Christel Baudet

1,25-Dihydroxyvitamin D3 regulates the synthesis of nerve growth factor in primary cultures of glial cells

The effect of 1,25-dihydroxyvitamin D3 (1,25-(OH)2 D3) on nerve growth factor (NGF) synthesis was investigated in primary cultures of astrocytes prepared from brain of neonatal rats. 1,25-(OH)2 D3 elicited a dose-dependent increase of NGF mRNA with a maximal effect at 10(-7) M, which persisted for at least 48 h. Northern blot analysis revealed an expression of the vitamin D3 receptor (VDR) gene in primary glial cells. Treatment of cells with 1,25-(OH)2 D3 led to an increase in the VDR mRNA levels. Similar results were obtained in C6 glioma cells. Exposure of primary glial cells to 10(-8) M 1,25-(OH)2 D3 caused only a 2-fold increase of the levels of cell-secreted NGF after 3 days of treatment. However, a 5-fold increase was observed three days after a second addition of vitamin D3. Likewise, a pretreatment with lower doses of hormone such as 10(-10) M or 10(-9) M enhanced the responsiveness of the cells to a 24 h treatment with 10(-8) M hormone. It appears, therefore, that the duration of the treatment influences the level of synthesis of NGF, possibly as a consequence of the increase of the VDR gene expression. The specificity of 1,25-(OH)2 D3 is supported by the fact that a concentration of 10(-7) M of an another vitamin D3 metabolite, 24,25-(OH)2 D3, had no effect on NGF synthesis. Several lines of evidence indicate that astrocytes constitute the major cell type responsive to 1,25-(OH)2 D3 in primary cultures of glial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

1,25-Dihydroxyvitamin D3 regulates NT-3, NT-4 but not BDNF mRNA in astrocytes

THE effect of 1,25-dihydroxyvitamin D3 on neurotrophin mRNA expression was studied in primary cultures of astrocytes. In addition to its known effects on NGF expression, 1,25-dihydroxy vitamin D3 was shown to upregulate NT-3 mRNA levels, while NT-4 expression was slightly but significantly downregulated. No effect was observed on BDNF mRNA expression. These data clearly show a differential regulation of the four neurotrophins by 1,25-dihydroxyvitamin D3 in primary cultures of astrocytes, and suggest that 1,25-dihydroxyvitamin D3 may participate in the expression of NGF, NT-3, and NT-4 in the central nervous system.

Differentially expressed genes in C6.9 glioma cells during vitamin D-induced cell death program

C6.9 rat glioma cells undergo a cell death program when exposed to 1,25-dihydroxyvitamin D3 (1,25-D3). As a global analytical approach, we have investigated gene expression in C6.9 engaged in this cell death program using differential screening of a rat brain cDNA library with probes derived from control and 1,25-D3-treated cells. Using this methodology we report the isolation of 61 differentially expressed cDNAs. Forty-seven cDNAs correspond to genes already characterized in rat cells or tissues. Seven cDNAs are homologous to yeast, mouse or human genes and seven are not related to known genes. Some of the characterized genes have been reported to be differentially expressed following induction of programmed cell death. These include PMP22/gas3, MGP and β-tubulin. For the first time, we also show a cell death program induced up-regulation of the c-myc associated primary response gene CRP, and of the proteasome RN3 subunit and TCTP/mortalin genes. Another interesting feature of this 1,25-D3 induced-cell death program is the down-regulated expression of transcripts for the microtubule motor dynein heavy chain/MAP 1C and of the calcium-binding S100β protein. Finally 15 upregulated cDNAs encode ribosomal proteins suggesting a possible involvement of the translational apparatus in this cell program. Alternatively, these ribosomal protein genes could be up-regulated in response to altered rates of cellular metabolism, as has been demonstrated for most of the other isolated genes which encode proteins involved in metabolic pathways. Thus, this study presents to our knowledge the first characterization of genes which are differentially expressed during a cell death program induced by 1,25-D3. Therefore, this data provides new information on the fundamental mechanisms which participate in the antineoplastic effects of 1,25-D3 and on the machinery of a cell death program in a glioma cell line.

Expression of 25(OH) vitamin D3 24-hydroxylase gene in glial cells.

The expression of the 25(OH) vitamin D3 24-hydroxylase gene was studied in C6 glioma and rat primary glial cell culture. The expression of the 25(OH)D3 24-hydroxylase gene was not detected in C6 glioma or glial cells cultured in a serum-free medium. However, the 25(OH)D3 24-hydroxylase mRNA was induced in a dose-dependent manner in cells treated with 1,25(OH)2D3. These findings provide further evidence for an involvement of vitamin D3 metabolites in brain function.

Retrograde Signaling onto Ret during Motor Nerve Terminal Maturation

Establishment of the neuromuscular synapse requires bidirectional signaling between the nerve and muscle. Although much is known on nerve-released signals onto the muscle, less is known of signals important for presynaptic maturation of the nerve terminal. Our results suggest that the Ret tyrosine kinase receptor transmits a signal in motor neuron synapses that contribute to motor neuron survival and synapse maturation at postnatal stages. Ret is localized specifically to the presynaptic membrane with its ligands, GDNF (glial cell line-derived neurotrophic factor)/NTN (neurturin), expressed in skeletal muscle tissue. Lack of Ret conditionally in cranial motor neurons results in a developmental deficit of maturation and specialization of presynaptic neuromuscular terminals. Regeneration of Ret-deficient adult hypoglossal motor neurons is unperturbed, but despite contact with the unaffected postsynaptic specializations, presynaptic axon terminal maturation is severely compromised in the absence of Ret signaling. Thus, Ret transmits a signal in motor nerve terminals that participate in the organization and maturation of presynaptic specializations during development and during regeneration in the adult.

Reactive Oxygen Species Influence Nerve Growth Factor Synthesis in Primary Rat Astrocytes

Abstract: Newborn rat brain astrocytes, cultured in a serum‐free medium, were exposed for 30 min to two types of reactive oxygen species. Cells were either treated with the xanthine/xanthine oxidase (X/XOD) system, which generates both H2O2 and the O2βradical, or to H2O2 alone. Both treatments induced a dose‐dependent accumulation of nerve growth factor (NGF) transcripts, 6 h after the exposure. Maximal effect was obtained with 6 mU/ml XOD, or 10–4M H2O2. A rapid expression of protooncogenes of the jun and fos families was also noticed in X/XOD‐or H2O2‐treated cells. This phenomenon was transient in cells exposed to X/XOD. However, in the case of H2O2‐treated cells, the accumulation of c‐fos or c‐jun mRNAs was still pronounced 6 h after the end of the treatment and the levels of cell‐secreted NGF appeared relatively reduced, when compared with those obtained after a shock with the X/XOD system. This raised the possibility that H2O2 at 10–4M could depress protein synthesis. Measurements of the incorporation of radiolabeled amino acids into trichloroacetic acid‐precipitable material supported this assumption. Level of radioactivity associated with cellular material was dramatically reduced in H2O2‐treated cells, when it was compared with control or XI XOD‐trreated cells. Furthermore, treatment of cells with the protein synthesis inhibitor anisomycin had an effect similar to that of H2O2 because it caused an accumulation of c‐fos, c‐jun, and NGF transcripts after 6 h of treatment. It is concluded that the effect of H2O2 results from an impairment of protein synthesis, which is accompanied by the “superinduction” of c‐fos, c‐jun, or NGF genes. To understand better the effect of X/XOD, the reaction was conducted in the presence of a large excess of catalase, which removes H2O2. The presence of catalase reduced the amplitude of the response of the NGF gene to the X/XOD treatment. Conversely, low doses of H2O2 and X/ XOD were more efficient for the production of NGF than each treatment alone. These results suggest that both O2β and H2O2 are important metabolites in the process of activation of the NGF gene, possibly via the formation of the OH* radical or other reactive products, which could constitute active regulatory molecules.

Attenuation of a caspase-3 dependent cell death in NT4- and p75-deficient embryonic sensory neurons.

Neuronal survival during the developmental period of naturally occurring cell death is mediated through a successful competition for limiting concentrations of neurotrophic factors, and the deprived neurons will die. New results show that induced death through the p75 neurotrophin receptor (p75(NTR)), a member of the p55TNF/Fas family of cell death receptors, may also influence survival during development. We find that eliminating p75(NTR) or neurotrophin 4 (NT4) in mice leads to a marked attenuation of apoptosis during the programmed cell death period of the trigeminal ganglion neurons, suggesting that NT4 can induce the death of these neurons through the p75(NTR). These in vivo findings were reproduced in primary cell cultures, where NT4 was found to induce death in a p75(NTR)-dependent pathway. Analysis of p75 deficient and wild-type cells revealed two separate cell death pathways, a p75(NTR)- and caspase-3-independent pathway activated by trophic factor deprivation, and a p75(NTR)- and caspase-3-dependent pathway initiated by NT4. Crossing in the NT4 null alleles in brain-derived neurotrophic factor (BDNF) null mutant mice led to a rescue of a large proportion of BDNF-dependent neurons from excessive cell death, indicating that trophic factor deprivation is not sufficient for the death of many neurons and that additional death inducing signals might be required. Our results suggest that NT4 competitively signals survival and death of sensory neurons through trkB and p75(NTR), respectively.

Activation by GDNF of a transcriptional program repressing neurite growth in dorsal root ganglia

Glial cell line-derived neurotrophic factor (GDNF) promotes the survival of postnatal—but not embryonic—mouse dorsal root ganglion cells in vitro, despite the fact that its receptors are expressed at both ages. To understand this difference, we have performed an oligonucleotide microarray experiment. We found that several hundred genes were regulated between embryonic and postnatal stages, and that several important classes of genes were differentially regulated by GDNF treatment, including genes related to translation and to phenotypic specification and maturation. Interestingly, a set of genes related to cell adhesion, cytoskeleton and cellular morphology were consistently down-regulated by GDNF, suggesting a previously uncharacterized role for GDNF in repressing neurite growth and/or branching. This nuclear program initiated by GDNF was functionally confirmed in cultures of embryonic wild-type neurons sustained with nerve growth factor and in bax−/− neurons that survive in the absence of trophic support.

Soluble and bound forms of GFRα1 elicit different GDNF-independent neurite growth responses in primary sensory neurons

We have investigated the role of glial cell‐line derived neurotrophic factor (GDNF) and the effect of soluble or immobilized localization of its GDNF family receptor α1 (GFRα1) on neurite growth in cultured embryonic Bax−/− dorsal root ganglion neurons, which survive in the absence of trophic support. Whereas GDNF alone has a moderate effect on neurite growth, soluble and immobilized GFRα1 elicit opposing and GDNF‐independent effects on neurite growth by a phospholipase C (PLC) γ‐dependent mechanism. Thus, GFRα1 elicits nerve growth responses independent of GDNF. However, GDNF in the presence of soluble or immobilized GFRα1 reverse the GDNF‐independent GFRα1 modulation of neurite growth. The different outcome of soluble and bound GFRα1 combined with our previous immunohistochemical data showing GFRα1‐protein in Schwann cells but not axons suggest terminal Schwann cells as a source of locally administered target‐derived GFRα1 and place this receptor in the path of axonal growth and guidance. Thus, target‐derived GFRα1 play opposing roles when presented alone and with GDNF and, therefore, can function as a nerve growth cue that both can promote and prevent growth in the developing peripheral nervous system. Developmental Dynamics 227:27–34, 2003