Essential role of ROS - 8-Nitro-cGMP signaling in long-term memory of motor learning and cerebellar synaptic plasticity.

Although reactive oxygen species (ROS) are known to have harmful effects in organisms, recent studies have demonstrated expression of ROS synthases at various parts of the organisms and the controlled ROS generation, suggesting possible involvement of ROS signaling in physiological events of individuals. However, physiological roles of ROS in the CNS, including functional roles in higher brain functions or neuronal activity-dependent ROS production, remain to be elucidated. Here, we demonstrated involvement of ROS - 8-NO2-cGMP signaling in motor learning and synaptic plasticity in the cerebellum. In the presence of inhibitors of ROS signal or ROS synthases, cerebellar motor learning was impaired, and the stimulus inducing long-term depression (LTD), cellular basis for the motor learning, failed to induce LTD but induced long-term potentiation (LTP)-like change at cerebellar synapses. Furthermore, ROS was produced by LTD-inducing stimulus in enzyme-dependent manner, and excess administration of the antioxidant vitamin E impaired cerebellar motor learning, suggesting beneficial roles of endogenous ROS in the learning. As a downstream signal, involvement of 8-NO2-cGMP in motor learning and cerebellar LTD were also revealed. These findings indicate that ROS - 8-NO2-cGMP signal is activated by neuronal activity and is essential for cerebellum-dependent motor learning and synaptic plasticity, demonstrating involvement of the signal in physiological function of brain systems.

In situ high-pressure pair distribution function measurement of liquid and glass by using 100 keV pink beam.

Understanding the pressure-induced structural changes in liquids and amorphous materials is fundamental in a wide range of scientific fields. However, experimental investigation of the structure of liquid and amorphous material under in situ high-pressure conditions is still limited due to the experimental difficulties. In particular, the range of the momentum transfer (Q) in the structure factor [S(Q)] measurement under high-pressure conditions has been limited at relatively low Q, which makes it difficult to conduct detailed structural analysis of liquid and amorphous material. Here, we show the in situ high-pressure pair distribution function measurement of liquid and glass by using the 100 keV pink beam. Structures of liquids and glasses are measured under in situ high-pressure conditions in the Paris-Edinburgh press by high-energy x-ray diffraction measurement using a double-slit collimation setup with a point detector. The experiment enables us to measure S(Q) of GeO2 and SiO2 glasses and liquid Ge at a wide range of Q up to 20-29 Å-1 under in situ high-pressure and high-temperature conditions, which is almost two times larger than that of the conventional high-pressure angle-dispersive x-ray diffraction measurement. The high-pressure experimental S(Q) precisely determined at a wide range of Q opens the way to investigate detailed structural features of liquids and amorphous materials under in situ high-pressure and high-temperature conditions, as well as ambient pressure study.

Biology of cognitive aging across species.

Aging is associated with cognitive decline, which can critically affect quality of life. Examining the biology of cognitive aging across species will lead to a better understanding of the fundamental mechanisms involved in this process, and identify potential interventions that could help to improve cognitive function in aging individuals. This minireview aimed to explore the mechanisms and processes involved in cognitive aging across a range of species, from flies to rodents, and covers topics, such as the role of reactive oxygen species and autophagy/mitophagy in cognitive aging. Overall, this literature provides a comprehensive overview of the biology of cognitive aging across species, highlighting the latest research findings and identifying potential avenues for future research. Geriatr Gerontol Int 2024; 24: 15-24.

Atypical cell death and insufficient matrix organization in long-bone growth plates from Tric-b-knockout mice.

TRIC-A and TRIC-B proteins form homotrimeric cation-permeable channels in the endoplasmic reticulum (ER) and nuclear membranes and are thought to contribute to counterionic flux coupled with store Ca2+ release in various cell types. Serious mutations in the TRIC-B (also referred to as TMEM38B) locus cause autosomal recessive osteogenesis imperfecta (OI), which is characterized by insufficient bone mineralization. We have reported that Tric-b-knockout mice can be used as an OI model; Tric-b deficiency deranges ER Ca2+ handling and thus reduces extracellular matrix (ECM) synthesis in osteoblasts, leading to poor mineralization. Here we report irregular cell death and insufficient ECM in long-bone growth plates from Tric-b-knockout embryos. In the knockout growth plate chondrocytes, excess pro-collagen fibers were occasionally accumulated in severely dilated ER elements. Of the major ER stress pathways, activated PERK/eIF2α (PKR-like ER kinase/ eukaryotic initiation factor 2α) signaling seemed to inordinately alter gene expression to induce apoptosis-related proteins including CHOP (CCAAT/enhancer binding protein homologous protein) and caspase 12 in the knockout chondrocytes. Ca2+ imaging detected aberrant Ca2+ handling in the knockout chondrocytes; ER Ca2+ release was impaired, while cytoplasmic Ca2+ level was elevated. Our observations suggest that Tric-b deficiency directs growth plate chondrocytes to pro-apoptotic states by compromising cellular Ca2+-handling and exacerbating ER stress response, leading to impaired ECM synthesis and accidental cell death.

Temperature dependence of nitrogen solubility in bridgmanite and evolution of nitrogen storage capacity in the lower mantle.

Relative nitrogen abundance normalized by carbonaceous chondrites in the bulk silicate Earth appears to be depleted compared to other volatile elements. Especially, nitrogen behavior in the deep part of the Earth such as the lower mantle is not clearly understood. Here, we experimentally investigated the temperature dependence of nitrogen solubility in bridgmanite which occupies 75 wt.% of the lower mantle. The experimental temperature ranged from 1400 to 1700 °C at 28 GPa in the redox state corresponding to the shallow lower mantle. The maximum nitrogen solubility in bridgmanite (MgSiO3) increased from 1.8 ± 0.4 to 5.7 ± 0.8 ppm with increasing temperature from 1400 to 1700 °C. The nitrogen storage capacity of Mg-endmember bridgmanite under the current temperature conditions is 3.4 PAN (PAN: mass of present atmospheric nitrogen). Furthermore, the nitrogen solubility of bridgmanite increased with increasing temperature, in contrast to the nitrogen solubility of metallic iron. Thus, the nitrogen storage capacity of bridgmanite can be larger than that of metallic iron during the solidification of the magma ocean. Such a "hidden" nitrogen reservoir formed by bridgmanite in the lower mantle may have depleted the apparent nitrogen abundance ratio in the bulk silicate Earth.

C-type natriuretic peptide facilitates autonomic Ca2+ entry in growth plate chondrocytes for stimulating bone growth.

The growth plates are cartilage tissues found at both ends of developing bones, and vital proliferation and differentiation of growth plate chondrocytes are primarily responsible for bone growth. C-type natriuretic peptide (CNP) stimulates bone growth by activating natriuretic peptide receptor 2 (NPR2) which is equipped with guanylate cyclase on the cytoplasmic side, but its signaling pathway is unclear in growth plate chondrocytes. We previously reported that transient receptor potential melastatin-like 7 (TRPM7) channels mediate intermissive Ca2+ influx in growth plate chondrocytes, leading to activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) for promoting bone growth. In this report, we provide evidence from experiments using mutant mice, indicating a functional link between CNP and TRPM7 channels. Our pharmacological data suggest that CNP-evoked NPR2 activation elevates cellular cGMP content and stimulates big-conductance Ca2+-dependent K+ (BK) channels as a substrate for cGMP-dependent protein kinase (PKG). BK channel-induced hyperpolarization likely enhances the driving force of TRPM7-mediated Ca2+ entry and seems to accordingly activate CaMKII. Indeed, ex vivo organ culture analysis indicates that CNP-facilitated bone growth is abolished by chondrocyte-specific Trpm7 gene ablation. The defined CNP signaling pathway, the NPR2-PKG-BK channel-TRPM7 channel-CaMKII axis, likely pinpoints promising target proteins for developing new therapeutic treatments for divergent growth disorders.

A novel RyR1-selective inhibitor prevents and rescues sudden death in mouse models of malignant hyperthermia and heat stroke.

Mutations in the type 1 ryanodine receptor (RyR1), a Ca2+ release channel in skeletal muscle, hyperactivate the channel to cause malignant hyperthermia (MH) and are implicated in severe heat stroke. Dantrolene, the only approved drug for MH, has the disadvantages of having very poor water solubility and long plasma half-life. We show here that an oxolinic acid-derivative RyR1-selective inhibitor, 6,7-(methylenedioxy)-1-octyl-4-quinolone-3-carboxylic acid (Compound 1, Cpd1), effectively prevents and treats MH and heat stroke in several mouse models relevant to MH. Cpd1 reduces resting intracellular Ca2+, inhibits halothane- and isoflurane-induced Ca2+ release, suppresses caffeine-induced contracture in skeletal muscle, reduces sarcolemmal cation influx, and prevents or reverses the fulminant MH crisis induced by isoflurane anesthesia and rescues animals from heat stroke caused by environmental heat stress. Notably, Cpd1 has great advantages of better water solubility and rapid clearance in vivo over dantrolene. Cpd1 has the potential to be a promising candidate for effective treatment of patients carrying RyR1 mutations.

Precise Regulation of the Basal PKCγ Activity by DGKγ Is Crucial for Motor Coordination.

Diacylglycerol kinase γ (DGKγ) is a lipid kinase to convert diacylglycerol (DG) to phosphatidic acid (PA) and indirectly regulates protein kinase C γ (PKCγ) activity. We previously reported that the basal PKCγ upregulation impairs cerebellar long-term depression (LTD) in the conventional DGKγ knockout (KO) mice. However, the precise mechanism in impaired cerebellar LTD by upregulated PKCγ has not been clearly understood. Therefore, we first produced Purkinje cell-specific DGKγ KO (tm1d) mice to investigate the specific function of DGKγ in Purkinje cells and confirmed that tm1d mice showed cerebellar motor dysfunction in the rotarod and beam tests, and the basal PKCγ upregulation but not PKCα in the cerebellum of tm1d mice. Then, the LTD-induced chemical stimulation, K-glu (50 mM KCl + 100 µM, did not induce phosphorylation of PKCα and dissociation of GluR2 and glutamate receptor interacting protein (GRIP) in the acute cerebellar slices of tm1d mice. Furthermore, treatment with the PKCγ inhibitor, scutellarin, rescued cerebellar LTD, with the phosphorylation of PKCα and the dissociation of GluR2 and GRIP. In addition, nonselective transient receptor potential cation channel type 3 (TRPC3) was negatively regulated by upregulated PKCγ. These results demonstrated that DGKγ contributes to cerebellar LTD by regulation of the basal PKCγ activity.

Functional maintenance of calcium store by ShcB adaptor protein in cerebellar Purkinje cells.

Intracellular Ca2+ levels are changed by influx from extracellular medium and release from intracellular stores. In the central nervous systems, Ca2+ release is involved in various physiological events, such as neuronal excitability and transmitter release. Although stable Ca2+ release in response to stimulus is critical for proper functions of the nervous systems, regulatory mechanisms relating to Ca2+ release are not fully understood in central neurons. Here, we demonstrate that ShcB, an adaptor protein expressed in central neurons, has an essential role in functional maintenance of Ca2+ store in cerebellar Purkinje cells (PCs). ShcB-knockout (KO) mice showed defects in cerebellar-dependent motor function and long-term depression (LTD) at cerebellar synapse. The reduced LTD was accompanied with an impairment of intracellular Ca2+ release. Although the expression of Ca2+ release channels and morphology of Ca2+ store looked intact, content of intracellular Ca2+ store and activity of sarco/endoplasmic reticular Ca2+-ATPase (SERCA) were largely decreased in the ShcB-deficient cerebellum. Furthermore, when ShcB was ectopically expressed in the ShcB-KO PCs, the Ca2+ release and its SERCA-dependent component were restored. These data indicate that ShcB plays a key role in the functional maintenance of ER Ca2+ store in central neurons through regulation of SERCA activity.

High nitrogen solubility in stishovite (SiO2) under lower mantle conditions.

Nitrogen is a crucial volatile element in the early Earth's evolution and the origin of life. Despite its importance, nitrogen's behavior in the Earth's interior remains poorly understood. Compared to other volatile elements, nitrogen is depleted in the Earth's atmosphere (the so-called "missing nitrogen"), calling for a hidden deep reservoir. To investigate nitrogen's behavior in the deep Earth including how the reservoir formed, high-pressure and high-temperature experiments were conducted at 28 GPa and 1,400-1,700 °C. To reproduce the conditions in the lower mantle, the redox was controlled using a Fe-FeO buffer. We observed that depending on the temperature conditions, stishovite can incorporate up to 90-404 ppm nitrogen, experimentally demonstrating that stishovite has the highest nitrogen solubility among the deep mantle minerals. Stishovite is the main mineral component of subducted nitrogen-rich sedimentary rocks and eroded continental crust that are eventually transported down to the lower mantle. Our results suggest that nitrogen could have been continuously transported into the lower mantle via subduction, ever since plate tectonics began.

DGKγ Knock-Out Mice Show Impairments in Cerebellar Motor Coordination, LTD, and the Dendritic Development of Purkinje Cells through the Activation of PKCγ.

Diacylglycerol kinase γ (DGKγ) regulates protein kinase C (PKC) activity by converting DG to phosphatidic acid (PA). DGKγ directly interacts with PKCγ and is phosphorylated by PKCγ, resulting in the upregulation of lipid kinase activity. PKC dysfunction impairs motor coordination, indicating that the regulation of PKC activity is important for motor coordination. DGKγ and PKC are abundantly expressed in cerebellar Purkinje cells. However, the physiological role of DGKγ has not been elucidated. Therefore, we developed DGKγ knock-out (KO) mice and tested their cerebellar motor coordination. In DGKγ KO mice, cerebellar motor coordination and long-term depression (LTD) were impaired, and the dendrites of Purkinje cells from DGKγ KO mice were significantly retracted. Interestingly, treatment with the cPKC inhibitor Gö6976 (Gö) rescued the dendritic retraction of primary cultured Purkinje cells from DGKγ KO mice. In contrast, treatment with the PKC activator 12-o-tetradecanoylphorbol 13-acetate (TPA) reduced morphologic alterations in the dendrites of Purkinje cells from wild-type (WT) mice. In addition, we confirmed the upregulation of PKCγ activity in the cerebellum of DGKγ KO mice and rescued impaired LTD in DGKγ KO mice with a PKCγ-specific inhibitor. Furthermore, impairment of motor coordination observed in DGKγ KO mice was rescued in tm1c mice with DGKγ reexpression induced by the FLP-flippase recognition target (FRT) recombination system. These results indicate that DGKγ is involved in cerebellar LTD and the dendritic development of Purkinje cells through the regulation of PKCγ activity, and thus contributes to cerebellar motor coordination.

TRPM7 channels mediate spontaneous Ca2+ fluctuations in growth plate chondrocytes that promote bone development.

During endochondral ossification of long bones, the proliferation and differentiation of chondrocytes cause them to be arranged into layered structures constituting the epiphyseal growth plate, where they secrete the cartilage matrix that is subsequently converted into trabecular bone. Ca2+ signaling has been implicated in chondrogenesis in vitro. Through fluorometric imaging of bone slices from embryonic mice, we demonstrated that live growth plate chondrocytes generated small, cell-autonomous Ca2+ fluctuations that were associated with weak and intermittent Ca2+ influx. Several genes encoding Ca2+-permeable channels were expressed in growth plate chondrocytes, but only pharmacological inhibitors of transient receptor potential cation channel subfamily M member 7 (TRPM7) reduced the spontaneous Ca2+ fluctuations. The TRPM7-mediated Ca2+ influx was likely activated downstream of basal phospholipase C activity and was potentiated upon cell hyperpolarization induced by big-conductance Ca2+-dependent K+ channels. Bones from embryos in which Trpm7 was conditionally knocked out during ex vivo culture exhibited reduced outgrowth and displayed histological abnormalities accompanied by insufficient autophosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) in the growth plate. The link between TRPM7-mediated Ca2+ fluctuations and CaMKII-dependent chondrogenesis was further supported by experiments with chondrocyte-specific Trpm7 knockout mice. Thus, growth plate chondrocytes generate spontaneous, TRPM7-mediated Ca2+ fluctuations that promote self-maturation and bone development.

[Physiological roles of redox signals in relation to synaptic plasticity and brain functions].

In our classical knowledge, redox molecules, including reactive oxygen species (ROS), nitric oxide (NO) and hydrogen sulfide, are considered to be generated as byproducts of aerobic metabolism and act as harmful oxidants of macromolecules, such as proteins and lipids. On the other hands, recently, expressions of enzymes producing redox molecules are identified and reported to be expressed in wide range of tissues, including brain. Moreover, activities of some of these enzymes are revealed to be regulated by physiological signals (e.g. calcium). These observations suggest that redox molecules act as physiological messengers and have biological functions. Actually, recent studies indicate possible involvement of redox signals in functional modification of proteins essential for synaptic plasticity in cultured cells and acute slice preparations. For example, S-nitrosylation of type 1 ryanodine receptor, an intracellular calcium-release channel, is revealed to be essential for NO-induced calcium release (NICR) and synaptic plasticity in cerebellar Purkinje cells. Further studies on mutant animals deficient in redox-modification site may clarify essential role of redox signals in brain functions in vivo.

[Functional roles of phosphotyrosine adaptor Shc in the brain].

Adaptor molecules (adaptor proteins) have indispensable roles in cellular signaling, essential for cellular proliferation, development and metabolism. Shc (Src homology and collagen homology)-family molecule is a group of adaptor molecules, and indicated to be involved in intracellular phosphotyrosine signaling. Shc family has 4 subtypes, ShcA-ShcD, and there are long and short isoforms in ShcA and ShcC whereas ShcB and ShcD have short isoform only. There are three domains conserved in all Shc-family isoforms: phosphotyrosine-binding (PTB) domain, collagen-homology 1 (CH1) domain and Src-homology 2 (SH2) domain, from the N-terminal to C-terminal. PTB and SH2 domains recognize and bind to phosphotyrosine in other molecules, and CH1 domain is recognized and bind to SH2 domain in Grb2, an adaptor molecule, when the tyrosine residues in the domain are phosphorylated. Expression of ShcA is observed in all tissues except for brain in adult animals, although ShcA mRNA is detected in brain during embryonic days. On the other hand, in adult brain, expressions of ShcB, ShcC, and ShcD are observed. Analysis of single knockout mice (ShcA (neuron specific), ShcB, ShcC) and double knockout mice for ShcB and C indicated essential roles of Shc-family molecules in proliferation and survival of cells in various brain regions as well as synaptic plasticity and higher brain functions such as learning and memory. Studies on multiple-knockout mice of Shc-family molecules may further clarify possible involvements of Shc family in physiological and pathophysiological functions in brain.

High-pressure rotational deformation apparatus to 135 GPa.

A large-strain, torsional deformation apparatus has been developed based on diamond anvil cells at high pressures, up to 135 GPa with a help of hard nano-polycrystalline diamond anvils. These pressure conditions correspond to the base of the Earth's mantle. An X-ray laminography technique is introduced for high-pressure in situ 3D observations of the strain markers. The technique developed in this study introduces the possibility of the in situ rheological measurements of the deep Earth materials under ultrahigh-pressure conditions.

Essential Roles of Natural Products and Gaseous Mediators on Neuronal Cell Death or Survival.

Although precise cellular and molecular mechanisms underlying neurodegeneration still remain enigmatic, key factors associated with degenerative disorders, such as glutamate toxicity and oxidative stress, have been recently identified. Accordingly, there has been growing interest in examining the effects of exogenous and endogenous molecules on neuroprotection and neurodegeneration. In this paper, we review recent studies on neuroprotective and/or neurodegenerative effects of natural products, such as caffeic acid and chlorogenic acid, and gaseous mediators, including hydrogen sulfide and nitric oxide. Furthermore, possible molecular mechanisms of these molecules in relation to glutamate signals are discussed. Insight into the pathophysiological role of these molecules will make progress in our understanding of molecular mechanisms underlying neurodegenerative diseases, and is expected to lead to potential therapeutic approaches.

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.

Involvement of the neuronal phosphotyrosine signal adaptor N-Shc in kainic acid-induced epileptiform activity.

BDNF-TrkB signaling is implicated in experimental seizures and epilepsy. However, the downstream signaling involved in the epileptiform activity caused by TrkB receptor activation is still unknown. The aim of the present study was to determine whether TrkB-mediated N-Shc signal transduction was involved in kainic acid (KA)-induced epileptiform activity. We investigated KA-induced behavioral seizures, epileptiform activities and neuronal cell loss in hippocampus between N-Shc deficient and control mice. There was a significant reduction in seizure severity and the frequency of epileptiform discharges in N-Shc deficient mice, as compared with wild-type and C57BL/6 mice. KA-induced neuronal cell loss in the CA3 of hippocampus was also inhibited in N-Shc deficient mice. This study demonstrates that the activation of N-Shc signaling pathway contributes to an acute KA-induced epileptiform activity and neuronal cell loss in the hippocampus. We propose that the N-Shc-mediated signaling pathway could provide a potential target for the novel therapeutic approaches of epilepsy.

Mice lacking the intracellular cation channel TRIC-B have compromised collagen production and impaired bone mineralization.

The trimeric intracellular cation (TRIC) channels TRIC-A and TRIC-B localize predominantly to the endoplasmic reticulum (ER) and likely support Ca(2+) release from intracellular stores by mediating cationic flux to maintain electrical neutrality. Deletion and point mutations in TRIC-B occur in families with autosomal recessive osteogenesis imperfecta. Tric-b knockout mice develop neonatal respiratory failure and exhibit poor bone ossification. We investigated the cellular defect causing the bone phenotype. Bone histology indicated collagen matrix deposition was reduced in Tric-b knockout mice. Osteoblasts, the bone-depositing cells, from Tric-b knockout mice exhibited reduced Ca(2+) release from ER and increased ER Ca(2+) content, which was associated with ER swelling. These cells also had impaired collagen release without a decrease in collagen-encoding transcripts, consistent with a defect in trafficking of collagen through ER. In contrast, osteoclasts, the bone-degrading cells, from Tric-b knockout mice were similar to those from wild-type mice. Thus, TRIC-B function is essential to support the production and release of large amounts of collagen by osteoblasts, which is necessary for bone mineralization.