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.

Microglial angiotensin type 2 receptors mediate sex-specific expression of inflammatory cytokines independently of circulating estrogen.

There are sex differences in microglia, which can maintain sex-related gene expression and functional differences in the absence of circulating sex steroids. The angiotensin type 2 (AT2) receptors mediate anti-inflammatory actions in different tissues, including brain. In mice, we performed RT-PCR analysis of microglia isolated from adult brains and RNA scope in situ hybridization from males, females, ovariectomized females, orchiectomized males and brain masculinized females. We also compared wild type and AT2 knockout mice. The expression of AT2 receptors in microglial cells showed sex differences with much higher AT2 mRNA expression in females than in males, and this was not dependent on circulating gonadal hormones, as observed using ovariectomized females, brain masculinized females and orchiectomized males. These results suggest genomic reasons, possibly related to sex chromosome complement, for sex differences in AT2 expression in microglia, as the AT2 receptor gene is located in the X chromosome. Furthermore, sex differences in expression of AT2 receptors were associated to sex differences in microglial expression of key anti-inflammatory cytokines such as interleukin-10 and pro-inflammatory cytokines such as interleukin-1ß and interleukin-6. In conclusion, sex differences in microglial AT2 receptor expression appear as a major factor contributing to sex differences in the neuroinflammatory responses beyond the effects of circulating steroids.

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.

NADPH-Oxidase, Rho-Kinase and Autophagy Mediate the (Pro)renin-Induced Pro-Inflammatory Microglial Response and Enhancement of Dopaminergic Neuron Death.

Dysregulation of the tissue renin-angiotensin system (RAS) is involved in tissue oxidative and inflammatory responses. Among RAS components, renin, its precursor (pro)renin and its specific receptor (PRR) have been less investigated, particularly in the brain. We previously showed the presence of PRR in neurons and glial cells in the nigrostriatal system of rodents and primates, including humans. Now, we used rat and mouse models and cultures of BV2 and primary microglial cells to study the role of PRR in microglial pro-inflammatory responses. PRR was upregulated in the nigral region, particularly in microglia during the neuroinflammatory response. In the presence of the angiotensin type-1 receptor blocker losartan, to exclude angiotensin-related effects, treatment of microglial cells with (pro)renin induces the expression of microglial pro-inflammatory markers, which is mediated by upregulation of NADPH-oxidase and Rho-kinase activities, downregulation of autophagy and upregulation of inflammasome activity. Conditioned medium from (pro)renin-treated microglia increased dopaminergic cell death relative to medium from non-treated microglia. However, these effects were blocked by pre-treatment of microglia with the Rho-kinase inhibitor fasudil. Activation of microglial PRR enhances the microglial pro-inflammatory response and deleterious effects of microglia on dopaminergic cells, and microglial NADPH-oxidase, Rho-Kinase and autophagy are involved in this process.

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.

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.

Angiotensin type 2 receptors: Role in aging and neuroinflammation in the substantia nigra.

Overactivity of the angiotensin-type-1 receptor (AT1)/NADPH-oxidase axis enhances aging processes, neuroinflammation and neurodegeneration. The role of AT2 receptors in the above-mentioned AT1-related effects in the aged brain, particularly substantia nigra, was investigated in this study. In the nigra, we observed a progressive decrease in AT2 mRNA expression with aging, and AT2 deletion led to changes in spontaneous motor behavior, dopamine receptors, renin-angiotensin system, and pro-oxidative and pro-inflammatory markers similar to those observed in aged wild type (WT) mice. Both aged WT mice and young AT2 KO mice showed an increased AT1, decreased MAS receptor and increased angiotensinogen mRNA and/or protein expression, as well as upregulation of pro-oxidative and pro-inflammatory markers. In cultures of microglial cells, activation of AT2 receptors inhibited the LPS-induced increase in AT1 mRNA and protein expression and neuroinflammatory markers. Both in AT2 KO microglial cultures and microglia obtained from adult AT2 KO mice, an increase in AT1 mRNA expression was observed. In cultured dopaminergic neurons, AT2 activation down-regulated AT1 mRNA and protein, and dopaminergic neurons from adult AT2 KO mice showed upregulation of AT1 mRNA expression. Both in microglia and dopaminergic neurons the pathway AT2/nitric oxide/cyclic guanosine monophosphate mediates the regulation of the AT1 mRNA and protein expression through downregulation of the Sp1 transcription factor. MAS receptors are also involved in the regulation of AT1 mRNA and protein expression by AT2. The results suggest that an aging-related decrease in AT2 expression plays a major role in the aging-related AT1 overexpression and AT1-related pro-inflammatory pro-oxidative effects.

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.