Efeitos da administração intranasal de HSP70 contra denervação dopaminérgica induzida por 6-hidroxidopamina

HSP70 é uma das principais chaperonas moleculares envolvidas na resposta ao estresse celular. Além de sua função como chaperona, a HSP70 pode modular respostas imunes. Quantidades insuficientes da forma induzível da HSP70 são encontradas em neurônios maduros, os tornando mais suscetíveis a alterações no ambiente intra- e extracelular. Por outro lado, a administração de HSP70 exógena demonstrou ter efeitos neuroprotetores em modelos animais de doenças relacionadas ao envelhecimento. Neste trabalho, nós investigamos os efeitos da HSP70 exógena em modelo animal de Doença Parkinson (PD). Após insulto com 6-hidroxidopamina (6-OHDA - injeção intraestriatal unilateral - 20 μg/rato), os animais receberam 15 dias de tratamento com HSP70 recombinante por via intranasal (2 µg/rato/dia). Nossos resultados indicam efeito neuroprotetor da HSP70 intranasal contra a denervação dopaminérgica induzida por 6-OHDA, uma vez que a HSP70 exógena reduziu a perda de neurônios dopaminérgicos e melhorou déficits comportamentais induzidos por 6-OHDA. Além disso, a HSP70 modulou a resposta neuroinflamatória na substância negra, que é uma característica importante da patogênese da PD. Especificamente o tratamento intranasal com HSP70 reduziu a ativação microglial e a astrogliose induzidas por 6-OHDA, bem como reduziu a expressão do mRNA de IL-1β nesta região. Além disso, o tratamento com HSP70 recombinante aumentou o conteúdo de HSP70 na substância negra de ratos induzidos por 6-OHDA. Portanto, nossos resultados indicam que este aumento do conteúdo de HSP70, após o estresse celular, estaria envolvido na proteção dos neurônios dopaminérgicos contra o insulto da 6-OHDA, através de uma modulação da resposta inflamatória.HSP70 is one of the main molecular chaperones involved in the cellular stress response. Beside its chaperone action, HSP70 also modulates immune response. Insufficient amounts of inducible HSP70 were observed in adult neurons, which caused an increased susceptible to toxic insults in intra- and extracellular environment. On the other hand, exogenous HSP70 administration has demonstrated neuroprotective effects in models of age-related disorders. In this study, we investigated the effects of exogenous HSP70 in an animal model of Parkinson's Disease (PD). After the insult with 6-hydroxydopamine (6-OHDA – unilateral intrastriatal injection – 20 μg/rat), the animals received purified recombinant HSP70 through intranasal administration (2 µg/rat/day), during 15 days. Our results indicated a neuroprotective effect of intranasal HSP70 against dopaminergic denervation induced by 6-OHDA, since exogenous HSP70 reduced the loss of dopaminergic neurons and improved behavior impairments induced by 6OHDA. Moreover, HSP70 modulated neuroinflammatory response in the substantia nigra, which is an important event in PD pathogenesis. Specifically, intranasal treatment reduced microglial activation and astrogliosis induced by 6-OHDA, as well reduced IL-1β mRNA expression in this region. In addition, HSP70 treatment increased the content of HSP70 in the substantia nigra of rats induced by 6-OHDA. Therefore, our results indicate that this increase in HSP70 content, after cellular stress, would be involved in the protection of dopaminergic neurons against the insult of 6-OHDA, through a modulation of the inflammatory response

Hypoxia-inducible factor-1α (HIF-1α) inhibition impairs retinoic acid-induced differentiation in SH-SY5Y neuroblastoma cells, leading to reduced neurite length and diminished gene expression related to cell differentiation

Neuroblastoma is the most common extracranial solid tumour in childhood, originated from cells of the neural crest during the development of the Sympathetic Nervous System. Retinoids are vitamin-A derived differentiating agents utilised to avoid disease resurgence in high-risk neuroblastoma treatment. Several studies indicate that hypoxia—a common feature of the tumoural environment—is a key player in cell differentiation and proliferation. Hypoxia leads to the accumulation of the hypoxia-inducible factor-1α (HIF-1α). This work aims to investigate the effects of the selective inhibition of HIF-1α on the differentiation induced by retinoic acid in human neuroblastoma cells from the SH-SY5Y lineage to clarify its role in cell differentiation. Our results indicate that HIF-1α inhibition impairs RA-induced differentiation by reducing neuron-like phenotype and diminished immunolabeling and expression of differentiation markers

Systemic Inflammation Changes the Site of RAGE Expression from Endothelial Cells to Neurons in Different Brain Areas

The receptor for advanced glycation endproducts (RAGE) is a transmembrane, immunoglobulin-like receptor that interacts with a broad repertoire of extracellular ligands. RAGE belongs to a family of cell adhesion molecules and is considered a key receptor in the inflammation axis and a potential contributor to the neurodegeneration. The present study aimed to investigate the content and cell localization of RAGE in the brain of Wistar rats subjected to systemic inflammation induced by a single dose of lipopolysaccharide (LPS, 5 mg/kg, i.p.). Fifteen days after LPS administration, the content of RAGE was analyzed in the prefrontal cortex (PFC), hippocampus (HIPP), cerebellum (CB), and substantia nigra (SN) were investigated. RAGE levels increased in all structures, except HIPP; however, immunohistochemistry analysis demonstrated that the cell site of RAGE expression changed from blood vessel-like structures to neuronal cells in all brain areas. Besides, the highest level of RAGE expression was found in SN. Immunofluorescence analysis in SN confirmed that RAGE expression was mainly co-localized in endothelial cells (RAGE/PECAM-1 co-staining) in untreated animals, while LPS-treated animals had RAGE expression predominantly in dopaminergic neurons (RAGE/TH co-staining). Decreased TH levels, as well as increased pro-inflammatory markers (TNF-α, IL-1β, Iba-1, GFAP, and phosphorylated ERK1/2) in SN, occurred concomitantly to RAGE stimulation in the same site. These results suggest a role for RAGE in the establishment of a neuroinflammation-neurodegeneration axis that develops as a long-term response to systemic inflammation by LPS

Neurobehavioral And Oxidative Stress Alterations Following Methylmercury And Retinyl Palmitate Co-Administration In Pregnant And Lactating Rats And Their Offspring

Fish consumption and ubiquitous methylmercury (MeHg) exposure represent a public health problem globally. Micronutrients presented in fish affects MeHg uptake/distribution. Vitamin A (VitA), another fish micronutrient is used in nutritional supplementation, especially during pregnancy. However, there is no information about the health effects arising from their combined exposure. The present study aimed to examine the effects of both MeHg and retinyl palmitate administered to pregnant and lactating rats. Thirty Wistar female rats were orally supplemented with MeHg (0,5 mg/Kg/day) and retinyl palmitate (7500 μg RAE1/Kg/day), either individually or in combination from the gestational day 0 to weaning. In dams, maternal behavior was scored. In neonatal and infant offspring, associative learning and neurodevelopment were evaluated. Further periadolescent male and female pups were assessed for open field, habituation and object recognition using episodic-like memory paradigm. Maternal and offspring redox parameters were evaluated. Our results showed no effects of MeHg-VitA co-administration in the quality of maternal care but showed subtle alterations in the pro-oxidant response of the hippocampus. In offspring, MeHg-VitA co-exposure affected early associative learning in neonatal pups, with no further modifications in neurodevelopment, and no locomotor or exploratory alterations in later developmental stages. Habituation was altered in a sex-dependent manner, but no overall memory disturbances were encountered

Targeted inhibition of RAGE in substantia nigra of rats blocks 6-OHDA-induced dopaminergic denervation

The receptor for advanced glycation endproducts (RAGE) is a pattern-recognition receptor associated with infammation in most cell types. RAGE up-regulates the expression of proinfammatory mediators and its own expression via activation of NF-kB. Recent works have proposed a role for RAGE in Parkinson’s disease (PD). In this study, we used the multimodal blocker of RAGE FPS-ZM1, which has become available recently, to selectively inhibit RAGE in the substantia nigra (SN) of rats intracranially injected with 6-hydroxydopamine (6-OHDA). FPS-ZM1 (40 μg per rat), injected concomitantly with 6-OHDA (10 μg per rat) into the SN, inhibited the increase in RAGE, activation of ERK1/2, Src and nuclear translocation of NF-kB p65 subunit in the SN. RAGE inhibition blocked glial fbrillary acidic protein and Iba-1 upregulation as well as associated astrocyte and microglia activation. Circulating cytokines in serum and CSF were also decreased by FPS-ZM1 injection. The loss of tyrosine hydroxylase and NeuN-positive neurons was signifcantly inhibited by RAGE blocking. Finally, FPS-ZM1 attenuated locomotory and exploratory defcits induced by 6-OHDA. Our results demonstrate that RAGE is an essential component in the neuroinfammation and dopaminergic denervation induced by 6-OHDA in the SN. Selective inhibition of RAGE may ofer perspectives for therapeutic approaches

Characterization and modulation of microglial phenotypes in an animal model of severe sepsis

We aim to characterize the kinetics of early and late microglial phenotypes after systemic inflammation in an animal model of severe sepsis and the effects of minocycline on these phenotypes. Rats were subjected to CLP, and some animals were treated with minocycline (10 ug/kg) by i.c.v. administration. Animals were killed 24 hours, 5, 10 and 30 days after sepsis induction, and serum and hippocampus were collected for subsequent analyses. Real‐time PCR was performed for M1 and M2 markers. TNF‐α, IL‐1β, IL‐6, IL‐10, CCL‐22 and nitrite/nitrate levels were measured. Immunofluorescence for IBA‐1, CD11b and arginase was also performed. We demonstrated that early after sepsis, there was a preponderant up‐regulation of M1 markers, and this was not switched to M2 phenotype markers later on. We found that up‐regulation of both M1 and M2 markers co‐existed up to 30 days after sepsis induction. In addition, minocycline induced a down‐regulation, predominantly, of M1 markers. Our results suggest early activation of M1 microglia that is followed by an overlap of both M1 and M2 phenotypes and that the beneficial effects of minocycline on sepsis‐associated brain dysfunction may be related to its effects predominantly on the M1 phenotype

High fat diet-induced obesity causes a reduction in brain tyrosine hydroxylase levels and non-motor features in rats through metabolic dysfunction, neuroinflammation and oxidative stress

Obesity is a health problem that has been associated with neuroinflammation, decreased cognitive functions and development of neurodegenerative diseases. Parkinson’s disease (PD) is a chronic neurodegenerative condition characterized by motor and non-motor abnormalities, increased brain inflammation, α-synuclein protein aggregation and dopaminergic neuron loss that is associated with decreased levels of tyrosine hydroxylase (TH) in the brain. Diet-induced obesity is a global epidemic and its role as a risk factor for PD is not clear. Herein, we showed that 25 weeks on a high-fat diet (HFD) promotes significant alterations in the nigrostriatal axis of Wistar rats. Obesity induced by HFD exposure caused a reduction in TH levels and increased TH phosphorylation at serine 40 in the ventral tegmental area. These effects were associated with insulin resistance, increased tumor necrosis factor-α levels, oxidative stress, astrogliosis and microglia activation. No difference was detected in the levels of α-synuclein. Obesity also induced impairment of locomotor activity, total mobility and anxiety-related behaviors that were identified in the open-field and light/dark tasks. There were no changes in motor coordination or memory. Together, these data suggest that the reduction of TH levels in the nigrostriatal axis occurs through an α-synuclein-independent pathway and can be attributed to brain inflammation, oxidative/nitrosative stress and metabolic disorders induced by obesit