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

Expression of X-chromosome linked inhibitor of apoptosis protein in mature purkinje cells and in retinal bipolar cells in transgenic mice induces neurodegeneration

Transgenic mice with overexpression of the caspase-inhibitor, X-chromosome-linked inhibitor of apoptosis protein (XIAP) in Purkinje cell (PC) and in retinal bipolar cells (RBCs) were produced to study the regulation of cell death. Unexpectedly, an increased neurodegeneration was observed in the PCs in these L7-XIAP mice after the third postnatal week with the mice exhibiting severe ataxia. The loss of PCs was independent of Bax as shown by crossing the L7-XIAP mice with Bax gene–deleted mice. Electron microscopy revealed intact organelles in PCs but with the stacking of ER cisterns indicative of cell stress. Immunostaining for cell death proteins showed an increased phosphorylation of c-Jun in the PCs, suggesting an involvement in cell degeneration. Apart from PCs, the number of RBCs was decreased in adult retina in line with the expression pattern for the L7 promoter. The data show that overexpression of the anti-apoptotic protein XIAP in vulnerable neurons leads to enhanced cell death. The mechanisms underlying this neurodegeneration can be related to the effects of XIAP on cell stress and altered cell signaling.Supported by Sigrid Juselius Foundation, Academy of Finland, EU Biotech Grant, Liv och Hälsa, Maud Kuistila, Ylppö Foundation, Uppsala University and Minerva Foundation. We thank Dr. Urmas Arumäe for discussions, and Dr. Patrik Ernfors for the Bax KO mice, and Eeva Lehto for technical assistance. L7AUG was a kind gift from Dr. J. Oberdick, Ohio State University, USA.Peer reviewe

c-Jun N-Terminal Kinase (JNK) and p38 Play Different Roles in Age-Related Purkinje Cell Death in Murine Organotypic Culture.

International audienceSeveral studies have shown that Purkinje cells die by apoptosis in organotypic slice cultures from postnatal 3-day-old (P3) mice. This cell death is age-dependent and has been proposed as indirect evidence for the programmed Purkinje cell death occurring in in vivo cerebellum. Here, we studied whether c-jun N-terminal kinase (JNK) and p38 kinase pathways contribute to the Purkinje cell death observed in cerebellar slice cultures obtained from P3 mice. Slice culture treatment with D-JNKI1 or SB203580, respectively inhibitors of JNK and p38 MAP kinases, results in a better survival of Purkinje cells. Interestingly, the combined treatment with the two inhibitors potentiated single treatment effects. These results suggest that p38 and JNK pathways might be differently implicated in this Purkinje cell death. Time course experiments found p38 activation immediately post-slicing, whereas JNK activation was detected only 2 h after the culture. We hypothesize that p38 activation might be due to the "sliced condition," and JNK activation might be more specific to P3 age-dependent cell death. The study of JNK and p38 activation in cerebellar lysates from P0 slice culture confirmed JNK activation being specific for the P3 explants, whereas p38 is activated both from P0 and P3 cerebellar slice culture lysates. These results suggest that p38 is activated by the slicing, whereas JNK activation is related to developmental Purkinje cell death

Postnatal Maturation of Na\u3csup\u3e+\u3c/sup\u3e, K\u3csup\u3e+\u3c/sup\u3e, 2Cl\u3csup\u3e–\u3c/sup\u3e Cotransporter Expression and Inhibitory Synaptogenesis in the Rat Hippocampus: An Immunocytochemical Analysis

GABA, a major inhibitory neurotransmitter, depolarizes hippocampal pyramidal neurons during the first postnatal week. These depolarizations result from an efflux of Cl– through GABAA-gated anion channels. The outward Cl– gradient that provides the driving force for Cl– efflux might be generated and maintained by the Na+, K+, 2Cl– cotransporter (NKCC) that keeps intracellular Cl– concentration above electrochemical equilibrium. The developmental pattern of expression of the cotransporter in the hippocampus is not known. We studied the postnatal distribution pattern of NKCC in the hippocampus using a monoclonal antibody (T4) against a conserved epitope in the C-terminus of the cotransporter molecule. We also examined the temporal relationships between the developmental pattern of NKCC expression and the formation of perisomatic GABAergic synapses. This study was aimed at determining, with antivesicular inhibitory amino acid transporter (VIAAT) antibodies, whether perisomatic GABAergic synapses are formed preferentially at the time when GABA is depolarizing. During the first postnatal week, NKCC immunolabelling was restricted to cell bodies in the pyramidal cell layer and in the strata oriens and radiatum. In contrast, at postnatal day 21 (P21) and in adult animals little or no labelling occurred in cell bodies; instead, a prominent dendritic labelling appeared in both pyramidal and nonpyramidal neurons. The ultrastructural immunogold study in P21 rat hippocampi corroborated the light-microscopy results. In addition, this study revealed that a portion of the silver-intensified colloidal gold particles were located on neuronal plasmalemma, as expected for a functional cotransporter. The formation of inhibitory synapses on perikarya of the pyramidal cell layer was a late process. The density of VIAAT-immunoreactive puncta in the stratum pyramidale at P21 reached four times the P7 value in CA3, and six times the P7 value in CA1. Electron microscopy revealed that the number of synapses per neuronal perikaryal profile in the stratum pyramidale of the CA3 area at P21 was three times higher than at P7, even if a concomitant 20% increase in the area of these neuronal perikaryal profiles occurred. It is concluded that, in hippocampal pyramidal cells, there is a developmental shift in the NKCC localization from a predominantly somatic to a predominantly dendritic location. The presence of NKCC during the first postnatal week is consistent with the hypothesis that this transporter might be involved in the depolarizing effects of GABA. The depolarizing effects of GABA may not be required for the establishment of the majority of GABAergic synapses in the stratum pyramidale, because their number increases after the first postnatal week, when GABA action becomes hyperpolarizing

Postnatal Maturation of Na\u3csup\u3e+\u3c/sup\u3e, K\u3csup\u3e+\u3c/sup\u3e, 2Cl\u3csup\u3e–\u3c/sup\u3e Cotransporter Expression and Inhibitory Synaptogenesis in the Rat Hippocampus: An Immunocytochemical Analysis

GABA, a major inhibitory neurotransmitter, depolarizes hippocampal pyramidal neurons during the first postnatal week. These depolarizations result from an efflux of Cl– through GABAA-gated anion channels. The outward Cl– gradient that provides the driving force for Cl– efflux might be generated and maintained by the Na+, K+, 2Cl– cotransporter (NKCC) that keeps intracellular Cl– concentration above electrochemical equilibrium. The developmental pattern of expression of the cotransporter in the hippocampus is not known. We studied the postnatal distribution pattern of NKCC in the hippocampus using a monoclonal antibody (T4) against a conserved epitope in the C-terminus of the cotransporter molecule. We also examined the temporal relationships between the developmental pattern of NKCC expression and the formation of perisomatic GABAergic synapses. This study was aimed at determining, with antivesicular inhibitory amino acid transporter (VIAAT) antibodies, whether perisomatic GABAergic synapses are formed preferentially at the time when GABA is depolarizing. During the first postnatal week, NKCC immunolabelling was restricted to cell bodies in the pyramidal cell layer and in the strata oriens and radiatum. In contrast, at postnatal day 21 (P21) and in adult animals little or no labelling occurred in cell bodies; instead, a prominent dendritic labelling appeared in both pyramidal and nonpyramidal neurons. The ultrastructural immunogold study in P21 rat hippocampi corroborated the light-microscopy results. In addition, this study revealed that a portion of the silver-intensified colloidal gold particles were located on neuronal plasmalemma, as expected for a functional cotransporter. The formation of inhibitory synapses on perikarya of the pyramidal cell layer was a late process. The density of VIAAT-immunoreactive puncta in the stratum pyramidale at P21 reached four times the P7 value in CA3, and six times the P7 value in CA1. Electron microscopy revealed that the number of synapses per neuronal perikaryal profile in the stratum pyramidale of the CA3 area at P21 was three times higher than at P7, even if a concomitant 20% increase in the area of these neuronal perikaryal profiles occurred. It is concluded that, in hippocampal pyramidal cells, there is a developmental shift in the NKCC localization from a predominantly somatic to a predominantly dendritic location. The presence of NKCC during the first postnatal week is consistent with the hypothesis that this transporter might be involved in the depolarizing effects of GABA. The depolarizing effects of GABA may not be required for the establishment of the majority of GABAergic synapses in the stratum pyramidale, because their number increases after the first postnatal week, when GABA action becomes hyperpolarizing

Purkinje Cell Maturation Participates in the Control of Oligodendrocyte Differentiation: Role of Sonic Hedgehog and Vitronectin

Oligodendrocyte differentiation is temporally regulated during development by multiple factors. Here, we investigated whether the timing of oligodendrocyte differentiation might be controlled by neuronal differentiation in cerebellar organotypic cultures. In these cultures, the slices taken from newborn mice show very few oligodendrocytes during the first week of culture (immature slices) whereas their number increases importantly during the second week (mature slices). First, we showed that mature cerebellar slices or their conditioned media stimulated oligodendrocyte differentiation in immature slices thus demonstrating the existence of diffusible factors controlling oligodendrocyte differentiation. Using conditioned media from different models of slice culture in which the number of Purkinje cells varies drastically, we showed that the effects of these differentiating factors were proportional to the number of Purkinje cells. To identify these diffusible factors, we first performed a transcriptome analysis with an Affymetrix array for cerebellar cortex and then real-time quantitative PCR on mRNAs extracted from fluorescent flow cytometry sorted (FACS) Purkinje cells of L7-GFP transgenic mice at different ages. These analyses revealed that during postnatal maturation, Purkinje cells down-regulate Sonic Hedgehog and up-regulate vitronectin. Then, we showed that Sonic Hedgehog stimulates the proliferation of oligodendrocyte precursor cells and inhibits their differentiation. In contrast, vitronectin stimulates oligodendrocyte differentiation, whereas its inhibition with blocking antibodies abolishes the conditioned media effects. Altogether, these results suggest that Purkinje cells participate in controlling the timing of oligodendrocyte differentiation in the cerebellum through the developmentally regulated expression of diffusible molecules such as Sonic Hedgehog and vitronectin. © 2012 Bouslama-Oueghlani et al.This work was supported by the Centre National de la Recherche Scientifique (CNRS): ATIP, University Pierre et Marie Curie (UPMC), the Institut National Scientifique pour la Recherche Médicale (INSERM), Association pour la Recherche sur le Cancer (ARC, contract 3532), RGN (Reseau Genopole National, microarray subvention) and the Agence National pour le Recherche (ANR, ANR-07-NEURO-043-01).Peer Reviewe

The RhoGEF DOCK10 is essential for dendritic spine morphogenesis

International audienceBy regulating actin cytoskeleton dynamics, Rho GTPases and their activators RhoGEFs are implicated in various aspects of neuronal differentiation, including dendritogenesis and synaptogenesis. Purkinje cells (PCs) of the cerebellum, by developing spectacular dendrites covered with spines, represent an attractive model system in which to decipher the molecular signaling underlying these processes. To identify novel regulators of dendritic spine morphogenesis among members of the poorly characterized DOCK family of RhoGEFs, we performed gene expression profiling of fluorescence-activated cell sorting (FACS)-purified murine PCs at various stages of their postnatal differentiation. We found a strong increase in the expression of the Cdc42-specific GEF DOCK10. Depleting DOCK10 in organotypic cerebellar cultures resulted in dramatic dendritic spine defects in PCs. Accordingly, in mouse hippocampal neurons, depletion of DOCK10 or expression of a DOCK10 GEF-dead mutant led to a strong decrease in spine density and size. Conversely, overexpression of DOCK10 led to increased spine formation. We show that DOCK10 function in spinogenesis is mediated mainly by Cdc42 and its downstream effectors N-WASP and PAK3, although DOCK10 is also able to activate Rac1. Our global approach thus identifies an unprecedented function for DOCK10 as a novel regulator of dendritic spine morphogenesis via a Cdc42-mediated pathway

The effects of CM (conditioned media) from mature slices on OPC differentiation in immature slices are correlated with the presence of Purkinje cells.

<p><b>A.</b> Schematic diagram of the co-culture experiment: P0 donor slices were grown for 14 days. The medium was changed at 3, 5, 7, 10, 12, and 14 DIV. The CM taken at 10 DIV was used for the culture of fresh recipient P0 slices from days 0 to 3; the CM taken at 12 DIV was used to culture recipient slices from days 3 to 5, and the CM taken at 14 DIV was used to culture recipient slices from days 5 to 7. <b>B.</b> The recipient P0–7 DIV slices grown in the presence of Ctrl-CM contained many MBP+ elements. <b>C.</b> The recipient P0 slice grown with BrdU-CM contains more MBP+ elements than that grown with Ctrl-CM (compare B and C). <b>D.</b> The recipient P0 slice grown with the Axt-CM contains few MBP+ elements (compare B and D). <b>E.</b> Quantitative evaluation of the density of MBP immunostaining in recipient P0-7DIV slices. Conditions: Ctrl corresponds to control P0–7DIV slices. Ctrl-CM corresponds to P0–7 DIV slices grown with the medium conditioned by untreated P0 7–14 DIV mature donor slices. BrdU-CM corresponds to P0–7 DIV slices grown with the medium conditioned by P0 7–14 DIV mature donor slices treated, for the first 3 DIV, with a high dose of BrdU. Axt-CM corresponds to P0–7 DIV slices grown with the medium conditioned by P0 7–14 DIV mature donor slices axotomized after 2 DIV. ** P<0.01 (Mann-Whitney tests). Scale bar is 390 µm for all images.</p