, and were associated with a stronger level of somatic depolarization. The model predicted that modulation of ryanodine receptors and of the Ca2+ extrusion mechanisms, such as the Na+/Ca2+-exchanger and SERCA pump, had a major impact on the magnitude of supralinear post-CaTs. These data reveal that supralinear Ca2+ summation is a common mechanism of Ca2+ signaling at CP-AMPAR-containing synapses. Shaped in a location-specific manner through modulation of ryanodine receptors and Ca2+ extrusion mechanisms, CP-AMPAR/CICR signaling is suitable for synapse-specific bidirectional modification of incoming inputs in the absence of active dendritic conductances.

Three-Dimensional Finite Element Model to Study Effect of RyR Calcium Channel, ER Leak and SERCA Pump on Calcium Distribution in Oocyte Cell

Calcium ions control many cellular processes by relaying signals in the form of their spatio-temporal distribution. Dynamics and patterns of calcium concentration such as repetitive waves, coherent oscillations or spatially localized elevations activate diverse physiological functions. Calcium is the most universal second messenger in cells and plays an important role in initiation, sustenance and termination of various activities in cells required for maintaining the structure and function of the cell. Calcium signal at fertilization is necessary for egg activation and exhibits specialized spatial and temporal dynamics. The specific calcium concentration distribution patterns in oocytes required for various activities like egg fertilization, maturation, etc. are not well understood. In this paper, a three-dimensional finite element model is proposed to study the spatio-temporal calcium distribution in oocytes. The parameters such as buffers, SERCA pump, ER Leak, ryanodine receptor (RyR) calcium channel, point source and line source of calcium are incorporated in the model. The appropriate initial and boundary conditions have been framed on the basis of the physical condition of the problem. A program is developed in MATLAB for simulation. The results have been used to study the effect of source geometry, RyR calcium channel, ER Leak, SERCA pump and buffers on cytosolic calcium concentration distribution in oocytes.

The heart of an acrobatic bird

The courtship behavior of some species of birds can be energetically demanding, but it is unknown if cardiovascular specializations enable such behaviors. While performing a highly acrobatic courtship dance, heart rate in male golden-collared manakins increases briefly to 1300 beats per minute, among the highest heart rates recorded in any bird or mammal. We hypothesize that male manakins have enhanced cardiovascular capabilities to meet these demands on the heart. Using histological and molecular techniques, we examined manakin heart structure as well as expression of genes involved in Ca2+ handling, action potential duration, steroidal signaling and cardiac growth. These measures were also made on the hearts of zebra finches, a similar-sized bird with limited cardiovascular demands. Compared to the zebra finch, the manakin had a significantly thicker left ventricular (LV) muscle (cross-sectional thickness of the free LV wall and septum) with a smaller LV chamber. In addition, compared to zebra finches, manakin hearts had significantly greater gene expression of ryanodine receptors as well as androgen receptors. Testosterone (T) treatment of non-breeding manakins (with low T) increased gene expression of the Ca2+ pump SERCA. These observations suggest that hearts of breeding male manakins require specialized Ca2+ handling and androgens may facilitate manakin cardiovascular function.

A Stem Cell-Based Screening Platform Identifies Compounds that Desensitize Motor Neurons to Endoplasmic Reticulum Stress.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease selectively targeting motor neurons in the brain and spinal cord. The reasons for differential motor neuron susceptibility remain elusive. We developed a stem cell-based motor neuron assay to study cell-autonomous mechanisms causing motor neuron degeneration, with implications for ALS. A small-molecule screen identified cyclopiazonic acid (CPA) as a stressor to which stem cell-derived motor neurons were more sensitive than interneurons. CPA induced endoplasmic reticulum stress and the unfolded protein response. Furthermore, CPA resulted in an accelerated degeneration of motor neurons expressing human superoxide dismutase 1 (hSOD1) carrying the ALS-causing G93A mutation, compared to motor neurons expressing wild-type hSOD1. A secondary screen identified compounds that alleviated CPA-mediated motor neuron degeneration: three kinase inhibitors and tauroursodeoxycholic acid (TUDCA), a bile acid derivative. The neuroprotective effects of these compounds were validated in human stem cell-derived motor neurons carrying a mutated SOD1 allele (hSOD1A4V). Moreover, we found that the administration of TUDCA in an hSOD1G93A mouse model of ALS reduced muscle denervation. Jointly, these results provide insights into the mechanisms contributing to the preferential susceptibility of ALS motor neurons, and they demonstrate the utility of stem cell-derived motor neurons for the discovery of new neuroprotective compounds.

Abstract P204: Induction of Endoplasmic Reticulum Stress Upregulates Renin Expression in Immortalized Mouse Hypothalamic N43/5 Cells

Dysregulation of the brain renin-angiotensin system (RAS) has been implicated in many forms of hypertension, including resistant hypertension. However, the mechanism by which the brain RAS is activated is unknown. We have recently published on the activation of brain RAS following disinhibition of renin-a expression after deletion of the brain-specific renin-b isoform. Endoplasmic reticulum (ER) stress, which is an accumulation of unfolded proteins in the ER, has been linked to hypertension as central angiotensin II administration induced ER stress markers. Here, we hypothesize that ER stress induces brain RAS activation by upregulating renin expression in the brain. We first tested if ER stress could induce renin expression in vitro in a mouse neuroblastoma hypothalamic cell line, N43/5 cells. After treating cells with ER stressor thapsigargin, a SERCA pump inhibitor, (100nM) for 8 hours we observed transcriptional upregulation of ER stress marker Bip (28.9±5.0 fold increase vs vehicle, p<0.05, n=6). This same treatment also induced an increase in renin expression (13.8±5.8 fold increase vs vehicle, p<0.05, n=6). We have previously shown that deletion of renin-b in the brain causes an increase in renin-a expression specifically in the rostral ventrolateral medulla (RVLM). This data suggests that ER stress may induce RAS activation through a similar renin-a disinhibitory mechanism. Under basal conditions, the RVLM of renin-b knockout mice showed no difference in gene expression of ER stress marker Bip (0.7±0.5 fold increase vs control, p=0.67, n=3-4) or Chop (0.7±0.35 fold increase vs control, p=0.64, n=3-4). This suggests that ER stress may be upstream of renin induction. To test this, we treated C57BL/6 mice with tunicamycin (40mg/mL, 2uL) via intracerebellar ventricle cannula. We detected a positive endogenous renin transcript signal via in situ hybridization bilaterally in the medulla in the vicinity of the RVLM (possibly the nucleus ambiguous) from vehicle and tunicamycin treated mice. We are currently testing if ER stress quantitatively increases renin expression using qPCR. These data extend previous studies showing that RAS activation in the brain causes ER stress by suggesting that ER stress may also induce the brain RAS.