Isabelle Neveu

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.

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.

A novel type I receptor serine-threonine kinase predominantly expressed in the adult central nervous system.

Receptor serine-threonine kinases (RSTK) mediate inhibitory as well as stimulatory signals for growth and differentiation by binding to members of the transforming growth factor-β (TGF-β) superfamily. Over 12 different RSTKs have been isolated so far, displaying wide expression in peripheral tissues and in the nervous system. Here we report the isolation and characterization of a novel type I RSTK termed activin receptor-like kinase-7 (ALK-7) that, unlike other members of this receptor family, is predominantly expressed in the adult central nervous system. The ALK-7 gene encodes a 55-kDa cell-surface protein that exhibits up to 78% amino acid sequence identity in the kinase domain to previously isolated type I receptors for TGF-β and activin. In the extracellular domain, however, ALK-7 is more divergent, displaying comparable similarities with all members of the ALK subfamily. RNase protection and in situ hybridization studies demonstrated a highly specific mRNA distribution restricted to neurons in several regions of the adult rat central nervous system, including cerebellum, hippocampus, and nuclei of the brainstem. Receptor reconstitution and cross-linking experiments indicated that ALK-7 can form complexes with type II RSTKs for TGF-β and activin in a ligand-dependent manner, although direct binding of ALK-7 to ligand in these complexes could not be demonstrated. The specific expression pattern of ALK-7, restricted to the postnatal central nervous system, indicates that this receptor may play an important role in the maturation and maintenance of several neuronal subpopulations.

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.

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.

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.

Intracerebral xenotransplantation: recent findings and perspectives for local immunosuppression.

Purpose of reviewCell therapy is a promising strategy for tissue repair in the central nervous system. In this perspective, several cell types are being considered, including allogenic neuroblasts, embryonic stem cells and induced pluripotent stem cells. The use of allogenic neuroblasts as cell source is limited by logistics and ethical problems whereas transplantation of the last two cell types is hampered by their propensity to generate tumour. In this context, transplantation of xenogeneic neural cells appears as an attractive approach for effective neuronal replacement in case of neurodegenerative disorders. Recent findingsWith the emergence of embryonic and induced pluripotent stem cells as potential cell source in regenerative medicine, little attention has been paid to the possibility of transplanting xenogenic neural cells in the central nervous system. However, recent progress to circumvent the host immune response in the brain has raised encouraging perspectives for intracerebral xenotransplantation as restorative strategy. SummaryTo date, most of the immunosuppressive strategies designed for long-term survival of intracerebral neural transplants were based on systemic immunosuppression that has detrimental side-effects. The immunological status of the brain and the presence of the blood–brain barrier raise the possibility of local immunosuppression. This article provides an overview of the strategies recently developed to protect intracerebral neural transplants with special focus on local immunosuppression.

Effects of Human Alpha-Synuclein A53T-A30P Mutations on SVZ and Local Olfactory Bulb Cell Proliferation in a Transgenic Rat Model of Parkinson Disease

A transgenic Sprague Dawley rat bearing the A30P and A53T α-synuclein (α-syn) human mutations under the control of the tyrosine hydroxylase promoter was generated in order to get a better understanding of the role of the human α-syn mutations on the neuropathological events involved in the progression of the Parkinsons disease (PD). This rat displayed olfactory deficits in the absence of motor impairments as observed in most early PD cases. In order to investigate the role of the mutated α-syn on cell proliferation, we focused on the subventricular zone (SVZ) and the olfactory bulbs (OB) as a change of the proliferation could affect OB function. The effect on OB dopaminergic innervation was investigated. The human α-syn co-localized in TH-positive OB neurons. No human α-syn was visualized in the SVZ. A significant increase in resident cell proliferation in the glomerular but not in the granular layers of the OB and in the SVZ was observed. TH innervation was significantly increased within the glomerular layer without an increase in the size of the glomeruli. Our rat could be a good model to investigate the role of human mutated α-syn on the development of olfactory deficits.

Minocycline promotes long-term survival of neuronal transplant in the brain by inhibiting late microglial activation and T-cell recruitment.

Background. Cell therapy in the brain is limited by the requirement of high doses of immunosuppressors that have harmful side effects, and often, it cannot prevent the ultimate rejection of the transplanted cells. Alternative treatments that replace or enable a reduction in the doses of usual immunosuppressors have to be found. In this regard, minocycline shows potential as therapeutic agent. This drug crosses the blood-brain barrier, has good safety records, and exhibits strong antiinflammatory effects. Methods. To study the impact of minocycline on the survival of intracerebral transplant, 400,000 porcine fetal neurons were transplanted into the striatum of rats treated daily with minocycline until sacrifice. Graft survival and immunologic reaction were evaluated by immunohistochemistry. Results. In the control groups, all the grafts were rejected at day 63, whereas healthy grafts exhibiting tyrosine hydroxylase+ neurons were observed in 40% of the treated rats. The low immunoreactivity for ED1 and R73 in treated rats when compared with the control groups suggests that minocycline promotes long-term survival of neuronal xenograft by inhibiting microglial activation and T-cell recruitment. Conclusions. Our present data provide the first evidence of an effect of minocycline on the host immune response after neuronal transplantation into the brain. This observation raises new perspectives concerning the use of minocycline and provides basis for the development of safe and efficient immunosuppressive protocols for intracerebral transplantation.