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The key link between renin–angiotensin system (RAS) and COVID-19 is ACE2 (angiotensin-converting enzyme 2), which acts as a double-edged sword, because ACE2 increases the tissue anti-inflammatory response but it is also the entry receptor for the virus. There is an important controversy on several drugs that regulate RAS activity and possibly ACE2, and are widely used, particularly by patients most vulnerable to severe COVID-19. In the lung of healthy rats, we observed that candesartan (an angiotensin type-1, AT1, receptor blocker; ARB) and captopril (an ACE inhibitor; ACEI) up-regulated expression of tissue ACE2 and RAS anti-inflammatory axis receptors (AT2 and Mas receptors). This effect was particularly pronounced in rats with metabolic syndrome (obesity, increased blood pressure and hyperglycemia) and aged rats. Treatment of cultures of human type-II pneumocytes with candesartan or captopril induced up-regulation of ACE2 expression in cells. Treatment with viral spike protein induced a decrease in full-length (i.e. transmembrane) ACE2, an increase in levels of a short intracellular ACE2 polypeptide and an increase in ADAM17 activity in cells, together with an increase in levels of soluble ACE2 and major proinflammatory cytokines in the culture medium. Spike protein-induced changes and levels of spike protein internalization in cells were inhibited by pretreatment with the above-mentioned drugs. The results suggest that these drugs increase ACE2 levels and promote the anti-inflammatory RAS axis in the lung. Furthermore, possible up-regulation of viral entry by the drug-induced increase in expression of transmembrane ACE2 is counteracted by additional mechanisms, particularly by drug-induced inhibition of ADAM17 activity.

The renin–angiotensin system (RAS) not only plays an important role in controlling blood pressure but also participates in almost every process to maintain homeostasis in mammals. Interest has recently increased because SARS viruses use one RAS component (ACE2) as a target-cell receptor. The occurrence of RAS in the basal ganglia suggests that the system may be targeted to improve the therapy of neurodegenerative diseases. RAS-related data led to the hypothesis that RAS receptors may interact with each other. The aim of this paper was to find heteromers formed by Mas and angiotensin receptors and to address their functionality in neurons and microglia. Novel interactions were discovered by using resonance energy transfer techniques. The functionality of individual and interacting receptors was assayed by measuring levels of the second messengers cAMP and Ca2+ in transfected human embryonic kidney cells (HEK-293T) and primary cultures of striatal cells. Receptor complex expression was assayed by in situ proximity ligation assay. Functionality and expression were assayed in parallel in primary cultures of microglia treated or not with lipopolysaccharide and interferon-γ (IFN-γ). The proximity ligation assay was used to assess heteromer expression in parkinsonian and dyskinetic conditions. Complexes formed by Mas and the angiotensin AT1 or AT2 receptors were identified in both a heterologous expression system and in neural primary cultures. In the heterologous system, we showed that the three receptors—MasR, AT1R, and AT2R—can interact to form heterotrimers. The expression of receptor dimers (AT1R-MasR or AT2R-MasR) was higher in microglia than in neurons and was differentially affected upon microglial activation with lipopolysaccharide and IFN-γ. In all cases, agonist-induced signaling was reduced upon coactivation, and in some cases just by coexpression. Also, the blockade of signaling of two receptors in a complex by the action of a given (selective) receptor antagonist (cross-antagonism) was often observed. Differential expression of the complexes was observed in the striatum under parkinsonian conditions and especially in animals rendered dyskinetic by levodopa treatment. The negative modulation of calcium mobilization (mediated by AT1R activation), the multiplicity of possibilities on RAS affecting the MAPK pathway, and the disbalanced expression of heteromers in dyskinesia yield new insight into the operation of the RAS system, how it becomes unbalanced, and how a disbalanced RAS can be rebalanced. Furthermore, RAS components in activated microglia warrant attention in drug-development approaches to address neurodegeneration. Supplementary Information The online version contains supplementary material available at 10.1007/s13311-020-00986-4.

According to Braak’s hypothesis, it is plausible that Parkinsońs disease (PD) starts in the enteric nervous system (ENS) to spread the brain via the vagus nerve. Thus, we were wondering whether human inflammatory bowel diseases (IBD) can progress with appearance of pathogenic α-synuclein (α-syn) in the gastrointestinal tract and midbrain dopaminergic neurons. Analysis of human gastrointestinal tract sections from IBD patients demonstrated the presence of pathogenic phosphorylated α-syn in both myenteric (Auerbach’s) and submucosal (Meissner’s) plexuses. Remarkably, PD subjects exhibit α-syn pathology in identical gastrointestinal locations. Analysis of human midbrain sections from IBD subjects revealed a clear displacement of neuromelanin in some nigral neurons from the ventral mesencephalon, which were inherently associated with presence of α-syn aggregates reminiscent of pale bodies. We also used different dextran sodium sulfate (DSS)-based rat models of gut inflammation (subchronic and chronic) to study the appearance of phosphorylated α-syn inclusions in both Auerbach’s and Meissner’s plexuses (gut), and in dopaminergic neuritic processes (brain) along with degeneration of nigral dopaminergic neurons, which are considered classical hallmarks of PD. Vagotomized DSS-treated animals exhibited pathological α-syn in the gut but failed to show dopaminergic cells degeneration and α-syn aggregation in the ventral mesencephalon. Taken together, these results strongly suggest that Braak’s hypothesis is plausible.

In the last decade, attention has become greater to the relationship between neurodegeneration and abnormal insulin signaling in the central nervous system, as insulin in the brain is implicated in neuronal survival, plasticity, oxidative stress and neuroinflammation. Diabetes mellitus and Parkinson’s disease are both aging-associated diseases that are turning into epidemics worldwide. Diabetes mellitus and insulin resistance not only increase the possibility of developing Parkinson’s disease but can also determine the prognosis and progression of Parkinsonian symptoms. Today, there are no available curative or disease modifying treatments for Parkinson’s disease, but the role of insulin and antidiabetic medications in neurodegeneration opens a door to treatment repurposing to fight against Parkinson’s disease, both in diabetic and nondiabetic Parkinsonian patients. Furthermore, it is essential to comprehend how a frequent and treatable disease such as diabetes can influence the progression of neurodegeneration in a challenging disease such as Parkinson’s disease. Here, we review the present evidence on the connection between Parkinson’s disease and diabetes and the consequential implications of the existing antidiabetic molecules in the severity and development of Parkinsonism, with a particular focus on glucagon-like peptide-1 receptor agonists.

The metabolic syndrome has been associated to chronic peripheral inflammation and related with neuroinflammation and neurodegeneration, including Parkinson's disease. However, the responsible mechanisms are unclear. Previous studies have involved the brain renin-angiotensin system in progression of Parkinson's disease and the angiotensin receptor type 1 (AT1) has been recently revealed as a major marker of dopaminergic vulnerability in humans. Dysregulation of tissue renin-angiotensin system is a key common mechanism for all major components of metabolic syndrome. Circulating AT1 agonistic autoantibodies have been observed in several inflammation-related peripheral processes, and activation of AT1 receptors of endothelial cells, dopaminergic neurons and glial cells have been observed to disrupt endothelial blood -brain barrier and induce neurodegeneration, respectively. Using a rat model, we observed that metabolic syndrome induces overactivity of nigral pro-inflammatory renin-angiotensin system axis, leading to increase in oxidative stress and neuroinflammation and enhancing dopaminergic neurodegeneration, which was inhibited by treatment with AT1 receptor blockers (ARBs). In rats, metabolic syndrome induced the increase in circulating levels of LIGHT and other major pro-inflammatory cytokines, and 27-hydroxycholesterol. Furthermore, the rats showed a significant increase in serum levels of proinflammatory AT1 and angiotensin converting enzyme 2 (ACE2) autoantibodies, which correlated with levels of several metabolic syndrome parameters. We also found AT1 and ACE2 autoantibodies in the CSF of these rats. Effects of circulating autoantibodies were confirmed by chronic infusion of AT1 autoantibodies, which induced blood-brain barrier disruption, an increase in the pro-inflammatory renin-angiotensin system activity in the substantia nigra and a significant enhancement in dopaminergic neuron death in two different rat models of Parkinson's disease. Observations in the rat models, were analyzed in a cohort of parkinsonian and non-parkinsonian patients with or without metabolic syndrome. Non-parkinsonian patients with metabolic syndrome showed significantly higher levels of AT1 autoantibodies than non-parkinsonian patients without metabolic syndrome. However, there was no significant difference between parkinsonian patients with metabolic syndrome or without metabolic syndrome, which showed higher levels of AT1 autoantibodies than non-parkinsonian controls. This is consistent with our recent studies, showing significant increase of AT1 and ACE2 autoantibodies in parkinsonian patients, which was related to dopaminergic degeneration and neuroinflammation. Altogether may lead to a vicious circle enhancing the progression of the disease that may be inhibited by strategies against production of these autoantibodies or AT1 receptor blockers (ARBs).

The tissue renin-angiotensin system (RAS) has been shown to be involved in prooxidative and proinflammatory changes observed in aging and aging-related diseases such as dopaminergic degeneration in Parkinson's disease (PD). We studied the activation of the NLRP3 inflammasome in the

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]. Keywords: NRF2, KLF9, Dopaminergic, Redox signaling, Oxidative stress, Renin-angiotensin system

Mutations in the GBA1 gene coding for glucocerebrosidase (GCase) are the main genetic risk factor for Parkinson’s disease (PD). Indeed, identifying reduced GCase activity as a common feature underlying the typical neuropathological signatures of PD—even when considering idiopathic forms of PD—has recently paved the way for designing novel strategies focused on enhancing GCase activity to reduce alpha-synuclein burden and preventing dopaminergic cell death. Here we have performed bilateral injections of a viral vector coding for the mutated form of alpha-synuclein (rAAV9-SynA53T) for disease modeling purposes, both in mice as well as in nonhuman primates (NHPs), further inducing a progressive neuronal death in the substantia nigra pars compacta (SNpc). Next, another vector coding for the GBA1 gene (rAAV9-GBA1) was unilaterally delivered in the SNpc of mice and NHPs one month after the initial insult, together with the contralateral delivery of an empty/null rAAV9 for control purposes. Obtained results showed that GCase enhancement reduced alpha-synuclein burden, leading to improved survival of dopaminergic neurons. Data reported here support using GCase gene therapy as a disease-modifying treatment for PD and related synucleinopathies, including idiopathic forms of these disorders.

The renin-angiotensin system (RAS) plays a major role in COVID-19. Severity of several inflammation-related diseases has been associated with autoantibodies against RAS, particularly agonistic autoantibodies for angiotensin type-1 receptors (AA-AT1) and autoantibodies against ACE2 (AA-ACE2). Disease severity of COVID-19 patients was defined as mild, moderate or severe following the WHO Clinical Progression Scale and determined at medical discharge. Serum AA-AT1 and AA-ACE2 were measured in COVID-19 patients (n = 119) and non-infected controls (n = 23) using specific solid-phase, sandwich enzyme-linked immunosorbent assays. Serum LIGHT (TNFSF14; tumor necrosis factor ligand superfamily member 14) levels were measured with the corresponding assay kit. At diagnosis, AA-AT1 and AA-ACE2 levels were significantly higher in the COVID-19 group relative to controls, and we observed significant association between disease outcome and serum AA-AT1 and AA-ACE2 levels. Mild disease patients had significantly lower levels of AA-AT1 (p, Graphical abstract Image 1

Fever in infections correlates with inflammation, macrophage infiltration into the affected organ, macrophage activation, and release of cytokines involved in immune response, hematopoiesis, and homeostatic processes. Angiotensin-converting enzyme 2 (ACE2) is the canonical cell surface receptor for SARS-CoV-2. ACE2 together with angiotensin receptor types 1 and 2 and ACE2 are components of the renin–angiotensin system (RAS). Exacerbated production of cytokines, mainly IL-6, points to macrophages as key to understand differential COVID-19 severity. SARS-CoV-2 may modulate macrophage-mediated inflammation events by altering the balance between angiotensin II, which activates angiotensin receptor types 1 and 2, and angiotensin 1–7 and alamandine, which activate MAS proto-oncogene and MAS-related D receptors, respectively. In addition to macrophages, lung cells express RAS components; also, some lung cells are able to produce IL-6. Addressing how SARS-CoV-2 unbalances RAS functionality via ACE2 will help design therapies to attenuate a COVID-19–related cytokine storm.

The physiopathological mechanisms that regulate menopausal and sex differences in colonic transit, inflammatory processes, and efficacy of treatments have not been clarified. The dopaminergic system and renin–angiotensin system coexist in the gut and regulate different processes such as motility, absorption/secretion, and inflammation. We investigated the changes in expression of major angiotensin and dopamine receptors in the colon of male, female, and ovariectomized female mice. Possible interaction between both systems was investigated using male and female mice deficient (ko) for major angiotensin and dopamine receptors. In wild-type mice, colonic tissue from females showed lower angiotensin type 1/angiotensin type 2 ratio (an index of pro-inflammatory/anti-inflammatory renin–angiotensin system balance), lower dopamine D1 and D2 receptor expression, and lower levels of pro-inflammatory and pro-oxidative markers relative to males. Interestingly, ovariectomy increased the expression of pro-inflammatory angiotensin type 1 receptor expression and decreased anti-inflammatory angiotensin type 2 receptor expression, increased D1 and D2 receptor expression, and increased the levels of pro-inflammatory and pro-oxidative markers. Ovariectomy-induced changes were blocked by estrogen replacement. The present results suggest a mutual regulation between colonic angiotensin and dopamine receptors and sex differences in this mutual regulation. Estrogen regulates changes in both angiotensin and dopamine receptor expression, which may be involved in sex- and surgical menopause-related effects on gut motility, permeability, and vulnerability to inflammatory processes.

Parkinson's disease and diabetes mellitus are two chronic disorders associated with aging that are becoming increasingly prevalent worldwide. Parkinson is a multifactorial progressive condition with no available disease modifying treatments at the moment. Over the last few years there is growing interest in the relationship between diabetes (and impaired insulin signaling) and neurodegenerative diseases, as well as the possible benefit of antidiabetic treatments as neuroprotectors, even in non-diabetic patients. Insulin regulates essential functions in the brain such as neuronal survival, autophagy of toxic proteins, synaptic plasticity, neurogenesis, oxidative stress and neuroinflammation. We review the existing epidemiological, experimental and clinical evidence that supports the interplay between insulin and neurodegeneration in Parkinson's disease, as well as the role of antidiabetic treatments in this disease.

Background/Aims : The renin-angiotensin system (RAS) is altered in Parkinson’s disease (PD), a disease due to substantia nigra neurodegeneration and whose dopamine-replacement therapy, using the precursor levodopa, leads to dyskinesias as the main side effect. Angiotensin AT 1 and AT 2 receptors, mainly known for their role in regulating water homeostasis and blood pressure and able to form heterodimers (AT 1/2 Hets), are present in the central nervous system. We assessed the functionality and expression of AT 1/2 Hets in Parkinson Disease (PD). Methods: Immunocytochemistry was used to analyze the colocalization between angiotensin receptors, bioluminescence resonance energy transfer was used to detect AT 1/2 Hets. Calcium and cAMP determination, MAPK activation and label-free assays were performed to characterize signaling. Proximity ligation assays was used to quantify receptor expression in microglial cells and brain striatal slices. Results: We confirmed that AT 1 and AT 2 receptors form AT 1/2 Hets that are expressed in cells of the central nervous system. AT 1/2 Hets are novel functional units with particular signaling properties. Importantly, the coactivation of the two receptors in the heteromer reduces the signaling output of angiotensin. Remarkably, AT 1/2 Hets that are expressed in both striatal neurons and microglia show a cross-potentiation, i.e. candesartan, the antagonist of AT 1 increases the effect of AT 2 receptor agonists. In addition, the level of expression in the unilateral 6-OH-dopamine lesion rat PD model increases upon disease progression and is maximal in dyskinetic animals. Conclusion: The results indicate that boosting the action of neuroprotective AT 2 receptors using an AT 1 receptor antagonist constitutes a promising therapeutic strategy in PD.

Background/aims The renin-angiotensin system (RAS) is altered in Parkinson’s disease (PD), a disease due to substantia nigra neurodegeneration and whose dopamine-replacement therapy, using the precursor levodopa, leads to dyskinesias as the main side effect. Angiotensin AT1 and AT2 receptors, mainly known for their role in regulating water homeostasis and blood pressure and able to form heterodimers (AT1/2Hets), are present in the central nervous system. We assessed the functionality and expression of AT1/2Hets in Parkinson disease (PD). Methods Immunocytochemistry was used to analyze the colocalization between angiotensin receptors; bioluminescence resonance energy transfer was used to detect AT1/2Hets. Calcium and cAMP determination, MAPK activation, and label-free assays were performed to characterize signaling in homologous and heterologous systems. Proximity ligation assays were used to quantify receptor expression in mouse primary cultures and in rat striatal sections. Results We confirmed that AT1 and AT2 receptors form AT1/2Hets that are expressed in cells of the central nervous system. AT1/2Hets are novel functional units with particular signaling properties. Importantly, the coactivation of the two receptors in the heteromer reduces the signaling output of angiotensin. Remarkably, AT1/2Hets that are expressed in both striatal neurons and microglia make possible that candesartan, the antagonist of AT1, increases the effect of AT2 receptor agonists. In addition, the level of striatal expression increased in the unilateral 6-OH-dopamine lesioned rat PD model and was markedly higher in parkinsonian-like animals that did not become dyskinetic upon levodopa chronic administration if compared with expression in those that became dyskinetic. Conclusion The results indicate that boosting the action of neuroprotective AT2 receptors using an AT1 receptor antagonist constitutes a promising therapeutic strategy in PD.

Highlights • In addition to the endocrine and paracrine RAS there is an intracellular RAS (iRAS). • The iRAS functions and interactions with the paracrine and endocrine RAS are unclear. • Observations in mitochondria and nucleus of neurons may clarify the role of the iRAS., The renin-angiotensin system (RAS) is one of the oldest hormone systems in vertebrate phylogeny. RAS was initially related to regulation of blood pressure and sodium and water homeostasis. However, local or paracrine RAS were later identified in many tissues, including brain, and play a major role in their physiology and pathophysiology. In addition, a major component, ACE2, is the entry receptor for SARS-CoV-2. Overactivation of tissue RAS leads several oxidative stress and inflammatory processes involved in aging-related degenerative changes. In addition, a third level of RAS, the intracellular or intracrine RAS (iRAS), with still unclear functions, has been observed. The possible interaction between the intracellular and extracellular RAS, and particularly the possible deleterious or beneficial effects of the iRAS activation are controversial. The dopaminergic system is particularly interesting to investigate the RAS as important functional interactions between dopamine and RAS have been observed in the brain and several peripheral tissues. Our recent observations in mitochondria and nucleus of dopaminergic neurons may clarify the role of the iRAS. This may be important for the developing of new therapeutic strategies, since the effects on both extracellular and intracellular RAS must be taken into account, and perhaps better understanding of COVID-19 cell mechanisms.

The renin-angiotensin system (RAS) is classically known as a circulating hormonal system that regulates blood pressure. A second level of RAS (the paracrine or tissular RAS) was identified in several tissues and organs, including brain. Finally, an intracellular or intracrine RAS has also been observed. In the nigrostriatal system, a decrease in dopamine levels induces RAS dysregulation. This may constitute a counterregulatory mechanism to increase dopamine levels. However, RAS overactivity also induces neuronal oxidative stress, enhances neuroinflammation, and contributes to progression of dopaminergic neuron death and Parkinson's disease. Additional factors may induce RAS dysregulation: aging, dopaminergic neurotoxins, a loss of estrogen and other mechanisms that inhibit neuroinflammation, and peripheral inflammatory processes. RAS dysregulation acts both on glial cells and dopaminergic neurons. The intraneuronal RAS counteracts the prooxidative effects of overactivation of the paracrine RAS. Experimental manipulation of brain RAS protects against dopaminergic neuron degeneration. However, additional clinical studies are necessary.

Angiotensin-converting enzyme 2 (ACE2) is a membrane peptidase and a component of the renin-angiotensin system (RAS) that has been found in cells of all organs, including the lungs. While ACE2 has been identified as the receptor for severe acute respiratory syndrome (SARS) coronaviruses, the mechanism underlying cell entry remains unknown. Human immunodeficiency virus infects target cells via CXC chemokine receptor 4 (CXCR4)-mediated endocytosis. Furthermore, CXCR4 interacts with dipeptidyl peptidase-4 (CD26/DPPIV), an enzyme that cleaves CXCL12/SDF-1, which is the chemokine that activates this receptor. By analogy, we hypothesized that ACE2 might also be capable of interactions with RAS-associated G-protein coupled receptors. Using resonance energy transfer and cAMP and mitogen-activated protein kinase signaling assays, we found that human ACE2 interacts with RAS-related receptors, namely the angiotensin II type 1 receptor (AT1R), the angiotensin II type 2 receptor (AT2R), and the MAS1 oncogene receptor (MasR). Although these interactions led to various alterations of signal transduction, but, more importantly, ligand binding to AT1R resulted in the downregulation of ACE2 cell surface expression, while ligand binding to AT2R, but not to MasR, resulted in upregulation of ACE2 cell surface expression. Proximity ligation assays performed in situ revealed macromolecular complexes containing ACE2 and AT1R, AT2R or MasR in adult but not fetal mouse lung tissue. These findings highlight the relevance of RAS in SARS-CoV-2 infection and the role of ACE2-containing complexes as potential therapeutic targets The Molecular Neurobiology Laboratory of the University of Barcelona is considered a research group of excellence by the Regional Catalonian Government (grup consolidat #2017 SGR 1497) SI

Background/Aims: The renin-angiotensin system (RAS) is altered in Parkinson’s disease (PD), a disease due to substantia nigra neurodegeneration and whose dopamine-replacement therapy, using the precursor levodopa, leads to dyskinesias as the main side effect. Angiotensin AT1 and AT2 receptors, mainly known for their role in regulating water homeostasis and blood pressure and able to form heterodimers (AT1/2Hets), are present in the central nervous system. We assessed the functionality and expression of AT1/2Hets in Parkinson Disease (PD).Methods: Immunocytochemistry was used to analyze the colocalization between angiotensin receptors, bioluminescence resonance energy transfer was used to detect AT1/2Hets. Calcium and cAMP determination, MAPK activation and label-free assays were performed to characterize signaling in homologous and heterologous systems. Proximity ligation assays was used to quantify receptor expression in mouse primary cultures and in rat striatal sections.Results: We confirmed that AT1 and AT2 receptors form AT1/2Hets that are expressed in cells of the central nervous system. AT1/2Hets are novel functional units with particular signaling properties. Importantly, the coactivation of the two receptors in the heteromer reduces the signaling output of angiotensin. Remarkably, AT1/2Hets that are expressed in both striatal neurons and microglia show a cross-potentiation, i.e. candesartan, the antagonist of AT1 increases the effect of AT2 receptor agonists. In addition, the level of striatal expression increased in the unilateral 6-OH-dopamine lesioned rat PD model and was markedly higher in parkinsonian-like animals that did not become dyskinetic upon levodopa chronic administration if compared with expression in those that became dyskinetic.Conclusion: The results indicate that boosting the action of neuroprotective AT2 receptors using an AT1 receptor antagonist constitutes a promising therapeutic strategy in PD.

The exact mechanism of gut dysfunction in Parkinson's disease and, conversely, the role of gut pathology in brain dopaminergic degeneration are controversial. We investigated the effects of nigral lesions on the colonic neurotransmission, the effect of gut inflammation on the nigrostriatal dopaminergic function, and the possible involvement of the vagus nerve and the local renin-angiotensin system (RAS). Nigrostriatal dopamine depletion was performed by bilateral injection 6-hydroxydopamine, and gut inflammation was induced by dextran sulfate sodium salt treatment in rats and mice, respectively, with or without vagal disruption. A decrease in central dopamine levels induced a decrease in colonic dopamine types 1 and 2 receptor expression together with an increase in the colonic levels of dopamine and a decrease in the levels of acetylcholine, which may explain a decrease in gut motility. Central dopaminergic depletion also induced an increase in the colonic levels of inflammatory and oxidative stress markers together with activation of the pro-inflammatory arm of the local RAS. Mice with acute (1 week) or subchronic (3 weeks) gut inflammation did not show a significant increase in colonic α-synuclein and phosphorylated α-synuclein expression during this relatively short survival period. Interestingly, we observed early changes in the nigrostriatal dopaminergic homeostasis, dopaminergic neuron death, and increased levels of nigral pro-inflammatory markers and RAS pro-inflammatory activity. The present results show that a dysregulation of the neural bidirectional gut-brain interaction may explain the early gut disturbances observed in parkinsonian patients, and also the increase in vulnerability of nigral dopaminergic neurons after gut inflammation.

Endocannabinoids are important regulators of neurotransmission and, acting on activated microglia, they are postulated as neuroprotective agents. Endocannabinoid action is mediated by CB1 and CB2 receptors, which may form heteromeric complexes (CB1-CB2Hets) with unknown function in microglia. We aimed at establishing the expression and signaling properties of cannabinoid receptors in resting and LPS/IFN-γ-activated microglia. In activated microglia mRNA transcripts increased (2 fold for CB1 and circa 20 fold for CB2), whereas receptor levels were similar for CB1 and markedly upregulated for CB2; CB1-CB2Hets were also upregulated. Unlike in resting cells, CB2 receptors became robustly coupled to Gi in activated cells, in which CB1-CB2Hets mediated a potentiation effect. Hence, resting cells were refractory while activated cells were highly responsive to cannabinoids. Interestingly, similar results were obtained in cultures treated with s-amyloid (As1-42). Microglial activation markers were detected in the striatum of a Parkinson's disease (PD) model and, remarkably, in primary microglia cultures from the hippocampus of mutant β-amyloid precursor protein (APPSw,Ind) mice, a transgenic Alzheimer's disease (AD) model. Also of note was the similar cannabinoid receptor signaling found in primary cultures of microglia from APPSw,Ind and in cells from control animals activated using LPS plus IFN-γ. Expression of CB1-CB2Hets was increased in the striatum from rats rendered dyskinetic by chronic levodopa treatment. In summary, our results showed sensitivity of activated microglial cells to cannabinoids, increased CB1-CB2Het expression in activated microglia and in microglia from the hippocampus of an AD model, and a correlation between levodopa-induced dyskinesia and striatal microglial activation in a PD model. Cannabinoid receptors and the CB1-CB2 heteroreceptor complex in activated microglia have potential as targets in the treatment of neurodegenerative diseases.

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.

ACE2 plays a pivotal role in the balance between the pro-oxidative pro-inflammatory and the anti-oxidative anti-inflammatory arms of the renin-angiotensin system. Furthermore, ACE2 is the entry receptor for SARS-CoV-2. Clarification of ACE2-related mechanisms is crucial for the understanding of COVID-19 and other oxidative stress and inflammation-related processes. In rat and monkey brain, we discovered that the intracellular ACE2 and its products Ang 1–7 and alamandine are highly concentrated in the mitochondria and bind to a new mitochondrial Mas-related receptor MrgE (MrgE) to produce nitric oxide. We found MrgE expressed in neurons and glia of rodents and primates in the substantia nigra and different brain regions. In the mitochondria, ACE2 and MrgE expressions decreased and NOX4 increased with aging. This new ACE2/MrgE/NO axis may play a major role in mitochondrial regulation of oxidative stress in neurons, and possibly other cells. Therefore, dysregulation of the mitochondrial ACE2/MrgE/NO axis may play a major role in neurodegenerative processes of dopaminergic neurons, where mitochondrial dysfunction and oxidative stress play a crucial role. Since ACE2 binds SARS-CoV-2 spike protein, the mitochondrial ACE2/MrgE/NO axis may also play a role in SARS-CoV-2 cellular effects., Graphical abstract Image 1, Highlights • ACE2 products Ang1-7 and alamandine (Ala) highly concentrate in brain mitochondria. • Ang1-7 and Ala bind to mitochondrial Mas-related receptor MrgE producing nitric oxide. • ACE2/MrgE may play a major role in mitochondrial function and oxidative stress. • Clarification of ACE2-related mechanisms is also crucial for understanding COVID-19.

Glucocerebrosidase (GCase) is a lysosomal enzyme encoded by the GBA1 gene. Mutations in GBA1 gene lead to Gaucher’s disease, the most prevalent lysosomal storage disorder. GBA1 mutations reduce GCase activity, therefore promoting the aggregation of alpha-synuclein, a common neuropathological finding underlying Parkinson’s disease (PD) and dementia with Lewy bodies. However, it is also worth noting that a direct link between GBA1 mutations and alpha-synuclein aggregation indicating cause and effect is still lacking, with limited experimental evidence to date. Bearing in mind that a number of strategies increasing GCase expression for the treatment of PD are currently under development, here we sought to analyze the baseline expression of GCase in the brain of Macaca fascicularis, which has often been considered as the gold-standard animal model of PD. Although as with other lysosomal enzymes, GCase is expected to be ubiquitously expressed, here a number of regional variations have been consistently found, together with several specific neurochemical phenotypes expressing very high levels of GCase. In this regard, the most enriched expression of GCase was constantly found in cholinergic neurons from the nucleus basalis of Meynert, dopaminergic cells in the substantia nigra pars compacta, serotoninergic neurons from the raphe nuclei, as well as in noradrenergic neurons located in the locus ceruleus. Moreover, it is also worth noting that moderate levels of expression were also found in a number of areas within the paleocortex and archicortex, such as the entorhinal cortex and the hippocampal formation, respectively.

The renin-angiotensin system (RAS) was initially considered as a circulating humoral system, which function is the regulation of blood pressure. However, it is now known that there exists local RAS in many tissues, including brain. In recent studies, we have demonstrated the presence of a local RAS in the substantia nigra of rodents and primates that modulates dopamine release and dopamine receptor expression. However, overactivation of local RAS exacerbates neuroinflammation, oxidative stress and dopaminergic cell death. In the striatum, it is not clear whether angiotensin receptors are located in dopaminergic terminals, glial cells and/or the projection neurons. The present study shows the location of major components of the RAS in striatal projection neurons of rats and monkeys (both in neurons of the direct and the indirect pathways). Striatal astrocytes and microglial cells also express major RAS components, which increase after induction of neuroinflammation by intrastriatal injection of lipopolysaccharide. Angiotensin receptors were located at the cell surface and also at cytoplasmic and nuclear levels. The results obtained by immunolabeling and confocal microscopy were confirmed with laser microdissection of striatal neurons and glial cells and detection of mRNA expression by PCR. The sequence of the resulting PCR products was verified by DNA sequencing. In addition to the interaction between angiotensin and dopamine receptors in dopaminergic neurons to regulate dopamine release, interaction between angiotensin and dopamine receptors in projection striatal neurons may further modulate the effects of dopamine on the direct and indirect pathways by fine-tuning striatal dopaminergic neurotransmission.

The field of gene therapy has recently witnessed a number of major conceptual changes. Besides the traditional thinking that comprises the use of viral vectors for the delivery of a given therapeutic gene, a number of original approaches have been recently envisaged, focused on using vectors carrying genes to further modify basal ganglia circuits of interest. It is expected that these approaches will ultimately induce a therapeutic potential being sustained by gene-induced changes in brain circuits. Among others, at present, it is technically feasible to use viral vectors to (1) achieve a controlled release of neurotrophic factors, (2) conduct either a transient or permanent silencing of any given basal ganglia circuit of interest, (3) perform an in vivo cellular reprogramming by promoting the conversion of resident cells into dopaminergic-like neurons, and (4) improving levodopa efficacy over time by targeting aromatic L-amino acid decarboxylase. Furthermore, extensive research efforts based on viral vectors are currently ongoing in an attempt to better replicate the dopaminergic neurodegeneration phenomena inherent to the progressive intraneuronal aggregation of alpha-synuclein. Finally, a number of incoming strategies will soon emerge over the horizon, these being sustained by the underlying goal of promoting alpha-synuclein clearance, such as, for instance, gene therapy initiatives based on increasing the activity of glucocerebrosidase. To provide adequate proof-of-concept on safety and efficacy and to push forward true translational initiatives based on these different types of gene therapies before entering into clinical trials, the use of non-human primate models undoubtedly plays an instrumental role.

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.

The local renin-angiotensin system (RAS) and insulin-like growth factor 1 (IGF-1) have been involved in longevity, neurodegeneration and aging-related dopaminergic degeneration. However, it is not known whether IGF-1 and angiotensin-II (AII) activate each other. In the present study, AII, via type 1 (AT1) receptors, exacerbated neuroinflammation and dopaminergic cell death. AII, via AT1 receptors, also increased the levels of IGF-1 and IGF-1 receptors in microglial cells. IGF-1 inhibited RAS activity in dopaminergic neurons and glial cells, and also inhibited the AII-induced increase in markers of the M1 microglial phenotype. Consistent with this, IGF-1 decreased dopaminergic neuron death induced by the neurotoxin MPP+ both in the presence and in the absence of glia. Intraventricular administration of AII to young rats induced a significant increase in IGF-1 expression in the nigral region. However, aged rats showed decreased levels of IGF-1 relative to young controls, even though RAS activity is known to be enhanced in aged animals. The study findings show that IGF-1 and the local RAS interact to inhibit or activate neuroinflammation (i.e. transition from the M1 to the M2 phenotype), oxidative stress and dopaminergic degeneration. The findings also show that this mechanism is impaired in aged animals.

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.

Redox properties enable copper to perform its essential role in many biological processes, but they can also convert it into a potentially hazardous element. Its dyshomeostasis may have serious neurological consequences, and its possible involvement in Parkinson’s disease and other neurodegenerative disorders has been suggested. The in vitro and ex vivo ability of copper to increase oxidative stress has already been demonstrated, and the aim of the present study was to assess in vivo the capacity of copper to cause brain oxidative damage and its ability to increase the dopaminergic degeneration induced by 6-hydroxydopamine. We found that chronic copper administration (10 mg Cu2+/kg/day, IP) causes its accumulation in different brain areas (cortex, striatum, nigra) and was accompanied by an increase in TBARS levels and a decrease in protein free-thiol content in the cortex. A decrease in catalase activity and an increase in glutathione peroxidase activity were also observed in the cortex. The intrastriatal administration of Cu2+ caused an increase in some indices of oxidative stress (TBARS and protein free-thiol content) in striatum and nigra, but was unable to induce dopaminergic degeneration. However, when copper was intrastriatally coadministered with 6-hydroxydopamine, it increased dopaminergic degeneration, a fact that was also accompanied by an increase in the assayed indices of oxidative stress, a decrease in catalase activity, and an augmentation in glutathione activity. Evidently, copper cannot cause neurodegeneration per se, but may potentiate the action of other factors involved in the pathogenesis of Parkinson’s disease through oxidative stress.

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.

The neuroprotective effects of menopausal hormonal therapy in Parkinson's disease have not yet been clarified, and it is not known whether there is a critical period. Estrogen induced significant protection against 6-hydroxydopamine-induced dopaminergic degeneration when administered immediately or 6 weeks, but not 20 weeks after ovariectomy. In the substantia nigra, ovariectomy induced a decrease in levels of estrogen receptor-α and increased angiotensin activity, NADPH-oxidase activity, and expression of neuroinflammatory markers, which were regulated by estrogen administered immediately or 6 weeks but not 20 weeks after ovariectomy. Interestingly, treatment with angiotensin receptor antagonists after the critical period induced a significant level of neuroprotection. In cultures, treatment with 1-methyl-4-phenylpyridinium induced an increase in astrocyte-derived angiotensinogen and dopaminergic neuron death, which were inhibited by estrogen receptor α agonists. In microglial cells, estrogen receptor β agonists inhibited the angiotensin-induced increase in inflammatory markers. The results suggest that there is a critical period for the neuroprotective effect of estrogen against dopaminergic cell death, and local estrogen receptor α and renin-angiotensin system play a major role.

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.

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.

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.