Biology subjects

Tominaga, T.

Publications and source records attributed to Tominaga, T..

17 recordsLinked to original sources

bioRxiv2025

Promotion of seed germination in Echinochloa crus-galli with atmospheric air plasma

Plasma technology has attracted increasing attention for potential agricultural applications, and it has been investigated for decades particularly for its ability to promote seed germination. However, most studies have focused on crop species, and little is known about its potential use for weed management. In this study, we examined whether air plasma influences the germination of Echinochloa crus-galli, a major paddy field weed. When seeds were cultivated without stratification, direct irradiation with air plasma did not enhance germination. In contrast, once dormancy was partially released by cold stratification, plasma treatment significantly promoted germination. Interestingly, the effect disappeared after prolonged chilling, when dormancy was completely released. These results suggest that plasma promotes germination by acting on the dormancy-breaking process. In addition, plasma-activated water (PAW) also stimulated germination, although its effect was weaker than that of gaseous plasma. Overall, this study demonstrates that air plasma can promote the germination of grass weed seeds, providing a potential strategy for weed control by inducing germination prior to crop seeding.

plant biology↗

bioRxiv2025

Endothelial Sphingosine-1-Phosphate Receptor (S1PR) 1, a Functional S1PR in the Human Cerebrovascular Endothelium, Limits Blood Brain Barrier Permeability and Neuronal Injury following Subarachnoid Hemorrhage in Mice.

Hypoxia-induced blood-brain barrier (BBB) permeability has been identified as a key contributor to the progression of ischemic-hypoxic brain injury and neuronal dysfunction in stroke and other cerebrovascular diseases. Emerging clinical evidence highlights that the vasoprotective signaling properties of high-density lipoprotein (HDL), mediated through its bioactive lipid component sphingosine-1-phosphate (S1P), may be impaired in cardiovascular and inflammatory conditions. Nonetheless, the precise contributions and mechanistic roles of S1P signaling within the cerebral microvasculature remain insufficiently characterized. In this study, we aimed to elucidate the role of S1P signaling via its endothelial receptor S1PR1 in the pathophysiology of early brain injury following subarachnoid hemorrhage (SAH), a particularly severe form of stroke. Additionally, we sought to evaluate the relevance of the endothelial S1PR1 pathway in the human cerebrovascular endothelium, its functional role in hypoxia-induced cerebral endothelial barrier dysfunction, and its underlying molecular mechanisms. To address these objectives, we utilized endothelial-specific S1PR1 knockout mice subjected to the endovascular rupture model of aneurysmal SAH, performed mechanistic studies in primary human cerebral microvascular endothelial cells, and characterized S1PR1 expression in human brain tissue using validated protocols. Our findings reveal robust expression of S1PR1 in the cerebrovascular endothelium of both mice and humans. Functional analyses demonstrated that S1PR1 is critical for maintaining BBB integrity and mitigating neuronal injury in the context of SAH. Mechanistic in vitro studies indicated that S1PR1 exerts a vasoprotective effect by limiting hypoxia-induced BBB dysfunction in human primary brain microvascular endothelial cells through inhibition of Rho-associated kinase (ROCK)-mediated phosphorylation of myosin light chain (MLC), suppression of stress fiber formation and caveolin-1-dependent endosomal trafficking. These results highlight the pivotal role of endothelial S1PR1 signaling in preserving cerebral vascular integrity and provide a strong scientific foundation for developing novel therapeutic approaches targeting the S1P pathway in the endothelium to enhance neurovascular protection.

neuroscience↗

bioRxiv2024

Induced systemic resistance by the root colonization of Trichoderma atroviride is independent from the chitin-mediated signaling pathway in Arabidopsis

Beneficial root endophytic fungi induce systemic responses, growth promotion, and induced systemic resistance (ISR) in the colonized host plants. Soil application of chitin, a main component of fungal cell walls, also systemically induces disease resistance. Thus, chitin recognition and its downstream signaling pathway are supposed to mediate ISR triggered by beneficial fungi colonizing the root. This study compared systemic disease resistance and transcriptional responses induced by Trichoderma, a representative beneficial root endophytic fungus, and chitin in Arabidopsis. A significant plant growth promotion was observed under root colonization by the three tested beneficial fungi, Trichoderma atroviride, Serendipita indica, and S. vermifera. Still, only T. atroviride and S. indica triggered ISR against the necrotrophic fungal pathogen Alternaria brassicicola. Induced systemic resistance triggered by T. atroviride was compromised in the chitin-receptor mutant, while systemic resistance caused by soil application of chitin was not. Transcriptome analysis demonstrated that the chitin-regulated genes are mostly shared with those regulated by T. atroviride, but many of the latter were specific. However, the commonly enriched gene ontologies for those regulated genes indicated that T. atroviride inoculation and chitin application systemically control similar transcriptional responses, mainly associated with cell wall functions. Taken together, Trichoderma could trigger ISR primarily independently from the chitin-mediated signaling pathway; however, chitin and Trichoderma would systemically induce similar cellular functions in ISR aboveground.

plant biology↗

bioRxiv2023

Dynamic mRNA stability changes buffer transcriptional activation during neuronal differentiation and are regulated by RNA binding proteins.

The steady state levels of mRNA are outcomes of a finely tuned interplay between RNA transcription and decay. Therefore, the modulation of RNA stability is generally assumed to influence RNA abundance in a positive direction. However, the correlation between mRNA transcription, translation and stability remains elusive. Here, we employed a newly developed simplified mRNA stability profiling technique to explore the role of mRNA stability in SH-SY5Y neuronal differentiation model. Transcriptome-wide mRNA stability analysis revealed neural-specific RNA stability kinetics, including stabilization of transcripts encoding regulators of neuronal morphogenesis and function and destabilization of mitochondrial electron transport and redox homeostasis. When we further examined the relationship between transcription, translation and mRNA stability, a bidirectional regulation of RNA stability was revealed, wherein mRNA stability could either exert the buffering effect on gene products or change in a same direction as transcription. Motif analysis unveiled SAMD4A as a major regulator of the dynamic changes in mRNA stability observed during differentiation. Knockdown of SAMD4A impaired neuronal differentiation and influenced the response to oxidative stress. Mechanistically, SAMD4A was found to alter the stability of several mRNAs to which it binds. Meanwhile, a dimorphic pattern of the correlation between gene expression and SAMD4A-regulated mRNA stability was observed, suggesting dynamic regulation mRNA stability during the neuronal differentiation guided by SAMD4A. The novel insights into the interplay between mRNA stability and cellular behaviors provide a foundation for understanding neurodevelopmental processes and neurodegenerative disorders and highlights dynamic mRNA stability as an important layer of gene expression regulation.

molecular biology↗

bioRxiv2023

Monoterpene glucosides accumulated in Eustoma grandiflorum roots promote hyphal branching in arbuscular mycorrhizal fungi

Host plant-derived strigolactones trigger hyphal branching in arbuscular mycorrhizal (AM) fungi, initiating a symbiotic interaction between land plants and AM fungi. However, our previous studies revealed that gibberellin-treated Eustoma grandiflorum (Gentianaceae) activates rhizospheric hyphal branching in AM fungi using unidentified molecules other than strigolactones. In this study, we analyzed independent transcriptomic data of E. grandiflorum and found that the gentiopicroside (GPS) and swertiamarin (SWM), which are characteristic monoterpene glucosides in Gentianaceae, were highly biosynthesized in gibberellin-treated E. grandiflorum roots. Moreover, these metabolites considerably promoted hyphal branching in the Glomeraceae AM fungi Rhizophagus irregularis and R. clarus. GPS treatment also enhanced R. irregularis colonization of the monocotyledonous crop Allium schoenoprasum. Interestingly, these metabolites did not provoke the germination of the root parasitic plant Orobanche minor. Altogether, our study unveiled the crucial role of GPS and SWM in activating the symbiotic relationship between AM fungi and E. grandiflorum.

plant biology↗

bioRxiv2022

RNF213 reshapes vascular transcriptome and epigenome; A potential role of Alternative splicing in Moyamoya pathology

RationaleMoyamoya disease (MMD) is a rare cerebrovascular occlusive disease that affects Asian population more often. The pathogenesis of MMD is related to mutation in RNF213 gene. However, why, and how RNF213 mutation leads to MMD is still not fully understood. ObjectiveAnalyze the impact of RNF213 loss of function on vascular cells and the observed changes correlate with MMD. Methods and resultsRNF213 KD was conducted in HUVEC and vascular smooth muscle cells (vSMCs). First, HUVEC cells showed alteration of angiogenesis, migration under LPS stimulation, Leukocyte endothelial transmigration, and endothelial-to-vSMCs communication. Transcriptome analysis revealed downregulation of genes regulating cell division and mitosis. Interestingly, Alternative splicing (AS) analysis revealed hundreds of AS events to occur after RNF213 KD in various types of AS. The alternatively spliced genes showed minimal overlap with transcriptome profiling. Pathway analysis revealed many processes and pathways to be regulated by AS events observed. Transcriptome profiling was also performed after LPS treatment and revealed the basis for increased sensitivity to LPS observed in our analysis. AS changes were also observed after LPS treatment in RNF213 KD HUVEC. Different types of AS showed different patterns of convergence or divergence in terms of the regulated pathways after LPS treatment. Finally, transcriptome and AS analysis was performed in vSMCs and showed various processes that impact vSMCs function and phenotype in RNF213 loss of function. ConclusionOur data provide a wealth of information on RNF213 gene function in vascular tissues and shed important light on different processes that contribute to MMD pathogenesis. Our results signify an immune-driven and hemodynamically linked process of MMD initiation and progression.

molecular biology↗

bioRxiv2022

Simultaneous overexpression of multiple herbicide-metabolizing genes for broad-spectrum resistance in an agricultural weed Echinochloa phyllopogon

O_LIPrevious research unveiled that the overexpression of catalytically promiscuous CYP81A cytochrome P450s underlies the multiple-herbicide resistance (MHR) in a Californian population of Echinochloa phyllopogon. However, it does not fully accommodate the resistance to diverse herbicides in MHR E. phyllopogon although the genetic inheritance of MHR was suggested as under a single gene control. C_LIO_LIWe investigated the high-level resistance to diclofop-methyl in MHR E. phyllopogon. Detailed diclofop-methyl metabolism was analyzed, followed by gene expression study and functional characterization of P450 genes. The generality of the MHR mechanism was investigated using another MHR line. C_LIO_LIThe MHR line rapidly produced two distinct hydroxylated-diclofop-acid, only one of which was the major metabolite produced by CYP81A12/21. Gene expression study identified the genetically linked overexpression of a novel gene CYP709C69 with CYP81A12/21 in the MHR line. The gene conferred diclofop-methyl resistance in plants and produced another hydroxylated-diclofop-acid in yeast. The activity was observed in some CYP709C in plants. Unlike the broad substrate-specificity in CYP81As, CYP709C69 showed narrow substrate-specificity. The overexpression of the CYP81A and CYP709C69 was also observed in another MHR line. C_LIO_LIThe present findings establish a novel concept that genetically-linked simultaneous overexpression of herbicide-metabolizing genes enhances and broadens the profile of metabolic resistance in weeds. C_LI

plant biology↗

bioRxiv2021

Mature Neurons sensitivity to oxidative stress is epigenetically programmed by alternative splicing and mRNA stability

Neuronal differentiation is a complex process that entails extensive morphological, transcriptional, metabolic, and functional changes that dictate neuronal lineage commitment. Much less understood is the role that epigenetic and epi-transcriptional reprogramming plays in the process of neuronal differentiation and maturation. To depict the whole landscape of transcriptomics and epigenetic changes during neuronal differentiation and maturation, we differentiated SH-SY5Y cells and performed RNA sequencing on differentiated and undifferentiated cells. 728 differentially expressed genes (DEGs) enriched in synaptic signaling and cell morphogenesis pathways were observed. Moreover, transcriptome-wide mRNA stability profiling revealed that genes with altered stability were exceptionally enriched for redox homeostasis pathways. Mature neurons are known to be highly sensitive to oxidative stress, which is crucial in the pathophysiology of neurodegenerative disease. Our results suggest that this heightened sensitivity is regulated at the mRNA stability level (i.e., epigenetic) rather than at the transcriptional level. Alternative splicing analysis revealed the exon skipping and alternative mRNA isoforms enriched for morphogenesis related pathway. Alternatively, alternative 5 and 3 prime splicing site, intron retention and mutually exclusive exon events exclusively clustered in the translation and translation initiation pathways, suggesting the potential effect of alternative splicing on translation following neuronal maturation. Splice motif analysis revealed enriched motifs for RBPs that regulate various splice types and can be further correlated to distinct phenotypical changes during neuronal differentiation and maturation. Here we present an extensive exploration of the transcriptional and epigenetic changes and their potential association with the process of neuronal differentiation, providing a new insight into understanding the molecular mechanism of neuronal function and behavior.

molecular biology↗

bioRxiv2021

Epigenetic regulation of translation repression in ferroptosis, and a role of Alternative splicing and tRNA methylation.

Ferroptosis is a non-apoptotic cell death mechanism characterized by the production of lipid peroxides. Ferroptosis plays important roles in many diseases such as cancer and neurodegenerative diseases. While many effectors in the ferroptosis pathway have been mapped, its epigenetic and epitranscriptional regulatory processes are not yet fully understood. Ferroptosis can be induced via system xCT inhibition (Class I) or GPX4 inhibition (Class II). Previous works have revealed important differences in cellular response to Class I and Class II ferroptosis inducers. Importantly, blocking mRNA transcription or translation appears to protect cells against Class I ferroptosis inducing agents but not Class II. Understanding these subtle differences is important in understanding ferroptosis as well as in developing therapeutics based on ferroptosis for various diseases. In this work, we examined the impact of blocking transcription (via Actinomycin D) or translation (via Cycloheximide) on Erastin (Class I) or RSL3 (Class II) induced ferroptosis. Blocking transcription or translation protected cells against Erastin but was detrimental against RSL3. Cycloheximide led to increased levels of GSH alone or when co-treated with Erastin and the activation of the reverse transsulfuration pathway. RNA sequencing analysis revealed an important and unexplored role of Alternative splicing (AS) in regulating ferroptosis stress response and mRNA translation repression. Our results indicated that translation repression is protective against Erastin but detrimental against RSL3. We tested this theory in Alkbh1 overexpressing glioma cells. Alkbh1 demethylates tRNA and represses translation and is associated with worse outcome in glioma patients. Our results showed that Alkbh1 overexpression protected glioma cells against Erastin but was detrimental against RSL3.

molecular biology↗

bioRxiv2021

Cross-scale Analysis of Temperature Compensation in the Cyanobacterial Circadian Clock System

Clock proteins maintain constant enzymatic activity regardless of temperature, even though thermal fluctuation is accelerated as temperature increases. We investigated temperature influences on the dynamics of KaiC, a temperature-compensated ATPase in the cyanobacterial circadian clock system, using quasielastic neutron scattering. The frequency of picosecond to sub-nanosecond incoherent local motions in KaiC was accelerated very slightly in a temperature-dependent manner. Our mutation studies revealed that internal motions of KaiC include several contributions of opposing temperature sensitivities. To take advantage of this balancing effect, the motional frequency of local dynamics in KaiC needs to exceed [~]0.3 ps-1. Some of the mutation sites may be in a pathway through which the motional frequency in the C-terminal domain of KaiC is fed back to the active site of ATPase in its N-terminal domain. The temperature-compensating ability at the dynamics level is likely crucial for circadian clock systems, into which the clock proteins are incorporated, to achieve reaction- or even system-level temperature compensation of the oscillation frequency.

biophysics↗

bioRxiv2021

Conservation and diversity in transcriptional responses among host plants forming distinct arbuscular mycorrhizal morphotypes

O_LIThe morphotype of arbuscular mycorrhizal (AM) roots is distinct mostly depending on AM host species: Arum, Paris, and Intermediate types. We previously reported that gibberellin (GA) promotes the establishment of Paris-type AM symbiosis in Eustoma grandiflorum despite its negative effects on Arum-type AM symbiosis in model plants. However, the molecular mechanisms underlying the differential effects of GA on different morphotypes, including Intermediate-type AM symbiosis, remain elusive. C_LIO_LIComparative transcriptomics revealed that several symbiosis-related genes were transcriptionally promoted upon AM fungal colonization in Lotus japonicus (Arum-type), Daucus carota (Intermediate-type), and E. grandiflorum (Paris-type). Interestingly, upon GA treatment, the fungal colonization levels and expression of symbiosis-related genes were suppressed in L. japonicus and D. carota but were promoted in E. grandiflorum. C_LIO_LIExogenous GA transcriptionally inhibited the biosynthetic process of a host-derived signal molecule involved in AM symbiosis, strigolactone, in L. japonicus and E. grandiflorum. Additionally, disaccharides mainly metabolized in AM roots would be different between L. japonicus and D. carota/E. grandiflorum. C_LIO_LIThis study uncovered the conserved transcriptional responses during mycorrhization and diverse responses to GA in AM roots with distinct morphotypes among phylogenetically distant host plants. C_LI

plant biology↗

bioRxiv2021

Gene expression shapes the patterns of parallel evolution of herbicide resistance in the agricultural weed Monochoria vaginalis

The evolution of herbicide resistance in weeds is an example of parallel evolution, through which genes encoding herbicide target proteins are repeatedly represented as evolutionary targets. The number of herbicide target-site genes differs among species, and little is known regarding the effects of duplicate gene copies on the evolution of herbicide resistance. We investigated the evolution of herbicide resistance in Monochoria vaginalis, which carries five copies of sulfonylurea target-site acetolactate synthase (ALS) genes. Suspected resistant populations collected across Japan were investigated for herbicide sensitivity and ALS gene sequences, followed by functional characterisation and ALS gene expression analysis. We identified over 60 resistant populations, all of which carried resistance-conferring amino acid substitutions exclusively in MvALS1 or MvALS3. All MvALS4 alleles carried a loss-of-function mutation. Although the enzymatic properties of ALS encoded by these genes were not markedly different, the expression of MvALS1 and MvALS3 was prominently higher among all ALS genes. The higher expression of MvALS1 and MvALS3 is the driving force of the biased representation of genes during the evolution of herbicide resistance in M. vaginalis. Our findings highlight that gene expression is a key factor in creating evolutionary hotspots.

plant biology↗

bioRxiv2020

Drastic shift in flowering phenology, an instant reproductive isolation mechanism, explains the population structure of Imperata cylindrica in Japan

Reproductive isolation plays an important role in population differentiation and speciation, thus enhancing biodiversity in wild plants. Hybridisation sometimes involves rapid reproductive isolation between parents and their hybrids through the novel traits of hybrids derived from a new combination of genomes. Here, we report how a hybrids new phenotype contributes to rapid reproductive isolation between two ecotypes of Imperata cylindrica. The two ecotypes differ in their flowering phenology and habitats. An analysis with genetic markers revealed that hybrid populations consisted of only F1 individuals. Both parental ecotypes flowered in spring, but F1s flowered in fall. This drastic shift in flowering phenology prevented backcrossing parental ecotypes to F1. F1s flowered in fall and dispersed seeds in winter. The germination percentage of seeds set on F1 was extremely low in their habitats, and seedlings did not survive due to the low temperatures in winter, resulting in the absence of a F2 generation. In conclusion, flowering phenology mismatch promotes reproductive isolation between parents and F1, resulting in a hybrid population consisting of only F1s.

evolutionary biology↗

bioRxiv2020

The cell and stress specific dynamics of in vivo and in vitro canonical and non-canonical tRNA cleavage.

Following stress, tRNA is cleaved to generate tRNA halves (tiRNAs). These stress-induced small RNAs have been shown to regulate translation during stress. To date, angiogenin is considered the main enzyme that cleaves tRNA at its anti-codon site to generate 35 ~ 45 nucleotide long 5' and 3' tiRNA halves, however recent reports indicate the presence of angiogenin-independent cleavage. We previously observed tRNA cleavage pattern occurring away from the anti-codon site. To explore this non-canonical cleavage, we analyze tRNA phenotypical cleavage patterns in rat model of ischemia reperfusion and in two rat cell lines. In vivo mitochondrial tRNAs were prone to this non-canonical cleavage pattern. In vitro, however, both cytosolic and mitochondrial tRNAs could be cleaved non-canonically. We also evaluated the roles of angiogenin and its inhibitor, RNH1, in regulating tRNA cleavage during stress. Our results suggest that mitochondrial stress has an important regulatory role in angiogenin-mediated tRNA cleavage. Angiogenin does not appear to regulate the non-canonical cleavage pattern of tRNA, and RNH1 does not affect it as well. Finally, we verified our previous findings of the stress-specific role of Alkbh1 in regulating tRNA cleavage and showed a strong influence of stress type on Alkbh1-mediated tRNA cleavage and that Alkbh1 impacts non-canonical tRNA cleavage.

molecular biology↗

bioRxiv2020

The stress specific impact of ALKBH1 on tRNA cleavage and tiRNA generation

tiRNAs are small non-coding RNAs produced when tRNA is cleaved under stress. tRNA methylation modifications has emerged in recent years as important regulators for tRNA structural stability and sensitivity to cleavage and tiRNA generation during stress, however, the specificity and higher regulation of such a process is not fully understood. Alkbh1 is a m1A demethylase that leads to destabilization of tRNA and enhanced tRNA cleavage. We examined the impact of Alkbh1 targeting via gene knockdown or overexpression on B35 rat neuroblastoma cell line fate following stresses and on tRNA cleavage. We show that Alkbh1 impact on cell fate and tRNA cleavage is a stress specific process that is impacted by the demethylating capacity of the cellular stress in question. We also show that not all tRNAs are cleaved equally following Alkbh1 manipulation and stress, and that Alkbh1 KD fails to rescue tRNAs from cleavage following demethylating stresses. These findings shed a light on the specificity and higher regulation of tRNA cleavage and should act as a guide for future work exploring the utility of Alkbh1 as a therapeutic target for cancers or ischemic insult.

molecular biology↗

bioRxiv2019

Instantaneous polarized light imaging reveals activity dependent structural changes of dendrites in mouse hippocampal slices

Intrinsic Optical Signal (IOS) imaging has been widely used to map patterns of brain activity in vivo in a label-free manner. Traditional IOS refers to changes in light transmission, absorption, and scattering, which have been correlated with neuronal swelling and volume changes in the observed tissue. Here we use polarized light for IOS imaging to monitor structural changes of cellular and sub-cellular architectures of neurons due to their synaptic activity in isolated brain slices. In order to reveal fast spatio-temporal changes of birefringence associated with neuronal activity, we developed the instantaneous PolScope. The instantaneous PolScope records changes in transmission, birefringence, and slow axis orientation in tissue at high spatial and temporal resolution using a single camera exposure. These capabilities enabled us to correlate polarization-sensitive IOS with traditional IOS on the same preparations. We detected reproducible spatio-temporal changes in both IOSs at the stratum radiatum in mouse hippocampal slices evoked by Schaffer collateral stimulation in the CA1 area. Upon stimulation, changes in traditional IOS signals were broadly similar across the area, while birefringence imaging revealed local variations not seen in traditional IOS. Locations with high resting birefringence produced larger stimulation-evoked birefringence changes than those with low resting birefringence. Local application of glutamate to the synaptic region in CA1 induced increase in both transmittance and birefringence signals. Blocking synaptic transmission with CNQX and D-APV (inhibitors of AMPA-and NMDA-type ionotropic glutamate receptors, respectively) reduced the peak amplitude of the optical signals. Changes in both IOSs were enhanced by an inhibitor of the membranous glutamate transporter, DL-TBOA. Our results indicate that birefringence imaging can monitor structural alterations of dendrites subjected to excitatory synaptic transmission also associated with neuronal activity in the brain.

biophysics↗

bioRxiv2018

Exacerbation of blood brain barrier dysfunction and brain injury after aneurysmal subarachnoid hemorrhage in endothelial specific S1PR1 knock out mice.

Blood brain barrier (BBB) dysfunction upon ischemia and hypoxia has been implicated in the exacerbation of neuronal injury in stroke. Despite the therapeutic potential of the cerebrovascular endothelium, the limited understanding of the endothelial signaling pathways governing BBB function restricts progress towards developing novel therapeutic approaches specifically targeting the endothelium. Sphingosine-1-phosphate (S1P) is a potent modulator of endothelial function via its receptors (S1PR). Recent human and mouse studies indicate that vasoprotective endothelial S1P signaling via S1PR1 may be impaired in cardiovascular and inflammatory diseases. Herein, we investigated the expression of S1PR1 in the mouse and human cerebrovascular endothelium and the role of endothelial-specific S1PR1 signaling in brain injury in a mouse model of aneurysmal subarachnoid hemorrhage (SAH), the most devastating type of stroke. We found that S1PR1 is the most abundant S1PR transcript in the mouse brain and in mouse and human brain endothelial cells (20-100 mRNA copies per cell). S1PR1 transcripts were significantly enriched (~6 fold) in mouse cortical microvessels compared to total brain. Using the S1PR1-eGFP knock in mouse, we found that S1PR1-eGFP is abundantly expressed in the cerebrovascular endothelium in the mouse brain. A similar pattern of expression was observed in human brain samples. Endothelial specific deletion of S1PR1 in adult mice (S1pr1 flox/floxxCdh5-CreERT2, referred to as S1pr1iECKO), resulted in exacerbation of brain edema, neuronal injury and worsened neurological outcomes upon SAH compared to S1pr1 flox/flox littermates. No differences in the subarachnoid blood, hemostasis or cerebral blood flow changes during and after SAH were found between groups. Mechanistically, S1pr1iECKO exhibited aggravated BBB dysfunction and increased phosphorylation of myosin light chain (MLC) in isolated cortical microvessels, a downstream effector of the Rho-ROCK pathway implicated in endothelial inflammation and barrier dysfunction. Taken together, our data indicate that S1PR1 is an endogenous protective signaling pathway in the endothelium, critical to maintain BBB function and to mitigate neuronal injury in pathological conditions. Thus, the therapeutic and diagnostic potential of the endothelial sphingosine-1-phosphate pathway in stroke deserves further study.

neuroscience↗