Epstein-Barr virus-induced gene 3 negatively regulates neuroinflammation and T cell activation following coronavirus-induced encephalomyelitis.

Epstein-Barr virus-induced gene 3 (EBI3) associates with p28 and p35 to form the immunomodulatory cytokines IL-27 and IL-35, respectively. Infection of EBI3-/- mice with the neuroadapted JHM strain of mouse hepatitis virus (JHMV) resulted in increased mortality that was not associated with impaired ability to control viral replication but enhanced T cell and macrophage infiltration into the CNS. IFN-γ secretion from virus-specific CD4+ and CD8+ T cells isolated from infected EBI3-/- mice was augmented while IL-10 expression muted in comparison to infected WT mice. These data demonstrate a regulatory role for EBI3-associated cytokines in controlling host responses following CNS viral infection.

IFN-γ-induced apoptosis of human embryonic stem cell derived oligodendrocyte progenitor cells is restricted by CXCR2 signaling.

Engraftment of human embryonic stem cell (hESC)-derived OPCs in animal models of demyelination results in remyelination and clinical recovery, supporting the feasibility of cell replacement therapies in promoting repair of damaged neural tissue. A critical gap in our understanding of the mechanisms associated with repair revolves around the effects of the local microenvironment on transplanted cell survival. We have determined that treatment of human ESC-derived OPCs with the pleiotropic cytokine IFN-γ promotes apoptosis that is associated with mitochondrial cytochrome c released into the cytosol with subsequent caspase 3 activation. IFN-γ-induced apoptosis is mediated, in part, by secretion of the CXC chemokine ligand 10 (CXCL10) from IFN-γ-treated cells. Signaling through the chemokine receptor CXCR2 by the ligand CXCL1 functions in a tonic manner by muting apoptosis and this is associated with reduced levels of cytosolic cytochrome c and impaired cleavage of caspase 3. These findings support a role for both IFN-γ and CXCL10 in contributing to neuropathology by promoting OPC apoptosis. In addition, these data suggest that hOPCs used for therapeutic treatment for human neurologic disease/damage are susceptible to death through exposure to local inflammatory cytokines present within the inflammatory milieu.

Dopamine D(2) Antagonist-Induced Striatal Nur77 Expression Requires Activation of mGlu5 Receptors by Cortical Afferents.

Dopamine D(2) receptor antagonists modulate gene transcription in the striatum. However, the molecular mechanism underlying this effect remains elusive. Here we used the expression of Nur77, a transcription factor of the orphan nuclear receptor family, as readout to explore the role of dopamine, glutamate, and adenosine receptors in the effect of a dopamine D(2) antagonist in the striatum. First, we investigated D(2) antagonist-induced Nur77 mRNA in D(2L) receptor knockout mice. Surprisingly, deletion of the D(2L) receptor isoform did not reduce eticlopride-induced upregulation of Nur77 mRNA levels in the striatum. Next, we tested if an ibotenic acid-induced cortical lesion could block the effect of eticlopride on Nur77 expression. Cortical lesions strongly reduced eticlopride-induced striatal upregulation of Nur77 mRNA. Then, we investigated if glutamatergic neurotransmission could modulate eticlopride-induced Nur77 expression. A combination of a metabotropic glutamate type 5 (mGlu5) and adenosine A(2A) receptor antagonists abolished eticlopride-induced upregulation of Nur77 mRNA levels in the striatum. Direct modulation of Nur77 expression by striatal glutamate and adenosine receptors was confirmed using corticostriatal organotypic cultures. Taken together, these results indicate that blockade of postsynaptic D(2) receptors is not sufficient to trigger striatal transcriptional activity and that interaction with corticostriatal presynaptic D(2) receptors and subsequent activation of postsynaptic glutamate and adenosine receptors in the striatum is required. Thus, these results uncover an unappreciated role of presynaptic D(2) heteroreceptors and support a prominent role of glutamate in the effect of D(2) antagonists.

CXCR2 signaling protects oligodendrocyte progenitor cells from IFN-γ/CXCL10-mediated apoptosis.

Infiltration of activated lymphocytes into the central nervous system (CNS) is potentially harmful by damaging resident cells through release of cytokines. Among these is IFN-γ that is secreted by activated natural killer (NK) cells and T lymphocytes and can exert a cytotoxic effect on resident glial populations including oligodendrocytes. Here we show that treatment of mouse oligodendrocyte progenitor cell (OPC)-enriched cultures with IFN-γ resulted in a dose-dependent increase in apoptosis. IFN-γ-induced apoptosis is mediated, in part, through induction of the CXC chemokine ligand 10 (CXCL10; IP-10) from cultured OPCs. Treatment of OPCs with CXCL10 resulted in cell death in a concentration-dependent manner and IFN-γ-treatment of CXCL10-/- OPCs resulted in >50% reduction in cell death. Further, treatment of CXCR3-/- OPC cultures with either IFN-γ or CXCL10 resulted in reduced cell death supporting an important role for CXCL10 signaling in IFN-γ-mediated OPC apoptosis. Data is also provided demonstrating that signaling through CXCR2 protects either IFN-γ or CXCL10-treated OPC cultures from apoptosis and this effect is abolished in CXCR2-/- OPCs. CXCR2-mediated protection from apoptosis is associated with impaired cleavage of caspase 3 and elevated expression of the anti-apoptotic protein Bcl-2. These findings demonstrate a previously unappreciated role for CXCL10 in contributing to neuropathology by promoting oligodendrocyte apoptosis and emphasize the potential relevance in targeting CXCL10 in treating human demyelinating diseases including multiple sclerosis (MS).

Oligodendrocytes as regulators of neuronal networks during early postnatal development.

Oligodendrocytes are the glial cells responsible for myelin formation. Myelination occurs during the first postnatal weeks and, in rodents, is completed during the third week after birth. Myelin ensures the fast conduction of the nerve impulse; in the adult, myelin proteins have an inhibitory role on axon growth and regeneration after injury. During brain development, oligodendrocytes precursors originating in multiple locations along the antero-posterior axis actively proliferate and migrate to colonize the whole brain. Whether the initial interactions between oligodendrocytes and neurons might play a functional role before the onset of myelination is still not completely elucidated. In this article, we addressed this question by transgenically targeted ablation of proliferating oligodendrocytes during cerebellum development. Interestingly, we show that depletion of oligodendrocytes at postnatal day 1 (P1) profoundly affects the establishment of cerebellar circuitries. We observed an impressive deregulation in the expression of molecules involved in axon growth, guidance and synaptic plasticity. These effects were accompanied by an outstanding increase of neurofilament staining observed 4 hours after the beginning of the ablation protocol, likely dependent from sprouting of cerebellar fibers. Oligodendrocyte ablation modifies localization and function of ionotropic glutamate receptors in Purkinje neurons. These results show a novel oligodendrocyte function expressed during early postnatal brain development, where these cells participate in the formation of cerebellar circuitries, and influence its development.

Contribution of Kv1.2 voltage-gated potassium channel to D2 autoreceptor regulation of axonal dopamine overflow.

Impairments in axonal dopamine release are associated with neurological disorders such as schizophrenia and attention deficit hyperactivity disorder and pathophysiological conditions promoting drug abuse and obesity. The D2 dopamine autoreceptor (D2-AR) exerts tight regulatory control of axonal dopamine (DA) release through a mechanism suggested to involve K(+) channels. To evaluate the contribution of Kv1 voltage-gated potassium channels of the Shaker gene family to the regulation of axonal DA release by the D2-AR, the present study employed expression analyses, real time measurements of striatal DA overflow, K(+) current measurements and immunoprecipitation assays. Kv1.1, -1.2, -1.3, and -1.6 mRNA and protein were detected in midbrain DA neurons purified by fluorescence-activated cell sorting and in primary DA neuron cultures. In addition, Kv1.1, -1.2, and -1.6 were localized to DA axonal processes in the dorsal striatum. By means of fast scan cyclic voltammetry in striatal slice preparations, we found that the inhibition of stimulation-evoked DA overflow by a D2 agonist was attenuated by Kv1.1, -1.2, and -1.6 toxin blockers. A particular role for the Kv1.2 subunit in the process whereby axonal D2-AR inhibits DA overflow was established with the use of a selective Kv1.2 blocker and Kv1.2 knock-out mice. Moreover, we demonstrate the ability of D2-AR activation to increase Kv1.2 currents in co-transfected cells and its reliance on Gßγ subunit signaling along with the physical coupling of D2-AR and Kv1.2-containing channels in striatal tissue. These findings underline the contribution of Kv1.2 in the regulation of nigrostriatal DA release by the D2-AR and thereby offer a novel mechanism by which DA release is regulated.

CXCR2 signaling protects oligodendrocytes and restricts demyelination in a mouse model of viral-induced demyelination.

BACKGROUND: The functional role of ELR-positive CXC chemokines during viral-induced demyelination was assessed. Inoculation of the neuroattenuated JHM strain of mouse hepatitis virus (JHMV) into the CNS of susceptible mice results in an acute encephalomyelitis that evolves into a chronic demyelinating disease, modeling white matter pathology observed in the human demyelinating disease Multiple Sclerosis. METHODOLOGY/PRINCIPAL FINDINGS: JHMV infection induced the rapid and sustained expression of transcripts specific for the ELR+ chemokine ligands CXCL1 and CXCL2, as well as their binding receptor CXCR2, which was enriched within the spinal cord during chronic infection. Inhibiting CXCR2 signaling with neutralizing antiserum significantly (p<0.03) delayed clinical recovery. Moreover, CXCR2 neutralization was associated with an increase in the severity of demyelination that was independent of viral recrudescence or modulation of neuroinflammation. Rather, blocking CXCR2 was associated with increased numbers of apoptotic cells primarily within white matter tracts, suggesting that oligodendrocytes were affected. JHMV infection of enriched oligodendrocyte progenitor cell (OPC) cultures revealed that apoptosis was associated with elevated expression of cleaved caspase 3 and muted Bcl-2 expression. Inclusion of CXCL1 within JHMV infected cultures restricted caspase 3 cleavage and increased Bcl-2 expression that was associated with a significant (p<0.001) decrease in apoptosis. CXCR2 deficient oligodendrocytes were refractory to CXCL1 mediated protection from JHMV-induced apoptosis, readily activating caspase 3 and down regulating Bcl-2. CONCLUSION/SIGNIFICANCE: These findings highlight a previously unappreciated role for CXCR2 signaling in protecting oligodendrocyte lineage cells from apoptosis during inflammatory demyelination initiated by viral infection of the CNS.

Cell replacement therapies to promote remyelination in a viral model of demyelination.

Persistent infection of the central nervous system (CNS) of mice with the neuroadapted JHM strain of mouse hepatitis (MHV) is characterized by ongoing demyelination mediated by inflammatory T cells and macrophages that is similar both clinically and histologically with the human demyelinating disease multiple sclerosis (MS). Although extensive demyelination occurs in mice persistently infected with MHV there is only limited remyelination. Therefore, the MHV model of demyelination is a relevant model for studying disease and evaluating therapeutic approaches to protect cells of the oligodendrocyte lineage and promote remyelination. This concept is further highlighted as the etiology of MS remains enigmatic, but viruses have long been considered as potential triggering agents in initiating and/or maintaining MS symptoms. As such, understanding mechanisms associated with promoting repair within the CNS in the context of a persistent viral infection is critical given the possible viral etiology of MS. This review focuses on recent studies using either mouse neural stem cells (NSCs) or human oligodendrocyte progenitor cells (OPCs) derived from human embryonic stem cell (hESC) to promote remyelination in mice persistently infected with MHV. In addition, the potential role for chemokines in positional migration of transplanted cells is addressed.

Genetically determined interaction between the dopamine transporter and the D2 receptor on prefronto-striatal activity and volume in humans.

Dopamine modulation of neuronal activity during memory tasks identifies a nonlinear inverted-U shaped function. Both the dopamine transporter (DAT) and dopamine D(2) receptors (encoded by DRD(2)) critically regulate dopamine signaling in the striatum and in prefrontal cortex during memory. Moreover, in vitro studies have demonstrated that DAT and D(2) proteins reciprocally regulate each other presynaptically. Therefore, we have evaluated the genetic interaction between a DRD(2) polymorphism (rs1076560) causing reduced presynaptic D(2) receptor expression and the DAT 3'-VNTR variant (affecting DAT expression) in a large sample of healthy subjects undergoing blood oxygenation level-dependent (BOLD)-functional magnetic resonance imaging (MRI) during memory tasks and structural MRI. Results indicated a significant DRD(2)/DAT interaction in prefrontal cortex and striatum BOLD activity during both working memory and encoding of recognition memory. The differential effect on BOLD activity of the DAT variant was mostly manifest in the context of the DRD(2) allele associated with lower presynaptic expression. Similar results were also evident for gray matter volume in caudate. These interactions describe a nonlinear relationship between compound genotypes and brain activity or gray matter volume. Complementary data from striatal protein extracts from wild-type and D(2) knock-out animals (D2R(-/-)) indicate that DAT and D(2) proteins interact in vivo. Together, our results demonstrate that the interaction between genetic variants in DRD(2) and DAT critically modulates the nonlinear relationship between dopamine and neuronal activity during memory processing.

Signaling by dopamine regulates D2 receptors trafficking at the membrane.

sGi2 is a spliced variant of the GTP-binding protein G(alpha i2). By difference with G(alpha i2), which is mainly present at the plasma membrane, sGi2 is localized in intracellular compartments. The splicing event generates a novel C-terminal region in sGi2, which is necessary for its intracellular localization. The role of sGi2 is presently unknown, although its intracellular localization might underlie a possible role in the regulation of trafficking of 7TM receptors. Here, we show that sGi2 complexes with dopamine D2 receptors (D2R) in striatal neurons. The sGi2-D2R complex is readily observed in immunoprecipitation studies using specific antibodies for both proteins on mouse striatal extracts, which identify D2-specific bands of >80 KDa suggesting sGi2 interactions with D2R dimers. Importantly, the sGi2-D2R complex in the absence of receptor stimulation is mostly found in intracellular perinuclear areas in primary neuronal cultures. Treatment of neurons with quinpirole, a D2-specific agonist, results into diffusion of D2R and sGi2 staining throughout the cell and into neurites and membranes. This suggests that dopamine could regulate availability of D2 receptors at the cell surface. The formation of sGi2-D2R complex is mediated through the interaction of sGi2 with the third intracellular loop of D2Rs. As functional consequence of the D2R-sGi2 interaction, we observed a reduction of D2 binding sites at the plasma membrane, when the two proteins are co-expressed in transfected cells. Altogether these studies identify sGi2 as a D2R interacting protein involved in the regulation of D2R at the membrane through a dopamine mediated mechanism.

Regulation of Akt signaling by D2 and D3 dopamine receptors in vivo.

The serine/threonine kinase Akt is a downstream target of dopamine receptor signaling that is inhibited/dephosphorylated in response to direct and indirect dopamine receptor agonists. Although pharmacological studies uncovered the involvement of D2-class dopamine receptors in Akt regulation, they did not identify the role of individual receptor subtypes in this process. Here we used knock-out mice lacking the D1, D2, D2 long, or D3 dopamine receptors as well as a D4 receptor-selective antagonist to address the function of each of these receptors in the regulation of Akt in vivo. Under basal conditions, D2, D2 long, and D3 knock-out mice display enhanced striatal Akt activation, whereas D1 knock-out mice and mice treated with the D4 receptor antagonist L745870 (3-[[4-(4-chlorophenyl)piperazin-1-yl]methyl]-1H-pyrrolo[2,3-b]pyridine trihydrochloride) have phospho-Akt levels comparable with those of normal control animals. Furthermore, both amphetamine and apomorphine lose their ability to inhibit Akt in D2 knock-out mice but retain their normal effect on this signaling molecule in D1 knock-out animals. Finally, D3 knock-out mice show a reduced sensitivity of Akt-mediated signaling to dopaminergic drugs but retain the action of these drugs on Akt at high dose regimens. These results indicate that D2 receptors are essential for the inhibition of Akt by dopamine and that D3 receptors also participate in this signaling potentially by enhancing D2 receptor response. Identification of the functions of individual dopamine receptor subtypes in Akt regulation may help the development of new pharmaceutical approaches for mental disorders related to abnormal dopamine transmission such as bipolar disorder and schizophrenia.

Impaired light masking in dopamine D2 receptor-null mice.

Disruption of overt circadian rhythms can occur without influencing the endogenous pacemaker, the so-called 'masking' effect classically elicited by light. As the physiological pathways involved in light masking remain elusive, we analyzed mice lacking the dopamine D2 receptor. Although circadian rhythmicity was normal, D2R-null mice showed a markedly deficient light masking response, indicating that D2R-mediated signaling is an essential component of the neuronal pathways leading to light masking of circadian rhythms.