Rossana Migheli

Enhancing effect of manganese on L-DOPA-induced apoptosis in PC12 cells: role of oxidative stress.

Abstract : L‐DOPA and manganese both induce oxidative stress‐mediated apoptosis in catecholaminergic PC12 cells. In this study, exposure of PC12 cells to 0.2 mM MnCl2 or 10‐20 μM L‐DOPA neither affected cell viability, determined by the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay, nor induced apoptosis, tested by flow cytometry, fluorescence microscopy, and the TUNEL technique. L‐DOPA (50 μM) induced decreases in both cell viability and apoptosis. When 0.2 mM MnCl2 was associated with 10, 20, or 50 μM L‐DOPA, a concentration‐dependent decrease in cell viability was observed. Apoptotic cell death also occurred. In addition, manganese inhibited L‐DOPA effects on dopamine (DA) metabolism (i.e., increases in DA and its acidic metabolite levels in both cell lysate and incubation medium). The antioxidant N‐acetyl‐L‐cysteine significantly inhibited decreases in cell viability, apoptosis, and changes in DA metabolism induced by the manganese association with L‐DOPA. An increase in autoxidation of L‐DOPA and of newly formed DA is suggested as a mechanism of manganese action. These data show that agents that induce oxidative stress‐mediated apoptosis in catecholaminergic cells may act synergistically.

Role of oxidative stress in the manganese and 1-methyl-4-(2′-ethylphenyl)-1,2,3,6-tetrahydropyridine-induced apoptosis in PC12 cells

Oxidative stress is thought to play a key role in the apoptotic death of several cellular systems, including neurons. Oxidative stress is proposed also as a mechanism of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)- and manganese (Mn)-induced neuronal death. We have recently shown that Mn and the MPTP analogue 1-methyl-4-(2-ethylphenyl)-1,2,3,6-tetrahydropyridine (2Et-MPTP), which is metabolized by MAO-A to 1-methyl-4-(2-ethylphenyl)-pyridinium ion, induce apoptosis in PC12 cells. In the present study, we evaluated the effects of deprenyl and the antioxidant drugs N-acetylcysteine (NAC) and ascorbic acid (AA) on Mn- and 2Et-MPTP-induced apoptosis in PC12 cells. Apoptosis was tested by terminal deoxynucleotidyl transferase-mediated 2-deoxy-uridine-5-triphosphate nick end labelling (TUNEL) technique, flow cytometry and fluorescence microscopy. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Mn-induced apoptosis and decrease in cell viability was inhibited by the antioxidants NAC and AA. Deprenyl failed to inhibit the above Mn effects. Neither NAC, AA nor deprenyl were able to inhibit both 2Et-MPTP-induced apoptosis and decrease in cell viability. These results confirm that apoptosis may be an important mechanism of cell death in MPTP- and Mn-induced parkinsonism. However, an oxidative stress mechanism may be recognized, at least in vitro, only in the Mn-induced apoptosis.

Cellular defence mechanisms in the striatum of young and aged rats subchronically exposed to manganese

A deficiency of striatal dopamine (DA) is generally accepted as an expression of manganese (Mn) toxicity in experimental animals. Since compromised cellular defence mechanisms may be involved in Mn neurotoxicity, we investigated the response of the neuronal antioxidant system [ascorbic acid (AA) oxidation, glutathione (GSH) and uric acid levels] and neurochemical changes in the striatum in aged rats exposed to Mn. Levels of dopamine (DA), dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), AA, dehydroascorbic acid (DHAA), GSH and uric acid were determined after subchronic oral exposure to MnCl2 200 mg/kg (3-month-old rats) and 30-100-200 mg/kg (20-month-old-rats). Aged rats had basal levels of striatal DA, DOPAC, HVA, 5-HT, 5-HIAA, GSH and AA lower than those of young rats. In the striatum of aged rats, Mn induced biphasic changes in the levels of DA, DOPAC, HVA (an increase at the lower dose and a decrease at the higher dose) and DHAA (opposite changes). Mn decreased GSH levels and increased uric acid levels both in the striatum and in synaptosomes in all groups of aged rats. All of these parameters were affected to a lesser extent in young rats. In conclusion, the response of cellular defence mechanisms in aged rats is consistent with a Mn-induced increase in the formation of reactive oxygen species. An age-related impairment of the neuronal antioxidant system may play an enabling role in Mn neurotoxicity.

Glucocorticoid receptor deficiency increases vulnerability of the nigrostriatal dopaminergic system: critical role of glial nitric oxide.

Glucocorticoids (GCs) exert via glucocorticoid receptors (GRs) potent anti‐inflammatory and immunosuppressive effects. Emerging evidence indicates that an inflammatory process is involved in dopaminergic nigro‐striatal neuronal loss in Parkinsons disease. We here report that the GR deficiency of transgenic (Tg) mice expressing GR antisense RNA from early embryonic life has a dramatic impact in “programming” the vulnerability of dopaminergic neurons to 1‐ methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP). The GR deficiency of Tg mice exacerbates MPTP‐induced toxicity to dopaminergic neurons, as revealed by both severe loss of tyrosine hydroxylase positive nigral neurons and sharp decreases in striatal levels of dopamine and its metabolites. In addition, the late increase in dopamine oxidative metabolism and ascorbic acid oxidative status in GR‐deficient mice was far greater than in wild‐type (Wt) mice. Inducible nitric oxide synthase (iNOS) was sharply increased in activated astrocytes, macrophages/microglia of GR‐deficient as compared with Wt mice. Moreover, GR‐deficient microglia produced three‐ to fourfold higher nitrite levels than Wt mice; these increases preceded the loss of dopaminergic function and were resistant to GR the inhibitory effect of GC, pointing to peroxynitrites as candidate neurotoxic effectors. The iNOS inhibitor N6‐(1‐ iminoethyl)‐L‐lysine normalized vulnerability of Tg mice, thus establishing a novel link between genetic impairment of GR function and vulnerability to MPTP.

Manganese and 1-methyl-4-(2′-ethylphenyl)-1,2,3,6-tetrahydropyridine induce apoptosis in PC12 cells

Oxidative stress is thought to play a key role both in the neurotoxin MPTP- and manganese (Mn)-induced neurotoxicity and in apoptotic cell death. In the present study, we report that Mn and the MPTP analogue 1-methyl-4-(2-ethylphenyl)-1,2,3,6-tetrahydropyridine (2Et-MPTP), which is metabolized by MAO-A to 1-methyl-4-(2-ethylphenyl)-pyridinium ion (at concentrations of 0.5 and 1.0 mM), induced apoptosis in PC12 cells. Apoptosis was tested by terminal deoxynucleotidyl transferase-mediated 2-deoxy-uridine-5-triphosphate nick end labelling (TUNEL) technique, flow cytometry and fluorescence microscopy. Both Mn and 2Et-MPTP induced also a time-dependent decrease in cell viability, as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Only Mn-induced apoptosis and decrease in cell viability were inhibited by the antioxidant ascorbic acid. We conclude that apoptosis may be an important mechanism of cell death in MPTP- and Mn-induced parkinsonism. However, an oxidative stress mechanism may be recognized only in the Mn-induced apoptosis.

Real-Time Monitoring of Brain Tissue Oxygen Using a Miniaturized Biotelemetric Device Implanted in Freely Moving Rats

A miniaturized biotelemetric device for the amperometric detection of brain tissue oxygen is presented. The new system, derived from a previous design, has been coupled with a carbon microsensor for the real-time detection of dissolved O(2) in the striatum of freely moving rats. The implantable device consists of a single-supply sensor driver, a current-to-voltage converter, a microcontroller, and a miniaturized data transmitter. The oxygen current is converted to a digital value by means of an analog-to-digital converter integrated in a peripheral interface controller (PIC). The digital data is sent to a personal computer using a six-byte packet protocol by means of a miniaturized 434 MHz amplitude modulation (AM) transmitter. The receiver unit is connected to a personal computer (PC) via a universal serial bus. Custom developed software allows the PC to store and plot received data. The electronics were calibrated and tested in vitro under different experimental conditions and exhibited high stability, low power consumption, and good linear response in the nanoampere current range. The in vivo results confirmed previously published observations on oxygen dynamics in the striatum of freely moving rats. The system serves as a rapid and reliable model for studying the effects of different drugs on brain oxygen and brain blood flow and it is suited to work with direct-reduction sensors or O(2)-consuming biosensors.

On the mechanism of d-amphetamine-induced changes in glutamate, ascorbic acid and uric acid release in the striatum of freely moving rats

The effects of systemic, intrastriatal or intranigral administration of d‐amphetamine on glutamate, aspartate, ascorbic acid (AA), uric acid, dopamine (DA), dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5‐hydroxyindoleacetic acid (5‐HIAA) concentrations in dialysates from the striatum of freely‐moving rats were evaluated using microdialysis. d‐Amphetamine (2u2003mgu2003kg−1) given subcutaneously (s.c.) increased DA, AA and uric acid and decreased DOPAC+HVA, glutamate and aspartate dialysate concentrations over a 3u2003h period after d‐amphetamine. 5‐HIAA concentrations were unaffected. Individual changes in glutamate and AA dialysate concentrations were negatively correlated. d‐Amphetamine (0.2u2003mM), given intrastriatally, increased DA and decreased DOPAC+HVA and aspartate dialysate concentrations, but failed to change those of glutamate, AA uric acid or 5‐HIAA, over a 2u2003h period after d‐amphetamine. Haloperidol (0.1u2003mM), given intrastriatally, increased aspartate concentrations without affecting those of glutamate or AA. d‐Amphetamine (0.2u2003mM), given intranigrally, increased AA and uric acid dialysate concentrations and decreased those of glutamate, aspartate and DA; DOPAC+HVA and 5‐HIAA concentrations were unaffected. These results suggest that d‐amphetamine‐induced increases in AA and uric acid and decreases in glutamate concentrations are triggered at nigral sites. The changes in aspartate levels may be evoked by at least two mechanisms: striatal (mediated by inhibitory dopaminergic receptors) and nigral (activation of amino acid carrier‐mediated uptake).

Effect of naloxone on morphine-induced changes in striatal dopamine metabolism and glutamate, ascorbic acid and uric acid release in freely moving rats

Recent findings have shown that systemic morphine increases extracellular dopamine (DA), dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), ascorbic acid (AA) and uric acid concentrations in the striatum of freely moving rats. The morphine-induced increase in DA oxidative metabolism is highly correlated with that of xanthine. In the present study, we evaluated the effects of subcutaneous (s.c.) naloxone (1 mg/kg) on morphine-induced changes in DA, DOPAC, HVA, 5-hydroxyindoleacetic acid (5-HIAA), AA, uric acid and glutamate in the striatum of freely moving rats using microdialysis. Dialysates were assayed by high performance liquid chromatography with electrochemical detection or (glutamate) ultraviolet detection. Morphine (5-20 mg/kg) given s.c. increased DA, DOPAC+HVA, 5-HIAA, AA and uric acid and decreased glutamate dialysate concentrations over a 3 h period after morphine. Morphine (1 mM), given intrastriatally, did not affect all the above parameters, with the exception of an early short-lasting decrease in AA concentration. Naloxone antagonised all morphine-induced changes with the exception of AA increase and glutamate decrease in dialysate concentrations. Systemic or intrastrial (0.2-2 mM) naloxone increased AA and decreased glutamate dialysate concentrations. When given intranigrally, morphine (1 mM) increased DOPAC+HVA, AA and uric acid and decreased glutamate dialysate concentrations over a 2 h period after morphine; DA and 5-HIAA concentrations were unaffected. These results suggest that: (i) morphine increases striatal DA release and 5-hydroxytryptamine oxidative metabolism by a micro-opioid receptor-mediated mechanism mainly at extranigrostriatal sites; (ii) morphine increases DA and xanthine oxidative metabolism and affects glutamate and AA release by a micro-opioid receptor mediated mechanism acting also at nigral sites; and (iii) a micro-opioid receptor-mediated mechanism tonically controls at striatal sites extracellular AA and glutamate concentrations.

Manganese increases L‐DOPA auto‐oxidation in the striatum of the freely moving rat: potential implications to L‐DOPA long‐term therapy of Parkinson's disease

We have previously shown that manganese enhances L‐dihydroxyphenylanine (L‐DOPA) toxicity to PC12 cells in vitro. The supposed mechanism of manganese enhancing effect [an increase in L‐DOPA and dopamine (DA) auto‐oxidation] was studied using microdialysis in the striatum of freely moving rats. Systemic L‐DOPA [25u2003mgu2003kg−1 intraperitoneally (i.p.) twice in a 12u2003h interval] significantly increased baseline dialysate concentrations of L‐DOPA, dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and uric acid, compared to controls. Conversely, DA and ascorbic acid concentrations were significantly decreased. A L‐DOPA oxidation product, presumptively identified as L‐DOPA semiquinone, was detected in the dialysate. The L‐DOPA semiquinone was detected also following intrastriatal infusion of L‐DOPA. In rats given L‐DOPA i.p., intrastriatal infusion of N‐acetylcysteine (NAC) significantly increased DA and L‐DOPA dialysate concentrations and lowered those of L‐DOPA semiquinone; in addition, NAC decreased DOPAC+HVA and uric acid dialysate concentrations. In rats given L‐DOPA either systemically or intrastriatally, intrastriatal infusion of manganese decreased L‐DOPA dialysate concentrations and greatly increased those of L‐DOPA semiquinone. These changes were inhibited by NAC infusion. These findings demonstrate that auto‐oxidation of exogenous L‐DOPA occurs in vivo in the rat striatum. The consequent reactive oxygen species generation may account for the decrease in dialysate DA and ascorbic acid concentrations and increase in enzymatic oxidation of xanthine and DA. L‐DOPA auto‐oxidation is inhibited by NAC and enhanced by manganese. These results may be of relevance to the L‐DOPA long‐term therapy of Parkinsons disease.

Simultaneous telemetric monitoring of brain glucose and lactate and motion in freely moving rats

A new telemetry system for simultaneous detection of extracellular brain glucose and lactate and motion is presented. The device consists of dual-channel, single-supply miniature potentiostat-I/V converter, a microcontroller unit, a signal transmitter, and a miniaturized microvibration sensor. Although based on simple and inexpensive components, the biotelemetry device has been used for accurate transduction of the anodic oxidation currents generated on the surface of implanted glucose and lactate biosensors and animal microvibrations. The device was characterized and validated in vitro before in vivo experiments. The biosensors were implanted in the striatum of freely moving animals and the biotelemetric device was fixed to the animals head. Physiological and pharmacological stimulations were given in order to induce striatal neural activation and to modify the motor behavior in awake, untethered animals.