Bioorthogonal chemical labeling of endogenous neurotransmitter receptors in living mouse brains

生きた動物脳内で発現する神経伝達物質受容体に目印を付ける新手法を開発 --遺伝子操作を伴わず、生体内でたんぱく質の機能解析が可能に--. 京都大学プレスリリース. 2024-02-05.Neurotransmitter receptors are essential components of synapses for communication between neurons in the brain. Because the spatiotemporal expression profiles and dynamics of neurotransmitter receptors involved in many functions are delicately governed in the brain, in vivo research tools with high spatiotemporal resolution for receptors in intact brains are highly desirable. Covalent labeling by chemical reaction (chemical labeling) of proteins without genetic manipulation is now a powerful method for analyzing receptors in vitro. However, selective target receptor labeling in the brain has not yet been achieved. This study shows that ligand-directed alkoxyacylimidazole (LDAI) chemistry can be used to selectively tether synthetic probes to target endogenous receptors in living mouse brains. The reactive LDAI reagents with negative charges were found to diffuse well over the whole brain and could selectively label target endogenous receptors, including AMPAR, NMDAR, mGlu1, and GABAAR. This simple and robust labeling protocol was then used for various applications: three-dimensional spatial mapping of endogenous receptors in the brains of healthy and disease-model mice; multi-color receptor imaging; and pulse–chase analysis of the receptor dynamics in postnatal mouse brains. Here, results demonstrated that bioorthogonal receptor modification in living animal brains may provide innovative molecular tools that contribute to the in-depth understanding of complicated brain functions

Versatile whole-organ/body staining and imaging based on electrolyte-gel properties of biological tissues

Whole-organ/body three-dimensional (3D) staining and imaging have been enduring challenges in histology. By dissecting the complex physicochemical environment of the staining system, we developed a highly optimized 3D staining imaging pipeline based on CUBIC. Based on our precise characterization of biological tissues as an electrolyte gel, we experimentally evaluated broad 3D staining conditions by using an artificial tissue-mimicking material. The combination of optimized conditions allows a bottom-up design of a superior 3D staining protocol that can uniformly label whole adult mouse brains, an adult marmoset brain hemisphere, an ~1 cm3 tissue block of a postmortem adult human cerebellum, and an entire infant marmoset body with dozens of antibodies and cell-impermeant nuclear stains. The whole-organ 3D images collected by light-sheet microscopy are used for computational analyses and whole-organ comparison analysis between species. This pipeline, named CUBIC-HistoVIsion, thus offers advanced opportunities for organ- and organism-scale histological analysis of multicellular systems

Activation of Sympathetic Signaling in Macrophages Blocks Systemic Inflammation and Protects against Renal Ischemia-Reperfusion Injury

Background: The sympathetic nervous system regulates immune cell dynamics. However, the detailed role of sympathetic signaling in inflammatory diseases is still unclear because it varies according to the disease situation and responsible cell types. This study focused on identifying the functions of sympathetic signaling in macrophages in LPS-induced sepsis and renal ischemia-reperfusion injury (IRI).Methods: We performed RNA sequencing of mouse macrophage cell lines to identify the critical gene that mediates the anti-inflammatory effect of β2-adrenergic receptor (Adrb2) signaling. We also examined the effects of salbutamol (a selective Adrb2 agonist) in LPS-induced systemic inflammation and renal IRI. Macrophage-specific Adrb2 conditional knockout (cKO) mice and the adoptive transfer of salbutamol-treated macrophages were used to assess the involvement of macrophage Adrb2 signaling.Results: In vitro, activation of Adrb2 signaling in macrophages induced the expression of T cell Ig and mucin domain 3 (Tim3), which contributes to anti-inflammatory phenotypic alterations. In vivo, salbutamol administration blocked LPS-induced systemic inflammation and protected against renal IRI; this protection was mitigated in macrophage-specific Adrb2 cKO mice. The adoptive transfer of salbutamol-treated macrophages also protected against renal IRI. Single-cell RNA sequencing revealed that this protection was associated with the accumulation of Tim3-expressing macrophages in the renal tissue.Conclusions: The activation of Adrb2 signaling in macrophages induces anti-inflammatory phenotypic alterations partially via the induction of Tim3 expression, which blocks LPS-induced systemic inflammation and protects against renal IRI

Lipopolysaccharides and Cellular Senescence: Involvement in Atherosclerosis

Atherosclerosis is a chronic inflammatory disease of the vascular walls related to aging. Thus far, the roles of cellular senescence and bacterial infection in the pathogenesis of atherosclerosis have been speculated to be independent of each other. Some types of macrophages, vascular endothelial cells, and vascular smooth muscle cells are in a senescent state at the sites of atherosclerotic lesions. Likewise, bacterial infections and accumulations of lipopolysaccharide (LPS), an outer-membrane component of Gram-negative bacteria, have also been observed in the atherosclerotic lesions of patients. This review introduces the integration of these two potential pathways in atherosclerosis. Previous studies have suggested that LPS directly induces cellular senescence in cultured monocytes/macrophages and vascular cells. In addition, LPS enhances the inflammatory properties (senescence-associated secretory phenotype [SASP]) of senescent endothelial cells. Thus, LPS derived from Gram-negative bacteria could exaggerate the pathogenesis of atherosclerosis by inducing and enhancing cellular senescence and the SASP-associated inflammatory properties of specific vascular cells in atherosclerotic lesions. This proposed mechanism can provide novel approaches to preventing and treating this common age-related disease

Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis

Systems-level identification and analysis of cellular circuits in the brain will require the development of whole-brain imaging with single-cell resolution. To this end, we performed comprehensive chemical screening to develop a whole-brain clearing and imaging method, termed CUBIC (clear, unobstructed brain imaging cocktails and computational analysis). CUBIC is a simple and efficient method involving the immersion of brain samples in chemical mixtures containing aminoalcohols, which enables rapid whole-brain imaging with single-photon excitation microscopy. CUBIC is applicable to multicolor imaging of fluorescent proteins or immunostained samples in adult brains and is scalable from a primate brain to subcellular structures. We also developed a whole-brain cell-nuclear counterstaining protocol and a computational image analysis pipeline that, together with CUBIC reagents, enable the visualization and quantification of neural activities induced by environmental stimulation. CUBIC enables time-course expression profiling of whole adult brains with single-cell resolution

Non-Enzymatic DNA Cleavage Reaction Induced by 5-Ethynyluracil in Methylamine Aqueous Solution and Application to DNA Concatenation

<div><p>DNA can be concatenated by hybridization of DNA fragments with protruding single-stranded termini. DNA cleavage occurring at a nucleotide containing a DNA base analogue is a useful method to obtain DNA with designed protruding termini. Here, we report a novel non-enzymatic DNA cleavage reaction for DNA concatenation. We found that DNA is cleaved at a nucleotide containing 5-ethynyluracil in a methylamine aqueous solution to generate 5′-phosphorylated DNA fragment as a cleavage product. We demonstrated that the reaction can be applied to DNA concatenation of PCR-amplified DNA fragments. This novel non-enzymatic DNA cleavage reaction is a simple practical approach for DNA concatenation.</p></div

Chemical structures of thymine (T) and 5-ethynyluracil (EU).

<p>Chemical structures of thymine (T) and 5-ethynyluracil (EU).</p

Chemical formula of the DNA cleavage reaction.

<p>R is expected to be an abasic sugar derivative.</p

Degradation of DNA oligonucleotides containing 5-ethynyluracil.

<p>(A), (B) HPLC charts of T₆(EU)T₆ before (gray) and after (black) the reaction in 14% NH₃aq (A) or 20% MeNH₂aq (B) at 70°C for 2 hours. (C), (D) (EU)T₂AT₂GT₂ (C) and T₂AT₂GT₂(EU)T (D) before (gray) and after (black) the reaction in 20% MeNH₂aq at 70°C for 2 hours.</p

Construction of plasmid from two PCR-amplified DNA fragments.

<p>(A) Scheme of plasmid construction. (B) Primer sequences used for PCR. The two sequences underlined in red and blue are complementary to each other. (C–G) Pictures of agarose gel electrophoresis. (C) PCR-amplified DNA fragments 1.5 (lane 2) and 2.2 kbp (lane 3). (D) 1.5 and 2.2 kbp DNA fragments before (lane 2,3) and after DNA cleavage at 25°C for 48 h (lane 4,5), 37°C for 10 h (lane 6,7), and 70°C for 0.5 h (lane 8,9). MeNH₂ was removed from the samples by speed-vac before electrophoresis. (E) Hybridized 1.5 and 2.2 kbp DNA fragments derived from those without cleavage reaction (lane 2) and cleaved at 25°C for 48 h (lane 3), 37°C for 10 h (lane 4), and 70°C for 0.5 h (lane 5). (F,G) Intact purified plasmids (F) and EcoRV-digested plasmids (G) derived from the DNA fragments cleaved at 25°C for 48 h (lane 2,3), 37°C for 10 h (lane 4–6), and 70°C for 0.5 h (lane 7–9). (H) Sequencing results of primer-derived regions of the plasmids. Underlined letters correspond to EU in the primers.</p