Kenji Ohba

Reactive astrocytes contribute to neuroinflammation and synaptic dysfunction, but it remains unclear whether transient inflammatory stimulation causes a persistent reactive state after the initial inflammatory stimulus is removed. Here, we investigated whether transient exposure to a defined inflammatory cytokine/complement cocktail induces a persistent reactive astrocyte state and examined the signaling mechanism underlying its maintenance. Human astrocytes were exposed to the inflammatory stimulus and subsequently subjected to stimulus washout, followed by time-course analyses to compare the reversibility of inflammatory gene expression after stimulus removal. Following washout, the expression of several inflammatory response genes, including CXCL10 and NF-κB-associated genes such as NFKBIA, TNFAIP3, and RELB, returned toward baseline levels. In contrast, C3 expression remained elevated, indicating persistence of a post-inflammatory C3-high astrocyte state after withdrawal of the inflammatory stimulus. Pharmacological inhibition of JAK signaling reduced persistent C3 expression to near-baseline levels, supporting the involvement of JAK-dependent signaling in maintenance of this persistent state. Together, these findings suggest that transient inflammatory stimulation induces a post-inflammatory persistent C3-high astrocyte state that is maintained even after broader inflammatory gene responses have subsided. This persistent C3-high component is pharmacologically attenuated by JAK inhibition, identifying JAK-dependent pathways as modulators of persistent astrocyte inflammatory reactivity.

Adeno-associated virus (AAV) vectors are widely used in gene therapy, whereas low manufacturing efficiency and a large proportion of empty capsids are major obstacles. This study focused on the Yin Yang 1 (YY1) binding motif (YY1-motif) and investigated the effect of its presence or insertion upstream of the Replicase (Rep)/Capsid (Cap) gene on AAV vector production. We found that the YY1-motif incidentally presented in a Rep/Cap plasmid was associated with high vector production. We then designed several modified Rep/Cap (RC2) constructs. The YY1-motif insertion in front of Rep/Cap gene increased vector yield in a repeat-number-dependent manner, and similar effects were not observed with other promoters insertion. Furthermore, the insertion of the YY1-motif reduced the amount of Cap protein per the same amount of full particle in supernatants on multiple serotypes, indicating the improvement in the empty/full capsid ratio. The YY1-motif insertion did not affect the AAV vector infectivity. These results denote that the YY1-motif has a universal regulatory function that optimizes the Rep/Cap expression balance, and simultaneously improves the production efficiency and full particle formation of AAV vectors. This finding could contribute to the development of highly efficient and high-quality AAV manufacturing processes.

Purpose: Treatment options of advanced oral squamous cell carcinomas (OSCC) are limited, and cisplatin toxicity and drug resistance are major clinical issues. Src is a central kinase that integrates multiple oncogenic pathways and a promising therapeutic target. However, Src inhibitors have shown suboptimal efficacy as monotherapies and their sensitivity in OSCC remains elusive. Experimental Design: We examined the activation of major oncogenic signaling pathways and the antitumor effects of six Src inhibitors (dasatinib, ponatinib, vandetanib, saracatinib, PP2, bosutinib) in seven human OSCC cell lines (HSC-2, HSC-3, HSC-4, SAS, HO-1-u-1, CAL27, SCC-25). BALB/cAJcl nu/nu mice bearing CAL27 xenografts received dasatinib (30 mg/kg, intraperitoneally, daily), bosutinib (50 mg/kg, intraperitoneally, daily), cisplatin (2 mg/kg or 4 mg/kg, intraperitoneally, weekly), or combinations. Tumor volume, bioluminescence imaging, and body weight were monitored for 17 or 21 days, followed by histopathological assessment. Results: The activation of the key pathways, including Src and MAPK, considerably differed among the cell lines and was linked to heterogeneous sensitivity to Src inhibitors. Effective growth suppression required Src dephosphorylation and downstream MAPK pathway inhibition, which vary depending on the cell line. Additionally, combination treatment with a Src inhibitor and cisplatin showed additive antitumor effects, allowing the reduction of cisplatin doses by half without efficacy loss. Notably, dasatinib alone and in combination with cisplatin decreased tumor burden with characteristic internal tumor death in vivo . Conclusions: These findings elucidate Src signaling dependency on OSCC and the potential of Src inhibition to decrease cisplatin toxicity, paving way for Src targeted therapeutic strategies.

It is established that neurogenesis of dentate gyrus is increased after ischemic insult, although the regulatory mechanisms have not yet been elucidated. In this study, we focused on Ezh2 which suppresses gene expression through catalyzing trimethylation of lysine 27 of histone 3. Male gerbils were injected with adeno-associated virus (AAV) carrying shRNA targeting to Ezh2 into right dentate gyrus 2 weeks prior to forebrain ischemia. One week after ischemia, animals were injected with thymidine analogue to label proliferating cells. Three weeks after ischemia, animals were killed for histological analysis. AAV-mediated knockdown of Ezh2 significantly decreased the ischemia-induced increment of proliferating cells, and the proliferated cells after ischemia showed significantly longer migration from subgranular zone (SGZ), compared to the control group. Furthermore, the number of neural stem cells in SGZ significantly decreased after ischemia with Ezh2 knockdown group. Of note, Ezh2 knockdown did not affect the number of proliferating cells or the migration from SGZ in the non-ischemic condition. Our data showed that, specifically after ischemia, Ezh2 knockdown shifted the balance between self-renewal and differentiation toward differentiation in adult dentate gyrus.

Fabry disease (FD) is an inherited lysosomal storage disease caused by deficiency of α-galactosidase A (α-Gal A), an enzyme that hydrolyzes glycosphingolipids in lysosome. Accumulation of glycosphingolipids, mainly globotriaosylceramide (Gb3) in tissues induces cellular dysfunction leading to multi-organ disorder. Gene therapy is a promising strategy that can overcome these problems, and virus vectors such as adeno-associated virus (AAV) have been used for study on gene therapy. We used human Gb3 synthetase-transgenic (TgG3S)/α-Gal A knockout (GLAko) mice. TgG3S/GLAko mice have elevated Gb3 accumulation in the major organs compared to GLAko mice, which have been widely used as a model for FD. At the age of 6 weeks, male TgG3S/GLAko were injected with 2 × 1012 vector genome AAV9 vectors containing human α-Gal A cDNA. Eight weeks after intravenous injection of AAV, α-Gal A enzymatic activity was elevated in the plasma, heart, and liver of TgG3S/GLAko mice to levels corresponding to 224%, 293%, and 105% of wild-type (WT), respectively. Gb3 amount 8 weeks after AAV injection in the heart and liver of this group was successfully reduced to levels corresponding to 16% and 3% of untreated TgG3S/GLAko mice. Although the brain and kidney of AAV9-treated TgG3S/GLAko mice showed no significant increases in α-Gal A activity, Gb3 amount was smaller than untreated littermates (48% and 44%, respectively). In this study, systemic AAV administration did not show significant extension of the lifespan of TgG3S/GLAko mice compared to the untreated littermates. The timing of AAV injection, capsid choice, administration route, and injection may be important to achieve sufficient expression of α-Gal A in the whole body for the amelioration of lifespan.

Background: Fabry disease (FD) is an inherited lysosomal storage disease caused by deficiency of α‐galactosidase A (α‐Gal A) encoded by the GLA gene. The symptoms of FD occur as a result of the accumulation of globotriaosylceramide (Gb3), comprising a substrate of α‐Gal A, in the organs. Adeno‐associated virus (AAV)‐mediated gene therapy is a promising treatment for FD.Methods: α‐Gal A knockout (GLAko) mice were injected intravenously with AAV2 (1 × 10¹¹ viral genomes [vg]) or AAV9 (1 × 10¹¹ or 2 × 10¹² vg) vectors carrying human GLA (AAV‐hGLA), and plasma, brain, heart, liver and kidney were tested for α‐Gal A activity. The vector genome copy numbers (VGCNs) and Gb3 content in each organ were also examined.Results: The plasma α‐Gal A enzymatic activity was three‐fold higher in the AAV9 2 × 10¹² vg group than wild‐type (WT) controls, which was maintained for up to 8 weeks after injection. In the AAV9 2 × 10¹² vg group, the level of α‐Gal A expression was high in the heart and liver, intermediate in the kidney, and low in the brain. VGCNs in the all organs of the AAV9 2 × 10¹² vg group significantly increased compared to the phosphate‐buffered‐saline (PBS) group. Although Gb3 in the heart, liver and kidney of the AAV9 2 × 10¹² vg was reduced compared to PBS group and AAV2 group, and the amount of Gb3 in the brain was not reduced.Conclusions: Systemic injection of AAV9‐hGLA resulted in α‐Gal A expression and Gb3 reduction in the organs of GLAko mice. To expect a higher expression of α‐Gal A in the brain, the injection dosage, administration route and the timing of injection should be reconsidered.

Epithelial cells have an ability termed ‘cell competition’, which is an immune surveillance-like function that extrudes precancerous cells from the epithelial layer, leading to apoptosis and clearance. However, it remains unclear how epithelial cells recognize and extrude transformed cells. Here, we discovered that a PirB family protein, leukocyte immunoglobulin-like receptor B3 (LILRB3), which is expressed on non-transformed epithelial cells, recognizes major histocompatibility complex class I (MHC class I) that is highly expressed on transformed cells. MHC class I interaction with LILRB3 expressed on normal epithelial cells triggers an SHP2–ROCK2 pathway that generates a mechanical force to extrude transformed cells. Removal of transformed cells occurs independently of natural killer (NK) cell or CD8⁺ cytotoxic T cell-mediated activity. This is a new mechanism in that the immunological ligand–receptor system generates a mechanical force in non-immune epithelial cells to extrude precancerous cells in the same epithelial layer.