BackgroundThe translocator protein (18 kDa) (TSPO) is a mitochondrial protein expressed on reactive glial cells and a biomarker for gliosis in the brain. TSPO ligands have been shown to reduce neuroinflammation in several mouse models of neurodegeneration. Here, we analyzed TSPO expression in mouse and human retinal microglia and studied the effects of the TSPO ligand XBD173 on microglial functions.MethodsTSPO protein analyses were performed in retinoschisin-deficient mouse retinas and human retinas. Lipopolysaccharide (LPS)-challenged BV-2 microglial cells were treated with XBD173 and TSPO shRNAs in vitro and pro-inflammatory markers were determined by qRT-PCR. The migration potential of microglia was determined with wound healing assays and the proliferation was studied with Fluorescence Activated Cell Sorting (FACS) analysis. Microglial neurotoxicity was estimated by nitrite measurement and quantification of caspase 3/7 levels in 661 W photoreceptors cultured in the presence of microglia-conditioned medium. The effects of XBD173 on filopodia formation and phagocytosis were analyzed in BV-2 cells and human induced pluripotent stem (iPS) cell-derived microglia (iPSdM). The morphology of microglia was quantified in mouse retinal explants treated with XBD173.ResultsTSPO was strongly up-regulated in microglial cells of the dystrophic mouse retina and also co-localized with microglia in human retinas. Constitutive TSPO expression was high in the early postnatal Day 3 mouse retina and declined to low levels in the adult tissue. TSPO mRNA and protein were also strongly induced in LPS-challenged BV-2 microglia while the TSPO ligand XBD173 efficiently suppressed transcription of the pro-inflammatory marker genes chemokine (C-C motif) ligand 2 (CCL2), interleukin 6 (IL6) and inducible nitric oxide (NO)-synthase (iNOS). Moreover, treatment with XBD173 significantly reduced the migratory capacity and proliferation of microglia, their level of NO secretion and their neurotoxic activity on 661 W photoreceptor cells. Furthermore, XBD173 treatment of murine and human microglial cells promoted the formation of filopodia and increased their phagocytic capacity to ingest latex beads or photoreceptor debris. Finally, treatment with XBD173 reversed the amoeboid alerted phenotype of microglial cells in explanted organotypic mouse retinal cultures after challenge with LPS.ConclusionsThese findings suggest that TSPO is highly expressed in reactive retinal microglia and a promising target to control microglial reactivity during retinal degeneration.

All biological systems exhibit cell-to-cell variability. Frameworks exist for understanding how stochastic fluctuations and transient differences in cell state contribute to experimentally observable variations in cellular responses. However, current methods do not allow identification of the sources of variability between and within stable subpopulations of cells. We present a data-driven modeling framework for the analysis of populations comprising heterogeneous subpopulations. Our approach combines mixture modeling with frameworks for distribution approximation, facilitating the integration of multiple single-cell datasets and the detection of causal differences between and within subpopulations. The computational efficiency of our framework allows hundreds of competing hypotheses to be compared. We initially validate our method using simulated data with an understood ground truth, then we analyze data collected using quantitative single-cell microscopy of cultured sensory neurons involved in pain initiation. This approach allows us to quantify the relative contribution of neuronal subpopulations, culture conditions, and expression levels of signaling proteins to the observed cell-to-cell variability in NGF/TrkA-initiated Erk1/2 signaling.

Genetic loss of the voltage-gated sodium channel Na1.7 (Na1.7) results in lifelong insensitivity to pain in mice and humans. One underlying cause is an increase in the production of endogenous opioids in sensory neurons. We analyzed whether Na1.7 deficiency altered nociceptive heterotrimeric guanine nucleotide-binding protein-coupled receptor (GPCR) signaling, such as initiated by GPCRs that respond to serotonin (pronociceptive) or opioids (antinociceptive), in sensory neurons. We found that the nociceptive neurons of Na1.7 knockout (Na1.7) mice, but not those of Na1.8 knockout (Na1.8) mice, exhibited decreased pronociceptive serotonergic signaling through the 5-HT receptors, which are Gα-coupled GPCRs that stimulate the production of cyclic adenosine monophosphate resulting in protein kinase A (PKA) activity, as well as reduced abundance of the RIIβ regulatory subunit of PKA. Simultaneously, the efficacy of antinociceptive opioid signaling mediated by the Gα-coupled mu opioid receptors was increased. Consequently, opioids inhibited more efficiently tetrodotoxin-resistant sodium currents, which are important for pain-initiating neuronal activity in nociceptive neurons. Thus, Na1.7 controls the efficacy and balance of GPCR-mediated pro- and antinociceptive intracellular signaling, such that without Na1.7, the balance is shifted toward antinociception, resulting in lifelong endogenous analgesia.

Hyperalgesic priming is characterized by enhanced nociceptor sensitization by pronociceptive mediators, prototypically PGE2. Priming has gained interest as a mechanism underlying the transition to chronic pain. Which stimuli induce priming and what cellular mechanisms are employed remains incompletely understood. In adult male rats, we present the cytokine Oncostatin M (OSM), a member of the IL‐6 family, as an inducer of priming by a novel mechanism. We used a high content microscopy based approach to quantify the activation of endogenous PKA‐II and ERK of thousands sensory neurons in culture. Incubation with OSM increased and prolonged ERK activation by agents that increase cAMP production such as PGE2, forskolin, and cAMP analogs. These changes were specific to IB4/CaMKIIα positive neurons, required protein translation, and increased cAMP‐to‐ERK signaling. In both, control and OSM‐treated neurons, cAMP/ERK signaling involved RapGEF2 and PKA but not Epac. Similar enhancement of cAMP‐to‐ERK signaling could be induced by GDNF, which acts mostly on IB4/CaMKIIα‐positive neurons, but not by NGF, which acts mostly on IB4/CaMKIIα‐negative neurons. In vitro, OSM pretreatment rendered baseline TTX‐R currents ERK‐dependent and switched forskolin‐increased currents from partial to full ERK‐dependence in small/medium sized neurons. In summary, priming induced by OSM uses a novel mechanism to enhance and prolong coupling of cAMP/PKA to ERK1/2 signaling without changing the overall pathway structure.

Depolarization drives neuronal plasticity. However, whether depolarization drives sensitization of peripheral nociceptive neurons remains elusive. By high-content screening (HCS) microscopy, we revealed that depolarization of cultured sensory neurons rapidly activates protein kinase A type II (PKA-II) in nociceptors by calcium influx through CaV1.2 channels. This effect was modulated by calpains but insensitive to inhibitors of cAMP formation, including opioids. In turn, PKA-II phosphorylated Ser1928 in the distal C terminus of CaV1.2, thereby increasing channel gating, whereas dephosphorylation of Ser1928 involved the phosphatase calcineurin. Patch-clamp and behavioral experiments confirmed that depolarization leads to calcium- and PKA-dependent sensitization of calcium currents ex vivo and local peripheral hyperalgesia in the skin in vivo. Our data suggest a local activity-driven feed-forward mechanism that selectively translates strong depolarization into further activity and thereby facilitates hypersensitivity of nociceptor terminals by a mechanism inaccessible to opioids.