Organellar stress intersects the astrocyte endoplasmic reticulum, mitochondria and nucleolus in HIV associated neurodegeneration

Primary human astrocytes were treated with IL-1β (20 ng/ml) and HIV-1 (p24, 20 ng/ml) alone or in combination (a) or with increasing concentrations of hydrogen peroxide (H2O2) (100？μM, 200？μM, and 400 μM) (b, c) and untreated astrocytes were maintained in parallel. RNA was isolated (8？h) and RRS1 mRNA levels were analyzed by RT2-PCR. Total cell lysates were immunoblotted for RRS1 and GAPDH following 24？h. GAPDH was used as housekeeping and loading controls. A representative blot is presented, while the average fold change from multiple independent donors is graphed. Statistical analyses were performed using one-way ANOVA with Tukey’s post-test for multiple comparisons (*p？<？0.05, **p？<？0.01, ***p？<？0.001). Data represent mean？±？standard error of the mean. d Astrocyte organelle stress responses during HAND (1) HIV-1 infected/activated CNS cells release HIV-1 proteins, ROS, and proinflammatory cytokines such as TNF-α and IL-1β. (2) Astrocytes become reactive in response to HIV-relevant stimuli. (3) Our data establish that IL-1β- and ARV-mediated increases in intracellular calcium initiate ER stress and mitochondrial dysfunction. (4) Activation of the UPR pathways PERK, IRE1α, and ATF6 result in expression of ER chaperones BiP and calnexin. (5) AEG-1 interaction with the calcium-binding chaperone calnexin suggests a role in calcium signaling. (6) HIV-1-relevant inflammation and oxidative stress significantly increase RRS1 expression, suggesting inhibition of rRNA transcription and nucleolar stress in astrocytes during HIV-1 infection. Taken together, we propose that the organelle network with interlinked stress responses may play a crucial role in HIV-associated neuropathogenesis and could be promising therapeutic targets in HAND. Scheme abbreviations: AEG-1 astrocyte elevated gene, ARV antiretroviral, ATF activating transcription factor, BiP binding immunoglobulin protein, Ca2+ calcium, ER endoplasmic reticulum, ERAD ER-associated degradation, f fragment, HIV human immunodeficiency virus, ROS reactive oxygen species, p-eIF phosphorylated-eukaryotic initiation factor, PERK protein kinase RNA-like ER kinase, IRE inositol-requiring enzyme, RRS regulator of ribosome synthesis, XBP x-box binding protei