NOX activation in reactive astrocytes regulates astrocytic LCN2 expression and neurodegeneration.

Reactive astrocytes (RA) secrete lipocalin-2 (LCN2) glycoprotein that regulates diverse cellular processes including cell death/survival, inflammation, iron delivery and cell differentiation. Elevated levels of LCN2 are considered as a biomarker of brain injury, however, the underlying regulatory mechanisms of its expression and release are not well understood. In this study, we investigated the role of astrocytic Na+/H+ exchanger 1 (NHE1) in regulating reactive astrocyte LCN2 secretion and neurodegeneration after stroke. Astrocyte specific deletion of Nhe1 in Gfap-CreER+/-;Nhe1f/f mice reduced astrogliosis and astrocytic LCN2 and GFAP expression, which was associated with reduced loss of NeuN+ and GRP78+ neurons in stroke brains. In vitro ischemia in astrocyte cultures triggered a significant increase of secreted LCN2 in astrocytic exosomes, which caused neuronal cell death and neurodegeneration. Inhibition of NHE1 activity during in vitro ischemia with its potent inhibitor HOE642 significantly reduced astrocytic LCN2+ exosome secretion. In elucidating the cellular mechanisms, we found that stroke triggered activation of NADPH oxidase (NOX)-NF-κB signaling and ROS-mediated LCN2 expression. Inhibition of astrocytic NHE1 activity attenuated NOX signaling and LCN2-mediated neuronal apoptosis and neurite degeneration. Our findings demonstrate for the first time that RA use NOX signaling to stimulate LCN2 expression and secretion. Blocking astrocytic NHE1 activity is beneficial to reduce LCN2-mediated neurotoxicity after stroke.

Functional Characterization of Brain Tumor-Initiating Cells and Establishment of GBM Preclinical Models that Incorporate Heterogeneity, Therapy, and Sex Differences.

Glioblastoma (GBM) is the most common primary brain cancer in adults where tumor cell heterogeneity and sex differences influence clinical outcomes. Here, we functionally characterize three male and three female patient-derived GBM cell lines, identify protumorigenic BTICs, and create novel male and female preclinical models of GBM. Cell lines were evaluated on the following features: proliferation, stemness, migration, tumorigenesis, clinical characteristics, and sensitivity to radiation, TMZ, rhTNFSF10 (rhTRAIL), and rhBMP4 All cell lines were classified as GBM according to epigenetic subtyping, were heterogenous and functionally distinct from one another, and re-capitulated features of the original patient tumor. In establishing male and female preclinical models, it was found that two male-derived GBM cell lines (QNS108 and QNS120) and one female-derived GBM cell line (QNS315) grew at a faster rate in female mice brains. One male-derived GBM cell line (QNS108) decreased survival in female mice in comparison with male mice. However, no survival differences were observed for mice injected with a female-derived cell line (QNS315). In summary, a panel of six GBM patient-derived cell lines were functionally characterized, and it was shown that BTIC lines can be used to construct sex-specific models with differential phenotypes for additional studies.

Poly(ethylene glycol)-Poly(beta-amino ester)-Based Nanoparticles for Suicide Gene Therapy Enhance Brain Penetration and Extend Survival in a Preclinical Human Glioblastoma Orthotopic Xenograft Model.

Glioblastoma (GBM) is the most devastating brain cancer, and cures remain elusive with currently available neurosurgical, pharmacological, and radiation approaches. While retrovirus- and adenovirus-mediated suicide gene therapy using DNA encoding herpes simplex virus-thymidine kinase (HSV-tk) and prodrug ganciclovir has been suggested as a promising strategy, a nonviral approach for treatment in an orthotopic human primary brain tumor model has not previously been demonstrated. Delivery challenges include nanoparticle penetration through brain tumors, efficient cancer cell uptake, endosomal escape to the cytosol, and biodegradability. To meet these challenges, we synthesized poly(ethylene glycol)-modified poly(beta-amino ester) (PEG-PBAE) polymers to improve extracellular delivery and coencapsulated plasmid DNA with end-modified poly(beta-amino ester) (ePBAE) polymers to improve intracellular delivery as well. We created and evaluated a library of PEG-PBAE/ePBAE nanoparticles (NPs) for effective gene therapy against two independent primary human stem-like brain tumor initiating cells, a putative target to prevent GBM recurrence. The optimally engineered PEG-PBAE/ePBAE NP formulation demonstrated 54 and 82% transfection efficacies in GBM1A and BTIC375 cells respectively, in comparison to 37 and 66% for optimized PBAE NPs without PEG. The leading PEG-PBAE NP formulation also maintained sub-250 nm particle size up to 5 h, while PBAE NPs without PEG showed aggregation over time to micrometer-sized complexes. The comparative advantage demonstrated in vitro successfully translated into improved in vivo diffusion, with a higher amount of PEG-PBAE NPs penetrating to a distance of 2 mm from the injection site. A significant increase in median survival from 53.5 to 67 days by PEG-PBAE/pHSV-tk NP and systemic ganciclovir treatment compared to a control group in orthotopic murine model of human glioblastoma demonstrates the potential of PEG-PBAE-based NPs as an effective gene therapy platform for the treatment of human brain tumors.