A highly photostable and bright green fluorescent protein.

The low photostability of fluorescent proteins is a limiting factor in many applications of fluorescence microscopy. Here we present StayGold, a green fluorescent protein (GFP) derived from the jellyfish Cytaeis uchidae. StayGold is over one order of magnitude more photostable than any currently available fluorescent protein and has a cellular brightness similar to mNeonGreen. We used StayGold to image the dynamics of the endoplasmic reticulum (ER) with high spatiotemporal resolution over several minutes using structured illumination microscopy (SIM) and observed substantially less photobleaching than with a GFP variant optimized for stability in the ER. Using StayGold fusions and SIM, we also imaged the dynamics of mitochondrial fusion and fission and mapped the viral spike proteins in fixed cells infected with severe acute respiratory syndrome coronavirus 2. As StayGold is a dimer, we created a tandem dimer version that allowed us to observe the dynamics of microtubules and the excitatory post-synaptic density in neurons. StayGold will substantially reduce the limitations imposed by photobleaching, especially in live cell or volumetric imaging.

A hypothalamic novelty signal modulates hippocampal memory.

The ability to recognize information that is incongruous with previous experience is critical for survival. Novelty signals have therefore evolved in the mammalian brain to enhance attention, perception and memory1,2. Although the importance of regions such as the ventral tegmental area3,4 and locus coeruleus5 in broadly signalling novelty is well-established, these diffuse monoaminergic transmitters have yet to be shown to convey specific information on the type of stimuli that drive them. Whether distinct types of novelty, such as contextual and social novelty, are differently processed and routed in the brain is unknown. Here we identify the supramammillary nucleus (SuM) as a novelty hub in the hypothalamus6. The SuM region is unique in that it not only responds broadly to novel stimuli, but also segregates and selectively routes different types of information to discrete cortical targets-the dentate gyrus and CA2 fields of the hippocampus-for the modulation of mnemonic processing. Using a new transgenic mouse line, SuM-Cre, we found that SuM neurons that project to the dentate gyrus are activated by contextual novelty, whereas the SuM-CA2 circuit is preferentially activated by novel social encounters. Circuit-based manipulation showed that divergent novelty channelling in these projections modifies hippocampal contextual or social memory. This content-specific routing of novelty signals represents a previously unknown mechanism that enables the hypothalamus to flexibly modulate select components of cognition.

A discrete neuronal circuit induces a hibernation-like state in rodents.

Hibernating mammals actively lower their body temperature to reduce energy expenditure when facing food scarcity1. This ability to induce a hypometabolic state has evoked great interest owing to its potential medical benefits2,3. Here we show that a hypothalamic neuronal circuit in rodents induces a long-lasting hypothermic and hypometabolic state similar to hibernation. In this state, although body temperature and levels of oxygen consumption are kept very low, the ability to regulate metabolism still remains functional, as in hibernation4. There was no obvious damage to tissues and organs or abnormalities in behaviour after recovery from this state. Our findings could enable the development of a method to induce a hibernation-like state, which would have potential applications in non-hibernating mammalian species including humans.

Visualizing and Modulating Mitophagy for Therapeutic Studies of Neurodegeneration.

Dysfunctional mitochondria accumulate in many human diseases. Accordingly, mitophagy, which removes these mitochondria through lysosomal degradation, is attracting broad attention. Due to uncertainties in the operational principles of conventional mitophagy probes, however, the specificity and quantitativeness of their readouts are disputable. Thorough investigation of the behaviors and fates of fluorescent proteins inside and outside lysosomes enabled us to develop an indicator for mitophagy, mito-SRAI. Through strict control of its mitochondrial targeting, we were able to monitor mitophagy in fixed biological samples more reproducibly than before. Large-scale image-based high-throughput screening led to the discovery of a hit compound that induces selective mitophagy of damaged mitochondria. In a mouse model of Parkinsons disease, we found that dopaminergic neurons selectively failed to execute mitophagy that promoted their survival within lesions. These results show that mito-SRAI is an essential tool for quantitative studies of mitochondrial quality control.

A spherical aberration-free microscopy system for live brain imaging.

The high-resolution in vivo imaging of mouse brain for quantitative analysis of fine structures, such as dendritic spines, requires objectives with high numerical apertures (NAs) and long working distances (WDs). However, this imaging approach is often hampered by spherical aberration (SA) that results from the mismatch of refractive indices in the optical path and becomes more severe with increasing depth of target from the brain surface. Whereas a revolving objective correction collar has been designed to compensate SA, its adjustment requires manual operation and is inevitably accompanied by considerable focal shift, making it difficult to acquire the best image of a given fluorescent object. To solve the problems, we have created an objective-attached device and formulated a fast iterative algorithm for the realization of an automatic SA compensation system. The device coordinates the collar rotation and the Z-position of an objective, enabling correction collar adjustment while stably focusing on a target. The algorithm provides the best adjustment on the basis of the calculated contrast of acquired images. Together, they enable the system to compensate SA at a given depth. As proof of concept, we applied the SA compensation system to in vivo two-photon imaging with a 25 × water-immersion objective (NA, 1.05; WD, 2 mm). It effectively reduced SA regardless of location, allowing quantitative and reproducible analysis of fine structures of YFP-labeled neurons in the mouse cerebral cortical layers. Interestingly, although the cortical structure was optically heterogeneous along the z-axis, the refractive index of each layer could be assessed on the basis of the compensation degree. It was also possible to make fully corrected three-dimensional reconstructions of YFP-labeled neurons in live brain samples. Our SA compensation system, called Deep-C, is expected to bring out the best in all correction-collar-equipped objectives for imaging deep regions of heterogeneous tissues.

Single-cell bioluminescence imaging of deep tissue in freely moving animals.

Bioluminescence is a natural light source based on luciferase catalysis of its substrate luciferin. We performed directed evolution on firefly luciferase using a red-shifted and highly deliverable luciferin analog to establish AkaBLI, an all-engineered bioluminescence in vivo imaging system. AkaBLI produced emissions in vivo that were brighter by a factor of 100 to 1000 than conventional systems, allowing noninvasive visualization of single cells deep inside freely moving animals. Single tumorigenic cells trapped in the mouse lung vasculature could be visualized. In the mouse brain, genetic labeling with neural activity sensors allowed tracking of small clusters of hippocampal neurons activated by novel environments. In a marmoset, we recorded video-rate bioluminescence from neurons in the striatum, a deep brain area, for more than 1 year. AkaBLI is therefore a bioengineered light source to spur unprecedented scientific, medical, and industrial applications.

HMGB1, a pathogenic molecule that induces neurite degeneration via TLR4-MARCKS, is a potential therapeutic target for Alzheimer's disease.

Alzheimer's disease (AD) is the most common neurodegenerative disease, but it remains an intractable condition. Its pathogenesis is predominantly attributed to the aggregation and transmission of two molecules, Aß and tau; however, other pathological mechanisms are possible. Here, we reveal that phosphorylation of MARCKS, a submembrane protein that regulates the stability of the actin network, occurs at Ser46 prior to aggregation of Aß and is sustained throughout the course of AD in human and mouse brains. Furthermore, HMGB1 released from necrotic or hyperexcitatory neurons binds to TLR4, triggers the specific phosphorylation of MARCKS via MAP kinases, and induces neurite degeneration, the classical hallmark of AD pathology. Subcutaneous injection of a newly developed monoclonal antibody against HMGB1 strongly inhibits neurite degeneration even in the presence of Aß plaques and completely recovers cognitive impairment in a mouse model. HMGB1 and Aß mutually affect polymerization of the other molecule, and the therapeutic effects of the anti-HMGB1 monoclonal antibody are mediated by Aß-dependent and Aß-independent mechanisms. We propose that HMGB1 is a critical pathogenic molecule promoting AD pathology in parallel with Aß and tau and a new key molecular target of preclinical antibody therapy to delay the onset of AD.

ScaleS: an optical clearing palette for biological imaging.

Optical clearing methods facilitate deep biological imaging by mitigating light scattering in situ. Multi-scale high-resolution imaging requires preservation of tissue integrity for accurate signal reconstruction. However, existing clearing reagents contain chemical components that could compromise tissue structure, preventing reproducible anatomical and fluorescence signal stability. We developed ScaleS, a sorbitol-based optical clearing method that provides stable tissue preservation for immunochemical labeling and three-dimensional (3D) signal rendering. ScaleS permitted optical reconstructions of aged and diseased brain in Alzheimer's disease models, including mapping of 3D networks of amyloid plaques, neurons and microglia, and multi-scale tracking of single plaques by successive fluorescence and electron microscopy. Human clinical samples from Alzheimer's disease patients analyzed via reversible optical re-sectioning illuminated plaque pathogenesis in the z axis. Comparative benchmarking of contemporary clearing agents showed superior signal and structure preservation by ScaleS. These findings suggest that ScaleS is a simple and reproducible method for accurate visualization of biological tissue.

Tool use specific adult neurogenesis and synaptogenesis in rodent (Octodon degus) hippocampus.

We previously demonstrated that degus (Octodon degus), which are a species of small caviomorph rodents, could be trained to use a T-shaped rake as a hand tool to expand accessible spaces. To elucidate the neurobiological underpinnings of this higher brain function, we compared this tool use learning task with a simple spatial (radial maze) memory task and investigated the changes that were induced in the hippocampal neural circuits known to subserve spatial perception and learning. With the exposure to an enriched environment in home cage, adult neurogenesis in the dentate gyrus of the hippocampus was augmented by tool use learning, but not radial maze learning, when compared to control conditions. Furthermore, the proportion of new synapses formed in the CA3 region of the hippocampus, the target area for projections of mossy fiber axons emanating from newborn neurons, was specifically increased by tool use learning. Thus, active tool use behavior by rodents, learned through multiple training sessions, requires the hippocampus to generate more novel neurons and synapses than spatial information processing in radial maze learning.

Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain.

Optical methods for viewing neuronal populations and projections in the intact mammalian brain are needed, but light scattering prevents imaging deep into brain structures. We imaged fixed brain tissue using Scale, an aqueous reagent that renders biological samples optically transparent but completely preserves fluorescent signals in the clarified structures. In Scale-treated mouse brain, neurons labeled with genetically encoded fluorescent proteins were visualized at an unprecedented depth in millimeter-scale networks and at subcellular resolution. The improved depth and scale of imaging permitted comprehensive three-dimensional reconstructions of cortical, callosal and hippocampal projections whose extent was limited only by the working distance of the objective lenses. In the intact neurogenic niche of the dentate gyrus, Scale allowed the quantitation of distances of neural stem cells to blood vessels. Our findings suggest that the Scale method will be useful for light microscopy-based connectomics of cellular networks in brain and other tissues.

Ipomotaosides A-D, resin glycosides from the aerial parts of Ipomoea batatas and their inhibitory activity against COX-1 and COX-2.

Four new resin glycosides, namely, ipomotaosides A-D (1-4), were isolated from the dried aerial parts of Ipomoea batatas. The structures of 1-4 were elucidated by analysis of their spectroscopic data and by chemical derivatization and were tested for their anti-inflammatory activity against cyclooxygenase (COX)-1 and -2.

Russujaponols G-L, illudoid sesquiterpenes, and their neurite outgrowth promoting activity from the fruit body of Russula japonica.

Six new illudoid sesquiterpene, russujaponols G-L (1-6) were isolated from the fruit bodies of Russula japonica. Their structures were established primarily by 2D NMR experiments. Furthermore, russujaponols I-K showed neurite outgrowth promoting activity in the primary cultured rat cortical neurons.

Tracing the silhouette of individual cells in S/G2/M phases with fluorescence.

The APC(Cdh1) E3 ligase is active in the late M and G(1) phases. Geminin is a direct substrate of the APC(Cdh1) complex, and accumulates during the S, G(2), and M phases. By fusing the amino-terminal region of Geminin to fluorescent proteins, we have developed cell cycle markers that accumulate in the S/G(2)/M phases in both the nucleus and the cytoplasm. These markers reveal the morphology of individual cells that have undergone DNA replication, allowing us to monitor cell growth relative to differentiation of various cell types. After electroporating the developing mouse embryos, we highlighted neuroepithelial progenitors in the S/G(2)/M phases, which possessed an elongated morphology with an apical and/or a basal attachment. We also show that nuclear localization of the ubiquitin ligase for Geminin is essential for full performance of the markers.

Oxidative stress-induced ubiquitination of RCAN1 mediated by SCFbeta-TrCP ubiquitin ligase.

A change in the protein level of RCAN1 (DSCR1/MCIP/Adapt78/CSP1) has been implicated in oxidative stress-induced cell death in neurons and in the pathogenesis of Alzheimer's disease. The pathogenic processes in neurodegenerative diseases are closely related to oxidative stress and the ubiquitin proteasome system (UPS). Therefore, we investigated whether oxidative stress induces a change in the protein level of RCAN1 through the UPS. H2O2 induced ubiquitination of RCAN1 at the same concentrations as those causing a decrease in RCAN1 in HEK293T cells. beta-TrCP, the F-box protein component of SCF ubiquitin ligase, interacted with RCAN1 in response to H2O2 stimulation. Although FBW4, another F-box protein, interacted with RCAN1, its interaction was independent of H2O2 stimulation. In vitro ubiquitination assay showed that SCFbeta-TrCP but not SCFFBW4 increased ubiquitination of RCAN1, dependent on H2O2 stimulation. In addition, knockdown of beta-TrCP by siRNA abolished the H2O2-induced decrease in RCAN1 in HEK293T cells. We further examined whether RCAN1 undergoes ubiquitination by H2O2 in primary neurons, similarly to that in HEK293T cells. An H2O2-induced decrease in RCAN1 was exhibited also in hippocampal and cortical neurons. Ubiquitination of RCAN1 was induced by 500 muM H2O2, the concentration at which H2O2 induced a decrease in RCAN1 in primary neurons. These results suggest that H2O2 induces SCF beta-TrCP-mediated ubiquitination of RCAN1, leading to a decrease in the protein level of RCAN1.

Visualizing spatiotemporal dynamics of multicellular cell-cycle progression.

The cell-cycle transition from G1 to S phase has been difficult to visualize. We have harnessed antiphase oscillating proteins that mark cell-cycle transitions in order to develop genetically encoded fluorescent probes for this purpose. These probes effectively label individual G1 phase nuclei red and those in S/G2/M phases green. We were able to generate cultured cells and transgenic mice constitutively expressing the cell-cycle probes, in which every cell nucleus exhibits either red or green fluorescence. We performed time-lapse imaging to explore the spatiotemporal patterns of cell-cycle dynamics during the epithelial-mesenchymal transition of cultured cells, the migration and differentiation of neural progenitors in brain slices, and the development of tumors across blood vessels in live mice. These mice and cell lines will serve as model systems permitting unprecedented spatial and temporal resolution to help us better understand how the cell cycle is coordinated with various biological events.

Involvement of p38alpha in kainate-induced seizure and neuronal cell damage.

We investigated how p38alpha mitogen-activated protein kinase (p38) is related to kainate-induced epilepsy and neuronal damages, by using the mice with a single copy disruption of the p38 alpha gene (p38alpha(+/-)). Mortality rate and seizure score of p38alpha(+/-) mice administered with kainate were significantly reduced compared with the case of wild-type (WT) mice. This was clearly supported by the electroencephalography data in which kainate-induced seizure duration and frequency in the brain of p38alpha(+/-) mice were significantly suppressed compared to those of WT mice. As a consequence of seizure, kainate induced delayed neuronal damages in parallel with astrocytic growth in the hippocampus and ectopic innervation of the mossy fibers into the stratum oriens in the CA3 region of hippocampus in WT mice, whose changes were moderate in p38alpha(+/-) mice. Likewise, kainate-induced phosphorylation of calcium/calmodulin-dependent kinase II in the hippocampus of p38alpha (+/-) mice was significantly decreased compared to that of WT mice. These results suggest that p38alpha signaling pathway plays an important role in epileptic seizure and excitotoxicity.

[Study for an allergic inflammation model using human lungs and its pharmacological application].

The complement system, which plays an important role in inmate immunity, is considered to be important in the pathophysiology of allergic asthma. A patient with allergic asthma shows the reversible characteristic system of bronchoconstriction, increased mucus secretion, and complicated airway inflammation. Various cytokines secreted from Th2 cells contribute to the system. Cysteinyl-leukotrienes (CysLTs) are also considered to be one of the important mediators involved in asthmatic pathophysiology. However, the effects of a drug on humans may not be the same as those on animals due to species differences in complement-related molecules. In this series of experiments, we tried to establish a model in which the effects of a drug on the production of CysLTs from human lung preparations were evaluated following an anaphylactic reaction. CysLT production increased when the passively sensitized lung tissues were stimulated with anti-IgE antibody. The coaddition of anaphylatoxin, C5a, with the anti-IgE antibody potentiated CysLT production. The response to C3a was weaker when compared with that to C5a. In addition, increased production of CysLTs by adding serum at a specific ratio was dose dependently inhibited by nonpeptide C5a receptor antagonist, W-54011, or a novel complementary peptide inhibitor of C5a, acetyl peptide A. From these results, it is suggested that C5a potentiates cysLT production from human lung tissues and contributes to allergic inflammation like asthma, and thus acetylated peptide A and W-54011 are useful for suppressing allergic inflammation in the lungs.

Russujaponols A-F, illudoid sesquiterpenes from the fruiting body of Russula japonica.

Six new illudoid sesquiterpenes, russujaponols A-F (1-6), were isolated from the fruiting bodies of Russula japonica Hongo. Their structures were established primarily by 2D NMR experiments, and the structure of the main compound, russujaponol A (1), was confirmed by X-ray crystallographic analysis of its benzoate (1a). Russujaponol A (1) suppressed invasion of human fibrosarcoma (HT1080) cells into Matrigel in a concentration-dependent manner and caused 63% inhibition at 3.73 microM.