Cell Therapy for Parkinson's Disease: A Translational Approach to Assess the Role of Local and Systemic Immunosuppression.

Neural transplantation is a promising therapeutic approach for neurodegenerative diseases; however, many patients receiving intracerebral fetal allografts exhibit signs of immunization to donor antigens that could compromise the graft. In this context, we intracerebrally transplanted mesencephalic pig xenografts into primates to identify a suitable strategy to enable long-term cell survival, maturation, and differentiation. Parkinsonian primates received WT or CTLA4-Ig transgenic porcine xenografts and different durations of peripheral immunosuppression to test whether systemic plus graft-mediated local immunosuppression might avoid rejection. A striking recovery of spontaneous locomotion was observed in primates receiving systemic plus local immunosuppression for 6 mo. Recovery was associated with restoration of dopaminergic activity detected both by positron emission tomography imaging and histological examination. Local infiltration by T cells and CD80/86+ microglial cells expressing indoleamine 2,3-dioxigenase were observed only in CTLA4-Ig recipients. Results suggest that in this primate neurotransplantation model, peripheral immunosuppression is indispensable to achieve the long-term survival of porcine neuronal xenografts that is required to study the beneficial immunomodulatory effect of local blockade of T cell costimulation.

IgG response to intracerebral xenotransplantation: specificity and role in the rejection of porcine neurons.

Xenogenic fetal neuroblasts are considered as a potential source of transplantable cells for the treatment of neurodegenerative diseases, but immunological barriers limit their use in the clinic. While considerable work has been performed to decipher the role of the cellular immune response in the rejection of intracerebral xenotransplants, there is much still to learn about the humoral reaction. To this end, the IgG response to the transplantation of fetal porcine neural cells (PNC) into the rat brain was analyzed. Rat sera did not contain preformed antibodies against PNC, but elicited anti-porcine IgG was clearly detected in the host blood once the graft was rejected. Only the IgG1 and IgG2a subclasses were up-regulated, suggesting a T-helper 2 immune response. The main target of these elicited IgG antibodies was porcine neurons, as determined by double labeling in vitro and in vivo. Complement and anti-porcine IgG were present in the rejecting grafts, suggesting an active role of the host humoral response in graft rejection. This hypothesis was confirmed by the prolonged survival of fetal porcine neurons in the striatum of immunoglobulin-deficient rats. These data suggest that the prolonged survival of intracerebral xenotransplants relies on the control of both cell-mediated and humoral immune responses.

The use of stem cells in regenerative medicine for Parkinson's and Huntington's Diseases.

Cell transplantation has been proposed as a means of replacing specific cell populations lost through neurodegenerative processes such as that seen in Parkinson's or Huntington's diseases. Improvement of the clinical symptoms has been observed in a number of Parkinson and Huntington's patients transplanted with freshly isolated fetal brain tissue but such restorative approach is greatly hampered by logistic and ethical concerns relative to the use of fetal tissue, in addition to potential side effects that remain to be controlled. In this context, stem cells that are capable of self-renewal and can differentiate into neurons, have received a great deal of interest, as demonstrated by the numerous studies based on the transplantation of neural stem/progenitor cells, embryonic stem cells or mesenchymal stem cells into animal models of Parkinson's or Huntington's diseases. More recently, the induction of pluripotent stem cells from somatic adult cells has raised a new hope for the treatment of neurodegenerative diseases. In the present article, we review the main experimental approaches to assess the efficiency of cell-based therapy for Parkinson's or Huntington's diseases, and discuss the recent advances in using stem cells to replace lost dopaminergic mesencephalic or striatal neurons. Characteristics of the different stem cells are extensively examined with a special attention to their ability of producing neurotrophic or immunosuppressive factors, as these may provide a favourable environment for brain tissue repair and long-term survival of transplanted cells in the central nervous system. Thus, stem cell therapy can be a valuable tool in regenerative medicine.

Assessing the potential clinical utility of transplantations of neural and mesenchymal stem cells for treating neurodegenerative diseases.

Treatments for neurodegenerative diseases have little impact on the long-term patient health. However, cellular transplants of neuroblasts derived from the aborted embryonic brain tissue in animal models of neurodegenerative disorders and in patients have demonstrated survival and functionality in the brain. However, ethical and functional problems due to the use of this fetal tissue stopped most of the clinical trials. Therefore, new cell sources were needed, and scientists focused on neural (NSCs) and mesenchymal stem cells (MSCs). When transplanted in the brain of animals with Parkinson's or Huntington's disease, NSCs and MSCs were able to induce partial functional recovery by promoting neuroprotection and immunomodulation. MSCs are more readily accessible than NSCs due to sources such as the bone marrow. However, MSCs are not capable of differentiating into neurons in vivo where NSCs are. Thus, transplantation of NSCs and MSCs is interesting for brain regenerative medicine. In this chapter, we detail the methods for NSCs and MSCs isolation as well as the transplantation procedures used to treat rodent models of neurodegenerative damage.

The neuropeptide Y receptors, Y1 and Y2, are transiently and differentially expressed in the developing cerebellum.

Neuropeptide Y (NPY), a peptide widely expressed in the brain, acts through the protein G-coupled receptors Y1, Y2 and Y5. In the adult rat, this peptide modulates many important functions such as the control of energy balance and anxiety. Its involvement in brain development has been less investigated. In the present study, we have analysed the expression of Y1 and Y2 in the developing rat cerebellum using RNase protection assay. Both receptors were detected in the embryo but at very low levels. Their expression then increased, reaching a peak at postnatal day 10. At later stages, we observed a down-regulation of both Y1 and Y2 mRNA levels. This pattern of expression was delayed in hypothyroid rats, suggesting that the regulation of NPY receptors was strictly related to cerebellar development stages. In situ hybridisation and immunohistochemistry analyses revealed specific localisations of the receptors. Y1 was exclusively expressed by Purkinje cells while Y2 was found mostly in granule cells of the internal granule cell layer. These observations argue in favour of specific roles for Y1 and Y2 in the developing cerebellum. In an initial attempt to characterise these roles, we have determined the number of apoptotic cells in the developing cerebellum of Y2(-/-) mice and analysed the effects of NPY on primary cultures of cerebellar granule neurones. Our data showed that the absence of Y2 did not increase cell death in the internal granule cell layer of the developing cerebellum, and that NPY by itself did not prevent the death of differentiated granule cells cultured in serum-free medium. However, we found that co-treatment of the cells by NPY and neuromediators such as NMDA or GABA strongly promoted the survival of granule neurones. Taken together, these observations suggest an involvement of the NPY receptors in cerebellar ontogenesis that remains to be demonstrated in vivo.

Different mechanisms mediate the rejection of porcine neurons and endothelial cells transplanted into the rat brain.

In order to investigate the early cellular responses mediating xenograft rejection in the brain, porcine aortic endothelial cells (PAEC) or porcine fetal mesencephalic neurons (PNEU) were transplanted into the striatum of LEW.1A rats. PAEC were detected with a specific anti-beta1 integrin antibody, and PNEU with an anti-porcine neurofilament antibody, or an antibody recognizing the NeuN antigen. PAEC grafts were massively infiltrated within 24 h by OX42-positive cells, which may correspond to polymorphonuclear (PMN) cells or macrophages. At that moment, the graft contained numerous cells expressing the inducible isoform of NO-synthase (iNOS). Infiltration by ED1-positive macrophages was effective after three days. The beta1-integrin labeling decreased from that time-point to day 7 post-implantation, and vanished after 11 days. Although some OX8-positive cells were present around the graft as soon as 3 days after transplantation, cells expressing the T-cell receptor (TCR)-beta chain infiltrated the graft after 7 days and their number remained low. A strong, diffuse OX8-and ED1-positive immunoreactive material remained in the scar up to the third week. In striking contrast, PNEU grafts remained poorly infiltrated by OX42- or ED1-positive cells during the first two weeks. A massive infiltration by macrophages and TCRbeta-positive lymphocytes occurred after 3 weeks. Natural killer (NK) cells were more scarce. The inflammation territory enlarged, and blood vessels were overloaded with macrophages or lymphocytes. Nevertheless, the graft contained NeuN-positive nuclei and neurites harbouring the porcine neurofilament protein. Hence, rejection was not completed at this time-point. These results suggest that the rapid rejection of PAEC is mainly driven by macrophages and possibly PMN cells, unlike PNEU, whose rejection is delayed and also involves lymphocytes. Differences in immunogenicity of grafted cells and/or patterns of production of pro-inflammatory cytokines may account for these contrasted rejection kinetics.

Differential regulation of GDNF, neurturin, and their receptors in primary cultures of rat glial cells.

Glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) bind to GFR alpha-1 and GFR alpha-2 receptors, respectively, and their neurotrophic activity is mediated by the tyrosine kinase receptor, Ret. All these molecules were found to be expressed in primary cultures of rat glial cells, which were largely composed of astrocytes and maintained in serum-free medium. Although GDNF, NTN and Ret mRNA levels were at the limit of detection, RNase protection assays revealed relatively high amounts of GFR alpha-1 and GFR alpha transcripts. To characterize signals controlling their expression, glial cells were exposed to serum or treated with hormones acting through nuclear receptors and by activators of the cAMP or protein kinase C (PKC)-dependent pathways. Retinoic acid or 1,25-dihydroxyvitamin D3 appeared ineffective. In contrast, the 5-fold increase in GFR alpha-2 mRNA after 24 hr of treatment with 10(-10) M of tri-iodothyronine, suggests a physiological role of thyroid hormone in the regulation of this receptor in vivo. The serum induced a 7-fold increase in GFR alpha-1 mRNA levels. These changes may be mediated by the cAMP or PKC pathways because both forskolin and TPA up-regulated the GFR alpha-1 gene. Interestingly, only TPA led to a coordinated increase in the levels of GDNF, GFR alpha-1 and GFR alpha-2 mRNAs. On the other hand, NTN transcripts remained constant, irrespective of the culture conditions. Taken together, these results indicate that GDNF family ligands and their receptors are regulated in glial cells by common or independent transductional pathways, which could modulate their specific expression during brain development or in the case of trauma.

Increased cell death and delayed development in the cerebellum of mice lacking the rev-erbA(alpha) orphan receptor.

The rev-erbA(alpha) gene, belonging to the steroid receptor superfamily of transcription factors, is highly conserved during evolution but little is known so far about its functions in development or in adult physiology. Here, we describe genetically altered mice lacking the rev-erbA(alpha) gene. These animals do not show any obvious phenotype in either fat tissue or skeletal muscle, despite the known regulation of rev-erbA(alpha) expression during adipocyte and myotube differentiation in vitro. However, during the second week of life, the cerebellum of rev-erbA(alpha) mutants presents several unexpected abnormalities, such as alterations in the development of Purkinje cells, delay in the proliferation and migration of granule cells from the external granule cell layer and increased apoptosis of neurons in the internal granule cell layer. Interestingly, the expression pattern of rev-erbA(alpha) suggests that the abnormalities observed in the external granule cell layer could be secondary to Purkinje cell alterations. Taken together, our data underline the importance of rev-erbA(alpha)expression for the appropriate balance of transcriptional activators and repressors during postnatal cerebellar development.

Brain-derived neurotrophic factor, neurotrophin-3 and neurotrophin-4/5 differentially regulate the phenotype and prevent degenerative changes in striatal projection neurons after excitotoxicity in vivo.

To determine whether growth factors of the neurotrophin family are able to regulate the phenotype of striatal projection neurons, cell lines overexpressing brain-derived neurotrophic factor, neurotrophin-3 or neurotrophin-4/5 were intrastriatally grafted. Striatal projection neurons were examined for the regulation of their soma areas and for the expression of glutamate decarboxylase 67, preprotachykinin A, preproenkephalin and prodynorphin messenger RNAs by in situ hybridization. Brain-derived neurotrophic factor, neurotrophin-3 and neurotrophin-4/5 differentially regulated the soma area of projection neurons at different distances from the graft, but did not modify their messenger RNA levels. Neurotrophin-3 induced an increase in the soma area of preproenkephalin- and preprotachykinin A-positive neurons, brain-derived neurotrophic factor increased the soma area of only preprotachykinin A-positive neurons, while neurotrophin-4/5 did not produce any effect. Because atrophy and neuronal loss are hallmarks of Huntington's disease, we next examined whether neurotrophins prevent degenerative changes in a quinolinate model of Huntington's disease. Seven days after intrastriatal quinolinate injection, we observed a halo of cell loss around the injection sites, reduced soma area of glutamate decarboxylase 67-, preproenkephalin- and preprotachykinin A-positive neurons bordering the lesion, and a decrease in the messenger RNA levels of glutamate decarboxylase 67 and these neuropeptides. Grafting of cell lines expressing brain-derived neurotrophic factor, neurotrophin-3 or neurotrophin-4/5 reduced the size of the lesion for preproenkephalin-, preprotachykinin- and glutamate decarboxylase 67-, but not for prodynorphin-positive neurons. Moreover, the three neurotrophins prevented the atrophy of all projection neurons, and the lesion-induced decrease in preproenkephalin and preprotachykinin A messenger RNA levels. We conclude that neurotrophins differentially regulate the phenotype of striatal projection neurons and prevent degenerative changes. The higher efficiency of neurotrophin-3 suggests a potential therapeutic application of this molecule in neurological disorders affecting striatal projection neurons, such as Huntington's disease.

1,25-Dihydroxyvitamin D3 regulates the expression of VDR and NGF gene in Schwann cells in vitro.

The vitamin D receptor (VDR) is a nuclear receptor that mediates the effect of the active metabolite of vitamin D3, the 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). To investigate the potential role of this hormone in the peripheral nervous system, we have studied the VDR expression in Schwann cells. The VDR mRNA was detected by Northern blot analysis in rat primary cultures of Schwann cells, and its levels were strongly increased in the presence of 1,25-(OH)2D3. Using the mouse Schwann cell line, MSC80, we showed that concentrations as low as 10(-10) M of hormone stimulated the expression of the VDR gene and strongly increased the amounts of activated VDR, capable of binding to the specific vitamin D responsive element (VDRE). We also found that 1,25-(OH)2D3 stimulated the expression of the nerve growth factor gene in MSC80. These data suggest a role for the hormone in the peripheral nervous system, possibly as a mediator active in trauma.

Complementary and overlapping expression of Y1, Y2 and Y5 receptors in the developing and adult mouse nervous system.

Neuropeptide Y, a 36 amino acid peptide, mediates its biological effects by activating the Y1, Y2, Y5 and Y6 receptors, which are also receptors for the structurally related peptide YY. Different classes of receptors have been suggested to be involved in different neuropeptide Y functions. In this report, we have characterized the developmental regulation and compared the cellular localization of these receptors in the developing and in the adult central and peripheral nervous systems of the mouse. RNase protection assays revealed that Y1, Y2 and Y5 messenger RNAs were expressed very early in spinal cord, brain, cerebellum and dorsal root ganglion development and were often down-regulated at times corresponding to their acquirement of the adult function in neurotransmission. In situ hybridization of the adult brain showed that Y1 was widely expressed, Y2 displayed a more restricted pattern, Y5 was expressed at very low levels and only in a few brain nuclei and Y6 was not expressed. Virtually all areas containing neurons positive for Y5 also expressed Y1, whereas many Y1-positive cells clearly did not express Y5. In contrast, Y2 was not expressed by the neurons expressing Y1 or Y5. These findings suggest that neuropeptide Y signaling in the brain could be mediated by simultaneous Y1 and Y5 activation. Similar results were also obtained in peripheral sensory neurons. Furthermore, our results suggest that neuropeptide Y/peptide YY receptors play an important role in nervous system development and that selective receptor combinations are responsible for signaling the different effects of neuropeptide Y in the peripheral and central nervous systems.

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-beta (TGF-beta) 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-beta 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-beta 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, 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 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.

Neurotrophins promote the survival and development of neurons in the cerebellum of hypothyroid rats in vivo.

The development of cerebellar cortex is strongly impaired by thyroid hormone (T3) deficiency, leading to altered migration, differentiation, synaptogenesis, and survival of neurons. To determine whether alteration in the expression of neurotrophins and/or their receptors may contribute to these impairments, we first analyzed their expression using a sensitive RNAse protection assay and in situ hybridization; second, we administered the deficient neurotrophins to hypothyroid animals. We found that early hypothyroidism disrupted the developmental pattern of expression of the four neurotrophins, leading to relatively higher levels of NGF and neurotrophin 4/5 mRNAs and to a severe deficit in NT-3 and brain-derived neurotrophic factor (BDNF) mRNA expression, without alteration in the levels of the full-length tyrosine kinase (trk) B and trkC receptor mRNAs. Grafting of P3 hypothyroid rats with cell lines expressing high levels of neurotrophin 3 (NT-3) or BDNF prevented hypothyroidism-induced cell death in neurons of the internal granule cell layer at P15. In addition, we found that NT-3, but not BDNF, induced the differentiation and/or migration of neurons in the external granule cell layer, stimulated the elaboration of the dendritic tree by Purkinje cells, and promoted the formation of the mature pattern of synaptic afferents to Purkinje cell somas. Thus, our results indicate that both granule and Purkinje neurons require appropriate levels of NT-3 for normal development in vivo and suggest that T3 may regulate the levels of neurotrophins to promote the development of cerebellum.

Regulation of NGF, BDNF and LNGFR gene expression in ROS 17/2.8 cells.

The secosteroid hormone 1.25-dihydroxyvitamin D3 (1,25(OH)2D3) has been recently shown to enhance the synthesis of NGF to mouse L929 fibroblasts. In view of the critical role of 1,25(OH)2D3 on bone metabolism, it has been investigated if ROS 17/2.8 osteoblastic cells were able to express the nerve growth factor (NGF) gene and if this process was responsive to 1,25(OH)2D3. Results indicate that these cells respond in a dose-dependent manner to the presence of 1,25(OH)2D3 by an increase in NGF mRNA levels. However, the phorbol ester PMA, previously reported to augment the synthesis of NGF via the recruitment of AP-1 complexes, depressed the expression of the NGF gene in ROS cells. In contrast, the mRNA levels of an NGF-related trophic factor, brain-derived neurotrophic factor (BDNF), was increased by PMA but not following 1,25(OH)2D3 treatment. Binding of 125I-NGF to ROS cells displayed the properties of a low affinity NGF receptor (dissociation constant Kd approximately 10(-9) M). In agreement with this result, the mRNA encoding the low affinity NGF receptor (LNGFR) was detected in ROS 17/2.8 cells, unlike trkA transcripts which encode the high affinity receptor. These data suggest that neurotrophins and their low affinity receptor could play an unsuspected role in bone tissue.

Interactions between second messenger pathways influence NGF synthesis in mouse primary astrocytes.

Primary mouse brain astrocytes were stimulated with phorbol 12-myristate 13-acetate (PMA), serum, forskolin and ionophore A23187, in order to investigate the effect of distinct signalling pathways on the expression of the nerve growth factor (NGF) gene and of proto-oncogenes encoding transcription factors of the Fos and Jun families. PMA, and to a lesser extent serum, induced a marked accumulation of NGF transcripts, in agreement with published observations [Brain Res., 570 (1992) 316-322]. The effect of A23187 was less pronounced and that of forskolin barely detectable. No relationship was observed between the expression of NGF gene and that of c-fos, fos-B, fra-1, jun-B proto-oncogenes. In contrast, changes in the levels of NGF transcripts were associated with corresponding modifications of the levels of c-jun transcripts, a fact which suggests that the c-Jun protein exerts a regulatory role on the expression of the NGF gene. In these cells, however, the regulation of NGF synthesis appears complex, since a pretreatment with forskolin or ionophore A23187 interfered with the promoting effect elicited by PMA or serum in inducing an early decline of the levels of NGF transcripts. This phenomenon was accompanied by a corresponding decrease in the amounts of cell-secreted NGF in cells treated with forskolin and PMA. A23187 had a much more striking effect on the production of mature NGF since this compound maintained the level of cell-secreted NGF to basal values, irrespective of the presence of PMA. A similar inhibitory effect was observed with thapsigargin, another compound able to increase the cytosolic concentration of calcium.(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-dihydroxyvitamin 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.

Decreased choline acetyltransferase activity in nerve growth factor-transgenic mice during brain development.

Activity of the synthetic enzyme for acetylcholine, choline acetyltransferase was investigated during development and in adult nerve growth factor-transgenic mice. A conspicuous reduction of choline acetyltransferase activity was observed in the anterior brain of nerve growth factor-transgenic embryos from embryonic days 13 to 16 (E13 to E16). Choline acetyltransferase activity levels subsequently resumed to normal levels, with the exception of a 15% increase in the adult hippocampus. Nerve growth factor contents followed a similar time-course and regional distribution in normal and nerve growth factor-transgenic animals and displayed significantly higher values from E14 to the early postnatal period. Nerve growth factor contents were normal in the adult brain. In vitro experiments confirmed the involvement of nerve growth factor in the decrease of choline acetyltransferase activity levels observed in transgenic neurons during development. These results suggest a role for nerve growth factor in the initial phase of the phenotypic differentiation of cholinergic neurons. They show that nerve growth factor may, under specific development conditions, lead to a paradoxical down-regulation of choline acetyltransferase activity.

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)