RESUMEN
It has been hypothesized that --Parkinson's disease (PD) may be initiated in the gastrointestinal tract, before manifesting in the central nervous system. In this respect, it was demonstrated that lipopolysaccharide (LPS), an endotoxin from gram-negative bacteria, accelerates the in vitro formation of α-synuclein (aSyn) fibrils, whose intracellular deposits is a histological hallmark of the degeneration of dopaminergic neurons in PD. Herein, N-terminal acetylation and missense mutations of aSyn (A30P, A53T, E46K, H50Q and G51D) linked to rare, early-onset forms of familial PD were investigated regarding their effect on aSyn aggregation stimulated by either LPS or small unilamellar lipid vesicles (SUVs). Our findings indicated that LPS as well as SUVs induce the fibrillation of N-terminally acetylated wild-type aSyn (Ac-aSyn-WT) more remarkably than the non-acetylated protein, while the LPS-free protein alone did not undergo fibrillation under our assay conditions. In addition, with the exception of A30P, PD mutations increased the fibrillation of Ac-aSyn in the presence of LPS compared with Ac-aSyn-WT. The most pronounced effect of LPS was noticed for A53T, as observed when either Thioflavin-T or JC-1 were used as fluorescent probes for fibrils. Overall, our results suggest for the first time the existence of a synergy between LPS and PD mutations/N-terminal acetylation toward aSyn fibrillation.
Asunto(s)
Enfermedad de Parkinson , alfa-Sinucleína , Humanos , alfa-Sinucleína/metabolismo , Enfermedad de Parkinson/patología , Lipopolisacáridos , Acetilación , MutaciónRESUMEN
Intestinal dysbiosis seems to play a role in neurodegenerative pathologies. Parkinson's disease (PD) patients have an altered gut microbiota. Moreover, mice treated orally with the gut microbe Proteus mirabilis developed Parkinson's-like symptoms. Here, the possible involvement of P. mirabilis urease (PMU) and its B subunit (PmUreß) in the pathogenesis of PD was assessed. Purified proteins were given to mice intraperitoneally (20 µg/animal/day) for one week. Behavioral tests were conducted, and brain homogenates of the treated animals were subjected to immunoassays. After treatment with PMU, the levels of TNF-α and IL-1ß were measured in Caco2 cells and cellular permeability was assayed in Hek 293. The proteins were incubated in vitro with α-synuclein and examined via transmission electron microscopy. Our results showed that PMU treatment induced depressive-like behavior in mice. No motor deficits were observed. The brain homogenates had an increased content of caspase-9, while the levels of α-synuclein and tyrosine hydroxylase decreased. PMU increased the pro-inflammatory cytokines and altered the cellular permeability in cultured cells. The urease, but not the PmUreß, altered the morphology of α-synuclein aggregates in vitro, forming fragmented aggregates. We concluded that PMU promotes pro-inflammatory effects in cultured cells. In vivo, PMU induces neuroinflammation and a depressive-like phenotype compatible with the first stages of PD development.
RESUMEN
Different SOD1 proteoforms are implicated## in both familial and sporadic cases of Amyotrophic Lateral Sclerosis (ALS), an aging-associated disease that affects motor neurons. SOD1 is crucial to neuronal metabolism and health, regulating the oxidative stress response and the shift between oxidative-fermentative metabolism, which is important for astrocyte-neuron metabolic cooperation. Neurons have a limited capacity to metabolize methylglyoxal (MGO), a potentially toxic side product of glycolysis. MGO is highly reactive and can readily posttranslationally modify proteins, in a reaction known as glycation, impacting their normal biology. Here, we aimed to investigate the effect of glycation on the aggregation and toxicity of human SOD1WT (hSOD1WT). Cells with deficiency in MGO metabolism showed increased levels of hSOD1WT inclusions, displaying also reduced hSOD1WT activity and viability. Strikingly, we also found that the presence of hSOD1WT in stress granules increased upon MGO treatment. The treatment of recombinant hSOD1WT with MGO resulted in the formation of SDS-stable oligomers, specially trimers, and thioflavin-T positive aggregates, which can promote cell toxicity and TDP-43 pathology. Together, our results suggest that glycation may play a still underappreciated role on hSOD1WT and TDP-43 pathologies in sporadic ALS, which could open novel perspectives for therapeutic intervention.
Asunto(s)
Esclerosis Amiotrófica Lateral , Humanos , Superóxido Dismutasa-1/genética , Esclerosis Amiotrófica Lateral/patología , Superóxido Dismutasa/metabolismo , Reacción de Maillard , Óxido de Magnesio , Neuronas Motoras/metabolismo , Proteínas de Unión al ADN/metabolismoRESUMEN
INTRODUCTION: Since the discovery of the presynaptic protein α-synuclein (aSyn) as a central player in Parkinson's disease (PD), several key questions on the function of the protein in neurodegeneration processes remain unclear, including: is there a synergy between dopamine metabolism and the formation of toxic aSyn species in neurons? What is the role of aSyn in the immunological system? AREAS COVERED: Herein, the authors revisit the intricate pathways related to dopamine metabolism and how it impacts on aSyn aggregation/function. Additionally, they discuss the importance of aSyn in the immune response to viral infections as well as the current findings on the possible protective role of certain virus vaccines against PD and other neuropathologies. EXPERT OPINION: The physiological function of aSyn seems to cover different pathways, such as immune response against infections and a neuroprotective role, besides the already-established regulation of synaptic vesicle trafficking. Clinical studies with monoclonal antibodies against aSyn aggregates have shown disappointing results in patients with early-stage PD. Alternatively, we could consider, as immunological target, specific neurotoxic oligomers of aSyn formed in the presence of dopamine metabolites, such as DOPAL. Nevertheless, the crucial question remains as to whether removing these protein deposits will affect the clinical course of the disease.
Asunto(s)
Enfermedad de Parkinson , Virosis , Humanos , alfa-Sinucleína , Enfermedad de Parkinson/metabolismo , Dopamina , Neuronas/metabolismo , Virosis/patologíaRESUMEN
Alpha-synuclein is a presynaptic protein linked to Parkinson's disease with a poorly characterized physiological role in regulating the synaptic vesicle cycle. Using RBL-2H3 cells as a model system, we earlier reported that wild-type alpha-synuclein can act as both an inhibitor and a potentiator of stimulated exocytosis in a concentration-dependent manner. The inhibitory function is constitutive and depends on membrane binding by the helix-2 region of the lipid-binding domain, while potentiation becomes apparent only at high concentrations. Using structural and functional characterization of conformationally selective mutants via a combination of spectroscopic and cellular assays, we show here that binding affinity for isolated vesicles similar in size to synaptic vesicles is a primary determinant of alpha-synuclein-mediated potentiation of vesicle release. Inhibition of release is sensitive to changes in the region linking the helix-1 and helix-2 regions of the N-terminal lipid-binding domain and may require some degree of coupling between these regions. Potentiation of release likely occurs as a result of alpha-synuclein interactions with undocked vesicles isolated away from the active zone in internal pools. Consistent with this, we observe that alpha-synuclein can disperse vesicles from in vitro clusters organized by condensates of the presynaptic protein synapsin-1.
Asunto(s)
Enfermedad de Parkinson , Membranas Sinápticas , Vesículas Sinápticas , alfa-Sinucleína , Humanos , alfa-Sinucleína/química , Lípidos/química , Enfermedad de Parkinson/metabolismo , Vesículas Sinápticas/metabolismo , Dominios Proteicos , Membranas Sinápticas/químicaRESUMEN
The misfolding and aggregation of the protein α-synuclein (aSyn) into potentially neurotoxic oligomers is believed to play a pivotal role in the neuropathogenesis of Parkinson's disease (PD). Herein, we explore how apomorphine (Apo), a nonselective dopamine D1 and D2 receptor agonist utilized in the therapy for PD, affects the aggregation and toxicity of aSyn in vitro. Our data indicated that Apo inhibits aSyn fibrillation leading to the formation of large oligomeric species (Apo-aSyn-O), which exhibit remarkable toxicity in mesencephalic dopaminergic neurons in primary cultures. Interestingly, purified Apo-aSyn-O, even at very low concentrations, seems to be capable of converting unmodified aSyn monomer into neurotoxic species. Collectively, our findings warn for a possible dangerous effect of Apo on aSyn misfolding/aggregation pathway.
Asunto(s)
alfa-SinucleínaRESUMEN
The current pandemic of coronavirus disease 2019 (COVID-19) has gained increased attention in the neuroscience community, especially taking into account the neuroinvasive potential of its causative agent, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the impact of its infection on the structure and function of the brain. Apart from the neurotropic properties of SARS-CoV-2, it is likewise important the observation that virus infection may perturb specific cellular processes that are believed to play an important role in the pathogenesis of diverse neurological disorders, particularly in Parkinson's disease (PD). In this scenario, viral infection-induced colon inflammation, gut microbial imbalance, and α-synuclein upregulation are of particular interest with regard to the interplay between the gastrointestinal tract and the central nervous system (microbiome-gut-brain axis). In this Perspective, we present a critical view on the different hypotheses that are recently being raised by neuroscientists about the relationship between SARS-CoV-2 infection and long-lasting neurodegenerative disorders, opening the question of whether COVID-19 might represent a risk factor for the development of PD.
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Disbiosis/virología , Enfermedades Neurodegenerativas/virología , SARS-CoV-2/patogenicidad , Virosis/virología , Encéfalo/patología , Encéfalo/virología , Disbiosis/complicaciones , Microbioma Gastrointestinal/fisiología , Humanos , Enfermedades Neurodegenerativas/patología , Virosis/complicaciones , Virosis/patologíaRESUMEN
The aggregation of α-synuclein (aSyn) is believed to be mechanistically linked to the degeneration of dopamine (DA)-producing neurons in Parkinson's disease (PD). In this respect, one crucial question that yet remains unsolved is whether aSyn aggregation is associated with either a gain- or loss-of-function of the protein in neuronal cells. Herein, we investigated the effect of monomeric versus fibrillar aSyn on mesencephalic dopaminergic neurons in primary cultures challenged with the neurotoxic catechols: salsolinol (SALSO; 1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline) and 3,4-dihydroxyphenylacetaldehyde (DOPAL). aSyn monomer protected cells against either SALSO- or DOPAL-induced toxicity via inhibition of caspase-3-mediated apoptosis. While fibrillar aSyn failed in attenuating SALSO neurotoxicity, it increased the viability of DOPAL-treated cells, which was apparently not associated with the inhibition of caspase-3 cleavage. The fact that DOPAL-derived aSyn adducts exhibit lower toxicity compared with DOPAL itself raises the question of whether the generation of these adducts could be part of or a collateral effect of aSyn-mediated protection in neurons exposed to DOPAL. Overall, our work provides important evidence on the impact of the fibrillation of aSyn on its protective role in neuronal cells exposed to the toxic catechols SALSO and DOPAL.
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Dopamina , Neuronas/efectos de los fármacos , alfa-Sinucleína , Ácido 3,4-Dihidroxifenilacético , Animales , Humanos , Isoquinolinas/farmacología , Ratones , Cultivo Primario de CélulasAsunto(s)
COVID-19/complicaciones , Disbiosis/complicaciones , Microbioma Gastrointestinal , Enfermedad de Parkinson/etiología , SARS-CoV-2/patogenicidad , Traslocación Bacteriana/inmunología , COVID-19/microbiología , Sistema Nervioso Entérico/fisiopatología , Humanos , Inflamación , Lipopolisacáridos/metabolismo , Lipopolisacáridos/toxicidad , Microglía/inmunología , Microglía/metabolismo , Modelos Biológicos , Neuroinmunomodulación , Enfermedad de Parkinson/microbiología , Agregación Patológica de Proteínas , Factores de Riesgo , Nervio Vago/fisiopatología , alfa-Sinucleína/metabolismoRESUMEN
The formation of neurotoxic oligomers of the presynaptic protein α-Synuclein (aSyn) is suggested to be associated with Parkinson's disease neurodegeneration. In this respect, it was demonstrated that the aldehyde 3,4-dihydroxyphenylacetaldehyde (DOPAL), a product from the enzymatic oxidation of dopamine, is capable of stabilizing potentially toxic aSyn oligomers via formation of covalent adducts with Lys residues of the protein. In addition, DOPAL-induced production of reactive oxygen species (ROS) leads to the oxidation of aSyn's Met residues to Met-sulfoxide. Recently, our group pointed out that the pre-oxidation of all-four Met residues of aSyn, upon treatment with H2O2, decreases the formation of large aSyn-DOPAL oligomers, which are suggested to be more toxic to neurons than the corresponding small oligomers (Carmo-Gonçalves et al., Biochem. Biophys. Res. Comm. 505, 295-301. 2018). By using a series of Met to Val mutants of aSyn, we demonstrated that the ability of aSyn to scavenge ROS/H2O2 generated from DOPAL oxidation is primarily dependent on Met residues located at the C-terminal domain of the protein, which contrasts with the reactivity of aSyn against H2O2 itself in which N-terminal Met residues (notably Met5) were more readily oxidized. Interestingly, the substitution of C-terminal Met residues (particularly Met127) by Val increased the formation of DOPAL-induced large oligomers in comparison with the wild-type protein. In this context, we demonstrated that the hydrophobicity of aSyn monomer, which is affected distinctively by the oxidation of N- versus C-terminal methionines, is correlated with the formation of large (but not small) oligomers of aSyn mediated by DOPAL.
Asunto(s)
Ácido 3,4-Dihidroxifenilacético/análogos & derivados , Peróxido de Hidrógeno/química , Metionina/química , Valina/química , alfa-Sinucleína/química , Ácido 3,4-Dihidroxifenilacético/química , Ácido 3,4-Dihidroxifenilacético/metabolismo , Sustitución de Aminoácidos , Naftalenosulfonatos de Anilina/química , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Humanos , Peróxido de Hidrógeno/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Metionina/metabolismo , Mutación , Oxidación-Reducción , Dominios Proteicos , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometría de Fluorescencia , Valina/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMEN
Identifying the mechanisms by which the presynaptic protein α-synuclein (aSyn) is associated with neurodegeneration of dopamine neurons is a major priority in the Parkinson's disease (PD) field. Studies indicate that DOPAL (3,4-dihydroxyphenylacetaldehyde), an aldehyde generated from the enzymatic oxidation of dopamine, may convert aSyn monomer into a neurotoxin via formation of covalently stabilized toxic oligomers. Herein we investigated the role of N-terminal acetylation and familial aSyn mutations (A30P, A53T, E46K, G51D, and H50Q) on DOPAL-induced oligomerization of the protein. Our results indicate that the wild-type (WT) N-terminally acetylated aSyn (Ac-aSyn) is less prone to form oligomers upon incubation with DOPAL than the non-N-terminally acetylated protein. On the other hand, familial mutants from Ac-aSyn, particularly A53T, E46K, and H50Q increased the formation of DOPAL-derived aSyn oligomers, especially large oligomers. Binding of aSyn to synaptic-like small unilamellar vesicles (SUVs) protected distinctive aSyn variants against the effects of DOPAL. While N-terminal acetylation increased the protective action of SUVs against DOPAL-induced aSyn oligomerization, A53T, A30P, and H50Q mutations in Ac-aSyn had an opposite effect. This means that PD-linked mutations may not only perturb the affinity of aSyn for membranes but also influence the formation of DOPAL-mediated oligomers. Overall, our findings provide important evidence for the existence of a connection between familial mutations of aSyn, their distinct affinity to lipid membranes, and the formation of potentially toxic oligomers of the protein mediated by DOPAL.
Asunto(s)
Ácido 3,4-Dihidroxifenilacético/análogos & derivados , Neuronas Dopaminérgicas/efectos de los fármacos , Enfermedad de Parkinson/genética , alfa-Sinucleína/genética , Ácido 3,4-Dihidroxifenilacético/farmacología , Acetilación/efectos de los fármacos , Dopamina/metabolismo , Humanos , Lípidos de la Membrana/metabolismo , Mutación/genética , Oxidación-Reducción/efectos de los fármacos , Enfermedad de Parkinson/metabolismo , Multimerización de Proteína/efectos de los fármacos , Procesamiento Proteico-Postraduccional/genética , alfa-Sinucleína/metabolismoRESUMEN
The dopamine metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL) is believed to play a central role in Parkinson's disease neurodegeneration by stabilizing potentially toxic oligomers of the presynaptic protein α-Synuclein (aSyn). Besides the formation of covalent DOPAL-Lys adducts, DOPAL promotes the oxidation of Met residues of aSyn, which is also a common oxidative post-translational modification found in the protein in vivo. Herein we set out to address the role of Met residues on the oligomerization and neurotoxic properties of DOPAL-modified aSyn. Our data indicate that DOPAL promotes the formation of two distinct types of aSyn oligomers: large and small (dimer and trimers) oligomers, which seem to be generated by independent mechanisms and cannot be interconverted by using denaturing agents. Interestingly, H2O2-treated aSyn monomer, which exhibits all-four Met residues oxidized to Met-sulfoxide, exhibited a reduced ability to form large oligomers upon treatment with DOPAL, with no effect on the population of small oligomers. In this context, triple Met-Val mutant M5V/M116V/M127V exhibited an increased population of large aSyn-DOPAL oligomers in comparison with the wild-type protein. Interestingly, the stabilization of large rather than small oligomers seems to be associated with an enhanced toxicity of DOPAL-aSyn adducts. Collectively, these findings indicate that Met residues may play an important role in modulating both the oligomerization and the neurotoxic properties of DOPAL-derived aSyn species.
Asunto(s)
Ácido 3,4-Dihidroxifenilacético/farmacología , Metionina/química , Neuronas/efectos de los fármacos , Multimerización de Proteína/efectos de los fármacos , alfa-Sinucleína/toxicidad , Ácido 3,4-Dihidroxifenilacético/análogos & derivados , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Humanos , Peróxido de Hidrógeno/farmacología , Metionina/genética , Ratones , Mutación , Neuronas/citología , Oxidantes/farmacología , Oxidación-Reducción/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , alfa-Sinucleína/química , alfa-Sinucleína/genéticaRESUMEN
BACKGROUND: Salsolinol (SALSO), a product from the reaction of dopamine (DA) with acetaldehyde, is found increased in dopaminergic neurons of Parkinson's disease (PD) patients. The administration of SALSO in rats causes myenteric neurodegeneration followed by the formation of deposits of the protein α-synuclein (aS), whose aggregation is intimately associated to PD. METHODS: NMR, isothermal titration calorimetry and MS were used to evaluate the interaction of SALSO with aS. The toxicity of SALSO and in vitro-produced aS-SALSO species was evaluated on mesencephalic primary neurons from mice. RESULTS: SALSO, under oxidative conditions, stabilizes the monomeric state besides a minor population of oligomers of aS, resulting in a strong inhibition of the fibrillation process. SALSO does not promote any chemical modification of the protein. Instead, the interaction of SALSO with aS seems to occur via hydrophobic effect, likely mediated by the NAC (non-amyloid component) domain of the protein. aS-SALSO species were found to be innocuous on primary neurons, while SALSO alone induces apoptosis via caspase-3 activation. Importantly, exogenous aS monomer was capable of protecting neurons against SALSO toxicity irrespective whether the protein was co-administered with SALSO or added until 2â¯h after SALSO, as evidenced by DAPI and cleaved-caspase 3 assays. Similar protective action of aS was found by pre-incubating neurons with aS before the administration of SALSO. CONCLUSIONS: Interaction of SALSO with aS leads to the formation of fibril-incompetent and innocuous adducts. SALSO toxicity is attenuated by aS monomer. SIGNIFICANCE: aS could exhibit a protective role against the neurotoxic effects of SALSO in dopaminergic neuron.
Asunto(s)
Neuronas Dopaminérgicas/efectos de los fármacos , Isoquinolinas/toxicidad , Sinapsis/metabolismo , alfa-Sinucleína/fisiología , Animales , Apoptosis/efectos de los fármacos , Calorimetría , Caspasa 3/metabolismo , Células Cultivadas , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Humanos , Espectrometría de Masas , Ratones , Oxidación-Reducción , Ratas , Espectrometría de Fluorescencia , alfa-Sinucleína/metabolismoRESUMEN
Glioblastoma multiform (GBM) is the most common and devastating type of primary brain tumor, being considered the deadliest of human cancers. In this context, extensive efforts have been undertaken to develop new drugs that exhibit both antiproliferation and antimetastasis effects on GBM. 1,4-Naphthoquinone (1,4-NQ) scaffold has been found in compounds able to inhibit important biological targets associated with cancer, which includes DNA topoisomerase, Hsp90 and monoamine oxidase. Among potential antineoplastic 1,4-NQs is the plant-derived lapachol (2-hydroxy-3-prenyl-1,4-naphthoquinone) that was found to be active against the Walker-256 carcinoma and Yoshida sarcoma. In the present study, we examined the effect of polyamine (PA)-conjugated derivatives of lapachol, nor-lapachol and lawsone on the growth and invasion of the human GBM cells. The conjugation with PA (a spermidine analog) resulted in dose-dependent and time-dependent increase of cytotoxicity of the 1,4-NQs. In addition, in-vitro inhibition of GBM cell invasion by lapachol was increased upon PA conjugation. Previous biochemical experiments indicated that these PA-1,4-NQs are capable of inhibiting DNA human topoisomerase II-α (topo2α), a major enzyme involved in maintaining DNA topology. Herein, we applied molecular docking to investigate the binding of PA-1,4-NQs to the ATPase site of topo2α. The most active molecules preferentially bind at the ATP-binding site of topo2α, which is energetically favored by the conjugation with PA. Taken together, these findings suggested that the PA-1,4-NQ conjugates might represent potential molecules in the development of new drugs in chemotherapy for malignant brain tumors.
Asunto(s)
Neoplasias Encefálicas/tratamiento farmacológico , Glioblastoma/tratamiento farmacológico , Naftoquinonas/farmacología , Poliaminas/farmacología , Adenosina Trifosfato/metabolismo , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Antineoplásicos/farmacología , Antineoplásicos Fitogénicos/síntesis química , Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/farmacología , Astrocitos/efectos de los fármacos , Astrocitos/patología , Sitios de Unión , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Corteza Cerebral/citología , Corteza Cerebral/efectos de los fármacos , ADN-Topoisomerasas de Tipo II/metabolismo , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Ratones , Naftoquinonas/síntesis química , Naftoquinonas/química , Poliaminas/síntesis química , Poliaminas/química , Cultivo Primario de CélulasRESUMEN
BACKGROUND: A series of perimidinone derivatives (7H-benzo[e]perimidin-7-one) were synthesized and assessed by means of in vitro assays as human MAO inhibitors. These compounds inhibited reversibly the enzymes with inhibitory constants in the range of 2 to 20 µM. In addition, the selectivity of inhibition of the MAO isoforms seems to be significantly dependent of the presence either of heteroatom or electron donating and withdrawing groups on the perimidinone framework, which was verified by using molecular docking simulation with the crystallized MAO receptors. Most of these inhibitors were highly selective: 9 and 11 inhibited selectively the MAO-B isoform while 12 had 10-fold selectivity for MAO-A isoform. Moreover, the compound 12 was both the most selective and potent MAO-A inhibitor among perimidinones. RESULT: These results have important implications for the drug design of molecules targeting depression and movement-related disorders.
Asunto(s)
Inhibidores de la Monoaminooxidasa/farmacología , Quinazolinas/farmacología , Humanos , Cinética , Simulación del Acoplamiento Molecular , Quinazolinas/químicaRESUMEN
Oxidative deamination of dopamine produces the highly toxic aldehyde 3,4-dihydroxyphenylacetaldehyde (DOPAL), enhanced production of which is found in post-mortem brains of Parkinson disease patients. When injected into the substantia nigra of rat brains, DOPAL causes the loss of dopaminergic neurons accompanied by the accumulation of potentially toxic oligomers of the presynaptic protein α-synuclein (aS), potentially explaining the synergistic toxicity described for dopamine metabolism and aS aggregation. In this work, we demonstrate that DOPAL interacts with aS via formation of Schiff-base and Michael-addition adducts with Lys residues, in addition to causing oxidation of Met residues to Met-sulfoxide. DOPAL modification leads to the formation of small aS oligomers that may be cross-linked by DOPAL. Both monomeric and oligomeric DOPAL adducts potently inhibit the formation of mature amyloid fibrils by unmodified aS. The binding of aS to either lipid vesicles or detergent micelles, which results in a gain of α-helix structure in its N-terminal lipid-binding domain, protects the protein against DOPAL adduct formation and, consequently, inhibits DOPAL-induced aS oligomerization. Functionally, aS-DOPAL monomer exhibits a reduced affinity for small unilamellar vesicles with lipid composition similar to synaptic vesicles, in addition to diminished membrane-induced α-helical content in comparison with the unmodified protein. These results suggest that DOPAL could compromise the functionality of aS, even in the absence of protein oligomerization, by affecting the interaction of aS with lipid membranes and hence its role in the regulation of synaptic vesicle traffic in neurons.
Asunto(s)
Ácido 3,4-Dihidroxifenilacético/análogos & derivados , Amiloide/química , Dopamina/metabolismo , Lípidos de la Membrana/química , Enfermedad de Parkinson/metabolismo , alfa-Sinucleína/química , Ácido 3,4-Dihidroxifenilacético/química , Ácido 3,4-Dihidroxifenilacético/metabolismo , Ácido 3,4-Dihidroxifenilacético/toxicidad , Amiloide/metabolismo , Animales , Membrana Celular/química , Neuronas Dopaminérgicas/efectos de los fármacos , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/patología , Humanos , Lisina/química , Lípidos de la Membrana/metabolismo , Oxidación-Reducción , Enfermedad de Parkinson/patología , Ratas , Bases de Schiff/química , Sustancia Negra/efectos de los fármacos , Sustancia Negra/metabolismo , Sustancia Negra/patología , alfa-Sinucleína/metabolismoRESUMEN
Epidemiological data have suggested that exposure to environmental toxins might be associated with the etiology of Parkinson's disease (PD). In this context, certain agrochemicals are able to induce Parkinsonism in different animal models via the inhibition of mitochondrial complex I, which leads to an increase in both oxidative stress and the death of nigrostriatal neurons. Additionally, in vitro experiments have indicated that pesticides are capable of accelerating the fibrillation of the presynaptic protein α-synuclein (aS) by binding directly to the protein. However, the molecular details of these interactions are poorly understood. In the present work we demonstrate that paraquat and rotenone, two agrochemicals that lead to a Parkinsonian phenotype in vivo, bind to aS via solvent effects rather than through specific interactions. In fact, these compounds produced no significant effects on aS fibrillation under physiological concentrations of NaCl. NMR data suggest that paraquat interacts with the C-terminal domain of the disordered aS monomer. This interaction was markedly reduced in the presence of NaCl, presumably due to the disruption of electrostatic interactions between the protein and paraquat. Interestingly, the effects produced by short-term incubation of paraquat with aS on the protein conformation resembled those produced by incubating the protein with NaCl alone. Taken together, our data indicate that the effects of these agrochemicals on PD cannot be explained via direct interactions with aS, reinforcing the idea that the role of these compounds in PD is limited to the inhibition of mitochondrial complex I and/or the up-regulation of aS.
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Paraquat/farmacología , Plaguicidas/farmacología , Rotenona/farmacología , Regulación hacia Arriba/efectos de los fármacos , alfa-Sinucleína/metabolismo , Animales , Relación Dosis-Respuesta a Droga , Humanos , Técnicas In Vitro , Microscopía Electrónica de Transmisión , Resonancia Magnética Nuclear Biomolecular , Unión Proteica/efectos de los fármacos , Radioisótopos/farmacocinética , Cloruro de Sodio/farmacología , alfa-Sinucleína/química , alfa-Sinucleína/ultraestructuraRESUMEN
Oxidative stress and the formation of cytotoxic aggregates of the presynaptic protein α-synuclein (AS) are two important events associated with the pathogenesis of Parkinson's disease (PD) and several other neurodegenerative diseases. In this context, extensive efforts have been made to elucidate the molecular basis of the cytotoxic synergy between oxidative stress and AS aggregation. In this study, we demonstrate that the exposure of AS to oxidative stress induced by UV radiation (ASUV) blocks the protein fibrillation, leading to the formation of highly toxic fibril-incompetent oligomers. In addition, ASUV exhibited stronger anti-fibrillogenic properties than H2O2-treated AS, inhibiting the fibrillation of unmodified AS at notably low concentrations. Mass spectrometry indicated that Met5 oxidation to Met-sulfoxide was the only modification promoted by UV exposure, which is reinforced by NMR data indicating that Met5 is the only residue whose amide resonance completely disappeared from the (1)H-(15)N HSQC spectrum after UV exposure. This result is supported by previous data that indicate that C-terminal Met residues (Met116 and Met127) and N-terminal Met1 are less susceptible to oxidation than Met5 because of the residual structure of the disordered AS monomer. Overall, our findings suggest that specific oxidation of Met5 might be sufficient to promote the formation of highly neurotoxic oligomers of AS.
Asunto(s)
Metionina/química , Agregado de Proteínas , alfa-Sinucleína/química , alfa-Sinucleína/toxicidad , Animales , Dicroismo Circular , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Humanos , Peróxido de Hidrógeno/farmacología , Ratones , Neuronas/química , Neuronas/citología , Neuronas/efectos de los fármacos , Oxidación-Reducción , Estrés Oxidativo , Cultivo Primario de Células , Agregado de Proteínas/efectos de los fármacos , Agregado de Proteínas/efectos de la radiación , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/toxicidad , Soluciones , Rayos Ultravioleta , alfa-Sinucleína/genéticaRESUMEN
The degeneration of dopaminergic neurons in Parkinson's disease (PD) is suggested to be associated with the generation of cytotoxic products from dopamine (DA) metabolism and the formation of fibrillar inclusions of the protein α-synuclein (AS). Despite of the role of AS in the pathogenesis of PD is not completely understood, the stabilization of nontoxic aggregates could represent a potential therapeutic route. In respect to the DA metabolism, a well-established strategy is the inhibition of the enzyme monoamine oxidase, which is responsible to catalyze the major route of inactivation of neurotransmitters. Although pharmacological strategies to treat different aspects of the parkinsonian condition are under investigation, the development of multifunctional molecules that act simultaneously on different targets associated to PD has gained attention only recently. In this work we examine the biochemical properties of synthetic and natural molecules that are capable of interfering on both DA system (via monoamine oxidase inhibition) and AS fibrillation.
Asunto(s)
Inhibidores de la Monoaminooxidasa/uso terapéutico , Monoaminooxidasa/metabolismo , Enfermedad de Parkinson/tratamiento farmacológico , alfa-Sinucleína/metabolismo , Neuronas Dopaminérgicas/efectos de los fármacos , Neuronas Dopaminérgicas/metabolismo , Humanos , Inhibidores de la Monoaminooxidasa/farmacología , Enfermedad de Parkinson/metabolismoRESUMEN
Thioflavin-T (ThT) is a cationic benzothiazole dye that displays enhanced fluorescence upon binding to amyloid fibrils. This property makes ThT the current reagent of choice for the quantification of amyloid fibrils. Herein, we investigate the main pitfalls associated with the use of ThT-based assays to monitor the fibrillation of α-synuclein (α-syn), a protein linked to Parkinson's disease and other α-synucleinopathies. We demonstrated for the first time that ThT interacts with α-syn disordered monomer and accelerates the protein fibrillation in vitro. As a consequence, misleading conclusions may arise from the use of ThT-based real-time assays in the evaluation of anti-fibrillogenic compounds. Interestingly, NMR experiments indicated that C-terminal domain of α-syn is the main region perturbed by ThT interaction, similarly to that found for the pesticide paraquat, a well-documented accelerator of α-syn fibrillation. Moreover, we demonstrated that certain potent inhibitors of α-syn fibrillation, such as oxidized catecholamines and polyphenols, undergo spontaneous oxidation in aqueous solution, generating compounds that strongly quench ThT fluorescence. In light of these findings, we alert for possible artifacts associated to the measure of the anti-fibrillogenic activity based only on ThT fluorescence approach.