Optimization, Characterization, and Comparison of Two Luciferase-Expressing Mouse Glioblastoma Models.

Glioblastoma (GBM) is the most aggressive brain cancer. To model GBM in research, orthotopic brain tumor models, including syngeneic models like GL261 and genetically engineered mouse models like TRP, are used. In longitudinal studies, tumor growth and the treatment response are typically tracked with in vivo imaging, including bioluminescence imaging (BLI), which is quick, cost-effective, and easily quantifiable. However, BLI requires luciferase-tagged cells, and recent studies indicate that the luciferase gene can elicit an immune response, leading to tumor rejection and experimental variation. We sought to optimize the engraftment of two luciferase-expressing GBM models, GL261 Red-FLuc and TRP-mCherry-FLuc, showing differences in tumor take, with GL261 Red-FLuc cells requiring immunocompromised mice for 100% engraftment. Immunohistochemistry and MRI revealed distinct tumor characteristics: GL261 Red-FLuc tumors were well-demarcated with densely packed cells, high mitotic activity, and vascularization. In contrast, TRP-mCherry-FLuc tumors were large, invasive, and necrotic, with perivascular invasion. Quantifying the tumor volume using the HALO® AI analysis platform yielded results comparable to manual measurements, providing a standardized and efficient approach for the reliable, high-throughput analysis of luciferase-expressing tumors. Our study highlights the importance of considering tumor engraftment when using luciferase-expressing GBM models, providing insights for preclinical research design.

Cognitive decline, Aß pathology, and blood-brain barrier function in aged 5xFAD mice.

BACKGROUND: Patients with Alzheimer's disease (AD) develop blood-brain barrier dysfunction to varying degrees. How aging impacts Aß pathology, blood-brain barrier function, and cognitive decline in AD remains largely unknown. In this study, we used 5xFAD mice to investigate changes in Aß levels, barrier function, and cognitive decline over time. METHODS: 5xFAD and wild-type (WT) mice were aged between 9.5 and 15.5 months and tested for spatial learning and reference memory with the Morris Water Maze (MWM). After behavior testing, mice were implanted with acute cranial windows and intravenously injected with fluorescent-labeled dextrans to assess their in vivo distribution in the brain by two-photon microscopy. Images were processed and segmented to obtain intravascular intensity, extravascular intensity, and vessel diameters as a measure of barrier integrity. Mice were sacrificed after in vivo imaging to isolate brain and plasma for measuring Aß levels. The effect of age and genotype were evaluated for each assay using generalized or cumulative-linked logistic mixed-level modeling and model selection by Akaike Information Criterion (AICc). Pairwise comparisons were used to identify outcome differences between the two groups. RESULTS: 5xFAD mice displayed spatial memory deficits compared to age-matched WT mice in the MWM assay, which worsened with age. Memory impairment was evident in 5xFAD mice by 2-threefold higher escape latencies, twofold greater cumulative distances until they reach the platform, and twice as frequent use of repetitive search strategies in the pool when compared with age-matched WT mice. Presence of the rd1 allele worsened MWM performance in 5xFAD mice at all ages but did not alter the rate of learning or probe trial outcomes. 9.5-month-old 15.5-month-old 5xFAD mice had twofold higher brain Aß40 and Aß42 levels (p < 0.001) and 2.5-fold higher (p = 0.007) plasma Aß40 levels compared to 9.5-month-old 5xFAD mice. Image analysis showed that vessel diameters and intra- and extravascular dextran intensities were not significantly different in 9.5- and 15.5-month-old 5xFAD mice compared to age-matched WT mice. CONCLUSION: 5xFAD mice continue to develop spatial memory deficits and increased Aß brain levels while aging. Given in vivo MP imaging limitations, further investigation with smaller molecular weight markers combined with advanced imaging techniques would be needed to reliably assess subtle differences in barrier integrity in aged mice.

Proteasome inhibition protects blood-brain barrier P-glycoprotein and lowers Aß brain levels in an Alzheimer's disease model.

BACKGROUND: Loss of P-glycoprotein (P-gp) at the blood-brain barrier contributes to amyloid-ß (Aß) brain accumulation in Alzheimer's disease (AD). Using transgenic human amyloid precursor protein (hAPP)-overexpressing mice (Tg2576), we previously showed that Aß triggers P-gp loss by activating the ubiquitin-proteasome pathway, which leads to P-gp degradation. Furthermore, we showed that inhibiting the ubiquitin-activating enzyme (E1) prevents P-gp loss and lowers Aß accumulation in the brain of hAPP mice. Based on these data, we hypothesized that repurposing the FDA-approved proteasome inhibitor, bortezomib (Velcade®; BTZ), protects blood-brain barrier P-gp from degradation in hAPP mice in vivo. METHODS: We treated hAPP mice with the proteasome inhibitor BTZ or a combination of BTZ with the P-gp inhibitor cyclosporin A (CSA) for 2 weeks. Vehicle-treated wild-type (WT) mice were used as a reference for normal P-gp protein expression and transport activity. In addition, we used the opioid receptor agonist loperamide as a P-gp substrate in tail flick assays to indirectly assess P-gp transport activity at the blood-brain barrier in vivo. We also determined P-gp protein expression by Western blotting, measured P-gp transport activity levels in isolated brain capillaries with live cell confocal imaging and assessed Aß plasma and brain levels with ELISA. RESULTS: We found that 2-week BTZ treatment of hAPP mice restored P-gp protein expression and transport activity in brain capillaries to levels found in WT mice. We also observed that hAPP mice displayed significant loperamide-induced central antinociception compared to WT mice indicating impaired P-gp transport activity at the blood-brain barrier of hAPP mice in vivo. Furthermore, BTZ treatment prevented loperamide-induced antinociception suggesting BTZ protected P-gp loss in hAPP mice. Further, BTZ-treated hAPP mice had lower Aß40 and Aß42 brain levels compared to vehicle-treated hAPP mice. CONCLUSIONS: Our data indicate that BTZ protects P-gp from proteasomal degradation in hAPP mice, which helps to reduce Aß brain levels. Our data suggest that the proteasome system could be exploited for a novel therapeutic strategy in AD, particularly since increasing Aß transport across the blood-brain barrier may prove an effective treatment for patients.

Virus-like particle expression and assembly in plants: hepatitis B and Norwalk viruses.

Expression of vaccine antigens in plants and delivery via ingestion of transgenic plant material has shown promise in numerous pre-clinical animal studies and in a few clinical trials. A number of different viral antigens have been tested, and among the most promising are those that can assemble virus-like particles (VLP), which mimic the form of authentic virions and display neutralizing antibody epitopes. We have extensively studied plant expression, VLP assembly, and immunogenicity of hepatitis B surface antigen (HBsAg) and Norwalk virus capsid protein (NVCP). The HBsAg small protein (S protein) was found by TEM to assemble tubular membrane complexes derived from endoplasmic reticulum in suspension cultured cells of tobacco and soybean, and in potato leaf and tuber tissues. The potato material was immunogenic in mice upon delivery by ingestion. Here we describe the plant expression and immunogenicity of HBsAg middle protein (M protein or pre-S2 + S) which contains additional 55 amino acid pre-S2 region at N-terminus of the S protein. Plant-derived recombinant M protein provoked stronger serum antibody responses against HBsAg than did S protein when injected systemically in mice. We discuss implications for use of fusion proteins for enhanced immunogenicity and mucosal targeting of HBsAg, as well as delivery of heterologous fused antigens. NVCP expressed in plants assembled 38 nm virion-size icosahedral (T = 3) VLP, similar to those produced in insect cells. The VLP stimulated serum IgG and IgA responses in mice and humans when they were delivered by ingestion of fresh potato tuber. Here we show that freeze-drying of transgenic NVCP tomato fruit yielded stable preparations that stimulated excellent IgG and IgA responses against NVCP when fed to mice. However, the predominant VLP form in tomato fruit was the small 23 nm particle also observed in insect cell-derived NVCP.

Challenges in creating a vaccine to prevent hepatitis E.

Recombinant hepatitis E virus capsid protein (HEV CP) assembles orally immunogenic virus-like particles (VLP) when expressed in an insect cell system. We used plant expression cassettes, pHEV101 and pHEV110, for transformation of potato to express HEV CP, and 10 independent transgenic lines of HEV101 and 6 lines of HEV110 were obtained. ELISA for HEV CP was performed on tuber extracts. Accumulation of HEV CP in tubers varied from about 5 to 30 microg/g fresh tuber depending on the transgenic plant line. We further compared the expression levels with the yield of tubers for each line. Tuber yield varied less than expression levels, and ranged from about 600 to 1000 g per pot. Although Western blot showed that apparently intact HEV CP accumulated, we observed very limited assembly of virus-like particles in potato tubers. Oral immunization of mice with transgenic potatoes failed to elicit detectable anti-CP antibody response in serum, suggesting that VLP assembly is a key factor in orally delivered HEV CP vaccines.