Micro-magnetic resonance imaging of ex vivo mouse embryos with potato starch suspension.

Potato starch suspension (PSS) holds promise as a solution to issues, such as air bubbles and specimen motion, associated with micro-magnetic resonance imaging (micro-MRI) of ex vivo embryos. Here, we present a protocol for using PSS when scanning specimens with micro-MRI. We describe steps for preparing samples and potato starch with phosphate-buffered saline. We then detail steps for specimen immersion and micro-MRI scanning. This protocol will enable micro-MRI of not only embryos but also other specimens, such as insects. For complete details on the use and execution of this protocol, please refer to Tsurugizawa et al.1.

Spontaneous and multifaceted ATP release from astrocytes at the scale of hundreds of synapses.

Astrocytes participate in information processing by releasing neuroactive substances termed gliotransmitters, including ATP. Individual astrocytes come into contact with thousands of synapses with their ramified structure, but the spatiotemporal dynamics of ATP gliotransmission remains unclear, especially in physiological brain tissue. Using a genetically encoded fluorescent sensor, GRABATP1.0 , we discovered that extracellular ATP increased locally and transiently in absence of stimuli in neuron-glia co-cultures, cortical slices, and the anesthetized mouse brain. Spontaneous ATP release events were tetrodotoxin-insensitive but suppressed by gliotoxin, fluorocitrate, and typically spread over 50-250 µm2 area at concentrations capable of activating purinergic receptors. Besides, most ATP events did not coincide with Ca2+ transients, and intracellular Ca2+ buffering with BAPTA-AM did not affect ATP event frequency. Clustering analysis revealed that these events followed multiple distinct kinetics, and blockade of exocytosis only decreased a minor group of slow events. Overall, astrocytes spontaneously release ATP through multiple mechanisms, mainly in non-vesicular and Ca2+ -independent manners, thus potentially regulating hundreds of synapses all together.

Utilization of potato starch suspension for MR-microimaging in ex vivo mouse embryos.

Magnetic resonance (MR) microimaging of the mouse embryo is a promising tool to noninvasively investigate the microstructure of the brain of a developing mouse. The proton-free fluid is used for the liquid surrounding the specimen in MR microimaging, but the potential issue of image quality remains due to the air bubbles on the specimen and the retained water proton in the curvature of the embryo. Furthermore, the specimen may move during the scanning, resulting in motion artifact. Here, we developed the new concept of the ex vivo microimaging protocol with the robust method using the potato starch-containing biological polymers. Potato starch suspension with PBS significantly reduced T1 and T2 signal intensity of the suspension and strongly suppressed the motion of the embryo. Furthermore, potato starch-PBS suspension is stable for long-time scanning at room temperature. These results indicate the utility of potato starch suspension for MR microimaging in mouse embryos.

Visualizing Cortical Development and Evolution: A Toolkit Update.

Visualizing the process of neural circuit formation during neurogenesis, using genetically modified animals or somatic transgenesis of exogenous plasmids, has become a key to decipher cortical development and evolution. In contrast to the establishment of transgenic animals, the designing and preparation of genes of interest into plasmids are simple and easy, dispensing with time-consuming germline modifications. These advantages have led to neuron labeling based on somatic transgenesis. In particular, mammalian expression plasmid, CRISPR-Cas9, and DNA transposon systems, have become widely used for neuronal visualization and functional analysis related to lineage labeling during cortical development. In this review, we discuss the advantages and limitations of these recently developed techniques.

Potential of Multiscale Astrocyte Imaging for Revealing Mechanisms Underlying Neurodevelopmental Disorders.

Astrocytes provide trophic and metabolic support to neurons and modulate circuit formation during development. In addition, astrocytes help maintain neuronal homeostasis through neurovascular coupling, blood-brain barrier maintenance, clearance of metabolites and nonfunctional proteins via the glymphatic system, extracellular potassium buffering, and regulation of synaptic activity. Thus, astrocyte dysfunction may contribute to a myriad of neurological disorders. Indeed, astrocyte dysfunction during development has been implicated in Rett disease, Alexander's disease, epilepsy, and autism, among other disorders. Numerous disease model mice have been established to investigate these diseases, but important preclinical findings on etiology and pathophysiology have not translated into clinical interventions. A multidisciplinary approach is required to elucidate the mechanism of these diseases because astrocyte dysfunction can result in altered neuronal connectivity, morphology, and activity. Recent progress in neuroimaging techniques has enabled noninvasive investigations of brain structure and function at multiple spatiotemporal scales, and these technologies are expected to facilitate the translation of preclinical findings to clinical studies and ultimately to clinical trials. Here, we review recent progress on astrocyte contributions to neurodevelopmental and neuropsychiatric disorders revealed using novel imaging techniques, from microscopy scale to mesoscopic scale.

De novo ATP1A3 variants cause polymicrogyria.

Polymicrogyria is a common malformation of cortical development whose etiology remains elusive. We conducted whole-exome sequencing for 124 patients with polymicrogyria and identified de novo ATP1A3 variants in eight patients. Mutated ATP1A3 causes functional brain diseases, including alternating hemiplegia of childhood (AHC), rapid-onset dystonia parkinsonism (RDP), and cerebellar ataxia, areflexia, pes cavus, optic nerve atrophy, and sensorineural deafness (CAPOS). However, our patients showed no clinical features of AHC, RDP, or CAPOS and had a completely different phenotype: a severe form of polymicrogyria with epilepsy and developmental delay. Detected variants had different locations in ATP1A3 and different functional properties compared with AHC-, RDP-, or CAPOS-associated variants. In the developing cerebral cortex of mice, radial neuronal migration was impaired in neurons overexpressing the ATP1A3 variant of the most severe patients, suggesting that this variant is involved in cortical malformation pathogenesis. We propose a previously unidentified category of polymicrogyria associated with ATP1A3 abnormalities.

Direct Readout of Neural Stem Cell Transgenesis with an Integration-Coupled Gene Expression Switch.

Stable genomic integration of exogenous transgenes is essential in neurodevelopmental and stem cell studies. Despite tools driving increasingly efficient genomic insertion with DNA vectors, transgenesis remains fundamentally hindered by the impossibility of distinguishing integrated from episomal transgenes. Here, we introduce an integration-coupled On genetic switch, iOn, which triggers gene expression upon incorporation into the host genome through transposition, thus enabling rapid and accurate identification of integration events following transfection with naked plasmids. In vitro, iOn permits rapid drug-free stable transgenesis of mouse and human pluripotent stem cells with multiple vectors. In vivo, we demonstrate faithful cell lineage tracing, assessment of regulatory elements, and mosaic analysis of gene function in somatic transgenesis experiments that reveal neural progenitor potentialities and interaction. These results establish iOn as a universally applicable strategy to accelerate and simplify genetic engineering in cultured systems and model organisms by conditioning transgene activation to genomic integration.

Dual-color deep-tissue three-photon microscopy with a multiband infrared laser.

Multiphoton microscopy combined with genetically encoded fluorescent indicators is a central tool in biology. Three-photon (3P) microscopy with excitation in the short-wavelength infrared (SWIR) water transparency bands at 1.3 and 1.7 µm opens up new opportunities for deep-tissue imaging. However, novel strategies are needed to enable in-depth multicolor fluorescence imaging and fully develop such an imaging approach. Here, we report on a novel multiband SWIR source that simultaneously emits ultrashort pulses at 1.3 and 1.7 µm that has characteristics optimized for 3P microscopy: sub-70 fs duration, 1.25 MHz repetition rate, and µJ-range pulse energy. In turn, we achieve simultaneous 3P excitation of green fluorescent protein (GFP) and red fluorescent proteins (mRFP, mCherry, tdTomato) along with third-harmonic generation. We demonstrate in-depth dual-color 3P imaging in a fixed mouse brain, chick embryo spinal cord, and live adult zebrafish brain, with an improved signal-to-background ratio compared to multicolor two-photon imaging. This development opens the way towards multiparametric imaging deep within scattering tissues.

A Sensitive and Versatile In Situ Hybridization Protocol for Gene Expression Analysis in Developing Amniote Brains.

The detection of specific RNA molecules in embryonic tissues has wide research applications including studying gene expression dynamics in brain development and evolution. Recent advances in sequencing technologies have introduced new animal models to explore the molecular principles underlying the assembly and diversification of brain circuits between different amniote species. Here, we provide a step-by-step protocol for a versatile in situ hybridization method that is immediately applicable to a range of amniote embryos including zebra finch and Madagascar ground gecko, two new model organisms that have rapidly emerged for comparative brain studies over recent years. The sensitive detection of transcripts from low to high abundance expression range using the same platform enables direct comparison of gene of interest among different amniotes, providing high-resolution spatiotemporal information of gene expression to dissect the molecular principles underlying brain evolution.

Evolutionary conservation and conversion of Foxg1 function in brain development.

Among the forkhead box protein family, Foxg1 is a unique transcription factor that plays pleiotropic and non-redundant roles in vertebrate brain development. The emergence of the telencephalon at the rostral end of the neural tube and its subsequent expansion that is mediated by Foxg1 was a key reason for the vertebrate brain to acquire higher order information processing, where Foxg1 is repetitively used in the sequential events of telencephalic development to control multi-steps of brain circuit formation ranging from cell cycle control to neuronal differentiation in a clade- and species-specific manner. The objective of this review is to discuss how the evolutionary changes in cis- and trans-regulatory network that is mediated by a single transcription factor has contributed to determining the fundamental vertebrate brain structure and its divergent roles in instructing species-specific neuronal circuitry and functional specialization.

Anti-inflammatory activity and molecular mechanism of delphinidin 3-sambubioside, a Hibiscus anthocyanin.

Delphinidin 3-sambubioside (Dp3-Sam), a Hibiscus anthocyanin, was isolated from the dried calices of Hibiscus sabdariffa L, which has been used for folk beverages and herbal medicine although the molecular mechanisms are poorly defined. Based on the properties of Dp3-Sam and the information of inflammatory processes, we investigated the anti-inflammatory activity and molecular mechanisms in both cell and animal models in the present study. In the cell model, Dp3-Sam and Delphinidin (Dp) reduced the levels of inflammatory mediators including iNOS, NO, IL-6, MCP-1, and TNF-α induced by LPS. Cellular signaling analysis revealed that Dp3-Sam and Dp downregulated NF-κB pathway and MEK1/2-ERK1/2 signaling. In animal model, Dp3-Sam and Dp reduced the production of IL-6, MCP-1 and TNF-α and attenuated mouse paw edema induced by LPS. Our in vitro and in vivo data demonstrated that Hibiscus Dp3-Sam possessed potential anti-inflammatory properties.

Comparison of the inhibitory effects of delphinidin and its glycosides on cell transformation.

Although anthocyanins are major forms distributed in many plant foods and promising as chemopreventive source, many molecular data are obtained from anthocyanidins, showing their low bioavailability. This study aims to clarify the inhibitory effects of delphinidin glycosides on cell transformation comparing them to those of delphinidin. Screening data revealed that delphinidin 3-sambubioside could directly bind to MAPK/ERK kinase 1. Affinity assay data confirmed that delphinidin 3-sambubioside had higher binding affinity to MAPK/ERK kinase 1 than ERK1/2 and B-Raf. Colony assay data further demonstrated that delphinidin 3-sambubioside inhibited 12-O- tetradecanoylphorbol-13-acetate-induced phosphorylation of MAPK/ERK kinase 1 and sequentially suppressed cell transformation. All of these effects caused by delphinidin 3-sambubioside were weaker than those by its aglycon, delphinidin. Our data suggested that the weaker anti- transformation activity of delphinidin glycosides compared to that of their aglycon is due to lower binding affinity to the target molecule MAPK/ERK kinase 1.

The timing of upper-layer neurogenesis is conferred by sequential derepression and negative feedback from deep-layer neurons.

The prevailing view of upper-layer (UL) neurogenesis in the cerebral cortex is that progenitor cells undergo successive rounds of asymmetric cell division that restrict the competence and production of UL neurons later in development. However, the recent discovery of UL fate-committed early progenitors raises an alternative perspective concerning their ontogeny. To investigate the emergence of UL progenitors, we manipulated the timing and extent of cortical neurogenesis in vivo in mice. We demonstrated that UL competence is tightly linked to deep-layer (DL) neurogenesis and that this sequence is determined primarily through derepression of Fezf2 by Foxg1 within a closed transcriptional cascade. We further demonstrated that the sequential acquisition of UL competence requires negative feedback, which is propagated from postmitotic DL neurons. Thus, neocortical progenitors integrate intrinsic and extrinsic cues to generate UL neurons through a system that controls the sequence of DL and UL neurogenesis and to scale the production of intracortical projection neurons based on the availability of their subcortical projection neuron counterparts during cortical development and evolution.

Neuronal subtype specification in establishing mammalian neocortical circuits.

The functional integrity of the neocortical circuit relies on the precise production of diverse neuron populations and their assembly during development. In recent years, extensive progress has been made in the understanding of the mechanisms that control differentiation of each neuronal type within the neocortex. In this review, we address how the elaborate neocortical cytoarchitecture is established from a simple neuroepithelium based on recent studies examining the spatiotemporal mechanisms of neuronal subtype specification. We further discuss the critical events that underlie the conversion of the stem amniotes cerebrum to a mammalian-type neocortex, and extend these key findings in the light of mammalian evolution to understand how the neocortex in humans evolved from common ancestral mammals.

Anti-inflammatory activity and molecular mechanism of Oolong tea theasinensin.

Oolong tea theasinensins are a group of tea polyphenols different from green tea catechins and black tea theaflavins, and they are considered as bioactive compounds in Oolong tea. In the present study, based on the properties of theasinensin and information about inflammatory processes, we investigated the anti-inflammatory activity and molecular mechanisms of theasinensin A (TSA) in both cell and animal models. In the cell model, TSA reduced the levels of pro-inflammatory mediators including inducible nitric oxide synthase (iNOS), nitric oxide (NO), interleukin-12 (IL-12) (p70), tumor necrosis factor alpha (TNF-α), and monocyte chemotactic protein-1 (MCP-1) induced by lipopolysaccharide (LPS). Cellular signaling analysis revealed that TSA downregulated MAPK/ERK kinase (MEK)-extracellular signal-regulated kinase (ERK) signaling. Pull-down assay and affinity data revealed that TSA might directly bind to MEK-ERK for the inhibitory action. In the animal model, TSA suppressed the production of IL-12 (p70), TNF-α, and MCP-1 and attenuated mouse paw edema induced by LPS.

Foxg1 coordinates the switch from nonradially to radially migrating glutamatergic subtypes in the neocortex through spatiotemporal repression.

The specification of neuronal subtypes in the cerebral cortex proceeds in a temporal manner; however, the regulation of the transitions between the sequentially generated subtypes is poorly understood. Here, we report that the forkhead box transcription factor Foxg1 coordinates the production of neocortical projection neurons through the global repression of a default gene program. The delayed activation of Foxg1 was necessary and sufficient to induce deep-layer neurogenesis, followed by a sequential wave of upper-layer neurogenesis. A genome-wide analysis revealed that Foxg1 binds to mammalian-specific noncoding sequences to repress over 12 transcription factors expressed in early progenitors, including Ebf2/3, Dmrt3, Dmrta1, and Eya2. These findings reveal an unexpected prolonged competence of progenitors to initiate corticogenesis at a progressed stage during development and identify Foxg1 as a critical initiator of neocorticogenesis through spatiotemporal repression, a system that balances the production of nonradially and radially migrating glutamatergic subtypes during mammalian cortical expansion.

Modulation of Nrf2/Keap1 system by Wasabi 6-methylthiohexyl isothiocyanate in ARE-mediated NQO1 expression.

SCOPE: 6-Methylthiohexyl isothiocyanate (6-MTITC), one of the major bioactive ingredients in Japanese Wasabi, has revealed cytoprotective and cancer chemopreventive effects. This study aims to clarify the molecular mechanisms how 6-MTITC modulates nuclear factor E2-related factor 2 (Nrf2)/Kelchlike ECH-associating protein 1 (Keap1) system in antioxidant-responsive element (ARE)-mediated nicotinamide adenine dinucleotide phosphate (NADP): quinone oxidoreductase 1 (NQO1) expression. METHODS AND RESULTS: HepG2 cells were treated with 6-MTITC with varying time and dose. NQO1, Nrf2, and Keap1 proteins were detected by Western blotting. ARE transactivation was detected by electrophilic mobility shift assay and reporter gene assay. Nuclear localization of Nrf2 was determined by immunocytochemistry assay. Ubiquitination of Nrf2 and Keap1 was detected using immunoprecipitation after treatment with MG132. Small interfering RNA was used to knockdown Nrf2 or Keap1. The results revealed that 6-MTITC modulated Nrf2/ARE pathway by stimulating Keap1 modification, and inhibiting Nrf2 ubiquitination and protein turnover. These actions finally increased nuclear Nrf2 accumulation and ARE-binding activity. Moreover, silencing Nrf2 markedly reduced ARE-driven activity induced by 6-MTITC. CONCLUSION: 6-MTITC modulated ARE-driven NQO1 expression by stabilizing Nrf2 with enhanced Keap1 modification and decreased Nrf2 degradation.

Flavonoids as protein kinase inhibitors for cancer chemoprevention: direct binding and molecular modeling.

Protein kinases play crucial roles in the regulation of multiple cell signaling pathways and cellular functions. Deregulation of protein kinase function has been implicated in carcinogenesis. The inhibition of protein kinases has emerged as an important target for cancer chemoprevention and therapy. Accumulated data revealed that flavonoids exert chemopreventive effects through acting at protein kinase signaling pathways, more than as conventional hydrogen-donating antioxidants. Recent studies show that flavonoids can bind directly to some protein kinases, including Akt/protein kinase B (Akt/PKB), Fyn, Janus kinase 1 (JAK1), mitogen-activated protein kinase kinase 1 (MEK1), phosphoinositide 3-kinase (PI3K), mitogen-activated protein (MAP) kinase kinase 4 (MKK4), Raf1, and zeta chain-associated 70-kDa protein (ZAP-70) kinase, and then alter their phosphorylation state to regulate multiple cell signaling pathways in carcinogenesis processes. In this review, we report recent results on the interactions of flavonoids and protein kinases, especially their direct binding and molecular modeling. The data suggest that flavonoids act as protein kinase inhibitors for cancer chemoprevention that were thought previously as conventional hydrogen-donating antioxidant. Moreover, the molecular modeling data show some hints for creating natural compound-based protein kinase inhibitors for cancer chemoprevention and therapy.

Akt is a direct target for myricetin to inhibit cell transformation.

Akt, a serine/threonine kinase, is a critical regulator in many cellular processes including cell growth, proliferation, and apoptosis. In this study, we found that myricetin, a typical flavonol existing in many fruits and vegetables, could directly target Akt to inhibit cell transformation. Binding assay revealed that myricetin bound to Akt directly by competing with ATP. In vitro and ex vivo data confirmed that myricetin inhibited the phosphorylation and kinase activity of Akt. Molecular modeling suggested that myricetin easily docks to the ATP-binding site of Akt with hydrogen bonds. Signaling analysis data further demonstrated that myricetin inhibited Akt-mediated activator protein-1 (AP-1) transactivation, cyclin D1 expression and cell transformation. Overall, our results indicate that Akt is a direct target for myricetin to inhibit cell transformation.

Myricetin directly targets JAK1 to inhibit cell transformation.

JAK1/STAT3 pathway has been suggested to play a role in cell transformation and carcinogenesis. In the present study, we found that myricetin (3, 3', 4', 5, 5', 7-hexahydroxyflavone), a typical flavonol existing in many fruits and vegetables, could directly bind to JAK1/STAT3 molecules to inhibit cell transformation in epidermal growth factor (EGF)-activated mouse JB6 P(+) cells. Colony assay revealed that myricetin had the strongest inhibitory effect on cell transformation among three flavonols including myricetin, quercetin and kaempferol. Molecular data revealed that myricetin inhibited DNA- binding and transcriptional activity of STAT3. Furthermore, myricetin inhibited the phosphorylation of STAT3 at Tyr705 and Ser727. Cellular signaling analyses revealed that EGF could induce the phosphorylation of Janus Kinase (JAK) 1, but not JAK2. Myricetin inhibited the phosphorylation of JAK1 and increased the autophosphorylation of EGF receptor (EGFR). Moreover, ex vivo and in vitro pull-down assay revealed that myricetin bound to JAK1 and STAT3, but not EGFR. Affinity data further demonstrated that myricetin had a higher affinity for JAK1 than STAT3. Thus, our data indicate that myricetin might directly target JAK1 to block cell transformation in mouse JB6 cells.