HIV-associated neurocognitive disorders (HAND) afflict about half of HIV-infected patients. HIV-infected cells shed viral proteins, such as the transactivator of transcription (Tat), which can cause neurotoxicity by over activation of NMDA receptors (NMDARs). Here, we show that Tat causes a time-dependent, biphasic change in NMDA-evoked increases in intracellular Ca2+ concentration ([Ca2+]i). NMDA-evoked responses were potentiated following 2 h exposure to Tat (50 ng/mL). Tat-induced potentiation of NMDA-evoked increases in [Ca2+]i peaked by 8 h and then adapted by gradually reversing to baseline by 24 h and eventually dropping below control by 48 h. Tat-induced potentiation of NMDA-evoked responses was blocked by inhibition of lipoprotein receptors (LRP) or Src tyrosine kinase. Potentiation was unaffected by inhibition of nitric oxide synthase (NOS). However, NOS activity was required for adaptation. Adaptation was also prevented by inhibition of soluble guanylate cyclase (sGC) and cGMP-dependent protein kinase (PKG). Together, these findings indicate that Tat potentiates NMDA-evoked increases in [Ca2+]i via LRP-dependent activation of Src and that this potentiation adapts via activation of the NOS/sGC/PKG pathway. Adaptation may protect neurons from excessive Ca2+ influx and could reveal targets for the treatment of HAND.

Inositol 1, 4, 5-trisphosphate receptor (IP3R)-mediated Ca2+ release from the endoplasmic reticulum (ER) triggers many physiological responses in neurons and when uncontrolled can cause ER stress that contributes to neurological disease. Here we show that the unfolded protein response (UPR) in neurons induces rapid translocation of nuclear receptor-interacting protein 140 (RIP140) to the cytoplasm. In the cytoplasm, RIP140 localizes to the ER by binding to the IP3R. The carboxyl-terminal RD4 domain of RIP140 interacts with the carboxyl-terminal gate-keeping domain of the IP3R. This molecular interaction disrupts the IP3R's “head-tail” interaction, thereby suppressing channel opening and attenuating IP3R-mediated Ca2+ release. This contributes to a rapid suppression of the ER stress response and provides protection from apoptosis in both hippocampal neurons in vitro and in an animal model of ER stress. Thus, RIP140 translocation to the cytoplasm is an early response to ER stress and provides protection against neuronal death.

Apolipoprotein E (apoE) has an important role in the pathogenesis of Alzheimer's disease with its three isoforms having distinct effects on disease risk. Here, we assessed the conformational differences between those isoforms using a novel flow cytometry-Forster resonance energy transfer (FRET) assay. We showed that the conformation of intracellular apoE within HEK cells and astrocytes adopts a directional pattern; in other words, E4 adopts the most closed conformation, E2 adopts the most open conformation, and E3 adopts an intermediate conformation. However, this pattern was not maintained upon secretion of apoE from astrocytes. Intermolecular interactions between apoE molecules were isoform-specific, indicating a great diversity in the structure of apoE lipoparticles. Finally, we showed that secreted E4 is the most lipidated isoform in astrocytes, suggesting that increased lipidation acts as a folding chaperone enabling E4 to adopt a closed conformation. In conclusion, this study gives insights into apoE biology and establishes a robust screening system to monitor apoE conformation.