Jannette Rodriguez-Pallares

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.

Localization and functional significance of striatal neurons immunoreactive to aromatic L-amino acid decarboxylase or tyrosine hydroxylase in rat Parkinsonian models.

Striatal neurons which are immunoreactive (ir) to aromatic L-amino-acid decarboxylase (AADC) or tyrosine hydrodroxylase (TH) may play a role in the decarboxylation of L-DOPA to dopamine (DA) in advanced stages of Parkinsons disease (PD). However, the functional significance of these neurons and the mechanisms responsible for their induction remain to be clarified. In this study, rats were subjected to different types of dopaminergic or serotonergic denervation and L-DOPA injection to study the effects on these neurons. AADC-ir neurons were found in both normal and DA-denervated striata, and no significant differences in their number and distribution were induced following different types of denervation or L-DOPA administration. TH-ir neurons were only found in DA-denervated striata. However, TH-ir neurons did not appear in those areas with maximal DA depletion, but rather were observed near spared or partially lesioned DA terminals. The population of AADC-ir neurons may make a significant contribution to the effects of exogenous L-DOPA in advanced stages of PD. In addition, TH-ir neurons may contribute to these effects, since we have detected AADC-ir in TH-ir neurons using confocal laser scanning microscopy. Finally, neither L-DOPA therapy nor serotonergic denervation induces significant changes in the number or distribution of these neurons.

Angiotensin II increases differentiation of dopaminergic neurons from mesencephalic precursors via angiotensin type 2 receptors.

In addition to the well‐known actions of the humoral renin–angiotensin system, all components of this system are present in many tissues, including the brain, and may play a major role in brain development and differentiation. We investigated the possible effects of angiotensin II on the generation of dopaminergic phenotype neurons from proliferating neurospheres of mesencephalic precursors. We observed immunoreactivity for both angiotensin type 1 and type 2 (AT1 and AT2) receptors in the cell aggregates. Double immunolabeling studies revealed that both receptor types are located in neurons and astrocytes. Interestingly, neurons with a dopaminergic phenotype (i.e. tyrosine hydroxylase activity) showed double labeling for AT1 and AT2 receptors although the labeling for AT2 was more intense. Treatment of the neurospheres with angiotensin II (100 nm) during the differentiation period induced a marked increase (about 400%) in the generation of dopaminergic neurons. This was not affected by treatment with the AT1 antagonist ZD 7155 but was blocked by treatment with the AT2 antagonist PD 123319. This suggests that AT2 receptors mediate the stimulatory effect of angiotensin II on the generation of dopaminergic neurons. Apoptotic cell death studies and bromodeoxyuridine immunohistochemistry indicated that the increase in generation of dopaminergic neurons is not due to increased survival or proliferation of dopaminergic cells during treatment with angiotensin and suggested that angiotensin induces increased differentiation of mesencephalic precursors towards the dopaminergic phenotype. Manipulation of the renin–angiotensin system may be useful for increasing production of dopaminergic neurons for transplantation in Parkinsons disease.

Nigral and striatal regulation of angiotensin receptor expression by dopamine and angiotensin in rodents: implications for progression of Parkinson's disease.

The basal ganglia have a local renin–angiotensin system and it has been shown that the loss of dopaminergic neurons induced by neurotoxins is amplified by local angiotensin II (AII) via angiotensin type 1 receptors (AT1) and nicotinamide adenine dinucleotide phosphate (NADPH) complex activation. Recent studies have revealed a high degree of counter‐regulatory interactions between dopamine and AII receptors in non‐neural cells such as renal proximal tubule cells. However, it is not known if this occurs in the basal ganglia. In the striatum and nigra, depletion of dopamine with reserpine induced a significant increase in the expression of AT1, angiotensin type 2 receptors (AT2) and the NADPH subunit p47phox, which decreased as dopamine function was restored. Similarly, 6‐hydroxydopamine‐induced chronic dopaminergic denervation induced a significant increase in expression of AT1, AT2 and p47phox, which decreased with L‐dopa administration. A significant reduction in expression of AT1 mRNA was also observed after administration of dopamine to cultures of microglial cells. Transgenic rats with very low levels of brain AII showed increased AT1, decreased p47 phox and no changes in AT2 expression, whereas mice deficient in AT1 exhibited a decrease in the expression of p47 phox and AT2. The administration of relatively high doses of AII (100 nm) decreased the expression of AT1, and the increased expression of AT2 and p47phox in primary mesencephalic cultures. The results reveal an important interaction between the dopaminergic and local renin–angiotensin system in the basal ganglia, which may be a major factor in the progression of Parkinson’s disease.

Striatal dopaminergic afferents concentrate in GDNF-positive patches during development and in developing intrastriatal striatal grafts

Glial cell line‐derived neurotrophic factor (GDNF) has potent trophic action on fetal dopaminergic neurons. We have used a double immunocytochemical approach with antibodies that recognize GDNF and tyroxine hydroxylase (TH) or the phosphoprotein DARPP‐32, to study the developmental pattern of their interactions in the rat striatum and in intrastriatal striatal transplants. Postnatally, at one day and also at 1 week, GDNF showed a patchy distribution in the striatum, together with a high level of expression in the lateral striatal border, similar to that observed for the striatal marker DARPP‐32 and also for TH. In the adult striatum, there was diffuse, weak immunopositivity for GDNF, together with widespread expression of DARPP‐32‐positive neurons and TH‐immunoreactive (TH‐ir) fibers. In 1‐week‐old intrastriatal striatal transplants, there were some GDNF immunopositive patches within the grafts and although there was not an abundance of TH‐positive fibers, the ones that were seen were located in GDNF‐positive areas. This was clearly evident in 2‐week‐old transplants, where TH‐ir fibers appeared selectively concentrated in GDNF‐positive patches. This pattern was repeated in 3‐week‐old grafts. In co‐transplants of mesencephalic and striatal fetal tissue (in a proportion of 1:4), TH‐ir somata were located mainly at the borders of areas that were more strongly immunostained for GDNF, and TH‐ir fibers were also abundant in these areas and were found in smaller numbers in regions that were weakly positive for GDNF.

Brain renin-angiotensin system and dopaminergic cell vulnerability

Although the renin-angiotensin system (RAS) was classically considered as a circulating system that regulates blood pressure, many tissues are now known to have a local RAS. Angiotensin, via type 1 receptors, is a major activator of the NADPH-oxidase complex, which mediates several key events in oxidative stress (OS) and inflammatory processes involved in the pathogenesis of major aging-related diseases. Several studies have demonstrated the presence of RAS components in the basal ganglia, and particularly in the nigrostriatal system. In the nigrostriatal system, RAS hyperactivation, via NADPH-oxidase complex activation, exacerbates OS and the microglial inflammatory response and contributes to progression of dopaminergic degeneration, which is inhibited by angiotensin receptor blockers and angiotensin converting enzyme (ACE) inhibitors. Several factors may induce an increase in RAS activity in the dopaminergic system. A decrease in dopaminergic activity induces compensatory upregulation of local RAS function in both dopaminergic neurons and glia. In addition to its role as an essential neurotransmitter, dopamine may also modulate microglial inflammatory responses and neuronal OS via RAS. Important counterregulatory interactions between angiotensin and dopamine have also been observed in several peripheral tissues. Neurotoxins and proinflammatory factors may also act on astrocytes to induce an increase in RAS activity, either independently of or before the loss of dopamine. Consistent with a major role of RAS in dopaminergic vulnerability, increased RAS activity has been observed in the nigra of animal models of aging, menopause and chronic cerebral hypoperfusion, which also showed higher dopaminergic vulnerability. Manipulation of the brain RAS may constitute an effective neuroprotective strategy against dopaminergic vulnerability and progression of Parkinson’s disease.

Interaction between NADPH‐oxidase and Rho‐kinase in angiotensin II‐induced microglial activation

Previous studies have shown that the brain renin–angiotensin system may play a major role, via angiotensin type 1 (AT1) receptors, in the regulation of neuroinflammation, oxidative stress and progression of dopaminergic degeneration. Angiotensin‐induced activation of the microglial nicotinamide adenine dinucleotide phosphate (NADPH)‐oxidase complex and microglial Rho‐kinase are particularly important in this respect. However, it is not known whether crosstalk between Rho‐kinase and NADPH‐oxidase leads to microglial activation. In the present study, we found that, in the substantia nigra of rats, NADPH‐oxidase activation was involved in angiotensin‐induced Rho‐kinase activation, which, in turn, was involved in angiotensin‐induced NADPH‐oxidase activation. In N9 microglial cell line and primary microglial cultures, a crosstalk signaling between NADPH‐oxidase and Rho‐kinase occurred in a positive feedback fashion during angiotensin‐induced microglial activation. Angiotensin‐induced NADPH‐oxidase activation and superoxide generation led to NF‐кB translocation and Rho‐kinase activation. Rho‐kinase activation was involved in regulation of NADPH‐oxidase activation via p38 mitogen‐activated protein kinase. Moreover, Rho‐kinase activation, via NF‐кB, upregulated AT1 receptor expression in microglial cells through a feed‐forward mechanism. NADPH‐oxidase and Rho‐kinase pathways are known to be responsible for major components of the microglial response, such as changes involving microglial motility and phagocytosis, generation of superoxide, and release of inflammatory cytokines. The present results show that both pathways are linked by a common mechanism that may constitute a basic means of coordinating the microglial response. GLIA 2015;63:466–482

Dopamine-angiotensin interactions in the basal ganglia and their relevance for Parkinson's disease.

Renin‐angiotensin systems are known to act in many tissues, for example, the blood vessel wall or kidney, where a close interaction between angiotensin and dopamine has been demonstrated. Regulatory interactions between the dopaminergic and renin‐angiotensin systems have recently been described in the substantia nigra and striatum. In animal models, dopamine depletion induces compensatory overactivation of the local renin‐angiotensin system, which primes microglial responses and neuron vulnerability by activating NADPH‐oxidase. Hyperactivation of the local renin‐angiotensin system exacerbates the inflammatory microglial response, oxidative stress, and dopaminergic degeneration, all of which are inhibited by angiotensin receptor blockers and inhibitors of angiotensin‐converting enzymes. In this review we provide evidence suggesting that the renin‐angiotensin system may play an important role in dopamines mediated neuroinflammation and oxidative stress changes in Parkinsons disease. We suggest that manipulating brain angiotensin may constitute an effective neuroprotective strategy for Parkinsons disease.