Biology subjects

Nagasawa, Y.

Publications and source records attributed to Nagasawa, Y..

7 recordsLinked to original sources

bioRxiv2024

Rem2 interacts with CaMKII at synapses and restricts long-term potentiation in hippocampus

Synaptic plasticity, the process whereby neuronal connections are either strengthened or weakened in response to stereotyped forms of stimulation, is widely believed to represent the molecular mechanism that underlies learning and memory. The holoenzyme CaMKII plays a well-established and critical role in the induction of a variety of forms of synaptic plasticity such as long-term potentiation (LTP), long-term depression (LTD) and depotentiation. Previously, we identified the GTPase Rem2 as a potent, endogenous inhibitor of CaMKII. Here, we report that knock out of Rem2 enhances LTP at the Schaffer collateral to CA1 synapse in hippocampus, consistent with an inhibitory action of Rem2 on CaMKII in vivo. Further, re-expression of WT Rem2 rescues the enhanced LTP observed in slices obtained from Rem2 conditional knock out (cKO) mice, while expression of a mutant Rem2 construct that is unable to inhibit CaMKII in vitro fails to rescue increased LTP. In addition, we demonstrate that CaMKII and Rem2 interact in dendritic spines using a 2pFLIM-FRET approach. Taken together, our data lead us to propose that Rem2 serves as a brake on runaway synaptic potentiation via inhibition of CaMKII activity. Further, the enhanced LTP phenotype we observe in Rem2 cKO slices reveals a previously unknown role for Rem2 in the negative regulation of CaMKII function.

neuroscience↗

bioRxiv2024

TANK binding kinase 1 promotes BACH1 degradation through both phosphorylation-dependent and -independent mechanisms without relying on heme and FBXO22

BTB and CNC homology 1 (BACH1) represses the expression of genes involved in the metabolism of iron, heme and reactive oxygen species. While BACH1 is rapidly degraded when it is bound to heme, it remains unclear how BACH1 degradation is regulated under other conditions. We found that FBXO22, a ubiquitin ligase previously reported to promote BACH1 degradation, polyubiquitinated BACH1 only in the presence of heme in a highly purified reconstitution assay. In parallel to this regulatory mechanism, TANK binding kinase 1 (TBK1), a protein kinase that activates innate immune response and regulates iron metabolism via ferritinophagy, was found to promote BACH1 degradation when overexpressed in 293T cells. While TBK1 phosphorylated BACH1 at multiple serine and threonine residues, BACH1 degradation was observed with not only the wild-type TBK1 but also catalytically impaired TBK1. The BACH1 degradation in response to catalytically impaired TBK1 was not dependent on FBXO22 but involved both autophagy-lysosome and ubiquitin-proteasome pathways judging from its suppression by using inhibitors of lysosome and proteasome. Chemical inhibition of TBK1 in hepatoma Hepa1 cells showed that TBK1 was not required for the heme-induced BACH1 degradation. Its inhibition in Namalwa B lymphoma cells increased endogenous BACH1 protein. These results suggest that TBK1 promotes BACH1 degradation in parallel to the FBXO22- and heme-dependent pathway, placing BACH1 as a downstream effector of TBK1 in iron metabolism or innate immune response.

biochemistry↗

bioRxiv2023

A novel vaccine strategy using quick and easy conversion of bacterial pathogens to unnatural amino acid-auxotrophic suicide derivatives

We propose a novel strategy for quick and easy preparation of suicide live vaccine candidates against bacterial pathogens. This method requires only the transformation of one or more plasmids carrying genes encoding for two types of biological devices, an unnatural amino acid (uAA) incorporation system and toxin-antitoxin systems in which translation of the antitoxins requires the uAA incorporation. Escherichia coli BL21-AI laboratory strains carrying the plasmids were viable in the presence of the uAA, whereas the free toxins killed these strains after removal of the uAA. The survival time after uAA removal could be controlled by the choice of uAA incorporation system and toxin-antitoxin systems. Multilayered toxin-antitoxin systems suppressed escape frequency to less than 1 escape per 109 generations in the best case. This conditional suicide system also worked in Salmonella enterica and E. coli clinical isolates. The S. enterica vaccine strains were attenuated with a >105-fold lethal dose. Serum IgG response and protection against the parental pathogenic strain were confirmed. In addition, the live E. coli vaccine strain was significantly more immunogenic and provided greater protection than a formalin-inactivated vaccine. The live E. coli vaccine was not detected after inoculation, presumably because the uAA is not present in the host animals or in the natural environment. These results suggest that this strategy provides a novel way to rapidly produce safe and highly immunogenic live bacterial vaccine candidates. SignificanceLive vaccines are the oldest vaccines with a history of more than 200 years. Due to their strong immunogenicity, live vaccines are still an important category of vaccines today. However, the development of live vaccines has been challenging due to the difficulties in achieving a balance between safety and immunogenicity. In recent decades, the frequent emergence of various new and old pathogens at risk of causing pandemics has highlighted the need for rapid vaccine development processes. We have pioneered the use of unnatural amino acids to control gene expression and to conditionally kill host bacteria as a biological containment system. This report highlights a quick and easy conversion of bacterial pathogens into live vaccine candidates using this containment system.

microbiology↗

bioRxiv2023

Activity of estrogen receptor beta expressing neurons in the medial amygdala regulates preference towards receptive females in male mice.

The processing of information regarding the sex and reproductive state of conspecific individuals is critical for successful reproduction and survival in males. Generally, male mice exhibit a preference towards sexually receptive (RF) over non-receptive females (XF) or gonadally intact males (IM). Previous studies suggested the involvement of estrogen receptor beta (ER{beta}) expressed in the medial amygdala (MeA) in male preference towards RF. To further delineate the role played by ER{beta} in the MeA in the neuronal network regulating male preference, we developed a new ER{beta}-iCre mouse line using the CRISPR-Cas9 system. Fiber-photometry Ca2+ imaging revealed that ER{beta} expressing neurons in the postero-dorsal part of the MeA (MeApd-ER{beta}+ neurons) were more active during social investigation towards RF compared to copresented XF or IM mice in a preference test. Chemogenetic inhibition of MeApd-ER{beta}+ neuronal activity abolished a preference to RF in "RF vs. XF", but not "RF vs. IM", tests. Analysis with cre-dependent retrograde tracing viral vectors identified the principal part of the bed nucleus of stria terminalis (BNSTp) as a primary projection site of MeApd-ER{beta}+ neurons. Fiber-photometry recording in the BNSTp during a preference test revealed that chemogenetic inhibition of MeApd-ER{beta}+ neurons abolished differential neuronal activity of BNSTp cells as well as a preference to RF against XF but not against IM mice. Collectively, these findings demonstrate for the first time that MeApd-ER{beta}+ neuronal activity is required for expression of receptivity-based preference (i.e., RF vs XF) but not sex-based preference (i.e., RF vs IM) in male mice. Significance StatementIn this study, by introducing a new Cre mice line for ER{beta}+ cells, we described the function of MeApd-ER{beta}+ neurons and characteristics of their neuronal activity during preference tests. Using fiber photometry and DREADD techniques we have found MeApd-ER{beta}+ neurons have a specific role in receptivity-based (receptive female vs. non-receptive female) preference but not in sexbased (receptive female vs. intact male) preference in male mice. We have also described this specific role of MeApd-ER{beta}+ neurons is achieved by regulating the neuronal activity of downstream BNSTp neurons during receptivity-based, but not sex-based, preference tests. Our findings contribute to a better understating of the function of estrogen receptor expressing neurons in the neuronal network for the male-typical reproductive behaviors.

neuroscience↗

bioRxiv2023

Evolutionarily acquired activity-dependent transformation of the CaMKII holoenzyme

Ca2+/calmodulin-dependent protein kinase II (CaMKII) has long been central in synaptic plasticity research. CaMKII is a dodecameric serine/threonine kinase that has been essentially conserved across metazoans for over a million years. While the mechanisms of CaMKII activation are well studied, its "behavior" at the molecular level has remained unobserved. Here, high-speed atomic force microscopy was used to visualize the activity-dependent structural dynamics of rat/hydra/C. elegans CaMKII in various states at nanometer resolution. Among the species, rat CaMKII underwent internal kinase domain aggregation in an activity-dependent manner and showed a higher tolerance to dephosphorylation by phosphatase. Our findings suggest that mammalian CaMKII has evolutionarily acquired a new structural form and a tolerance to phosphatase to maintain robust CaMKII activity for proper neuronal function. One-Sentence SummaryHigh-speed atomic force microscopy reveals the activity-dependent structural dynamics of rat/hydra/C. elegans CaMKII

neuroscience↗

bioRxiv2022

Transcriptional lability of brain oxytocin receptor (Oxtr) generates diversity in brain OXTR distribution and social behaviors

Although oxytocin (OXT) exhibits a highly conserved neuroanatomical pattern among vertebrates, the distribution of OXT receptor (OXTR) in brain varies considerably across species and is associated with species-typical social behavior. To investigate the genomic basis of the phylogenetic plasticity in brain Oxtr expression and its social behavioral consequences, we generated transgenic mice carrying a bacteria artificial chromosome (BAC) harboring the entire prairie vole Oxtr locus and flanking intergenic regulatory regions. We established eight independent "volized" mouse lines expressing prairie vole Oxtr (pvOxtr). Strikingly, despite conserved Oxtr expression in mammary gland of all transgenic mouse lines, each line displayed a unique pattern of brain expression distinct from both mice and prairie voles. Together with topologically associating domain (TAD) structure analysis with mouse genome, our findings suggest that unlike Oxt, Oxtr expression patterns in brain, involve contributions of distal regulatory elements beyond the BAC insert. In contrast, Oxtr expression in peripheral tissues appears resistant to such distal influences. Moreover, the "volized" mouse lines with different brain Oxtr expression patterns showed differences in partner preference and maternal behaviors, providing direct functional evidence that variation in brain Oxtr expression can drive differences in social behaviors. We propose that brain Oxtr expression is transcriptionally sensitive to long-range interactions with distal genomic elements, rendering it more susceptible to diverse regulatory influences. This supports a model in which regulatory flexibility facilitates the evolutionary diversification of social behavior, while maintaining essential peripheral Oxtr expression.

neuroscience↗

bioRxiv2020

Limb-clasping, cognitive deficit and increased vulnerability to kainic acid -induced seizures in neuronal GPI anchor deficiency mouse models

Post-translational modification of a protein with glycosylphosphatidylinositol (GPI) is a conserved mechanism exists in all eukaryotes. Thus far, more than 150 human GPI anchored proteins have been discovered and about 30 enzymes have been reported to be involved in the biosynthesis and maturation of mammalian GPI. Phosphatidylinositol glycan biosynthesis class A protein (PIGA) catalyzes the very first step of GPI anchor biosynthesis. Patients carrying a mutation of the PIGA gene usually suffer from intractable epilepsy and intellectual developmental disorder. We generated three mouse models with PIGA deficits specifically in telencephalon excitatory neurons (Ex-M-cko), inhibitory neurons (In-M-cko), or thalamic neurons (Th-H-cko), respectively. Both Ex-M-cko and In-M-cko mice showed impaired long-term fear memory and were more susceptible to kainic acid (KA)-induced seizures. In addition, In-M-cko demonstrated a severe limb-clasping phenotype. Hippocampal synapse changes were observed in Ex-M-cko mice. Our Piga conditional knockout mouse models provide powerful tools to understand the cell-type specific mechanisms underlying inherited GPI deficiency and to test different therapeutic modalities.

neuroscience↗