Aggregation of alpha-synuclein (ASYN) in Lewy bodies and Lewy neurites is the typical pathological hallmark of Parkinson's disease (PD) and other synucleinopathies. Furthermore, mutations in the gene encoding for ASYN are associated with familial and sporadic forms of PD, suggesting this protein plays a central role in the disease. However, the precise contribution of ASYN to neuronal dysfunction and death is unclear. There is intense debate about the nature of the toxic species of ASYN and little is known about the molecular determinants of oligomerization and aggregation of ASYN in the cell. In order to clarify the effects of different mutations on the propensity of ASYN to oligomerize and aggregate, we assembled a panel of 19 ASYN variants and compared their behaviour. We found that familial mutants linked to PD (A30P, E46K, H50Q, G51D and A53T) exhibited identical propensities to oligomerize in living cells, but had distinct abilities to form inclusions. While the A30P mutant reduced the percentage of cells with inclusions, the E46K mutant had the opposite effect. Interestingly, artificial proline mutants designed to interfere with the helical structure of the N-terminal domain, showed increased propensity to form oligomeric species rather than inclusions. Moreover, lysine substitution mutants increased oligomerization and altered the pattern of aggregation. Altogether, our data shed light into the molecular effects of ASYN mutations in a cellular context, and established a common ground for the study of genetic and pharmacological modulators of the aggregation process, opening new perspectives for therapeutic intervention in PD and other synucleinopathies.
Alpha-synuclein (aSyn) misfolding and aggregation are pathological features common to several neurodegenerative diseases, including Parkinson's disease (PD). Mounting evidence suggests that aSyn can be secreted and transferred from cell to cell, participating in the propagation and spreading of pathological events. Rab11, a small GTPase, is an important regulator in both endocytic and secretory pathways. Here, we show that Rab11 is involved in regulating aSyn secretion. Rab11 knockdown or overexpression of either Rab11a wild-type (Rab11a WT) or Rab11a GDP-bound mutant (Rab11a S25N) increased secretion of aSyn. Furthermore, we demonstrate that Rab11 interacts with aSyn and is present in intracellular inclusions together with aSyn. Moreover, Rab11 reduces aSyn aggregation and toxicity. Our results suggest that Rab11 is involved in modulating the processes of aSyn secretion and aggregation, both of which are important mechanisms in the progression of aSyn pathology in PD and other synucleinopathies.
Alpha-synuclein (?S) misfolding is associated with Parkinson's disease (PD) but little is known about the mechanisms underlying ?S toxicity. Increasing evidence suggests that defects in membrane transport play an important role in neuronal dysfunction. Here we demonstrate that the GTPase Rab8a interacts with ?S in rodent brain. NMR spectroscopy reveals that the C-terminus of ?S binds to the functionally important switch region as well as the C-terminal tail of Rab8a. In line with a direct Rab8a/?S interaction, Rab8a enhanced ?S aggregation and reduced ?S-induced cellular toxicity. In addition, Rab8 - the Drosophila ortholog of Rab8a - ameliorated ?S-oligomer specific locomotor impairment and neuron loss in fruit flies. In support of the pathogenic relevance of the ?S-Rab8a interaction, phosphorylation of ?S at S129 enhanced binding to Rab8a, increased formation of insoluble ?S aggregates and reduced cellular toxicity. Our study provides novel mechanistic insights into the interplay of the GTPase Rab8a and ?S cytotoxicity, and underscores the therapeutic potential of targeting this interaction.
Acrylamide (AA) is a well-known industrial chemical classified as a probable human carcinogen. Benign and malignant tumours at different sites, including the mammary gland, have been reported in rodents exposed to AA. This xenobiotic is also formed in many carbohydrate-rich foods prepared at high temperatures. For this reason, AA is an issue of concern in terms of human cancer risk. The epoxide glycidamide (GA) is thought to be the ultimate genotoxic AA metabolite. Despite extensive experimental and epidemiological data focused on AA-induced breast cancer, there is still lack of information on the deleterious effects induced by GA in mammary cells. The work reported here addresses the characterisation and modulation of cytotoxicity, generation of reactive oxygen species, formation of micronuclei (MN) and quantification of specific GA-DNA adducts in human MCF10A epithelial cells exposed to GA. The results show that GA significantly induces MN, impairs cell proliferation kinetics and decreases cell viability at high concentrations by mechanisms not involving oxidative stress. KU55933, an inhibitor of ataxia telangiectasia mutated kinase, enhanced the cytotoxicity of GA (P < 0.05), supporting a role of this enzyme in regulating the repair of GA-induced DNA lesions. Moreover, even at low GA levels, N7-GA-Gua adducts were generated in a linear dose-response manner in MCF10A cells. These results confirm that human mammary cells are susceptible to GA toxicity and reinforce the need for additional studies to clarify the potential correlation between dietary AA exposure and breast cancer risk in human populations.
Pharmacological inhibition of DNA repair is a promising approach to increase the effectiveness of anticancer drugs. The chemotherapeutic drug doxorubicin (Dox) may act, in part, by causing oxidative DNA damage. The base excision repair (BER) pathway effects the repair of many DNA lesions induced by reactive oxygen species (ROS). Methoxyamine (MX) is an indirect inhibitor of apurinic/apyrimidinic endonuclease 1 (APE1), a multifunctional BER protein. We have evaluated the effects of MX on the cytotoxicity and genotoxicity of Dox in MDA-MB-231 metastatic breast cancer cells. MX has little effects on the viability and proliferation of Dox-treated cells. However, as assessed by the cytokinesis-block micronucleus assay (CBMN), MX caused a significant 1.4-fold increase (P<0.05) in the frequency of micronucleated binucleated cells induced by Dox, and also altered the distribution of the numbers of micronuclei. The fluorescence probe dihydroethidium (DHE) indicated little production of ROS by Dox. Overall, our results suggest differential outcomes for the inhibition of APE1 activity in breast cancer cells exposed to Dox, with a sensitizing effect observed for genotoxicity but not for cytotoxicity.
Deleterious sustained inflammation mediated by activated microglia is common to most of neurologic disorders. Here, we identified sirtuin 2 (SIRT2), an abundant deacetylase in the brain, as a major inhibitor of microglia-mediated inflammation and neurotoxicity. SIRT2-deficient mice (SIRT2(-/-)) showed morphological changes in microglia and an increase in pro-inflammatory cytokines upon intracortical injection of lipopolysaccharide (LPS). This response was associated with increased nitrotyrosination and neuronal cell death. Interestingly, manipulation of SIRT2 levels in microglia determined the response to Toll-like receptor (TLR) activation. SIRT2 overexpression inhibited microglia activation in a process dependent on serine 331 (S331) phosphorylation. Conversely, reduction of SIRT2 in microglia dramatically increased the expression of inflammatory markers, the production of free radicals, and neurotoxicity. Consistent with increased NF-?B-dependent transcription of inflammatory genes, NF-?B was found hyperacetylated in the absence of SIRT2, and became hypoacetylated in the presence of S331A mutant SIRT2. This finding indicates that SIRT2 functions as a gatekeeper, preventing excessive microglial activation through NF-?B deacetylation. Our data uncover a novel role for SIRT2 opening new perspectives for therapeutic intervention in neuroinflammatory disorders.
Cerebral Palsy (CP) is an encephalic static lesion defined as a non-progressive disorder of movements and posture. It is usually associated with epilepsy, speaking, hearing and sight disorders and also mental retardation. Even though, people who have CP need special care for the prevention of factors linked to oral problems, in the odontological context, the literary information is contradictory when it comes to the incidence of oral diseases on patients who have cerebral palsy. In order to determine the oral health and associated factors conditions, an epidemiological research has been made in 41 children who have cerebral palsy. The variables taken into account were: social-economical aspects, risk factors for the development of oral diseases, access to odontological care, caries index, periodontal disease, malocclusion and dental fluorose. Children who were examined showed a higher level of gingivitis and caries experience, mainly on the primary dentition, and also severe malocclusions, factors that indicate the need of early intervention, whether with educational programs or healing assistance. The study has shown that, in addition to the quantitative necessity of care, it is also necessary to improve the quality of these patients consultations.
Mutations in the genes encoding leucine-rich repeat kinase 2 (LRRK2) and ?-synuclein are associated with both autosomal dominant and idiopathic forms of Parkinsons disease (PD). ?-Synuclein is the main protein in Lewy bodies, hallmark inclusions present in both sporadic and familial PD. We show that in PD brain tissue, the levels of LRRK2 are positively related to the increase in ?-synuclein phosphorylation and aggregation in affected brain regions (amygdala and anterior cingulate cortex), but not in the unaffected visual cortex. In disease-affected regions, we show co-localization of these two proteins in neurons and Lewy body inclusions. Further, in vitro experiments show a molecular interaction between ?-synuclein and LRRK2 under endogenous and over-expression conditions. In a cell culture model of ?-synuclein inclusion formation, LRRK2 co-localizes with the ?-synuclein inclusions, and knocking down LRRK2 increases the number of smaller inclusions. In addition to providing strong evidence for an interaction between LRRK2 and ?-synuclein, our results shed light on the complex relationship between these two proteins in the brains of patients with PD and the underlying molecular mechanisms of the disease.
Parkinsons disease (PD) is the most common representative of a group of disorders known as synucleinopathies, in which misfolding and aggregation of ?-synuclein (a-syn) in various brain regions is the major pathological hallmark. Indeed, the motor symptoms in PD are caused by a heterogeneous degeneration of brain neurons not only in substantia nigra pars compacta but also in other extrastriatal areas of the brain. In addition to the well known motor dysfunction in PD patients, cognitive deficits and memory impairment are also an important part of the disorder, probably due to disruption of synaptic transmission and plasticity in extrastriatal areas, including the hippocampus. Here, we investigated the impact of a-syn aggregation on AMPA and NMDA receptor-mediated rat hippocampal (CA3-CA1) synaptic transmission and long-term potentiation (LTP), the neurophysiological basis for learning and memory. Our data show that prolonged exposure to a-syn oligomers, but not monomers or fibrils, increases basal synaptic transmission through NMDA receptor activation, triggering enhanced contribution of calcium-permeable AMPA receptors. Slices treated with a-syn oligomers were unable to respond with further potentiation to theta-burst stimulation, leading to impaired LTP. Prior delivery of a low-frequency train reinstated the ability to express LTP, implying that exposure to a-syn oligomers drives the increase of glutamatergic synaptic transmission, preventing further potentiation by physiological stimuli. Our novel findings provide mechanistic insight on how a-syn oligomers may trigger neuronal dysfunction and toxicity in PD and other synucleinopathies.
Human exposure to cadmium (Cd) occurs via different routes, including diet. The increasing amount of data linking Cd with different cellular effects in the mammary gland justifies additional toxicological assessments using human mammary epithelial cells. This work aimed therefore to assess the cytotoxic effects of Cd in MCF10A cells and to characterize the cytoprotective role of the macrocycle pyN(5) in the form of calcium salt. Cadmium chloride revealed to be cytotoxic to MCF10A cells, decreasing cell viability and proliferation in a concentration-dependent manner. Comparable dose-response curves and IC50 values (57-63 ?M, 24h treatment) were obtained using the MTT reduction, crystal violet and BrdU assays. In terms of reactive oxygen species formation, only a slight increase in superoxide radical anion was observed at very high Cd concentrations (?100 ?M). Chelation should thus constitute the primary strategy to mitigate the cytotoxic effects induced by Cd in mammary cells. In this context, pyN(5) which presents appropriate chemical and thermodynamic features was studied as a Cd chelator. This macrocycle (25 and 50 ?M) significantly reduced or even abolished Cd-induced cytotoxicity. Protective effects were observed in terms of cell viability, cell proliferation and morphological alterations, being the protection mostly attributed to a chelating-based mechanism.
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