José A. Rodríguez-Gómez

Dopamine neurons derived from embryonic stem cells function in an animal model of Parkinson's disease

Parkinsons disease is a widespread condition caused by the loss of midbrain neurons that synthesize the neurotransmitter dopamine. Cells derived from the fetal midbrain can modify the course of the disease, but they are an inadequate source of dopamine-synthesizing neurons because their ability to generate these neurons is unstable. In contrast, embryonic stem (ES) cells proliferate extensively and can generate dopamine neurons. If ES cells are to become the basis for cell therapies, we must develop methods of enriching for the cell of interest and demonstrate that these cells show functions that will assist in treating the disease. Here we show that a highly enriched population of midbrain neural stem cells can be derived from mouse ES cells. The dopamine neurons generated by these stem cells show electrophysiological and behavioural properties expected of neurons from the midbrain. Our results encourage the use of ES cells in cell-replacement therapy for Parkinsons disease.

Low selenium diet increases the dopamine turnover in prefrontal cortex of the rat

It has been proposed that interaction of catecholamines and indoleamines with free radicals may result in the formation of endogenous neurotoxins. In order to better understand the mechanisms involved in neurodegenerative disorders showing evidence of oxidative stress, we have studied the basal concentrations and the turnover rates of dopamine, noradrenaline, serotonin and their metabolites in the prefrontal cortex of rats that were fed on control or low selenium diets. Nutritional deficit of selenium decreases the brain antioxidant protection in experimental conditions by the decrease in glutathione peroxidase activity. The dopamine and serotonin turnover increased and noradrenaline and 5-hydroxy-3-indoleacetic acid turnover decreased compared to experimental control animals. The increase of dopamine turnover in experimental rats was accompanied by an increase in tyrosine hydroxylase activity. These results suggest that the decrease of brain protection against oxidative damage could induce brain damage by disturbing the turnover rate of some monoamines.

Protection of the aged substantia nigra of the rat against oxidative damage by (−)−deprenyl

1 We have studied the effect of (−)−deprenyl on the oxidative damage that the rat substantia nigra suffers during aging. 2 (−)−Deprenyl (2 mg kg−1, three times a week) administered for two months, beginning at 22 months of age, produced a significant increase in tyrosine hydroxylase (TH) activity (2.67±0.40 and 3.64±0.38 nmol mg−1 protein h−1 in untreated aged rats and treated aged rats respectively, P<0.05) and in TH amount (0.072±0.012 and 0.128±0.38 absorbance 405 nm in untreated aged and treated aged rats respectively, P<0.05). 3 The proteins of aged rat substantia nigra showed a significant decrease of carbonyl groups in treated animals compared with saline‐injected control rats (136.2±21.8 and 71.5±13.2 c.p.m. μg−1 protein in untreated aged and treated aged rats respectively, P<0.05). 4 The carbonyl groups measured in TH enzyme showed a statistically significant decrease (42.3%) after (−)−deprenyl treatment (471.4±73.0 and 271.9±50.00 c.p.m. in untreated aged and treated aged rats respectively, P<0.001). 5 All these results suggest that oxidative damage produced during aging is prevented by (−)−deprenyl treatment and could explain the effect of this drug in Parkinsons disease (PD) and other degenerative diseases such as Alzheimers disease.

Deprenyl induces the tyrosine hydroxylase enzyme in the rat dopaminergic nigrostriatal system

Chronic treatment of aged rats with deprenyl prevents age-induced protein oxidation in substantia nigra and protects tyrosine hydroxylase (TH) enzyme against inactivation [11]. With these precedents, we treated adult rats with deprenyl for 3 weeks in order to get further insight in the mechanism by which deprenyl exerts such actions. After completing the treatment, dopamine (DA) levels markedly increased in both striatum and substantia nigra while levels of the acid DA metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), decreased in the two brain areas, thus proving MAO-inhibiting properties of the treatment. We then studied the cellular expression of TH mRNA by in situ hybridization. Following treatment with deprenyl, levels of TH mRNA were significantly higher in individual dopaminergic nigral cell bodies than in those of control rats (+74%). Western blotting analysis of TH enzyme amount revealed a positive effect of the treatment in both the terminal field (+44%) and the cell body region (+31%). This correlation between TH mRNA and amount was also extended to TH enzyme activity in the two brain areas studied, which significantly increased in striatum (+57%) and substantia nigra (+35%) following deprenyl treatment. Taken together, our results clearly suggest a TH-inducing effect of deprenyl in the dopaminergic nigrostriatal system, which seems to be independent of its protective action against oxidative stress described previously. These results expand our knowledge about the beneficial effect of deprenyl in the therapy of Parkinsons disease.

T-type Ca2+ channels in mouse embryonic stem cells: modulation during cell cycle and contribution to self-renewal

Ion channels participate in cell homeostasis and are involved in the regulation of proliferation and differentiation in several cell types; however, their presence and function in embryonic stem (ES) cells are poorly studied. We have investigated the existence of voltage-dependent inward currents in mouse ES cells and their ability to modulate proliferation and self-renewal. Patch-clamped ES cells had inactivating tetrodotoxin (TTX)-sensitive Na(+) currents as well as transient Ca(2+) currents abolished by the external application of Ni(2+). Biophysical and pharmacological data indicated that the Ca(2+) current is predominantly mediated by T-type (Ca(v)3.2) channels. The number of cells expressing T-type channels and Ca(v)3.2 mRNA levels increased at the G1/S transition of the cell cycle. TTX had no effect on ES cell proliferation. However, blockade of T-type Ca(2+) currents with Ni(2+) induced a decrease in proliferation and alkaline phosphatase positive colonies as well as reduced expression of Oct3/4 and Nanog, all indicative of loss in self-renewal capacity. Decreased alkaline phosphatase and Oct3/4 expression were also observed in cells subjected to small interfering RNA-induced knockdown for T-type (Ca(v)3.2) Ca(2+) channels, thus partially recapitulating the pharmacological effects on self-renewal. These results indicate that Ca(v)3.2 channel expression in ES cells is modulated along the cell cycle being induced at late G1 phase. They also suggest that these channels are involved in the maintenance of the undifferentiated state of mouse ES cells. We propose that Ca(2+) entry mediated by Ca(v)3.2 channels might be one of the intracellular signals that participate in the complex network responsible for ES cell self-renewal.

The effect of age on the monoamines of the hypothalamus

Measurement of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) homovanillic acid (HVA), 3-methoxytyramine (3-MT), noradrenaline (NA), 3-methoxy-4-hydroxyphenyl glycol (MHPG) and serotonin (5-HT) and its main metabolite, 5-hydroxyindol-3-acetic acid (5-HIAA) was assessed in hypothalamus and median eminence of aged rats. Age-related changes were not observed in the concentration of NA and its metabolites in median eminence. In contrast, there was a significant NA decrease in aged hypothalamus compared with 12 months (no differences were found compared with 3 months). No significant differences were found in DA concentration and its metabolites in hypothalamus but DA decreased significantly in aged median eminence compared with 12 months. The ratio 5-HIAA/5-HT, indicative of 5-HT turnover, appeared to increase in the hypothalamus and median eminence of the aged rat. Morphological dissimilarities between hypothalamus of young and aged rats were demonstrated using serotonin-immunocytochemistry. A degeneration of the serotoninergic system, denoted by the appearance of enlarged or swollen varicosities, was observed in the hypothalamus of the aged rat. These aberrant serotoninergic fibers may reflect the local degeneration of serotoninergic hypothalamic afferents during ageing. Such differential age-dependent alterations of the serotoninergic system might be responsible for at least some of the functional deficits in aged animals.

Chronic and progressive Parkinson's disease MPTP model in adult and aged mice.

Despite the different animal models of Parkinsons disease developed during the last years, they still present limitations modelling the slow and progressive process of neurodegeneration. Here, we undertook a histological, neurochemical and behavioural analysis of a new chronic parkinsonian mouse model generated by the subcutaneous administration of low doses of MPTP (20 mg/kg, 3 times per week) for 3 months, using both young adult and aged mice. The MPTP‐induced nigrostriatal neurodegeneration was progressive and was accompanied by a decrease in striatal dopamine levels and motor impairment. We also demonstrated the characteristic neuroinflammatory changes (microglial activation and astrogliosis) associated with the neurodegenerative process. Aged animals showed both a faster time course of neurodegeneration and an altered neuroinflammatory response. The long‐term systemic application of low MPTP doses did not induce any increase in mortality in either young adult or aged mice and better resembles the slow evolution of the neurodegenerative process. This treatment could be useful to model different stages of Parkinsons disease, providing a better understanding of the pathophysiology of the disease and facilitating the testing of both protective and restorative treatments.

CHRONIC INHIBITION OF THE HIGH-AFFINITY DOPAMINE UPTAKE SYSTEM INCREASES OXIDATIVE DAMAGE TO PROTEINS IN THE AGED RAT SUBSTANTIA NIGRA

The effect of chronic treatment of aged rats with nomifensine has been studied in the rat nigrostriatal dopaminergic system. The rat substantia nigra suffers an oxidative damage during aging that results in both an increase in carbonyl groups of its total proteins and the oxidative inactivation of tyrosine hydroxylase (TH) enzyme, which are partially reversed by chronic treatment with deprenyl. Different mechanisms may account for this effect, including inhibition of the high-affinity dopamine uptake system. We treated aged rats chronically with nomifensine for 2 months and found some significant effects. Nomifensine treatment significantly increased TH enzyme amount in substantia nigra (39.2%), which was accompanied by a significant increase in TH enzyme activity (47.8%). However, these effects were not observed in the terminal field (striatum). As a further step we quantified the oxidative level of proteins by measuring the number of carbonyl groups coupled either to total proteins or specifically to TH enzyme. The proteins of aged rat substantia nigra showed a significant increase of carbonyl groups following nomifensine treatment. The number of carbonyl groups coupled to nigral TH enzyme also increased in the nomifensine-treated animals. However, this increase was lower than that found in the total homogenate proteins. All these results show that the oxidative damage produced during aging in tyrosine hydroxylase enzyme and total proteins is not reduced by nomifensine treatment. On the contrary, the nomifensine treatment increased the oxidative damage to proteins. These results suggest the capability of deprenyl to induce TH enzyme could be due to inhibition of the high-affinity dopamine uptake system, but its ability to protect against oxidative damage is not produced by this mechanism.

(-)-Deprenyl treatment restores serum insulin-like growth factor-I (IGF-I) levels in aged rats to young rat level.

We studied the effects of treatment with (-)-deprenyl, a monoamine oxidase B inhibitor, on plasma levels of insulin-like growth factor-I (IGF-I) (as indicator of growth hormone (GH) secretion), levels of monoamines and their metabolites, and the activity and content of tyrosine hydroxylase - the rate-limiting enzyme in the biosynthesis of catecholamines - in the hypothalamus and hypophysis of old male rats. Male Wistar rats (22 months old) were treated with 2 mg deprenyl/kg body weight s.c. three times a week for 2 months. At the end of the treatment period, blood was collected for measurement of plasma IGF-I levels by radioimmunoassay (RIA). The concentrations of dopamine, serotonin (5-HT) and their main metabolites were determined by high performance liquid chromatography (HPLC) with electrochemical detection, and the tyrosine hydroxylase content in hypothalamus and hypophysis was determined by enzyme-linked immunoabsorbent assay (ELISA). (-)-Deprenyl treatment produced a pronounced increase in dopamine and 5-HT in both the hypothalamus and hypophysis (P < 0.01). The main dopaminergic metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), decreased in hypothalamus but not in hypophysis, and treatment had no effect on the concentration of 5-hydroxyindole-3-acetic acid (5-HIAA). The tyrosine hydroxylase activity and tyrosine hydroxylase content increased in hypothalamus and hypophysis (P < 0.05). In the hypophysis the increase in tyrosine hydroxylase activity was consistent with the increase in tyrosine hydroxylase amount. Moreover, (-)-deprenyl treatment restored the IGF-I plasma levels in old rats to a concentration similar to those found in young animals. Postulated anti-aging effects of (-)-deprenyl could hence be due to restoration of hypothalamic hormones such as GH.

Decreased messenger RNA expression of key markers of the nigrostriatal dopaminergic system following vitamin E deficiency in the rat

We have evaluated the effect of a vitamin E-deficient diet on the rat nigrostriatal dopaminergic system. After 15 days of deficient diet, the amount and activity of striatal and nigral tyrosine hydroxylase increased, which contrasted with a decreased messenger RNA expression for tyrosine hydroxylase and the dopamine transporter in the ventral mesencephalon. When we prolonged the deficiency of vitamin E for 30 days, dopamine levels did not differ in both areas. In contrast, messenger RNA levels for tyrosine hydroxylase and the dopamine transporter were markedly reduced in 30-day deficient rats. In addition, the number of oxidatively modified proteins significantly increased in the striatal and nigral areas studied. Overall, we propose that these changes suggest an important role of vitamin E in maintaining the normal equilibrium of the dopaminergic nigrostriatal system.