Background: The ubiquitin-proteasome system (UPS) and autophagy are 2 major protein degradation pathways in eukaryotic cells. We previously identified a switch from UPS to autophagy with changes in BAG3 (B-cell lymphoma 2-associated-athanogene 3) expression after cerebral ischemia in mice. BAG3 is an antiapoptotic-cochaperone that is directly involved in cellular protein quality control as a mediator for selective macroautophagy. Here, we aimed to investigate the role of BAG3 in ischemic stroke. Methods: Middle cerebral artery occlusion/reperfusion (MCAO/R) and oxygen-glucose deprivation/reoxygenation were used to mimic cerebral ischemia in vivo and in vitro. The UPS inhibitor MG132 and autophagy inhibitor 3-MA (3-methyladenine) were administered to mice to identify how BAG3 was involved after MCAO/R. Adeno-associated virus and lentiviral vector were used to regulate BAG3 expression in vivo and in vitro, respectively. Behavioral tests, 2,3,5-triphenyltetrazolium chloride staining, and Hematoxylin & Eosin staining were performed to evaluate cerebral injury following MCAO/R, and a Cell Counting kit-8 assay was conducted to assess oxygen-glucose deprivation/reoxygenation–induced injury in cells. Brain tissues and cell lysates were collected and analyzed for UPS activation, autophagy, and apoptosis. Results: The UPS inhibitor alleviated MCAO injury in mice and increased autophagy and BAG3 expression, whereas the autophagy inhibitor exacerbated MCAO/R-induced injury. In addition, BAG3 overexpression significantly improved neurological outcomes, reduced infarct volume in vivo, and enhanced cell survival by activating autophagy and suppressing apoptosis in vitro. Conclusions: Our findings indicate that BAG3 overexpression activates autophagy and inhibits apoptosis to prevent cerebral ischemia/reperfusion and hypoxia/reoxygenation injury, suggesting a potential therapeutic benefit of BAG3 expression in cerebral ischemia.

Background: The transfer of mitochondria from healthy mesenchymal stem cells (MSCs) to injured MSCs has been shown to have potential therapeutic benefits for neural cell post-ischemic stroke. Specifically, functional mitochondria can perform their normal functions after being internalized by stressed cells, leading to host cell survival. However, while this approach shows promise, there is still a lack of understanding regarding which neural cells can internalize functional mitochondria and the regulatory mechanisms involved. To address this gap, we investigated the ability of different neural cells to internalize exogenous functional mitochondria extracted from MSCs.Methods: Functional mitochondria (F-Mito) isolated from umbilical cord derived-MSCs (UCMSCs) were labeled with lentivirus of HBLV-mito-dsred-Null-PURO vector. The ability of stressed cells to internalize F-Mito was analyzed using a mouse (C57BL/6 J) middle cerebral artery occlusion (MCAO) model and an oxygen-glucose deprivation/reoxygenation (OGD/R) cell model. The cell viability was measured by CCK-8 kit. Time-course of intracellular ROS levels in stressed cells were analyzed by DCFH-DA staining after OGD/R and F-Mito treatment. MitoSOX, Mitotracker and WGA labeling were used to assess the relationship between ROS levels and the uptake of F-Mito at the single-cell level. Pharmacological modulation of ROS was performed using acetylcysteine (ROS inhibitor).Results: Our findings demonstrate that neurons and endothelial cells are more effective at internalizing mitochondria than astrocytes, both in vitro and in vivo, using an ischemia-reperfusion model. Additionally, internalized F-Mito decreases host cell reactive oxygen species (ROS) levels and rescues survival. Importantly, we found that the ROS response in stressed cells after ischemia is a crucial determinant in positively mediating the internalization of F-Mito by host cells, and inhibiting the generation of ROS chemicals in host cells may decrease the internalization of F-Mito. These results offer insight into how exogenous mitochondria rescue neural cells via ROS response in an ischemic stroke model. Overall, our study provides solid evidence for the translational application of MSC-derived mitochondria as a promising treatment for ischemic stroke.

Background and Purpose There has been increasing interest in the causes and pathogenesis of depression, which is a common psychiatric disorder. We aimed to investigate the protective effect of hops ethyl acetate extract (HEA) on neuroinflammation-mediated lipopolysaccharide (LPS)-induced depression-like symptoms. Experimental Approach A battery of behavior tests, including the open field test (OFT), elevated plus maze (EPM), tail suspension test (TST), and forced swimming test (FST), was used to evaluate the effects of HEA on LPS-induced depression. Furthermore, the levels of inflammatory factors (tumor necrosis factor-α [TNF-α], interleukin-1β) and norepinephrine were evaluated through enzyme-linked immunosorbent assay. The density of hippocampal dendritic spines was assessed through Golgi staining. Finally, the toxicological effects of hops extract on depression in mice were further analyzed through hematoxylin and eosin staining and blood biochemistry. Key Results Based on the OFT, EPM, TST, and FST results, oral gavage HEA prevented LPS-induced depression-like behaviors in the mice. Further, HEA reduced neuroinflammation, increased norepinephrine levels, and increased the density of hippocampal dendritic spines. Finally, blood biochemistry and HE staining did not reveal any side effects or toxicity of HEA. Conclusion and Implications Our findings indicated that HEA is a potential compound for treating depression.