Blanca Cutillas

Lymphocyte populations in Parkinson's disease and in rat models of parkinsonism.

To assess the involvement of the immune system in Parkinsons disease we studied the phenotype of circulating lymphocytes in 30 untreated and 34 treated patients. We found a numeric decrease in helper T cells (higher in CD4(+)CD45RA(+) than in CD4(+)CD29(+)) and B cells, and a rise in activated, CD4(+)CD25(+) lymphocytes that was correlated with lymphocyte depletion. All these alterations were independent of levodopa treatment. In addition, we performed striatal dopamine depletion in rats with either MPP(+) or 6-OHDA, showing that MPP(+) but not 6-OHDA can increase CD4(+)CD25(+) lymphocytes. Thus, mechanisms other than dopamine deficit may explain the immune activation in Parkinsons disease.

Fas and Fas‐L expression in Huntington's disease and Parkinson's disease

The Fas/Fas‐L signalling system plays a role in the control of cell death and the survival of lymphocytes, in the regulation of the immune system, and in the progression of autoimmune diseases. Studies in the nervous system have shown Fas/Fas‐L activation in multiple sclerosis and in various paradigms leading to neuronal death. Enhanced Fas and Fas‐L expression has also been documented in astrocytomas and glioma cell lines. However, little is known about the possible implication of Fas/Fas‐L signals in primary human neurodegenerative diseases. In an attempt to gain understanding of the mechanisms commanding cell death and neurone loss in Huntingtons disease (HD) and Parkinsons disease (PD), Fas and Fas‐L expression has been examined in the brains of patients with HD and PD with Western blotting and immunohistochemistry. Fas and Fas‐L expression levels are reduced in the caudate and putamen, but not in the parietal cortex, in HD, as revealed in Western blots. Moreover, Fas and Fas‐L immunoreactivity is reduced in striatal neurones in HD. Fas and Fas‐L immunoreactivity is also decreased in neurones of the substantia nigra pars compacta in PD. Reduced Fas and Fas‐L expression is observed equally in Lewy body‐bearing and non‐Lewy body‐bearing neurones. Yet increased Fas and Fas‐L immunoreactivity occurs in normal astrocytes in control brains and in reactive astrocytes in diseased brains. The meaning of increased Fas and Fas‐L expression in astrocytes is still unclear. However, the present results suggest that Fas/Fas‐L signals are minimized in sensitive neurones in HD and PD.

Increased survival of dopaminergic neurons in striatal grafts of fetal ventral mesencephalic cells exposed to neurotrophin-3 or glial cell line-derived neurotrophic factor.

The transplantation of fetal mesencephalic cell suspensions into the brain striatal system is an emerging treatment for Parkinsons disease. However, one objection to this procedure is the relatively poor survival of implanted cells. The ability of neurotrophic factors to regulate developmental neuron survival and differentiation suggests they could be used to enhance the success of cerebral grafts. We studied the effects of neurotrophin-3 (NT-3) or glial cell line-derived neurotrophic factor (GDNF) on the survival of dopaminergic neurons from rat fetal ventral mesencephalic cells (FMCs) implanted into the rat striatum. Two conditions were tested: (a) incubation of FMCs in media containing NT-3 and GDNF, prior to grafting, and (b) co-grafting of FMCs with cells engineered to overexpress high levels of NT-3 or GDNF. One week after grafting into the rat striatum, the survival of TH+ neurons was significantly increased by pretreatment of ventral mesencephalic cells with NT-3 or GDNF. Similarly, co-graft of ventral mesencephalic cells with NT-3- or GDNF-overexpressing cells, but not the mock-transfected control cell line, increased the survival of graft-derived dopaminergic neurons. Interestingly, we also found that co-grafting of GDNF-overexpressing cells was less effective than NT-3 at improving the survival of fetal dopaminergic neurons in the grafts, and that only GDNF induced intense TH immunostaining in fibers and nerve endings of the host tissue surrounding the implant. Thus, our results suggest that NT-3, by strongly enhancing survival, and GDNF, by promoting both survival and sprouting, may improve the efficiency of fetal transplants in the treatment of Parkinsons disease.

Caspase inhibition protects nigral neurons against 6-OHDA-induced retrograde degeneration.

6-Hydroxydopamine (6-OHDA) administered intrastriatally to adult rats in a single injection causes neurodegeneration of the nigrostriatal pathway and loss of > 50% of dopamine neurons in substantia nigra pars compacta 30 days after administration. The death of nigral neurons occurs, at least partially, by a caspase-mediated mechanism. The nigral loss of dopaminergic neurons could be prevented by stereotaxical administration of zVAD.fmk, a caspase inhibitor, into the substantia nigra, indicating that 6-OHDA-induced nigrostriatal degeneration involves caspase activation. These results suggest that caspases are probably involved in neurodegenerative chronic processes such as Parkinsons disease and might be considered as possible targets in the treatment of such neurological disorders.

Chronic effects of single intrastriatal injections of 6-hydroxydopamine or 1-methyl-4-phenylpyridinium studied by microdialysis in freely moving rats

Extracellular dopamine (DA) and its main cerebral metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), were measured by bilateral striatal microdialysis in rats at different times (2, 7, 15 and 60 days) after unilateral administration into the right striatum of 1-methyl-4-phenylpyridinium ion (MPP+) or 6-hydroxydopamine (6-OHDA). In both cases the decrease in extracellular dopamine did not exceed 40% of control values. The response of DOPAC and HVA depended on the treatment: MPP+ caused a marked acute decrease in the dopamine metabolites but allowed a progressive recovery that was very evident after 60 days; 6-OHDA caused a progressive decrease in the dopamine metabolites throughout the two months of the study. Tyrosine hydroxylase immunostaining revealed severe neuronal loss in substantia nigra two months after striatal administration of 6-OHDA, whereas no significant neuronal loss was found at the same time after MPP+ administration. A bilateral challenge infusion of MPP+ through the microdialysis probe was used to assess the dopaminergic capacity of both striata: at all the times studied there was a sharp depletion of DA on the non-lesioned side; both MPP(+)- and 6-OHDA-treated striata were unresponsive after a short time (2 days); after 2 months the response in MPP(+)-lesioned rats was similar on both sides, whereas 6-OHDA-lesioned striata were still unresponsive to MPP+. In rats, then, the effects of MPP+ could be partly reversed whereas the effects of 6-OHDA were not. These results suggest that neurotoxins causing striatal dopamine loss may act through different mechanisms, which could be significant for the etiopathogenic development of Parkinsons disease.

Exposure of foetal mesencephalic cells to bone morphogenetic protein-2 enhances the survival of dopaminergic neurones in rat striatal grafts.

The transplantation of foetal mesencephalic cells (FMC) into the brain striatal system is an emerging treatment for Parkinsons disease, despite of the relatively poor survival of implanted cells. The ability of neurotrophic factors to regulate neurone survival and differentiation suggests they could be used to enhance the success of cerebral grafts. We analyzed the effect of pre-treatment of FMC suspensions with bone morphogenetic protein-2 (BMP-2) (50 ng/ml) prior to grafting into the striatum of 6-hydroxydopamine lesioned rats. The viability of a FMC suspension was enhanced in vitro by BMP-2. Four weeks after transplantation, the number of dopaminergic neurones was higher and their morphology more developed in grafts pre-treated with BMP-2, compared with non-pre-treated grafts and rats showed a significant reduction in the turning behaviour test. Thus, the pre-treatment of FMCs with BMP-2 should be considered, together with other neurotrophic factors, as a procedure for transplantational treatment of Parkinsons disease.

The targets of acetone cyanohydrin neurotoxicity in the rat are not the ones expected in an animal model of konzo

Konzo is a neurotoxic motor disease caused by excess consumption of insufficiently processed cassava. Cassava contains the cyanogenic glucoside linamarin, but konzo does not present the known pathological effects of cyanide. We hypothesized that the aglycone of linamarin, acetone cyanohydrin, may be the cause of konzo. This nitrile rapidly decomposes into cyanide and acetone, but the particular exposure and nutrition conditions involved in the emergence of konzo may favor its stabilization and subsequent acute neurotoxicity. A number of preliminary observations were used to design an experiment to test this hypothesis. In the experiment, young female Long-Evans rats were given 10mM acetone cyanohydrin in drinking water for 2 weeks, and then 20mM for 6 weeks. Nutrition deficits associated with konzo were modeled by providing tapioca (cassava starch) as food for the last 3 of these weeks. After this period, rats were fasted for 24h in order to increase endogenous acetone synthesis, and then exposed to 0 (control group) or 50 micromol/kg-h of acetone cyanohydrin for 24h (treated group) through subcutaneous osmotic minipump infusion (n=6/group). Motor activity and gait were evaluated before exposure (pre-test), and 1 and 6 days after exposure. Brains (n=4) were stained for neuronal degeneration by fluoro-jade B. Rats exposed to 50 micromol/kg-h of acetone cyanohydrin showed acute signs of toxicity, but no persistent motor deficits. Two animals showed fluoro-jade staining in discrete thalamic nuclei, including the paraventricular and the ventral reuniens nuclei; one also exhibited labeling of the dorsal endopiriform nucleus. Similar effects were not elicited by equimolar KCN exposure. Therefore, acetone cyanohydrin may cause selective neuronal degeneration in the rat, but the affected areas are not those expected in an animal model of konzo.

Neuroprotective effect of the monoamine oxidase inhibitor PF 9601N [N-(2-propynyl)-2-(5-benzyloxy-indolyl) methylamine] on rat nigral neurons after 6-hydroxydopamine-striatal lesion.

Monoamine oxidase B (MAO-B) inhibitors are potentially useful in the therapeutic treatment of Parkinsons disease. L-Deprenyl has been shown to slow nigrostriatal tract degeneration in human idiopathic Parkinsonism and to be an effective neuroprotector in experimental 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxicity models. However, L-amphetamine and (-)methamphetamine, the metabolites generated by L-deprenyl, can have adverse and severe side-effects. Therefore, the search for new MAO-B inhibitors without potential amphetamine-like properties is a matter of great therapeutic interest. The present report is the first to describe the neuroprotective effect--following chronic intraperitoneal (i.p.) treatment--of a novel and non-amphetaminic MAO-B inhibitor, [N-(2-propynyl)-2-(5-benzyloxy-indolyl) methylamine] (PF 9601N), on the neurodegeneration of nigral dopaminergic neurons caused by administration of intrastriatal 6-hydroxydopamine (6-OHDA). Two groups of six animals were unilaterally injected with 6-OHDA in the right striatum. One group was treated daily with 60 mg/kg PF 9601N i.p., starting before stereotaxic lesion and continuing for 18 days thereafter. The other group was treated with vehicle solution. Coronal slabs including the substantia nigra pars compacta (SNpc) were processed for tyrosine hydroxylase immunohistochemistry (TH). The number of TH positive (TH+) neurons in the SNpc was 60% lower in 6-OHDA lesioned rats. However, the loss of TH+ neurons in the SNpc was only 30% in PF 9601N i.p.-treated animals. Therefore, treatment with the specific MAO-B inhibitor significantly reduced the 6-OHDA-induced degeneration to about 50%.

MPP+ toxicity in rat striatal slices: relationship between non-selective effects and free radical production.

Incubations of rat striatal slices have been used to assay MPP+ neurotoxicity. MPP+, at concentrations of 1 mM or higher, caused a marked increase in hydroxyl radicals, measured as malondialdehyde (MDA) accumulation, but not in nitric oxide production. At these doses, MPP+ showed an effect on dopamine terminals, causing a massive dopamine decrease, and on non-neuronal glial cells, where a marked reduction in glutamine synthetase activity was detected. At lower concentrations (25 μM), the toxic effect on dopaminergic endings was maintained without increasing malondialdehyde concentrations or inhibiting glutamine synthetase activity. The effect on glutamine synthetase was prevented by the addition to the medium of 0.5% dimethyl sulfoxide, a hydroxyl-radical scavenger, but this did not protect the effect of dopamine depletion. We propose that non-selective effects of MPP+, at doses of 1 mM or higher, are mediated by extracellular overproduction of hydroxyl radicals. The main factor responsible for this overproduction would not be the released dopamine but rather the MPP+ itself, through non selective inhibition of the mitochondrial respiratory chain or through a redox cycling that can trigger oxygen radical production.

Intrastriatal Grafts of Fetal Mesencephalic Cell Suspensions in MPP+-Lesioned Rats: A Microdialysis Study In Vivo

The striatum of rats was lesioned by unilateral administration of MPP+. Two weeks later, a suspension of fetal mesencephalic cells (FMC), obtained from 14-day rat embryos, was injected into the lesioned striatum. Two weeks after grafting, the success of implantation and recovery of dopamine function were assessed by tyrosine hydroxylase immunocytochemistry (TH) and the measurement of striatal dopamine content. In addition, the extracellular concentrations of dopamine and dopamine metabolites were studied by microdialysis in vivo before and after perfusion of MPP+ to induce dopamine release from vesicular stores. TH+ cell bodies were seen in the lesioned grafted striata, indicating that fetal cells survived in these striata. In addition, there was a marked increase in TH-immunoreactivity in the neuronal fibers and terminals in the area surrounding the cell implant, suggesting a compensatory response of the host tissue which may involve fiber sprouting. Grafting induced a recovery in indices of dopamine function, including recovery in dopamine content, and basal and MPP+-induced dopamine release. Thus, grafts of FMC may provide a significant recovery of dopamine function in MPP+-lesioned striata.