Nuclear Calcium Transients Research Articles

Decoding Arc transcription: a live-cell study of stimulation patterns and transcriptional output.

Activity-regulated cytoskeleton-associated protein (Arc) plays a crucial role in synaptic plasticity, a process integral to learning and memory. Arc transcription is induced within a few minutes of stimulation, making it a useful marker for neuronal activity. However, the specific neuronal activity patterns that initiate Arc transcription have remained elusive due to the inability to observe mRNA transcription in live cells in real time. Using a genetically encoded RNA indicator (GERI) mouse model that expresses endogenous Arc mRNA tagged with multiple GFPs, we investigated Arc transcriptional activity in response to various electrical field stimulation patterns. The GERI mouse model was generated by crossing the Arc-PBS knock-in mouse, engineered with binding sites in the 3' untranslated region (UTR) of Arc mRNA, and the transgenic mouse expressing the cognate binding protein fused to GFP. In dissociated hippocampal neurons, we found that the pattern of stimulation significantly affects Arc transcription. Specifically, theta-burst stimulation consisting of high-frequency (100 Hz) bursts delivered at 10 Hz frequency induced the highest rate of Arc transcription. Concurrently, the amplitudes of nuclear calcium transients also reached their peak with 10 Hz burst stimulation, indicating a correlation between calcium concentration and transcription. However, our dual-color single-cell imaging revealed that there were no significant differences in calcium amplitudes between Arc-positive and Arc-negative neurons upon 10 Hz burst stimulation, suggesting the involvement of other factors in the induction of Arc transcription. Our live-cell RNA imaging provides a deeper insight into the complex regulation of transcription by activity patterns and calcium signaling pathways.

Autophagy-related gene 5 (Atg5) is required for maintaining subcellular calcium homeostasis during acute cardiac stress

Abstract Background Autophagy exerts protective effects during cardiac stress inflicted by β-adrenergic stimulation and pressure overload. Contrarily, deletion of the cardiac-specific Atg5 gene contributes to left ventricular hypertrophy and contractile dysfunction, resulting in dilated cardiomyopathy. Heart failure is characterized by perturbations of excitation-contraction coupling (ECC) underlying alterations in intracellular calcium signaling. Purpose In this study, we aimed to understand whether loss of basal autophagy due to Atg5 deletion specifically in cardiomyocytes contributes directly to disturbances in subcellular calcium cycling. Methods Cardiac-specific Atg5−/− and Atg5+/+ mice were used to isolate ventricular cardiomyocytes, which were loaded with 8 μM Ca2+ indicator Fluo4/AM. Isolated cells were electrically stimulated and subjected to elevated workload by increasing pacing frequencies from 1 to 4 Hz. Cytosolic and nuclear calcium transients (CaTs) were recorded in line-scan mode using a laser scanning confocal microscope. Pharmacological inhibition of autophagic flux was performed by an intraperitoneal injection of 40 mg/kg leupeptin to Atg5+/+ mice. Results At baseline (1 Hz) stimulation the amplitude and kinetics of calcium transients were comparable between Atg5−/− and Atg5+/+ cells. However, after progressive increase of the pacing rate from 1 Hz to 4 Hz, Atg5−/− cardiomyocytes displayed reduced cytoplasmic and nuclear CaT amplitude, but increased nuclear time-averaged CaTs compared to controls. Such increase in nuclear calcium load stimulates Ca2+-dependent transcriptional activity, and likely involves CaMKII-mediated pro-hypertrophic gene program thus contributing to cardiac remodelling. In addition, we observed increased occurrence of arrhythmic events in Atg5−/− hearts at high pacing frequency, indicative of intracellular calcium overload and disturbed ECC. Acute pharmacological autophagy inhibition failed to elicit direct changes in time-averaged CaTs in the cytoplasm and nucleoplasm, suggesting that loss of Atg5-dependent autophagy seems to induce adverse cardiac remodelling that involves increased CaMKII activity due to the imbalances in nuclear Ca2+ levels. Conclusion Specific loss of ATG5 protein impairs calcium cycling during acute stress exposure by reducing CaT amplitudes, promoting arrhythmia, and increasing nuclear calcium load, which may induce hypertrophic gene expression. Further work is needed to identify key components mediating autophagy-related cardioprotection to develop strategies against cardiac remodeling and its progression to heart failure. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Austrian Science Fund (FWF)

Measuring nuclear calcium and actin assembly in living cells.

Nuclear calcium signalling has emerged as a critical mechanism regulating processes like chromatin organization and gene expression. Recently, we have shown that nuclear calcium elevation triggers rapid and transient actin filament assembly inside the nucleus. Here, we constructed and employed a nuclear-specific calcium sensor based upon the new generation of genetically encoded probes jGCaMP7f. By fusing a nuclear localization signal to jGCaMP7f, we achieved highly efficient nuclear-specific targeting. Comparing the jGCaMP7f-NLS probe with the previous GCaMP6f-NLS calcium sensor showed clearly that jGCaMP7f-NLS is more sensitive and reverses significantly quicker thereby reflecting rapid nuclear calcium transients in a closely physiological manner. We further confirm that nuclear calcium transients precede nuclear actin polymerization by several seconds. Our data show that calcium-triggered nuclear actin assembly in fibroblasts is independent of the actin nucleating Arp2/3 complex. Together, jGCaMP7f-NLS represents an easy to use, reliable and highly sensitive nuclear calcium sensor that allows to tightly interrogate real-time, spatiotemporal calcium signalling and calcium-elicited effects in the nucleus of living cells.

Reciprocal Interaction of Dendrite Geometry and Nuclear Calcium-VEGFD Signaling Gates Memory Consolidation and Extinction.

Nuclear calcium is an important signaling end point in synaptic excitation-transcription coupling that is critical for long-term neuroadaptations. Here, we show that nuclear calcium acting via a target gene, VEGFD, is required for hippocampus-dependent fear memory consolidation and extinction in mice. Nuclear calcium-VEGFD signaling upholds the structural integrity and complexity of the dendritic arbor of CA1 neurons that renders those cells permissive for the efficient generation of synaptic input-evoked nuclear calcium transients driving the expression of plasticity-related genes. Therefore, the gating of memory functions rests on the reciprocally reinforcing maintenance of an intact dendrite geometry and a functional synapse-to-nucleus communication axis. In psychiatric and neurodegenerative disorders, therapeutic application of VEGFD may help to stabilize dendritic structures and network connectivity, which may prevent cognitive decline and could boost the efficacy of extinction-based exposure therapies.SIGNIFICANCE STATEMENT This study uncovers a reciprocal relationship between dendrite geometry, the ability to generate nuclear calcium transients in response to synaptic inputs, and the subsequent induction of expression of plasticity-related and dendritic structure-preserving genes. Insufficient nuclear calcium signaling in CA1 hippocampal neurons and, consequently, reduced expression of the nuclear calcium target gene VEGFD, a dendrite maintenance factor, leads to reduced-complexity basal dendrites of CA1 neurons, which severely compromises the animals' consolidation of both memory and extinction memory. The structure-protective function of VEGFD may prove beneficial in psychiatric disorders as well as neurodegenerative and aging-related conditions that are associated with loss of neuronal structures, dysfunctional excitation-transcription coupling, and cognitive decline.

One nuclear calcium transient induced by a single burst of action potentials represents the minimum signal strength in activity-dependent transcription in hippocampal neurons

Neurons undergo dramatic changes in their gene expression profiles in response to synaptic stimulation. The coupling of neuronal excitation to gene transcription is well studied and is mediated by signaling pathways activated by cytoplasmic and nuclear calcium transients. Despite this, the minimum synaptic activity required to induce gene expression remains unknown. To address this, we used cultured hippocampal neurons and cellular compartment analysis of temporal activity by fluorescence in situ hybridization (catFISH) that allows detection of nascent transcripts in the cell nucleus. We found that a single burst of action potentials, consisting of 24.4±5.1 action potentials during a 6.7±1.9s depolarization of 19.5±2.0mV causing a 9.3±0.9s somatic calcium transient, is sufficient to activate transcription of the immediate early gene arc (also known as Arg3.1). The total arc mRNA yield produced after a single burst-induced nuclear calcium transient was very small and, compared to unstimulated control neurons, did not lead to a significant increase in arc mRNA levels measured using quantitative reverse transcriptase PCR (qRT-PCR) of cell lysates. Significantly increased arc mRNA levels became detectable in hippocampal neurons that had undergone 5–8 consecutive burst-induced nuclear calcium transients at 0.05–0.15Hz. These results indicate that a single burst-induced nuclear calcium transient can activate gene expression and that transcription is rapidly shut off after synaptic stimulation has ceased.

Abstract 388: Functional Role of Inositol 1,4,5-Trisphosphate Receptors in Cardiac Hypertrophy

Calcium is a second messenger that is essential for the control of a variety of cellular functions. In the heart, calcium plays an integral role in many cellular processes including energy metabolism, contraction, gene expression, and cell death. The inositol 1,4,5-trisphosphate receptor (IP 3 R) is a calcium channel that is ubiquitously expressed throughout the body. There are three IP 3 R isoforms encoded by separate genes. In the heart, the IP 3 R-2 isoform is reported to being most predominant with regards to both expression levels and functional significance. Several groups have shown that perinuclear IP 3 R-2 may play a key role in the progression of hypertrophy by mediating nuclear calcium release in response to endothelin-1 (ET-1) and angiotensin II. However, the functional roles of IP 3 R-1 and IP 3 R-3 in the heart are essentially unexplored despite measureable expression levels. We hypothesize that all three IP 3 R isoforms can contribute to cytosolic calcium transients and cardiac stress induced by ET-1. As our model we used dissociated neonatal and adult rat ventricular cardiomyocytes. We found readily detectable levels of expression of all three IP 3 Rs in both neonatal and adult rat ventricular cardiomyocytes. We found that in addition to the perinuclear area, all three IP 3 R isoforms were expressed throughout the cardiomyocyte including the sarcoplasmic reticulum. Using isoform specific siRNA, we found that knocking down individual IP 3 Rs did not alter ET-1 induced calcium responses in neonatal cardiomyocytes. However, knocking down all three isoforms abolished the effects of ET-1 on calcium homeostasis. We next tested whether IP 3 Rs mediate nuclear-specific calcium elevations. Using a genetically encoded calcium indicator targeted to the nucleus we were able to specifically discriminate nuclear versus cytosolic calcium transients after ET-1 treatment. Contrary to previous reports, we found no evidence that IP 3 R-mediated calcium release is confined to the nucleus. Our findings that IP 3 R isoforms are functionally redundant in the heart have significant implications for animal models of hypertrophy and human disease.

Computational Analysis of Heart-Failure-Related Remodeling of Cytosolic and Nuclear Calcium Handling in the Canine Atrial Cardiomyocyte

Background: Heart failure (HF) results in extensive remodeling of ventricular cytosolic and nucleoplasmic calcium handling (CaCyt / CaNucl) and produces atrial calcium-handling dysregulation, promoting atrial fibrillation. The molecular mechanisms underlying arrhythmogenic HF-related atrial calcium-handling abnormalities, notably for atrial CaNucl, are largely unknown. Computational modeling has emerged as a tool to study arrhythmogenic mechanisms, but no currently available atrial model can simulate CaNucl, which was addressed here.Methods: CaCyt and CaNucl were measured using confocal microscopy in atrial cardiomyocytes from control or ventricular-tachypacing-induced HF dogs loaded with Fluo4 (1 Hz stimulation). We extended the Ramirez et al. 2000 canine atrial cardiomyocyte model with a nuclear compartment with nuclear-pore channel-mediated calcium diffusion from the cytosol, perinuclear calcium-store (periCs) linked to the sarcoplasmic reticulum (SR), and IP3-receptor-mediated calcium release from periCs to nucleus. IP3-receptor expression in control and HF dogs was evaluated using Western blot.Results: Nucleoplasmic calcium fluorescence ratios exceeded cytosolic levels in experiments (+28% diastolic, +9% systolic) and model (+32% diastolic, +9% systolic). HF significantly increased nuclear and cytosolic fluorescence, and IP3-receptor expression (+20%; P<0.05). We simulated the relative contribution of increased IP3-receptor expression and HF-related atrial CaCyt remodeling (−60% ryanodine-receptor expression, increased SR calcium-uptake; Yeh et al. 2008) to CaNucl remodeling. Increased IP3-receptor expression slightly augmented CaNucl (+5% diastolic, +2% systolic), without affecting CaCyt, whereas CaCyt remodeling strongly increased CaNucl (+28% diastolic, +41% systolic) in the model. The combined effect (IP3-receptor+Cacyt remodeling) was overadditive (+38% diastolic, +46% systolic).Conclusion: We developed the first computational model to simulate atrial CaNucl. HF-related CaCyt and CaNucl remodeling synergistically modulate nuclear calcium transients. The model provides new insights into atrial CaNucl determinants and their regulation in HF, and may help uncover the mechanisms of proarrhythmic atrial gene-expression changes.

Regional Variation of Calcium Transients within Neonatal Cardiomyocyte

Cardiomyocyte contraction is preceded by a calcium transient, an influx of calcium into the cytosol causing a contraction to occur followed by relaxation which occurs as the intracellular calcium returns to resting level.We are investigating how calcium transient signals from the nuclear, perinuclear and more distant cytosolic regions of neonatal cardiomyocytes differ. Calcium transients exhibit significant differences in the rise and decay time courses between regions close to and relatively distant from the nucleus. Nuclear calcium transients are slower to occur and have broader peaks than cytosolic transients more distant from the nucleus. The distribution of SR around nuclear and perinuclear regions influences the behavior of calcium transient signals. We targeted four regions of interest (ROI) in the cell: (1) ROI 1 is the region with the least amount of nuclear calcium contribution; (2) ROI 2 refers to regions adjacent the nucleus where there is significant nuclear calcium contribution; (3) ROI 3 is within the nucleus; (4) ROI 4 refers to the signal averaged over the entire cell. In order to accurately define these four regions of interest the cells were stained with ER‐TrackerTM, a dye that binds to the SR, and with Fluo‐3. Calcium transients were recorded using Leica SP2 Fluorescent Laser Scanning Confocal Microscope. XT Scans were used to record all three regions of interest simultaneously for 30 sec and to compare these transients from an averaged signal recorded from the entire cell when cells were paced at 0.3 Hz. Neonatal ventricular myocyte measurements were obtained in an in‐line scan mode at 200 lines per second. Data files were exported in text format and graphed as bar charts and scatter plots. An averaged time to peak and averaged time to 50 percent signal decay were used to compare signal behavior between regions.

Nuclear Calcium Signaling Regulates Nuclear Export of a Subset of Class IIa Histone Deacetylases following Synaptic Activity

In neurons, dynamic changes in the subcellular localization of histone deacetylases (HDACs) are thought to contribute to signal-regulated gene expression. Here we show that in mouse hippocampal neurons, synaptic activity-dependent nucleo-cytoplasmic shuttling is a common feature of all members of class IIa HDACs, which distinguishes them from other classes of HDACs. Nuclear calcium, a key regulator in neuronal gene expression, is required for the nuclear export of a subset of class IIa HDACs. We found that inhibition of nuclear calcium signaling using CaMBP4 or increasing the nuclear calcium buffering capacity by means of expression of a nuclear targeted version of parvalbumin (PV.NLS-mC) led to a build-up of HDAC4 and HDAC5 in the cell nucleus, which in the case of PV.NLS-mC can be reversed by nuclear calcium transients triggered by bursts of action potential firing. A similar nuclear accumulation of HDAC4 and HDAC5 was observed in vivo in the mouse hippocampus following stereotaxic delivery of recombinant adeno-associated viruses expressing either CaMBP4 or PV.NLS-mC. The modulation of HDAC4 activity either by RNA interference-mediated reduction of HDAC4 protein levels or by expression of a constitutively nuclear localized mutant of HDAC4 leads to changes in the mRNA levels of several nuclear calcium-regulated genes with known functions in acquired neuroprotection (atf3, serpinb2), memory consolidation (homer1, arc), and the development of chronic pain (ptgs2, c1qc). These results identify nuclear calcium as a regulator of nuclear export of HDAC4 and HDAC5. The reduction of nuclear localized HDACs represents a novel transcription-promoting pathway stimulated by nuclear calcium.

Nuclear Calcium Signaling in Spinal Neurons Drives a Genomic Program Required for Persistent Inflammatory Pain

Persistent pain induced by noxious stimuli is characterized by the transition from normosensitivity to hypersensitivity. Underlying mechanisms are not well understood, although gene expression is considered important. Here, we show that persistent nociceptive-like activity triggers calcium transients in neuronal nuclei within the superficial spinal dorsal horn, and that nuclear calcium is necessary for the development of long-term inflammatory hypersensitivity. Using a nucleus-specific calcium signal perturbation strategy in vivo complemented by gene profiling, bioinformatics, and functional analyses, we discovered a pain-associated, nuclear calcium-regulated gene program in spinal excitatory neurons. This includes C1q, a modulator of synaptic spine morphogenesis, which we found to contribute to activity-dependent spine remodelling on spinal neurons in a manner functionally associated with inflammatory hypersensitivity. Thus, nuclear calcium integrates synapse-to-nucleus communication following noxious stimulation and controls a spinal genomic response that mediates the transition between acute and long-term nociceptive sensitization by modulating functional and structural plasticity.

A Signaling Cascade of Nuclear Calcium-CREB-ATF3 Activated by Synaptic NMDA Receptors Defines a Gene Repression Module That Protects against Extrasynaptic NMDA Receptor-Induced Neuronal Cell Death and Ischemic Brain Damage

Synapse-to-nucleus signaling triggered by synaptic NMDA receptors can lead to the buildup of a neuroprotective shield. Nuclear calcium activating the cAMP response element binding protein (CREB) plays a key role in neuroprotection acquired by synaptic activity. Here we show that in mouse hippocampal neurons, the transcription factor Atf3 (activating transcription factor 3) is a direct target of CREB. Induction of ATF3 expression by CREB in hippocampal neurons was initiated by calcium entry through synaptic NMDA receptors and required nuclear calcium transients and calcium/calmodulin-dependent protein kinase IV activity. Acting as a transcriptional repressor, ATF3 protects cultured hippocampal neurons from apoptosis and extrasynaptic NMDA receptor-induced cell death triggered by bath application of NMDA or oxygen-glucose deprivation. Expression of ATF3 in vivo using stereotaxic delivery of recombinant adeno-associated virus reduces brain damage following a cerebral ischemic insult in mice. Conversion of ATF3 to a transcriptional activator transforms ATF3 into a potent prodeath protein that kills neurons in cell culture and, when expressed in vivo in the hippocampus, ablates the neuronal cell layer. These results link nuclear calcium-CREB signaling to an ATF3-mediated neuroprotective gene repression program, indicating that activity-dependent shutoff of genes is an important process for survival. ATF3 supplementation may counteract age- and disease-related neuronal cell loss caused by a reduction in synaptic activity, malfunctioning of calcium signaling toward and within the nucleus ("nuclear calciopathy"), or increases in death signaling by extrasynaptic NMDA receptors.

Nuclear Calcium Sensors Reveal that Repetition of Trains of Synaptic Stimuli Boosts Nuclear Calcium Signaling in CA1 Pyramidal Neurons

Nuclear calcium is a key signal in the dialogue between synapse and nucleus that controls the genomic responses required for persistent adaptations, including memory and acquired neuroprotection. The amplitude and duration of nuclear calcium transients specify activity-induced transcriptional changes. However, the precise relationship between synaptic input and nuclear calcium output is unknown. Here, we used stereotaxic delivery to the rat brain of recombinant adeno-associated viruses encoding nuclear-targeted calcium sensors to assess nuclear calcium transients in CA1 pyramidal neurons after stimulation of the Schaffer collaterals. We show that in acute hippocampal slices, a burst of synaptic activity elicits a nuclear calcium signal with a regenerative component at above-threshold stimulation intensities. Using classical stimulation paradigms (i.e., high-frequency stimulation (HFS) and θ burst stimulation (TBS)) to induce early LTP (E-LTP) and transcription-dependent late LTP (L-LTP), we found that the magnitude of nuclear calcium signals and the number of action potentials activated by synaptic stimulation trains are greatly amplified by their repetition. Nuclear calcium signals and action potential generation were reduced by blockade of either NMDA receptors or L-type voltage-gated calcium channels, but not by procedures that lead to internal calcium store depletion or by blockade of metabotropic glutamate receptors. These findings identify a repetition-induced switch in nuclear calcium signaling that correlates with the transition from E-LTP to L-LTP, and may explain why the transcription-dependent phase of L-LTP is not induced by a single HFS or TBS but requires repeated trains of activity. Recombinant, nuclear-targeted indicators may prove useful for further analysis of nuclear calcium signaling in vivo.

Endoplasmic reticulum generates calcium signalling microdomains around the nucleus and spindle in syncytial Drosophila embryos

Cell cycle calcium signals are generated by inositol trisphosphate-mediated release of calcium from internal stores [Ciapa, Pesando, Wilding and Whitaker (1994) Nature (London) 368, 875-878; Groigno and Whitaker (1998) Cell 92, 193-204]. The major internal calcium store is the ER (endoplasmic reticulum): the spatial organization of the ER during mitosis is important in defining a microdomain around the nucleus and mitotic spindle in early Drosophila embryos [Parry, McDougall and Whitaker (2005) J. Cell Biol. 171, 47-59]. Nuclear divisions in syncytial Drosophila embryos are accompanied by both cortical and nuclear localized calcium transients. Mitosis is prevented by the InsP(3) antagonists Xestospongin C and heparin. Nuclear-localized transients and cortical transients rely on extraembryonic calcium, suggesting that ER calcium levels are maintained by calcium influx.

Microdomains bounded by endoplasmic reticulum segregate cell cycle calcium transients in syncytial Drosophila embryos

Cell cycle calcium signals are generated by the inositol trisphosphate (InsP3)–mediated release of calcium from internal stores (Ciapa, B., D. Pesando, M. Wilding, and M. Whitaker. 1994. Nature. 368:875–878; Groigno, L., and M. Whitaker. 1998. Cell. 92:193–204). The major internal calcium store is the endoplasmic reticulum (ER); thus, the spatial organization of the ER during mitosis may be important in shaping and defining calcium signals. In early Drosophila melanogaster embryos, ER surrounds the nucleus and mitotic spindle during mitosis, offering an opportunity to determine whether perinuclear localization of ER conditions calcium signaling during mitosis. We establish that the nuclear divisions in syncytial Drosophila embryos are accompanied by both cortical and nuclear localized calcium transients. Constructs that chelate InsP3 also prevent nuclear division. An analysis of nuclear calcium concentrations demonstrates that they are differentially regulated. These observations demonstrate that mitotic calcium signals in Drosophila embryos are confined to mitotic microdomains and offer an explanation for the apparent absence of detectable global calcium signals during mitosis in some cell types.

Calcium goes nuclear

![Graphic][1] Nuclear calcium transients send PKC to the nuclear envelope (top right) rather than the plasma membrane (bottom right). Nathanson/MacmillanAfine reticulum that pokes its fingers across the nucleus is capable of storing and releasing calcium, according to Wihelma

Transcription-dependent neuronal plasticity: The nuclear calcium hypothesis.

In neurons, calcium ions control gene transcription induced by synaptic activity. The states and histories of neuronal activity are represented by a calcium code that comprises the site of calcium entry, and the amplitude, duration and spatial properties of signal-evoked calcium transients. The calcium code is used to transform specific firing patterns into qualitatively and quantitatively distinct transcriptional responses. The following hypothesis is proposed: electrical activity causes long-lasting, transcription-dependent changes in neuronal functions when synaptically evoked calcium transients associated with the stimulation propagate to the nucleus; gene transcription activated by dendritic calcium signals only is insufficient to consolidate functional alterations long-term. Similar to enduring increases in synaptic efficacy, nuclear calcium transients are induced by high-frequency firing patterns or by weak synaptic inputs coinciding with backpropagating dendritic action potentials. Nuclear calcium stimulates CREB-mediated transcription and, through inducing the activity of the transcriptional coactivator CREB-binding protein (CBP), may modulate the expression of numerous genes including neurotransmitter receptors and scaffolding proteins. Increases in the transcription rate of target genes are predicted to be transient and in many cases small, however, they collectively contribute to the maintenance of changes in synaptic efficacy. Nuclear calcium may be the common regulator of diverse transcription-dependent forms of neuronal plasticity.