Isoform- and cell type-specific structure of apolipoprotein E lipoparticles as revealed by a novel Forster resonance energy transfer assay.

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.

Interleukin-1ß activates an Src family kinase to stimulate the plasma membrane Ca2+ pump in hippocampal neurons.

The plasma membrane Ca(2+) ATPase (PMCA) plays a major role in clearing Ca(2+) from the neuronal cytoplasm. The cytoplasmic Ca(2+) clearance rate affects neuronal excitability, synaptic plasticity, and neurotransmission. Here, we examined the modulation of PMCA activity by PTKs in hippocampal neurons. PMCA-mediated Ca(2+) clearance slowed in the presence of pyrazolopyrimidine 2, an inhibitor of Src family kinases (SFKs), and accelerated in the presence of C2-ceramide, an activator of PTKs. Ca(2+) clearance kinetics were attenuated in cells expressing a dominant-negative Src mutant, suggesting that the pump is tonically stimulated by a PTK. Tonic stimulation was reduced in hippocampal neurons expressing short hairpin (sh)RNA directed to mRNA for Yes. shRNA-mediated knockdown of PMCA isoform 1 (PMCA1) removed tonic stimulation of Ca(2+) clearance, indicating that the kinase stimulates PMCA1. IL-1ß accelerated Ca(2+) clearance in a manner blocked by an IL-1ß receptor antagonist or by an inhibitor of neutral sphingomyelinase, the enzyme that produces ceramide. Thus IL-1ß activates an SFK to stimulate the plasma membrane Ca(2+) pump, decreasing the duration of Ca(2+) transients in hippocampal neurons.

HIV-1 Tat activates a RhoA signaling pathway to reduce NMDA-evoked calcium responses in hippocampal neurons via an actin-dependent mechanism.

HIV-associated neurocognitive disorders afflict approximately half of HIV-infected patients. HIV-infected cells within the CNS release neurotoxic viral proteins such as the transactivator of transcription (Tat). Tat caused a biphasic change in NMDAR function; NMDA-evoked increases in intracellular Ca(2+) were initially potentiated following 16 h exposure to Tat and then adapted by gradually returning to baseline by 24 h. Following Tat-induced NMDAR potentiation, a RhoA/Rho-associated protein kinase (ROCK) signaling pathway was activated; a subsequent remodeling of the actin cytoskeleton reduced NMDA-evoked increases in intracellular Ca(2+) . Pharmacologic or genetic inhibition of RhoA or ROCK failed to affect potentiation, but prevented adaptation of NMDAR function. Activation of RhoA/ROCK signaling increases the formation of filamentous actin. Drugs that prevent changes to filamentous actin blocked adaptation of NMDAR function following Tat-induced potentiation, whereas stimulating either depolymerization or polymerization of actin attenuated NMDAR function. These findings indicate that Tat activates a RhoA/ROCK signaling pathway resulting in actin remodeling and subsequent reduction of NMDAR function. Adaptation of NMDAR function may be a mechanism to protect neurons from excessive Ca(2+) influx and could reveal targets for the treatment of HIV-associated neurocognitive disorders.

Receptor-interacting protein 140 attenuates endoplasmic reticulum stress in neurons and protects against cell death.

Inositol 1, 4, 5-trisphosphate receptor (IP3R)-mediated Ca(2+) 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 Ca(2+) 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.

HIV-1 protein Tat produces biphasic changes in NMDA-evoked increases in intracellular Ca2+ concentration via activation of Src kinase and nitric oxide signaling pathways.

HIV-associated neurocognitive disorders 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. Here, we show that Tat causes a time-dependent, biphasic change in NMDA-evoked increases in intracellular Ca(2+) concentration ([Ca(2+)]i). NMDA-evoked responses were potentiated following 2-h exposure to Tat (50 ng/mL). Tat-induced potentiation of NMDA-evoked increases in [Ca(2+)]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 receptor-related protein (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 cyclic guanosine monophosphate-dependent protein kinase G (PKG). Together, these findings indicate that Tat potentiates NMDA-evoked increases in [Ca(2+)]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 Ca(2+) influx and could reveal targets for the treatment of HIV-associated neurocognitive disorders. 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). We show that HIV-1 Tat evoked biphasic changes in NMDA-evoked [Ca(2+) ]i responses. Initially, Tat potentiated NMDA-evoked responses following LRP-mediated activation of Src kinase. Subsequently, Tat-induced NMDAR potentiation adapted by activation of a NOS/sGC/PKG pathway that attenuated NMDA-evoked increases in [Ca(2+)]i . Adaptation may be a novel neuroprotective mechanism to prevent excessive Ca(2+) influx. Solid and dashed arrows represent direct and potentially indirect connections, respectively.

HIV-1 Tat-induced changes in synaptically-driven network activity adapt during prolonged exposure.

HIV-associated neurocognitive disorders (HAND) afflict approximately half of HIV-infected patients. The HIV-1 transactivator of transcription (Tat) protein is released by infected cells and contributes to the pathogenesis of HAND, but many of the underlying mechanisms remain poorly understood. Here we used fura-2-based Ca(2+) imaging and whole-cell patch-clamp recording to study the effects of Tat on the spontaneous synaptic activity that occurs in networked rat hippocampal neurons in culture. Tat triggered aberrant network activity that exhibited a decrease in the frequency of spontaneous action potential bursts and Ca(2+) spikes with a simultaneous increase in burst duration and Ca(2+) spike amplitude. These network changes were apparent after 4 h treatment with Tat and required the low-density lipoprotein receptor-related protein (LRP). Interestingly, Tat-induced changes in network activity adapted during 24 h exposure. The activity returned to control levels in the maintained presence of Tat for 24 h. These observations indicate that Tat causes aberrant network activity, which is dependent on LRP, and adapts following prolonged exposure. Changes in network excitability may contribute to Tat-induced neurotoxicity in vitro and seizure disorders in vivo. Adaptation of neural networks may be a neuroprotective response to the sustained presence of the neurotoxic protein Tat and could underlie the behavioral and electrophysiological changes observed in HAND.

Epileptiform stimulus increases Homer 1a expression to modulate synapse number and activity in hippocampal cultures.

Neurons adapt to seizure activity structurally and functionally to attenuate hyperactive neural circuits. Homer proteins provide a scaffold in the postsynaptic density (PSD) by binding to ligands through an EVH1 domain and to other Homer proteins by a coiled-coil domain. The short Homer isoform 1a (H1a) has a ligand-binding domain but lacks a coiled-coil domain and thus acts in a dominant-negative manner to uncouple Homer scaffolds. Here, we show that treating rat hippocampal cultures with bicuculline and 4-aminopyridine (Bic+4-AP) evoked epileptiform activity and synchronized Ca(2+) spiking, measured with whole cell current-clamp and fura-2-based digital imaging; Bic+4-AP increased H1a mRNA through the activation of metabotropic glutamate receptor 5 (mGluR5). Treatment with Bic+4-AP for 4 h attenuated burst firing and induced synapse loss. Synaptic changes were measured using a confocal imaging-based assay that quantified clusters of PSD-95 fused to green fluorescent protein. Treatment with an mGluR5 antagonist blocked H1a expression, synapse loss, and burst attenuation. Overexpression of H1a inhibited burst firing similar to Bic+4-AP treatment. Furthermore, knockdown of H1a using a short hairpin RNA (shRNA) strategy reduced synapse loss and burst attenuation induced by Bic+4-AP treatment. Thus an epileptiform stimulus applied to hippocampal neurons in culture induced burst firing and H1a expression through the activation of mGluR5; a 4-h exposure to this stimulus resulted in synapse loss and burst attenuation. These results suggest that H1a expression functions in a negative-feedback manner to reduce network excitability by regulating the number of synapses.

Epileptic stimulus increases Homer 1a expression to modulate endocannabinoid signaling in cultured hippocampal neurons.

Endocannabinoid (eCB) signaling serves as an on-demand neuroprotective system. eCBs are produced postsynaptically in response to depolarization or activation of metabotropic glutamate receptors (mGluRs) and act on presynaptic cannabinoid receptor-1 to suppress synaptic transmission. Here, we examined the effects of epileptiform activity on these two forms of eCB signaling in hippocampal cultures. Treatment with bicuculline and 4-aminopyridine (Bic + 4-AP), which induced burst firing, inhibited metabotropic-induced suppression of excitation (MSE) and prolonged the duration of depolarization-induced suppression of excitation (DSE). The Homer family of proteins provides a scaffold for signaling molecules including mGluRs. It is known that seizures induce the expression of the short Homer isoform 1a (H1a) that acts in a dominant negative manner to uncouple Homer scaffolds. Bic + 4-AP treatment increased H1a mRNA. A group I mGluR antagonist blocked the Bic + 4-AP-evoked increase in burst firing, the increase in H1a expression, and the inhibition of MSE. Bic + 4-AP treatment reduced mGluR-mediated Ca(2+) mobilization from inositol trisphosphate-sensitive stores relative to untreated cells. Expression of H1a, but not a mutant form that cannot bind Homer ligands, mimicked Bic + 4-AP inhibition of MSE and mGluR-mediated Ca(2+) mobilization. In cells expressing shRNA targeted to Homer 1 mRNA, Bic + 4-AP did not affect mGluR-mediated Ca(2+) release. Furthermore, knockdown of H1a prevented the inhibition of MSE induced by Bic + 4-AP. Thus, an epileptic stimulus increased H1a expression, which subsequently uncoupled mGluR-mediated eCB production. These results indicate that seizure activity modulates eCB-mediated synaptic plasticity, suggesting a changing role for the eCB system following exposure to aberrant patterns of excitatory synaptic activity.