Microglia Express Mu Opioid Receptor: Insights From Transcriptomics and Fluorescent Reporter Mice

Background: Microglia activation contributes to chronic pain and to the adverse effects of opiate use such as analgesic tolerance and opioid-induced hyperalgesia. Both mu opioid receptor (MOR) encoded by Oprm1/OPRM1 gene and toll like receptor 4 (TLR4) have been reported to mediate these morphine effects and a current question is whether microglia express the Oprm1 transcript and MOR protein. The aim of this study was to characterize Oprm1-MOR expression in naive murine and human microglia, combining transcriptomics datasets previously published by other groups with our own imaging study using the Cx3cr1-eGFP-MOR-mCherry reporter mouse line.Methods: We analyzed microglial Oprm1/OPRM1 expression obtained from transcriptomics datasets, focusing on ex vivo studies from adult wild-type animals and adult post-mortem human cerebral cortex. Oprm1, as well as co-regulated gene sets were examined. The expression of MOR in microglia was also investigated using our novel fluorescent Cx3cr1-eGFP-MOR-mcherry reporter mouse line. We determined whether CX3cR1-eGFP positive microglial cells expressed MOR-mCherry protein by imaging various brain areas including the Frontal Cortex, Nucleus Accumbens, Ventral Tegmental Area, Central Amygdala, and Periaqueductal Gray matter, as well as spinal cord.Results:Oprm1 expression was found in all 12 microglia datasets from mouse whole brain, in 7 out of 8 from cerebral cortex, 3 out of 4 from hippocampus, 1 out of 1 from striatum, and 4 out of 5 from mouse or rat spinal cord. OPRM1 was expressed in 16 out of 17 microglia transcriptomes from human cerebral cortex. In Cx3cr1-eGFP-MOR-mCherry mice, the percentage of MOR-positive microglial cells ranged between 35.4 and 51.6% in the different brain areas, and between 36.8 and 42.4% in the spinal cord.Conclusion: The comparative analysis of the microglia transcriptomes indicates that Oprm1/OPRM1 transcripts are expressed in microglia. The investigation of Cx3cr1-eGFP-MOR-mCherry mice also shows microglial expression of MOR proteinin the brain and spine. These results corroborate functional studies showing the actions of MOR agonists on microglia and suppression of these effects by MOR-selective antagonists or MOR knockdown

Efficacy and Tolerance of Post-operative Hypo-Fractionated Stereotactic Radiotherapy in a Large Series of Patients With Brain Metastases

Purpose: The aim of this study was to assess, in a large series, the efficacy and tolerance of post-operative adjuvant hypofractionated stereotactic radiation therapy (HFSRT) for brain metastases (BMs).Materials and Methods: Between July 2012 and January 2017, 160 patients from 2 centers were operated for BM and treated by HFSRT. Patients had between 1 and 3 BMs, no brainstem lesions or carcinomatous meningitis. The primary endpoint was local control. Secondary endpoints were distant brain control, overall survival (OS) and tolerance to HFSRT.Results: 73 patients (46%) presented with non-small cell lung cancer (NSCLC), 23 (14%) had melanoma and 21 (13%) breast cancer. Median age was 58 years (range, 22–83 years). BMs were synchronous in 50% of the cases. The most frequent prescription regimens were 24 Gy in 3 fractions (n = 52, 33%) and 30 Gy in 5 fractions (n = 37, 23%). Local control rates at 1 and 2 years were 88% [95%CI, 81–93%] and 81% [95%CI, 70–88%], respectively. Distant control rate at 1 year was 48% [95%CI, 81–93%]. In multivariate analysis, primary NSCLC was associated with a significant reduction in the risk of death compared to other primary sites (HR = 0.57, p = 0.007), the number of extra-cerebral metastatic sites (HR = 1.26, p = 0.003) and planning target volumes (HR = 1.15, p = 0.012) were associated with a lower OS. There was no prognostic factor of time to local progression. Median OS was 15.2 months [95%CI, 12.0–17.9 months] and the OS rate at 1 year was 58% [95% CI, 50–65%]. Salvage radiotherapy was administered to 72 patients (45%), of which 49 received new HFSRT. Ten (7%) patients presented late grade 2 and 4 (3%) patients late grade 3 toxicities. Thirteen (8.9%) patients developed radiation necrosis.Conclusions: This large multicenter retrospective study shows that HFSRT allows for good local control of metastasectomy tumor beds and that this technique is well-tolerated by patients

Rôles tardifs du facteur de transcription HNF-6 dans le développement des jonctions neuromusculaires et du cervelet

In humans, a large number of diseases and lesions of the CNS, leading to abnormalities of locomotion have been described. Locomotor activity is regulated at three levels in the CNS. Motor areas receive information that will be transmitted to the spinal cord via the pyramidal or extrapyramidal tracts, resulting in an appropriate motor response by effector muscles. In addition, the cerebellum and basal ganglia are involved in the coordination, planning and execution of motor programs. It is important to understand the cells, structures and circuits involved in locomotion in order to better understand the mechanisms of these lesions and diseases and to apply them in humans. The family of the Onecut transcription factors comprises three members in mammals, namely HNF-6, OC-2 and OC-3. They play important roles in the CNS including cell differentiation, maintenance of cell identity and cell migration. Hnf6-/- mice display a paralysis of the hindlimbs at birth. The goal of my thesis was to understand the origin of the paralysis in these mice. To assess locomotor defects in adult Hnf6-/- mice, we performed behavioral tests. These mice display a reduction of muscular strength and an abnormal positioning of the hindlimbs, and a defect in the coordination and in the balance. These defects suggest that the neuromuscular junction and the cerebellum are altered in Hnf6-/- mice. Therefore, we aimed at identifying abnormalities of the neuromuscular junctions and of the cerebellum in mice Hnf6-/- and to characterize the roles of HNF-6 in these two structures. The morphology of the neuromuscular junctions and the localization of synaptophysin were abnormal in Hnf6-/- mice. HNF-6 regulates, directly or indirectly, the expression of agrin and neuregulin, two genes necessary for proper formation and maintenance of the neuromuscular junction. In addition, the organization of Purkinje cells in the cerebellum was abnormal and characterized by a superposition of cells, a localization of cells in the granular layer or the absence of cells, depending on the location. These results show that HNF-6 is required for the organization of Purkinje cells. To conclude, we identified two new roles of HNF-6 during this work, namely in the formation of the NMJ and in the organization of Purkinje cells.Chez l’homme, il existe un grand nombre de pathologies et de lésions du SNC qui conduisent à des altérations de locomotion. L’activité locomotrice est régulée à trois niveaux dans le SNC. Les aires motrices reçoivent des informations nerveuses qui vont être transmises à la moelle épinière par la voie pyramidale ou extrapyramidale, induisant une réponse motrice appropriée de la part des muscles effecteurs. De plus, le cervelet et les ganglions de la base sont des structures qui sont impliqués dans la coordination, la planification et l’exécution des ordres moteurs. Il est important de mieux comprendre les cellules, les structures et les circuits impliqués dans la locomotion afin de mieux comprendre ce qui se passe dans ces pathologies et de pouvoir l’appliquer chez l’homme à des fins thérapeutiques. La famille de facteurs de transcription Onecut comprend trois membres chez les mammifères, HNF-6 (OC-1), OC-2 et OC-3 et sont décrits comme ayant un rôle important dans la différenciation de certaines populations neuronales, le maintien de leur identité cellulaire et dans la migration de ces cellules. Les souris Hnf6-/- ont la particularité de présenter à la naissance une paralysie des pattes postérieures. Le but de ce travail a été de comprendre l’origine de cette paralysie. Afin d’évaluer les défauts locomoteurs présents à l’âge adulte chez ces souris, nous avons réalisé des tests comportementaux. Nous avons montré que les souris Hnf6-/- présentent, une diminution de la force musculaire, un positionnement anormal des pattes postérieures et un défaut de coordination et d’équilibre. Ce type de défaut locomoteur suggère que les motoneurones et/ou les jonctions neuromusculaires (JNM) soient à l’origine des anomalies motrices et que le cervelet celles de la coordination et de l’équilibre. Mon objectif a donc été d’identifier les anomalies présentes au sein des JNM ainsi que dans le cervelet chez les souris Hnf6-/- afin d’identifier les rôles du facteur HNF-6 dans ces deux structures. Nous avons tout d’abord montré que la morphologie de la plaque motrice et la localisation de la synaptophysine étaient anormales chez ces souris. De plus, nous avons montré que HNF-6 est nécessaire pour le développement des JNM en régulant directement ou indirectement l’expression de l’agrine et de la neuréguline. Dans le cervelet des souris Hnf6-/-, nous avons montré une organisation anormale des cellules de Purkinje caractérisée par une superposition des cellules, une localisation des cellules dans la couche granulaire ou une absence de cellules. HNF-6 semble donc jouer un rôle dans l’organisation des cellules de Purkinje. En conclusion de ce travail, nous avons montré deux nouveaux rôles du facteur HNF-6: un rôle dans la formation des JNM et dans l’organisation des cellules de Purkinje.(SBIM 3) -- UCL, 201

Motor Deficit in a Tauopathy Model Is Induced by Disturbances of Axonal Transport Leading to Dying-Back Degeneration and Denervation of Neuromuscular Junctions

Several neurodegenerative diseases are characterized by both cognitive and motor deficits associated with accumulation of tau aggregates in brain, brainstem, and spinal cord. The Tg30 murine tauopathy model expresses a human tau protein bearing two frontotemporal dementia with Parkinsonism linked to chromosome 17 pathogenic mutations and develops a severe motor deficit and tau aggregates in brain and spinal cord. To investigate the origin of this motor deficit, we analyzed the age-dependent innervation status of the neuromuscular junctions and mutant tau expression in Tg30 mice. The human transgenic tau was detected from postnatal day 7 onward in motoneurons, axons in the sciatic nerve, and axon terminals of the neuromuscular junctions. The development and maturation of neuromuscular junctions were not disrupted in Tg30 mice, but their maintenance was disturbed in adult Tg30 mice, resulting in a progressive and severe muscle denervation. This muscle denervation was associated with early electrophysiological signs of muscle spontaneous activities and histological signs of muscle degeneration. Early loss of synaptic vesicles in axon terminals preceding motor deficits, accumulation of Gallyas-positive aggregates, and cathepsin-positive vesicular clusters in axons in the sciatic nerve suggest that this denervation results from disturbances of axonal transport. This physiopathological mechanism might be responsible for motor signs observed in some human tauopathies, and for synaptic dysfunction resulting from alterations at the presynaptic level in these diseases.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Mu opioid receptor in microglia contributes to morphine analgesic tolerance, hyperalgesia, and withdrawal in mice

A major challenge in medicine is developing potent pain therapies without the adverse effects of opiates. Neuroinflammation and in particular microglial activation have been shown to contribute to these effects. However, the implication of the microglial mu opioid receptor (MOR) is not known. We developed a novel conditional knockout (cKO) mouse line, wherein MOR is deleted in microglia. Morphine analgesic tolerance was delayed in both sexes in cKO mice in the hot plate assay. Opioid-induced hyperalgesia (OIH) as measured in the tail immersion assay was abolished in male cKO mice, and physical dependence to morphine as assessed by naloxone-induced withdrawal was attenuated in female cKO mice. Our results show a sex-dependent contribution of microglial MOR in morphine analgesic tolerance, OIH, and physical dependence. In conclusion, our data suggest that blockade of microglial MOR could represent a therapeutic target for opiate analgesia without the opiate adverse effects

Optimized Protocol for Subcutaneous Implantation of Encapsulated Cells Device and Evaluation of Biocompatibility

Improving a drug delivery system is critical to treat central nervous system disorders. Here we studied an innovative approach based on implantation of a wireless-powered cell-based device in mice. This device, coupling biologic material and electronics, is the first of its kind. The advantage of this technology is its ability to control the secretion of a therapeutic molecule and to switch the classical permanent delivery to activation on demand. In diseases with relapsing-remitting phases such as multiple sclerosis, such activation could be selectively achieved in relapsing phases. However, the safety (tolerance to biomaterials and surgical procedure) of such a clinical device needs to be verified. Therefore, the development of tools to assess the biocompatibility of the system in animal models is an essential step. We present the development of this new therapeutic approach, the challenges we encountered during the different steps of its development (such as cell loading in the chamber, surgery protocol for subcutaneous implantation of the device) and the tools we used to evaluate cell viability and biocompatibility of the device.ISSN:2296-418

The Onecut transcription factor HNF-6 contributes to proper reorganization of Purkinje cells during postnatal cerebellum development

The Onecut (OC) family of transcription factors comprises three members in mammals, namely HNF-6 (or OC-1), OC-2 and OC-3. During embryonic development, these transcriptional activators control cell differentiation in pancreas, in liver and in the nervous system. Adult Hnf6 mutant mice exhibit locomotion defects characterized by hindlimb muscle weakness, abnormal gait and defective balance and coordination. Indeed, HNF-6 is required in spinal motor neurons for proper formation of the hindlimb neuromuscular junctions, which likely explain muscle weakness observed in corresponding mutant animals. The goal of the present study was to determine the cause of the balance and coordination defects in Hnf6 mutant mice. Coordination and balance deficits were quantified by rotarod and runway tests. Hnf6 mutant animals showed an increase in the fall frequency from the beam and were unable to stay on the rotarod even at low speed, indicating a severe balance and coordination deficit. To identify the origin of this abnormality, we assessed whether the development of the main CNS structure involved in the control of balance and coordination, namely the cerebellum, was affected by the absence of HNF-6. Firstly, we observed that Hnf6 was expressed transiently during the first week after birth in the Purkinje cells of wild type newborn mice. Secondly, we showed that, in Hnf6-/- mice, the organization of Purkinje cells became abnormal during a second phase of their development. Indeed, Purkinje cells were produced normally but part of them failed to reorganize as a regular continuous monolayer at the interface between the molecular and the granular layer of the cerebellum. Thus, the Onecut factor HNF-6 contributes to the reorganization of Purkinje cells during a late phase of cerebellar development

Complete Correction of Brain and Spinal Cord Pathology in Metachromatic Leukodystrophy Mice

International audienceMetachromatic leukodystrophy (MLD) is a lysosomal storage disorder characterized by accumulation of sulfatides in both glial cells and neurons. MLD results from an inherited deficiency of arylsulfatase A (ARSA) and myelin degeneration in the central and peripheral nervous systems. Currently, no effective treatment is available for the most frequent late infantile (LI) form of MLD after symptom onset. The LI form results in rapid neurological degradation and early death. ARSA enzyme must be rapidly and efficiently delivered to brain and spinal cord oligodendrocytes of patients with LI MLD in order to potentially stop the progression of the disease. We previously showed that brain gene therapy with adeno-associated virus serotype rh10 (AAVrh10) driving the expression of human ARSA cDNA alleviated most long-term disease manifestations in MLD mice but was not sufficient in MLD patient to improve disease progression. Herein, we evaluated the short-term effects of intravenous AAVPHP.eB delivery driving the expression of human ARSA cDNA under the control of the cytomegalovirus/b-actin hybrid (CAG) promoter in 6-month-old MLD mice that already show marked sulfatide accumulation and brain pathology. Within 3 months, a single intravenous injection of AAVPHP.eB-hARSA-HA resulted in correction of brain and spinal cord sulfatide storage, and improvement of astrogliosis and microgliosis in brain and spinal cord of treated animals. These results strongly support to consider the use of AAVPHP.eB-hARSA vector for intravenous gene therapy in symptomatic rapidly progressing forms of MLD

Locomotor deficit and recovery in Hnf6 knockout mice. E. Audouard, O. Schakman,

The Onecut (OC) family of transcription factors comprises three members in mammals, namely HNF-6 (or OC-1), OC-2 and OC-3. During embryonic development, these transcriptional activators control cell differentiation in pancreas, in liver and in the nervous system. In particular, they are collectively required for proper differentiation of the spinal motor neurons. The Hnf6-/- newborn mice suffer from paralysis of the hindlimbs. However, some of these animals show with age a progressive recovery of locomotor capacities. The goal of this project is to identify the causes of paralysis and mechanisms that are activated during locomotor recovery in Hnf6-/- mice. Spinal motor neurons were identified by immunofluorescence for motor neuron markers (Isl1, Hb9, VAChT) or by retrograde FluoroGold labeling. Neuromuscular junctions (NMJ) were visualized after alpha-bungarotoxin labeling and immunofluorescence for synaptophysin and neurofilaments on hindlimb muscle sections. Locomotor recovery was quantified by four complementary tests (neurological score, Grip Strength system, open field videotracking and catwalk analyses). In Hnf6-/- newborns, the amount of motor neurons was normal, suggesting that altered generation of motor neurons is not the cause of the paralysis. In contrast, the amount of astrocytes was reduced, indicating that the roles of HNF-6 during spinal cord development extend to astroglial differentiation. Interestingly, the morphology of the NMJ was severely altered in the absence of HNF-6, resulting in a ±40% reduction in hindlimb muscular strength. Preliminary observations suggested that locomotor improvement characterized by a partial recovery of muscle strength was initiated in some Hnf6-/- mice around 5-6 months of age. Current investigations aim to identify how HNF-6 controls NMJ formation and whether locomotor recovery correlates with any improvement in neuromuscular connections and/or with changes in the neuronal or glial compartments in the spinal cor

Propagation of tau pathology by alzheimer PHF: seeding of tau isoforms in wild-type mouse and in cells.

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