RÉSUMÉ
Midbrain dopaminergic (mDA) neurons are diverse in their projection targets, effect on behavior, and susceptibility to neurodegeneration. Little is known about the molecular mechanisms establishing this diversity during development. We show that the transcription factor BCL11A is expressed in a subset of mDA neurons in the developing and adult murine brain and in a subpopulation of pluripotent-stem-cell-derived human mDA neurons. By combining intersectional labeling and viral-mediated tracing, we demonstrate that Bcl11a-expressing mDA neurons form a highly specific subcircuit within the murine dopaminergic system. In the substantia nigra, the Bcl11a-expressing mDA subset is particularly vulnerable to neurodegeneration upon α-synuclein overexpression or oxidative stress. Inactivation of Bcl11a in murine mDA neurons increases this susceptibility further, alters the distribution of mDA neurons, and results in deficits in skilled motor behavior. In summary, BCL11A defines mDA subpopulations with highly distinctive characteristics and is required for establishing and maintaining their normal physiology.
Sujet(s)
Neurones dopaminergiques/métabolisme , Protéines de répression/métabolisme , Animaux , Comportement animal , Encéphale/métabolisme , Dopamine/métabolisme , Humains , Cellules souches pluripotentes induites/cytologie , Cellules souches pluripotentes induites/métabolisme , Mâle , Souris , Souris knockout , Protéines de répression/déficit , Protéines de répression/génétique , Substantia nigra/métabolisme , Substantia nigra/anatomopathologie , Transcriptome , Aire tegmentale ventrale/métabolisme , Aire tegmentale ventrale/anatomopathologie , alpha-Synucléine/génétique , alpha-Synucléine/métabolismeRÉSUMÉ
Spinocerebellar ataxia type 2 (SCA2) is caused by polyglutamine expansion in Ataxin-2 (ATXN2). This factor binds RNA/proteins to modify metabolism after stress, and to control calcium (Ca2+) homeostasis after stimuli. Cerebellar ataxias and corticospinal motor neuron degeneration are determined by gain/loss in ATXN2 function, so we aimed to identify key molecules in this atrophic process, as potential disease progression markers. Our Atxn2-CAG100-Knock-In mouse faithfully models features observed in patients at pre-onset, early and terminal stages. Here, its cerebellar global RNA profiling revealed downregulation of signaling cascades to precede motor deficits. Validation work at mRNA/protein level defined alterations that were independent of constant physiological ATXN2 functions, but specific for RNA/aggregation toxicity, and progressive across the short lifespan. The earliest changes were detected at three months among Ca2+ channels/transporters (Itpr1, Ryr3, Atp2a2, Atp2a3, Trpc3), IP3 metabolism (Plcg1, Inpp5a, Itpka), and Ca2+-Calmodulin dependent kinases (Camk2a, Camk4). CaMKIV-Sam68 control over alternative splicing of Nrxn1, an adhesion component of glutamatergic synapses between granule and Purkinje neurons, was found to be affected. Systematic screening of pre/post-synapse components, with dendrite morphology assessment, suggested early impairment of CamKIIα abundance together with the weakening of parallel fiber connectivity. These data reveal molecular changes due to ATXN2 pathology, primarily impacting excitability and communication.
Sujet(s)
Ataxine-2/génétique , Signalisation calcique/génétique , Calcium-Calmodulin-Dependent Protein Kinase Type 2/génétique , Régulation négative/génétique , Cellules de Purkinje/physiologie , Animaux , Protéines de liaison au calcium/génétique , Cellules cultivées , Cervelet/physiologie , Souris , Souris knockout , ARN messager/génétique , Synapses/génétiqueRÉSUMÉ
The regenerative capacity of peripheral nerves declines during aging, contributing to the development of neuropathies, limiting organism function. Changes in Schwann cells prompt failures in instructing maintenance and regeneration of aging nerves; molecular mechanisms of which have yet to be delineated. Here, we identified an altered inflammatory environment leading to a defective Schwann cell response, as an underlying mechanism of impaired nerve regeneration during aging. Chronic inflammation was detected in intact uninjured old nerves, characterized by increased macrophage infiltration and raised levels of monocyte chemoattractant protein 1 (MCP1) and CC chemokine ligand 11 (CCL11). Schwann cells in the old nerves appeared partially dedifferentiated, accompanied by an activated repair program independent of injury. Upon sciatic nerve injury, an initial delayed immune response was followed by a persistent hyperinflammatory state accompanied by a diminished repair process. As a contributing factor to nerve aging, we showed that CCL11 interfered with Schwann cell differentiation in vitro and in vivo. Our results indicate that increased infiltration of macrophages and inflammatory signals diminish regenerative capacity of aging nerves by altering Schwann cell behavior. The study identifies CCL11 as a promising target for anti-inflammatory therapies aiming to improve nerve regeneration in old age.
Sujet(s)
Vieillissement/anatomopathologie , Inflammation/anatomopathologie , Régénération nerveuse , Nerfs périphériques/anatomopathologie , Nerfs périphériques/physiopathologie , Animaux , Anti-inflammatoires/pharmacologie , Anti-inflammatoires/usage thérapeutique , Acide acétylsalicylique/pharmacologie , Acide acétylsalicylique/usage thérapeutique , Chimiokine CCL11/pharmacologie , Ganglions sensitifs des nerfs spinaux/effets des médicaments et des substances chimiques , Ganglions sensitifs des nerfs spinaux/métabolisme , Souris de lignée C57BL , Gaine de myéline/métabolisme , Écrasement de nerf , Régénération nerveuse/effets des médicaments et des substances chimiques , Lésions des nerfs périphériques/anatomopathologie , Lésions des nerfs périphériques/physiopathologie , Nerfs périphériques/effets des médicaments et des substances chimiques , Cellules de Schwann/effets des médicaments et des substances chimiques , Cellules de Schwann/métabolisme , Cellules de Schwann/anatomopathologie , Nerf ischiatique/effets des médicaments et des substances chimiques , Nerf ischiatique/métabolisme , Nerf ischiatique/anatomopathologie , Nerf ischiatique/physiopathologieRÉSUMÉ
The neurofibromatosis type 2 (NF2) tumor suppressor protein Merlin functions as a negative regulator of cell growth and actin dynamics in different cell types amongst which Schwann cells have been extensively studied. In contrast, the presence and the role of Merlin in oligodendrocytes, the myelin forming cells within the CNS, have not been elucidated. In this work, we demonstrate that Merlin immunoreactivity was broadly distributed in the white matter throughout the central nervous system. Following Merlin expression during development in the cerebellum, Merlin could be detected in the cerebellar white matter tract at early postnatal stages as shown by its co-localization with Olig2-positive cells as well as in adult brain sections where it was aligned with myelin basic protein containing fibers. This suggests that Merlin is expressed in immature and mature oligodendrocytes. Expression levels of Merlin were low in oligodendrocytes as compared to astrocytes and neurons throughout development. Expression of Merlin in oligodendroglia was further supported by its identification in either immortalized cell lines of oligodendroglial origin or in primary oligodendrocyte cultures. In these cultures, the two main splice variants of Nf2 could be detected. Merlin was localized in clusters within the nuclei and in the cytoplasm. Overexpressing Merlin in oligodendrocyte cell lines strengthened reduced impedance in XCELLigence measurements and Ki67 stainings in cultures over time. In addition, the initiation and elongation of cellular projections were reduced by Merlin overexpression. Consistently, cell migration was retarded in scratch assays done on Nf2-transfected oligodendrocyte cell lines. These data suggest that Merlin actively modulates process outgrowth and migration in oligodendrocytes.
Sujet(s)
Prolifération cellulaire/physiologie , Neurofibromatose de type 2/métabolisme , Neurofibromine-2/métabolisme , Oligodendroglie/métabolisme , Animaux , Astrocytes/métabolisme , Lignée cellulaire , Mouvement cellulaire/physiologie , Noyau de la cellule/métabolisme , Système nerveux central/métabolisme , Cytoplasme/métabolisme , Souris , Souris de lignée C57BL , Protéines de tissu nerveux/métabolisme , Neurogenèse/physiologie , Neurones/métabolisme , Cellules de Schwann/métabolisme , Transfection/méthodesRÉSUMÉ
Efficient coupling of the actin cytoskeleton to the cell membrane is crucial for histogenesis and maintenance of the nervous system. At this critical interface, BAR (Bin-Amphiphysin-Rvs) proteins regulate membrane bending, shown to be instrumental for mobility and morphogenesis of individual cells. Yet, the systemic significance of these proteins remains largely unexplored. Here, we probe the role of a prominent member of this protein family, the inverse-BAR protein Mtss1, for the development and function of a paradigmatic neuronal circuit, the cerebellar cortex. Mtss1-null mice show granule cell ectopias, dysmorphic Purkinje cells, malformed axons, and a protracted neurodegeneration entailing age-dependent motor deficits. In postmitotic granule cells, which transiently express Mtss1 while they migrate and form neurites, Mtss1 impinges on directional persistence and neuritogenesis. The latter effect can be specifically attributed to its exon 12a splice variant. Targeted re-expression of Mtss1 in Mtss1-null animals indicated that these pathologies were largely due to cell type-specific and intrinsic effects. Together, our results provide a mechanistic perspective on Mtss1 function for brain development and degeneration and relate it to structural features of this protein.
Sujet(s)
Axones/métabolisme , Cervelet/métabolisme , Protéines des microfilaments/métabolisme , Activité motrice , Motoneurones/métabolisme , Protéines tumorales/métabolisme , Dégénérescence nerveuse , Cellules de Purkinje/métabolisme , Facteurs âges , Animaux , Axones/anatomopathologie , Cellules cultivées , Cervelet/anatomopathologie , Cervelet/physiopathologie , Exons , Régulation de l'expression des gènes au cours du développement , Génotype , Souris de lignée C57BL , Souris knockout , Protéines des microfilaments/déficit , Protéines des microfilaments/génétique , Motoneurones/anatomopathologie , Protéines tumorales/déficit , Protéines tumorales/génétique , Phénotype , Isoformes de protéines , Cellules de Purkinje/anatomopathologie , Test du rotarodRÉSUMÉ
In contrast to axons of the central nervous system (CNS), axons of the peripheral nervous system (PNS) show better, but still incomplete and often slow regeneration following injury. The tumor suppressor protein merlin, mutated in the hereditary tumor syndrome Neurofibromatosis type 2 (NF2), has recently been shown to have RhoA regulatory functions in PNS neurons-in addition to its well-characterized, growth-inhibitory activity in Schwann cells. Here we report that the conditional knockout of merlin in PNS neurons leads to impaired functional recovery of mice following sciatic nerve crush injury, in a gene-dosage dependent manner. Gross anatomical or electrophysiological alterations of sciatic nerves could not be detected. However, correlating with attenuated RhoA activation due to merlin deletion, ultrastructural analysis of nerve samples indicated enhanced sprouting of axons with reduced caliber size and increased myelination compared to wildtype animals. We conclude that deletion of the tumor suppressor merlin in the neuronal compartment of peripheral nerves results in compromised functional regeneration after injury. This mechanism could explain the clinical observation that NF2 patients suffer from higher incidences of slowly recovering facial nerve paralysis after vestibular schwannoma surgery.
Sujet(s)
Délétion de gène , Gènes nf2 , Régénération nerveuse/physiologie , Animaux , Souris , Souris knockoutRÉSUMÉ
Schwannomas are predominantly benign nerve sheath neoplasms caused by Nf2 gene inactivation. Presently, treatment options are mainly limited to surgical tumor resection due to the lack of effective pharmacological drugs. Although the mechanistic understanding of Nf2 gene function has advanced, it has so far been primarily restricted to Schwann cell-intrinsic events. Extracellular cues determining Schwann cell behavior with regard to schwannoma development remain unknown. Here we show pro-tumourigenic microenvironmental effects on Schwann cells where an altered axonal microenvironment in cooperation with injury signals contribute to a persistent regenerative Schwann cell response promoting schwannoma development. Specifically in genetically engineered mice following crush injuries on sciatic nerves, we found macroscopic nerve swellings in mice with homozygous nf2 gene deletion in Schwann cells and in animals with heterozygous nf2 knockout in both Schwann cells and axons. However, patient-mimicking schwannomas could only be provoked in animals with combined heterozygous nf2 knockout in Schwann cells and axons. We identified a severe re-myelination defect and sustained macrophage presence in the tumor tissue as major abnormalities. Strikingly, treatment of tumor-developing mice after nerve crush injury with medium-dose aspirin significantly decreased schwannoma progression in this disease model. Our results suggest a multifactorial concept for schwannoma formation-emphasizing axonal factors and mechanical nerve irritation as predilection site for schwannoma development. Furthermore, we provide evidence supporting the potential efficacy of anti-inflammatory drugs in the treatment of schwannomas.
Sujet(s)
Axones/anatomopathologie , Neurinome/anatomopathologie , Cellules de Schwann/anatomopathologie , Nerf ischiatique/anatomopathologie , Microenvironnement tumoral/physiologie , Animaux , Souris transgéniques , Gaine de myéline/anatomopathologie , Neurinome/génétique , Neurofibromatose de type 2/génétique , Microenvironnement tumoral/génétiqueRÉSUMÉ
Lentiviral vectors are valuable tools to express genes of interest in living animals and stem cell cultures. The use of promoters in lentiviral constructs has been successfully used to drive gene expression in particular cell types including neurons and glia of the central nervous system in vivo. However, their suitability in cell culture is less well documented. In this paper, we describe lentiviral vectors containing neuronal promoters of the murine stem cell virus, of the synapsin 1 gene, the tubulin alpha 1 gene, and the calmodulin kinase II gene, and the glial promoter of the glial fibrillary acidic protein gene to drive reporter gene expression in primary dissociated cerebellar cell cultures and in slice cultures. While the glial promoter was highly specific for glia, the neuronal promoters were active in neurons and glia of dissociated cultures to a comparable extent. In slice cultures, neuronal and glial promoters demonstrated higher, but not absolute selectivity for particular cell types. In addition, the promoters allowed for an efficient and graded expression of genes in dissociated cultures. By using selected combinations of vectors, it was also possible to drive the expression of two genes in one cell type with high efficiency. A gene of interest in combination with a reporter gene can thus be expressed in a graded manner to reveal gene function in a rather short time and in a complex cellular environment.
Sujet(s)
Cervelet/cytologie , Expression des gènes/génétique , Vecteurs génétiques/génétique , Lentivirus/génétique , Névroglie/métabolisme , Neurones/métabolisme , Transduction génétique , Animaux , Cellules cultivées , Techniques de transfert de gènes , Protéine gliofibrillaire acide/génétique , Protéine gliofibrillaire acide/métabolisme , Lentivirus/physiologie , Souris , Lignées consanguines de souris , Névroglie/cytologie , Névroglie/virologie , Neurones/cytologie , Neurones/virologie , Régions promotrices (génétique)/génétiqueRÉSUMÉ
Axonal surface proteins encompass a group of heterogeneous molecules, which exert a variety of different functions in the highly interdependent relationship between axons and Schwann cells. We recently revealed that the tumour suppressor protein merlin, mutated in the hereditary tumour syndrome neurofibromatosis type 2, impacts significantly on axon structure maintenance in the peripheral nervous system. We now report on a role of neuronal merlin in the regulation of the axonal surface protein neuregulin 1 important for modulating Schwann cell differentiation and myelination. Specifically, neuregulin 1 type III expression is reduced in sciatic nerve tissue of neuron-specific knockout animals as well as in biopsies from seven patients with neurofibromatosis type 2. In vitro experiments performed on both the P19 neuronal cell line and primary dorsal root ganglion cells demonstrate the influence of merlin on neuregulin 1 type III expression. Moreover, expression of ERBB2, a Schwann cell receptor for neuregulin 1 ligands is increased in nerve tissue of both neuron-specific merlin knockout animals and patients with neurofibromatosis type 2, demonstrating for the first time that axonal merlin indirectly regulates Schwann cell behaviour. Collectively, we have identified that neuronally expressed merlin can influence Schwann cell activity in a cell-extrinsic manner.
Sujet(s)
Neuréguline-1/physiologie , Neurofibromine-2/physiologie , Neurones/physiologie , Récepteur ErbB-2/biosynthèse , Cellules de Schwann/métabolisme , Transduction du signal/physiologie , Adulte , Sujet âgé , Animaux , Lignée cellulaire , Cellules cultivées , Femelle , Humains , Mâle , Souris de lignée C57BL , Souris knockout , Souris transgéniques , Adulte d'âge moyen , Neurofibromatose de type 2/métabolisme , Neurofibromatose de type 2/anatomopathologie , Neurones/anatomopathologie , Cellules de Schwann/anatomopathologieRÉSUMÉ
The autosomal dominant disorder neurofibromatosis type 2 (NF2) is a hereditary tumor syndrome caused by inactivation of the NF2 tumor suppressor gene, encoding merlin. Apart from tumors affecting the peripheral and central nervous systems, most NF2 patients develop peripheral neuropathies. This peripheral nerve disease can occur in the absence of nerve-damaging tumors, suggesting an etiology that is independent of gross tumor burden. We discovered that merlin isoform 2 (merlin-iso2) has a specific function in maintaining axonal integrity and propose that reduced axonal NF2 gene dosage leads to NF2-associated polyneuropathy. We identified a merlin-iso2-dependent complex that promotes activation of the GTPase RhoA, enabling downstream Rho-associated kinase to promote neurofilament heavy chain phosphorylation. Merlin-iso2-deficient mice exhibited impaired locomotor capacities, delayed sensory reactions and electrophysiological signs of axonal neuropathy. Sciatic nerves from these mice and sural nerve biopsies from NF2 patients revealed reduced phosphorylation of the neurofilament H subunit, decreased interfilament spacings and irregularly shaped axons.
Sujet(s)
Neurofibromatose de type 2/métabolisme , Neurofibromine-2/physiologie , Polyneuropathies/métabolisme , Adulte , Séquence d'acides aminés , Animaux , Animaux nouveau-nés , Lignée cellulaire tumorale , Cellules cultivées , Femelle , Ganglions sensitifs des nerfs spinaux/métabolisme , Ganglions sensitifs des nerfs spinaux/anatomopathologie , Cellules HEK293 , Humains , Mâle , Souris , Souris de lignée C57BL , Souris knockout , Adulte d'âge moyen , Données de séquences moléculaires , Neurofibromatose de type 2/génétique , Neurofibromatose de type 2/anatomopathologie , Neurofibromine-2/génétique , Phosphorylation/physiologie , Polyneuropathies/génétique , Polyneuropathies/anatomopathologie , Grossesse , Isoformes de protéines/génétique , Isoformes de protéines/physiologieRÉSUMÉ
The neurofibromatosis type 2 gene product merlin is known to provoke gliogenic tumors as a result of its mutagenic loss. Merlin's physiological anti-mitogenic function makes it unique among its ezrin-radixin-moesin (ERM) family members. Although ERM proteins and merlin are known to be expressed in glial cells of the peripheral nervous system and CNS, the neuronal expression pattern and function of merlin have been less well investigated. We report here expression of merlin in developing and mature neurons of the murine CNS. Within cerebellar Purkinje cells (PCs), merlin was localized in the soma, sprouting dendrites and axons. Merlin expression in PCs was high during the period of initial dendrite regression and declined during later phases of dendrite elongation. Consistently, merlin expression in vivo was increased in Engrailed-2-overexpressing PCs, which are characterized by a reduced dendritic extension. Furthermore, overexpression of merlin in dissociated cerebellar cultures and in neurogenic P19 cells caused a significant decline in neurite outgrowth, while, conversely, inhibition of merlin expression increased process formation. This effect was dependent on phosphorylation of serine 518 and involved the inactivation of the growth-promoting GTPase Rac. We thus provide evidence that merlin plays a pivotal role in controlling the neuronal wiring in the developing CNS.
Sujet(s)
Système nerveux central/cytologie , Régulation de l'expression des gènes au cours du développement/physiologie , Neurites/physiologie , Neurofibromine-2/métabolisme , Neurones/métabolisme , Alanine/génétique , Animaux , Animaux nouveau-nés , Acide aspartique/génétique , Calbindines , Différenciation cellulaire/génétique , Cellules cultivées , Femelle , Protéine GAP-43/métabolisme , Régulation de l'expression des gènes au cours du développement/génétique , Protéines à fluorescence verte/génétique , Protéines à homéodomaine/génétique , Mâle , Souris , Souris de lignée C57BL , Souris transgéniques , Protéines associées aux microtubules/métabolisme , Mutation/génétique , Protéine basique de la myéline/métabolisme , Protéines de tissu nerveux/génétique , Neurites/effets des médicaments et des substances chimiques , Neurofibromine-2/génétique , Neurones/cytologie , Petit ARN interférent/pharmacologie , Protéine G liant le calcium S100/métabolisme , Transduction du signal/effets des médicaments et des substances chimiques , Transduction du signal/génétique , Transfection/méthodesRÉSUMÉ
Hyaluronan is an unsulfated linear glycosaminoglycan with the ability to nucleate extracellular matrices by the formation of aggregates with lecticans. These matrices are essential during development of the central nervous system. In the prospective white matter of the developing brain hyaluronan is organized into fiber-like structures according to confocal microscopy of fixed slices which may guide the migration of neural precursor cells [Baier, C., S.L. Baader, J. Jankowski, V. Gieselmann, K. Schilling, U. Rauch, and J. Kappler. 2007. Hyaluronan is organized into fiber-like structures along migratory pathways in the developing mouse cerebellum. Matrix Biol. 26: 348-58]. By using plasmon surface resonance, microinjection into brain slices and fluorescence correlation spectroscopy, we show that the brain-specific lecticans bind to, but also dissociate rather rapidly from hyaluronan. After microinjection into native cerebellar slices a GFP-tagged hyaluronan-binding neurocan fragment was enriched at binding sites in the prospective white matter, which had a directional orientation and formed local stationary concentration gradients in areas where binding sites are abundant. Fluorescence correlation spectroscopy measurements at fixed brain slices revealed that fiber-bound neurocan-GFP was mobile with D(fiber(neurocan-GFP))=4x10(-10)cm(2)/s. Therefore, we propose that hyaluronan-rich fibers in the prospective white matter of the developing mouse cerebellum can guide the diffusion of lecticans. Since lecticans bind a variety of growth and mobility factors, their guided diffusion may contribute to the transport of these polypeptides and to the formation of concentration gradients. This mechanism could serve to encode positional information during development.
Sujet(s)
Cervelet/métabolisme , Antigènes CD44/métabolisme , Animaux , Brévicane , Cervelet/cytologie , Protéoglycanes à chondroïtine sulfate/génétique , Protéoglycanes à chondroïtine sulfate/métabolisme , Glycosaminoglycanes/métabolisme , Protéines à fluorescence verte/génétique , Protéines à fluorescence verte/métabolisme , Antigènes CD44/génétique , Acide hyaluronique/métabolisme , Lectines/métabolisme , Lectines de type C/génétique , Lectines de type C/métabolisme , Souris , Protéines de tissu nerveux/génétique , Protéines de tissu nerveux/métabolisme , Protéoglycane-3 à chondroïtine sulfate , Liaison aux protéines , Protéoglycanes/génétique , Protéoglycanes/métabolisme , Protéines de fusion recombinantes/génétique , Protéines de fusion recombinantes/métabolisme , Résonance plasmonique de surfaceRÉSUMÉ
Physiological cell death is crucial for matching defined cellular populations within the central nervous system. Whereas the time course of developmental cell death in the central nervous system is well analyzed, information about its precise spatial patterning is scarce. Yet, the latter one is needed to appraise its contribution to circuit formation and refinement. Here, we document that during normal cerebellar development, dying Purkinje cells were highly localized within the vermal midline and in a lobule specific, parasagittal pattern along the whole mediolateral axis. In addition, single hot spots of cell death localized to the caudal declive and ventral lobule IX within the posterolateral fissure. These hot spots of dying Purkinje cells partly overlapped with gaps within the Purkinje cell layer which supports the classification of different gaps based on histological and molecular criteria, i.e., midline gap, patchy gaps, and raphes. Areas characterized by a high incidence of Purkinje cell death and gaps colocalize with known molecular and functional boundaries within the cerebellar cortex. Physiological cell death can thus be considered to serve as an important regulator of cerebellar histogenesis.
Sujet(s)
Cervelet/cytologie , Cervelet/croissance et développement , Régulation de l'expression des gènes au cours du développement/physiologie , Cellules de Purkinje/physiologie , Animaux , Animaux nouveau-nés , Mort cellulaire/physiologie , Mouvement cellulaire , Souris , Souris de lignée C57BL , Microscopie électronique à transmission/méthodes , Protéines de tissu nerveux/métabolisme , Cellules de Purkinje/ultrastructure , Facteurs tempsRÉSUMÉ
Ever since the groundbreaking work of Ramon y Cajal, the cerebellar cortex has been recognized as one of the most regularly structured and wired parts of the brain formed by a rather limited set of distinct cells. Its rather protracted course of development, which persists well into postnatal life, the availability of multiple natural mutants, and, more recently, the availability of distinct molecular genetic tools to identify and manipulate discrete cell types have suggested the cerebellar cortex as an excellent model to understand the formation and working of the central nervous system. However, the formulation of a unifying model of cerebellar function has so far proven to be a most cantankerous problem, not least because our understanding of the internal cerebellar cortical circuitry is clearly spotty. Recent research has highlighted the fact that cerebellar cortical interneurons are a quite more diverse and heterogeneous class of cells than generally appreciated, and have provided novel insights into the mechanisms that underpin the development and histogenetic integration of these cells. Here, we provide a short overview of cerebellar cortical interneuron diversity, and we summarize some recent results that are hoped to provide a primer on current understanding of cerebellar biology.
Sujet(s)
Cortex cérébelleux/cytologie , Cortex cérébelleux/physiologie , Interneurones/cytologie , Interneurones/physiologie , Animaux , Humains , Modèles neurologiques , Cellules de Purkinje/physiologieRÉSUMÉ
Engrailed transcription factors regulate survival, cell fate decisions and axon pathfinding in central neurons. En-2 can also attenuate Purkinje cell (PC) maturation. Here, we use array analysis to scrutinize gene expression in developing PCs overexpressing Engrailed-2 (L7En-2). The majority (70%) of regulated genes was found down-regulated in L7En-2 cerebella, consistent with the known repressive function of Engrailed-2. Differential gene expression, verified by in situ hybridization or Western blotting, was particularly evident during the first postnatal week, when L7En-2 PCs display conspicuous deficits in dendritogenesis. Functional classification revealed clusters of genes linked to vesicle formation and transport. Consistently, Golgi stacks located at the axonal pole of wild type PC somata were rarely detected in L7En-2 PCs. In addition, long continuous stretches of endoplasmic reticulum typically found around the axonal pole of wild type PCs were less frequently observed in transgenic cells. Engrailed-2 might therefore orchestrate PC survival and process formation as a regulator of subcellular organization.
Sujet(s)
Cervelet/physiologie , Protéines à homéodomaine/physiologie , Protéines de tissu nerveux/physiologie , Cellules de Purkinje/physiologie , Vésicules synaptiques/génétique , Vésicules synaptiques/métabolisme , Animaux , Animaux nouveau-nés , Survie cellulaire/génétique , Cervelet/cytologie , Régulation négative/génétique , Régulation de l'expression des gènes/physiologie , Protéines à homéodomaine/antagonistes et inhibiteurs , Protéines à homéodomaine/biosynthèse , Protéines à homéodomaine/génétique , Souris , Souris transgéniques , Protéines de tissu nerveux/antagonistes et inhibiteurs , Protéines de tissu nerveux/biosynthèse , Protéines de tissu nerveux/génétique , Transport des protéines/génétique , Cellules de Purkinje/cytologieRÉSUMÉ
BACKGROUND: Mtss1 encodes an actin-binding protein, dysregulated in a variety of tumors, that interacts with sonic hedgehog/Gli signaling in epidermal cells. Given the prime importance of this pathway for cerebellar development and tumorigenesis, we assessed expression of Mtss1 in the developing murine cerebellum and human medulloblastoma specimens. RESULTS: During development, Mtss1 is transiently expressed in granule cells, from the time point they cease to proliferate to their synaptic integration. It is also expressed by granule cell precursor-derived medulloblastomas. In the adult CNS, Mtss1 is found exclusively in cerebellar Purkinje cells. Neuronal differentiation is accompanied by a switch in Mtss1 splicing. Whereas immature granule cells express a Mtss1 variant observed also in peripheral tissues and comprising exon 12, this exon is replaced by a CNS-specific exon, 12a, in more mature granule cells and in adult Purkinje cells. Bioinformatic analysis of Mtss1 suggests that differential exon usage may affect interaction with Fyn and Src, two tyrosine kinases previously recognized as critical for cerebellar cell migration and histogenesis. Further, this approach led to the identification of two evolutionary conserved nuclear localization sequences. These overlap with the actin filament binding site of Mtss1, and one also harbors a potential PKA and PKC phosphorylation site. CONCLUSION: Both the pattern of expression and splicing of Mtss1 is developmentally regulated in the murine cerebellum. These findings are discussed with a view on the potential role of Mtss1 for cytoskeletal dynamics in developing and mature cerebellar neurons.
Sujet(s)
Transformation cellulaire néoplasique/anatomopathologie , Cervelet/croissance et développement , Régulation de l'expression des gènes au cours du développement , Protéines des microfilaments/génétique , Protéines tumorales/génétique , Animaux , Tumeurs du cervelet/anatomopathologie , Cervelet/anatomopathologie , Exons , Humains , Souris , Souris de lignée C57BL , Souris transgéniques , Réaction de polymérisation en chaîne , Épissage des protéines/génétique , Cellules de Purkinje/anatomopathologie , Cellules cancéreuses en cultureRÉSUMÉ
Sox proteins of group C are strongly expressed in the developing nervous system and have been associated with maturation of neurons and glia. Here, we overexpressed the group C protein Sox4 in transgenic mice under the control of the human GFAP promoter. Transgene expression was detected in radial glia and astrocytes throughout the CNS. The transgenic mice were ataxic and exhibited hydrocephaly as well as cerebellar malformations. In the cerebellum, fissures were not formed and neuronal layering was dramatically disturbed. Nevertheless, all neuronal cell types of the cerebellum were present as well as cells with characteristics of early radial glia, astrocytes, and oligodendrocytes. However, radial glia failed to migrate into the position normally taken by Bergmann glia and did not extend radial fibers toward the pial surface. The cerebellar malformations can therefore be explained by the absence of functional Bergmann glia. We conclude that Sox4 expression counteracts differentiation of radial glia and has to be downregulated before full maturation can occur.
Sujet(s)
Ataxie/métabolisme , Ataxie/anatomopathologie , Cervelet/métabolisme , Régulation de l'expression des gènes au cours du développement/physiologie , Protéines HMG/biosynthèse , Protéines HMG/génétique , Névroglie/métabolisme , Névroglie/anatomopathologie , Transactivateurs/biosynthèse , Transactivateurs/génétique , Animaux , Ataxie/génétique , Cervelet/malformations , Régulation négative/génétique , Femelle , Protéines HMG/antagonistes et inhibiteurs , Protéines HMG/physiologie , Humains , Mâle , Souris , Souris transgéniques , Rats , Facteurs de transcription SOX-C , Transactivateurs/antagonistes et inhibiteurs , Transactivateurs/physiologieRÉSUMÉ
Hyaluronan is a free glycosaminoglycan which is abundant in the extracellular matrix of the developing brain. Although not covalently linked to any protein it can act as a backbone molecule forming aggregates with chondroitin sulfate proteoglycans of the lectican family and link proteins. Using neurocan-GFP as a direct histochemical probe we analyzed the distribution and organization of hyaluronan in the developing mouse cerebellum, and related its fine structure to cell types of specified developmental stages. We observed a high affinity of this probe to fiber-like structures in the prospective white matter which are preferentially oriented parallel to the cerebellar cortex during postnatal development suggesting a specially organized form of hyaluronan. In other layers of the cerebellar cortex, the hyaluronan organization seemed to be more diffuse. During the second postnatal week, the overall staining intensity of hyaluronan in the white matter declined but fiber-like structures were still present at the adult stage. This type of hyaluronan organization is different from perineuronal nets e.g. found in deep cerebellar nuclei. Double staining experiments with cell type specific markers indicated that these fiber-like structures are predominantly situated in regions where motile cells such as Pax2-positive inhibitory interneuron precursors and MBP-positive oligodendroglial cells are located. In contrast, more stationary cells such as mature granule cells and Purkinje cells are associated with lower levels of hyaluronan in their environment. Thus, hyaluronan-rich fibers are concentrated at sites where specific neural precursor cell types migrate, and the anisotropic orientation of these fibers suggests that they may support guided neural migration during brain development.
Sujet(s)
Cervelet/embryologie , Cervelet/métabolisme , Acide hyaluronique/composition chimique , Animaux , Matrice extracellulaire/composition chimique , Protéines à fluorescence verte/métabolisme , Immunohistochimie , Souris , Lignées consanguines de souris , Microscopie confocale , Protéines de tissu nerveux/génétique , Protéines de tissu nerveux/métabolisme , Cellules de Purkinje/métabolisme , Protéines de fusion recombinantes/métabolismeRÉSUMÉ
Cell death after stroke involves apoptotic, autophagocytic and necrotic mechanisms which may cause the release of cytosolic proteins to the extracellular space. Aldolase C (AldC) is the brain specific isoform of the glycolytic enzyme fructose-1,6-bisphosphate aldolase. According to its characteristic striped expression pattern in the adult cerebellum AldC is also termed zebrin II. Here, we demonstrate release of AldC into the cerebrospinal fluid (CSF) after stroke in vivo. Studies with cell cultures confirmed that AldC is released to the extracellular space after hypoxia. Moreover, addition of purified recombinant AldC to networks of cortical neurons plated on multielectrode arrays reversibly inhibited the spontaneous generation of action potentials at AldC concentrations which can be expected to occur after lesions of the human cerebral cortex. This mechanism could be relevant in the pathogenesis of the electrophysiological changes in the penumbra region after stroke.
Sujet(s)
Cortex cérébral/anatomopathologie , Cortex cérébral/physiopathologie , Espace extracellulaire/métabolisme , Réseau nerveux/effets des médicaments et des substances chimiques , Protéines de tissu nerveux/liquide cérébrospinal , Protéines de tissu nerveux/physiologie , Neurones/anatomopathologie , Accident vasculaire cérébral/liquide cérébrospinal , Accident vasculaire cérébral/physiopathologie , Adulte , Animaux , Aphasie/étiologie , Technique de Western , Mort cellulaire , Lignée cellulaire tumorale , Cortex cérébral/métabolisme , Infarctus cérébral/complications , Infarctus cérébral/étiologie , Clonage moléculaire , Électrophorèse sur gel de polyacrylamide , Électrophysiologie , Escherichia coli/effets des médicaments et des substances chimiques , Escherichia coli/métabolisme , Femelle , Hémiplégie/étiologie , Humains , Cinétique , Mâle , Maladie de Moya-Moya/complications , Maladie de Moya-Moya/anatomopathologie , Neurones/effets des médicaments et des substances chimiques , Plasmides/génétique , Rats , Accident vasculaire cérébral/métabolismeRÉSUMÉ
Collapsin response mediator proteins (CRMPs) form a family of cytosolic phosphoproteins which are involved in the signal transduction of semaphorin 3A leading to growth cone collapse. These proteins interact with a variety of cytosolic proteins including tubulin heterodimers. Here, we show that CRMP-4 co-localizes with F-actin in regular rib-like structures within lamellipodia of B35 neuroblastoma cells. Furthermore, depolymerization of actin fibers changed the distribution of GFP-CRMP-4 in vivo. In vitro, recombinant CRMP-4 formed homo-oligomers, bound to F-actin and organized F-actin into tight bundles. Both oligomerization and F-actin bundling depended on the C-terminal part of CRMP-4. The stoichiometry of actin and CRMP-4 in bundles was approximately 1:1 and the apparent equilibrium constant of the microfilament-CRMP-4 interaction was estimated from bundling assays as K(app) = 730 mM(-1). CRMP-4 was abundant in the cytosol of B35 neuroblastoma cells and its concentration was measured as approximately 1.7 microM. Overexpression of CRMP-4 inhibited the migration of B35 neuroblastoma cells, while knockdown of CRMP-4 enhanced cell migration and disturbed rib-like actin-structures in lamellipodia. Taken together, our data indicate that CRMP-4 promotes bundling of F-actin in vitro, that it is an important component of rib-like actin bundles in lamellipodia in vivo and that it functionally regulates the actin cytoskeleton in motile cells. These findings suggest a specific regulatory role of CRMP-4 towards the actin cytoskeleton which may by be relevant for growth cone collapse.