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Structural plasticity of dendritic spines in the nucleus accumbens (NAc) is crucial for learning from aversive experiences. Activation of NMDA receptors (NMDARs) stimulates Ca 2+ -dependent signaling that leads to changes in the actin cytoskeleton, mediated by the Rho family of GTPases, resulting in postsynaptic remodeling essential for learning. We investigated how phosphorylation events downstream of NMDAR activation drive the changes in synaptic morphology that underlie aversive learning. Large-scale phosphoproteomic analyses of protein kinase targets in mouse striatal/accumbal slices revealed that NMDAR activation resulted in the phosphorylation of 194 proteins, including RhoA regulators such as ARHGEF2 and ARHGAP21. Phosphorylation of ARHGEF2 by the Ca 2+ -dependent protein kinase CaMKII enhanced its RhoGEF activity, thereby activating RhoA and its downstream effector Rho-associated kinase (ROCK/Rho-kinase). Further phosphoproteomic analysis identified 221 ROCK targets, including the postsynaptic scaffolding protein SHANK3, which is crucial for its interaction with NMDARs and other postsynaptic scaffolding proteins. ROCK-mediated phosphorylation of SHANK3 in the NAc was essential for spine growth and aversive learning. These findings demonstrate that NMDAR activation initiates a phosphorylation cascade crucial for learning and memory.

9-cyanopyronin is a promising scaffold that exploits resonance Raman enhancement to enable sensitive, highly multiplexed biological imaging. Here, we developed cyano-Hydrol Green (CN-HG) derivatives as resonance Raman scaffolds to expand the color palette of 9-cyanopyronins. CN-HG derivatives exhibit sufficiently long wavelength absorption to produce strong resonance Raman enhancement for near-infrared (NIR) excitation, and their nitrile peaks are shifted to a lower frequency than those of 9-cyanopyronins. The fluorescence of CN-HG derivatives is strongly quenched due to the lack of the 10th atom, unlike pyronin derivatives, and this enabled us to detect spontaneous Raman spectra with high signal-to-noise ratios. CN-HG derivatives are powerful candidates for high performance vibrational imaging., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Natural products are important sources of seed compounds for drug discovery. However, it has become difficult in recent years to discover new compounds with valuable pharmacological activities. On the other hand, among the vast number of natural products that have been isolated so far, a considerable number of compounds with specific biological activities are thought to be overlooked in screening that uses biological activity as an index. Therefore, it is conceivable that such overlooked useful compounds may be found by screening compound libraries that have been amassed previously through specific assays. Previously, NPD723, a member of the Natural Products Depository library comprised of a mixture of natural and non-natural products developed at RIKEN, and its metabolite H-006 were found to inhibit growth of various cancer cells at low nanomolar half-maximal inhibitory concentration. Subsequent analysis revealed that H-006 strongly inhibited human dihydroorotate dehydrogenase (DHODH), the rate-limiting enzyme in the de novo pyrimidine biosynthetic pathway. Here, we elucidated the crystal structure of the DHODH-flavin mononucleotide-orotic acid-H-006 complex at 1.7 Å resolution to determine that furocoumavirin, the S-enantiomer of H-006, was the actual inhibitor. The overall mode of interaction of furocoumavirin with the inhibitor binding pocket was similar to that described for previously reported tight-binding inhibitors. However, the structural information together with kinetic characterizations of site-specific mutants identified key unique features that are considered to contribute to the sub-nanomolar inhibition of DHODH by furocoumavirin. Our finding identified new chemical features that could improve the design of human DHODH inhibitors.

Body movements influence brain-wide neuronal activities. In the sensory cortex, thalamocortical bottom-up inputs and motor-sensory top-down inputs are thought to affect the dynamics of membrane potentials ( V m ) of neurons and change their processing of sensory information during movements. However, direct perturbation of the axons projecting to the sensory cortex from other remote areas during movements has remained unassessed, and therefore the interareal circuits generating motor-related signals in sensory cortices remain unclear. Using a G i/o -coupled opsin, eOPN3, we here inhibited interareal signals incoming to the whisker primary somatosensory barrel cortex (wS1) of awake male mice and tested their effects on whisking-related changes in neuronal activities in wS1. Spontaneous whisking in air induced the changes in spike rates of a subset of wS1 neurons, which were accompanied by depolarization and substantial reduction of slow-wave oscillatory fluctuations of V m Despite an extensive innervation, inhibition of inputs from the whisker primary motor cortex (wM1) to wS1 did not alter the spike rates and V m dynamics of wS1 neurons during whisking. In contrast, inhibition of axons from the whisker-related thalamus (wTLM) and the whisker secondary somatosensory cortex (wS2) to wS1 largely attenuated the whisking-related supra- and sub-threshold V m dynamics of wS1 neurons. Notably, silencing inputs from wTLM markedly decreased the modulation depth of whisking phase-tuned neurons in wS1, while inhibiting wS2 inputs did not impact the whisking variable tuning of wS1 neurons. Thus, sensorimotor integration in wS1 during spontaneous whisking is predominantly facilitated by direct synaptic inputs from wTLM and wS2 rather than from wM1., (Copyright © 2024 Kawatani et al.)

Carboxypeptidases (CPs) are a family of hydrolases that cleave one or more amino acids from the C-terminal of peptides or proteins and play indispensable roles in various physiological and pathological processes. However, only a few highly activatable fluorescence probes for CPs have been reported, and there is a need for a flexibly tunable molecular design platform to afford a range of fluorescence probes for CPs for biological and medical research. Here, we focused on the unique activation mechanism of ProTide-based prodrugs and established a modular design platform for CP-targeting florescence probes based on ProTide chemistry. In this design, probe properties such as fluorescence emission wavelength, reactivity/stability, and target CP can be readily tuned and optimized by changing the four probe modules: the fluorophore, the substituent on the phosphorus atom, the linker amino acid at the P1 position, and the substrate amino acid at the P1' position. In particular, switching the linker amino acid at position P1 enabled us to precisely optimize the reactivity for target CPs. As a proof-of-concept, we constructed probes for carboxypeptidase M (CPM) and prostate-specific membrane antigen (also known as glutamate carboxypeptidase II). The developed probes were applicable for the imaging of CP activities in live cells and in clinical specimens from patients. This design strategy should be useful in studying CP-related biological and pathological phenomena.

Measuring the membrane potential dynamics of neurons offers a comprehensive understanding of the molecular and cellular mechanisms that form their spiking activity, thus playing a crucial role in unraveling the mechanistic processes governing brain function. Techniques for intracellular recordings of membrane potentials pioneered in the 1940s have witnessed significant advancements since their inception. Among these, whole-cell patch-clamp recording has emerged as a leading method for measuring neuronal membrane potentials due to its high stability and broad applicability ranging from cultured cells to brain slices and even behaving animals. This chapter provides a detailed protocol to acquire stable whole-cell recordings from neurons in the cerebral cortex of awake, head-restrained mice. Significant enhancements to our protocol include implanting a metal head-post using adhesive resin cement and preparing a recording pipette with a long shank for targeting deeper brain regions. This protocol, once implemented, enables whole-cell recordings up to 2.5 mM beneath the cortical surface., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Small-molecule based activatable fluorescence probes for detecting specific enzyme activity with high sensitivity can visualize the expression site of marker genes and cancers where the enzyme is highly expressed. However, the enzyme-catalyzed fluorescent hydrolysis product easily leaks out and diffuses from the reaction site, making it difficult to perform long-term tracking and immunohistochemical analysis which needs washing/fixation procedure. Our group have focused on quinone methide chemistry and developed series of activatable fluorescence probes with excellent intracellular retention that are converted to quinone-methide or aza-quinone-methide intermediates upon reaction with enzymes, which are then react with intracellular nucleophiles such as proteins and glutathione to be retained in cells and to exhibit significant increase in fluorescence. Based on this molecular design, we have developed fluorescence probes targeting β-galactosidase and γ-glutamyltranspeptidase with different colors. We also developed photo-functional probes such as activatable photosensitizers and caged fluorophores. These probes can visualize or kill target enzyme-expressing cells with high selectivity by suppressing the leakage of hydrolysis products from target cells, and fluorescence imaging in combination with immunostaining was possible due to the high tolerance of the obtained fluorescence signal even after washing and fixation.