Development of Axon-Target Specificity of Ponto-Cerebellar Afferents

The function of neuronal networks relies on selective assembly of synaptic connections during development. We examined how synaptic specificity emerges in the pontocerebellar projection. Analysis of axon-target interactions with correlated light-electron microscopy revealed that developing pontine mossy fibers elaborate extensive cell-cell contacts and synaptic connections with Purkinje cells, an inappropriate target. Subsequently, mossy fiber–Purkinje cell connections are eliminated resulting in granule cell-specific mossy fiber connectivity as observed in mature cerebellar circuits. Formation of mossy fiber-Purkinje cell contacts is negatively regulated by Purkinje cell-derived BMP4. BMP4 limits mossy fiber growth in vitro and Purkinje cell-specific ablation of BMP4 in mice results in exuberant mossy fiber–Purkinje cell interactions. These findings demonstrate that synaptic specificity in the pontocerebellar projection is achieved through a stepwise mechanism that entails transient innervation of Purkinje cells, followed by synapse elimination. Moreover, this work establishes BMP4 as a retrograde signal that regulates the axon-target interactions during development

Induction of early Purkinje cell dendritic differentiation by thyroid hormone requires RORα

<p>Abstract</p> <p>Background</p> <p>The active form (T₃) of thyroid hormone (TH) controls critical aspects of cerebellar development, such as migration of postmitotic neurons and terminal dendritic differentiation of Purkinje cells. The effects of T₃on early dendritic differentiation are poorly understood.</p> <p>Results</p> <p>In this study, we have analyzed the influence of T₃on the progression of the early steps of Purkinje cell dendritic differentiation in postnatal day 0 organotypic cerebellar cultures. These steps include, successively, regression of immature neuritic processes, a stellate cell stage, and the extension of several long and mature perisomatic protrusions before the growth of the ultimate dendritic tree. We also studied the involvement of RORα, a nuclear receptor controlling early Purkinje cell dendritic differentiation. We show that T₃treatment leads to an accelerated progression of the early steps of dendritic differentiation in culture, together with an increased expression of RORα (mRNA and protein) in both Purkinje cells and interneurons. Finally, we show that T₃failed to promote early dendritic differentiation in <it>staggerer </it>RORα-deficient Purkinje cells.</p> <p>Conclusions</p> <p>Our results demonstrate that T₃action on the early Purkinje cell dendritic differentiation process is mediated by RORα.</p

Transcriptomics reveal an integrative role for maternal thyroid hormones during zebrafish embryogenesis

Thyroid hormones (THs) are essential for embryonic brain development but the genetic mechanisms involved in the action of maternal THs (MTHs) are still largely unknown. As the basis for understanding the underlying genetic mechanisms of MTHs regulation we used an established zebrafish monocarboxylic acid transporter 8 (MCT8) knock-down model and characterised the transcriptome in 25hpf zebrafish embryos. Subsequent mapping of differentially expressed genes using Reactome pathway analysis together with in situ expression analysis and immunohistochemistry revealed the genetic networks and cells under MTHs regulation during zebrafish embryogenesis. We found 4,343 differentially expressed genes and the Reactome pathway analysis revealed that TH is involved in 1681 of these pathways. MTHs regulated the expression of core developmental pathways, such as NOTCH and WNT in a cell specific context. The cellular distribution of neural MTH-target genes demonstrated their cell specific action on neural stem cells and differentiated neuron classes. Taken together our data show that MTHs have a role in zebrafish neurogenesis and suggest they may be involved in cross talk between key pathways in neural development. Given that the observed MCT8 zebrafish knockdown phenotype resembles the symptoms in human patients with Allan-Herndon-Dudley syndrome our data open a window into understanding the genetics of this human congenital condition.Portuguese Fundacao para Ciencia e Tecnologia (FCT) [PTDC/EXPL/MARBIO/0430/2013]; CCMAR FCT Plurianual financing [UID/Multi/04326/2013]; FCT [SFRH/BD/111226/2015, SFRH/BD/108842/2015, SFRH/BPD/89889/2012]; FCT-IF Starting Grant [IF/01274/2014]info:eu-repo/semantics/publishedVersio

Homozygous staggerer (sg/sg) mice display improved insulin sensitivity and enhanced glucose uptake in skeletal muscle

Homozygous staggerer (sg/sg) mice, which have decreased and dysfunctional Ror alpha (also known as Rora) expression in all tissues, display a lean and dyslipidaemic phenotype. They are also resistant to (high fat) diet-induced obesity. We explored whether retinoic acid receptor-related orphan receptor (ROR) alpha action in skeletal muscle was involved in the regulation of glucose metabolism

Rôle du récepteur nucléaire RORα dans la survie et la différenciation<br />neuronale

RORα is a transcription factor belonging to the nuclear receptor superfamily. RORα disruption in homozygous staggerer mutants leads to a variety of age-related phenotypes, including neurodegeneration, immunodeficiencies, muscular atrophy, osteoporosis and atherosclerosis (for review, see ). Most obviously, staggerer mutants display a strong ataxic phenotype, associated with severe cerebellar degeneration. Most cerebellar Purkinje cells (PCs) (82%) undergo cell death during postnatal development, whilst those remaining PCs display an immature shape. However, whether this immature shape is due to the absence of RORα or to secondary events consecutive to PC death was unknown. During a first part of my thesis, I studied the implication of RORα in the neuronal survival in RORα overexpressing experiments. I have constructed a lentiviral vector to perform efficient RORα overexpression in both cortical and cerebellar cultures. By FACS quantification, the survival rate of RORα-overexpressing cortical neurons was evaluated in response to different stressors disturbing redox homeostasis, such as Aβ peptide, c2 ceramide and H2O2. We have shown that hRORα1 overexpression provides neuroprotection against reactive oxygen species (ROS)-induced apoptosis and real-time RT-PCR revealed an overexpression of the anti-oxidant enzymes glutathione peroxidase 1 and peroxiredoxin 6. Down-regulation of these enzymes by si-RNA experiments partially suppressed the RORα-mediated neuroprotection, further demonstrating their implication in the protection provided by RORα . RORα appears thus as a factor controlling the oxidative stress in neurons. To test whether an overexpression of RORα could protect Purkinje cells from death during development, we studied the survival of RORα-overexpressing PCs in a model which reproduces a developmental cell-death in organotypic cultures. Quantification of the PC number showed an increased survival of lentiviral-mediated RORα-overexpressing PCs, suggesting that the age-dependant apoptotic cell death in culture is decreased when RORα is overexpressed, indicating a neuroprotective role of RORα against developmental cell death. We are currently investigating the ROS pathway in the RORα-overexpressing PCs . In a second part of my thesis, I studied the role of RORα in the PC differentiation process in organotypic culture. As observed in vivo, in these cultures fusiform PCs with embryonic bipolar shape undergo regression prior to the outgrowth of the ultimate dendritic tree. We show that lentiviral-mediated overexpression of RORα in fusiform PCs leads to a cell-autonomous accelerated progression of dendritic differentiation. In addition, we demonstrate that RORα is necessary for the regressive events of early differentiation: while staggerer RORα-deficient PCs remain in the embryonic fusiform stage, replacement of RORα restores normal dendritogenesis. In this study, we have thus demonstrated that RORα controls initial dendritic remodelling during development, particularly the first phase of dendritic regression of the fusiform PCs . In a third part of my thesis, I studied the implication of RORα in the thyroid hormone (TH) signalling during early PC dendritic differentiation. We show that TH treatment leads to an upregulation of RORα expression in PCs. Moreover, action of TH requires RORα expression, since we show that, unlike controls, TH has no effect on the PC differentiation in RORα-deficient PCs. Our results strongly suggest that TH action on the early PC dendritic is mediated by RORα . We are now exploring whether RORα is a target gene of thyroid nuclear receptor in PCs.RORα (Retinoic acid receptor related Orphan Receptor α) est un récepteur nucléairedont la perte de fonction entraîne chez la souris staggerer - entre autres phénotypes - unesévère ataxie cérébelleuse. RORα a longtemps été considéré comme un récepteur nucléaireorphelin, cependant, le cholestérol (ou un de ses dérivés) semble être un ligand physiologiquede ce récepteur.Le mutant staggerer, identifié dès 1962 par Sidman, Lane et Dickie, présente unehypoplasie cérébelleuse liée à l'absence de la grande majorité des cellules de Purkinje, ainsique des grains du cervelet. L'expression de la mutation staggerer dans les cellules de Purkinjeconduit à leur mort massive pendant le développement, et les cellules de Purkinje survivanteschez l'adulte présentent d'importantes anomalies de différenciation. Par ailleurs, à l'étathétérozygote, le mutant staggerer présente une diminution de la survie des cellules dePurkinje ainsi que des anomalies de différenciation au cours du vieillissement.Le phénotype cérébelleux des mutants homozygote et hétérozygote staggerersuggère que RORα est impliqué dans la survie et/ou la différenciation des cellules de Purkinjeau cours du développement et du vieillissement. Au cours de cette thèse, nous avons donctenté de déterminer le rôle de RORα dans la survie et la différenciation dans des neuronescorticaux et/ou dans des cellules de Purkinje, en étudiant l'effet de sa surexpression dans cesprocessus.Afin de surexprimer RORα, nous avons construit un vecteur lentiviral exprimantl'isoforme humaine RORα1. Ce vecteur nous a permis de transduire avec une très grandeefficacité des neurones corticaux en culture primaire ainsi que les cellules de Purkinje enculture organotypique.Dans une première étude, nous avons cherché à déterminer si RORα pouvait exercerun rôle dans la survie neuronale. Dans ce but, nous avons évalué la survie de neuronescorticaux surexprimant ou non RORα et soumis à un stress oxydatif entraînant l'apoptose desneurones. RORα protège les neurones en diminuant le stress oxydatif causé par ces inducteurspro-apoptotiques. L'expression des deux enzymes Glutathion peroxydase 1 et Peroxiredoxine6 est augmentée dans les neurones qui surexpriment hRORα1, et semble partiellement médierl'effet neuroprotecteur de RORα. Nous avons par ailleurs évalué et comparé la survie descellules de Purkinje en culture organotypique qui expriment RORα de façon endogène, ou quisurexpriment hRORα1, et nos résultats suggèrent que la surexpression de RORα a égalementun effet neuroprotecteur dans ces cellules.Dans une seconde étude, afin de déterminer le rôle de RORα dans la différenciationdes cellules de Purkinje, nous avons analysé en culture la progression de la différenciationdendritique précoce de cellules de Purkinje en fonction de l'expression de RORα. RORα estcrucial pour l'étape de régression des neurites des cellules de Purkinje au stade bipolaireembryonnaire. Alors que l'absence de RORα chez les mutants homozygotes staggererentraîne un arrêt de la différenciation des cellules de Purkinje à ce stade (les cellules dePurkinje ne parviennent pas à entamer la régression des neurites), la surexpression dehRORα1 accélère l'étape de régression. Cette étape de régression est totalement dépendantede l'expression de protéines RORα fonctionnelles et cet effet est vraisemblablementintrinsèque. Nous montrons enfin que l'hormone thyroïdienne accélère la différenciationdendritique précoce des cellules de Purkinje, et que RORα semble contribuer à ce processus.L'ensemble de ces résultats montre que RORα intervient de façon cruciale à la foisdans la survie et dans la différenciation des cellules de Purkinje dans une étape très précoce deleur développement post-natal

Rôle du récepteur nucléaire RORa dans la survie et la différentiation des neurones

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Ablation of the mTORC2 component rictor in brain or Purkinje cells affects size and neuron morphology

The mammalian target of rapamycin (mTOR) assembles into two distinct multi-protein complexes called mTORC1 and mTORC2. Whereas mTORC1 is known to regulate cell and organismal growth, the role of mTORC2 is less understood. We describe two mouse lines that are devoid of the mTORC2 component rictor in the entire central nervous system or in Purkinje cells. In both lines neurons were smaller and their morphology and function were strongly affected. The phenotypes were accompanied by loss of activation of Akt, PKC, and SGK1 without effects on mTORC1 activity. The striking decrease in the activation and expression of several PKC isoforms, the subsequent loss of activation of GAP-43 and MARCKS, and the established role of PKCs in spinocerebellar ataxia and in shaping the actin cytoskeleton strongly suggest that the morphological deficits observed in rictor-deficient neurons are mediated by PKCs. Together our experiments show that mTORC2 has a particularly important role in the brain and that it affects size, morphology, and function of neurons

Activation of the JNK-c-Jun pathway during the early phase of neuronal apoptosis induced by PrP106-126 and prion infection.

Prion diseases are neurodegenerative pathologies characterized by apoptotic neuronal death. Although the late execution phase of neuronal apoptosis is beginning to be characterized, the sequence of events occurring during the early decision phase is not yet well known. In murine cortical neurons in primary culture, apoptosis was first induced by exposure to a synthetic peptide homologous to residues 106-126 of the human prion protein (PrP), PrP106-126. Exposure to its aggregated form induced a massive neuronal death within 24 h. Apoptosis was characterized by nuclear fragmentation, neuritic retraction and fragmentation and activation of caspase-3. During the early decision phase, reactive oxygen species were detected after 3 h. Using immunocytochemistry, we showed a peak of phosphorylated c-Jun-N-terminal kinase (JNK) translocation into the nucleus after 8 h, along with the activation of the nuclear c-Jun transcription factor. Both pharmacological inhibition of JNK by SP600125 and overexpression of a dominant negative form of c-Jun significantly reduced neuronal death, while the MAPK p38 inhibitor SB203580 had no effect. Apoptosis was also studied after exposure of tg338 cortical neurons in primary culture to sheep scrapie agent. In this model, prion-induced neuronal apoptosis gradually increased with time and induced a 40% cell death after 2 weeks exposure. Immunocytochemical analysis showed early c-Jun activation after 7 days. In summary, the JNK-c-Jun pathway plays an important role in neuronal apoptosis induced by PrP106-126. This pathway is also activated during scrapie infection and may be involved in prion-induced neuronal death. Pharmacological blockade of early pathways opens new therapeutic prospects for scrapie PrP-based pathologies