Jose L. Labandeira-Garcia

Autoxidation and neurotoxicity of 6-hydroxydopamine in the presence of some antioxidants: potential implication in relation to the pathogenesis of Parkinson's disease.

Abstract: 6‐Hydroxydopamine (6‐OHDA) is a dopaminergic neurotoxin putatively involved in the pathogenesis of Parkinsons disease (PD). Its neurotoxicity has been related to the production of reactive oxygen species. In this study we examine the effects of the antioxidants ascorbic acid (AA), glutathione (GSH), cysteine (CySH), and N‐acetyl‐CySH (NAC) on the autoxidation and neurotoxicity of 6‐OHDA. In vitro, the autoxidation of 6‐OHDA proceeds rapidly with the formation of H2O2 and with the participation of the H2O2 produced in the reaction. The presence of AA induced a reduction in the consumption of O2 during the autoxidation of 6‐OHDA and a negligible presence of the p‐quinone, which demonstrates the efficiency of AA to act as a redox cycling agent. The presence of GSH, CySH, and NAC produced a significant reduction in the autoxidation of 6‐OHDA. In vivo, the presence of sulfhydryl antioxidants protected against neuronal degeneration in the striatum, which was particularly remarkable in the case of CySH and was attributed to its capacity to remove the H2O2 produced in the autoxidation of 6‐OHDA. These results corroborate the involvement of oxidative stress as the major mechanism in the neurotoxicity of 6‐OHDA and the putative role of CySH as a scavenger in relation to PD.

The overall rod performance test in the MPTP-treated-mouse model of Parkinsonism

We investigated the usefulness of the Overall Rotarod Performance (ORP) test for evaluating overall locomotory ability in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-injected-mouse model of Parkinsons disease (PD). For this procedure, the mice are pretrained on the rotarod and then tested at a series of increasing speeds, recording the time that the animal remains on the rod at each speed; the overall rod performance (ORP) of each animal is then calculated as the area under the curve in a plot of time-on-the-rod against rotation speed. At 15-day intervals, C57BL/6 mice were injected (or sham-injected) with MPTP, with ORP testing 7-10 days after each injection. After the fourth injection (day 45), mice in the treated group showed clearly lower ORP than mice in the control group (70-90% reduction in ORP), and were thus considered effectively lesioned. Subsequently, we investigated the short-term effects of apomorphine and L-DOPA on ORP in MPTP-treated mice. Apomorphine (at 0.5 or 2.5 mg/kg) had no significant effect, while L-DOPA (at 80 but not at 40 mg/kg) caused almost complete short-term recovery of pretreatment ORP. By about 100 days after the last MPTP injection, MPTP-treated mice showed partial long-term recovery of ORP; at this stage the mice were killed for tyrosine hydroxylase (TH) immunohistochemistry studies. TH immunoreactivity in the striatum showed a strong positive correlation with ORP as tested on day 100. We conclude that the ORP test is useful for evaluating motor deficit in MPTP-treated mice, and the effects of subsequent treatments.

Mechanism of 6-hydroxydopamine neurotoxicity: the role of NADPH oxidase and microglial activation in 6-hydroxydopamine-induced degeneration of dopaminergic neurons

Cell death induced by 6‐hydroxydopamine (6‐OHDA) is thought to be caused by reactive oxygen species (ROS) derived from 6‐OHDA autooxidation and by a possible direct effect of 6‐OHDA on the mitochondrial respiratory chain. However, the process has not been totally clarified. In rat primary mesencephalic cultures, we observed a significant increase in dopaminergic (DA) cell loss 24 h after administration of 6‐OHDA (40 μmol/L) and a significant increase in NADPH subunit expression, microglial activation and superoxide anion/superoxide‐derived ROS in DA cells that were decreased by the NADPH inhibitor apocynin. Low doses of 6‐OHDA (10 μmol/L) did not induce a significant loss of DA cells or a significant increase in NADPH subunit expression, microglial activation or superoxide‐derived ROS. However, treatment with the NADPH complex activator angiotensin II caused a significant increase in all the latter. Forty‐eight hours after intrastriatal 6‐OHDA injection in rats, there was still no loss of DA neurons although there was an increase in NADPH subunit expression and NADPH oxidase activity. The results suggest that in addition to the autooxidation‐derived ROS and the inhibition of the mitochondrial respiratory chain, early microglial activation and NADPH oxidase‐derived ROS act synergistically with 6‐OHDA and constitute a relevant and early component of the 6‐OHDA‐induced cell death.

Brain angiotensin enhances dopaminergic cell death via microglial activation and NADPH-derived ROS

Angiotensin II (AII) plays a major role in the progression of inflammation and NADPH-derived oxidative stress (OS) in several tissues. The brain possesses a local angiotensin system, and OS and inflammation are key factors in the progression of Parkinsons disease. In rat mesencephalic cultures, AII increased 6-OHDA-induced dopaminergic (DA) cell death, generation of superoxide in DA neurons and microglial cells, the expression of NADPH-oxidase mRNA, and the number of reactive microglial cells. These effects were blocked by AII type-1 (AT1) antagonists, NADPH inhibitors, or elimination of glial cells. DA degeneration increased angiotensin converting enzyme activity and AII levels. In rats, 6-OHDA-induced dopaminergic cell loss and microglial activation were reduced by treatment with AT1 antagonists. The present data suggest that AII, via AT1 receptors, increases the dopaminergic degeneration process by amplifying the inflammatory response and intraneuronal levels of OS, and that glial cells play a major role in this process.

The inflammatory response in the MPTP model of Parkinson’s disease is mediated by brain angiotensin: relevance to progression of the disease

The neurotoxin MPTP reproduces most of the biochemical and pathological hallmarks of Parkinson’s disease. In addition to reactive oxygen species (ROS) generated as a consequence of mitochondrial complex I inhibition, microglial NADPH‐derived ROS play major roles in the toxicity of MPTP. However, the exact mechanism regulating this microglial response remains to be clarified. The peptide angiotensin II (AII), via type 1 receptors (AT1), is one of the most important inflammation and oxidative stress inducers, and produces ROS by activation of the NADPH‐oxidase complex. Brain possesses a local angiotensin system, which modulates striatal dopamine (DA) release. However, it is not known if AII plays a major role in microglia‐derived oxidative stress and DA degeneration. The present study indicates that in primary mesencephalic cultures, DA degeneration induced by the neurotoxin MPTP/MPP+ is amplified by AII and inhibited by AT1 receptor antagonists, and that protein kinase C, NADPH‐complex activation and microglial activation are involved in this effect. In mice, AT1 receptor antagonists inhibited both DA degeneration and early microglial and NADPH activation. The brain angiotensin system may play a key role in the self‐propelling mechanism of Parkinson’s disease and constitutes an unexplored target for neuroprotection, as previously reported for vascular diseases.

Effects of (-)-nicotine and (-)-cotinine on 6-hydroxydopamine-induced oxidative stress and neurotoxicity: relevance for Parkinson's disease

In view of the apparent controversial properties of (-)-nicotine (NIC) in relation to both oxidative stress and neuroprotection, we studied the effects of NIC on hydroxyl radical (*OH) formation, oxidative stress production by 6-hydroxydopamine (6-OHDA) autoxidation in the presence and absence of ascorbate, and 6-OHDA neurotoxicity. Both NIC and (-)-cotinine (COT) exhibited increased *OH production during 6-OHDA autoxidation. Although the same effect was observed in *OH generation by the Fenton reaction (H2O2 + Fe2+), this reaction was completely prevented with the previous incubation of Fe2+ with NIC or COT. Furthermore, both NIC and COT demonstrated a capacity to be able to reduce the TBARS formation provoked in rat brain mitochondrial preparations by 6-OHDA autoxidation. This effect is assumed as a consequence of the action of NIC and COT on lipid peroxidation propagation. We treated with NIC (1mg/kg, i.p.) two 6-OHDA-induced rat models of Parkinsons disease. However, only in one of these models did we obtain clear evidence of a neuroprotective effect of NIC on nigrostriatal terminals, as revealed by immunohistochemistry against tyrosine hydroxylase. Thus, the antioxidant properties of both NIC and COT in relation to the lipid peroxidation induced by 6-OHDA autoxidation, together with their reported capacity to prevent the Fenton reaction, probably by sequestration of Fe2+, may contribute to an understanding of its neuroprotective properties. In addition, the reported capacity of both NIC and COT to increase the production of *OH by 6-OHDA autoxidation might help explain the controversial observation found under different experimental conditions.

Sprouting of the serotonergic afferents into striatum after selective lesion of the dopaminergic system by MPTP in adult mice

Neonatal destruction of the nigrostrial dopaminergic (DA) system with 6-hydroxydopamine leads to serotonergic (5-HT) hyperinnervation of the striatum. However, it is not clear whether this occurs in adult animals. We investigated whether serotonergic sprouting occurs in adult mice subjected to bilateral lesion of the DA system by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The effects of the lesion were evaluated using a new rotarod test and immunohistochemistry. One hundred days after the last MPTP lesion, there was a clear bilateral serotonergic hyperinnervation throughout the striatum. Additionally, those mice showing the highest reductions in striatal tyrosine hydroxylase (TH) immunoreactivity and in rotarod performance showed the highest density of serotonergic innervation (116% increase). The functional consequences of this process in Parkinsons disease and secondary parkinsonism remain to be clarified.

MECHANISMS OF THE EFFECTS OF EXOGENOUS LEVODOPA ON THE DOPAMINE-DENERVATED STRIATUM

The efficacy of exogenous levodopa (L-DOPA) is attributed to its conversion to dopamine by the enzyme aromatic L-amino-acid decarboxylase in striatal dopaminergic terminals. However, there is controversy about the mechanisms underlying the therapeutic and adverse effects of L-DOPA after almost all striatal dopaminergic afferents have disappeared (i.e. in the later stages of Parkinsons disease). After administration of 30mg/kg or 100mg/kg of L-DOPA, rats subjected to unilateral dopaminergic denervation showed intense contraversive rotation and a high density of Fos-immunoreactive nuclei throughout the denervated striatum, with no significant induction of Fos in the intact striatum. Injection of the central aromatic L-amino-acid decarboxylase inhibitor NSD-1015 30min before and 15min after the injection of L-DOPA suppressed the rotational behavior and the striatal induction of Fos. Comparison of results obtained in rats subjected to unilateral and bilateral dopaminergic denervation indicated that the presence of contralateral dopaminergic innervation does not significantly modulate the effects of L-DOPA on the denervated striatum. Serotonergic denervation led to slight and statistically non-significant decrease in the rotational behavior and Fos expression induced by high doses of L-DOPA (100mg/kg) in the dopamine-denervated striatum, but totally suppressed the rotational behavior and Fos expression induced by low doses of L-DOPA (30mg/kg). The present data indicate that the major effects observed after administration of exogenous L-DOPA are not due to a direct action of L-DOPA on dopamine receptors, or to extrastriatal release of dopamine, but to conversion of L-DOPA to dopamine by serotonergic terminals and probably some intrastriatal cells. Given that serotonergic neurons appear to play an important role in the action of L-DOPA in the later stages of Parkinsons disease, strategies targeting the serotonergic system should be considered for the treatment of Parkinsons disease and for combating undesirable side effects of L-DOPA therapy.

Angiotensin type-1-receptor antagonists reduce 6-hydroxydopamine toxicity for dopaminergic neurons

Angiotensin II activates (via type 1 receptors) NAD(P)H-dependent oxidases, which are a major source of superoxide, and is relevant in the pathogenesis of several cardiovascular diseases and certain degenerative changes associated with ageing. Given that there is a brain renin-angiotensin system and that oxidative stress is a key contributor to Parkinsons disease, we investigated the effects of angiotensin II and angiotensin type 1 (AT(1)) receptor antagonists in the 6-hydroxydopamine model of Parkinsons disease. Rats subjected to intraventricular injection of 6-hydroxydopamine showed bilateral reduction in the number of dopaminergic neurons and terminals. Injection of angiotensin alone did not induce any significant effect. However, angiotensin increased the toxic effect of 6-hydroxydopamine. Rats treated with the AT(1) receptor antagonist ZD 7155 and then 6-hydroxydopamine (with or without exogenous administration of angiotensin) showed a significant reduction in 6-hydroxydopamine-induced oxidative stress (lipid peroxidation and protein oxidation) and dopaminergic degeneration. Dopaminergic degeneration was also reduced by the NAD(P)H inhibitor apocynin. Angiotensin may play a pivotal role, via AT(1) receptors, in increasing the oxidative damage of dopaminergic cells, and treatment with AT(1) antagonists may reduce the progression of Parkinsons disease.

Treadmill running induces striatal Fos expression via NMDA glutamate and dopamine receptors.

Abstract Several non-physiological stimuli (i.e. pharmacological or electrical stimuli) have been shown to induce Fos expression in striatal neurons. In this work, striatal Fos (i.e. Fos-like) expression was studied after physiological stimulation, i.e. motor activity (treadmill running at 36 m/min for 20 min). In rats killed 2 h after the treadmill session, Fos expression was observed in the medial region of the rostral and central striatum, and in the dorsal region of the caudal striatum. Fos expression was prevented by pretreatment with the non-competitive N-methyl-D-aspartate (NMDA) glutamate receptor antagonist MK-801 (0.1 mg/kg) or the D1 dopamine receptor antagonist SCH-23390 (0.1 mg/kg), but not by pretreatment with the D2 receptor antagonist eticlopride (0.5 mg/kg). Thirty-six hours after 6-hydroxydopamine lesion, a considerable reduction in treadmill-induced Fos expression was observed in both sides; however, Fos expression in the lesioned striatum was higher than in the contralateral intact striatum. Several weeks after unilateral 6-hydroxydopamine lesion of the nigrostriatal system, treadmill-induced Fos expression was significantly, but not totally, reduced in the lesioned striatum. Corticostriatal deafferentation also led to considerable reduction in treadmill-induced Fos expression. The present results indicate that exercise induces striatal Fos expression and that, under physiological stimulation, concurrent activation of D1 and NMDA receptors is necessary for such expression to occur. Reduction of Fos expression is practically absolute after acute blockage of these receptors, but not after lesions, possibly due partially to compensatory changes.