G Protein-coupled Receptor 17 Research Articles

Montelukast improves disease outcome in SOD1G93A female mice by counteracting oligodendrocyte dysfunction and aberrant glial reactivity.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by progressive motor neuron (MN) loss and consequent muscle atrophy, for which no effective therapies are available. Recent findings reveal that disease progression is fuelled by early aberrant neuroinflammation and the loss of oligodendrocytes with neuroprotective and remyelinating properties. On this basis, pharmacological interventions capable of restoring a pro-regenerative local milieu and re-establish proper oligodendrocyte functions may be beneficial. Here, we evaluated the in vivo therapeutic effects of montelukast (MTK), an antagonist of the oligodendroglial G protein-coupled receptor 17 (GPR17) and of cysteinyl-leukotriene receptor 1 (CysLT1R) receptors on microglia and astrocytes, in the SOD1G93A ALS mouse model. We chronically treated SOD1G93A mice with MTK, starting from the early symptomatic disease stage. Disease progression was assessed by behavioural and immunohistochemical approaches. Oral MTK treatment significantly extended survival probability, delayed body weight loss and ameliorated motor functionalityonly in female SOD1G93A mice. Noteworthy, MTK significantly restored oligodendrocyte maturation and induced significant changes in the reactive phenotype and morphological features of microglia/macrophages and astrocytes in the spinal cord of female SOD1G93A mice, suggesting enhanced pro-regenerative functions. Importantly, concomitant MN preservation has been detected after MTK administration. No beneficial effects were observed in male mice, highlighting a sex-based difference in the protective activity of MTK. Our results provide the first preclinical evidence indicating that repurposing of MTK, a safe and marketed anti-asthmatic drug, may be a promising sex-specific strategy for personalized ALS treatment.

Buyang huanwu decoction promotes remyelination via miR-760-3p/GPR17 axis after intracerebral hemorrhage

Ethnopharmacological relevanceThe repairment of myelin sheaths is crucial for mitigating neurological impairments of intracerebral hemorrhage (ICH). However, the current research on remyelination processes in ICH remains limited. A representative traditional Chinese medicine, Buyang Huanwu decoction (BYHWD), shows a promising therapeutic strategy for ICH treatment. Aim of the studyTo investigate the pro-remyelination effects of BYHWD on ICH and explore the underlying mechanisms. Materials and methodsThe collagenase-induced mice ICH model was created for investigation. BYHWD's protective effects were assessed by behavioral tests and histological staining. Transmission electron microscopy was used for displaying the structure of myelin sheaths. The remyelination and oligodendrocyte differentiation were evaluated by the expressions of myelin proteolipid protein (PLP), myelin basic protein (MBP), MBP/TAU, Olig2/CC1, and PDGFRα/proliferating cell nuclear antigen (PCNA) through RT-qPCR and immunofluorescence. Transcriptomics integrated with disease database analysis and experiments in vivo and in vitro revealed the microRNA-related underlying mechanisms. ResultsHere, we reported that BYHWD promoted the neurological function of ICH mice and improved remyelination by increasing PLP, MBP, and TAU, as well as restoring myelin structure. Besides, we showed that BYHWD promoted remyelination by boosting the differentiation of PDGFRα+ oligodendrocyte precursor cells into olig2+/CC1+ oligodendrocytes. Additionally, we demonstrated that the remyelination effects of BYHWD worked by inhibiting G protein-coupled receptor 17 (GPR17). miRNA sequencing integrated with miRNA database prediction screened potential miRNAs targeting GPR17. By applying immunofluorescence, RNA in situ hybridization and dual luciferase reporter gene assay, we confirmed that BYHWD suppressed GPR17 and improved remyelination by increasing miR-760-3p. ConclusionsBYHWD improves remyelination and neurological function in ICH mice by targeting miR-760-3p to inhibit GPR17. This study may shed light on the orchestration of remyelination mechanisms after ICH, thus providing novel insights for developing innovative prescriptions with brain-protective properties.

G protein-coupled receptor 17 is regulated by WNT pathway during oligodendrocyte precursor cell differentiation

G protein-coupled receptor 17 (GPR17) and the WNT pathway are critical players of oligodendrocyte (OL) differentiation acting as essential timers in developing brain to achieve fully-myelinating cells. However, whether and how these two systems are related to each other is still unknown. Of interest, both factors are dysregulated in developing and adult brain diseases, including white matter injury and cancer, making the understanding of their reciprocal interactions of potential importance for identifying new targets and strategies for myelin repair. Here, by a combined pharmacological and biotechnological approach, we examined regulatory mechanisms linking WNT signaling to GPR17 expression in OLs. We first analyzed the relative expression of mRNAs encoding for GPR17 and the T cell factor/Lymphoid enhancer-binding factor-1 (TCF/LEF) transcription factors of the canonical WNT/β-CATENIN pathway, in PDGFRα+ and O4+ OLs during mouse post-natal development. In O4+ cells, Gpr17 mRNA level peaked at post-natal day 14 and then decreased concomitantly to the physiological uprise of WNT tone, as shown by increased Lef1 mRNA level. The link between WNT signaling and GPR17 expression was further reinforced in vitro in primary PDGFRα+ cells and in Oli-neu cells. High WNT tone impaired OL differentiation and drastically reduced GPR17 mRNA and protein levels. In Oli-neu cells, WNT/β-CATENIN activation repressed Gpr17 promoter activity through both putative WNT response elements (WRE) and upregulation of the inhibitor of DNA-binding protein 2 (Id2). We conclude that the WNT pathway influences OL maturation by repressing GPR17, which could have implications in pathologies characterized by dysregulations of the OL lineage including multiple sclerosis and oligodendroglioma.

The committed oligodendrocyte precursor cell, a newly-defined intermediate progenitor cell type in oligodendroglial lineage.

In the central nervous system, oligodendrocytes (OLs) produce myelin sheaths that provide trophic support to neuronal axons and increase the propagation speed of action potential. OLs are constantly generated from OL precursor cells (OPCs) throughout life span. The production of myelinating OLs consists of three canonical stages: OPCs, newly-formed OLs (NFOs), and mature myelinating OLs. Recently, single-cell RNA transcriptomic analyses identified a new population of oligodendroglial cells, namely differentiation committed OPCs (COPs). COPs represent a critical intermediate population between OPCs and NFOs, as revealed by specific expression of G-protein coupled receptor 17 (GPR17). The dysregulation of COPs leads to the remyelination failure in demyelinating diseases and impairs the replacement of lost myelin sheaths due to aging. Hence, understanding the development of COPs and their underlying regulatory network will be helpful in establishing new strategies for promoting myelin repair in demyelinating diseases. This review summarizes the current knowledge on the development and functions of COPs under both physiological and pathological conditions. Overall, COPs function as "checkpoints" to prevent inappropriate precocious OL differentiation and myelination through expressing distinct regulatory factors. Deepening our understanding of COPs may not only advance our knowledge of how OL lineage progresses during development, but also open the door to new treatments for demyelinating diseases.

Analysis of the GPR17 receptor in NG2-glia under physiological conditions unravels a new subset of oligodendrocyte progenitor cells with distinct functions.

NG2-glia comprise a heterogeneous population of cycling cells that give rise to mature, myelinating oligodendrocytes. The mechanisms that regulate the process of differentiation from NG2-glia into oligodendrocytes are still not fully understood but over the last years the G Protein-coupled Receptor 17 (GPR17) has been on the spotlight as a possible key regulator. Interestingly, GPR17-expressing NG2-glia show under physiological conditions a slower and lower level of differentiation compared to NG2-glia without GPR17. In contrast, after a CNS insult these react with proliferation and differentiation in a high rate, pointing towards a role in repair processes. However, the role of GPR17+ NG2-glia under healthy conditions in adulthood has not been addressed yet. Therefore, we aimed here to characterize the GPR17-expressing NG2-glia. Using transgenic mouse models, we showed restricted GPR17 expression in only some NG2-glia. Furthermore, we found that these cells constitute a distinct subset within the NG2-glia population, which shows a different gene expression profile and behavior when compared to the total NG2-glia population. Genetic depletion of GPR17+ cells showed that these are not contributing to the dynamic and continuous generation of new oligodendrocytes in the adult brain. Taken together, GPR17+ NG2-glia seem to play a distinct role under physiological conditions that goes beyond their classic differentiation control, that needs to be further elucidated. These results open new avenues for using the GPR17 receptor as a target to change oligodendrogenesis under physiological and pathological conditions, highlighting the importance of further characterization of this protein for future pharmacological studies.

Identification and characterization of select oxysterols as ligands for GPR17.

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. 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. 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. 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.

G Protein-Coupled Receptor 17 Inhibition as a Prospective Treatment for Multiple Sclerosis: A Research Protocol

Introduction: Multiple sclerosis (MS) is a chronic inflammatory neurodegenerative disease. MS is the most prevalent neurological disability that often leads to severe cognitive or physical incapacitations in young adults. As MS is currently an incurable disease, more effective treatments need to be investigated. The disease is associated with axonal degenerations and the development of demyelinated plaques, due to episodic autoimmune destruction of oligodendrocytes. Usually, demyelination is followed by remyelination as the brain attempts to reconstruct the myelin sheaths. Oligodendrocyte precursor cells are essential for remyelination as these cells proliferate and differentiate into mature oligodendrocytes. An important regulator of oligodendrocyte development is G protein-coupled receptor 17 (GPR17), whose inhibition has been suggested in previous studies to the promote oligodendrocyte differentiation and remyelination. As such, a potential therapy for MS patients is through decreasing GPR17 expression via inhibition of one of its ligands, LTC4. Methods: We aim to promote remyelination in MS patients using the LTC4 synthase inhibitor AZD9898 to indirectly partially inhibit GPR17 in proteolipid protein-induced experimental autoimmune encephalomyelitis (PLP-induced EAE) MS mice models. There will be 6 experimental groups and 8 control groups. All experimental groups will receive a three mg dose of AZD9898. After approximately zero, one, five and ten days, the mice will be sacrificed, and magnetic resonance imaging will be conducted. The myelin water fraction will then be determined to visualize in vivo myelination of the central nervous system through myelin water imaging. Western blotting will be used to verify AZD9898’s indirect inhibition of GPR17. The resulting data will be analyzed with the Kolomogorov–Smirnov test, Pearson’s correlation coefficient (linear) and two-tailed paired t-tests and ImageJ software. Anticipated Results: It is expected that AZD9898 administration in PLP-induced EAE mice models will result in an increased myelin water fraction, indicating remyelination, alongside decreased GPR17 expression. Discussion: These results will provide a potential treatment for MS by illustrating how AZD9898 is effective at indirectly inhibiting GPR17 in mice models, and thus promoting remyelination. Conclusion: This study will provide insight on the treatment of demyelinating diseases by demonstrating how pharmacological inhibition of GPR17 ligand LTC4 can promote remyelination in MS patients.

Rewiring of Glucose and Lipid Metabolism Induced by G Protein-Coupled Receptor 17 Silencing Enables the Transition of Oligodendrocyte Progenitors to Myelinating Cells.

In the mature central nervous system (CNS), oligodendrocytes (OLs) provide support and insulation to axons thanks to the production of a myelin sheath. During their maturation to myelinating cells, OLs require energy and building blocks for lipids, which implies a great investment of energy fuels and molecular sources of carbon. The oligodendroglial G protein-coupled receptor 17 (GPR17) has emerged as a key player in OL maturation; it reaches maximal expression in pre-OLs, but then it has to be internalized to allow terminal maturation. In this study, we aim at elucidating the role of physiological GPR17 downregulation in OL metabolism by applying transcriptomics, metabolomics and lipidomics on differentiating OLs. After GPR17 silencing, we found a significant increase in mature OL markers and alteration of several genes involved in glucose metabolism and lipid biosynthesis. We also observed an increased release of lactate, which is partially responsible for the maturation boost induced by GPR17 downregulation. Concomitantly, GPR17 depletion also changed the kinetics of specific myelin lipid classes. Globally, this study unveils a functional link between GPR17 expression, lactate release and myelin composition, and suggests that innovative interventions targeting GPR17 may help to foster endogenous myelination in demyelinating diseases.

Activation of A2B adenosine receptor protects against demyelination in a mouse model of schizophrenia.

The purpose of the present study was to explore the effects of A2B adenosine receptor (A2BAR) on learning, memory and demyelination in a dizocilpine maleate (MK-801)-induced mouse model of schizophrenia (SCZ). BAY 60-6583, an agonist of A2BAR, or PSB 603, an antagonist of A2BAR, was used to treat SCZ in this model. The Morris Water Maze (MWM) was utilized to determine changes in cognitive function. Moreover, western blotting, immunohistochemistry and immunofluorescence were conducted to investigate the myelination and oligodendrocyte (OL) alterations at differentiation and maturation stages. The MWM results showed that learning and memory were impaired in SCZ mice, while subsequent treatment with BAY 60-6583 alleviated these impairments. In addition, western blot analysis revealed that myelin basic protein (MBP) and chondroitin sulphate proteoglycan 4 (NG2) expression levels were significantly decreased in MK-801-induced mice, while the expression of G protein-coupled receptor 17 (GPR17) was increased. Additionally, the number of anti-adenomatous polyposis coli clone CC-1/OL transcription factor 2 (CC-1+/Olig2+) cells was also decreased. Notably, BAY 60-6583 administration could reverse these changes, resulting in a significant increase in MBP and NG2 protein expression, and in the number of CC-1+/Olig2+ cells, while GPR17 protein expression levels were decreased. The present study indicated that the selective activation of A2BAR using BAY 60-6583 could improve the impaired learning and memory of SCZ mice, as well as protect the myelin sheath from degeneration by regulating the survival and maturation of OLs.

Insulin-like Growth Factor 1 Promotes Cell Proliferation by Downregulation of G-Protein-Coupled Receptor 17 Expression via PI3K/Akt/FoxO1 Signaling in SK-N-SH Cells.

Insulin-like growth factor 1 (IGF-1) not only regulates neuronal function and development but also is neuroprotective in the setting of acute ischemic stroke. G-protein-coupled receptor 17 (GPR17) expression in brain tissue serves as an indicator of brain damage. As whether IGF-1 regulates GPR17 expression remains unknown, the aim of this study is to investigate how IGF-1 regulates GPR17 expression in vitro. Human neuroblastoma SK-N-SH cells were used. Lentivirus-mediated short hairpin RNA (shRNA) was constructed to mediate the silencing of FoxO1, while adenoviral vectors were used for its overexpression. Verification of the relevant signaling cascade was performed using a FoxO1 inhibitor (AS1842856), a phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002), and a GPR17 antagonist (cangrelor). Cell proliferation was analyzed using EdU staining; immunofluorescence staining was used to detect the expression and subcellular localization of FoxO1. Chromatin immunoprecipitation was used to analyze the binding of FoxO1 to the GPR17 promoter in SK-N-SH cells. The expression of FoxO1, GPR17, and protein kinase B (also known as Akt) mRNA and protein as well as the levels of FoxO1 and Akt phosphorylation were investigated in this study. IGF-1 was found to downregulate FoxO1 and GPR17 expression in SK-N-SH cells while promoting cell viability and proliferation. Inhibition of FoxO1 and antagonism of GPR17 were found to play a role similar to that of IGF-1. Silencing of FoxO1 by lentivirus-mediated shRNA resulted in the downregulation of FoxO1 and GPR17 expression. The overexpression of FoxO1 via adenoviral vectors resulted in the upregulation of FoxO1 and GPR17 expression. Blocking of PI3K signaling by LY294002 inhibited the effect of IGF-1 on GPR17 suppression. Results from chromatin immunoprecipitation revealed that IGF-1 promotes FoxO1 nuclear export and reduces FoxO1 binding to the GPR17 promoter in SK-N-SH cells. Here, we conclude that IGF-1 enhances cell viability and proliferation in SK-N-SH cells via the promotion of FoxO1 nuclear export and reduction of FoxO1 binding to the GPR17 promoter via PI3K/Akt signaling. Our findings suggest that the enhancement of IGF-1 signaling to antagonize GPR17 serves as a potential therapeutic strategy in the management of acute ischemic stroke.

G protein-coupled receptor 17 restricts rabies virus replication via BAK-mediated apoptosis

Rabies, caused by rabies virus (RABV), is an ancient zoonotic disease that significantly affects human and animal health throughout the world. RABV causes acute encephalitis in mammals with a high fatality rate in developing countries. G protein-coupled receptor 17 (GPR17) is a vital gene in the central nervous system (CNS) that plays important roles in demyelinating diseases and ischemia brain. However, it is still unclear whether GPR17 participates in the regulation of RABV infection. Here, we found that upregulation or activation of GPR17 can reduce the virus titer; conversely, the inactivation or silence of GPR17 led to increased RABV replication in N2a cells. The recombinant RABV expressing GPR17 (rRABV-GPR17) showed reduced replication capacity compared to the parent virus rRABV. Moreover, overexpression of GPR17 can attenuate RABV pathogenicity in mice. Further study demonstrated that GPR17 suppressed RABV replication via BAK-mediated apoptosis. Our findings uncover an unappreciated role of GPR17 in suppressing RABV infection, where GPR17 mediates cell apoptosis to limit RABV replication and may be an attractive candidate for new therapeutic interventions in the treatment of rabies.

Inhibition of GPR17 with cangrelor improves cognitive impairment and synaptic deficits induced by Aβ1–42 through Nrf2/HO-1 and NF-κB signaling pathway in mice

The accumulation of amyloid beta (Aβ) in the brain is thought to be associated with cognitive deficits in Alzheimer's disease (AD). However, current methods to combat Aβ neurotoxicity are still lacking. G protein-coupled receptor 17 (GPR17) has become a target for treating inflammation in brain diseases, but it is unclear whether it has a role in AD. Here, we investigated the effects of cangrelor, a GPR17 antagonist, on neurotoxicity and memory impairment induced by intracerebroventricular (i.c.v.) injection of Aβ1–42 in mice. The behavior results showed that cangrelor (2.0 or 4.0 μg/mouse, i.c.v.) treatment reversed the deficits in memory and learning ability induced by Aβ1–42 in mice. Importantly, we demonstrated for the first time that GPR17 expression in the hippocampus and frontal cortex is increased in response to Aβ1–42 exposures. We also found that cangrelor treatment reduced the activity of β-secretase 1 (BACE1) and the levels of soluble Aβ1–42 in the hippocampus and frontal cortex. Meanwhile, cangrelor treatment suppressed oxidative stress induced by Aβ1–42, as proved by reduced production of malondialdehyde (MDA), and increased glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT), and promoted the expression of nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1). Furthermore, cangrelor also suppressed Aβ1–42-induced neuroinflammation, characterized by suppressed activation of microglia, decreased the levels of pro-inflammatory cytokines, and nuclear translocation of NF-κB p65, as well as ameliorated synaptic deficits by promoting the upregulation of synaptic proteins, and increasing the number of Golgi-Cox stained dendritic spines. These results suggest that cangrelor may reverse Aβ1–42-induced cognition deficits via inhibiting oxidative stress, neuroinflammation, and synaptic dysfunction mediated by Nrf2/HO-1 and NF-κB signaling.

Involvement of GPR17 in Neuronal Fibre Outgrowth.

Characterization of new pharmacological targets is a promising approach in research of neurorepair mechanisms. The G protein-coupled receptor 17 (GPR17) has recently been proposed as an interesting pharmacological target, e.g., in neuroregenerative processes. Using the well-established ex vivo model of organotypic slice co-cultures of the mesocortical dopaminergic system (prefrontal cortex (PFC) and substantia nigra/ventral tegmental area (SN/VTA) complex), the influence of GPR17 ligands on neurite outgrowth from SN/VTA to the PFC was investigated. The growth-promoting effects of Montelukast (MTK; GPR17- and cysteinyl-leukotriene receptor antagonist), the glial cell line-derived neurotrophic factor (GDNF) and of two potent, selective GPR17 agonists (PSB-16484 and PSB-16282) were characterized. Treatment with MTK resulted in a significant increase in mean neurite density, comparable with the effects of GDNF. The combination of MTK and GPR17 agonist PSB-16484 significantly inhibited neuronal growth. qPCR studies revealed an MTK-induced elevated mRNA-expression of genes relevant for neuronal growth. Immunofluorescence labelling showed a marked expression of GPR17 on NG2-positive glia. Western blot and RT-qPCR analysis of untreated cultures suggest a time-dependent, injury-induced stimulation of GPR17. In conclusion, MTK was identified as a stimulator of neurite fibre outgrowth, mediating its effects through GPR17, highlighting GPR17 as an interesting therapeutic target in neuronal regeneration.

Synthesis and Preclinical Validation of Novel Indole Derivatives as a GPR17 Agonist for Glioblastoma Treatment.

The discovery of a potential ligand-targeting G protein-coupled receptor 17 (GPR17) is important for developing chemotherapeutic agents against glioblastoma multiforme (GBM). We used the integration of ligand- and structure-based cheminformatics and experimental approaches for identifying the potential GPR17 ligand for GBM treatment. Here, we identified a novel indoline-derived phenolic Mannich base as an activator of GPR17 using molecular docking of over 6000 indoline derivatives. One of the top 10 hit molecules, CHBC, with a glide score of −8.390 was synthesized through a multicomponent Petasis borono–Mannich reaction. The CHBC–GPR17 interaction leads to a rapid decrease of cAMP and Ca2+. CHBC exhibits the cytotoxicity effect on GBM cells in a dose-dependent manner with an IC50 of 85 μM, whereas the known agonist MDL29,951 showed a negligible effect. Our findings suggest that the phenolic Mannich base could be a better GPR17 agonist than MDL29,951, and further uncovering their pharmacological properties could potentiate an inventive GBM treatment.

Cangrelor ameliorates CLP-induced pulmonary injury in sepsis by inhibiting GPR17

BackgroundSepsis is a common complication of severe wound injury and infection, with a very high mortality rate. The P2Y12 receptor inhibitor, cangrelor, is an antagonist anti-platelet drug.MethodsIn our study, we investigated the protective mechanisms of cangrelor in CLP-induced pulmonary injury in sepsis, using C57BL/6 mouse models.ResultsTdT-mediated dUTP Nick-End Labeling (TUNEL) and Masson staining showed that apoptosis and fibrosis in lungs were alleviated by cangrelor treatment. Cangrelor significantly promoted surface expression of CD40L on platelets and inhibited CLP-induced neutrophils in Bronchoalveolar lavage fluid (BALF) (p < 0.001). We also found that cangrelor decreased the inflammatory response in the CLP mouse model and inhibited the expression of inflammatory cytokines, IL-1β (p < 0.01), IL-6 (p < 0.05), and TNF-α (p < 0.001). Western blotting and RT-PCR showed that cangrelor inhibited the increased levels of G-protein-coupled receptor 17 (GPR17) induced by CLP (p < 0.001).ConclusionOur study indicated that cangrelor repressed the levels of GPR17, followed by a decrease in the inflammatory response and a rise of neutrophils in BALF, potentially reversing CLP-mediated pulmonary injury during sepsis.

HGG-35. IDENTIFICATION OF G PROTEIN-COUPLED RECEPTOR 17 (GPR17) UP-REGULATION IN PAEDIATRIC H3 K27M-MUTANT DIFFUSE MIDLINE GLIOMA AND EXAMINATION OF ITS ROLE IN DIFFUSE MIDLINE GLIOMA CELL LINES

Paediatric H3 K27M-mutant diffuse midline glioma (pDMG) is an incurable, aggressive childhood brain malignancy, that arises in a region- and age-specific nature. The underlying pathophysiology suggests dysregulation of postnatal neurodevelopmental processes causing aborted cell differentiation. The cell of origin is unclear, but data suggests an oligodendrocytic lineage (OPC), supported by the over-expression of transcription factors such as Olig1 and Olig2 in 80% of DIPG cases. In-depth bioinformatics and principal component analyses (PCA) of genes involved in brain development and pDMG support reports of OPC gene dysregulation and led to the identification of the G-protein coupled receptor 17 (GPR17) and its association with pDMG. GPR17, a rhodopsin-like orphan GPCR (unknown ligand), has the typical features of the GPCR superfamily, seven transmembrane domains (TM1-TM7), eight amphipathic helices, an extracellular N-terminal domain, and an intracellular C-terminus. GPR17 has been implicated in several normal physiological and pathological processes, such as oligodendrocyte differentiation, spinal cord injury and brain injury. GPR17 mRNA and protein expression was confirmed in all pDMG cell lines tested. Using a well-characterised agonist (MDL 299,51) and antagonist (HAMI3379) to modulate GPR17 function in pDMG cell lines resulted in phenotypic and genomic changes as well as in cell growth and migration. HAMI3379, a GPR17 specific antagonist resulted in a significant reduction in GPR17 mRNA and protein expression (p<0.006) and a significant reduction in migration (p<0.0025). When pDMG cells were pretreated with HAMI3379 in combination with known cytotoxic agents (Bleomycin, a radiation mimic, Panobinostat or Vincristine), there was a decrease in cell viability compare to cytotoxic agent alone. There are no current effective therapies for pDMG patients and the ability of blocking GPR17 function to enhance sensitivity to standard therapies is appealing and warrants further investigation.

Quetiapine promotes oligodendroglial process outgrowth and membrane expansion by orchestrating the effects of Olig1.

Oligodendroglial lineage cells go through a series of morphological changes before myelination. Prior to myelination, cell processes and membrane structures enlarge by approximately 7,000 times, which is required to support axonal wrapping and myelin segment formation. Failure of these processes leads to maldevelopment and impaired myelination. Quetiapine, an atypical antipsychotic drug, was proved to promote oligodendroglial differentiation and (re)myelination, pending detailed effects and regulatory mechanism. In this study, we showed that quetiapine promotes morphological maturation of oligodendroglial lineage cells and myelin segment formation, and a short-term quetiapine treatment is sufficient to induce these changes. To uncover the underlying mechanism, we examined the effect of quetiapine on the Oligodendrocyte transcription factor 1 (Olig1). We found that quetiapine upregulates Olig1 expression level and promotes nuclear Olig1 translocation to the cytosol, where it functions not as a transcription modulator, but in a way that highly correlates with oligodendrocyte morphological transformation. In addition, quetiapine treatment reverses the negative regulatory effect of the Olig1-regulated G protein-coupled receptor 17 (GPR17) on oligodendroglial morphological maturation. Our results demonstrate that quetiapine enhances oligodendroglial differentiation and myelination by promoting cell morphological transformation. This would shed light on the orchestration of oligodendroglia developmental mechanisms, and provides new targets for further therapeutic research.

Knockdown of G protein-coupled receptor-17 (GPR17) facilitates the regeneration and repair of myelin sheath post-periventricular leukomalacia (PVL)

ABSTRACT The G protein-coupled receptor-17 (GPR17) plays an important role in regulating the differentiation of oligodendrocytes and remyelination, which is a key negative regulator of oligodendrocyte differentiation. The present study aimed to investigate the function of GPR17 in the white matter of periventricular leukomalacia (PVL) neonatal rats. The PVL model was established in 2-day old neonatal rats by intracerebral injection of LPS (1 mg/kg). Compared to sham, GPR17 was significantly upregulated, while Olig1 was significantly downregulated in the PVL group at 1 d, 3 days, and 7 days post-modeling. Compared to the negative control (NC) group, the expression of GPR17 was suppressed, while that of Olig1 was elevated in the siRNA-GPR17 group as time progressed; the opposite results were observed in the GPR17-overexpressed group. Decreased formation of myelin sheaths as well as poor structure and loose arrangement were observed in the PVL group. Similar observations were found in the PVL + siRNA-GPR17 group at 1 d and 3 days post-modeling. However, on day 7 post-modeling, a dramatic increase in the formation of myelin sheath as well as thicker myelin sheaths were observed in the PVL + siRNA-GPR17 group. The migration ability of oligodendrocyte progenitor cells (OPCs) isolated from animals was found to be significantly suppressed in the GPR17-overexpressed group, accompanied by the downregulation of Olig1. Taken together, the regeneration and repair of myelin sheaths post-PVL white matter injury were induced by downregulating the GPR17 gene, which elevated the expression of Olig1.

Microglial vesicles improve post-stroke recovery by preventing immune cell senescence and favoring oligodendrogenesis

Contrasting myelin damage through the generation of new myelinating oligodendrocytes represents a promising approach to promote functional recovery after stroke. Here, we asked whether activation of microglia and monocyte-derived macrophages affects the regenerative process sustained by G protein-coupled receptor 17 (GPR17)-expressing oligodendrocyte precursor cells (OPCs), a subpopulation of OPCs specifically reacting to ischemic injury. GPR17-iCreERT2:CAG-eGFP reporter mice were employed to trace the fate of GPR17-expressing OPCs, labeled by the green fluorescent protein (GFP), after permanent middle cerebral artery occlusion. By microglia/macrophages pharmacological depletion studies, we show that innate immune cells favor GFP+ OPC reaction and limit myelin damage early after injury, whereas they lose their pro-resolving capacity and acquire a dystrophic "senescent-like" phenotype at later stages. Intracerebral infusion of regenerative microglia-derived extracellular vesicles (EVs) restores protective microglia/macrophages functions, limiting their senescence during the post-stroke phase, and enhances the maturation of GFP+ OPCs at lesion borders, resulting in ameliorated neurological functionality. Invitro experiments show that EV-carried transmembrane tumor necrosis factor (tmTNF) mediates the pro-differentiating effects on OPCs, with future implications for regenerative therapies.

Mechanisms underlying remyelination with special focus on demyelination models of multiple sclerosis

Failure to remyelinate and rewrap the demyelinated axons has been revealed as the major hurdle for treatment of multiple sclerosis (MS), and the bottleneck is the inability of oligodendrocyte progenitor cell (OPC) to differentiate into mature oligodendrocyte. Remyelination is a spontaneous regenerative process, which includes activation, migration and differentiation of OPC, and is believed to protect the axon and further halt neurodegeneration. In recent years, studies have identified many potential drug targets for efficiently promoting OPC differentiation in in vivo demyelination models, such as metformin, clemostine, and drug targets as myelin transcription factor 1-like protein (Myt1L), N-methyl-D-aspartic acid (NMDA) receptor, connexin 43 (Cx43), G protein coupled receptor 17 (GPR17), κ opioid receptor (KOR), sterol 14α-demethylase (CYP51), Δ14-sterol reductase (TM7SF2), emopamil-binding protein (EBP). This review summarizes the recent progress on the mechanisms underlying the activation, migration and differentiation of OPC in remyelination with special focus on studies using demyelination models of MS, which may provide insights of further exploring new therapeutic strategies for MS.