RESUMEN
Major depressive disorder ranks as a major burden of disease worldwide, yet the current antidepressant medications are limited by frequent non-responsiveness and significant side effects. The lateral septum (LS) is thought to control of depression, however, the cellular and circuit substrates are largely unknown. Here, we identified a subpopulation of LS GABAergic adenosine A2A receptors (A2AR)-positive neurons mediating depressive symptoms via direct projects to the lateral habenula (LHb) and the dorsomedial hypothalamus (DMH). Activation of A2AR in the LS augmented the spiking frequency of A2AR-positive neurons leading to a decreased activation of surrounding neurons and the bi-directional manipulation of LS-A2AR activity demonstrated that LS-A2ARs are necessary and sufficient to trigger depressive phenotypes. Thus, the optogenetic modulation (stimulation or inhibition) of LS-A2AR-positive neuronal activity or LS-A2AR-positive neurons projection terminals to the LHb or DMH, phenocopied depressive behaviors. Moreover, A2AR are upregulated in the LS in two male mouse models of repeated stress-induced depression. This identification that aberrantly increased A2AR signaling in the LS is a critical upstream regulator of repeated stress-induced depressive-like behaviors provides a neurophysiological and circuit-based justification of the antidepressant potential of A2AR antagonists, prompting their clinical translation.
Asunto(s)
Trastorno Depresivo Mayor , Habénula , Ratones , Animales , Masculino , Habénula/fisiología , Adenosina/farmacología , Neuronas/metabolismo , Hipotálamo/metabolismo , Receptor de Adenosina A2A/metabolismoRESUMEN
The choroid plexus (CP) is one of the key gateways regulating the entry of peripheral immune cells into the CNS. However, the neuromodulatory mechanisms of maintaining its gateway activity are not fully understood. Here, we identified adenosine A2A receptor (A2AR) activity as a regulatory signal for the activity of CP gateway under physiological conditions. In association with a tightly closed CP gateway, we found that A2AR was present at low density in the CP. The RNA-seq analysis revealed that the A2AR antagonist KW6002 affected the expression of the cell adhesion molecules' (CAMs) pathway and cell response to IFN-γ in the CP. Furthermore, blocking or activating A2AR signaling in the CP resulted in a decreased and an increased, respectively, expression of lymphocyte trafficking determinants and disruption of the tight junctions (TJs). Furthermore, A2AR signaling regulates the CP permeability. Thus, A2AR activity in the CP may serve as a therapeutic target for remodeling the immune homeostasis in the CNS with implications for the treatment of neuroimmunological disorders.
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Plexo Coroideo , Receptor de Adenosina A2A , Receptor de Adenosina A2A/metabolismo , Plexo Coroideo/metabolismo , Adenosina/metabolismo , Transducción de SeñalRESUMEN
The adenosine A2A receptor (A2AR), a G protein-coupled receptor, is involved in numerous and varied physiological and pathological processes, including inflammation, immune responses, blood flow, and neurotransmission. Accordingly, it has become an important drug target for the treatment of neuropsychiatric disorders. However, the exact brain distribution of A2AR in regions outside the striatum that display relatively low levels of endogenous A2AR expression has hampered the exploration of A2AR functions under both physiological and pathological conditions. To further study the detailed distribution of the A2AR in low-expression regions, we have generated A2AR knock-in mice in which the 3xHA-2xMyc epitope tag sequence was fused to the C-terminus of A2AR (A2AR-tag mice) via CRISPR/Cas9 technology. Here, using CRISPR/Cas9 technology, we have generated A2AR knock-in mice in which the 3xHA-2xMyc epitope tag sequence was fused to the C-terminus of A2AR (A2AR-tag mice). The A2AR-tag mice exhibited normal locomotor activity and emotional state. Consistent with previous studies, A2AR fluorescence was widely detected in the striatum, nucleus accumbens, and olfactory tubercles, with numerous labeled cells being evident in these regions in the A2AR-tag mouse. Importantly, we also identified the presence of a few but clearly labeled cells in heterogeneous brain regions where A2AR expression has not previously been unambiguously detected, including the lateral septum, hippocampus, amygdala, cerebral cortex, and gigantocellular reticular nucleus. The A2AR-tag mouse represents a novel useful genetic tool for monitoring the expression of A2AR and dissecting its functions in brain regions other than the striatum.
RESUMEN
BACKGROUND: Multiple sclerosis (MS) is one of the most common autoimmune disorders characterized by the infiltration of immune cells into the brain and demyelination. The unwanted immunosuppressive side effect of therapeutically successful natalizumab led us to focus on the choroid plexus (CP), a key site for the first wave of immune cell infiltration in experimental autoimmune encephalomyelitis (EAE), for the control of immune cells trafficking. Adenosine A2A receptor (A2AR) is emerging as a potential pharmacological target to control EAE pathogenesis. However, the cellular basis for the A2AR-mediated protection remains undetermined. METHODS: In the EAE model, we assessed A2AR expression and leukocyte trafficking determinants in the CP by immunohistochemistry and qPCR analyses. We determined the effect of the A2AR antagonist KW6002 treatment at days 8-12 or 8-14 post-immunization on T cell infiltration across the CP and EAE pathology. We determined the critical role of the CP-A2AR on T cell infiltration and EAE pathology by focal knock-down of CP-A2AR via intracerebroventricular injection of CRE-TAT recombinase into the A2ARflox/flox mice. In the cultured CP epithelium, we also evaluated the effect of overexpression of A2ARs or the A2AR agonist CGS21680 treatment on the CP permeability and lymphocytes migration. RESULTS: We found the specific upregulation of A2AR in the CP associated with enhanced CP gateway activity peaked at day 12 post-immunization in EAE mice. Furthermore, the KW6002 treatment at days 8-12 or 8-14 post-immunization reduced T cell trafficking across the CP and attenuated EAE pathology. Importantly, focal CP-A2AR knock-down attenuated the pathogenic infiltration of Th17+ cells across the CP via inhibiting the CCR6-CCL20 axis through NFκB/STAT3 pathway and protected against EAE pathology. Lastly, activation of A2AR in the cultured epithelium by A2AR overexpression or CGS21680 treatment increased the permeability of the CP epithelium and facilitated lymphocytes migration. CONCLUSION: These findings define the CP niche as one of the primary sites of A2AR action, whereby A2AR antagonists confer protection against EAE pathology. Thus, pharmacological targeting of the CP-A2AR represents a novel therapeutic strategy for MS by controlling immune cell trafficking across CP.
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Encefalomielitis Autoinmune Experimental , Adenosina/farmacología , Adenosina/uso terapéutico , Animales , Plexo Coroideo/metabolismo , Encefalomielitis Autoinmune Experimental/metabolismo , Ratones , Ratones Endogámicos C57BL , Receptor de Adenosina A2A/metabolismo , Receptor de Adenosina A2A/uso terapéuticoRESUMEN
Despite the progress in deorphanization of G Protein-Coupled Receptors (GPCRs), ≈100 GPCRs are still classified as orphan receptors without identified endogenous ligands and with unknown physiological functions. The lack of endogenous ligands triggering GPCR signaling has hampered the study of orphan GPCR functions. Using GPR37 as an example, we provide here the first demonstration of the channelrhodopsin 2 (ChR2)-GPCR approach to bypass the endogenous ligand and selectively activate the orphan GPCR signal by optogenetics. Inspired by the opto-XR approach, we designed the ChR2-GPR37 chimera, in which the corresponding parts of GPR37 replaced the intracellular portions of ChR2. We showed that optogenetic activation of ChR2/opto-GPR37 elicited specific GPR37 signaling, as evidenced by reduced cAMP level, enhanced ERK phosphorylation and increased motor activity, confirming the specificity of opto-GPR37 signaling. Besides, optogenetic activation of opto-GPR37 uncovered novel aspects of GPR37 signaling (such as IP-3 signaling) and anxiety-related behavior. Optogenetic activation of opto-GPR37 permits the causal analysis of GPR37 activity in the defined cells and behavioral responses of freely moving animals. Importantly, given the evolutionarily conserved seven-helix transmembrane structures of ChR2 and orphan GPCRs, we propose that opto-GPR37 approach can be readily applied to other orphan GPCRs for their deorphanization in freely moving animals.
RESUMEN
Perinatal inflammatory insult in preterm babies is associated with vision impairment, but the underlying cellular mechanism is still unknown. In this study, we set out to explore whether systemic inflammatory stress affects the development of retinal ganglion cells (RGCs). Neonatal inflammation was induced by single and systemic injection of lipopolysaccharide (LPS, 1â¯mg/kg) at postnatal day 4 (P4). Morphological changes of RGCs were investigated by using 3D neuron reconstruction technique in Thy-1 YFPH transgenic mice at P21, of which a fraction of RGCs selectively expresses the yellow fluorescent protein (YFP). Three types (Type I, II, III) of RGCs were distinguished and classified according to the characteristic features in their dendritic field area and dendrite density. Neonatal exposure to LPS did not alter the composition of the three RGC types but induced a reorganization of dendritic architecture in the RGC Type I and II (but not Type III). The average diameter, surface area and volume of dendrites in both RGC Type I and II were increased after systemic inflammation compared with those in the control group. However, soma sizes of all three RGC types were not affected by neonatal inflammation. Meanwhile, using anterograde labeling of the retinal cells, we found that neonatal exposure to LPS also did not affect the pattern of RGC projections in the dorsal lateral geniculate nucleus of the thalamus (dLGN). These results indicate that RGC dendrite reorganization induced by neonatal inflammation may contribute to the retinal cell dysfunctions associated with systemic inflammation in premature babies.
Asunto(s)
Dendritas/patología , Cuerpos Geniculados/patología , Inflamación/patología , Células Ganglionares de la Retina/patología , Vías Visuales/patología , Animales , Animales Recién Nacidos , Inflamación/inducido químicamente , Lipopolisacáridos/administración & dosificación , Ratones Endogámicos C57BLRESUMEN
Adeno-associated virus (AAV) vectors are important gene delivery tools for the treatment of many recessively inherited retinal diseases. For example, a wild-type (WT) AAV5 vector can deliver a full-length Cnga3 (cyclic nucleotide-gated channel alpha-3) cDNA to target cells of the cone photoreceptor function loss 5 (cpfl5) mouse, a spontaneous animal model of achromatopsia with a Cnga3 mutation. Gene therapy restores cone-mediated function and blocks cone degeneration in the mice. However, since transgene expression delivered by an AAV vector shows relatively short-term effectiveness, this cannot be regarded as a very successful therapy. AAV2 and AAV8 vectors with capsid mutations have significantly enhanced transduction efficiency in retinas compared to WT AAV controls. In this study, AAV8 (Y447, 733F+T494V)-treated cpfl5 retinas showed greater preservation of short-term cone electroretinogram (ERG) responses than AAV8 (Y447, 733F)- or AAV2 (Y272, 444, 500, 730F+T491V)-mediated treatments. To explore the long-term rescue effect, AAV8 (Y447, 733F+T494V)-treated cpfl5 retinas were evaluated at 9 months following postnatal day 14 (P14) treatment. Rescued ERG responses in the cones of treated cpfl5 eyes decreased with increasing age, but still maintained more than 60% of the WT mouse responses at the oldest time point examined. Expression of CNGA3 and M/S-opsins was maintained in cone outer segments of the treated cpfl5 eyes and was equal to expression in age-matched WT retinas. Near-normal cone-mediated water maze behavior was observed in the treated cpfl5 mice. As these are the longest follow-up data reported thus far, AAV8 with capsid Y-F and T-V mutations may be one of the most effective AAV vectors for long-term treatment in a naturally occurring mouse model of CNGA3 achromatopsia.