Kazunori Kanemaru

Imaging intraorganellar Ca2+ at subcellular resolution using CEPIA

The endoplasmic reticulum (ER) and mitochondria accumulate Ca2+ within their lumens to regulate numerous cell functions. However, determining the dynamics of intraorganellar Ca2+ has proven to be difficult. Here we describe a family of genetically encoded Ca2+ indicators, named calcium-measuring organelle-entrapped protein indicators (CEPIA), which can be utilized for intraorganellar Ca2+ imaging. CEPIA, which emit green, red or blue/green fluorescence, are engineered to bind Ca2+ at intraorganellar Ca2+ concentrations. They can be targeted to different organelles and may be used alongside other fluorescent molecular markers, expanding the range of cell functions that can be simultaneously analysed. The spatiotemporal resolution of CEPIA makes it possible to resolve Ca2+ import into individual mitochondria while simultaneously measuring ER and cytosolic Ca2+. We have used these imaging capabilities to reveal differential Ca2+ handling in individual mitochondria. CEPIA imaging is a useful new tool to further the understanding of organellar functions.

In Vivo Visualization of Subtle, Transient, and Local Activity of Astrocytes Using an Ultrasensitive Ca2+ Indicator

Astrocytes generate local calcium (Ca(2+)) signals that are thought to regulate their functions. Visualization of these signals in the intact brain requires an imaging method with high spatiotemporal resolution. Here, we describe such a method using transgenic mice expressing the ultrasensitive ratiometric Ca(2+) indicator yellow Cameleon-Nano 50 (YC-Nano50) in astrocytes. In these mice, we detected a unique pattern of Ca(2+) signals. These occur spontaneously, predominantly in astrocytic fine processes, but not the cell body. Upon sensory stimulation, astrocytes initially responded with Ca(2+) signals at fine processes, which then propagated to the cell body. These observations suggest that astrocytic fine processes function as a high-sensitivity detector of neuronal activities. Thus, the method provides a useful tool for studying the activity of astrocytes in brain physiology and pathology.

Regulation of Neurite Growth by Spontaneous Ca2+ Oscillations in Astrocytes

Astrocytes play a pivotal role in the regulation of neurite growth, but the intracellular signaling mechanism in astrocytes that mediates this regulation remains unclarified. We studied the relationship between spontaneous Ca2+ oscillations in astrocytes and the astrocyte-mediated neurite growth. We generated Ca2+ signal-deficient astrocytes in which spontaneous Ca2+ oscillations were abolished by a chronic inhibition of IP3 signaling. When hippocampal neurons were cultured on a monolayer of Ca2+ signal-deficient astrocytes, the growth of dendrites and axons was inhibited. Time-lapse imaging of the advancement of axonal growth cones indicated the involvement of membrane-bound molecules for this inhibition. Among six candidate membrane-bound molecules that may modulate neuronal growth, N-cadherin was downregulated in Ca2+ signal-deficient astrocytes. Although a blocking antibody to N-cadherin suppressed the axonal growth on control astrocytes, extrinsic N-cadherin expression rescued the suppressed axonal growth on Ca2+ signal-deficient astrocytes. These findings suggest that spontaneous Ca2+ oscillations regulate the astrocytic function to promote neurite growth by maintaining the expression of specific growth-enhancing proteins on their surface, and that N-cadherin is one of such molecules.

Calcium-dependent N-cadherin up-regulation mediates reactive astrogliosis and neuroprotection after brain injury

Brain injury induces phenotypic changes in astrocytes, known as reactive astrogliosis, which may influence neuronal survival. Here we show that brain injury induces inositol 1,4,5-trisphosphate (IP3)-dependent Ca2+ signaling in astrocytes, and that the Ca2+ signaling is required for astrogliosis. We found that type 2 IP3 receptor knockout (IP3R2KO) mice deficient in astrocytic Ca2+ signaling have impaired reactive astrogliosis and increased injury-associated neuronal death. We identified N-cadherin and pumilio 2 (Pum2) as downstream signaling molecules, and found that brain injury induces up-regulation of N-cadherin around the injured site. This effect is mediated by Ca2+-dependent down-regulation of Pum2, which in turn attenuates Pum2-dependent translational repression of N-cadherin. Furthermore, we show that astrocyte-specific knockout of N-cadherin results in impairment of astrogliosis and neuroprotection. Thus, astrocytic Ca2+ signaling and the downstream function of N-cadherin play indispensable roles in the cellular responses to brain injury. These findings define a previously unreported signaling axis required for reactive astrogliosis and neuroprotection following brain injury.

Genetically Encoded Fluorescent Indicators for Organellar Calcium Imaging

Optical Ca(2+) indicators are powerful tools for investigating intracellular Ca(2+) signals in living cells. Although a variety of Ca(2+) indicators have been developed, deciphering the physiological functions and spatiotemporal dynamics of Ca(2+) in intracellular organelles remains challenging. Genetically encoded Ca(2+) indicators (GECIs) using fluorescent proteins are promising tools for organellar Ca(2+) imaging, and much effort has been devoted to their development. In this review, we first discuss the key points of organellar Ca(2+) imaging and summarize the requirements for optimal organellar Ca(2+) indicators. Then, we highlight some of the recent advances in the engineering of fluorescent GECIs targeted to specific organelles. Finally, we discuss the limitations of currently available GECIs and the requirements for advancing the research on intraorganellar Ca(2+) signaling.

Neuronal Regulation of Schwann Cell Mitochondrial Ca2+ Signaling during Myelination

Schwann cells (SCs) myelinate peripheral neurons to promote the rapid conduction of action potentials, and the process of myelination is known to be regulated by signals from axons to SCs. Given that SC mitochondria are one of the potential regulators of myelination, we investigated whether SC mitochondria are regulated by axonal signaling. Here, we show a purinergic mechanism that sends information from neurons to SC mitochondria during myelination. Our results show that electrical stimulation of rat sciatic nerve increases extracellular ATP levels enough to activate purinergic receptors. Indeed, electrical stimulation of sciatic nerves induces Ca(2+) increases in the cytosol and the mitochondrial matrix of surrounding SCs via purinergic receptor activation. Chronic suppression of this pathway during active myelination suppressed the longitudinal and radial development of myelinating SCs and caused hypomyelination. These results demonstrate a neuron-to-SC mitochondria signaling, which is likely to have an important role in proper myelination.

Visualization of Ca2+ Filling Mechanisms upon Synaptic Inputs in the Endoplasmic Reticulum of Cerebellar Purkinje Cells

The endoplasmic reticulum (ER) plays crucial roles in intracellular Ca2+ signaling, serving as both a source and sink of Ca2+, and regulating a variety of physiological and pathophysiological events in neurons in the brain. However, spatiotemporal Ca2+ dynamics within the ER in central neurons remain to be characterized. In this study, we visualized synaptic activity-dependent ER Ca2+ dynamics in mouse cerebellar Purkinje cells (PCs) using an ER-targeted genetically encoded Ca2+ indicator, G-CEPIA1er. We used brief parallel fiber stimulation to induce a local decrease in the ER luminal Ca2+ concentration ([Ca2+]ER) in dendrites and spines. In this experimental system, the recovery of [Ca2+]ER takes several seconds, and recovery half-time depends on the extent of ER Ca2+ depletion. By combining imaging analysis and numerical simulation, we show that the intraluminal diffusion of Ca2+, rather than Ca2+ reuptake, is the dominant mechanism for the replenishment of the local [Ca2+]ER depletion immediately following the stimulation. In spines, the ER filled almost simultaneously with parent dendrites, suggesting that the ER within the spine neck does not represent a significant barrier to Ca2+ diffusion. Furthermore, we found that repetitive climbing fiber stimulation, which induces cytosolic Ca2+ spikes in PCs, cumulatively increased [Ca2+]ER. These results indicate that the neuronal ER functions both as an intracellular tunnel to redistribute stored Ca2+ within the neurons, and as a leaky integrator of Ca2+ spike-inducing synaptic inputs. SIGNIFICANCE STATEMENT Ca2+ is a key messenger that regulates neuronal functions in the brain. Although the endoplasmic reticulum (ER) plays indispensable roles as a source and sink of Ca2+, technical difficulties have impeded the analysis of Ca2+ dynamics within the ER. In this study, we have used a genetically encoded ER Ca2+ indicator to visualize Ca2+ dynamics within the neuronal ER. We found that Ca2+-mobilizing synaptic inputs locally decreased the ER Ca2+ concentration, followed by Ca2+ replenishment by intraluminal Ca2+ diffusion throughout the ER of dendrites and spines. Furthermore, Ca2+ spike-inducing synaptic inputs cumulatively increased the Ca2+ content of the ER. Thus, our study indicates that the ER functions both as a tunnel to redistribute stored Ca2+ and as a leaky integrator of synaptic inputs.

Divergent Activity Profiles of Type 1 Ryanodine Receptor Channels Carrying Malignant Hyperthermia and Central Core Disease Mutations in the Amino-Terminal Region.

The type 1 ryanodine receptor (RyR1) is a Ca2+ release channel in the sarcoplasmic reticulum of skeletal muscle and is mutated in several diseases, including malignant hyperthermia (MH) and central core disease (CCD). Most MH and CCD mutations cause accelerated Ca2+ release, resulting in abnormal Ca2+ homeostasis in skeletal muscle. However, how specific mutations affect the channel to produce different phenotypes is not well understood. In this study, we have investigated 11 mutations at 7 different positions in the amino (N)-terminal region of RyR1 (9 MH and 2 MH/CCD mutations) using a heterologous expression system in HEK293 cells. In live-cell Ca2+ imaging at room temperature (~25 °C), cells expressing mutant channels exhibited alterations in Ca2+ homeostasis, i.e., an enhanced sensitivity to caffeine, a depletion of Ca2+ in the ER and an increase in resting cytoplasmic Ca2+. RyR1 channel activity was quantitatively evaluated by [3H]ryanodine binding and three parameters (sensitivity to activating Ca2+, sensitivity to inactivating Ca2+ and attainable maximum activity, i.e., gain) were obtained by fitting analysis. The mutations increased the gain and the sensitivity to activating Ca2+ in a site-specific manner. The gain was consistently higher in both MH and MH/CCD mutations. Sensitivity to activating Ca2+ was markedly enhanced in MH/CCD mutations. The channel activity estimated from the three parameters provides a reasonable explanation to the pathological phenotype assessed by Ca2+ homeostasis. These properties were also observed at higher temperatures (~37 °C). Our data suggest that divergent activity profiles may cause varied disease phenotypes by specific mutations. This approach should be useful for diagnosis and treatment of diseases with mutations in RyR1.

Genotype–Phenotype Correlations of Malignant Hyperthermia and Central Core Disease Mutations in the Central Region of the RYR1 Channel

Type 1 ryanodine receptor (RYR1) is a Ca2+ release channel in the sarcoplasmic reticulum of skeletal muscle and is mutated in some muscle diseases, including malignant hyperthermia (MH) and central core disease (CCD). Over 200 mutations associated with these diseases have been identified, and most mutations accelerate Ca2+‐induced Ca2+ release (CICR), resulting in abnormal Ca2+ homeostasis in skeletal muscle. However, it remains largely unknown how specific mutations cause different phenotypes. In this study, we investigated the CICR activity of 14 mutations at 10 different positions in the central region of RYR1 (10 MH and four MH/CCD mutations) using a heterologous expression system in HEK293 cells. In live‐cell Ca2+ imaging, the mutant channels exhibited an enhanced sensitivity to caffeine, a reduced endoplasmic reticulum Ca2+ content, and an increased resting cytoplasmic Ca2+ level. The three parameters for CICR (Ca2+ sensitivity for activation, Ca2+ sensitivity for inactivation, and attainable maximum activity, i.e., gain) were obtained by [3H]ryanodine binding and fitting analysis. The mutant channels showed increased gain and Ca2+ sensitivity for activation in a site‐specific manner. Genotype–phenotype correlations were explained well by the near‐atomic structure of RYR1. Our data suggest that divergent CICR activity may cause various disease phenotypes by specific mutations.

Nitric Oxide-induced Activation of the Type 1 Ryanodine Receptor Is Critical for Epileptic Seizure-induced Neuronal Cell Death

Status epilepticus (SE) is a life-threatening emergency that can cause neurodegeneration with debilitating neurological disorders. However, the mechanism by which convulsive SE results in neurodegeneration is not fully understood. It has been shown that epileptic seizures produce markedly increased levels of nitric oxide (NO) in the brain, and that NO induces Ca2 + release from the endoplasmic reticulum via the type 1 ryanodine receptor (RyR1), which occurs through S-nitrosylation of the intracellular Ca2 + release channel. Here, we show that through genetic silencing of NO-induced activation of the RyR1 intracellular Ca2 + release channel, neurons were rescued from seizure-dependent cell death. Furthermore, dantrolene, an inhibitor of RyR1, was protective against neurodegeneration caused by SE. These results demonstrate that NO-induced Ca2 + release via RyR is involved in SE-induced neurodegeneration, and provide a rationale for the use of RyR1 inhibitors for the prevention of brain damage following SE.