Hiroyuki Hosokawa

Because cytokine storm syndromes such as sepsis, acute respiratory distress syndrome (ARDS), and coagulopathy, including those seen in COVID-19, are fatal, development of highly effective therapeutics is urgently needed. Recent evidence suggests that the hydrolysis of phospholipids in extracellular vesicles (EVs) by secreted phospholipase A ₂ (sPLA ₂ ) can augment the ability of EVs to modulate inflammation, allergy, and cancer. We aimed to apply this phenomenon technically using “sPLA ₂ -reacted EVs (SPLEVs),” which showed high therapeutic efficacies against ARDS and other diseases. Mechanistically, SPLEVs bind to type II alveolar epithelial cells, increase membrane fluidity by reorganizing phospholipid building blocks with more polyunsaturated fatty acids through the sterol regulatory element–binding protein 1 pathway, and elicit “lipid counterstorm” by increasing tissue-protective lipid mediators. The potent therapeutic effects of SPLEVs depend partly on sPLA ₂ -driven generation of lysophosphatidylglycerol. Thus, SPLEVs are expected to be an effective therapeutic tool for treatment of broad ranges of inflammatory diseases including COVID-19 and other new pandemic diseases.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder with diverse genetic and environmental origins, yet whether these factors converge on common molecular pathways remains unclear. This study identifies dysregulation of the Notch signaling pathway as a shared mechanism in both hereditary and nonhereditary ASD models. Aberrant histone deacetylase 3-mediated epigenetic regulation of Notch signaling during embryonic forebrain development disrupts the specification of vasoactive intestinal peptide (VIP + ) GABAergic interneuron subtypes (VIP-INs), which originate in the caudal ganglionic eminence (CGE). CGE-specific ablation of Notch1/2 genes in ASD models restores the loss of VIP-INs, normalizes maladaptive excitatory and inhibitory balance, and selectively improves social behaviors. A single antenatal dose of a γ-secretase inhibitor ameliorates multiple ASD-associated neuronal, behavioral, and transcriptomic changes in adult models. The study indicates a strong convergence of ASD-related factors on Notch signaling dysregulation and establishes this pathway as a promising therapeutic target for developmental and behavioral deficits in ASD.

Plasminogen activator inhibitor-1 (PAI-1) is significantly upregulated during inflammatory responses, and elevated PAI-1 levels are associated with poor prognosis in various diseases. However, the precise mechanism through which PAI-1 exacerbates inflammation remains unclear. In the present study, we have investigated the role of PAI-1 in inflammation using a mouse model of skeletal muscle injury. We found that CCR2⁺Ly6C⁺ inflammatory macrophages infiltrated the injured tissues and produced substantial amounts of PAI-1. Notably, PAI-1 deficiency specifically in these macrophages resulted in attenuated inflammation and accelerated tissue repair despite the continued presence of PAI-1 in body fluids, indicating a local macrophage-driven effect. Low-density lipoprotein receptor-related protein-1 (LRP-1), expressed on macrophages, is a common receptor for both PAI-1 and calreticulin (CRT). CRT is exposed on the surface of dying cells and functions as an “eat me” signal recognized by macrophages via LRP-1. We found that PAI-1 binds to LRP-1 with higher affinity than that to CRT, thereby competitively inhibiting CRT recognition and suppressing efferocytosis, the process by which macrophages clear dead cells, ultimately leading to prolonged inflammation. Importantly, administration of a PAI-1 inhibitor, TM5614, restored efferocytosis and significantly improved tissue regeneration. These findings therefore reveal that PAI-1 produced by infiltrating inflammatory macrophages contributes to sustained inflammation by blocking efferocytosis, and that PAI-1 is a promising therapeutic target for the treatment of inflammatory diseases.

Accumulated studies have reported that hematopoietic differentiation was primarily regulated by transcription factors. Early B cell factor 1 (EBF1) is an essential transcription factor for B lymphopoiesis. Contrary to the canonical notion, we found that a single miRNA, miRNA-195 ( Mir195 ) transduction let Ebf1 -deficient hematopoietic progenitor cells (HPCs) express CD19, carry out V(D)J recombination and class switch recombination, which implied that B cell matured without EBF1. A part of the mechanism was caused by FOXO1 accumulation via inhibition of FOXO1 phosphorylation pathways in which targets of Mir195 are enriched. These results suggested that some miRNA transductions could function as alternatives to transcription factors.

Accumulated studies have reported that hematopoietic differentiation was primarily regulated by transcription factors. Early B cell factor 1 (EBF1) is an essential transcription factor for B lymphopoiesis. Contrary to the canonical notion, we found that a single miRNA, miRNA-195 (miR-195) transduction let EBF1 deficient hematopoietic progenitor cells (HPCs) express CD19, carry out V(D)J recombination and class switch recombination, which implied that B cell matured without EBF1. A part of the mechanism was caused by FOXO1 accumulation via inhibition of FOXO1 phosphorylation pathways in which targets of miR-195 are enriched. These results suggested that some miRNA transductions could function as alternatives to transcription factors.

Runt-related transcription (RUNX) factors play a key role in T cell development. At the T-lineage commitment checkpoint, RUNX1 undergoes dynamic partner switching, resulting in its redeployment. Here, we investigated the functional differences in RUNX factors between the lymphoid progenitor (LP)– and Notch-stimulated earliest T progenitor stages (Phase 1). We identified CCCTC-binding factor (CTCF) as an LP-specific RUNX1-interacting partner, with LP-specific RUNX1-binding genomic sites significantly enriched for CTCF consensus motifs and co-occupied by CTCF. On Notch stimulation, Notch1 intracellular domain directly interacts with RUNX1 and recruits the RUNX1/Mediator/p300 transcriptional activation complex to Notch-regulated T-signature gene loci. CRISPR/Cas9-mediated stage-specific deletion of RUNX factors and their binding partners revealed that the RUNX1/CTCF complex in LP negatively regulates T-signature gene expression, whereas the RUNX1/Mediator/p300 complex in Phase 1 promotes it. Our findings highlight the crucial role of Notch-mediated functional conversion of RUNX factors, including protein complex reorganization and genomic redeployment in initiating T-lineage program.

Three types of O -linked glycosylation— O -glucose, O -fucose, and O - N -acetylglucosamine— are crucial for the function of Notch receptors, which regulate critical cell fate determination processes in a wide variety of contexts. O -Glucose glycans are transferred to serine residues located between the first and second conserved cysteines within the epidermal growth factor-like (EGF) repeats in the Notch extracellular domain. Previously, O -glucose glycans were shown to be extended to a trisaccharide structure with two xyloses via α1-3 linkages. Our recent studies, however, indicated that the O -glucose glycan on NOTCH1 EGF10 can be extended by hexose and Neu5Ac. Here, we demonstrated that this hexose- and Neu5Ac-extended glycan has a 3’-sialyllactose-like structure synthesized by specific members of two isoenzyme families, B4GALT1 and ST3GAL4. Using mass spectrometry, we identified this modification exclusively on NOTCH1 EGF10 and the analogous NOTCH3 EGF9 domain, with no detection in any other EGF domains in NOTCH1, NOTCH2, and NOTCH3. Sequence comparison and mutagenesis experiments identified one amino acid at position -2 of the fourth cysteine (C ⁴ -2) in the EGF domain as crucial for the galactose elongation of O -glucose glycans. We further demonstrated that this site-specific elongation of O -glucose on NOTCH1 EGF10 significantly impacts ligand binding and signal transduction of NOTCH1. In the context of early T cell development, the C ⁴ -2 mutants NOTCH1 A396Y and A396F enhance T cell differentiation through DLL1- and DLL4-dependent NOTCH1 signaling. Our findings contribute to the understanding of the intricate regulatory mechanisms of Notch receptor function mediated by distinct positions and structures of O -glycans.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder with diverse genetic and environmental origins, yet whether these factors converge on common molecular pathways remains unclear. Here we identify dysregulation of the Notch signaling pathway as a shared mechanism in both hereditary and non-hereditary ASD models. Aberrant HDAC3-mediated epigenetic regulation of Notch signaling during embryonic forebrain development disrupts the specification of caudal ganglionic eminence (CGE) progenitors into VIP+ GABAergic interneuron subtypes (VIP-INs). CGE-specific ablation of Notch1/2 genes in ASD models restores the loss of VIP-INs and E/I imbalance, and selectively improves social behaviors. Remarkably, a single antenatal dose of a γ-secretase inhibitor ameliorates multiple ASD-associated neuronal, behavioral, and transcriptomic changes in adult models. Thus, this study confers a strong convergence of ASD-related factors on Notch signaling dysregulation and establishes this pathway as a promising therapeutic target for developmental and behavioral deficits in ASD.

Accumulated studies have reported that hematopoietic differentiation was primarily regulated by transcription factors. Early B cell factor 1 (EBF1) is an essential transcription factor for B lymphopoiesis. Contrary to the canonical notion, we found that a single miRNA, miRNA-195 (miR-195) transduction let EBF1 deficient hematopoietic progenitor cells (HPCs) express CD19, carry out V(D)J recombination and class switch recombination, which implied that B cell matured without EBF1. A part of the mechanism was caused by FOXO1 accumulation via inhibition of FOXO1 phosphorylation pathways in which targets of miR-195 are enriched. These results suggested that some miRNA transductions could function as alternatives to transcription factors.