María Angeles Mena

Levodopa in the treatment of Parkinson's disease: Current controversies

Levodopa is the most effective symptomatic agent in the treatment of Parkinsons disease (PD) and the “gold standard” against which new agents must be compared. However, there remain two areas of controversy: (1) whether levodopa is toxic, and (2) whether levodopa directly causes motor complications. Levodopa is toxic to cultured dopamine neurons, and this may be a problem in PD where there is evidence of oxidative stress in the nigra. However, there is little firm evidence to suggest that levodopa is toxic in vivo or in PD. Clinical trials have not clarified this situation. Levodopa is also associated with motor complications. Increasing evidence suggests that they are related, at least in part, to the short half‐life of the drug (and its potential to induce pulsatile stimulation of dopamine receptors) rather than to specific properties of the molecule. Treatment strategies that provide more continuous stimulation of dopamine receptors provide reduced motor complications in MPTP monkeys and PD patients. These studies raise the possibility that more continuous and physiological delivery of levodopa might reduce the risk of motor complications. Clinical trials to test this hypothesis are underway. We review current evidence relating to these areas of controversy.

Trehalose ameliorates dopaminergic and tau pathology in parkin deleted/tau overexpressing mice through autophagy activation.

Tauopathies are neurodegenerative diseases, sporadic or familial, mainly characterized by dementia and parkinsonism associated to atrophy of the frontotemporal cortex and the basal ganglia, with deposition of abnormal tau in brain. Hereditary tauopathies are related with mutations of the tau gene. Up to the present, these diseases have not been helped by any disease-modifying treatment, and patients die a few years after the onset of symptoms. We have developed and characterized a mouse model of tauopathy with parkinsonism, overexpressing human mutated tau protein with deletion of parkin (PK(-/-)/Tau(VLW)). At 3 months of age, these mice present abnormal dopamine-related behavior, severe dropout of dopamine neurons in the ventral midbrain, reduced dopamine levels in the striatum and abundant phosphorylated tau-positive neuritic plaques, neurofibrillary tangles, astrogliosis, and, at 12 months old, plaques of murine beta-amyloid in the hippocampus. Trehalose is a natural disaccharide that increases the removal of abnormal proteins through enhancement of autophagy. In this work, we tested if 1% trehalose in the drinking water reverts the PK(-/-)/Tau(VLW) phenotype. The treatment with trehalose of 3-month-old PK(-/-)/Tau(VLW) mice for 2.5 months reverted the dropout of dopamine neurons, which takes place in the ventral midbrain of vehicle treated PK(-/-)/Tau(VLW) and the reduced dopamine-related proteins levels in the midbrain and striatum. The number of phosphorylated tau-positive neuritic plaques and the levels of phosphorylated tau decreased, as well as astrogliosis in brain regions. The autophagy markers in the brain, the autophagic vacuoles isolated from the liver, and the electron microscopy data indicate that these effects of trehalose are mediated by autophagy. The treatment with trehalose for 4 months of 3-month-old PK(-/-)/Tau(VLW) mice maintained the amelioration of the tau pathology and astrogliosis but failed to revert DA-related pathology in the striatum. Furthermore, the 3-week treatment with trehalose of 14-month-old PK(-/-)/Tau(VLW) mice, at the limit of their life expectancy, improved the motor behavior and anxiety of these animals, and reduced their levels of phosphorylated tau and the number of murine beta-amyloid plaques. Trehalose is neuroprotective in this model of tauopathy. Since trehalose is free of toxic effects at high concentrations, this study opens the way for clinical studies of the effects of trehalose in human tauopathies.

Role of extracellular signal‐regulated protein kinase in neuronal cell death induced by glutathione depletion in neuron/glia mesencephalic cultures

To date, glutathione (GSH) depletion is the earliest biochemical alteration shown in brains of Parkinsons disease patients, but the role of GSH in dopamine cell survival is debated. In this study we show that GSH depletion, produced with GSH synthesis inhibitor, l‐buthionine‐(S,R)‐sulfoximine (BSO), induces selectively neuronal cell death in neuron/glia, but not in neuronal‐enriched midbrain cultures and that cell death occurs with characteristics of necrosis and apoptosis. BSO produces a dose‐ and time‐dependent generation of reactive oxygen species (ROS) in neurons. BSO activates extracellular signal‐regulated kinases (ERK‐1/2), 4 and 6 h after treatment. MEK‐1/2 and lipoxygenase (LOX) inhibitors, as well as ascorbic acid, prevent ERK‐1/2 activation and neuronal loss, but the inhibition of nitric oxide sintase (NOS), cyclo‐oxygenase (COX), c‐Jun N‐terminal kinase (JNK) and p38 mitogen‐activated protein kinase (p38 MAPK) does not have protective effects. Co‐localization studies show that p‐ERK‐1/2 expression after BSO treatment increased in astrocytes and microglial cells, but not in neurons. Selective metabolic impairment of glial cells with fluoroacetate decreased ERK activation. However, blockade of microglial activation with minocycline did not. Our results indicate that neuronal death induced by GSH depletion is due to ROS‐dependent activation of the ERK‐1/2 signalling pathway in glial cells. These data may be of relevance in Parkinsons disease, where GSH depletion and glial dysfunction have been documented.

Glial Cells as Players in Parkinsonism: The “Good,” the “Bad,” and the “Mysterious” Glia

The role of glia in Parkinsons disease (PD) is very interesting because it may open new therapeutic strategies in this disease. Traditionally it has been considered that astrocytes and microglia play different roles in PD: Astroglia are considered the “good” glia and have traditionally been supposed to be neuroprotective due to their capacity to quench free radicals and secrete neurotrophic factors, whereas microglia, considered the “bad” glia, are thought to play a critical role in neuroinflammation. The proportion of astrocytes surrounding dopamine (DA) neurons in the substantia nigra, the target nucleus for neurodegeneration in PD, is the lowest for any brain area, suggesting that DA neurons are more vulnerable in terms of glial support than any neuron in other brain areas. Astrocytes are critical in the modulation of the neurotoxic effects of many toxins that induce experimental parkinsonism and they produce substances in vitro that could modify the effects of L-DOPA from neurotoxic to neurotrophic. There is a great interest in the role of inflammation in PD, and in the brains of these patients there is evidence for microglial production of cytokines and other substances that could be harmful to neurons, suggesting that microglia of the substantia nigra could be actively involved, primarily or secondarily, in the neurodegeneration process. There is, however, evidence in favor of the role of neurotoxic diffusible signals from microglia to DA neurons. More recently a third glial player, oligodendroglia, has been implicated in the pathogenesis of PD. Oligodendroglia play a key role in myelination of the nervous system. Recent neuropathological studies suggested that the nigrostriatal dopamine neurons, which were considered classically as the primary target for neurodegeneration in PD, degenerate at later stages than other neurons with poor myelination. Therefore, the role of oligodendroglia, which also secrete neurotrophic factors, has entered the center of interest of neuroscientists.

Drug-induced parkinsonism

Drug-induced parkinsonism (DIP) is the second cause of akinetic rigid syndrome in the Western world and its prevalence is increasing and approaching that of idiopathic Parkinson’s disease due to the ageing of the population and to the rising of polypharmacotherapy. DIP was initially reported as a complication of neuroleptics in psychiatric patients, but it has also been described with a great diversity of compounds such as antiemetics, drugs used for the treatment of vertigo, antidepressants, calcium channel antagonists, antiarrythmics, antiepileptics, cholinomimetics and other drugs. Although traditionally considered reversible, DIP may persist after drug withdrawal. At least 10% of patients with DIP develop persistent and progressive parkinsonism in spite of the discontinuation of the causative drug. Irreversible or progressive DIP has been considered as an indication of presymptomatic parkinsonian deficit, unmasked but not caused by the offending drug, but it could be explained by persistent toxicity of the responsible pharmacological agents on the nigrostriatal dopamine pathway. The best treatment of DIP is prevention, including the avoidance of prescription of causative drugs whenever it is not strictly necessary. In patients who require potentially risky medication, it is necessary to perform adequate monitoring for early parkinsonian deficits and early discontinuation if these deficits appear. Atypical neuroleptics are associated with lower risk than first generation antipsychotic drugs. Special precautions are needed in elderly subjects, in patients treated with multiple drugs for prolonged periods of time and in those with familial risk factors including familial parkinsonism or tremor, or in those with genetic variants of genes involved in idiopathic Parkinson’s disease.

Effects of Wild‐Type and Mutated Copper/Zinc Superoxide Dismutase on Neuronal Survival and l‐DOPA‐Induced Toxicity in Postnatal Midbrain Culture

Abstract: Mutations in the free radical‐scavenging enzyme copper/zinc superoxide dismutase (Cu/Zn‐SOD) are associated with neuronal death in humans and mice. Here, we examine the effects of human wild‐type (WT SOD) and mutant (Gly93→ Ala; G93A) Cu/Zn‐SOD enzyme on the fate of postnatal midbrain neurons. One‐week‐old cultures from transgenic mice expressing WT SOD enzyme had significantly more midbrain neurons and fewer necrotic and apoptotic neurons than non‐transgenic cultures. In contrast, 1‐week‐old cultures from transgenic G93A mice expressing mutant SOD enzyme had significantly fewer midbrain neurons and more necrotic and apoptotic neurons than nontransgenic cultures. To subject postnatal midbrain neurons to oxidative stress, cultures were incubated with l‐DOPA. l‐DOPA at 200 µM caused ∼50% loss of tyrosine hydroxylase (TH)‐positive neurons in nontransgenic cultures and even greater loss in transgenic G93A cultures; no alterations were noted in GABA neuron numbers. In contrast, 200 µMl‐DOPA did not cause any significant reductions in TH‐positive or GABA neuron numbers in transgenic WT SOD cultures. l‐DOPA at 50 µM had opposite effects, in that it significantly increased TH‐positive, but not GABA neuron numbers in transgenic WT SOD and G93A and in nontransgenic cultures. These results indicate that increased amounts of WT SOD enzyme promote cell survival and protect against l‐DOPA‐induced dopaminergic neurotoxicity, whereas increased amounts of mutated Cu/Zn‐SOD enzyme have inverse effects. As the spontaneous loss and l‐DOPA‐induced loss of postnatal dopaminergic midbrain neurons appear to be mediated by free radicals, our study supports the view that mutated Cu/Zn‐SOD enzyme kills cells by oxidative stress.

Glial Dysfunction in Parkin Null Mice: Effects of Aging

Parkin mutations in humans produce parkinsonism whose pathogenesis is related to impaired protein degradation, increased free radicals, and abnormal neurotransmitter release. The role of glia in parkin deficiency is little known. We cultured midbrain glia from wild-type (WT) and parkin knock-out (PK-KO) mice. After 18–20 d in vitro, PK-KO glial cultures had less astrocytes, more microglia, reduced proliferation, and increased proapoptotic protein expression. PK-KO glia had greater levels of intracellular glutathione (GSH), increased mRNA expression of the GSH-synthesizing enzyme γ-glutamylcysteine synthetase, and greater glutathione S-transferase and lower glutathione peroxidase activities than WT. The reverse happened in glia cultured in serum-free defined medium (EF12) or in old cultures. PK-KO glia was more susceptible than WT to transference to EF12 or neurotoxins (1-methyl-4-phenylpyridinium, blockers of GSH synthesis or catalase, inhibitors of extracellular signal-regulated kinase 1/2 and phosphatidylinositol 3 kinases), aging of the culture, or combination of these insults. PK-KO glia was less susceptible than WT to Fe2+ plus H2O2 and less responsive to protection by deferoxamine. Old WT glia increased the expression of heat shock protein 70, but PK-KO did not. Glia conditioned medium (GCM) from PK-KO was less neuroprotective and had lower levels of GSH than WT. GCM from WT increased the levels of dopamine markers in midbrain neuronal cultures transferred to EF12 more efficiently than GCM from PK-KO, and the difference was corrected by supplementation with GSH. PK-KO-GCM was a less powerful suppressor of apoptosis and microglia in neuronal cultures. Our data prove that abnormal glial function is critical in parkin mutations, and its role increases with aging.

Mortality, oxidative stress and tau accumulation during ageing in parkin null mice

Young parkin null (pk−/−) mice have subtle abnormalities of behaviour, dopamine (DA) neurotransmission and free radical production, but no massive loss of DA neurons. We investigated whether these findings are maintained while ageing. Pk−/− mice have reduced life span and age‐related reduced exploratory behaviour, abnormal walking and posture, and behaviours similar to those of early Parkinson’s disease (PD), reduced number of nigrostriatal DA neurons and proapoptotic shifts in the survival/death proteins in midbrain and striatum. Contrary to young pk−/− animals 24‐month‐old pk−/− mice do not have compensatory elevation of GSH in striatum, glutathione reductase (GR) and glutathione peroxidase (GPx) activities are increased and catalase unchanged. Aged pk−/− mice accumulate high levels of tau and fail to up‐regulate CHIP and HSP70. Our results suggest that aged pk−/− mice lack of the compensatory mechanisms that maintain a relatively normal DA function in early adulthood. This study could help to explain the effects of ageing in patients with genetic risks for Parkinson’s disease.

Neurotrophic and neurotoxic effects of nitric oxide on fetal midbrain cultures

There is evidence suggesting that nitric oxide (NO) may play an important role in dopamine (DA) cell death. Thus, the aim of this study was to investigate the effects of NO on apoptosis and functionality of DA neurones and glial cells. The experiments were carried out in neuronal‐enriched midbrain cultures treated with the NO donor diethylamine–nitric oxide complexed sodium (DEA–NO). DEA–NO, at doses of 25 and 50 µm, exerted neurotrophic effects on dopamine cells, increasing the number of tyrosine hydroxylase positive (TH+) cells, TH+ neurite processes, DA levels and [3H]DA uptake. A dose of 25 µm DEA–NO protected DA cells from apoptosis. In addition, it induced de novo TH synthesis and increased intracellular reduced glutathione (GSH) levels, indicating a possible neuroprotective role for GSH. However, in doses ranging from 200 to 400 µm, DEA–NO decreased TH+ cells, DA levels, [3H]DA uptake and the number of mature oligodendrocytes (O1+ cells). No changes in either the amount or morphology of astrocytes and glial progenitors were detected. A dose‐ and time‐dependent increase in apoptotic cells in the DEA–NO‐treated culture was also observed, with a concomitant increase in the proapoptotic Bax protein levels and a reduction in the ratio between Bcl‐xL and Bcl‐xS proteins. In addition, DEA–NO induced a dose‐ and time‐dependent increase in necrotic cells. 1H‐[1,2,4]oxadiazolo[4,3a]quinoxaline‐1‐one (ODQ, 0.5 µm), a selective guanylate cyclase inhibitor, did not revert the NO‐induced effect on [3H]DA uptake. Glia‐conditioned medium, obtained from fetal midbrain astrocyte cultures, totally protected neuronal‐enriched midbrain cultures from NO‐induced apoptosis and rescued [3H]DA uptake and TH+ cell number. In conclusion, our results show that low NO concentrations have neurotrophic effects on DA cells via a cGMP‐independent mechanism that may implicate up‐regulation of GSH. On the other hand, higher levels of NO induce cell death in both dopamine neurones and mature oligodendrocytes that is totally reverted by soluble factors released from glia.

The role of astroglia on the survival of dopamine neurons.

Glial cells play a key role in the function of dopamine (DA) neurons and regulate their differentiation, morphology, physiological and pharmacological properties, survival, and resistance to different models of DA lesion. Several studies suggest that glial cells may be important in the pathogenesis of Parkinson’s disease (PD), a common neurodegenerative disorder characterized by degeneration of the nigrostriatal DA system. In this disease the role of glia could be due to the excessive production of toxic products such as nitric oxide (NO) or cytokines characteristic of inflammatory process, or related to a defective release of neuroprotective agents, such as small antioxidants with free radical scavenging properties or peptidic neurotrophic factors.