EWI-2 Inhibits Cell-Cell Fusion at the HIV-1 Virological Presynapse.

Cell-to-cell transfer of virus particles at the Env-dependent virological synapse (VS) is a highly efficient mode of HIV-1 transmission. While cell-cell fusion could be triggered at the VS, leading to the formation of syncytia and preventing exponential growth of the infected cell population, this is strongly inhibited by both viral (Gag) and host (ezrin and tetraspanins) proteins. Here, we identify EWI-2, a protein that was previously shown to associate with ezrin and tetraspanins, as a host factor that contributes to the inhibition of Env-mediated cell-cell fusion. Using quantitative fluorescence microscopy, shRNA knockdowns, and cell-cell fusion assays, we show that EWI-2 accumulates at the presynaptic terminal (i.e., the producer cell side of the VS), where it contributes to the fusion-preventing activities of the other viral and cellular components. We also find that EWI-2, like tetraspanins, is downregulated upon HIV-1 infection, most likely by Vpu. Despite the strong inhibition of fusion at the VS, T cell-based syncytia do form in vivo and in physiologically relevant culture systems, but they remain small. In regard to that, we demonstrate that EWI-2 and CD81 levels are restored on the surface of syncytia, where they (presumably) continue to act as fusion inhibitors. This study documents a new role for EWI-2 as an inhibitor of HIV-1-induced cell-cell fusion and provides novel insight into how syncytia are prevented from fusing indefinitely.

Axonal and subcellular labelling using modified rabies viral vectors.

An important aspect of any neural circuit is the placement of its output synapses, at levels ranging from macroscopic to subcellular. The many new molecular tools for locating and manipulating synapses are limited by the viral vectors available for delivering them. Adeno-associated viruses are the best current means of labelling and manipulating axons and synapses, but they have never expressed more than one transgene highly enough to label fine axonal structure while also labelling or perturbing synapses. Their slow expression also makes them incompatible with retrograde and transsynaptic vectors, preventing powerful combinatorial experiments. Here we show that deletion-mutant rabies virus can be specifically targeted to cells local to an injection site, brightly labelling axons even when coexpressing two other transgenes. We demonstrate several novel capabilities: simultaneously labelling axons and presynaptic terminals, labelling both dendrites and postsynaptic densities, and simultaneously labelling a region's inputs and outputs using co-injected vectors.

Targeting single neuronal networks for gene expression and cell labeling in vivo.

To understand fine-scale structure and function of single mammalian neuronal networks, we developed and validated a strategy to genetically target and trace monosynaptic inputs to a single neuron in vitro and in vivo. The strategy independently targets a neuron and its presynaptic network for specific gene expression and fine-scale labeling, using single-cell electroporation of DNA to target infection and monosynaptic retrograde spread of a genetically modifiable rabies virus. The technique is highly reliable, with transsynaptic labeling occurring in every electroporated neuron infected by the virus. Targeting single neocortical neuronal networks in vivo, we found clusters of both spiny and aspiny neurons surrounding the electroporated neuron in each case, in addition to intricately labeled distal cortical and subcortical inputs. This technique, broadly applicable for probing and manipulating single neuronal networks with single-cell resolution in vivo, may help shed new light on fundamental mechanisms underlying circuit development and information processing by neuronal networks throughout the brain.

Dynamic changes in presynaptic and axonal transport proteins combined with striatal neuroinflammation precede dopaminergic neuronal loss in a rat model of AAV alpha-synucleinopathy.

Little is known about key pathological events preceding overt neuronal degeneration in Parkinson's disease (PD) and alpha-synucleinopathy. Recombinant adeno-associated virus 2-mediated delivery of mutant (A53T) human alpha-synuclein into the substantia nigra (SN) under a neuron-specific synapsin promoter resulted in protracted neurodegeneration with significant dopaminergic (DA) neuron loss by 17 weeks. As early as 4 weeks, there was an increase in a dopamine metabolite, DOPAC and histologically, DA axons in the striatum were dystrophic with degenerative bulbs. Before neuronal loss, significant changes were identified in levels of proteins relevant to synaptic transmission and axonal transport in the striatum and the SN. For example, striatal levels of rabphilin 3A and syntaxin were reduced. Levels of anterograde transport motor proteins (KIF1A, KIF1B, KIF2A, and KIF3A) were decreased in the striatum, whereas retrograde motor proteins (dynein, dynamitin, and dynactin1) were increased. In contrast to reduced levels in the striatum, KIF1A and KIF2A levels were elevated in the SN. There were dramatic changes in cytoskeletal protein levels, with actin levels increased and alpha-/gamma-tubulin levels reduced. In addition to these alterations, a neuroinflammatory response was observed at 8 weeks in the striatum, but not in the SN, demonstrated by increased levels of Iba-1, activated microglia and increased levels of proinflammatory cytokines, including IL-1beta, IFN-gamma and TNF-alpha. These results demonstrate that changes in proteins relevant to synaptic transmission and axonal transport coupled with neuroinflammation, precede alpha-synuclein-mediated neuronal death. These findings can provide ideas for antecedent biomarkers and presymptomatic interventions in PD.

Inhibition of tumor necrosis factor-alpha signaling prevents human immunodeficiency virus-1 protein Tat and methamphetamine interaction.

Our previous studies demonstrated that the psychostimulant methamphetamine (MA) and the human immunodeficiency virus-1 (HIV-1) protein Tat interacted to cause enhanced dopaminergic neurotoxicity. The present study examined whether tumor necrosis factor-alpha (TNF-alpha) mediates the interaction between Tat and MA. In Sprague-Dawley rats, injections of Tat caused a small but significant increase in striatal TNF-alpha level, whereas MA resulted in no change. The increase in TNF-alpha induced by Tat + MA was not significantly different from that induced by Tat alone. Temporal analysis of TNF-alpha levels revealed a 50-fold increase 4 h after Tat administration. In C57BL/6 mice, Tat + MA induced a 50% decline in striatal dopamine levels, which was significantly attenuated in mice lacking both receptors for TNF-alpha. TNF-alpha synthesis inhibitors significantly attenuated Tat + MA neurotoxicity in hippocampal neuronal culture. The results suggest that Tat-induced elevation of TNF-alpha may predispose the dopaminergic terminals to subsequent damage by MA.

Alpha-herpesvirus glycoprotein D interaction with sensory neurons triggers formation of varicosities that serve as virus exit sites.

Alpha-herpesviruses constitute closely related neurotropic viruses, including herpes simplex virus in man and pseudorabies virus (PRV) in pigs. Peripheral sensory neurons, such as trigeminal ganglion (TG) neurons, are predominant target cells for virus spread and lifelong latent infections. We report that in vitro infection of swine TG neurons with the homologous swine alpha-herpesvirus PRV results in the appearance of numerous synaptophysin-positive synaptic boutons (varicosities) along the axons. Nonneuronal cells that were juxtaposed to these varicosities became preferentially infected with PRV, suggesting that varicosities serve as axonal exit sites for the virus. Viral envelope glycoprotein D (gD) was found to be necessary and sufficient for the induction of varicosities. Inhibition of Cdc42 Rho GTPase and p38 mitogen-activated protein kinase signaling pathways strongly suppressed gD-induced varicosity formation. These data represent a novel aspect of the cell biology of alpha-herpesvirus infections of sensory neurons, demonstrating that virus attachment/entry is associated with signaling events and neuronal changes that may prepare efficient egress of progeny virus.

Numerous GABAergic afferents to locus ceruleus in the pericerulear dendritic zone: possible interneuronal pool.

Most nuclei in the CNS are composed of principal neurons that project to other areas and interneurons that serve to integrate information among afferents. The noradrenergic brain nucleus locus ceruleus (LC) has appeared to be an exception to this general rule, because the LC is composed almost entirely of noradrenergic principal neurons. Here, we report that numerous small neurons in the peri-LC region become retrogradely labeled after focal injections of wheat germ agglutinin-apo (inactivated) horseradish peroxidase conjugated to colloidal gold, or pseudorabies virus (PRV), into the nuclear core of the rat LC. A substantial number of these neurons were routinely found within the dendritic field of the LC, in the area surrounding the compact cell-dense region classically defined as LC. Double labeling revealed that a large percentage of these cells stained for GABA. Ultrastructural analyses revealed axodendritic and axosomatic contacts between PRV-labeled afferents and LC neurons labeled with tyrosine hydroxylase immunohistochemistry. In addition, PRV-labeled neurons or axons were immunopositive for GABA in ultrastructural localizations. Analysis of the synaptology of immunopositive profiles demonstrated that these LC afferents in the peri-LC region receive several non-LC synaptic inputs. These results indicate that a population of small GABAergic neurons in the peri-LC dendritic zone may provide interneuronal integration for LC noradrenergic neurons.

Directional spread of an alpha-herpesvirus in the nervous system.

Pseudorabies virus (PRV), an alpha-herpesvirus, is capable of spreading between synaptically connected neurons in diverse hosts. In this report, two lines of experimentation are summarized that provide insight into the mechanism of virus spread in neurons. First, techniques were developed to measure the transport dynamics of capsids in infected neurons. Individual viral capsids labeled with green fluorescent protein (GFP) were visualized and tracked as they moved in axons away from infected neuronal cell bodies in culture during egress. Second, the effects of three viral membrane proteins (gE, gI and Us9) on the localization of envelope, tegument, and capsid proteins in infected, cultured sympathetic neurons were determined. These three proteins are necessary for spread of infection from pre-synaptic neurons to post-synaptic neurons in vivo (anterograde spread). Us9 mutants apparently are defective in anterograde spread in neural circuits because essential viral membrane proteins such as gB are not transported to axon terminals to facilitate spread to the connected neuron. By contrast, gE and gI mutants manifest their phenotype because these proteins most likely function at the axon terminal of the infected neuron to promote spread. These two sets of experiments are consistent with a model for herpesvirus spread in neurons first suggested by Cunningham and colleagues where capsids and envelope proteins, but not whole virions, are transported separately into the axon.

Oestrogen-dependent tracing in the rat CNS after pseudorabies virus infection.

This study examines the hypothesis that neuronal infectivity and the spreading of the pseudorabies virus (PRV) through the synapses in the central nervous system (CNS) are influenced by the oestrogen levels. The arcuate nucleus (ARC) and the subfornical organ (SFO) were chosen as models for analysis; the neurons in both structures possess oestrogen receptors and are mutually connected. A genetically engineered pseudorabies virus (Ba-DupLac) was used as a transneuronal tract tracer. This virus is taken up preferably by axon terminals, and transported very specifically through the synapses in a retrograde manner. Ba-DupLac was injected into the ARC of rats, followed by monitoring of the PRV-immunoreactivity (PRV-IR) in the SFO 72 h following inoculation. We found no PRV immunolabelling in the SFO of ovariectomized (OVX) rats, or in those OVX animals that received oestrogen shortly (4 h) before PRV infection (OVX + E 4 h). In contrast, in those OVX animals that received oestrogen 12 h before PRV infection (OVX + E 12 h), and also in intact control animals, PRV-IR was demonstrated in the SFO in all cases. Surprisingly, a reverse labelling was observed in the OVX rats; PRV-IR appeared in the pyriform cortex, whereas PRV-IR could not be detected in the control and OVX + E 12 h animals. As far as we are aware, this is the first study to demonstrate that transneuronal PRV labelling depends on the effects of oestrogen on certain CNS structures and connections.

Rabies virus entry into cultured rat hippocampal neurons.

Rabies virus entry into cultured hippocampal neurons was investigated by immunofluorescence and electron microscopy. Hippocampal neurons were susceptible to rabies virus infection and became filled with viral antigen 1 day after infection. Infection was inhibited by the lysosomotropic agents chloroquine and ammonium chloride. To study entry, neurons were adsorbed with rabies virus at 4 degrees C and warmed to 37 degrees C for short periods of time prior to fixation and localization of viral antigen by immunofluorescence microscopy By 5 min at 37 degrees C, viral antigen was localized to puncta in the cell body and dendrites and in synapses along dendrites. Little viral antigen was present in axons. Cells adsorbed with rabies virus were incubated with tracers for early endosomes. The endocytic tracers or markers Lucifer Yellow, transferrin receptor, dextran, and wheat germ agglutinin co-localized with rabies virus, indicating that rabies virus enters an endosome compartment shortly after uptake. Rabies virus also co-localized with LysoTracker Red, an acidotropic probe, indicating that some of the virus-containing endosomes are acidified. Rabies virus also co-localized with synapsin I, a synaptic vesicle marker, in nerve terminals but did not co-localize with lysosomal glycoprotein. By electron microscopy, after adsorption of virus and warming for 10 min, virus particles were present in coated pits, coated vesicles, and vacuolar membrane compartments in processes and axon terminals. It is concluded that rabies virus enters the somatodendritic domain and axon terminals of cultured hippocampal neurons by adsorptive endocytosis and is located in endosomes shortly after uptake.

Retrograde and anterograde labeling of cerebellar afferent projection by the injection of recombinant adenoviral vectors into the mouse cerebellar cortex.

Adenoviral vectors have recently been recognized as highly efficient systems for gene delivery into various tissues. We show that a reporter gene introduced into nerve terminals via an adenovirus can be used to label cell bodies retrogradely and then label the axons and nerve terminals of the infected neurons anterogradely in vivo. We injected a replication-defective recombinant adenovirus carrying the E. coli beta-galactosidase gene (lacZ) into the cerebellar cortex of the adult mouse. The first evidence of retrograde labeling was obtained at 2 days after the infection when neurons in the pontine nuclei and the reticulotegmental nucleus of the pons weakly expressed beta-galactosidase, and at 3 days post-infection when neurons in all precerebellar nuclei, known to project to the cerebellar cortex, were strongly stained with X-gal in a Golgi-like manner. Anterograde transport of lacZ gene products was recognized at 3 days post-infection; beta-galactosidase-positive axons arose from somata or dendrites of retrogradely labeled neurons, passed through the middle or inferior cerebellar peduncles, and entered the cerebellum. Anterogradely labeled mossy terminals were recognized on the injection side at 8 days post-infection, and on the contralateral side at 14 days post-infection. Beta-galactosidase expression persisted for up to two months, with a decrease in the total number of labeled cells over time. We could not find any signs of anterograde or retrograde transsynaptic labeling in the nuclei synaptically linked to the cerebellar cortex at any time point after injection up to 58 days post-infection.