Identification and characterization of select oxysterols as ligands for GPR17.

BACKGROUND AND PURPOSE: G-protein coupled receptor 17 (GPR17) is an orphan receptor involved in the process of myelination, due to its ability to inhibit the maturation of oligodendrocyte progenitor cells (OPCs) into myelinating oligodendrocytes. Despite multiple claims that the biological ligand has been identified, it remains an orphan receptor. EXPERIMENTAL APPROACH: Seventy-seven oxysterols were screened in a cell-free [35 S]GTPγS binding assay using membranes from cells expressing GPR17. The positive hits were characterized using adenosine 3',5' cyclic monophosphate (cAMP), inositol monophosphate (IP1) and calcium mobilization assays, with results confirmed in rat primary oligodendrocytes. Rat and pig brain extracts were separated by high-performance liquid chromatography (HPLC) and endogenous activator(s) were identified in receptor activation assays. Gene expression studies of GPR17, and CYP46A1 (cytochrome P450 family 46 subfamily A member 1) enzymes responsible for the conversion of cholesterol into specific oxysterols, were performed using quantitative real-time PCR. KEY RESULTS: Five oxysterols were able to stimulate GPR17 activity, including the brain cholesterol, 24(S)-hydroxycholesterol (24S-HC). A specific brain fraction from rat and pig extracts containing 24S-HC activates GPR17 in vitro. Expression of Gpr17 during mouse brain development correlates with the expression of Cyp46a1 and the levels of 24S-HC itself. Other active oxysterols have low brain concentrations below effective ranges. CONCLUSIONS AND IMPLICATIONS: Oxysterols, including but not limited to 24S-HC, could be physiological activators for GPR17 and thus potentially regulate OPC differentiation and myelination through activation of the receptor.

Screening for modulators of neural network activity in 3D human iPSC-derived cortical spheroids.

Human induced Pluripotent Stem Cells (iPSCs) are a powerful tool to dissect the biology of complex human cell types such as those of the central nervous system (CNS). However, robust, high-throughput platforms for reliably measuring activity in human iPSC-derived neuronal cultures are lacking. Here, we assessed 3D cultures of cortical neurons and astrocytes displaying spontaneous, rhythmic, and highly synchronized neural activity that can be visualized as calcium oscillations on standard high-throughput fluorescent readers as a platform for CNS-based discovery efforts. Spontaneous activity and spheroid structure were highly consistent from well-to-well, reference compounds such as TTX, 4-AP, AP5, and NBQX, had expected effects on neural spontaneous activity, demonstrating the presence of functionally integrated neuronal circuitry. Neurospheroid biology was challenged by screening the LOPAC®1280 library, a collection of 1280 pharmacologically active small molecules. The primary screen identified 111 compounds (8.7%) that modulated neural network activity across a wide range of neural and cellular processes and 16 of 17 compounds chosen for follow-up confirmed the primary screen results. Together, these data demonstrate the suitability and utility of human iPSC-derived neurospheroids as a screening platform for CNS-based drug discovery.

The Zinc Transporter SLC39A7 (ZIP7) Is Essential for Regulation of Cytosolic Zinc Levels.

Zinc homeostasis is a highly regulated process in mammalian cells that is critical for normal growth and development. Movement of zinc across cell compartments is controlled by two classes of transporters: Slc39a family members transport zinc into the cytosol from either the extracellular space or intracellular stores such as the endoplasmic reticulum (ER), whereas the SLC30A family mediates zinc efflux from the cytosol. In this study, we report that genetic ablation of SLC39A7 (ZIP7) results in decreased cytosolic zinc levels, increased ER zinc levels, impaired cell proliferation, and induction of ER stress. Confirmatory of impaired zinc transport as the causal mechanism, both the increased ER stress and impaired cell proliferation were rescued by increasing cytosolic zinc. Furthermore, using these robust cellular phenotypes, we implemented a small-molecule library screen with 2800 compounds and identified one small molecule capable of rescuing ER stress and cell proliferation in ZIP7-deficient cells in the low micromolar range.

NACHO Mediates Nicotinic Acetylcholine Receptor Function throughout the Brain.

Neuronal nicotinic acetylcholine receptors (nAChRs) participate in diverse aspects of brain function and mediate behavioral and addictive properties of nicotine. Neuronal nAChRs derive from combinations of α and ß subunits, whose assembly is tightly regulated. NACHO was recently identified as a chaperone for α7-type nAChRs. Here, we find NACHO mediates assembly of all major classes of presynaptic and postsynaptic nAChR tested. NACHO acts at early intracellular stages of nAChR subunit assembly and then synergizes with RIC-3 for receptor surface expression. NACHO knockout mice show profound deficits in binding sites for α-bungarotoxin, epibatidine, and conotoxin MII, illustrating essential roles for NACHO in proper assembly of α7-, α4ß2-, and α6-containing nAChRs, respectively. By contrast, GABAA receptors are unaffected consistent with NACHO specifically modulating nAChRs. NACHO knockout mice show abnormalities in locomotor and cognitive behaviors compatible with nAChR deficiency and underscore the importance of this chaperone for physiology and disease associated with nAChRs.

Brain α7 Nicotinic Acetylcholine Receptor Assembly Requires NACHO.

Nicotine exerts its behavioral and additive actions through a family of brain nicotinic acetylcholine receptors (nAChRs). Enhancing α7-type nAChR signaling improves symptoms in Alzheimer's disease and schizophrenia. The pharmaceutical study of α7 receptors is hampered because these receptors do not form their functional pentameric structure in cell lines, and mechanisms that underlie α7 receptor assembly in neurons are not understood. Here, a genomic screening strategy solves this long-standing puzzle and identifies NACHO, a transmembrane protein of neuronal endoplasmic reticulum that mediates assembly of α7 receptors. NACHO promotes α7 protein folding, maturation through the Golgi complex, and expression at the cell surface. Knockdown of NACHO in cultured hippocampal neurons or knockout of NACHO in mice selectively and completely disrupts α7 receptor assembly and abolishes α7 channel function. This work identifies NACHO as an essential, client-specific chaperone for nAChRs and has implications for physiology and disease associated with these widely distributed neurotransmitter receptors.

Coronin-1 is a neurotrophin endosomal effector that is required for developmental competition for survival.

Retrograde communication from axonal targets to neuronal cell bodies is critical for both the development and function of the nervous system. Much progress has been made in recent years linking long-distance, retrograde signaling to a signaling endosome, yet the mechanisms governing the trafficking and signaling of these endosomes remain mostly uncharacterized. Here we report that in mouse sympathetic neurons, the target-derived nerve growth factor (NGF)-tropomyosin-related kinase type 1 (TrkA, also called Ntrk1) signaling endosome, on arrival at the cell body, induces the expression and recruitment of a new effector protein known as Coronin-1 (also called Coro1a). In the absence of Coronin-1, the NGF-TrkA signaling endosome fuses to lysosomes sixfold to tenfold faster than when Coronin-1 is intact. We also define a new Coronin-1-dependent trafficking event in which signaling endosomes recycle and re-internalize on arrival at the cell body. Beyond influencing endosomal trafficking, Coronin-1 is also required for several NGF-TrkA-dependent signaling events, including calcium release, calcineurin activation and phosphorylation of cAMP responsive element binding protein (CREB). These results establish Coronin-1 as an essential component of a feedback loop that mediates NGF-TrkA endosome stability, recycling and signaling as a critical mechanism governing developmental competition for survival.

Long-distance retrograde neurotrophic factor signalling in neurons.

The specialized architecture of neurons necessitates unique modes of intracellular communication to allow for cell survival, the ability to detect and respond to injury and aspects of neuronal development, such as axon and dendrite growth, plasticity, and synapse and circuit formation. Many of these neuronal processes rely on signal transduction pathways and transcriptional programmes that are activated by retrograde signals originating from target-derived cues that act on distal axons. Here, we review the many functions of long-range distal axon-to-cell body signalling and discuss mechanisms of retrograde target-derived growth factor signalling.

Recruitment of actin modifiers to TrkA endosomes governs retrograde NGF signaling and survival.

The neurotrophins NGF and NT3 collaborate to support development of sympathetic neurons. Although both promote axonal extension via the TrkA receptor, only NGF activates retrograde transport of TrkA endosomes to support neuronal survival. Here, we report that actin depolymerization is essential for initiation of NGF/TrkA endosome trafficking and that a Rac1-cofilin signaling module associated with TrkA early endosomes supports their maturation to retrograde transport-competent endosomes. These actin-regulatory endosomal components are absent from NT3/TrkA endosomes, explaining the failure of NT3 to support retrograde TrkA transport and survival. The inability of NT3 to activate Rac1-GTP-cofilin signaling is likely due to the labile nature of NT3/TrkA complexes within the acidic environment of TrkA early endosomes. Thus, TrkA endosomes associate with actin-modulatory proteins to promote F-actin disassembly, enabling their maturation into transport-competent signaling endosomes. Differential control of this process explains how NGF but not NT3 supports retrograde survival of sympathetic neurons.

Long-distance control of synapse assembly by target-derived NGF.

We report a role for long-distance retrograde neurotrophin signaling in the establishment of synapses in the sympathetic nervous system. Target-derived NGF is both necessary and sufficient for formation of postsynaptic specializations on dendrites of sympathetic neurons. This, in turn, is a prerequisite for formation of presynaptic specializations, but not preganglionic axonal ingrowth from the spinal cord into sympathetic ganglia. We also find that NGF-TrkA signaling endosomes travel from distal axons to cell bodies and dendrites where they promote PSD clustering. Furthermore, the p75 neurotrophin receptor restricts PSD formation, suggesting an important role for antagonistic NGF-TrkA and p75 signaling pathways during retrograde control of synapse establishment. Thus, in addition to defining the appropriate number of sympathetic neurons that survive the period of developmental cell death, target-derived NGF also exerts control over the degree of connectivity between the spinal cord and sympathetic ganglia through retrograde control of synapse assembly.

The role of Kalirin9 in p75/nogo receptor-mediated RhoA activation in cerebellar granule neurons.

p75 and the Nogo receptor form a signaling unit for myelin inhibitory molecules, with p75 being responsible for RhoA activation. Because p75 lacks the GDP/GTP exchange factor domain, it has remained unclear how p75 activates RhoA. Here, we report that Kalirin9, a dual RhoGEF, binds p75 directly and regulates p75-Nogo receptor-dependent RhoA activation and neurite inhibition in response to myelin-associated glycoprotein. The region of p75 that Kalirin9 binds includes its mastoparan-like fifth helix, which was shown to recruit RhoGDI-RhoA. As predicted from the presence of a shared binding site, we found that Kalirin9 competes with RhoGDI for p75 binding in a dose-dependent manner in vitro. In line with these data, myelin-associated glycoprotein addition to cerebellar granule neurons resulted in a reduction in the association of Kalirin9 with p75, and a simultaneous increase in the binding of RhoGDI to p75. These results reveal a mechanism by which the fifth helix of p75 regulates RhoA activation.

Small, nonpeptide p75NTR ligands induce survival signaling and inhibit proNGF-induced death.

Studies showing that neurotrophin binding to p75NTR can promote cell survival in the absence of Trk (tropomyosin-related kinase) receptors, together with recent structural data indicating that NGF may bind to p75NTR in a monovalent manner, raise the possibility that small molecule p75NTR ligands that positively regulate survival might be found. A pharmacophore designed to capture selected structural and physical chemical features of a neurotrophin domain known to interact with p75NTR was applied to in silico screening of small molecule libraries. Small, nonpeptide, monomeric compounds were identified that interact with p75NTR. In cells showing trophic responses to neurotrophins, the compounds promoted survival signaling through p75NTR-dependent mechanisms. In cells susceptible to proneurotrophin-induced death, compounds did not induce apoptosis but inhibited proneurotrophin-mediated death. These studies identify a unique range of p75NTR behaviors that can result from isolated receptor liganding and establish several novel therapeutic leads.

ProNGF induces p75-mediated death of oligodendrocytes following spinal cord injury.

The neurotrophin receptor p75 is induced by various injuries to the nervous system, but its role after injury has remained unclear. Here, we report that p75 is required for the death of oligodendrocytes following spinal cord injury, and its action is mediated mainly by proNGF. Oligodendrocytes undergoing apoptosis expressed p75, and the absence of p75 resulted in a decrease in the number of apoptotic oligodendrocytes and increased survival of oligodendrocytes. ProNGF is likely responsible for activating p75 in vivo, since the proNGF from the injured spinal cord induced apoptosis among p75(+/+), but not among p75(-/-), oligodendrocytes in culture, and its action was blocked by proNGF-specific antibody. Together, these data suggest that the role of proNGF is to eliminate damaged cells by activating the apoptotic machinery of p75 after injury.

Activation of Rac GTPase by p75 is necessary for c-jun N-terminal kinase-mediated apoptosis.

The neurotrophin receptor p75 can induce apoptosis both in vitro and in vivo. The mechanisms by which p75 induces apoptosis have remained mostly unknown. Here, we report that p75 activates Rac GTPase, which in turn activates c-jun N-terminal kinase (JNK), including an injury-specific JNK3, in an NGF-dependent manner. N17Rac blocks this JNK activation and subsequent NGF-dependent apoptosis, indicating that activation of Rac GTPase is required for JNK activation and apoptosis induced by p75. In addition, p75-mediated Rac activation is modulated by coactivation of Trk, identifying Rac GTPase as one of the key molecules whose activity is critical for cell survival and death in neurotrophin signaling. The crucial role of the JNK pathway in p75 signaling is further confirmed by the results that blocking p75 from signaling via the JNK pathway or suppressing the JNK activity itself led to inhibition of NGF-dependent death. Together, these results indicate that the apoptotic machinery of p75 comprises Rac GTPase and JNK.