Ana Clara Cristóvão

NADPH Oxidase 1-Mediated Oxidative Stress Leads to Dopamine Neuron Death in Parkinson's Disease

AIM Oxidative stress has long been considered as a major contributing factor in the pathogenesis of Parkinsons disease. However, molecular sources for reactive oxygen species in Parkinsons disease have not been clearly elucidated. Herein, we sought to investigate whether a superoxide-producing NADPH oxidases (NOXs) are implicated in oxidative stress-mediated dopaminergic neuronal degeneration. RESULTS Expression of various Nox isoforms and cytoplasmic components were investigated in N27, rat dopaminergic cells. While most of Nox isoforms were constitutively expressed, Nox1 expression was significantly increased after treatment with 6-hydroxydopamine. Rac1, a key regulator in the Nox1 system, was also activated. Striatal injection of 6-hydroxydopamine increased Nox1 expression in dopaminergic neurons in the rat substantia nigra. Interestingly, it was localized into the nucleus, and immunostaining for DNA oxidative stress marker, 8-oxo-dG, was increased. Nox1 expression was also found in the nucleus of dopaminergic neurons in the substantia nigra of Parkinsons disease patients. Adeno-associated virus-mediated Nox1 knockdown or Rac1 inhibition reduced 6-hydroxydopamine-induced oxidative DNA damage and dopaminergic neuronal degeneration significantly. INNOVATION Nox1/Rac1 could serve as a potential therapeutic target for Parkinsons disease. CONCLUSION We provide evidence that dopaminergic neurons are equipped with the Nox1/Rac1 superoxide-generating system. Stress-induced Nox1/Rac1 activation causes oxidative DNA damage and neurodegeneration. Reduced dopaminergic neuronal death achieved by targeting Nox1/Rac1, emphasizes the impact of oxidative stress caused by this system on the pathogenesis and therapy in Parkinsons disease.

Astrocyte-derived GDNF is a potent inhibitor of microglial activation.

Neuroinflammation is recognized as a major factor in Parkinsons disease (PD) pathogenesis and increasing evidence propose that microglia is the main source of inflammation contributing to the dopaminergic degeneration observed in PD. Several studies suggest that astrocytes could act as physiological regulators preventing excessive microglia responses. However, little is known regarding how astrocytes modulate microglial activation. In the present study, using Zymosan A-stimulated midbrain microglia cultures, we showed that astrocytes secrete factors capable of modulating microglial activation, namely its phagocytic activity and the production of reactive oxygen species since both parameters were highly diminished in cells incubated with astrocytes conditioned media (ACM). Glial cell line-derived neurotrophic factor (GDNF), cerebral dopamine neurotrophic factor (CDNF) and brain-derived neurotrophic factor (BDNF), known to have a neuroprotective role in the nigrostriatal system, are among the candidates to be astrocyte-secreted molecules involved in the modulation of microglial activation. The effect of ACM on Zymosan A-induced microglial activation was abolished when the GDNF present in the ACM was abrogated using a specific antibody, but not when ACM was neutralized with anti-CDNF, anti-BDNF or with a heat-inactivated GDNF antibody. In addition, media conditioned by astrocytes silenced for GDNF were not able to prevent microglial activation, whereas supplementation of non-conditioned media with GDNF prevented the activation of microglia evoked by Zymosan A. Taken together, these results indicate that astrocyte-derived GDNF plays a major contribution to the control of midbrain microglial activation, suggesting that GDNF can protect from neurodegeneration through the inhibition of neuroinflammation.

Neuroprotective and anti-inflammatory properties of a coffee component in the MPTP model of Parkinson's disease.

Consumption of coffee is associated with reduced risk of Parkinson’s disease (PD), an effect that has largely been attributed to caffeine. However, coffee contains numerous components that may also be neuroprotective. One of these compounds is eicosanoyl-5-hydroxytryptamide (EHT), which ameliorates the phenotype of α-synuclein transgenic mice associated with decreased protein aggregation and phosphorylation, improved neuronal integrity and reduced neuroinflammation. Here, we sought to investigate if EHT has an effect in the MPTP model of PD. Mice fed a diet containing EHT for four weeks exhibited dose-dependent preservation of nigral dopaminergic neurons following MPTP challenge compared to animals given control feed. Reductions in striatal dopamine and tyrosine hydroxylase content were also less pronounced with EHT treatment. The neuroinflammatory response to MPTP was markedly attenuated, and indices of oxidative stress and JNK activation were significantly prevented with EHT. In cultured primary microglia and astrocytes, EHT had a direct anti-inflammatory effect demonstrated by repression of lipopolysaccharide-induced NFκB activation, iNOS induction, and nitric oxide production. EHT also exhibited a robust anti-oxidant activity in vitro. Additionally, in SH-SY5Y cells, MPP+-induced demethylation of phosphoprotein phosphatase 2A (PP2A), the master regulator of the cellular phosphoregulatory network, and cytotoxicity were ameliorated by EHT. These findings indicate that the neuroprotective effect of EHT against MPTP is through several mechanisms including its anti-inflammatory and antioxidant activities as well as its ability to modulate the methylation and hence activity of PP2A. Our data, therefore, reveal a strong beneficial effect of a novel component of coffee in multiple endpoints relevant to PD.

NADPH Oxidase 1 Mediates α-Synucleinopathy in Parkinson's Disease

Accumulation of misfolded α-synuclein is the pathological hallmark of Parkinsons disease (PD). Nevertheless, little is known about the mechanism contributing to α-synuclein aggregation and its further toxicity to dopaminergic neurons. Since oxidative stress can increase the expression and aggregation levels of α-synuclein, NADPH oxidases (Noxs), which are responsible for reactive oxygen species generation, could be major players in α-synucleinopathy. Previously, we demonstrated that Nox1 is expressed in dopaminergic neurons of the PD animal models as well as postmortem brain tissue of PD patients, and is responsible for oxidative stress and subsequent neuronal degeneration. Here, using paraquat (PQ)-based in vitro and in vivo PD models, we show that Nox1 has a crucial role in modulating the behavior of α-synuclein expression and aggregation in dopaminergic neurons. We observed in differentiated human dopaminergic cells that Nox1 and α-synuclein expressions are increased under PQ exposure. Nox1 knockdown significantly reduced both α-synuclein expression and aggregation, supporting the role of Nox1 in this process. Furthermore, in rats exposed to PQ, the selective knockdown of Nox1 in the substantia nigra, using adeno-associated virus encoding Nox1-specific shRNA, largely attenuated the PQ-mediated increase of α-synuclein and ubiquitin expression levels as well as α-synuclein aggregates (proteinase K resistant) and A11 oligomers. Significant reductions in oxidative stress level and dopaminergic neuronal loss were also observed. Our data reveal a new mechanism by which α-synuclein becomes a neuropathologic protein through Nox1-mediated oxidative stress. This finding may be used to generate new therapeutic interventions that slower the rate of α-synuclein aggregation and the progression of PD pathogenesis.

Rodent models of Parkinson's disease: beyond the motor symptomatology

Parkinsons disease (PD) is classically characterized by motor symptoms; however, non-motor symptoms (NMS) are increasingly recognized as relevant in disease-state, given the associated alterations in mood (depression and anxiety) and cognition. Here, particularly in regards to NMS, we aimed to compare the motor, emotional and cognitive behavior of three animal models of PD that trigger dopaminergic (DAergic) degeneration on both brain hemispheres: (i) the 6-hydroxydopamine (6-OHDA, 8 or 6 μg) lesion model; (ii) the paraquat (PQ) induced model, and (iii) a genetic model based on α-synuclein overexpression (α-syn). 6-OHDA and α-syn vector were injected bilaterally in the substantia nigra pars compacta (SNpc) of adult male Wistar rats; as for PQ delivery, micro-osmotic pumps were implanted in the interscapular region. Motor deficits were observed in all models, with histological analysis of tyrosine hydroxylase positive cells in the SNpc revealing a significant loss of DAergic neurons in all animal models. In addition, the α-syn animal model also presented a reduction in exploratory activity, and the 6-OHDA and PQ animals displayed a significant increase in both depressive- and anxiety-like behavior. Interestingly, cognitive impairment (working memory) was only observed in the 6-OHDA model. Overall, these PD models are suitable for mimicking the motor symptoms associated to PD, with each encompassing other relevant NMS components of the disorder that may prove beneficial for further studies in PD.

GDNF contributes to oestrogen-mediated protection of midbrain dopaminergic neurones.

Parkinson’s disease (PD) is characterised by the preferential loss of dopaminergic neurones from the substantia nigra (SN) that leads to the hallmark motor disturbances. Animal and human studies suggest a beneficial effect of oestrogen to the nigrostriatal system, and the regulation of neurotrophic factor expression by oestrogens has been suggested as a possible mechanism contributing to that neuroprotective effect. The present study was designed to investigate whether the neuroprotection exerted by 17β‐oestradiol on nigrostriatal dopaminergic neurones is mediated through the regulation of glial cell line‐derived neurotrophic factor (GDNF) expression. Using an in vivo rat model of PD, we were able to confirm the relevance of 17β‐oestradiol in defending dopaminergic neurones against 6‐hydroxydopamine (6‐OHDA) toxicity. 17β‐oestradiol, released by micro‐osmotic pumps, implanted 10 days before intrastriatal 6‐OHDA injection, prevented the loss of dopaminergic neurones induced by 6‐OHDA. 17β‐oestradiol treatment also promoted an increase in GDNF protein levels both in the SN and striatum. To explore the relevance of GDNF increases to 17β‐oestradiol neuroprotection, we analysed, in SN neurone‐glia cultures, the effect of GDNF antibody neutralisation and RNA interference‐mediated GDNF knockdown. The results showed that both GDNF neutralisation and GDNF silencing abolished the dopaminergic protection provided by 17β‐oestradiol against 6‐OHDA toxicity. Taken together, these results strongly identify GDNF as an important player in 17β‐oestradiol‐mediated dopaminergic neuroprotection.

Retinoic acid-loaded polymeric nanoparticles induce neuroprotection in a mouse model for Parkinson's disease

Retinoic acid (RA) plays an important role in the commitment, maturation and survival of neural cells. Recently, RA was pointed as a therapeutic option for some neurodegenerative diseases, including Parkinsons disease (PD). The administration of RA has been defying, and in this sense we have previously developed novel RA-loaded polymeric nanoparticles (RA-NPs) that ensure the efficient intracellular transport and controlled release of RA. Herein, we show that nanoformulation as an efficient neuroprotective effect on dopaminergic (DA) neurons in the 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) induced mouse model for PD. The results showed that the RA-NPs administration induced a significant reduction of DA neuron loss in the substantia nigra (SN) as well as their neuronal fiber/axonal innervations in the striatum. Furthermore, we observed an increase in the expression levels of the transcription factors Pitx3 and Nurr1 induced by RA-NPs, showing its supportive effect on the development and functional maintenance of DA neurons in PD. This is the first study showing that RA-NPs can be an innovative strategy to halt the progression of PD pathogenesis, suggesting that this nanoformulation could be of particular interest for the development of new approaches for PD therapeutics.

Histamine induces microglia activation and dopaminergic neuronal toxicity via H1 receptor activation

BackgroundHistamine is an amine widely known as a peripheral inflammatory mediator and as a neurotransmitter in the central nervous system. Recently, it has been suggested that histamine acts as an innate modulator of microglial activity. Herein, we aimed to disclose the role of histamine in microglial phagocytic activity and reactive oxygen species (ROS) production and to explore the consequences of histamine-induced neuroinflammation in dopaminergic (DA) neuronal survival.MethodsThe effect of histamine on phagocytosis was assessed both in vitro by using a murine N9 microglial cell line and primary microglial cell cultures and in vivo. Cells were exposed to IgG-opsonized latex beads or phosphatidylserine (PS) liposomes to evaluate Fcγ or PS receptor-mediated microglial phagocytosis, respectively. ROS production and protein levels of NADPH oxidases and Rac1 were assessed as a measure of oxidative stress. DA neuronal survival was evaluated in vivo by counting the number of tyrosine hydroxylase-positive neurons in the substantia nigra (SN) of mice.ResultsWe found that histamine triggers microglial phagocytosis via histamine receptor 1 (H1R) activation and ROS production via H1R and H4R activation. By using apocynin, a broad NADPH oxidase (Nox) inhibitor, and Nox1 knockout mice, we found that the Nox1 signaling pathway is involved in both phagocytosis and ROS production induced by histamine in vitro. Interestingly, both apocynin and annexin V (used as inhibitor of PS-induced phagocytosis) fully abolished the DA neurotoxicity induced by the injection of histamine in the SN of adult mice in vivo. Blockade of H1R protected against histamine-induced Nox1 expression and death of DA neurons in vivo.ConclusionsOverall, our results highlight the relevance of histamine in the modulation of microglial activity that ultimately may interfere with neuronal survival in the context of Parkinson’s disease (PD) and, eventually, other neurodegenerative diseases which are accompanied by microglia-induced neuroinflammation. Importantly, our results also open promising new perspectives for the therapeutic use of H1R antagonists to treat or ameliorate neurodegenerative processes.

Daily consumption of white tea ( Camellia sinensis (L.)) improves the cerebral cortex metabolic and oxidative profile in prediabetic Wistar rats

Diabetes mellitus (DM) is a major public health problem and its incidence is rising dramatically. The brain, particularly the cerebral cortex, is very susceptible to glucose fluctuations and hyperglycaemia-induced oxidative stress. Tea (Camellia sinensis (L.)) is widely consumed; however, the antidiabetic properties of white tea remain largely unexplored. In the present study, we investigated the effects of daily consumption of white tea on the cerebral cortex of prediabetic rats. The cerebral cortex metabolic profile was evaluated, and the expression levels of GLUT, phosphofructokinase-1, lactate dehydrogenase (LDH) and monocarboxylate transporter 4 were assessed. LDH activity was also determined. The cerebral cortex oxidative profile was determined by evaluating its antioxidant power, lipid peroxidation and protein oxidation levels. Catalase, glutathione, glutamate, N-acetylaspartate, aspartate, choline, γ-aminobutyric acid, taurine and valine contents were determined. Daily consumption of white tea ameliorated glucose tolerance and insulin sensitivity. Moreover, white tea altered the cortex glycolytic profile, modulating GLUT expression and lactate and alanine contents. Finally, white tea consumption restored protein oxidation and lipid peroxidation levels and catalase expression, and improved antioxidant capacity. In conclusion, daily consumption of white tea improved the cerebral cortex metabolic and oxidative profile in prediabetic rats, suggesting it as a good, safe and inexpensive strategy to prevent DM-related effects in the cerebral cortex.

Microglia of rat ventral midbrain recovers its resting state over time in vitro: Let microglia rest before work

Cortical or total brain cultures of microglia are commonly used as a model to study the inflammatory processes in Parkinsons disease. Here we characterize microglia cultures from rat ventral midbrain and evaluate their response to zymosan A. We used specific markers of microglia and evaluated the morphology, the phagocytic activity and reactive oxygen species (ROS) levels of the cells. During the first 10 days in vitro (DIV), cultures presented predominantly cells with a round morphology, expressing CD68 and with high phagocytic activity and ROS production. After 13 DIV, this tendency was reversed, with cultures showing higher number of ramified cells and fewer CD68+ cells along with lower phagocytic and ROS production capability, suggesting that microglia must be kept in vitro for at least 13 days to recover its resting state. The exposure of cultures with less than 10 DIV to zymosan A significantly decreased cell viability. Exposure of cultures with 13 DIV to zymosan A (0.05, 0.5, or 5 μg/ml) increased the total cell number, the percentage of CD68+ cells, and the phagocytic activity. Concentrations of zymosan A higher than 5 μg/ml were also effective in activating microglia but significantly decreased the number of viable cells. In summary, microglial cells remain in the activated state for several days after the isolation process and, thus, stimulation of microglia recently isolated can compromise interpretation of the results. However, upon 13 DIV, cells achieve properties of nonactivated microglia and present a characteristic response to a proinflammatory agent.