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1.
Oncogene ; 37(2): 241-254, 2018 01 11.
Article in English | MEDLINE | ID: mdl-28925399

ABSTRACT

Glioblastomas (GBM) are lethal primitive brain tumours characterized by a strong intra-tumour heterogeneity. We observed in GBM tissues the coexistence of functionally divergent micro-territories either enriched in more differentiated and non-mitotic cells or in mitotic undifferentiated OLIG2 positive cells while sharing similar genomic abnormalities. Understanding the formation of such functionally divergent micro-territories in glioblastomas (GBM) is essential to comprehend GBM biogenesis, plasticity and to develop therapies. Here we report an unexpected anti-proliferative role of beta-catenin in non-mitotic differentiated GBM cells. By cell type specific stimulation of miR-302, which directly represses cyclin D1 and stemness features, beta-catenin is capable to change its known proliferative function. Nuclear beta-catenin accumulation in non-mitotic cells is due to a feed forward mechanism between DOCK4 and beta-catenin, allowed by increased GSK3-beta activity. DOCK4 over expression suppresses selfrenewal and tumorigenicity of GBM stem-like cells. Accordingly in the frame of GBM median of survival, increased level of DOCK4 predicts improved patient survival.


Subject(s)
GTPase-Activating Proteins/metabolism , Glioblastoma/pathology , MicroRNAs/metabolism , Neoplastic Stem Cells/pathology , beta Catenin/metabolism , Adult , Animals , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Brain/pathology , Cell Nucleus/metabolism , Cell Proliferation , Feedback, Physiological , GTPase-Activating Proteins/genetics , Glioblastoma/genetics , Glioblastoma/mortality , Glycogen Synthase Kinase 3 beta/genetics , Glycogen Synthase Kinase 3 beta/metabolism , Humans , Male , Mice , Mice, Inbred NOD , MicroRNAs/genetics , Mitosis , Neoplastic Stem Cells/cytology , Oligodendrocyte Transcription Factor 2/metabolism , Primary Cell Culture , RNA, Small Interfering/metabolism , Tumor Cells, Cultured , Xenograft Model Antitumor Assays , Young Adult , beta Catenin/genetics
2.
Oncogene ; 26(19): 2695-706, 2007 Apr 26.
Article in English | MEDLINE | ID: mdl-17057735

ABSTRACT

An instability of the mature cell phenotype is thought to participate to the formation of gliomas, primary brain tumors deriving from astrocytes and/or neural stem cells. Transforming growth factor alpha (TGFalpha) is an erbB1 ligand overexpressed in the earliest stages of gliomas, and exerts trophic effects on gliomal cells and astrocytes. Here, we questioned whether prolonged TGFalpha exposure affects the stability of the normal mature astrocyte phenotype. We first developed astrocyte cultures devoid of residual neural stem cells or progenitors. We demonstrate that days of TGFalpha treatment result in the functional conversion of a population of mature astrocytes into radial glial cells, a population of neural progenitors. TGFalpha-generated radial glial cells support embryonic neurons migration, and give birth to cells of the neuronal lineage, expressing neuronal markers and the electrophysiological properties of neuroblasts. Lengthening TGFalpha treatment to months results in the delayed appearance of cells with neural stem cells properties: they form floating cellular spheres that are self-renewing, can be clonally derived from a single cell and differentiated into cells of the neuronal lineage. This study uncovers a novel population of mature astrocytes capable, in response to a single epigenetic factor, to regress progressively into a neural stem-like cell stage via an intermediate progenitor stage.


Subject(s)
Astrocytes/cytology , Cell Differentiation/drug effects , Neurons/metabolism , Stem Cells/cytology , Transforming Growth Factor alpha/pharmacology , Animals , Astrocytes/metabolism , Cell Lineage , Cell Movement , Cells, Cultured , Electrophysiology , Embryo, Mammalian/cytology , Embryo, Mammalian/metabolism , ErbB Receptors/metabolism , Female , Fetus/cytology , Fetus/metabolism , Humans , Immunoblotting , Immunoprecipitation , Mice , Mice, Inbred C57BL , Neuroglia/cytology , Neurons/cytology , Receptor, ErbB-2/metabolism , Recombinant Proteins/pharmacology , Stem Cells/metabolism
3.
Oncogene ; 25(29): 4076-85, 2006 Jul 06.
Article in English | MEDLINE | ID: mdl-16532035

ABSTRACT

Astrocyte death has been implicated in several neuropathological diseases, but the identification of molecules susceptible of promoting astrocyte survival has been elusive. We investigated whether transforming growth factor alpha (TGFalpha), an erbB1/EGFR ligand, which promotes glioma progression and affects astrocyte metabolism at embryonic and adult stages, regulates astrocyte survival. Primary serum-free astrocyte cultures from post-natal mouse and fetal human cortices were used. Transforming growth factor alpha protected both species of astrocytes from staurosporine-induced apoptosis. In serum-free medium, mouse astrocytes did not survive beyond 2 months while TGFalpha-treated astrocytes survived up to 12 months. Transforming growth factor alpha also promoted long-term survival of human astrocytes. We additionally extended TGFalpha proliferative effects to human astrocytes. After 3 days of permanent application, TGFalpha induced a major downregulation of both erbB1 and erbB2. This downregulation did not impair the functional activation of the receptors, as ascertained by their tyrosine phosphorylation and the continuous stimulation of both ERK/MAPK and PI3K/Akt pathways up to 7 days, the longest time examined. The full cellular effects of TGFalpha required activation of both transduction pathways. Enhanced proliferation and survival thus define TGFalpha as a gliatrophin for mammalian astrocytes. These results demonstrate that in normal, non-transformed astrocytes, sustained and functional erbBs activation is achieved without bypassing ligand-induced receptors downregulation.


Subject(s)
Astrocytes/metabolism , Down-Regulation/drug effects , ErbB Receptors/metabolism , MAP Kinase Signaling System/drug effects , Receptor, ErbB-2/metabolism , Transforming Growth Factor alpha/pharmacology , Aging/metabolism , Animals , Astrocytes/cytology , Cell Death/drug effects , Cell Survival/drug effects , Cells, Cultured , Cerebellar Cortex/cytology , Cerebellar Cortex/embryology , Extracellular Signal-Regulated MAP Kinases/metabolism , Glioma/metabolism , Humans , Mice , Oncogene Protein v-akt/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Time Factors , Transforming Growth Factor alpha/metabolism
4.
Neuroscience ; 126(2): 263-75, 2004.
Article in English | MEDLINE | ID: mdl-15207344

ABSTRACT

Phosphoprotein enriched in astrocytes of 15 kDa (PEA-15) is an abundant phosphoprotein in primary cultures of mouse brain astrocytes. Its capability to interact with members of the apoptotic and mitogen activated protein (MAP) kinase cascades endows PEA-15 with anti-apoptotic and anti-proliferative properties. We analyzed the in vivo cellular sources of PEA-15 in the normal adult mouse brain using a novel polyclonal antibody. Immunohistochemical assays revealed numerous PEA-15-immunoreactive cells throughout the brain of wild-type adult mice while no immunoreactive signal was observed in the brain of PEA-15 -/- mice. Cell morphology and double immunofluorescent staining showed that both astrocytes and neurons could be cellular sources of PEA-15. Closer examination revealed that in a given area only part of the astrocytes expressed the protein. The hippocampus was the most striking example of this heterogeneity, a spatial segregation restricting PEA-15 positive astrocytes to the CA1 and CA3 regions. A PEA-15 immunoreactive signal was also observed in a few cells within the subventricular zone and the rostral migratory stream. In vivo analysis of an eventual PEA-15 regulation in astrocytes was performed using a model of astrogliosis occurring along motor neurons degeneration, the transgenic mouse expressing the mutant G93A human superoxyde-dismutase-1, a model of amyotrophic lateral sclerosis. We observed a marked up-regulation of PEA-15 in reactive astrocytes that had developed throughout the ventral horn of the lumbar spinal cord of the transgenic mice. The heterogeneous cellular expression of the protein and its increased expression in pathological situations, combined with the known properties of PEA-15, suggest that PEA-15 expression is associated with a particular metabolic status of cells challenged with potentially apoptotic and/or proliferative signals.


Subject(s)
Astrocytes/metabolism , Brain/metabolism , Neurons/metabolism , Phosphoproteins/biosynthesis , 3T3 Cells , Amino Acid Sequence , Animals , Apoptosis Regulatory Proteins , Astrocytes/cytology , Brain/cytology , Cells, Cultured , Female , Gene Expression Regulation/physiology , Humans , Immunohistochemistry , Intracellular Signaling Peptides and Proteins , Male , Mice , Mice, Transgenic , Molecular Sequence Data , Neurons/cytology , Phosphoproteins/immunology
6.
Neuroscience ; 113(4): 825-35, 2002.
Article in English | MEDLINE | ID: mdl-12182889

ABSTRACT

Prevention of protein misfolding is ensured by chaperone proteins, including the heat shock proteins (HSP) of the DNAJ/HSP40 family. Detection of abnormal protein aggregates in various neurodegenerative diseases has led to the proposal that altered chaperone activity contributes to neurodegeneration. Msj-1, a DNAJ/HSP40 protein located around the spermatozoa acrosome, was recently found to be down-regulated in the testis of wobbler mutant mice. Wobbler is an unidentified recessive mutation which triggers progressive motoneuron degeneration with abnormal intracellular protein accumulations, and defective spermatozoa maturation. Here, we examined Msj-1 expression in the spinal cord of the mutants and their controls. Msj-1 transcripts were amplified by reverse transcription-polymerase chain reaction from mutant and wild-type spinal cord RNA. Sequencing of Msj-1 coding region revealed no change in the mutant. In contrast, decreased Msj-1 mRNA levels were observed in five to six-week-old wobbler mice spinal cord, when motoneuron degeneration is at its apex, as compared to controls. A similar decrease was observed in two-week-old wobbler spinal cord, when the number of motoneurons is still unaltered, indicating that the decreased mRNA content is intrinsic to the mutant and not simply related to the loss of cells expressing Msj-1. Assays of Msj-1 protein levels yielded similar results. Immunofluorescent labeling revealed numerous Msj-1-ir motoneurons in five-week-old control spinal cord while no signal was observed in age-matched wobbler. Our results show, therefore, that Msj-1 expression is down-regulated in both organs affected by the wobbler mutation, the CNS and the testis, and that this defect precedes the first histological signs of motoneuron degeneration. These results provide the first example of an association between transcriptional repression of a chaperone protein and a neurodegenerative process.


Subject(s)
Heat-Shock Proteins/biosynthesis , Motor Neuron Disease/metabolism , Spermatozoa/metabolism , Spinal Cord/metabolism , Animals , Down-Regulation/physiology , HSP40 Heat-Shock Proteins , Heat-Shock Proteins/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Inbred NZB , Mice, Neurologic Mutants , Motor Neuron Disease/genetics , Mutation/physiology , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Testis/metabolism
7.
Exp Neurol ; 172(1): 17-28, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11681837

ABSTRACT

Excitotoxic lesions in the gray matter induce profuse demyelination of passage and afferent fibers in areas of neuronal loss, independent of Wallerian degeneration. The time course of this phenomenon, which extends over weeks after the excitotoxin injection, suggests that demyelination is not related only to a direct effect of the toxin. In order to define mechanisms at work, a parallel study of myelin and oligodendrocytes was carried out following kainate injections into the adult rat thalamus. Within the 1st day postlesion, myelin alteration appeared throughout the area exhibiting neuronal loss, while the number of oligodendrocytes fell by 45%. No apoptotic oligodendrocytes were identified at that time. Over the following 2 days, there was no further loss of myelin and oligodendrocytes, but there was an increase in the number of oligodendrocytes displaying typical signs of apoptosis as revealed with TUNEL-end-labeled nuclei, Hoechst-labeled condensed chromatin bodies, or bax immunoreactivity. This resulted in a second, progressive loss of both myelin and oligodendrocytes leading to their almost complete disappearance 2 weeks postlesion. These results demonstrate two temporal stages of oligodendroglial cell death. The excitotoxin injection resulted in the rapid destruction of a first oligodendroglial population, most probably by necrosis. A second population died in a delayed manner from apoptosis. This second wave of death coincided with an activated microglia/macrophage invasion of the lesion, suggesting that delayed oligodendroglial death results from toxic microglia/macrophage effects. In addition, the longest surviving oligodendrocytes were located next to reactive astrocytes, suggesting the existence of trophic interactions between these two glial populations.


Subject(s)
Brain/pathology , Oligodendroglia/pathology , Proto-Oncogene Proteins c-bcl-2 , Animals , Apoptosis , Astrocytes/pathology , Brain/drug effects , Brain/metabolism , Cell Count , Demyelinating Diseases/chemically induced , Demyelinating Diseases/pathology , Disease Progression , Female , In Situ Nick-End Labeling , Kainic Acid , Macrophages/pathology , Microglia/pathology , Myelin Sheath/drug effects , Myelin Sheath/pathology , Myelin Sheath/ultrastructure , Necrosis , Oligodendroglia/drug effects , Oligodendroglia/metabolism , Proliferating Cell Nuclear Antigen/biosynthesis , Proto-Oncogene Proteins/biosynthesis , Rats , Rats, Sprague-Dawley , Thalamus/drug effects , Thalamus/pathology , Time Factors , bcl-2-Associated X Protein
8.
J Neurosci ; 21(18): 7079-88, 2001 Sep 15.
Article in English | MEDLINE | ID: mdl-11549718

ABSTRACT

Expression of transforming growth factor alpha (TGFalpha), a member of the epidermal growth factor (EGF) family, is a general response of adult murine motoneurons to genetic and experimental lesions, TGFalpha appearing as an inducer of astrogliosis in these situations. Here we address the possibility that TGFalpha expression is not specific to pathological situations but may participate to the embryonic development of motoneurons. mRNA of TGFalpha and its receptor, the EGF receptor (EGFR), were detected by ribonuclease protection assay in the ventral part of the cervical spinal cord from embryonic day 12 (E12) until adult ages. Reverse transcription-PCR amplification of their transcripts from immunopurified E15 motoneurons, associated with in situ double-immunohistological assays, identified embryonic motoneurons as cellular sources of the TGFalpha-EGFR couple. In vitro, TGFalpha promoted the survival of immunopurified E15 motoneurons in a dose-dependent manner, with a magnitude similar to BDNF neuroprotective effects at equivalent concentrations. In a transgenic mouse expressing a human TGFalpha transgene under the control of the metallothionein 1 promoter, axotomy of the facial nerve provoked significantly less degeneration in the relevant motor pool of 1-week-old mice than in wild-type animals. No protection was observed in neonates, when the transgene exhibits only weak expression levels in the brainstem. In conclusion, our results point to TGFalpha as a physiologically relevant candidate for a neurotrophic role on developing motoneurons. Its expression by the embryonic motoneurons, which also synthesize its receptor, suggests that this chemokine is endowed with the capability to promote motoneuron survival in an autocrine-paracrine manner.


Subject(s)
Motor Neurons/drug effects , Transforming Growth Factor alpha/pharmacology , Animals , Animals, Newborn , Anterior Horn Cells/cytology , Anterior Horn Cells/metabolism , Axotomy , Cell Survival/drug effects , Culture Media, Serum-Free/pharmacology , Dose-Response Relationship, Drug , ErbB Receptors/genetics , ErbB Receptors/metabolism , Facial Nerve/physiology , Gene Expression Regulation, Developmental , Humans , In Situ Hybridization , Metallothionein/genetics , Mice , Mice, Transgenic , Motor Neurons/cytology , Neck , RNA, Messenger/analysis , RNA, Messenger/biosynthesis , Rats , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction , Spinal Cord/cytology , Spinal Cord/embryology , Spinal Cord/metabolism , Transforming Growth Factor alpha/genetics , Transforming Growth Factor alpha/metabolism , Transgenes
9.
Mol Cell Neurosci ; 17(1): 107-26, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11161473

ABSTRACT

TTF-1 is a member of the Nkx family of homeodomain genes required for morphogenesis of the hypothalamus. Whether TTF-1, or other Nkx genes, contributes to regulating differentiated hypothalamic functions is not known. We now report that postnatal hypothalamic TTF-1 expression is developmentally regulated and associated with the neuroendocrine process of female sexual development. Lesions of the hypothalamus that cause sexual precocity transiently activate neuronal TTF-1 expression near the lesion site. In intact animals, hypothalamic TTF-1 mRNA content also increases transiently, preceding the initiation of puberty. Postnatal expression of the TTF-1 gene was limited to subsets of hypothalamic neurons, including LHRH neurons, which control sexual maturation, and preproenkephalinergic neurons of the lateroventromedial nucleus of the basal hypothalamus, which restrain sexual maturation and facilitate reproductive behavior. TTF-1 mRNA was also detected in astrocytes of the median eminence and ependymal/subependymal cells of the third ventricle, where it colocalized with erbB-2, a receptor involved in facilitating sexual development. TTF-1 binds to and transactivates the erbB-2 and LHRH promoters, but represses transcription of the preproenkephalin gene. The singular increase in hypothalamic TTF-1 gene expression that precedes the initiation of puberty, its highly specific pattern of cellular expression, and its transcriptional actions on genes directly involved in neuroendocrine reproductive regulation suggest that TTF-1 may represent one of the controlling factors that set in motion early events underlying the central activation of mammalian puberty.


Subject(s)
Gene Expression Regulation, Developmental , Hypothalamus/metabolism , Neurosecretory Systems/metabolism , Nuclear Proteins/biosynthesis , Transcription Factors/biosynthesis , Aging/metabolism , Animals , Astrocytes/cytology , Astrocytes/metabolism , Cell Line , Diencephalon/cytology , Diencephalon/embryology , Diencephalon/metabolism , Enkephalins/genetics , Enkephalins/metabolism , Ependyma/cytology , Ependyma/metabolism , Female , Gene Silencing , Gonadotropin-Releasing Hormone/genetics , Gonadotropin-Releasing Hormone/metabolism , Hypothalamus/cytology , Hypothalamus/surgery , Median Eminence/cytology , Median Eminence/metabolism , Neurons/classification , Neurons/cytology , Neurons/metabolism , Neurosecretory Systems/cytology , Nuclear Proteins/genetics , Nuclear Proteins/pharmacology , Promoter Regions, Genetic/drug effects , Protein Precursors/genetics , Protein Precursors/metabolism , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Receptor, ErbB-2/genetics , Receptor, ErbB-2/metabolism , Thyroid Gland/cytology , Thyroid Gland/metabolism , Thyroid Nuclear Factor 1 , Transcription Factors/genetics , Transcription Factors/pharmacology , Transcriptional Activation
10.
Pain ; 90(1-2): 113-25, 2001 Feb 01.
Article in English | MEDLINE | ID: mdl-11166977

ABSTRACT

In the adult brain, neurotrophins play a key role in adaptive processes linked to increased neuronal activity. A growing body of evidence suggests that chronic pain results from long-term plasticity of central pathways involved in nociception. We have investigated the involvement of nerve growth factor (NGF) in adaptive responses of primary sensory neurons during the course of a long-lasting inflammatory pain model. The amount and distribution of the NGF receptors p75(NTR) and TrkA were measured in the dorsal horn and dorsal root ganglia (DRG) of animals subjected to Freund's adjuvant-induced arthritis (AIA). We observed an increased immunoreactivity of both receptors in the central terminals of primary sensory neurons in the arthritic state. The increases were seen in the same population of afferent terminals in deep dorsal horn laminae. These changes paralleled the variations of clinical and behavioral parameters that characterize the course of the disease. They occurred in NGF-sensitive, but not GDNF-sensitive, nerve terminals. However, p75(NTR) and TrkA protein levels in the DRG (in the cell body of these neurons) showed different response patterns. An immediate rise of p75(NTR) was seen in parallel with the initial inflammation that developed after administration of Freund's adjuvant in hindpaws. In contrast, increases of the mature (gp140(trk)) form of TrkA occurred later and seemed to be linked to the development of the long-lasting inflammatory response. The changes in receptor expression were observed exclusively at lumbar levels, L3-L5, somatotopically appropriate for the inflammation. Together, these results implicate NGF in long-term mechanisms accompanying chronic inflammatory pain, via the up-regulation of its high affinity receptor, and offer additional evidence for differential processes underlying short- versus long-lasting inflammatory pain.


Subject(s)
Arthritis, Experimental/metabolism , Ganglia, Spinal/metabolism , Posterior Horn Cells/metabolism , Receptor, trkA/metabolism , Receptors, Nerve Growth Factor/metabolism , Animals , Calcitonin Gene-Related Peptide/metabolism , Male , Neurons, Afferent/metabolism , Proteins/metabolism , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Receptor, Nerve Growth Factor , Substance P/metabolism
11.
Prog Neurobiol ; 62(5): 443-73, 2000 Dec.
Article in English | MEDLINE | ID: mdl-10869779

ABSTRACT

Transforming growth factor alpha (TGFalpha) is a member of the epidermal growth factor (EGF) family with which it shares the same receptor, the EGF receptor (EGFR or erbB1). Identified since 1985 in the central nervous system (CNS), its functions in this organ have started to be determined during the past decade although numerous questions remain unanswered. TGFalpha is widely distributed in the nervous system, both glial and neuronal cells contributing to its synthesis. Although astrocytes appear as its main targets, mediating in part TGFalpha effects on different neuronal populations, results from different studies have raised the possibility for a direct action of this growth factor on neurons. A large array of experimental data have thus pointed to TGFalpha as a multifunctional factor in the CNS. This review is an attempt to present, in a comprehensive manner, the very diverse works performed in vitro and in vivo which have provided evidences for (i) an intervention of TGFalpha in the control of developmental events such as neural progenitors proliferation/cell fate choice, neuronal survival/differentiation, and neuronal control of female puberty onset, (ii) its role as a potent regulator of astroglial metabolism including astrocytic reactivity, (iii) its neuroprotective potential, and (iv) its participation to neuropathological processes as exemplified by astroglial neoplasia. In addition, informations regarding the complex modes of TGFalpha action at the molecular level are provided, and its place within the large EGF family is precised with regard to the potential interactions and substitutions which may take place between TGFalpha and its kindred.


Subject(s)
Central Nervous System/chemistry , Central Nervous System/physiology , Transforming Growth Factor alpha/physiology , Animals , Astrocytes/chemistry , Astrocytes/physiology , Central Nervous System/cytology , Humans , Molecular Sequence Data , Neurons/chemistry , Neurons/physiology , Sequence Homology, Amino Acid , Transforming Growth Factor alpha/chemistry
12.
J Neurosci ; 18(24): 10541-52, 1998 Dec 15.
Article in English | MEDLINE | ID: mdl-9852591

ABSTRACT

TGFalpha is a member of the epidermal growth factor (EGF) family with which it shares the same receptor, the EGF receptor (EGFR). Synthesis of TGFalpha and EGFR in reactive astrocytes developing after CNS insults is associated with the differentiative and mitogenic effects of TGFalpha on cultured astrocytes. This suggests a role for TGFalpha in the development of astrogliosis. We evaluated this hypothesis using transgenic mice bearing the human TGFalpha cDNA under the control of the zinc-inducible metallothionein promoter. Expression levels of glial fibrillary acidic protein (GFAP) and vimentin and morphological features of astrocytes were used as indices of astroglial reactivity in adult transgenic versus wild-type mice provided with ZnCl2 in their water for 3 weeks. In the striatum, the hippocampus, and the cervical spinal cord, the three CNS areas monitored, transgenic mice displayed enhanced GFAP mRNA and protein levels and elevated vimentin protein levels. GFAP-immunoreactive astrocytes exhibited numerous thick processes and hypertrophied somata, which are characteristic aspects of reactive astrocytes. Their number increased additionally in the striatum and the spinal cord, but no astrocytic proliferation was observed using bromodeoxyuridine immunohistochemistry. Neither the morphology nor the number of microglial cells appeared modified. A twofold increase in phosphorylated EGFR was detected in the striatum and was associated with the immunohistochemical detection of numerous GFAP-positive astrocytes bearing the EGFR, suggesting a direct action of TGFalpha on astrocytes. Altogether, these results demonstrate that enhanced TGFalpha synthesis is sufficient to trigger astrogliosis throughout the CNS, whereas microglial metabolism is unaffected.


Subject(s)
Astrocytes/metabolism , Astrocytes/pathology , Gliosis/metabolism , Transforming Growth Factor alpha/physiology , Animals , Corpus Striatum/metabolism , Corpus Striatum/pathology , ErbB Receptors/metabolism , Glial Fibrillary Acidic Protein/biosynthesis , Gliosis/pathology , Hippocampus/metabolism , Hippocampus/pathology , Humans , Immunohistochemistry , Metallothionein/genetics , Mice , Mice, Transgenic , Microglia/cytology , Spinal Cord/metabolism , Spinal Cord/pathology , Transforming Growth Factor alpha/genetics , Transgenes , Vimentin/biosynthesis
13.
Mol Cell Neurosci ; 12(3): 168-77, 1998 Oct.
Article in English | MEDLINE | ID: mdl-9790737

ABSTRACT

Motoneuronal degenerative diseases are characterized by their progressivity; once affected, the motoneurons remain in altered states during an intermediate phase of degeneration prior to their final disappearance. Whether this survival period coincides with active metabolic rearrangements in the affected neuron remains unknown. As a first step toward the elucidation of this question, we developed cDNA pooled samples obtained from degenerating and control motoneuron mRNA populations through cellular patch sampling and RT-PCR, using the murine wobbler mutant as a model of spinal atrophy. Hybridization of the cDNA pools to various markers of intact or degenerating motoneurons allowed us to verify the cellular specificity of the patch sampling and indicated conservation of the original mRNA population complexity. Exploration of transcriptional alterations of genes encoding growth factors thought to be involved in motoneuronal development revealed that gene expression of the neurotrophin BDNF was induced in affected motoneurons, while expression of neurotrophin-3 was present in both neuronal types. Likewise, expression of a member of the epidermal growth factor (EGF) family, the neuregulin transcript sensory motor neuron-derived factor, was detected in both control and degenerating motoneurons, while transforming growth factor alpha, the functional homolog of EGF, was present only in the affected motoneurons. Immunohistochemical detection of corresponding proteins corroborated these observations. These results demonstrate that, during the course of their degeneration, motoneurons can initiate expression of novel genes which lead to the production of molecules endowed with trophic and/or differentiative properties for the neurons themselves and their glial environment. They also validate the use of the developed cDNA pooled samples for further exploration of transcriptional alterations taking place in degenerating motoneurons.


Subject(s)
Gene Expression Regulation/physiology , Growth Substances/genetics , Motor Neurons/physiology , Nerve Degeneration/physiopathology , Animals , In Vitro Techniques , Mice , Mice, Neurologic Mutants , Motor Neurons/metabolism , Nerve Growth Factors/metabolism , Reverse Transcriptase Polymerase Chain Reaction
14.
J Neuropathol Exp Neurol ; 56(5): 459-71, 1997 May.
Article in English | MEDLINE | ID: mdl-9143258

ABSTRACT

We previously showed that degenerating adult motor neurons of the murine mutant wobbler, a model of spinal muscular atrophy, express Transforming Growth Factor alpha (TGF alpha), a growth factor endowed with glio- and neurotrophic activities. Here, we evaluated whether TGF alpha expression is a general response of adult motor neurons to injury. Synthesis of its precursor (pro-TGF alpha) was investigated in another model of motoneuronal degeneration, the murine mutant muscle deficient, and in hypoglossal motor neurons following axonal crush and cut. In control conditions, motor neurons were devoid of pro-TGF alpha immunoreactivity. In the mutant lumbar spinal cord, pro-TGF alpha immunoreactive motor neurons appeared as soon as the disease developed and pro-TGF alpha expression persisted until the latest stages of degeneration. Motor neurons and astrocytes of the white matter weakly immunoreactive for the TGF alpha receptor were also present in both control and mutant lumbar spinal cords. Following hypoglossal nerve crush and cut, motoneuronal pro-TGF alpha expression was precocious and transient, visible at one day post-injury and lasting for only 3 days, during which time astrocyte-like cells immunoreactive for both TGF alpha and its receptor appeared within the injured nucleus. Enhanced TGF alpha mRNA levels following nerve crush showed that activation occurred at the transcriptional level. These results show that upregulation of TGF alpha is an early and common response of adult murine motor neurons to injury, regardless of its experimental or genetic origin.


Subject(s)
Axons/physiology , Hypoglossal Nerve Injuries , Motor Neurons/metabolism , Mutation , Nerve Degeneration , Transforming Growth Factor alpha/metabolism , Animals , Denervation , Hypoglossal Nerve/pathology , Hypoglossal Nerve/physiopathology , Male , Mice , Mice, Inbred Strains , Mice, Mutant Strains , Muscles/abnormalities , Nerve Crush , Protein Precursors/biosynthesis , RNA, Messenger/metabolism , Spinal Cord/abnormalities , Spinal Cord/metabolism , Spinal Cord/pathology , Transforming Growth Factor alpha/biosynthesis , Transforming Growth Factor alpha/genetics
15.
J Neurosci ; 16(19): 5897-904, 1996 Oct 01.
Article in English | MEDLINE | ID: mdl-8815872

ABSTRACT

The molecular events leading to motoneuronal death are still poorly understood. In mammals, the bcl-2 proto-oncogene, which encodes a membrane-associated protein, has been shown to suppress both developmental motoneuronal death and experimental axotomy-induced motoneuronal death. We assessed a potential protective effect of Bcl-2 on pathological motoneuronal death processes in adult rodents. We took advantage of the murine mutant wobbler, which undergoes progressive degeneration of the spinal and brainstem motoneurons. A hybrid carrying both the wobbler mutation and the human bcl-2 transgene under the control of the neuron-specific enolase promoter was produced. Although Bcl-2 protected spinal and brainstem motoneurons from developmental death and the postnatal motoneurons of the facial nucleus from axotomy-induced death, the pathological motoneuronal death was not altered in the adult hybrid. These results demonstrate that Bcl-2 sensitivity distinguishes at least two different motoneuronal death pathways in the wobbler mutant. They support the hypothesis that experimental and pathological motoneuronal death are dependent on different cellular mechanisms.


Subject(s)
Motor Neurons/physiology , Proto-Oncogene Proteins c-bcl-2/metabolism , Animals , Cell Death , Gene Expression , Humans , Mice , Mice, Neurologic Mutants , Motor Neurons/pathology , Phenotype , Proto-Oncogene Mas , Proto-Oncogene Proteins c-bcl-2/genetics , Transgenes
16.
Neuroscience ; 64(3): 665-74, 1995 Feb.
Article in English | MEDLINE | ID: mdl-7715779

ABSTRACT

Intraperitoneal or intrahippocampal injections of kainate induce both hippocampal cell death and axonal remodeling of the dentate gyrus granular neurons. We report here that injection of kainate into the dorsal hippocampus of adult mice may also trigger a conspicuous and long-lasting global trophic response of granule cells. Morphological changes include somatic and dendritic growth and increased nuclear volume with ultrastructural features characteristic of neuronal development. The trophic response is correlated with a specific overexpression of brain-derived neurotrophic factor that is maintained for at least six months. This shows that plasticity in adult neurons can, in addition to axonal remodeling, extend to generalized cell growth. Our results further suggest that brain-derived neurotrophic factor could be involved in the activation and/or maintenance of this phenomenon.


Subject(s)
Hippocampus/drug effects , Kainic Acid/pharmacology , Nerve Tissue Proteins/metabolism , Neuronal Plasticity/drug effects , Animals , Brain-Derived Neurotrophic Factor , Gene Expression Regulation/drug effects , Hippocampus/chemistry , Hippocampus/physiology , In Situ Hybridization , Male , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Nerve Growth Factors/drug effects , Nerve Growth Factors/metabolism , Neuronal Plasticity/physiology , Neurotrophin 3 , RNA, Messenger/analysis , Receptor Protein-Tyrosine Kinases/metabolism , Receptor, Ciliary Neurotrophic Factor , Receptor, trkA/metabolism , Receptor, trkC , Receptors, Nerve Growth Factor/metabolism , Seizures/chemically induced , Time Factors
17.
J Neurosci ; 14(7): 4206-16, 1994 Jul.
Article in English | MEDLINE | ID: mdl-8027772

ABSTRACT

The enhanced expression of the trophic factor transforming growth factor alpha (TGF alpha) in reactive astrocytes following CNS injury suggests that TGF alpha has a role in the development of astrogliosis. We explored this hypothesis in the murine mutant wobbler, which presents a progressive motoneuronal degeneration associated with an astrogliosis. Evolution of astrogliosis, and expression of TGF alpha precursor (pro-TGF alpha) and of its receptor were examined over the course of the disease, using genetically diagnosed animals and immunocytochemical techniques. We report here that degenerating motoneurons of the cervical spinal cord and a subset of astrocytes express pro-TGF alpha, prior to the onset of astrogliosis, when the first clinical manifestations of the disease are observed at 4 weeks of age. TGF alpha expression appeared strongly correlated with motoneuronal degeneration. All pro-TGF alpha-immunoreactive neurons exhibited a degenerative morphology, and the number of pro-TGF alpha-immunoreactive neurons increased with the progression of the disease. At the glial level, we observed that astrogliosis was a transitory phenomenon in the wobbler mice, developing in coordination with the motoneuronal expression of pro-TGF alpha. Astrogliosis became evident in 6-week-old wobbler mice, when the number of pro-TGF alpha-immunoreactive motoneurons was maximal, and regressed in older mutant mice in correlation with the disappearance of pro-TGF alpha-immunoreactive motoneurons. Furthermore, TGF alpha/EGF receptor immunoreactivity was exclusively localized in a subset of reactive astrocytes, its expression following closely the course of the astrogliosis. These data show that TGF alpha synthesis by the affected motoneurons is an early event in the course of the wobbler disease, and suggest a role for TGF alpha as a neuronal inducer of astrocytic reactivity.


Subject(s)
Astrocytes/metabolism , Gliosis/metabolism , Motor Neurons/metabolism , Nerve Degeneration , Transforming Growth Factor alpha/metabolism , Animals , Base Sequence , ErbB Receptors/metabolism , Mice , Mice, Neurologic Mutants , Molecular Probes/genetics , Molecular Sequence Data , Neck , Polymerase Chain Reaction , Spinal Cord/metabolism , Time Factors
18.
Brain Res Mol Brain Res ; 24(1-4): 247-60, 1994 Jul.
Article in English | MEDLINE | ID: mdl-7968364

ABSTRACT

Reactive gliosis is part of the response of central nervous system to injury and neurodegeneration. Cellular components of the reactive gliosis have the capability to synthesize neurotrophic factors, and thus are capable of affecting the fate of neuronal populations in the injured tissue. In this study, we explored the putative involvement of reactive glia-derived neurotrophins in sustaining the axonal projections of target-deprived neurons. Neuronal targets of the dorsal column nuclei neurons were suppressed through excitotoxic lesion of the ventrobasal complex of the rat thalamus (VB). Despite the development of reactive gliosis, neither up-regulation of NGF, nor BDNF or NT3 mRNA could be detected by solution hybridization in the lesioned site at all times tested. In contrast, expression of the LNGFR gene increased progressively up to 90 days post-lesion. Immunocytochemical studies localized the LNGFR protein in a subset of small cells with ramified processes resembling microglia at 7 and 20 days post-lesion. At longer times, double immunolabelling studies revealed that a substantial part of LNGFR-immunoreactive cells filling the area of neuronal loss were neither microglial cells nor astrocytes although presence of LNGFR in a subset of microglial cells could not be excluded. Previous ultrastructural studies of the kainate-lesioned VB suggest that these LNGFR-immunoreactive cells correspond to oligodendrocytes and/or Schwann cells. At 2 months post-lesion, when LNGFR expression was maximal, increased levels of trkA mRNA were detected in the lesioned site. Immunocytochemical studies revealed the presence of numerous trkA-immunoreactive astrocytes. TrkB mRNA, encoding the full-length high-affinity receptor for BDNF, remained undetectable by non-isotopic in situ hybridization. In contrast to the lack of neurotrophin gene expression by glial components of the lesioned VB, dorsal column nuclei neurons contained NGF mRNA as revealed by in situ hybridization studies at 10 days--prior to enhanced LNGFR expression in the lesion--and 2 months post-lesion. In addition, the number and the staining intensity of NGF mRNA-positive neurons was increased in the target-deprived neurons, as compared with the contra-lateral nucleus projecting to intact targets. These results show that glial cells present in a reactive gliosis which develops in the kainic acid-lesioned thalamus, do not synthesize neurotrophins but instead produce high levels of both low- and high-affinity NGF receptors, LNGFR by Schwann cells/oligodendrocytes and possibly a subset of microglial cells, and trkA by reactive astrocytes.(ABSTRACT TRUNCATED AT 400 WORDS)


Subject(s)
Axons/metabolism , Brain/metabolism , Gene Expression , Nerve Growth Factors/biosynthesis , Neuroglia/metabolism , Neurons/metabolism , Receptors, Nerve Growth Factor/biosynthesis , Animals , Brain-Derived Neurotrophic Factor , Female , Immunohistochemistry , In Situ Hybridization , Kainic Acid/toxicity , Nerve Tissue Proteins/biosynthesis , Neurotrophin 3 , Proto-Oncogene Proteins/biosynthesis , RNA Probes , RNA, Messenger/analysis , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Receptor Protein-Tyrosine Kinases/biosynthesis , Receptor, Ciliary Neurotrophic Factor , Receptor, trkA , Receptors, Nerve Growth Factor/analysis , Schwann Cells/metabolism , Thalamus/drug effects , Thalamus/metabolism
19.
Mol Cell Neurosci ; 5(3): 246-62, 1994 Jun.
Article in English | MEDLINE | ID: mdl-8087423

ABSTRACT

Recent findings have led to the concept that transforming growth factor alpha (TGF alpha) contributes to the neuroendocrine regulation of female puberty by stimulating the release of luteinizing hormone-releasing hormone (LHRH), the neurohormone controlling sexual development. It was postulated that this effect is mediated by epidermal growth factor receptors (EGFR) and that EGFR may not be located on LHRH neurons, so that TGF alpha-induced LHRH release would require an intermediate cell-to-cell interaction, presumably of glial-neuronal nature. The present study was undertaken to characterize the presence of EGFR in rat hypothalamus and to determine if changes in EGFR gene expression and EGFR protein occur at the time of puberty. RNA blot hybridization demonstrated that the hypothalamus expresses all mRNA species known to encode EGFR. RNase protection assays revealed that alternative splicing of the EGFR primary mRNA transcript occurs in the hypothalamus and produces a predominant transcript encoding the full-length EGFR and a much less abundant, shorter mRNA encoding a truncated, and presumably secreted form of EGFR. EGFR-like immunoreactive material was found in several hypothalamic regions including the organum vasculosum of the lamina terminalis, supraoptic, suprachiasmatic, and paraventricular nuclei, ependymal cells lining the third ventricle, some astrocytes associated with blood vessels, astrocytes of the pial surface, and tanycytes and glial cells of the median eminence (ME). Low levels of EGFR mRNA were detected by hybridization histochemistry in cells of the same areas containing EGFR-like immunoreactivity. Double-immunohistochemistry revealed that even though LHRH neurons are in close proximity to EGFR-positive cells, they do not contain EGFR. In the ME, EGFR-immunonegative LHRH nerve terminals tightly coexist with EGFR-positive cells, presumably tanycytes and glial astrocytes. EGFR mRNA levels measured by quantitative reverse transcription-polymerase chain reaction assay (RT-PCR) in the ME-arcuate nucleus region at the time of puberty decreased in the morning of the first proestrus, i.e., preceding the first preovulatory surge of gonadotropins, and rebounded at the time of the surge. Functional EGFR protein levels, detected by the ability of the receptor to autophosphorylate in response to ligand or divalent antibody-induced activation, changed in a similar manner at the time of puberty. No such changes were observed in the cerebellum, a brain region irrelevant to neuroendocrine reproductive control. These results demonstrate the existence of EGF receptors in the prepubertal female rat hypothalamus and suggest that changes in EGFR gene expression and biologically active EGFR protein contributes to the neuroendocrine process underlying the first preovulatory surge of gonadotropins.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Astrocytes/metabolism , ErbB Receptors/biosynthesis , Gene Expression Regulation , Hypothalamus/metabolism , Sexual Maturation , Animals , Base Sequence , Epidermal Growth Factor/physiology , ErbB Receptors/genetics , Female , Gonadotropin-Releasing Hormone/metabolism , Hypothalamus/cytology , Molecular Sequence Data , Neurons/metabolism , Phosphorylation , Polymerase Chain Reaction , Proestrus , Protein Processing, Post-Translational , Rats , Rats, Sprague-Dawley , Transforming Growth Factor alpha/physiology
20.
J Neurosci ; 13(2): 703-13, 1993 Feb.
Article in English | MEDLINE | ID: mdl-8426232

ABSTRACT

Injury of the nervous system triggers a complex series of repair mechanisms that include production of neurotrophic and mitogenic factors by cells neighboring the injured area. While trauma of most parts of the brain results in loss of function, lesions of certain regions of the female hypothalamus enhance the secretory activity of a group of specialized neurons that produce luteinizing hormone-releasing hormone (LHRH), the neuropeptide that controls sexual development. The increased output of LHRH causes sexual precocity by prematurely activating the neuroendocrine reproductive axis. Recent studies have implicated transforming growth factor alpha (TGF alpha) produced by reactive astrocytes in the process by which lesions hasten sexual maturation, and have suggested that the stimulatory actions of TGF alpha on LHRH neurons require the intermediacy of epidermal growth factor receptors (EGFRs). In the present study, we examined the changes in EGFR gene expression following lesions of the preoptic-anterior hypothalamic area (POA-AHA) of immature female rats, identified the cell types where EGFR synthesis increases, and assessed the biochemical activity of the newly formed EGFR protein. RNase protection assays demonstrated that the lesion significantly increased the levels of a predominant mRNA transcript encoding the full-length, membrane-spanning EGFR, but did not affect those of a much less abundant, alternatively spliced mRNA that encodes a truncated, presumably secreted form of EGFR. Following lesions, antibody-induced EGFR kinase activity increased twofold. Antibodies directed against a peptide sequence contained within the carboxy terminus of EGFR showed intense EGFR immunoreactivity in cells surrounding the lesion site; double immunohistochemistry identified these cells as astrocytes since EGFR immunoreactivity was colocalized with that of glial fibrillary acidic protein, an astrocytic marker. That these changes result from an increase in EGFR gene expression was indicated by the elevated levels of EGFR mRNA detected by in situ hybridization in cells of the same area. Although POA-AHA lesions did not result in appearance of EGFR in LHRH neurons themselves, EGFR-positive cells and processes were seen in close proximity to LHRH neurons and their nerve terminals, particularly in the area surrounding the lesion. Since TGF alpha gene expression is also increased in reactive astrocytes of POA-AHA lesions and blockade of EGFR prevented the advancing effect of the lesion on puberty (Junier et al., 1991b), the present results support the concept that, in lesioned animals, TGF alpha stimulates LHRH secretion indirectly via a paracrine mechanism that involves its interaction with EGFRs located on astroglial cells.


Subject(s)
ErbB Receptors/genetics , Gene Expression Regulation/physiology , Hypothalamus/injuries , Neuroglia/metabolism , RNA Splicing , Sexual Maturation/physiology , Animals , ErbB Receptors/analysis , ErbB Receptors/metabolism , Female , Gonadotropin-Releasing Hormone/analysis , Hypothalamus, Anterior/chemistry , Hypothalamus, Anterior/injuries , Hypothalamus, Anterior/metabolism , Immunohistochemistry , Nucleic Acid Hybridization , Preoptic Area/chemistry , Preoptic Area/injuries , Preoptic Area/metabolism , RNA, Messenger/analysis , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley
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