Didier Wion

New clues about vitamin D functions in the nervous system

Accumulating data have provided evidence that 1 alpha,25 dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] is involved in brain function. Thus, the nuclear receptor for 1,25-(OH)(2)D(3) has been localized in neurons and glial cells. Genes encoding the enzymes involved in the metabolism of this hormone are also expressed in brain cells. The reported biological effects of 1,25-(OH)(2)D(3) in the nervous system include the biosynthesis of neurotrophic factors and at least one enzyme involved in neurotransmitter synthesis. 1,25-(OH)(2)D(3) can also inhibit the synthesis of inducible nitric oxide synthase and increase glutathione levels, suggesting a role for the hormone in brain detoxification pathways. Neuroprotective and immunomodulatory effects of this hormone have been described in several experimental models, indicating the potential value of 1,25-(OH)(2)D(3) pharmacological analogs in neurodegenerative and neuroimmune diseases. In addition, 1,25-(OH)(2)D(3) induces glioma cell death, making the hormone of potential interest in the management of brain tumors. These results reveal previously unsuspected roles for 1,25-(OH)(2)D(3) in brain function and suggest possible areas of future research.

N6-methyl-adenine: an epigenetic signal for DNA-protein interactions.

N6-methyl-adenine is found in the genomes of bacteria, archaea, protists and fungi. Most bacterial DNA adenine methyltransferases are part of restriction–modification systems. Certain groups of Proteobacteria also harbour solitary DNA adenine methyltransferases that provide signals for DNA–protein interactions. In γ-proteobacteria, Dam methylation regulates chromosome replication, nucleoid segregation, DNA repair, transposition of insertion elements and transcription of specific genes. In Salmonella, Haemophilus, Yersinia and Vibrio species and in pathogenic Escherichia coli, Dam methylation is required for virulence. In α-proteobacteria, CcrM methylation regulates the cell cycle in Caulobacter, Rhizobium and Agrobacterium, and has a role in Brucella abortus infection.

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, an inducer of glial cell line-derived neurotrophic factor

Glial cell line-derived neurotrophic factor (GDNF) has significant therapeutic potentials, in particular for neurodegenerative disorders. To determine factors that would enhance GDNF expression, we analysed the effect of 1,25-(OH)2 D3 in C6 glioma cells. Treatment of C6 cells with 10−7 M, 1,25-(OH)2 D3 for 48 h elicited an 18.5-fold increase in the level of GDNF mRNA. In addition, our results indicate that 1,25-(OH)2 D3 is effective at concentrations as low as 10−10 M and that retinoic acid has additive effects. These data indicate that 1,25-(OH)2 D3 is a potent inducer of GDNF expression and suggest that 1,25-(OH)2 D3 may contribute to the regulation of GDNF in vivo.

MicroRNA and Target Protein Patterns Reveal Physiopathological Features of Glioma Subtypes

Gliomas such as oligodendrogliomas (ODG) and glioblastomas (GBM) are brain tumours with different clinical outcomes. Histology-based classification of these tumour types is often difficult. Therefore the first aim of this study was to gain microRNA data that can be used as reliable signatures of oligodendrogliomas and glioblastomas. We investigated the levels of 282 microRNAs using membrane-array hybridisation and real-time PCR in ODG, GBM and control brain tissues. In comparison to these control tissues, 26 deregulated microRNAs were identified in tumours and the tissue levels of seven microRNAs (miR-21, miR-128, miR-132, miR-134, miR-155, miR-210 and miR-409-5p) appropriately discriminated oligodendrogliomas from glioblastomas. Genomic, epigenomic and host gene expression studies were conducted to investigate the mechanisms involved in these deregulations. Another aim of this study was to better understand glioma physiopathology looking for targets of deregulated microRNAs. We discovered that some targets of these microRNAs such as STAT3, PTBP1 or SIRT1 are differentially expressed in gliomas consistent with deregulation of microRNA expression. Moreover, MDH1, the target of several deregulated microRNAs, is repressed in glioblastomas, making an intramitochondrial-NAD reduction mediated by the mitochondrial aspartate-malate shuttle unlikely. Understanding the connections between microRNAs and bioenergetic pathways in gliomas may lead to identification of novel therapeutic targets.

Enhancement of the Synthesis and Secretion of Nerve Growth Factor in Primary Cultures of Glial Cells by Proteases: A Possible Involvement of Thrombin

Abstract: Newborn rat brain astrocytes cultured in vitro in a chemically defined medium are shown to secrete enhanced levels of nerve growth factor (NGF) when they are exposed to various types of proteases. Proteolytic enzymes such as α‐thrombin or collagenase induce a continuous, dose‐dependent enhancement of the levels of cell‐secreted NGF. Incubation of astrocytes for a 24‐h period with 300 ng/ml of α‐thrombin (∼9 nM, or 1 U/ml) results in an increase of the levels of cell‐secreted NGF by a factor of three‐ to fourfold, and at doses 10 times higher, stimulation by a factor of up to four‐to fivefold was observed. This phenomenon reflects an enhancement of the cellular pool of NGF mRNA, already noticeable after 3 h of treatment, which is preceded by a temporary activation of protooncogenes encoding transcription factors of the AP‐1 family, such as c‐fos, c‐jun or junB. Trypsin, plasmin, α‐chymotrypsin, or elastase also enhanced, to different extents, the levels of cell‐secreted NGF. However, unlike α‐thrombin or collagenase, these enzymes cause, above a critical concentration, an extensive cell detachment from the solid support, and this is accompanied by a decrease of their activity on the production of NGF, so that their dose‐response curves are bell shaped. Stimulation was maximal at those concentrations that cause a limited loosening of the cell‐substratum interactions, as evidenced by a retraction of some cell processes after 24 h of treatment. Studies of the effect of α‐thrombin‐indicate that the proteolytic activity itself is required to enhance the production of NGF by astrocytes. Inactivation of α‐thrombin with D‐phenyl‐alanyl‐L‐propyl‐L‐arginine chloromethyl ketone, phenylmethylsulfonyl fluoride, antithrombin III, or hirudin results in a marked decrease of the stimulatory effect. Furthermore, the prolonged presence of α‐thrombin is required to elicit a maximal effect on the levels of extracellular NGF, which was observed after 48 h of treatment. It is known that some effects of α‐thrombin require binding to the cell surface. We found that γ‐thrombin, which still has some proteolytic activity but has lost its ability to bind to the cell surface, is almost as potent as α‐thrombin in promoting the release of NGF. It is concluded that the effect of thrombin on NGF synthesis is essentially mediated by its proteolytic activity. Part of this effect may be due to the cleavage of a transmembrane receptor for a heptapeptide that acts as an agonist of this receptor and displays a limited but significant action on the levels of cell‐secreted NGF. These data suggest that proteolytic enzymes might induce astrocytes to produce NGF in vivo.

Development of gliomas: potential role of asymmetrical cell division of neural stem cells

Asymmetrical cell division is a mechanism that gives rise to two daughter cells with different proliferative and differentiative fates. It occurs mainly during development and in adult stem cells. Accumulating evidence suggests that tumour cells arise from the transformation of normal stem cells. Here, we propose that the asymmetrical mitosis potential of stem cells is associated with the generation of migrating tumour progenitors. Application of this speculative model to glioma proposes that the sites where tumour-initiating stem cells reside are indolent and distinct from the tumour mass, and implies that the tumour mass is continuously replenished with new migrating tumour cells from these clinically silent regions. This hypothesis offers explanations for our inability to cure glioblastoma and points to asymmetrical division as a new potential therapeutic target.

1,25-Dihydroxyvitamin D3 regulates the expression of the low-affinity neurotrophin receptor

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) is known to regulate the expression of neurotrophins [45,46]. Here, we report that 1,25-(OH)2D3 does not influence the expression of truncated or full-length forms of trkB and trkC receptors mRNAs in primary cultures of astrocytes and in C6 glioma cells. In contrast, low concentrations of 1,25-(OH)2D3 increased low-affinity neurotrophin receptor (P75NTR) mRNA and protein levels in C6 glioma cells. Putative vitamin D responsive elements (VDRE) in the P75NTR promoter have been investigated by transfecting plasmids containing sequences from P75NTR promoter fused to a cat reporter gene. A region between -610 and -860 bp upstream from the translation start codon was found to respond to 1,25-(OH)2D3. Interestingly, 1,25-(OH)2D3 does not regulate P75NTR in primary cultures of astrocytes even at concentration as high as 10(-7) M. Since long-term treatment of 1,25-(OH)2D3 induces cell death in C6 glioma cells but not in primary astrocytes [41], the possible involvement of P75NTR in 1,25-(OH)2D3-induced cell death is discussed. Finally, in-vivo studies show that treatment of 15-day-old and adult rats with 1,25-(OH)2D3 leads to a decrease in the level of P75NTR mRNA in the spinal cord but does not influence its expression in dorsal root ganglion or sciatic nerve. These results suggest that 1,25-(OH)2D3 may have a role in the specific regulation of P75NTR in vivo.

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.

Activation of nerve growth factor synthesis in primary glial cells by phorbol 12-myristate 13-acetate : role of protein kinase C

Phorbol 12-myristate 13-acetate (PMA) induces a dramatic production of nerve growth factor (NGF) in primary cultures of newborn mouse astrocytes maintained in a serum-free medium. This stimulation is dose-dependent and a maximal effect on the levels of cell-secreted factor was observed at a concentration of 10 nM. At this concentration, the promoting effect of PMA appears much more important than that elicited by 10% fetal calf serum (FCS) under the same culture conditions. PMA acts primarily on the accumulation of NGF mRNA, which was detected by northern blot analysis after 6 h of treatment. This accumulation may be totally or partially prevented when PMA-treated glial cells are concomitantly exposed to the protein kinase inhibitors H-7, H-9, and to a lesser degree, HA-1004. The known specificity of these inhibitors agrees with the possibility that protein kinase C (PKC), which constitutes so far the sole known target of PMA, represents a key element involved in the stimulation of NGF gene. The role of PKC is further supported by the observation that alpha phorbol didecanoate, which has no activity on PKC, is depleted of effect on the synthesis of NGF. Likewise, 1,2-dioctanoylglycerol (1,2-DOG) has a weak, but significant promoting action on the production of NGF, unlike the 1,3-isomer which is not active on PKC. Finally, a treatment of 15 min with 100 nM PMA is sufficient to stimulate the cells, suggesting that the activation phase of PKC, rather than its down regulation, constitutes an important trigger leading to an increased expression of the NGF gene.(ABSTRACT TRUNCATED AT 250 WORDS)