María José Casarejos

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.

Susceptibility to rotenone is increased in neurons from parkin null mice and is reduced by minocycline

Parkinsons disease is a neurodegenerative disorder which is in most cases of unknown etiology. Mutations of the Park‐2 gene are the most frequent cause of familial parkinsonism and parkin knockout (PK‐KO) mice have abnormalities that resemble the clinical syndrome. We investigated the interaction of genetic and environmental factors, treating midbrain neuronal cultures from PK‐KO and wild‐type (WT) mice with rotenone (ROT). ROT (0.025–0.1 µm) produced a dose‐dependent selective reduction of tyrosine hydroxylase‐immunoreactive cells and of other neurons, as shown by the immunoreactivity to microtubule‐associated protein 2 in PK‐KO cultures, suggesting that the toxic effect of ROT involved dopamine and other types of neurons. Neuronal death was mainly apoptotic and suppressible by the caspase inhibitor t‐butoxycarbonyl‐Asp(OMe)‐fluoromethyl ketone (Boc‐D‐FMK). PK‐KO cultures were more susceptible to apoptosis induced by low doses of ROT than those from WT. ROT increased the proportion of astroglia and microglia more in PK‐KO than in WT cultures. Indomethacin, a cyclo‐oxygenase inhibitor, worsened the effects of ROT on tyrosine hydroxylase cells, apoptosis and astroglial (glial fibrillary acidic protein) cells. N‐nitro‐l‐arginine methyl ester, an inhibitor of nitric oxide synthase, increased ROT‐induced apoptosis but did not change tyrosine hydroxylase‐immunoreactive or glial fibrillary acidic protein area. Neither indomethacin nor N‐nitro‐l‐arginine methyl ester had any effect on the reduction by ROT of the mitochondrial potential as measured by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide. Microglial NADPH oxidase inhibition, however, protected against ROT. The roles of p38 MAPK and extracellular signal‐regulated kinase signaling pathways were tested by treatment with SB20358 and PD98059, respectively. These compounds were inactive in ROT‐naive cultures but PD98059 slightly increased cellular necrosis, as measured by lactate dehydrogenase levels, caused by ROT, without changing mitochondrial activity. SB20358 increased the mitochondrial failure and lactate dehydrogenase elevation induced by ROT. Minocycline, an inhibitor of microglia, prevented the dropout of tyrosine hydroxylase and apoptosis by ROT; the addition of microglia from PK‐KO to WT neuronal cultures increased the sensitivity of dopaminergic neurons to ROT. PK‐KO mice were more susceptible than WT to ROT and the combined effects of Park‐2 suppression and ROT reproduced the cellular events observed in Parkinsons disease. These events were prevented by minocycline.

Toxic effects of L-DOPA on mesencephalic cell cultures : protection with antioxidants

The toxicity of L-3,4-dihydroxyphenylalanine (L-DOPA) was studied in neuronal cultures from rat mesencephalon. The survival and function of DA neurons were assessed by the number of tyrosine hydroxylase-positive (TH+) cells and 3H-DA uptake and those non-DA neurons by the exclusion of Trypan blue and the high-affinity 3H-GABA uptake. L-DOPA was toxic for both DA and non-DA neurons. DA neurons were more severely affected than non-DA neurons after short periods of treatment and with exposure to a low dose of L-DOPA (25 vs. 100 microM) and less selectively affected after 1 or 2 days of treatment. After incubation with L-DOPA, a disruption of the neuritic network and an overall deterioration were observed, more evident for TH+ cells in the whole culture. Auto-oxidation to quinones is responsible in part for L-DOPA toxicity in non-DA neurons since the levels of quinones correlated well with the severity of cell death in the cultures. The damage of DA neurons took place before the rising of quinones, suggesting that quinones are not essential in L-DOPA toxicity for DA neurons. Antioxidants, such as ascorbic acid and sodium metabisulfite, completely prevented L-DOPA-induced quinone formation as well as the death of non-DA neurons. In contrast, they could only partially prevent the damage produced by L-DOPA in DA neurons. Mazindol, a selective inhibitor of DA uptake, protected TH+ cells from L-DOPA.

Glia protect fetal midbrain dopamine neurons in culture from L-DOPA toxicity through multiple mechanisms

SummaryMesencephalic glia produce soluble factors that protect dopamine neurons from L-DOPA toxicity. The chemical composition of these soluble factors is unknown. We investigated the protective effect against L-DOPA neurotoxicity in midbrain dopamine neurons of fractions of different molecular size of glia conditioned medium and candidate neuroprotective agents produced by glia including neurotrophic factors and antioxidants. Protective effects were evaluated according to the number of tyrosine hydroxylase immunoreactive cells, high affinity dopamine uptake and levels of quinones. Both fractions of glia conditioned medium, smaller and larger than 10kD, protected against L-DOPA, but the fraction of smaller molecular size, that contains small free radical scanvenger molecules, was more effective than the fraction of larger molecular size, that contains large neurotrophic peptides. Among the neurotrophic factors GDNF and BDNF totally prevented L-DOPA neurotoxicity, while NGF and bFGF were less effective. However, only NGF significantly reduced the elevation of quinones induced by L-DOPA. Ascorbic acid, at the concentration found in glia conditioned medium, provided partial protective effect against L-DOPA toxicity. Glutathione, had neurotrophic effects on untreated midbrain dopamine neurons and prevented the effect of L-DOPA. In conclusion, the protective effect against L-DOPA neurotoxicity by glia conditioned medium is mediated by several compounds including neurotrophic factors and small antioxidants.

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 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.

The accumulation of neurotoxic proteins, induced by proteasome inhibition, is reverted by trehalose, an enhancer of autophagy, in human neuroblastoma cells

Neurodegenerative diseases like Parkinsons disease, Alzheimers disease, Huntingtons disease and others are due to accumulation of abnormal proteins which fold improperly and impair neuronal function. Accumulation of these proteins could be achieved by several mechanisms including mutation, overproduction or impairment of its degradation. Inhibition of the normal protein degradation is produced by blockade of the ubiquitin proteasome system. We have shown that epoxomicin, a proteasome inhibitor, increases the levels of proteins involved in neurodegenerative disorders such as α-synuclein and hyper phosphorylated tau in NB69 human neuroblastoma cells and that such increase correlates with an enhanced rate of cell death. We then investigated whether the stimulation of autophagy, an alternative mechanism for elimination of abnormal proteins, by treatment with trehalose, counteracts the effects of proteasomal blockade. Trehalose, a disaccharide present in many non-mammalian species, known to enhance autophagy, protects cells against various environmental stresses. Treatment with trehalose produced a dose and time-dependent increase in the number of autophagosomes and markers of autophagy in NB69 cells. Trehalose did not change the number of total neither the number of dividing cells in the culture but it completely prevented the necrosis of NB69 induced by epoxomicin. In addition, the treatment with trehalose reverted the accumulation, induced by epoxomicin, of polyubiquitinated proteins, total and phosphorylated tau, p-GSK-3, and α-synuclein, as well as the α-synuclein intracellular aggregates. The effects of trehalose were not mediated through activation of free radical scavenging compounds, like GSH, or mitochondrial proteins, like DJ1, but trehalose reduced the activation of ERK and chaperone HSP-70 induced by epoxomicin. Inhibition of ERK phosphorylation prevented the epoxomicin-induced cell death. Inhibition of autophagy reverted the neuroprotective effects of trehalose in epoxomicin-induced cell death. These results suggest that trehalose is a powerful modifier of abnormal protein accumulation in neurodegenerative diseases.

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.

Glutathione depletion switches nitric oxide neurotrophic effects to cell death in midbrain cultures: implications for Parkinson's disease

Nitric oxide (NO) exerts neurotrophic and neurotoxic effects on dopamine (DA) function in primary midbrain cultures. We investigate herein the role of glutathione (GSH) homeostasis in the neurotrophic effects of NO. Fetal midbrain cultures were pretreated with GSH synthesis inhibitor, l‐buthionine‐(S,R)‐sulfoximine (BSO), 24 h before the addition of NO donors (diethylamine/nitric oxide‐complexed sodium and S‐nitroso‐N‐acetylpenicillamine) at doses tested previously as neurotrophic. Under these conditions, the neurotrophic effects of NO disappeared and turned on highly toxic. Reduction of GSH levels to 50% of baseline induced cell death in response to neurotrophic doses of NO. Soluble guanylate cyclase (sGC) and cyclic GMP‐dependent protein kinase (PKG) inhibitors protected from cell death for up to 10 h after NO addition; the antioxidant ascorbic acid also protected from cell death but its efficacy decreased when it was added after NO treatment (40% protection 2 h after NO addition). The pattern of cell death was characterized by an increase in chromatin condensed cells with no DNA fragmentation and with breakdown of plasmatic membrane. The inhibition of RNA and protein synthesis and of caspase activity also protected from cell death. This study shows that alterations in GSH levels change the neurotrophic effects of NO in midbrain cultures into neurotoxic. Under these conditions, NO triggers a programmed cell death with markers of both apoptosis and necrosis characterized by an early step of free radicals production followed by a late requirement for signalling on the sGC/cGMP/PKG pathway.