Antonio Dominguez-Meijide

Involvement of microglial RhoA/Rho-kinase pathway activation in the dopaminergic neuron death. Role of angiotensin via angiotensin type 1 receptors.

It has recently been shown that the dopaminergic cell loss induced by neurotoxins is enhanced by brain angiotensin II (AII) via type 1 receptors (AT1). However, the mechanisms involved in the dopaminergic degeneration and the brain inflammatory effects of AII have not been clarified. The RhoA-Rho-Kinase (ROCK) pathway may play a critical role in the inflammatory and oxidative effects of AII. In the substantia nigra of mice, administration of the dopaminergic neurotoxin MPTP induced an increase in the expression of RhoA and ROCK II mRNA levels and ROCK activity, which were inhibited by AT1 receptor deletion (i.e., in AT1a null mice treated with MPTP). Administration of the ROCK inhibitor Y-27632 or AT1 deletion induced a significant decrease in MPTP-induced microglial activation and dopaminergic cell death. In rat primary mesencephalic cultures treated with MPP(+), the increase in dopaminergic cell loss induced by AII administration was also inhibited by treatment with Y27632. Intense expression of ROCK II was observed in the microglial cells in the substantia nigra of mice treated with MPTP, and the major role of the microglial ROCK was confirmed by comparing mesencephalic cultures with and without microglia. Activation of the RhoA/ROCK pathway is involved in the MPTP-induced dopaminergic degeneration, and in the enhancing effect of AII/AT1 activation on the microglial response and dopaminergic degeneration. ROCK inhibitors and AT1 receptor antagonists may provide new neuroprotective strategies against the progression of Parkinsons disease.

Inhibition of Rho kinase mediates the neuroprotective effects of estrogen in the MPTP model of Parkinson's disease.

The mechanism by which estrogen protects dopaminergic neurons has not yet been clarified. It is not known if changes in RhoA/Rho kinase activity are involved in the enhanced vulnerability of dopaminergic neurons observed after estrogen depletion. The present study shows that the MPTP-induced loss of dopaminergic neurons is increased by estrogen depletion and inhibited by estrogen replacement, the Rho kinase inhibitor Y27632 and deletion of the angiotensin type-1 receptor. In ovariectomized mice, treatment with MPTP induced a marked increase in Rho kinase activity, and RhoA and RhocK II mRNA and protein expression, which were significantly higher than in ovariectomized mice treated with MPTP and estrogen replacement or type-1 receptor deletion. Estrogen depletion increased Rho kinase activity, via enhancement of the angiotensin type-1 receptor pathway, and Rho kinase activation increased type-1 receptor expression suggesting a vicious cycle in which Rho kinase and type-1 receptor activate each other and promote the degenerative process. The results suggest that type-1 receptor antagonists and Rho kinase inhibitors may provide a new neuroprotective strategy, which may circumvent the potential risks of estrogen replacement therapy and be particularly useful in elderly women or women affected by long-term lack of estrogen.

Aging-related dysregulation of dopamine and angiotensin receptor interaction

It is not known whether the aging-related decrease in dopaminergic function leads to the aging-related higher vulnerability of dopaminergic neurons and risk for Parkinsons disease. The renin-angiotensin system (RAS) plays a major role in the inflammatory response, neuronal oxidative stress, and dopaminergic vulnerability via type 1 (AT1) receptors. In the present study, we observed a counterregulatory interaction between dopamine and angiotensin receptors. We observed overexpression of AT1 receptors in the striatum and substantia nigra of young adult dopamine D1 and D2 receptor-deficient mice and young dopamine-depleted rats, together with compensatory overexpression of AT2 receptors or compensatory downregulation of angiotensinogen and/or angiotensin. In aged rats, we observed downregulation of dopamine and dopamine receptors and overexpression of AT1 receptors in aged rats, without compensatory changes observed in young animals. L-Dopa therapy inhibited RAS overactivity in young dopamine-depleted rats, but was ineffective in aged rats. The results suggest that dopamine may play an important role in modulating oxidative stress and inflammation in the substantia nigra and striatum via the RAS, which is impaired by aging.

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.

Angiotensin type 1 receptor blockage reduces l-dopa-induced dyskinesia in the 6-OHDA model of Parkinson's disease. Involvement of vascular endothelial growth factor and interleukin-1β.

Non-neuronal factors such as angiogenesis and neuroinflammation may play a role in l-dopa induced dyskinesias (LID). Vascular endothelial growth factor (VEGF) and proinflammatory cytokines such as interleukin-1β (IL-1β) have been found to be involved in LID. The renin-angiotensin system (RAS) is involved in the inflammatory response and VEGF synthesis via type 1 (AT1) receptors. However, it is not known whether the RAS plays a role in LID and whether AT1 antagonists could constitute a useful therapy against LID. In this study, we investigated whether manipulation of brain RAS is effective in preventing LID. Blocking AT1 receptors with candesartan significantly reduces LID in the 6-OHDA rat model. Chronic dopaminergic denervation induces an increase in striatal levels of VEGF and IL-1β. Dyskinetic animals showed significantly higher levels of VEGF and IL-1β in the lateral striatum and the substantia nigra, as revealed by western blot and real time-PCR analyses. Interestingly, animals treated with both candesartan and l-dopa displayed significantly lower levels of VEGF, IL-1β and dyskinesia than those treated with l-dopa alone. The stimulatory effect of angiotensin II (AII) on VEGF expression was confirmed by the addition of AII to primary mesencephalic cultures and intraventricular administration of AII in rats. The results of the present study reveal for the first time that blockage of AT-1 receptors reduces LID. A candesartan-induced decrease in VEGF and IL-1β may be responsible for the beneficial effects, suggesting the brain RAS as a new target for LID treatment in PD patients.

Brain angiotensin regulates iron homeostasis in dopaminergic neurons and microglial cells

Dysfunction of iron homeostasis has been shown to be involved in ageing, Parkinsons disease and other neurodegenerative diseases. Increased levels of labile iron result in increased reactive oxygen species and oxidative stress. Angiotensin II, via type-1 receptors, exacerbates oxidative stress, the microglial inflammatory response and progression of dopaminergic degeneration. Angiotensin activates the NADPH-oxidase complex, which produces superoxide. However, it is not known whether angiotensin affects iron homeostasis. In the present study, administration of angiotensin to primary mesencephalic cultures, the dopaminergic cell line MES23.5 and to young adult rats, significantly increased levels of transferrin receptors, divalent metal transporter-1 and ferroportin, which suggests an increase in iron uptake and export. In primary neuron-glia cultures and young rats, angiotensin did not induce significant changes in levels of ferritin or labile iron, both of which increased in neurons in the absence of glia (neuron-enriched cultures, dopaminergic cell line) and in the N9 microglial cell line. In aged rats, which are known to display high levels of angiotensin activity, ferritin levels and iron deposits in microglial cells were enhanced. Angiotensin-induced changes were inhibited by angiotensin type-1 receptor antagonists, NADPH-oxidase inhibitors, antioxidants and NF-kB inhibitors. The results demonstrate that angiotensin, via type-1 receptors, modulates iron homeostasis in dopaminergic neurons and microglial cells, and that glial cells play a major role in efficient regulation of iron homeostasis in dopaminergic neurons.

Rho Kinase and Dopaminergic Degeneration: A Promising Therapeutic Target for Parkinson’s Disease

The small GTP-binding protein Rho plays an important role in several cellular functions. RhoA, which is a member of the Rho family, initiates cellular processes that act on its direct downstream effector Rho-associated kinase (ROCK). ROCK inhibition protects against dopaminergic cell death induced by dopaminergic neurotoxins. It has been suggested that ROCK inhibition activates neuroprotective survival cascades in dopaminergic neurons. Axon-stabilizing effects in damaged neurons may represent another mechanism of neuroprotection of dopaminergic neurons by ROCK inhibition. However, it has been shown that microglial cells play a crucial role in neuroprotection by ROCK inhibition and that activation of microglial ROCK mediates major components of the microglial inflammatory response. Additional mechanisms such as interaction with autophagy may also contribute to the neuroprotective effects of ROCK inhibition. Interestingly, ROCK interacts with several brain factors that play a major role in dopaminergic neuron vulnerability such as NADPH-oxidase, angiotensin, and estrogen. ROCK inhibition may provide a new neuroprotective strategy for Parkinson’s disease. This is of particular interest because ROCK inhibitors are currently used against vascular diseases in clinical practice. However, it is necessary to develop more potent and selective ROCK inhibitors to reduce side effects and enhance the efficacy.

Dopamine modulates astroglial and microglial activity via glial renin-angiotensin system in cultures

Dopamine is an immunomodulatory molecule that acts on immune effector cells both in the CNS and peripheral tissues. However, the role of changes in dopamine levels in the neuroinflammatory response is controversial. The local/paracrine renin-angiotensin system (RAS) plays a major role in inflammatory processes in peripheral tissues and brain. In the present study, we investigated the possible role of the brain RAS in the effects of dopamine on the glial inflammatory responses. Astrocytes are the major source of the precursor protein angiotensinogen and angiotensin II (AII) in the brain. Neurotoxins such as MPP+ (1-methyl-4-phenylpyridinium) can act directly on astrocytes to increase levels of angiotensinogen and AII. Conversely, dopamine, via type-2 (D2) receptors, inhibited production of angiotensinogen, decreased expression of angiotensin type-1 (AT1) receptors and increased expression of AT2 receptors. In microglia, dopamine and dopamine agonists also regulated RAS activity. First, indirectly, via downregulation of the astrocyte-derived AII. Second, via dopamine-induced regulation of microglial angiotensin receptors. Dopamine decreased the microglial AT1/AT2 ratio leading to inhibition of the pro-inflammatory AT1/NADPH-oxidase/superoxide axis. D2 receptors were particularly responsible for microglial RAS inhibition in basal culture conditions. However, both D1 and D2 agonists inhibited the AT1/NADPH-oxidase axis in lipopolysaccharide-treated (LPS; i.e. activated) microglia. The results indicate that the decrease in dopamine levels observed in early stages of Parkinsons disease and aging may promote neuroinflammation and disease progression via glial RAS exacerbation.

Aging-related dysregulation in enteric dopamine and angiotensin system interactions: implications for gastrointestinal dysfunction in the elderly

Gastrointestinal dysfunction is a common problem in the elderly. Aging-related changes in interactions between local dopaminergic and renin-angiotensin systems (RAS) have been observed in the brain, renal and vascular tissues. However, it is not known if these interactions also occur in the gut, and are dysregulated with aging. We showed a mutual regulation between the colonic dopaminergic system and RAS using young and aged mice deficient for major angiotensin and dopamine receptors. Aged rats showed a marked decrease in colonic dopamine D2 receptor expression, together with an increase in angiotensin type 1 (AT1) receptor expression, a decrease in angiotensin type 2 (AT2) receptor expression (i.e. an increase in the RAS pro-inflammatory arm activity), and increased levels of inflammatory and oxidative markers. Aged rats also showed increased levels of colonic dopamine and noradrenalin, and a marked decrease in acetylcholine and serotonin levels. The present observations contribute to explain an aging-related pro-inflammatory state and dysregulation in gastrointestinal function, which may be counteracted by treatment of aged animals with the AT1 receptor blocker candesartan.