Non-HLA angiotensin-type-1 receptor autoantibodies mediate the long-term loss of grafted neurons in Parkinson's disease models.

BACKGROUND: Clinical trials have provided evidence that transplants of dopaminergic precursors, which may be replaced by new in vitro stem cell sources, can integrate into the host tissue, and alleviate motor symptoms in Parkinson´s disease (PD). In some patients, deterioration of graft function occurred several months after observing a graft-derived functional improvement. Rejection of peripheral organs was initially related to HLA-specific antibodies. However, the role of non-HLA antibodies is now considered also relevant for rejection. Angiotensin-II type-1 receptor autoantibodies (AT1-AA) act as agonists of the AT1 receptors. AT1-AA are the non-HLA antibodies most widely associated with graft dysfunction or rejection after transplantation of different solid organs and hematopoietic stem cells. However, it is not known about the presence and possible functional effects of AT1-AA in dopaminergic grafts, and the effects of treatment with AT1 receptor blockers (ARBs) such as candesartan on graft survival. METHODS: In a 6-hydroxydopamine PD rat model, we studied the short-term (10 days)- and long-term (3 months) effects of chronic treatment with the ARB candesartan on survival of grafted dopaminergic neurons and microglial graft infiltration, as well as the effects of dopaminergic denervation and grafting on serum and CSF AT1-AA levels. The expression of AT1 receptors in grafted neurons was determined by laser capture microdissection. RESULTS: At the early period post-grafting, the number of grafted dopaminergic neurons that survived was not significantly different between treated and untreated hosts (i.e., control rats and rats treated with candesartan), probably because, just after grafting, other deleterious factors are predominant for dopaminergic cell death, such as mechanical trauma, lack of growth factors/nutrients and ischemia. However, several months post-grafting, we observed a significantly higher number of surviving dopaminergic neurons and a higher density of striatal dopaminergic terminals in the candesartan-treated group. For several months, grafted rats showed blood and cerebrospinal fluid levels of AT1-AA higher than normal controls, and also higher AT1-AA levels than non-grafted parkinsonian rats. CONCLUSIONS: The results suggest the use of ARBs such as candesartan in PD patients, particularly before and after dopaminergic grafts, and the need to monitor AT1-AA levels in PD patients, particularly in those candidates for dopaminergic grafting.

Combined cell-based therapy strategies for the treatment of Parkinson's disease: focus on mesenchymal stromal cells.

Parkinson's disease is a neurodegenerative condition characterized by motor impairments caused by the selective loss of dopaminergic neurons in the substantia nigra. Levodopa is an effective and well-tolerated dopamine replacement agent. However, levodopa provides only symptomatic improvements, without affecting the underlying pathology, and is associated with side effects after long-term use. Cell-based replacement is a promising strategy that offers the possibility to replace lost neurons in Parkinson's disease treatment. Clinical studies of transplantation of human fetal ventral mesencephalic tissue have provided evidence that the grafted dopaminergic neurons can reinnervate the striatum, release dopamine, integrate into the host neural circuits, and improve motor functions. One of the limiting factors for cell therapy in Parkinson's disease is the low survival rate of grafted dopaminergic cells. Different factors could cause cell death of dopaminergic neurons after grafting such as mechanical trauma, growth factor deprivation, hypoxia, and neuroinflammation. Neurotrophic factors play an essential role in the survival of grafted cells. However, direct, timely, and controllable delivery of neurotrophic factors into the brain faces important limitations. Different types of cells secrete neurotrophic factors constitutively and co-transplantation of these cells with dopaminergic neurons represents a feasible strategy to increase neuronal survival. In this review, we provide a general overview of the pioneering studies on cell transplantation developed in patients and animal models of Parkinson's disease, with a focus on neurotrophic factor-secreting cells, with a particular interest in mesenchymal stromal cells; that co-implanted with dopaminergic neurons would serve as a strategy to increase cell survival and improve graft outcomes.

NRF2 Activation and Downstream Effects: Focus on Parkinson's Disease and Brain Angiotensin.

Reactive oxygen species (ROS) are signalling molecules used to regulate cellular metabolism and homeostasis. However, excessive ROS production causes oxidative stress, one of the main mechanisms associated with the origin and progression of neurodegenerative disorders such as Parkinson's disease. NRF2 (Nuclear Factor-Erythroid 2 Like 2) is a transcription factor that orchestrates the cellular response to oxidative stress. The regulation of NRF2 signalling has been shown to be a promising strategy to modulate the progression of the neurodegeneration associated to Parkinson's disease. The NRF2 pathway has been shown to be affected in patients with this disease, and activation of NRF2 has neuroprotective effects in preclinical models, demonstrating the therapeutic potential of this pathway. In this review, we highlight recent advances regarding the regulation of NRF2, including the effect of Angiotensin II as an endogenous signalling molecule able to regulate ROS production and oxidative stress in dopaminergic neurons. The genes regulated and the downstream effects of activation, with special focus on Kruppel Like Factor 9 (KLF9) transcription factor, provide clues about the mechanisms involved in the neurodegenerative process as well as future therapeutic approaches.

Dopamine regulates adult neurogenesis in the ventricular-subventricular zone via dopamine D3 angiotensin type 2 receptor interactions.

Adult neurogenesis is a dynamic and highly regulated process, and different studies suggest that dopamine modulates ventricular-subventricular zone (V-SVZ) neurogenesis. However, the specific role of dopamine and the mechanisms/factors underlying its effects on physiological and pathological conditions such as Parkinson's disease (PD) are not fully understood. Recent studies have described counter-regulatory interactions between renin-angiotensin system (RAS) and dopamine in peripheral tissues and in the nigrostriatal system. We have previously demonstrated that angiotensin receptors regulate proliferation and generation of neuroblasts in the rodent V-SVZ. However, possible interactions between dopamine receptors and RAS in the V-SVZ and their role in alterations of neurogenesis in animal models of PD have not been investigated. In V-SVZ cultures, activation of dopamine receptors induced changes in the expression of angiotensin receptors. Moreover, dopamine, via D2-like receptors and particularly D3 receptors, increased generation of neurospheres derived from the V-SVZ and this effect was mediated by angiotensin type-2 (AT2) receptors. In rats, we observed a marked reduction in proliferation and generation of neuroblasts in the V-SVZ of dopamine-depleted animals, and inhibition of AT1 receptors or activation of AT2 receptors restored proliferation and generation of neuroblasts to control levels. Moreover, intrastriatal mesencephalic grafts partially restored proliferation and generation of neuroblasts observed in the V-SVZ of dopamine-depleted rats. Our data revealed that dopamine and angiotensin receptor interactions play a major role in the regulation of V-SVZ and suggest potential beneficial effects of RAS modulators on the regulation of adult V-SVZ neurogenesis.

Dose-dependent effect of mesenchymal stromal cells co-grafted with dopaminergic neurons in a Parkinson's disease rat model.

A major limiting factor for cell therapy in Parkinson's disease is the poor survival and reinnervation capacity of grafted dopaminergic neurons, independently of the cell source. Mesenchymal stromal cells (MSCs) have high capability to regulate the local environment through the release of trophic, antiapoptotic and immunomodulatory factors. In this work, we investigated whether co-grafting of MSCs could improve the survival and reinnervation ability of dopaminergic precursors transplanted in animal models of Parkinson's disease. Rats with total unilateral dopaminergic denervation were grafted with a cell suspension of rat dopaminergic precursors (500,000 cells) with or without a high (200,000 cells) or low (25,000 cells) number of MSCs. Eight weeks after grafting, rats were tested for motor behaviour and sacrificed for histological analysis. Our results showed that the survival of dopaminergic neurons and graft-derived striatal dopaminergic innervation was higher in rats that received co-grafts containing a low number of MSCs than in non-co-grafted controls. However, the survival of dopaminergic neurons and graft-derived dopaminergic reinnervation was lower in rats receiving co-grafts with high number of MSCs than in non-co-grafted controls. In conclusion, co-grafting with MSCs or MSCs-derived products may constitute a useful strategy to improve dopaminergic graft survival and function. However, a tight control of MSCs density or levels of MSCs-derived products is necessary.

Interaction between Angiotensin Type 1, Type 2, and Mas Receptors to Regulate Adult Neurogenesis in the Brain Ventricular-Subventricular Zone.

The renin-angiotensin system (RAS), and particularly its angiotensin type-2 receptors (AT2), have been classically involved in processes of cell proliferation and maturation during development. However, the potential role of RAS in adult neurogenesis in the ventricular-subventricular zone (V-SVZ) and its aging-related alterations have not been investigated. In the present study, we analyzed the role of major RAS receptors on neurogenesis in the V-SVZ of adult mice and rats. In mice, we showed that the increase in proliferation of cells in this neurogenic niche was induced by activation of AT2 receptors but depended partially on the AT2-dependent antagonism of AT1 receptor expression, which restricted proliferation. Furthermore, we observed a functional dependence of AT2 receptor actions on Mas receptors. In rats, where the levels of the AT1 relative to those of AT2 receptor are much lower, pharmacological inhibition of the AT1 receptor alone was sufficient in increasing AT2 receptor levels and proliferation in the V-SVZ. Our data revealed that interactions between RAS receptors play a major role in the regulation of V-SVZ neurogenesis, particularly in proliferation, generation of neuroblasts, and migration to the olfactory bulb, both in young and aged brains, and suggest potential beneficial effects of RAS modulators on neurogenesis.

Data on the effect of Angiotensin II and 6-hydroxydopamine on reactive oxygen species production, antioxidant gene expression and viability of different neuronal cell lines.

This article describes the effect of the oxidative stress inducers Angiotensin II and 6-hydroxydopamine (6-OHDA) on different cell lines. The levels of expression Angiotensin type 1 and type 2 receptors in different dopaminergic cell lines are shown. The data indicate that treatment with Angiotensin II and 6-OHDA increases the production of reactive oxygen species (ROS) and decreases cell viability. NRF2 is a transcription factor induced by ROS. We provide data that NRF2 overexpression increases cell viability in response to oxidative stress inducers compared to control cells, and that these inducers can, both separately and in combination, enhance the expression of NRF2-regulated genes heme oxygenase 1 (Hmox1), NAD(P)H quinone dehydrogenase 1 (Nqo1) and Kruppel like factor 9 (Klf9). Interpretation of these data and additional information is presented in the research article "Angiotensin II induces oxidative stress and upregulates neuroprotective signaling from the NRF2 and KLF9 pathway in dopaminergic cells" (Parga et al., 2018) [1].

Angiotensin II induces oxidative stress and upregulates neuroprotective signaling from the NRF2 and KLF9 pathway in dopaminergic cells.

Nuclear factor-E2-related factor 2 (NRF2) is a transcription factor that activates the antioxidant cellular defense in response to oxidative stress, leading to neuroprotective effects in Parkinson's disease (PD) models. We have previously shown that Angiotensin II (AngII) induces an increase in reactive oxygen species (ROS) via AngII receptor type 1 and NADPH oxidase (NOX), which may activate the NRF2 pathway. However, controversial data suggest that AngII induces a decrease in NRF2 signaling leading to an increase in oxidative stress. We analyzed the effect of AngII and the dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA) in culture and in vivo, and examined the effects on the expression of NRF2-related genes. Treatment of neuronal cell lines Mes23.5, N27 and SH-SY5Y with AngII, 6-OHDA or a combination of both increased ROS production and reduced cell viability. Simultaneously, these treatments induced an increase in expression in the NRF2-regulated genes heme oxygenase 1 (Hmox1), NAD(P)H quinone dehydrogenase 1 (Nqo1) and Kruppel like factor 9 (Klf9). Moreover, overexpression of KLF9 transcription factor caused a reduction in the production of ROS induced by treatment with AngII or 6-OHDA and improved the survival of these neuronal cells. Rats treated with AngII, 6-OHDA or a combination of both also showed an increased expression of NRF2 related genes and KLF9. In conclusion, our data indicate that AngII induces a damaging effect in neuronal cells, but also acts as a signaling molecule to activate NRF2 and KLF9 neuroprotective pathways in cellular and animal models of PD.

Prostaglandin EP2 Receptors Mediate Mesenchymal Stromal Cell-Neuroprotective Effects on Dopaminergic Neurons.

Mesenchymal stromal cells (MSCs) have been shown to have useful properties for cell therapy and have been proposed for treatment of neurodegenerative diseases, including Parkinson's disease. However, the mechanisms involved in recovering dopaminergic neurons are not clear. The present study aims to evaluate the pathways and molecules involved in the neuroprotective effect of MSCs. We analyzed the viability of dopaminergic cells from different sources in response to conditioned medium derived from bone marrow MSC (MSC-CM). MSC-CM increased the viability of dopaminergic cells of rat and human origins, having both neuroprotective and neurorescue activities against effects of dopaminergic neurotoxin 6-hydroxydopamine. We found that lipid removal, inhibition of the prostaglandin E2 receptor 2 (EP2), and its signaling pathway were able to block the effects of MSC-CM on a pure population of dopaminergic neurons. Moreover, in primary mesencephalic cultures and hiPSC-derived neurons, inhibition of EP2 signaling caused a reduction in the number of dopaminergic neurons obtained in culture. Taken together, our results demonstrate for the first time the involvement of prostaglandin signaling from MSC in dopaminergic neuron survival through EP2 receptors, and suggest new approaches for treatment of Parkinson's disease.

Brain Renin-Angiotensin System and Microglial Polarization: Implications for Aging and Neurodegeneration.

Microglia can transform into proinflammatory/classically activated (M1) or anti-inflammatory/alternatively activated (M2) phenotypes following environmental signals related to physiological conditions or brain lesions. An adequate transition from the M1 (proinflammatory) to M2 (immunoregulatory) phenotype is necessary to counteract brain damage. Several factors involved in microglial polarization have already been identified. However, the effects of the brain renin-angiotensin system (RAS) on microglial polarization are less known. It is well known that there is a "classical" circulating RAS; however, a second RAS (local or tissue RAS) has been observed in many tissues, including brain. The locally formed angiotensin is involved in local pathological changes of these tissues and modulates immune cells, which are equipped with all the components of the RAS. There are also recent data showing that brain RAS plays a major role in microglial polarization. Level of microglial NADPH-oxidase (Nox) activation is a major regulator of the shift between M1/proinflammatory and M2/immunoregulatory microglial phenotypes so that Nox activation promotes the proinflammatory and inhibits the immunoregulatory phenotype. Angiotensin II (Ang II), via its type 1 receptor (AT1), is a major activator of the NADPH-oxidase complex, leading to pro-oxidative and pro-inflammatory effects. However, these effects are counteracted by a RAS opposite arm constituted by Angiotensin II/AT2 receptor signaling and Angiotensin 1-7/Mas receptor (MasR) signaling. In addition, activation of prorenin-renin receptors may contribute to activation of the proinflammatory phenotype. Aged brains showed upregulation of AT1 and downregulation of AT2 receptor expression, which may contribute to a pro-oxidative pro-inflammatory state and the increase in neuron vulnerability. Several recent studies have shown interactions between the brain RAS and different factors involved in microglial polarization, such as estrogens, Rho kinase (ROCK), insulin-like growth factor-1 (IGF-1), tumor necrosis factor α (TNF)-α, iron, peroxisome proliferator-activated receptor gamma, and toll-like receptors (TLRs). Metabolic reprogramming has recently been involved in the regulation of the neuroinflammatory response. Interestingly, we have recently observed a mitochondrial RAS, which is altered in aged brains. In conclusion, dysregulation of brain RAS plays a major role in aging-related changes and neurodegeneration by exacerbation of oxidative stress (OS) and neuroinflammation, which may be attenuated by pharmacological manipulation of RAS components.

Effects of Rho Kinase Inhibitors on Grafts of Dopaminergic Cell Precursors in a Rat Model of Parkinson's Disease.

UNLABELLED: In models of Parkinson's disease (PD), Rho kinase (ROCK) inhibitors have antiapoptotic and axon-stabilizing effects on damaged neurons, decrease the neuroinflammatory response, and protect against dopaminergic neuron death and axonal retraction. ROCK inhibitors have also shown protective effects against apoptosis induced by handling and dissociation of several types of stem cells. However, the effect of ROCK inhibitors on dopaminergic cell grafts has not been investigated. In the present study, treatment of dopaminergic cell suspension with ROCK inhibitors yielded significant decreases in the number of surviving dopaminergic neurons, in the density of graft-derived dopaminergic fibers, and in graft vascularization. Dopaminergic neuron death also markedly increased in primary mesencephalic cultures when the cell suspension was treated with ROCK inhibitors before plating, which suggests that decreased angiogenesis is not the only factor leading to cell death in grafts. Interestingly, treatment of the host 6-hydroxydopamine-lesioned rats with ROCK inhibitors induced a slight, nonsignificant increase in the number of surviving neurons, as well as marked increases in the density of graft-derived dopaminergic fibers and the size of the striatal reinnervated area. The study findings discourage treatment of cell suspensions before grafting. However, treatment of the host induces a marked increase in graft-derived striatal reinnervation. Because ROCK inhibitors have also exerted neuroprotective effects in several models of PD, treatment of the host with ROCK inhibitors, currently used against vascular diseases in clinical practice, before and after grafting may be a useful adjuvant to cell therapy in PD. SIGNIFICANCE: Cell-replacement therapy is one promising therapy for Parkinson's disease (PD). However, many questions must be addressed before widespread application. Rho kinase (ROCK) inhibitors have been used in a variety of applications associated with stem cell research and may be an excellent strategy for improving survival of grafted neurons and graft-derived dopaminergic innervation. The present results discourage the treatment of suspensions of dopaminergic precursors with ROCK inhibitors in the pregrafting period. However, treatment of the host (patients with PD) with ROCK inhibitors, currently used against vascular diseases, may be a useful adjuvant to cell therapy in PD.

Reciprocal regulation between sirtuin-1 and angiotensin-II in the substantia nigra: implications for aging and neurodegeneration.

Local angiotensin II (AII) and sirtuin 1 (SIRT1) play a major role in the modulation of neuroinflammation, oxidative stress and aging-related dopaminergic vulnerability to damage. However, it is not known whether the modulation is related to reciprocal regulation between SIRT1 and AII. In the present study, a single intraventricular injection of AII increased nigral SIRT1 levels in young adult rats. Although AII activity is known to be increased in aged rats, levels of SIRT1 were significantly lower than in young controls. Treatment with the SIRT1-activating compound resveratrol increased nigral SIRT1 levels in aged rats. Levels of SIRT1 were significantly higher in aged wild type mice than in AII type-1 receptor (AT1) deficient mice. In cell culture studies, treatment with AII also induced a transitory increase in levels of SIRT1 in the MES 23.5 dopaminergic neuron and the N9 microglial cell lines. In aged rats, treatment with resveratrol induced a significant decrease in the expression of AT1 receptors and markers of NADPH-oxidase activation (p47phox). In aged transgenic mice over-expressing SIRT1, levels of AT1 and p47 phox were lower than in aged wild type controls. In vitro, the inhibitory effects of resveratrol on AII/AT1/NADPH-oxidase activity were confirmed in primary mesencephalic cultures, the N9 microglial cell line, and the dopaminergic neuron cell line MES 23.5, and they were blocked by the SIRT1 specific inhibitor EX527. The present findings show that SIRT1 and the axis AII/AT1/NADPH-oxidase regulate each other. This is impaired in aged animals and may be mitigated with sirtuin-activating compounds.

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.

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.

Brain angiotensin regulates iron homeostasis in dopaminergic neurons and microglial cells.

Dysfunction of iron homeostasis has been shown to be involved in ageing, Parkinson's 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.

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 dopamine's mediated neuroinflammation and oxidative stress changes in Parkinson's disease. We suggest that manipulating brain angiotensin may constitute an effective neuroprotective strategy for Parkinson's disease.

Cografting of carotid body cells improves the long-term survival, fiber outgrowth and functional effects of grafted dopaminergic neurons.

AIMS: A major limiting factor for cell therapy in Parkinson's disease is that the survival of grafted dopaminergic neurons is very poor, which may be improved by administration of GDNF, for which the carotid body is a good source. MATERIALS & METHODS: Rats with total unilateral dopaminergic denervation were grafted with a cell suspension of rat dopaminergic neuroblasts with or without cell aggregates from the rat carotid body. At 1, 2 and 3 months after grafting, the rats were tested in the cylinder and the rotometer and killed 4 months after grafting. RESULTS: We observed that the survival of dopaminergic neurons and graft-derived dopaminergic innervation were higher in rats that received mixed grafts. Both grafted groups showed complete recovery in the amphetamine-induced rotation test. However, rats with cografts performed significantly better in the cylinder test. CONCLUSION: Cografting of carotid body cells may constitute a useful strategy for cell therapy in Parkinson's disease.

2-Benzazepine nitrones protect dopaminergic neurons against 6-hydroxydopamine-induced oxidative toxicity.

A number of C-3 spirocyclic 2-benzazepine analogs of α-phenyl-N-tert-butyl nitrone (PBN) were synthesized and tested for their activity in protecting rat brain mitochondria and dopaminergic (DA) neurons against 6-hydroxydopamine (6-OHDA), a toxin inducing destruction of the DA nigro-striatal pathway in rodent models of Parkinson's disease. The newly synthesized nitrone derivatives were firstly investigated for their activity in decreasing the level of hydroxyl radicals generated during 6-OHDA oxidation, and inhibit lipid peroxidation (TBARS assay) and protein carbonyl content (PCC) in rat brain mitochondria. Most of the studied 2-benzazepine nitrones showed inhibitory potencies in both TBARS and PCC assays at least two magnitude orders higher than that of PBN. The data obtained usefully complemented the known structure-activity relationships. In particular, 5 and 10, bearing C-3 spiro cyclopentyl and tetrahydropyranyl moieties, respectively, at 8 µM concentration proved to be significantly more effective than PBN in protecting cultured DA neurons exposed to 6-OHDA, which alone causes about 45% cell loss in 24 h. In addition, we found that 5 inhibited butyrylcholinesterase with an IC(50) value of 16.8 µM, which would enhance its potential as neuroprotective agent in Alzheimer's neurodegeneration. These findings extend the utility of benzazepine-based PBN analogs in the treatment of age-related free radical-mediated disorders.

Mitochondrial ATP-sensitive potassium channels enhance angiotensin-induced oxidative damage and dopaminergic neuron degeneration. Relevance for aging-associated susceptibility to Parkinson's disease.

Recent studies have shown that renin-angiotensin system overactivation is involved in the aging process in several tissues as well as in longevity and aging-related degenerative diseases by increasing oxidative damage and inflammation. We have recently shown that angiotensin II enhances dopaminergic degeneration by increasing levels of reactive oxygen species and neuroinflammation, and that there is an aging-related increase in angiotensin II activity in the substantia nigra in rats, which may constitute a major factor in the increased risk of Parkinson's disease with aging. The mechanisms involved in the above mentioned effects and particularly a potential angiotensin-mitochondria interaction have not been clarified. The present study revealed that activation of mitochondrial ATP-sensitive potassium channels [mitoK(ATP)] may play a major role in the angiotensin II-induced effects on aging and neurodegeneration. Inhibition of mitoK(ATP) channels with 5-hydroxydecanoic acid inhibited the increase in dopaminergic cell death induced by angiotensin II, as well as the increase in superoxide/superoxide-derived reactive oxygen species levels and the angiotensin II-induced decrease in the mitochondrial inner membrane potential in cultured dopaminergic neurons. The present study provides data for considering brain renin-angiotensin system and mitoK(ATP) channels as potential targets for protective therapy in aging-associated diseases such as Parkinson's disease.

Brain angiotensin and dopaminergic degeneration: relevance to Parkinson's disease.

The pathogenic mechanism of Parkinson's disease (PD) appears to be multifactorial. However, oxidative stress and neuroinflammation, including activation of NADPH-dependent oxidases, play a major role in the progression of dopaminergic cell death. The renin-angiotensin system (RAS) was described as a circulating humoral system that regulates blood pressure and water homeostasis. However, there exist local RAS in many tissues, and locally formed angiotensin activates NADPH-dependent oxidases, which are a major source of superoxide and are upregulated in major aging-related diseases such as hypertension, diabetes and atherosclerosis. Furthermore, an intracellular or intracrine RAS, with still unknown functions, has been identified in several cell types. The brain has an independent local RAS, which has been involved in several brain disorders, including neurodegenerative diseases. It is particularly interesting for PD the important interaction observed between angiotensin and dopamine, which counterregulate each other in renal cells and also in the striatum and substantia nigra. In recent studies, we have observed both a local and an intracellular RAS in the rodent, monkey and human substantia nigra, and that dopamine depletion induced RAS upregulation possibly as a compensatory mechanism. However, RAS hyperactivation also exacerbated oxidative stress and neuroinflammation, which contributed to progression of dopaminergic degeneration. In addition, we observed increased RAS activity in the nigra of animals with higher vulnerability of dopaminergic neurons to degeneration, such as aged males, menopausal females and rats subjected to chronic brain hypoperfusion. RAS activity and dopaminergic vulnerability were significantly reduced by treatment with angiotensin type I receptor antagonists. Manipulation of the brain RAS may constitute an effective neuroprotective strategy against dopaminergic degeneration in PD.