Antiretroviral Tenofovir Induces Senescence-Associated ß-Galactosidase Activity in Primary Human Brain Vascular Cells in Multi-Layer Three-Dimensional Co-Culture.

Introduction In the current context of early diagnosis of HIV infection, immediate initiation of antiretroviral (ARV) therapy, and lifelong chronic treatment, the potential ARV toxicity is of particular concern. Emtricitabine (FTC) and tenofovir (TFV) are commonly used as backbone drugs in ARV regimens recommended for initial therapy of HIV infection. Here we assessed the effects of FTC and TFV exposure on senescence-associated ß-galactosidase (SA-ß-Gal) activity, a marker of cellular senescence, in human brain vascular cells. Design Multi-layer three-dimensional cell co-cultures and in vitro assays. Methods To mimic the small vessel wall structure in vivo, three types of primary human brain vascular cells (endothelial cells, smooth muscle cells, and pericytes) were co-cultured on three Alvetex Scaffold disks placed on top of each other in order (three-layer three-dimensional cell co-cultures) and exposed to clinically relevant concentrations of ARV drugs (FTC, TFV, or FTC+TFV combination) or vehicle for eight days (four or five biological replicates per condition, 18 replicates totally). The SA-ß-Gal activity was quantitatively assayed in vitro by using the chemiluminescent Galacto-Star System (T1012; Applied Biosystems, Thermo Fisher Scientific, Waltham, MA) in 54 protein lysates extracted from individual cell-culture disks. Three-factor analysis of variance (cell type, FTC, TFV) was used to assess differences in the SA-ß-Gal activity levels normalized by the corresponding total protein concentrations. Results There was a trend for the FTC by TFV interaction effect on SA-ß-Gal activity (P = 0.058). The effects of FTC and TFV were not significantly different among the three cell types. The overall effect of FTC was not significant when controlling for TFV and cell type. The overall effect of TFV was significant when controlling for FTC and cell type (F(1,48) = 30.61, P < 0.001, partial η2 = 0.389). In the absence of FTC, TFV raised SA-ß-Gal activity by 0.631 units on average, regardless of cell type (P < 0.001, partial η2 = 0.368). In the presence of FTC, TFV raised SA-ß-Gal activity by 0.303 units on average, regardless of cell type (P = 0.015, partial η2 = 0.118). Conclusion Our preliminary findings suggest that primary human brain vascular cells exposed to TFV at clinically relevant concentrations undergo cellular senescence. This potential adverse effect of TFV should be further studied in animal models of HIV infection.

Loss of Immunohistochemical Reactivity in Association With Handling-Induced Dark Neurons in Mouse Brains.

The handling-induced dark neuron is a histological artifact observed in brain samples handled before fixation with aldehydes. To explore associations between dark neurons and immunohistochemical alterations in mouse brains, we examined protein products encoded by Cav3 (neuronal perikarya/neurites), Rbbp4 (neuronal nuclei), Gfap (astroglia), and Aif1 (microglia) genes in adjacent tissue sections. Here, dark neurons were incidental findings from our prior project, studying the effects of age and high-fat diet on metabolic homeostasis in male C57BL/6N mice. Available were brains from 4 study groups: middle-aged/control diet, middle-aged/high-fat diet, old/control diet, and old/high-fat diet. Young/control diet mice were used as baseline. The hemibrains were immersion-fixed with paraformaldehyde and paraffin-embedded. In the hippocampal formation, we found negative correlations between dark neuron hyperbasophilia and immunoreactivity for CAV3, RBBP4, and glial fibrillary acidic protein (GFAP) using quantitative image analysis. There was no significant difference in dark neuron hyperbasophilia or immunoreactivity for any protein examined among all groups. In contrast, in the hippocampal fimbria, old age seemed to be associated with higher immunoreactivity for GFAP and allograft inflammatory factor-1. Our findings suggest that loss of immunohistochemical reactivity for CAV3, RBBP4, and GFAP in the hippocampal formation is an artifact associated with the occurrence of dark neurons. The unawareness of dark neurons may lead to misinterpretation of immunohistochemical reactivity alterations.

Protein disulphide isomerase is associated with mutant SOD1 in canine degenerative myelopathy.

Canine degenerative myelopathy (DM) is a fatal neurodegenerative disorder prevalent in the canine population. It may represent a unique, naturally occurring disease model for human amyotrophic lateral sclerosis (ALS) because of similar clinical signs and association with superoxide dismutase 1 gene (SOD1) mutations. Misfolded SOD1 aggregates and endoplasmic reticulum (ER) stress are major pathophysiological features associated with ALS. Interestingly, an ER foldase, protein disulphide isomerase (PDI) is upregulated during ALS and it co-localizes with SOD1 inclusions in ALS patient tissues. Furthermore, mutations in the gene encoding PDI were recently associated with ALS. Given the genetic similarity between DM and ALS, we investigated whether ER stress and PDI were associated with DM. Protein extracts from spinal cord tissue of DM-affected dogs bearing a SOD1 mutation were examined for ER stress by western blotting. Immunohistochemical staining was also carried out to examine co-localization between endogenous PDI and SOD1 inclusions in spinal cord tissues of dogs affected with DM. PDI and CHOP, the proapoptotic protein induced during ER stress, were significantly upregulated in DM-affected dogs compared with controls. Furthermore, PDI co-localized with intracellular SOD1 aggregates in DM-affected dogs in all motor neurons examined, indicating that PDI may be a cellular defence mechanism against SOD1 misfolding in DM. Our results imply that ER stress is induced in DM-affected dogs; hence, it is a common pathological mechanism associated with both ALS and DM. The possibility that PDI may be a therapeutic target to inhibit SOD1 aggregation in DM dogs is also raised by this study.