Molecular pathways involved in the neurotoxicity of 6-OHDA, dopamine and MPTP: contribution to the apoptotic theory in Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by a preferential loss of the dopaminergic neurons of the substantia nigra pars compacta. Although the etiology of PD is unknown, major biochemical processes such as oxidative stress and mitochondrial inhibition are largely described. However, despite these findings, the actual therapeutics are essentially symptomatical and are not able to block the degenerative process. Recent histological studies performed on brains from PD patients suggest that nigral cell death could be apoptotic. However, since post-mortem studies do not allow precise determination of the sequence of events leading to this apoptotic cell death, the molecular pathways involved in this process have been essentially studied on experimental models reproducing the human disease. These latter are created by using neurotoxic compounds such as 6-hydroxydopamine (6-OHDA), 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or dopamine (DA). Extensive study of these models have shown that they mimick, in vitro and in vivo, the histological and/or the biochemical characteristics of PD and thus help to define important cellular actors of cell death presumably critical for the nigral degeneration. This review reports recent data concerning the biochemical and molecular apoptotic mechanisms underlying the experimental models of PD and correlates them to the phenomena occurring in human disease.

Immunodetection of endogenous opioid peptides in human brain tumors and associated cyst fluids.

The antitumorigenic effects of endogenous opioid peptides and their presence in extracerebral tumors are well documented. In this study, methionine-enkephaline (met-enkephalin) was measured by radioimmunoassay in 108 glial and nonglial brain tumors and in 44 associated cyst fluids. By immunohistochemistry, the distribution of the peptide and its precursor, preproenkephalin A, was also analyzed. Met-enkephalin and preproenkephalin were detected in the cytoplasm and cell processes of all tumors. Moreover, for neuroectodermal tumors (i.e., gliomas, gangliogliomas, and dysembryoplastic neuroepithelial tumors), a strong inverse correlation (P < 0.0001) was observed between the met-enkephalin levels and the degree of malignancy (242.9, 148.3, 55.3, and 30.3 pg/mg protein for grade 1, 2, 3, and 4, respectively). When compared to normal tissue, this differential expression mainly results from a decrease in the opioid peptide content in high-grade neuroectodermal tumors. Meningiomas and cerebral metastases displayed low met-enkephalin levels, similar to those of grade 4 neuroectodermal tumors. Large amounts of met-enkephalin were found in all cyst fluids. These data suggest that the endogenous opioid system is an integral component of brain tumors and that met-enkephalin may represent a useful malignancy marker in neuroectodermal tumors.

Increased bax expression is associated with cell death induced by ganciclovir in a herpes thymidine kinase gene-expressing glioma cell line.

The herpes simplex virus thymidine kinase gene (HSV-tk) was stably transfected into rat C6 glioma cells (C6tk) in order to characterize the mechanisms underlying cell toxicity induced in vitro by the guanosine analog ganciclovir (GCV). The results demonstrate the efficiency of the HSV-tk/GCV system in ablating most of the tumoral cells within 7 to 8 days of treatment with 20 mivroM GCV; however, a few cells still survive. C6tk cells arrest in the S phase of the cell cycle after 2 days of drug treatment before undergoing cell death. Microscopic analysis reveals dying cells with ultrastructural characteristics consistent with apoptosis; we cannot rule out, however, that necrotic cell death may also be occurring. The cytotoxicity induced by GCV is not associated with changes in the expression of p53 protein, suggesting that cell cycle arrest and cell death may occur through a p53-independent pathway. C6tk cells constitutively express Bcl-xL and Bax proteins; when exposed to GCV, Bcl-xL levels do not change but Bax accumulation is rapidly induced. These findings suggest that the balance between Bcl-xL and Bax proteins may be of importance in determining the sensitivity of tumoral cells to GCV.

Identification and characterization of an anti-tyrosine kinase factor in cystic gliomas.

In view of the frequent activation of the epidermal growth factor receptor (EGF-R) in gliomas and autocrine hypothesis, we searched for 'EGF-like' factor(s) in cystic fluids (CFs) associated with gliomas. Membranes of A431 cells, which overexpress EGF-R, were used to explore such activity in 20 CFs. In all cases CFs induced inhibition of EGF-R phosphorylation. Biochemical analysis revealed an anti-tyrosine kinase activity which was identified as a 18 kDa proteic factor. Effectiveness at high dilution and anti-proliferative effect on living cells in culture suggest that this factor may be involved in the negative regulation of glial oncogenesis.

6-hydroxydopamine-induced nuclear factor-kappa B activation in PC12 cells.

The involvement of nuclear Factor-kappa B (NF-kappa B) transcription factor in PC12 cell death triggered by the dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA) was investigated. Results show that oxidative stress generated by 6-OHDA activates NF-kappa B. When the NF-kappa B activation was inhibited by parthenolide, PC12 cell death induced by 6-OHDA was significantly increased, thus suggesting an involvement of this transcription factor in a protective mechanism against 6-OHDA toxicity. To further assess this hypothesis, we studied the involvement of NF-kappa B in the protective effect of two anti-apoptotic genes, bcl-2 and bfl-1. Although Bcl-2 and Bfl-1 expression normally protects PC12 cells from 6-OHDA, parthenolide strongly decreased the beneficial effects afforded by transgene expression. These results suggest: (1) that the transcription factor NF-kappa B is likely associated with the protection of catecholaminergic PC12 cells and (2) that the protective effects afforded by bcl-2 and bfl-1 expression may be dependent on NF-kappa activation.

Evidence for dopamine D2 receptor mRNA expression by striatal astrocytes in culture: in situ hybridization and polymerase chain reaction studies.

The expression of dopamine D2 receptor mRNA in cultured rat striatal and cerebellar astrocytes was examined by in situ hybridization (ISH) and polymerase chain reaction (PCR). Cells double-labelled for glial fibrillary acidic protein (GFAP) immuno-histochemistry and dopamine D2 receptor mRNA (ISH) provide evidence that striatal but not cerebellar astrocytes express the dopamine D2 gene in vitro. These results were confirmed by polymerase chain reaction studies. As judged by GFAP immunostaining and morphology of the cells, this gene is almost exclusively expressed by astrocytes type 1. The expression of dopamine D2 receptor mRNA by striatal astrocytes in vitro, as found in this study, brings thus evidences for the existence of dopamine D2 receptors in such glial cells. This had been previously suggested from ligand binding studies but the typical dopaminergic nature of the binding to striatal astrocytes was left questionable. Our results with molecular biological techniques thus suggest that striatal dopamine might modulate the functions of striatal astrocytes.

Met-enkephalin receptors in human gliomas.

Endogenous opioid systems (opioid peptides and receptors) are involved in many functions including the regulation of cell growth. We investigated the presence of Met-enkephalin binding sites in gliomas by displacement assays. Results demonstrated that few gliomas exhibit Met-enkephalin binding sites and that the percentage of tumours which express these binding sites strongly decreases with increasing malignancy. Moreover, we observed a shift from mu Met-enkephalin binding sites in low grade gliomas to delta Met-enkephalin binding sites in high grade gliomas. These results suggest an inactivation of the Met-enkephalinergic system in gliomas which could lead to loss of the inhibitory effect exerted by Met-enkephalin on normal astrocyte growth and thus favour progression of malignancy.

p53 and Bax activation in 6-hydroxydopamine-induced apoptosis in PC12 cells.

p53, Bax and Bcl-xL proteins have been implicated in apoptotic neuronal cell death. We have investigated whether those proteins are involved in 6-OHDA-induced PC12 cell death. After a 24-h exposure to the neurotoxin (100 microM), morphological evidence for apoptosis was observed in PC12 cells. Up-regulation of p53 and Bax proteins was demonstrated 4 and 6 h, respectively, after 6-OHDA treatment; in contrast, no change in Bcl-xL levels was found. These findings suggest that p53 and Bax could be relevant markers of neuronal apoptosis as previously described in kainic acid- or ischemia-induced neuronal cell death and may participate to neuronal degeneration in Parkinson's disease.

Influence of glycosaminoglycans on neurite morphology and outgrowth patterns in vitro.

The neuritic growth patterns obtained on substrates made of several glycosaminoglycans (GAGs) bound to type I collagen were analysed and compared in primary cultures of chick embryo dorsal root ganglion grown in serum-free supplemented medium. In 2-day cultures grown on type I collagen or heparan sulphate (HS)-collagen surfaces, ganglionic explants exhibit a dense, symmetrical network of long, parallel neuritic processes and very few flat migrating non-neuronal cells. In contrast, on either dermatan sulphate (DS), chondroitin-6-sulphate (C6S) or hyaluronic acid (HA)-bound collagen substrates, neurons form irregular nerve fibre patterns; indeed, neurites follow convoluted paths and often, after abrupt turns, totally reverse their direction of extension. Experiments were carried out in which a choice was given to growing neural processes between collagen or GAG-collagen substrates. While growth cones elongating over type I collagen easily cross the border with HS-bound collagen surface and indiscriminately extend on this substrate, in contrast, neurites generally avoid surfaces coated with DS, C6S or HA and change their direction of growth in order to stay on collagen. The binding of DS, C6S or HA, but not HS, to type I collagen thus decreases its ability to promote neurite elongation. The interaction of neuronal cells with these extracellular matrix components by restricting neurites in their paths of extension may, therefore, play a role in the patterning of the nervous circuitry.

Effects of tunicamycin on the avoidance reaction of epidermis by sensory neurites in co-cultures.

In 7-day chick embryo dorsal root ganglia and epidermis or dermis co-cultures, nerve fibres establish contacts with dermis while avoiding epidermis. Previous results have indicated that factor(s) secreted by epidermis could be involved in this avoidance reaction. The present study demonstrates that the avoidance reaction is abolished when epidermal cells are treated by the N-linked glycoproteins synthesis inhibitor, tunicamycin. The same result is obtained after monensin treatment. The epidermal cell viability, development and total protein secretion are not significantly affected by tunicamycin, as demonstrated by trypan blue exclusion, electron microscopy and SDS-PAGE electrophoresis after 35S-methionine labelling. It has thus been concluded that the avoidance factor is glycoproteic in nature. It is also suggested that this factor possibly contains chondroitin-6-sulphate moieties.

Unlike MPP+, apoptosis induced by 6-OHDA in PC12 cells is independent of mitochondrial inhibition.

The mechanisms of 6-hydroxydopamine (6-OHDA) cytotoxicity were studied in vitro using the PC12 cell line. Following a 24 h exposure, this neurotoxin induced apoptosis and a dose-dependent decrease in cell survival. The presence of monoamine oxidase inhibitors, tranylcypromine and clorgyline, together with 6-OHDA had neither synergistic nor protective effects. Unlike 1-methyl-4-phenylpyridinium (MPP+), 6-OHDA toxicity to PC12 cells remained unchanged when glycolysis was prevented by either depleting glucose from the culture medium or growing the cells in low-glucose medium containing 2-deoxy-glucose. These results suggest that the inhibition of mitochondrial respiration is not responsible for the cell death induced by 6-OHDA.

Extracellular toxicity of 6-hydroxydopamine on PC12 cells.

6-hydroxydopamine (6-OHDA) is usually thought to cross cell membrane through dopamine uptake transporters, to inhibit mitochondrial respiration and to generate intracellular reactive oxygen species. In this study, we show that the anti-oxidants catalase, glutathione and N-acetyl-cysteine are able to reverse the toxic effects of 6-OHDA. These two latter compounds considerably slow down 6-OHDA oxidation in a cell free system suggesting a direct chemical interaction with the neurotoxin. Moreover, desipramine does not protect PC12 cells and 6-OHDA is also strongly toxic towards non-catecholaminergic C6 and NIH3T3 cells. These results thus suggest that 6-OHDA toxicity on PC12 cells mainly involves an extracellular process.

Nuclear factor-kappa B activation in permanent intraluminal focal cerebral ischemia in the rat.

Nuclear factor-kappa B (NF-kappa B) is an oxidative stress responsive transcription factor known to be activated in response to transient middle cerebral artery intraluminal occlusion. Since oxidative stress activation may largely occur during reperfusion, the aim of this study was to determine if permanent middle cerebral artery intraluminal occlusion without reperfusion induces NF-kappa B activation and the relationship of NF-kappa B activation to HSP70 expression and neuronal cell death. Our results suggest that permanent intraluminal occlusion is sufficient to induce NF-kappa B activation 7 h after the onset of occlusion. Interestingly, this activation seems to occur specifically in dying neurons of the penumbra area devoid of any HSP70 neuronal immunoreactivity. These findings are consistent with the suggested protective role of HSP70 expression and suggest that NF-kappa B activation observed in the penumbra area has a role in neuronal cell death after permanent intraluminal cerebral ischemia.

[The EGF receptor pathway in human cerebral tumors]. / Etude de la voie du récepteur a l'E.G.F. dans les tumeurs cérébrales humaines.

The epidermal growth factor receptor gene is the most frequently involved proto-oncogene in human glial brain tumors, in the present series in agreement with previous reports in literature. It is therefore important to study this gene from DNA to the protein product. The vicinity of cystic fluid (C.F.) to tumor cells of the cystic wall has suggested investigation of possible "E.G.F.-like" autocrine activities in C.F. In 40% of gliomas, E.G.F.-R. gene is amplified and overexpressed. This is never observed in low grade astrocytomas. In 12% of the cases, mutations of the E.G.F.-R. gene are observed. In correlation with genomic abnormalities, E.G.F.-R. is immunoprecipitated in 40% gliomas. The basal phosphorylation of the receptor is increased in 50% gliomas. In C.F., unexpectedly, E.G.F.-R. phosphorylation inhibitory effect is observed. Its biochemical analysis suggests an anti-tyrosine kinase activity. The observation of anti-tyrosine kinase activity in C.Fs suggests the presence of negative modulatory factors of the proto-oncogene activation in tumor tissues. This could have therapeutical interest.

In vitro analysis of interactions between sensory neurons and skin: evidence for selective innervation of dermis and epidermis.

Axons from dorsal root ganglion cells cultured in a serum-free medium on poly-L-lysine or collagen substrates interact differently with dermis and epidermis. The orientation of neurite growth is not changed by encountering mesenchymal cells migrating from the outgrowth zone of a dermal explant, and neurites form close membrane associations with some dermal cells; in contrast, neurites strongly avoid epidermis and deviate around the edge of an epidermal explant. When cultures are grown on polylysine this avoidance behaviour occurs at a distance from the epidermis. It is suppressed in the presence of necrotic epidermal cells. We suggest that this avoidance is due to epidermal diffusible factor(s) which bind preferentially to polylysine. The possibility of an absence of specific recognition cues between neurites and epidermal cells is discussed.

[Development of cutaneous innervation in the chick: ultrastructural and quantitative analysis (author's transl)]. / Développement de l'innervation cutanée chez le poulet: analyse ultrastructurale et quantitative.

In the chick, at the thoracic level, the dorsal branches of spinal nerves form at 4 days of incubation (stage 25) and reach the skin between 5 1/2 and 6 days (stages 28-29). At 6 days, the growing nervous peripheral processes ("axons") form large bundles (200-1,000 fibers). At 10 days, young Schwann cells divide the bundles into groups of axons. The perineurium and endoneurium differentiate between 10 and 14 days (but epineurium is formed after hatching). At 14 days of incubation, the adult pattern of cutaneous innervation is established. At this same stage, myelogenesis begins but develops mainly after hatching : 1% of the axons is myelinated at 16 days of incubation, 4% at hatching, 40% in 6-week old chickens and 60% in adults. Thus, less than 10% of myelinated axons of the adult are already myelinated at hatching. Two modes of myelogenesis were observed: 1) early myelination, starting in the embryo around axons measuring about 1 micrometer in diameter: 2) delayed myelination, occurring in the older chickens after an increase in axon diameter. These observations suggest that there is, in the development of chick skin innervation, a critical stage (14-15 days of incubation) apparently corresponding to the stabilization of cutaneous nerve supply.

[Formation of cutaneous nerve trunks in the chick. Ultrastructural and quantitative analysis]. / Formation des troncs nerveux cutanés chez le poulet. Analyse ultrastructurale et quantitative.

In chick skin structuration of nervous trunks takes place at 14-15 days of incubation, at the time of formation of the adult pattern of cutaneous innervation. At this same stage, myelogenesis begins, but develops mainly after hatching: only 4% of axons are myelinated at hatching, 40% in 6-week old Chickens and 60% in adults. This "critical" stage (14-15 days of incubation) apparently corresponds to the stabilization of cutaneous nerve supply.

[Quantitative microcinematographic analysis of interactions between sensory nerve cells and cutaneous tissues in vitro: demonstration of a difference in the behavior of nerve fibers with respect to dermal and epidermal cells]. / Analyse microcinématographique quantitative des interactions entre cellules nerveuses sensorielles et tissus cutanés in vitro: mise en évidence d'une différence de comportement des fibres nerveuses vis-à-vis des cellules dermiques et épidermiques.

Dynamic and morphometric analysis of interactions between sensory nerve cells and cutaneous tissue of 7 day Chick embryos in co-culture show that nerve endings behave differently when interacting with dermis of epidermis. In particular, nerve fibers display a deviation reaction in the proximity of epidermis.

Differentiation of pure chick embryo epidermis grown in primary serum-free culture.

The differentiation of precocious embryonic epidermis in serum-free primary culture was analyzed by light and electron microscopic methods. Explants of 7-day chick embryo epidermis were grown on collagen or poly-L-lysine substrates in the absence of dermal mesenchyme. The serum substitute consisted of a mixture of insulin, transferrin, putrescine and seleneous acid together with (or without) Nerve Growth Factor. These culture conditions were shown to support proliferation, growth and development (evaluated using morphological criteria) of the epidermal explants up to 4-5 days; during this period, the epidermis underwent stratification; well-developed desmosomes as well as tonofilaments were formed and the epidermis achieved a morphology close to that of 10-11 day epidermis in ovo. However long-term survival of the explants was not obtained as cellular death, starting on day 5, progressively led to the necrosis of most parts of the explant. This morphological study demonstrates that the early phases of epidermal growth and maturation can occur to some extent in the virtual absence of dermal elements and serum factors. Chick embryo epidermal cells may thus possess the intrinsic ability to go through, at least for short periods in vitro, their differentiation programme. Then, at the onset of epidermal keratinization (12 days in ovo), they require specific exogenous factors to fully differentiate in vitro.

Involvement of a chondroitin sulfate proteoglycan in the avoidance of chick epidermis by dorsal root ganglia fibers: a study using beta-D-xyloside.

In 7-day chick embryo dorsal root ganglia and epidermis cocultures, nerve fibers avoid the epidermis. Previous studies have indicated that glycoproteic factors, secreted by epidermis, could be involved in this phenomenon. Treatment of epidermis by beta-D-xyloside, a specific proteoglycan synthesis inhibitor, abolishes the avoidance reaction. The same result is obtained when anti-chondroitin sulfate antibodies are added to the culture medium. Using HPLC and 35SO4 labeling combined with chondroitinase and hyaluronidase treatment, it has been demonstrated that chondroitin sulfate is present in the epidermal conditioned medium. This suggests that a chondroitin sulfate proteoglycan secreted by the epidermis is implicated in the neurite avoidance reaction and that epidermis could therefore control its own "noninnervation". In vivo, inhibitory influences by local extracellular components may control the guidance of growth cones during nerve pattern formation.