CHCHD2 P14L, found in amyotrophic lateral sclerosis, exhibits cytoplasmic mislocalization and alters Ca2+ homeostasis.

CHCHD2 and CHCHD10, linked to Parkinson's disease and amyotrophic lateral sclerosis-frontotemporal dementia (ALS), respectively, are mitochondrial intermembrane proteins that form a heterodimer. This study aimed to investigate the impact of the CHCHD2 P14L variant, implicated in ALS, on mitochondrial function and its subsequent effects on cellular homeostasis. The missense variant of CHCHD2, P14L, found in a cohort of patients with ALS, mislocalized CHCHD2 to the cytoplasm, leaving CHCHD10 in the mitochondria. Drosophila lacking the CHCHD2 ortholog exhibited mitochondrial degeneration. In contrast, human CHCHD2 P14L, but not wild-type human CHCHD2, failed to suppress this degeneration, suggesting that P14L is a pathogenic variant. The mitochondrial Ca2+ buffering capacity was reduced in Drosophila neurons expressing human CHCHD2 P14L. The altered Ca2+-buffering phenotype was also observed in cultured human neuroblastoma SH-SY5Y cells expressing CHCHD2 P14L. In these cells, transient elevation of cytoplasmic Ca2+ facilitated the activation of calpain and caspase-3, accompanied by the processing and insolubilization of TDP-43. These observations suggest that CHCHD2 P14L causes abnormal Ca2+ dynamics and TDP-43 aggregation, reflecting the pathophysiology of ALS.

Palliative Radiation Therapy without Chemotherapy for a Patient with Monomorphic Epitheliotropic Intestinal T Cell Lymphoma: A Case Report.

Monomorphic epitheliotropic intestinal T cell lymphoma (MEITL), which used to be known as type 2 enteropathy-associated T cell lymphoma, is a rare lymphoma and is generally treated with chemotherapy. However, the MEITL prognosis is poor, and intestinal lymphoma including MEITL has the risk of bowel perforation not only at presentation but also during chemotherapy. A 67-year-old man was diagnosed with MEITL after presenting in our emergency room with bowel perforation. He and his family did not opt for the administration of anticancer drugs because of the risk of bowel perforation. However, they wanted the patient to receive palliative radiation therapy without chemotherapy. This treatment shrunk the tumor size without causing severe complications or decline in the quality of life, until he accidentally died due to traumatic intracranial hematoma. Considering the potential efficacy and safety of this treatment, it should be studied in more patients with MEITL.

Investigation of Brain Functions with Fluorescence Imaging Techniques.

An intricate interplay of complex spatio-temporal events underlies brain functions. Therefore, clarifying these dynamic processes is indispensable for revealing the mechanisms of brain functions. Fluorescence imaging is a powerful technique for visualizing cellular and molecular dynamics in the brain. Recent developments in fluorescent indicators and specialized optics have advanced research in the field of neuroscience. In this review, I will exemplify the power and beauty of fluorescence imaging by discussing my work focusing on the molecular dynamics of metabotropic glutamate receptor (mGluR) signaling at the synapse. By developing novel fluorescent indicators for glutamate, inositol 1,4,5-trisphosphate and Ca2+ within the endoplasmic reticulum, I succeeded in imaging the spatio-temporal dynamics of synaptic mGluR signaling, which led to the identification of novel mechanisms of mGluR-mediated glutamatergic neurotransmission. These discoveries highlight the importance of the development and application of novel fluorescence imaging techniques for the investigation of brain functions.

Astrocytic Ca2+ signaling mediated by the endoplasmic reticulum in health and disease.

Astrocytes generate robust intracellular Ca2+ signals that are assumed to be key regulators of astrocytic function. Among various Ca2+ mobilization mechanisms, Ca2+ release from the endoplasmic reticulum (ER) via the inositol 1,4,5-trisphosphate receptor (IP3R) has attracted attention as a major component of astrocytic Ca2+ signaling. Manipulation of astrocytic IP3-Ca2+ signaling, such as genetic deletion of the type 2 IP3R, has revealed multifaceted roles of astrocytic ER Ca2+ release in health and disease. Recent developments in Ca2+ imaging techniques including ER intraluminal Ca2+ imaging have been indispensable in determining the physiological and pathophysiological significance of astrocytic ER Ca2+ release via IP3Rs. Beneficial and detrimental roles of IP3R-dependent Ca2+ release in astrocytes have been revealed in wide variety of disorders in the brain, strongly suggesting astrocytic IP3-Ca2+ signaling as a novel and promising therapeutic target.

Rational Design of a Near-infrared Fluorescence Probe for Ca2+ Based on Phosphorus-substituted Rhodamines Utilizing Photoinduced Electron Transfer.

Fluorescence imaging in the near-infrared (NIR) region (650-900 nm) is useful for bioimaging because background autofluorescence is low and tissue penetration is high in this range. In addition, NIR fluorescence is useful as a complementary color window to green and red for multicolor imaging. Here, we compared the photoinduced electron transfer (PeT)-mediated fluorescence quenching of silicon- and phosphorus-substituted rhodamines (SiRs and PRs) in order to guide the development of improved far-red to NIR fluorescent dyes. The results of density functional theory calculations and photophysical evaluation of a series of newly synthesized PRs confirmed that the fluorescence of PRs was more susceptible than that of SiRs to quenching via PeT. Based on this, we designed and synthesized a NIR fluorescence probe for Ca2+ , CaPR-1, and its membrane-permeable acetoxymethyl derivative, CaPR-1 AM, which is distributed to the cytosol, in marked contrast to our previously reported Ca2+ far-red to NIR fluorescence probe based on the SiR scaffold, CaSiR-1 AM, which is mainly localized in lysosomes as well as cytosol in living cells. CaPR-1 showed longer-wavelength absorption and emission (up to 712 nm) than CaSiR-1. The new probe was able to image Ca2+ at dendrites and spines in brain slices, and should be a useful tool in neuroscience research.

Visualization of astrocytic intracellular Ca2+ mobilization.

Astrocytes generate robust intracellular Ca2+ concentration changes (Ca2+ signals), which are assumed to regulate astrocytic functions that play crucial roles in the regulation of brain functions. One frequently used strategy for exploring the role of astrocytic Ca2+ signalling is the use of mice deficient in the type 2 inositol 1,4,5-trisphosphate receptor (IP3 R2). These IP3 R2-knockout (KO) mice are reportedly devoid of Ca2+ mobilization from the endoplasmic reticulum (ER) in astrocytes. However, they have shown no functional deficits in several studies, causing a heated debate as to the functional relevance of ER-mediated Ca2+ signalling in astrocytes. Recently, the assumption that Ca2+ mobilization from the ER is absent in IP3 R2-KO astrocytes has been re-evaluated using intraorganellar Ca2+ imaging techniques. The new results indicated that IP3 R2-independent Ca2+ release may generate Ca2+ nanodomains around the ER, which may help explain the absence of functional deficits in IP3 R2-KO mice.

Store-operated Ca2+ entry-dependent Ca2+ refilling in the endoplasmic reticulum in astrocytes.

Astrocytes regulate various brain functions, for which Ca2+ release from the endoplasmic reticulum (ER) often play crucial roles. Because astrocytic ER Ca2+ release is robust and frequent, the ER Ca2+ refilling mechanism should be critical for ongoing Ca2+ signaling in astrocytes. In this study, we focused on the putative functional significance of store-operated Ca2+ entry (SOCE) in ER Ca2+ refilling. We expressed the ER luminal Ca2+ indicator G-CEPIA1er in astrocytes in acute cortical slices to directly monitor the decrease and recovery of ER Ca2+ concentration upon spontaneous or norepinephrine-induced Ca2+ release. Inhibition of SOCE significantly slowed the recovery of ER Ca2+ concentration after Ca2+ release in astrocytes. This delayed recovery resulted in a prolonged decrease in the ER Ca2+ content in astrocytes with periodic spontaneous Ca2+ release, followed by the attenuation of cytosolic Ca2+ responses upon Ca2+ release. Therefore, our results provide direct evidence for the physiological significance of SOCE in ER Ca2+ refilling after ER Ca2+ release.

[Neuronal cell death induced by the disruption of endoplasmic reticulum-mediated Ca2+ signaling].

For the function and survival of neurons in the central nervous system, it is indispensable that the intracellular Ca2+ dynamics are properly controlled. The endoplasmic reticulum (ER), a major intracellular Ca2+ store, plays an important role in the control of the intracellular Ca2+ dynamics in neurons through the supply and uptake of Ca2+. It has been suggested that the disruption of ER Ca2+ signaling is involved in neuronal cell death in various pathological conditions. Therefore, the disruption of ER Ca2+ signaling has attracted attention as a novel mechanism for neurodegenerative diseases including Alzheimer's disease. In this review, we introduce the latest findings including our research on the relationship between the disruption of ER Ca2+ signaling and neuronal cell death. In addition, we will introduce recent developments on the technology for visualizing intraluminal Ca2+ dynamics within the ER, which is indispensable for promoting research in this field.

Generation of a common innate lymphoid cell progenitor requires interferon regulatory factor 2.

Innate lymphoid cells (ILCs), composed of heterogeneous populations of lymphoid cells, contribute critically to immune surveillance at mucosal surfaces. ILC subsets develop from common lymphoid progenitors through stepwise lineage specification. However, the composition and temporal regulation of the transcription factor network governing such a process remain incompletely understood. Here, we report that deletion of the transcription factor interferon regulatory factor 2 (IRF-2), known also for its importance in the maturation of conventional NK cells, resulted in an impaired generation of ILC1, ILC2 and ILC3 subsets with lymphoid tissue inducer (LTi)-like cells hardly affected. In IRF-2-deficient mice, PD-1hi ILC precursors (ILCPs) that generate these three ILCs but not LTi-like cells were present at normal frequency, while their sub-population expressing high amounts of PLZF, another marker for ILCPs, was severely reduced. Notably, these IRF-2-deficient ILCPs contained normal quantities of PLZF-encoding Zbtb16 messages, and PLZF expression in developing invariant NKT cells within the thymus was unaffected in these mutant mice. These results point to a unique, cell-type selective role for IRF-2 in ILC development, acting at a discrete step critical for the generation of functionally competent ILCPs.

Inositol 1,4,5-trisphosphate receptor type 2-independent Ca2+ release from the endoplasmic reticulum in astrocytes.

Accumulating evidence indicates that astrocytes are actively involved in the physiological and pathophysiological functions of the brain. Intracellular Ca2+ signaling, especially Ca2+ release from the endoplasmic reticulum (ER), is considered to be crucial for the regulation of astrocytic functions. Mice with genetic deletion of inositol 1,4,5-trisphosphate receptor type 2 (IP3 R2) are reportedly devoid of astrocytic Ca2+ signaling, and thus widely used to explore the roles of Ca2+ signaling in astrocytic functions. While functional deficits in IP3 R2-knockout (KO) mice have been found in some reports, no functional deficit was observed in others. Thus, there remains a controversy regarding the functional significance of astrocytic Ca2+ signaling. To address this controversy, we re-evaluated the assumption that Ca2+ release from the ER is abolished in IP3 R2-KO astrocytes using a highly sensitive imaging technique. We expressed the ER luminal Ca2+ indicator G-CEPIA1er in cortical and hippocampal astrocytes to directly visualize spontaneous and stimulus-induced Ca2+ release from the ER. We found attenuated but significant Ca2+ release in response to application of norepinephrine to IP3 R2-KO astrocytes. This IP3 R2-independent Ca2+ release induced only minimal cytosolic Ca2+ transients but induced robust Ca2+ increases in mitochondria that are frequently in close contact with the ER. These results indicate that ER Ca2+ release is retained and is sufficient to increase the Ca2+ concentration in close proximity to the ER in IP3 R2-KO astrocytes.

Role of Endoplasmic Reticulum-Mediated Ca2+ Signaling in Neuronal Cell Death.

SIGNIFICANCE: Properly controlled intracellular Ca2+ dynamics is crucial for regulation of neuronal function and survival in the central nervous system. The endoplasmic reticulum (ER), a major intracellular Ca2+ store, plays a critical role as a source and sink for neuronal Ca2+. Recent Advances: Accumulating evidence indicates that disrupted ER Ca2+ signaling is involved in neuronal cell death under various pathological conditions, providing novel insight into neurodegenerative disease mechanisms. CRITICAL ISSUES: We summarize current knowledge concerning the relationship between abnormal ER Ca2+ dynamics and neuronal cell death. We also introduce recent technical advances for probing ER intraluminal Ca2+ dynamics with unprecedented spatiotemporal resolution. FUTURE DIRECTIONS: Further studies on ER Ca2+ signaling are expected to provide progress for unmet medical needs in neurodegenerative disease. Antioxid. Redox Signal. 29, 1147-1157.

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.

Whisker experience-dependent mGluR signaling maintains synaptic strength in the mouse adolescent cortex.

Sensory experience-dependent plasticity in the somatosensory cortex is a fundamental mechanism of adaptation to the changing environment not only early in the development but also in adolescence and adulthood. Although the mechanisms underlying experience-dependent plasticity during early development have been well documented, the corresponding understanding in the mature cortex is less complete. Here, we investigated the mechanism underlying whisker deprivation-induced synaptic plasticity in the barrel cortex in adolescent mice. Layer 4 (L4) to L2/3 excitatory synapses play a crucial role for whisker experience-dependent plasticity in rodent barrel cortex and whisker deprivation is known to depress synaptic strength at L4-L2/3 synapses in adolescent and adult animals. We found that whisker deprivation for 5 days or longer decreased the presynaptic glutamate release probability at L4-L2/3 synapses in the barrel cortex in adolescent mice. This whisker deprivation-induced depression was restored by daily administration of a positive allosteric modulator of the type 5 metabotropic glutamate receptor (mGluR5). On the other hand, the administration of mGluR5 antagonists reproduced the effect of whisker deprivation in whisker-intact mice. Furthermore, chronic and selective suppression of inositol 1,4,5-trisphosphate (IP3 ) signaling in postsynaptic L2/3 neurons decreased the presynaptic release probability at L4-L2/3 synapses. These findings represent a previously unidentified mechanism of cortical plasticity, namely that whisker experience-dependent mGluR5-IP3 signaling in the postsynaptic neurons maintains presynaptic function in the adolescent barrel cortex.

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 Ca(2+) signaling, serving as both a source and sink of Ca(2+), and regulating a variety of physiological and pathophysiological events in neurons in the brain. However, spatiotemporal Ca(2+) dynamics within the ER in central neurons remain to be characterized. In this study, we visualized synaptic activity-dependent ER Ca(2+) dynamics in mouse cerebellar Purkinje cells (PCs) using an ER-targeted genetically encoded Ca(2+) indicator, G-CEPIA1er. We used brief parallel fiber stimulation to induce a local decrease in the ER luminal Ca(2+) concentration ([Ca(2+)]ER) in dendrites and spines. In this experimental system, the recovery of [Ca(2+)]ER takes several seconds, and recovery half-time depends on the extent of ER Ca(2+) depletion. By combining imaging analysis and numerical simulation, we show that the intraluminal diffusion of Ca(2+), rather than Ca(2+) reuptake, is the dominant mechanism for the replenishment of the local [Ca(2+)]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 Ca(2+) diffusion. Furthermore, we found that repetitive climbing fiber stimulation, which induces cytosolic Ca(2+) spikes in PCs, cumulatively increased [Ca(2+)]ER. These results indicate that the neuronal ER functions both as an intracellular tunnel to redistribute stored Ca(2+) within the neurons, and as a leaky integrator of Ca(2+) spike-inducing synaptic inputs.

Neuronal Regulation of Schwann Cell Mitochondrial Ca(2+) 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.

Impact of advanced age on the short- and long-term outcomes in patients undergoing hepatectomy for hepatocellular carcinoma: a single-center analysis over a 20-year period.

BACKGROUND: The purpose of this study was to analyze the influence of age on both the risk of hepatectomy and the prognosis in patients with hepatocellular carcinoma (HCC). METHODS: Patients undergoing an initial hepatectomy for HCC were classified into 2 age groups: 75 years or over (n = 113) and less than 75 years (n = 499). RESULTS: A zero 90-day mortality was achieved in the elderly. Although the recurrence rate and recurrence sites were almost similar between the 2 groups, the 5-year survival rate in the elderly patients was significantly lower than that in the younger patients (46.0% vs 57.6%; P = .018), possibly because of the higher incidence of deaths from other causes (26.8% vs 10.4%; P = .011) in the elderly. CONCLUSION: Selected elderly HCC patients can undergo a hepatectomy safely and can benefit from long-term HCC control comparable with that of their younger counterparts.

In vivo visualization of subtle, transient, and local activity of astrocytes using an ultrasensitive Ca(2+) 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.

Imaging intraorganellar Ca2+ at subcellular resolution using CEPIA.

The endoplasmic reticulum (ER) and mitochondria accumulate Ca(2+) within their lumens to regulate numerous cell functions. However, determining the dynamics of intraorganellar Ca(2+) has proven to be difficult. Here we describe a family of genetically encoded Ca(2+) indicators, named calcium-measuring organelle-entrapped protein indicators (CEPIA), which can be utilized for intraorganellar Ca(2+) imaging. CEPIA, which emit green, red or blue/green fluorescence, are engineered to bind Ca(2+) at intraorganellar Ca(2+) 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 Ca(2+) import into individual mitochondria while simultaneously measuring ER and cytosolic Ca(2+). We have used these imaging capabilities to reveal differential Ca(2+) handling in individual mitochondria. CEPIA imaging is a useful new tool to further the understanding of organellar functions.