Biology subjects

Ohshima, T.

Publications and source records attributed to Ohshima, T..

6 recordsLinked to original sources

bioRxiv2026

Intranuclear Quantum Sensing with Fluorescent Nanodiamond Enabled by Electron-Irradiation and Surface-Chemical Optimization for Microinjection

Fluorescent nanodiamonds (FNDs) containing nitrogen-vacancy (NV) centers are promising quantum sensors for intracellular measurements, yet nuclear applications remained out of reach because optically detected magnetic resonance (ODMR) signals are weak and capillary delivery is inefficient. This study addresses both constraints by optimizing the electron irradiation dose to balance NV creation and charge-state stability, and by grafting hyperbranched polyglycerol with terminal carboxyl groups (HPGCOOH) to suppress aggregation and prevent needle clogging. The optimized dose yields strong ODMR contrast while preserving fluorescence suitable for microscopy. HPGCOOH surfaces enable smooth and reproducible microinjection through fine capillaries. Using this strategy, the microinjection of ODMR-active FNDs into the nuclei of living COS7 cells is achieved, and clear intranuclear spectra comparable to cytoplasmic readouts are obtained. Furthermore, field-of-view temperature sensing across multiple cell nuclei is demonstrated, enabling quantitative and spatially resolved thermal mapping within the genomic environment. This methodology provides a practical route to nuclear quantum sensing and opens opportunities for nanoscale physicochemical measurements within the genomic environment.

biophysics↗

bioRxiv2025

Deep-brain thermo-endomicroscopy

Subtle deviations in biophysical parameters, particularly temperature, within deep-brain regions exert substantial effects on whole-body homeostasis and metabolism. However, the underlying mechanisms remain poorly understood because cellular-resolution measurements of these parameters in the deep brain have been technically inaccessible. Here, we present a lensless fiber-optic quantum-sensing endomicroscopy that enables cellular-resolution temperature mapping in the mouse deep brain. A microwire-coupled, lensless fiber bundle permits simultaneous temperature readout from optically detected magnetic resonance spectra of >50 fluorescent nanodiamonds, achieving cellular resolution (4.88 m) with a precision of 0.49 (minimum precision, 0.18 ) within a 0.28 mm2 field of view. We demonstrated temperature mapping of multiple brain depths in head-fixed and vigorously behaving mice subjected to nociceptive stimulation, detecting lower temperatures during anesthesia compared to the awake state in ventromedial hypothalamic nucleus (VMH) at a 5 mm depth. These mappings reveal that spatio-temporal temperature distribution and its anesthesia-dependent reduction are heterogeneous rather than uniform in VMH. This endomicroscopy provides a promising platform to dissect how deviations in biophysical parameters modulate deep-brain function.

biophysics↗

bioRxiv2022

NCOA4 drives ferritin phase separation to facilitate macroferritinophagy and endosomal microferritinophagy

A ferritin particle consists of 24 ferritin proteins (FTH1 and FTL) and stores iron ions within it. During iron deficiency, ferritin particles are transported to lysosomes to release iron ions. Two transport pathways have been reported: macroautophagy and ESCRT-dependent endosomal microautophagy. Although the membrane dynamics of these pathways differ, both require NCOA4, which is thought to be an autophagy receptor for ferritin. However, the exact function of NCOA4 remains elusive. Here, we found that ferritin particles form liquid-like condensates in a NCOA4-dependent manner. Homodimerization of NCOA4 and interaction between FTH1 and NCOA4 (i.e., multivalent interactions between ferritin particles and NCOA4) were required for the formation of ferritin condensates. Disruption of these interactions impaired ferritin degradation. Time-lapse imaging and three-dimensional correlative light and electron microscopy revealed that these ferritin-NCOA4 condensates were directly engulfed by autophagosomes and endosomes. In contrast, TAX1BP1 was not required for the formation of ferritin-NCOA4 condensates but was required for their incorporation into autophagosomes and endosomes. These results suggest that NCOA4 acts not only as a canonical autophagy receptor but also as a driver to form ferritin condensates to facilitate the degradation of these condensates by macroautophagy (i.e., macroferritinophagy) and endosomal microautophagy (i.e., microferritinophagy).

cell biology↗

bioRxiv2021

An isogenic panel of single App knock-in mouse models of Alzheimer's disease confers differential profiles of β-secretase inhibition and endosomal abnormalities

We previously developed single App knock-in mouse models of Alzheimers disease (AD) that harbor the Swedish and Beyreuther/Iberian mutations with or without the Arctic mutation (AppNL- G-F and AppNL-F mice). These models showed the development of amyloid {beta} peptide (A{beta}) pathology, neuroinflammation and cognitive impairment with aging. We have now generated App knock-in mice devoid of the Swedish mutations (AppG-F mice) and some additional mutants to address the following two questions: [1] Do the Swedish mutations influence the mode of {beta}-secretase inhibitor action in vivo? [2] Does the quantity of C-terminal fragment of amyloid precursor protein (APP) generated by {beta}-secretase (CTF-{beta}) affect endosomal properties as previously reported as well as other pathological events? A{beta} pathology was exhibited by AppG-F mice from 6 to 8 months of age, and was accompanied by microglial and astrocyte activation. We found that a {beta}-secretase inhibitor, verubecestat, inhibited A{beta} production in AppG-F mice, but not in AppNL-G-F mice, indicating that the AppG-F mice are more suitable for preclinical studies of {beta}-secretase inhibition given that most AD patients do not carry Swedish mutations. We also found that the quantity of CTF-{beta} generated by various App knock-in mutants failed to correlate with endosomal alterations or enlargement, implying that CTF-{beta}, endosomal abnormalities, or both are unlikely to play a major role in AD pathogenesis. This is the first AD mouse model ever described that recapitulates amyloid pathology in the brain without the presence of Swedish mutations and without relying on the overexpression paradigm. Thus, experimental comparisons between different App knock-in mouse lines will potentially provide new insights into our understanding of the etiology of AD.

neuroscience↗

bioRxiv2021

Neprilysin-sensitive amyloidogenic Aβ versus IDE-sensitive soluble Aβ: a probable mechanistic cause for sporadic Alzheimer's disease

Neprilysin (NEP) and insulin-degrading enzyme (IDE) are considered the two major catabolic enzymes that degrade amyloid {beta} peptide (A{beta}), the primary cause of Alzheimers disease (AD). However, their roles in A{beta} metabolism in vivo have never been compared in an impartial and side-by-side manner. Here, we crossbred single App knock-in mice with NEP (Mme) KO mice and with IDE (Ide) KO mice to generate double mutants that were analyzed for their biochemical and A{beta} pathology properties. We found that NEP is responsible for the metabolism of amyloidogenic insoluble A{beta} whereas IDE affects soluble A{beta}. A deficiency of NEP, but not of IDE, augmented the formation of A{beta} plaques, dystrophic neurites, and astrocytic and microglial activation, all of which are key pathological events in the development of AD. In addition, a deficiency of NEP had no significant impact on the levels of various neuropeptides (somatostatin, substance P, cholecystokinin, and neuropeptide Y), well known to be in vitro substrates for NEP, presumably because NEP is expressed in secretory vesicles and on the presynaptic membranes of excitatory neurons while most if not all neuropeptides are secreted from inhibitory neurons. This argues against the concern that NEP up-regulation for treatment of preclinical AD would reduce the levels of these neuropeptides. These findings indicate that NEP relatively selectively degrades A{beta} in the brain. Whereas familial AD (FAD) is unambiguously caused by an increased anabolism of A{beta}, and of A{beta} 42 and A{beta} 43 in particular, the anabolism of A{beta} appears unaffected before its deposition in the brain that subsequently leads to the onset of sporadic AD (SAD). These observations thus suggest that NEP-sensitive amyloidogenic A{beta} likely plays a primary pathogenic role in the etiology of SAD. Our findings are consistent with the aging-dependent decline of NEP expression in human brain and with recent genome-wide association studies (GWAS) indicating that variants of the gene encoding NEP (MME) are associated with the risk of SAD development. Taken together, our results imply that the aging-associated decrease in NEP expression is a primary cause of SAD and could thus be a target for the treatment of preclinical AD once other factors such as apolipoprotein E genotypes have also been considered.

neuroscience↗

bioRxiv2021

New App knock-in mice that accumulate wild-type human Aβ as rapidly as AppNL-G-F mice exhibit intensive cored plaque pathology and neuroinflammation.

We previously developed single App knock-in mouse models of Alzheimers disease (AD), harboring the Swedish and Beyreuther/Iberian mutations with or without the Arctic mutation (AppNL-G-F and AppNL-F mice). These models showed amyloid {beta} peptide (A{beta}) pathology, neuroinflammation and cognitive impairment in an age-dependent manner. The former line exhibits extensive pathology as early as 6 months but is unsuitable for investigating A{beta} metabolism and clearance because the Arctic mutation renders A{beta} resistant to proteolytic degradation and prone to aggregation. In particular, it is inapplicable to preclinical immunotherapy studies due to its discrete affinity for anti-A{beta} antibodies. The weakness of the latter model is that it may take as long as 18 months for the pathology to become prominent. We have thus generated a new model that exhibits early deposition of wild-type human A{beta} by crossbreeding the AppNL-F line with the Psen1P117L/WT line. We show that the effects of the pathogenic mutations in the App and Psen1 genes are additive or synergistic. This new mouse model showed more cored plaque pathology and neuroinflammation than AppNL-G-F mice and will help accelerate the development of disease-modifying therapies to treat preclinical AD.

neuroscience↗