RESUMO
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.
Assuntos
Ácido 3,4-Di-Hidroxifenilacético/farmacologia , Metionina/química , Neurônios/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , alfa-Sinucleína/toxicidade , Ácido 3,4-Di-Hidroxifenilacético/análogos & derivados , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Peróxido de Hidrogênio/farmacologia , Metionina/genética , Camundongos , Mutação , Neurônios/citologia , Oxidantes/farmacologia , Oxirredução/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , alfa-Sinucleína/química , alfa-Sinucleína/genéticaRESUMO
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.
Assuntos
Doença de Parkinson , Viroses , Humanos , alfa-Sinucleína , Doença de Parkinson/metabolismo , Dopamina , Neurônios/metabolismo , Viroses/patologiaRESUMO
Alzheimer's disease (AD) is related to the presence of extracellular aggregated amyloid-ß peptide (Aß), which binds copper(II) with high affinity in its N-terminal region. In this sense, two new 1-methylimidazole-containing N-acylhydrazonic metallophores, namely, X1TMP and X1Benz, were synthesized as hydrochlorides and characterized. The compound X1TMP contains the 3,4,5-trimethoxybenzoyl moiety present in the structure of mescaline, a natural hallucinogenic protoalkaloid that occurs in some species of cacti. Single crystals of X1Benz, the unsubstituted derivative of X1TMP, were obtained. The experimental partition coefficients of both compounds were determined, as well as their apparent affinity for Cu2+ in aqueous solution. Ascorbate consumption assays showed that these N-acylhydrazones are able to lessen the production of ROS by the Cu(Aß)-system, and a short-time scale aggregation study, measured through turbidity and confirmed by TEM images, revealed their capacity in preventing Aß fibrillation at equimolar conditions in the presence and absence of copper. 1H15N HSQC NMR experiments demonstrated a direct interaction between Aß and X1Benz, the most soluble of the compounds. The Cu2+ sequestering potential of this hydrazone towards Aß was explored by 1H NMR. Although increasing amounts of X1Benz were unexpectedly not efficient at removing the metal-induced perturbations in Aß backbone amides, the broadening effects observed on the compound's signals indicate the formation of a ternary Aßcopper-X1Benz species, which can be responsible for the observed ROS-lessening and aggregation-preventing activities. Overall, the N-acylhydrazones X1TMP and X1Benz have shown promising prospects as agents for the treatment of AD.
Assuntos
Doença de Alzheimer , Humanos , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Cobre/química , Mescalina , Espécies Reativas de Oxigênio/metabolismo , Peptídeos beta-Amiloides/químicaRESUMO
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.
Assuntos
alfa-SinucleínaRESUMO
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.
Assuntos
Dopamina , Neurônios/efeitos dos fármacos , alfa-Sinucleína , Ácido 3,4-Di-Hidroxifenilacético , Animais , Humanos , Isoquinolinas/farmacologia , Camundongos , Cultura Primária de CélulasRESUMO
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.
Assuntos
Neurônios Dopaminérgicos/efeitos dos fármacos , Isoquinolinas/toxicidade , Sinapses/metabolismo , alfa-Sinucleína/fisiologia , Animais , Apoptose/efeitos dos fármacos , Calorimetria , Caspase 3/metabolismo , Células Cultivadas , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Humanos , Espectrometria de Massas , Camundongos , Oxirredução , Ratos , Espectrometria de Fluorescência , alfa-Sinucleína/metabolismoRESUMO
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.
Assuntos
Metionina/química , Agregados Proteicos , alfa-Sinucleína/química , alfa-Sinucleína/toxicidade , Animais , Dicroísmo Circular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Humanos , Peróxido de Hidrogênio/farmacologia , Camundongos , Neurônios/química , Neurônios/citologia , Neurônios/efeitos dos fármacos , Oxirredução , Estresse Oxidativo , Cultura Primária de Células , Agregados Proteicos/efeitos dos fármacos , Agregados Proteicos/efeitos da radiação , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/toxicidade , Soluções , Raios Ultravioleta , alfa-Sinucleína/genéticaRESUMO
Fibrillization of the protein α-synuclein (α-syn) is a hallmark of Parkinson's disease and other α-synucleinopathies. The well-established idea that α-syn is a natively disordered monomer prone to forming fibrils was recently challenged by data showing that the protein mostly exists in vitro and in vivo as helically folded tetramers that are resistant to fibrillization. These apparently conflicting findings may be reconciled by the idea that α-syn exists as a disordered monomer in equilibrium with variable amounts of dynamic oligomeric species. In this context, varying the approaches used for protein purification, such as the method used to lyse cells or the inclusion of denaturing agents, could dramatically perturb this equilibrium and hence alter the relative abundance of the disordered monomer. In the present study, we investigated how the current methods for α-syn purification affect the structure and oligomeric state of the protein, and we discuss the main pitfalls associated with the production of recombinant α-syn in Escherichia coli. We demonstrate that α-syn was expressed in E. coli as a disordered monomer independent of both the cell lysis method and the use of heating/acidification for protein purification. In addition, we provide convincing evidence that the disordered monomer exists in equilibrium with a dynamic dimer, which is not an artefact of the cross-linking protocol as previously suggested. Unlike the helically folded tetramer, α-syn dimer is prone to fibrillate and thus it may be an interesting target for anti-fibrillogenic molecules.