Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 21
Filtrar
1.
Mol Cell ; 73(5): 1001-1014.e8, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30527540

RESUMO

In Parkinson's disease (PD), α-synuclein (αS) pathologically impacts the brain, a highly lipid-rich organ. We investigated how alterations in αS or lipid/fatty acid homeostasis affect each other. Lipidomic profiling of human αS-expressing yeast revealed increases in oleic acid (OA, 18:1), diglycerides, and triglycerides. These findings were recapitulated in rodent and human neuronal models of αS dyshomeostasis (overexpression; patient-derived triplication or E46K mutation; E46K mice). Preventing lipid droplet formation or augmenting OA increased αS yeast toxicity; suppressing the OA-generating enzyme stearoyl-CoA-desaturase (SCD) was protective. Genetic or pharmacological SCD inhibition ameliorated toxicity in αS-overexpressing rat neurons. In a C. elegans model, SCD knockout prevented αS-induced dopaminergic degeneration. Conversely, we observed detrimental effects of OA on αS homeostasis: in human neural cells, excess OA caused αS inclusion formation, which was reversed by SCD inhibition. Thus, monounsaturated fatty acid metabolism is pivotal for αS-induced neurotoxicity, and inhibiting SCD represents a novel PD therapeutic approach.


Assuntos
Antiparkinsonianos/farmacologia , Descoberta de Drogas/métodos , Inibidores Enzimáticos/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Metabolômica/métodos , Neurônios/efeitos dos fármacos , Doença de Parkinson/tratamento farmacológico , Estearoil-CoA Dessaturase/antagonistas & inibidores , alfa-Sinucleína/toxicidade , Animais , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/genética , Linhagem Celular , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/enzimologia , Córtex Cerebral/patologia , Diglicerídeos/metabolismo , Modelos Animais de Doenças , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/enzimologia , Neurônios Dopaminérgicos/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/enzimologia , Células-Tronco Pluripotentes Induzidas/patologia , Gotículas Lipídicas/efeitos dos fármacos , Gotículas Lipídicas/enzimologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Terapia de Alvo Molecular , Degeneração Neural , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/enzimologia , Células-Tronco Neurais/patologia , Neurônios/enzimologia , Neurônios/patologia , Ácido Oleico/metabolismo , Doença de Parkinson/enzimologia , Doença de Parkinson/genética , Doença de Parkinson/patologia , Ratos Sprague-Dawley , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Estearoil-CoA Dessaturase/metabolismo , Triglicerídeos/metabolismo , alfa-Sinucleína/genética
2.
J Neurosci ; 42(10): 2116-2130, 2022 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-35086904

RESUMO

α-Synuclein (αS) plays a key role in Parkinson's disease. Although Parkinson's disease is typically "sporadic," inherited αS missense mutations provide crucial insights into molecular mechanisms. Here, we examine two clinical mutants, E46K and G51D, which are both in the conserved N-terminus that mediates transient αS-membrane interactions. However, E46K increases and G51D decreases αS-membrane interactions. Previously, we amplified E46K via the 11-residue repeat motifs, creating "3K" (E35K+E46K+E61K). Here, we engineered these motifs to amplify G51D (V40D+G51D+V66D = "3D") and systematically compared E46K/3K versus G51D/3D. We found that G51D increased cytosolic αS in neural cells and 3D aggravates this. G51D, and 3D even more, reduced αS multimer-to-monomer (αS60:αS14) ratio. Both amplified variants caused cellular stress in rat primary neurons and reduced growth in human neuroblastoma cells. Importantly, both 3K- and 3D-induced stress was ameliorated by pharmacologically inhibiting stearoyl-CoA desaturase or by conditioning the cells in palmitic (16:0) or myristic (14:0) acid. SCD inhibition lowered lipid-droplet accumulation in both 3D- and 3K-expressing cells and benefitted G51D by normalizing multimer:monomer ratio, as reported previously for E46K. Our findings suggest that, despite divergent cytosol/membrane partitioning, both G51D and E46K neurotoxicity can be prevented by decreasing fatty-acid unsaturation as a common therapeutic approach.SIGNIFICANCE STATEMENT α-Synuclein (αS) dyshomeostasis is linked to Parkinson's disease. Here we focus on two contrasting familial-Parkinson's disease αS mutants, E46K and G51D, that alter αS membrane association in opposite directions (E46K increases, G51D decreases it). Taking advantage of αS repeat structure, here we designed αS "3D," an amplified G51D variant (V40D+G51D+V66D). αS 3D further enhanced G51D's cytosolic enrichment. Systematic comparison of G51D/3D with membrane-enriched E46K/its amplified variant 3K revealed that both can elicit stress in human neural cells and primary rodent neurons. This toxicity can be ameliorated by inhibiting stearoyl-CoA desaturase or by saturated fatty acid conditioning. Thus, despite divergent membrane binding, both G51D and E46K αS dyshomeostasis are mitigated by altering fatty acid saturation as a shared target.


Assuntos
Ácidos Graxos , Doença de Parkinson , alfa-Sinucleína , Animais , Citosol/metabolismo , Ácidos Graxos/metabolismo , Homeostase , Doença de Parkinson/metabolismo , Ratos , Estearoil-CoA Dessaturase/metabolismo , alfa-Sinucleína/metabolismo
3.
J Neurochem ; 165(2): 246-258, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36625497

RESUMO

Alpha-synuclein (αS), the key protein in Parkinson's disease, is typically described as an intrinsically disordered protein. Consistent with this notion, several context-dependent folding states may coexist in neurons. Unfolded soluble monomers, helical monomers at membranes and helical multimers (soluble or at membranes) have all been reported and may be in an equilibrium with each other. We previously found that αS can be stabilized in its membrane-associated monomeric form by genetically increasing the hydrophobicity of the membrane-embedded half of the αS helix. αS amphipathic helix formation at membranes is governed by up to nine 11-amino acid repeats with the core motif KTKEGV. However, this repeat is only imperfectly conserved; for example, it consists of KAKEGV in repeat #1, KTKEQV in repeat #5, and AVVTGV in the poorly conserved repeat #6. Here we explored the effect of perfecting the αS core repeat to nine times KTKEGV ("9KV") and found by sequential protein extraction that this engineered mutant accumulates in the cytosolic phase of neural cells. Intact-cell cross-linking trapped a part of the cytosolic portion at multimeric positions (30, 60, 80, 100 kDa). Thus, compared to wild-type αS, αS 9KV seems less prone to populating the membrane-associated monomeric form. Removing the "ATVA" intervening amino-acid sequence between repeats 4 and 5 slightly increased cytosolic localization while adding "ATVA" in between all repeats 1-8 caused αS to be trapped as a monomer in membrane fractions. Our results contribute to an ongoing debate on the dynamic structure of αS, highlighting that wild-type αS is unlikely to be fully multimeric/monomeric or fully cytosolic/membrane-associated in cells, but protein engineering can create αS variants that preferentially adopt a certain state. Overall, the imperfect nature of the KTKEGV repeat motifs and the presence of ATVA in between repeats 4 and 5 seem to prevent a strong cytosolic localization of αS and thus play a major role in the protein's ability to dynamically populate cytosolic vs. membrane-associated and monomeric vs. multimeric states.


Assuntos
Doença de Parkinson , alfa-Sinucleína , Humanos , alfa-Sinucleína/metabolismo , Solubilidade , Mutação , Doença de Parkinson/metabolismo , Sequência de Aminoácidos
4.
Hum Mol Genet ; 30(23): 2332-2346, 2021 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-34254125

RESUMO

α-Synuclein (αS) has been well-documented to play a role in human synucleinopathies such as Parkinson's disease (PD) and dementia with Lewy bodies (DLB). First, the lesions found in PD/DLB brains-Lewy bodies and Lewy neurites-are rich in aggregated αS. Second, genetic evidence links missense mutations and increased αS expression to familial forms of PD/DLB. Third, toxicity and cellular stress can be caused by αS under certain experimental conditions. In contrast, the homologs ß-synuclein (ßS) and γ-synuclein (γS) are not typically found in Lewy bodies/neurites, have not been clearly linked to brain diseases and have been largely non-toxic in experimental settings. In αS, the so-called non-amyloid-ß component of plaques (NAC) domain, constituting amino acids 61-95, has been identified to be critical for aggregation in vitro. This domain is partially absent in ßS and only incompletely conserved in γS, which could explain why both homologs do not cause disease. However, αS in vitro aggregation and cellular toxicity have not been firmly linked experimentally, and it has been proposed that excess αS membrane binding is sufficient to induce neurotoxicity. Indeed, recent characterizations of Lewy bodies have highlighted the accumulation of lipids and membranous organelles, raising the possibility that ßS and γS could also become neurotoxic if they were more prone to membrane/lipid binding. Here, we increased ßS and γS membrane affinity by strategic point mutations and demonstrate that these proteins behave like membrane-associated monomers, are cytotoxic and form round cytoplasmic inclusions that can be prevented by inhibiting stearoyl-CoA desaturase.


Assuntos
Membrana Celular/metabolismo , Corpos de Inclusão/metabolismo , alfa-Sinucleína/metabolismo , beta-Sinucleína/metabolismo , gama-Sinucleína/metabolismo , Sequência de Aminoácidos , Sequência Conservada , Humanos , Mutagênese , Agregação Patológica de Proteínas , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Solubilidade , alfa-Sinucleína/química , alfa-Sinucleína/genética , beta-Sinucleína/química , beta-Sinucleína/genética , gama-Sinucleína/química , gama-Sinucleína/genética
5.
Ann Neurol ; 89(1): 74-90, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32996158

RESUMO

OBJECTIVE: Parkinson disease (PD) has useful symptomatic treatments that do not slow the neurodegenerative process, and no significant disease-modifying treatments are approved. A key therapeutic target in PD is α-synuclein (αS), which is both genetically implicated and accumulates in Lewy bodies rich in vesicles and other lipid membranes. Reestablishing αS homeostasis is a central goal in PD. Based on previous lipidomic analyses, we conducted a mouse trial of a stearoyl-coenzyme A desaturase (SCD) inhibitor ("5b") that prevented αS-positive vesicular inclusions and cytotoxicity in cultured human neurons. METHODS: Oral dosing and brain activity of 5b were established in nontransgenic mice. 5b in drinking water was given to mice expressing wild-type human αS (WT) or an amplified familial PD αS mutation (E35K + E46K + E61K ["3K"]) beginning near the onset of nigral and cortical neurodegeneration and the robust PD-like motor syndrome in 3K. Motor phenotypes, brain cytopathology, and SCD-related lipid changes were quantified in 5b- versus placebo-treated mice. Outcomes were compared to effects of crossing 3K to SCD1-/- mice. RESULTS: 5b treatment reduced αS hyperphosphorylation in E46K-expressing human neurons, in 3K neural cultures, and in both WT and 3K αS mice. 5b prevented subtle gait deficits in WT αS mice and the PD-like resting tremor and progressive motor decline of 3K αS mice. 5b also increased αS tetramers and reduced proteinase K-resistant lipid-rich aggregates. Similar benefits accrued from genetically deleting 1 SCD allele, providing target validation. INTERPRETATION: Prolonged reduction of brain SCD activity prevented PD-like neuropathology in multiple PD models. Thus, an orally available SCD inhibitor potently ameliorates PD phenotypes, positioning this approach to treat human α-synucleinopathies. ANN NEUROL 2021;89:74-90.


Assuntos
Doença de Parkinson/prevenção & controle , alfa-Sinucleína/genética , Animais , Encéfalo/patologia , Humanos , Corpos de Lewy/patologia , Camundongos Transgênicos , Neurônios/metabolismo , Doença de Parkinson/genética , Fenótipo , alfa-Sinucleína/metabolismo
6.
Proc Natl Acad Sci U S A ; 116(41): 20760-20769, 2019 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-31548371

RESUMO

Microscopy of Lewy bodies in Parkinson's disease (PD) suggests they are not solely filamentous deposits of α-synuclein (αS) but also contain vesicles and other membranous material. We previously reported the existence of native αS tetramers/multimers and described engineered mutations of the αS KTKEGV repeat motifs that abrogate the multimers. The resultant excess monomers accumulate in lipid membrane-rich inclusions associated with neurotoxicity exceeding that of natural familial PD mutants, such as E46K. Here, we use the αS "3K" (E35K+E46K+E61K) engineered mutation to probe the mechanisms of reported small-molecule modifiers of αS biochemistry and then identify compounds via a medium-throughput automated screen. αS 3K, which forms round, vesicle-rich inclusions in cultured neurons and causes a PD-like, l-DOPA-responsive motor phenotype in transgenic mice, was fused to YFP, and fluorescent inclusions were quantified. Live-cell microscopy revealed the highly dynamic nature of the αS inclusions: for example, their rapid clearance by certain known modulators of αS toxicity, including tacrolimus (FK506), isradipine, nilotinib, nortriptyline, and trifluoperazine. Our automated 3K cellular screen identified inhibitors of stearoyl-CoA desaturase (SCD) that robustly prevent the αS inclusions, reduce αS 3K neurotoxicity, and prevent abnormal phosphorylation and insolubility of αS E46K. SCD inhibition restores the E46K αS multimer:monomer ratio in human neurons, and it actually increases this ratio for overexpressed wild-type αS. In accord, conditioning 3K cells in saturated fatty acids rescued, whereas unsaturated fatty acids worsened, the αS phenotypes. Our cellular screen allows probing the mechanisms of synucleinopathy and refining drug candidates, including SCD inhibitors and other lipid modulators.


Assuntos
Corpos de Inclusão/efeitos dos fármacos , Lipídeos/análise , Mutação , Neuroblastoma/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/farmacologia , Estearoil-CoA Dessaturase/antagonistas & inibidores , alfa-Sinucleína/química , Animais , Linhagem Celular , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Estearoil-CoA Dessaturase/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo
7.
Mov Disord ; 36(2): 348-359, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33103814

RESUMO

BACKGROUND: Synucleinopathies, including Parkinson's disease (PD), are characterized by α-synuclein (αS) cytoplasmic inclusions. αS-dependent vesicle-trafficking defects are important in PD pathogenesis, but their mechanisms are not well understood. Protein palmitoylation, post-translational addition of the fatty acid palmitate to cysteines, promotes trafficking by anchoring specific proteins to the vesicle membrane. αS itself cannot be palmitoylated as it lacks cysteines, but it binds to membranes, where palmitoylation occurs, via an amphipathic helix. We hypothesized that abnormal αS membrane-binding impairs trafficking by disrupting palmitoylation. Accordingly, we investigated the therapeutic potential of increasing cellular palmitoylation. OBJECTIVES: We asked whether upregulating palmitoylation by inhibiting the depalmitoylase acyl-protein-thioesterase-1 (APT1) ameliorates pathologic αS-mediated cellular phenotypes and sought to identify the mechanism. METHODS: Using human neuroblastoma cells, rat neurons, and iPSC-derived PD patient neurons, we examined the effects of pharmacologic and genetic downregulation of APT1 on αS-associated phenotypes. RESULTS: APT1 inhibition or knockdown decreased αS cytoplasmic inclusions, reduced αS serine-129 phosphorylation (a PD neuropathological marker), and protected against αS-dependent neurotoxicity. We identified the APT1 substrate microtubule-associated-protein-6 (MAP6), which binds to vesicles in a palmitoylation-dependent manner, as a key mediator of these effects. Mechanistically, we found that pathologic αS accelerated palmitate turnover on MAP6, suggesting that APT1 inhibition corrects a pathological αS-dependent palmitoylation deficit. We confirmed the disease relevance of this mechanism by demonstrating decreased MAP6 palmitoylation in neurons from αS gene triplication patients. CONCLUSIONS: Our findings demonstrate a novel link between the fundamental process of palmitoylation and αS pathophysiology. Upregulating palmitoylation represents an unexplored therapeutic strategy for synucleinopathies. © 2020 International Parkinson and Movement Disorder Society.


Assuntos
Doença de Parkinson , alfa-Sinucleína , Animais , Humanos , Lipoilação , Neurônios/metabolismo , Ratos , Regulação para Cima , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo
8.
PLoS Pathog ; 14(9): e1007283, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30235355

RESUMO

Synaptic degeneration is one of the earliest pathological correlates of prion disease, and it is a major determinant of the progression of clinical symptoms. However, the cellular and molecular mechanisms underlying prion synaptotoxicity are poorly understood. Previously, we described an experimental system in which treatment of cultured hippocampal neurons with purified PrPSc, the infectious form of the prion protein, induces rapid retraction of dendritic spines, an effect that is entirely dependent on expression of endogenous PrPC by the target neurons. Here, we use this system to dissect pharmacologically the underlying cellular and molecular mechanisms. We show that PrPSc initiates a stepwise synaptotoxic signaling cascade that includes activation of NMDA receptors, calcium influx, stimulation of p38 MAPK and several downstream kinases, and collapse of the actin cytoskeleton within dendritic spines. Synaptic degeneration is restricted to excitatory synapses, spares presynaptic structures, and results in decrements in functional synaptic transmission. Pharmacological inhibition of any one of the steps in the signaling cascade, as well as expression of a dominant-negative form of p38 MAPK, block PrPSc-induced spine degeneration. Moreover, p38 MAPK inhibitors actually reverse the degenerative process after it has already begun. We also show that, while PrPC mediates the synaptotoxic effects of both PrPSc and the Alzheimer's Aß peptide in this system, the two species activate distinct signaling pathways. Taken together, our results provide powerful insights into the biology of prion neurotoxicity, they identify new, druggable therapeutic targets, and they allow comparison of prion synaptotoxic pathways with those involved in other neurodegenerative diseases.


Assuntos
Príons/metabolismo , Príons/patogenicidade , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Sinalização do Cálcio , Células Cultivadas , Espinhas Dendríticas/metabolismo , Espinhas Dendríticas/patologia , Potenciais Pós-Sinápticos Excitadores , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Mutantes , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Neurônios/metabolismo , Neurônios/patologia , Proteínas PrPSc/genética , Proteínas PrPSc/metabolismo , Proteínas PrPSc/patogenicidade , Doenças Priônicas/metabolismo , Doenças Priônicas/patologia , Príons/genética , Receptores de AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Transmissão Sináptica , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores
9.
Int J Mol Sci ; 21(15)2020 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-32707907

RESUMO

Genetic and biochemical evidence attributes neuronal loss in Parkinson's disease (PD) and related brain diseases to dyshomeostasis of the 14 kDa protein α-synuclein (αS). There is no consensus on how αS exerts toxicity. Explanations range from disturbed vesicle biology to proteotoxicity caused by fibrillar aggregates. To probe these mechanisms further, robust cellular toxicity models are needed, but their availability is limited. We previously reported that a shift from dynamic multimers to monomers is an early event in αS dyshomeostasis, as caused by familial PD (fPD)-linked mutants such as E46K. Excess monomers accumulate in round, lipid-rich inclusions. Engineered αS '3K' (E35K+E46K+E61K) amplifies E46K, causing a PD-like, L-DOPA-responsive motor phenotype in transgenic mice. Here, we present a cellular model of αS neurotoxicity after transducing human neuroblastoma cells to express yellow fluorescent protein (YFP)-tagged αS 3K in a doxycycline-dependent manner. αS-3K::YFP induction causes pronounced growth defects that accord with cell death. We tested candidate compounds for their ability to restore growth, and stearoyl-CoA desaturase (SCD) inhibitors emerged as a molecule class with growth-restoring capacity, but the therapeutic window varied among compounds. The SCD inhibitor MF-438 fully restored growth while exerting no apparent cytotoxicity. Our αS bioassay will be useful for elucidating compound mechanisms, for pharmacokinetic studies, and for compound/genetic screens.


Assuntos
Proliferação de Células/efeitos dos fármacos , Neuroblastoma/metabolismo , Piridazinas/farmacologia , Estearoil-CoA Dessaturase/antagonistas & inibidores , Tiadiazóis/farmacologia , alfa-Sinucleína/genética , alfa-Sinucleína/toxicidade , Proteínas de Bactérias , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Doença por Corpos de Lewy/tratamento farmacológico , Doença por Corpos de Lewy/metabolismo , Proteínas Luminescentes , Mutação , Neuroblastoma/tratamento farmacológico , Neuroblastoma/genética , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/metabolismo , Estearoil-CoA Dessaturase/metabolismo , alfa-Sinucleína/metabolismo
10.
PLoS Pathog ; 12(5): e1005623, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-27227882

RESUMO

Synaptic pathology is an early feature of prion as well as other neurodegenerative diseases. Although the self-templating process by which prions propagate is well established, the mechanisms by which prions cause synaptotoxicity are poorly understood, due largely to the absence of experimentally tractable cell culture models. Here, we report that exposure of cultured hippocampal neurons to PrPSc, the infectious isoform of the prion protein, results in rapid retraction of dendritic spines. This effect is entirely dependent on expression of the cellular prion protein, PrPC, by target neurons, and on the presence of a nine-amino acid, polybasic region at the N-terminus of the PrPC molecule. Both protease-resistant and protease-sensitive forms of PrPSc cause dendritic loss. This system provides new insights into the mechanisms responsible for prion neurotoxicity, and it provides a platform for characterizing different pathogenic forms of PrPSc and testing potential therapeutic agents.


Assuntos
Técnicas de Cultura de Células/métodos , Espinhas Dendríticas/patologia , Proteínas PrPSc/toxicidade , Animais , Western Blotting , Modelos Animais de Doenças , Hipocampo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/patologia , Doenças Priônicas/patologia , Sinapses/patologia
11.
J Biol Chem ; 291(50): 26164-26176, 2016 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-27803163

RESUMO

Prion diseases are devastating neurodegenerative disorders with no known cure. One strategy for developing therapies for these diseases is to identify compounds that block conversion of the cellular form of the prion protein (PrPC) into the infectious isoform (PrPSc). Most previous efforts to discover such molecules by high-throughput screening methods have utilized, as a read-out, a single kind of cellular assay system: neuroblastoma cells that are persistently infected with scrapie prions. Here, we describe the use of an alternative cellular assay based on suppressing the spontaneous cytotoxicity of a mutant form of PrP (Δ105-125). Using this assay, we screened 75,000 compounds, and identified a group of phenethyl piperidines (exemplified by LD7), which reduces the accumulation of PrPSc in infected neuroblastoma cells by >90% at low micromolar doses, and inhibits PrPSc-induced synaptotoxicity in hippocampal neurons. By analyzing the structure-activity relationships of 35 chemical derivatives, we defined the pharmacophore of LD7, and identified a more potent derivative. Active compounds do not alter total or cell-surface levels of PrPC, and do not bind to recombinant PrP in surface plasmon resonance experiments, although at high concentrations they inhibit PrPSc-seeded conversion of recombinant PrP to a misfolded state in an in vitro reaction (RT-QuIC). This class of small molecules may provide valuable therapeutic leads, as well as chemical biological tools to identify cellular pathways underlying PrPSc metabolism and PrPC function.


Assuntos
Piperidinas/química , Piperidinas/farmacologia , Proteínas PrPSc/antagonistas & inibidores , Proteínas PrPSc/metabolismo , Ressonância de Plasmônio de Superfície/métodos , Linhagem Celular Tumoral , Células HEK293 , Humanos , Proteínas PrPSc/genética
12.
Am J Pathol ; 186(4): 761-4, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26854642

RESUMO

This Commentary highlights the article by Makarava et al that discusses the formation of synthetic prions and the role of substrate levels in their evolution.


Assuntos
Doenças Priônicas , Príons , Humanos
14.
J Neurosci ; 31(42): 14882-92, 2011 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-22016521

RESUMO

Accumulation of PrP(Sc), an abnormal form of cellular prion protein (PrP), in the brain of animals and humans leads to fatal neurodegenerative disorders known as prion diseases. Limited protease digestion of PrP(Sc) produces a truncated form called PrP(27-30) that retains prion infectivity and is the main marker of disease targeted in most diagnostic tests. In the search for new anti-prion molecules, drug-screening assays on prion-infected murine cells have been oriented toward decreasing levels of PrP(27-30). In contrast, we screened for drugs promoting multimers of PrP(27-30), illustrating a possible stabilization of mouse PrP(Sc) species, because recent studies aiming to characterize the conformational stability of various prion strains showed that stable recombinant amyloids produced more stable prion strain, leading to longest incubation time. We identified a family of thienyl pyrimidine derivatives that induce SDS-resistant dimers and trimers of PrP(27-30). Bioassays performed on mice brain homogenates treated with these compounds showed that these thienyl pyrimidine derivatives diminished prion infectivity in vivo. Oligomeric-induced activity by thienyl pyrimidine compounds is a promising approach not only to understanding the pathogenesis of prions but also for prion diagnostics. This approach could be extended to other neurodegenerative "prionopathies," such as Alzheimer's, Huntington, or Parkinson's diseases.


Assuntos
Encéfalo/efeitos dos fármacos , Proteínas PrPC/química , Proteínas PrPC/metabolismo , Doenças Priônicas/patologia , Pirimidinas/farmacologia , Anilidas/farmacologia , Anilidas/uso terapêutico , Animais , Encéfalo/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Endopeptidase K/farmacologia , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Camundongos , Modelos Moleculares , Neuroblastoma/patologia , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Peptídeo Hidrolases/metabolismo , Proteínas PrPC/análise , Doenças Priônicas/tratamento farmacológico , Conformação Proteica/efeitos dos fármacos , Pirimidinas/uso terapêutico , Silício , Estatísticas não Paramétricas , Fatores de Tempo , Transfecção/métodos
15.
Methods Mol Biol ; 1948: 77-91, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30771172

RESUMO

ß-Sheet-rich aggregates of α-synuclein (αS) are the hallmark neuropathology of Parkinson's disease (PD) and related synucleinopathies, whereas the native conformations of αS in healthy cells are under debate. Cross-linking analyses in intact cells detect a large portion of endogenous αS in apparent multimeric states, most notably as putative tetramers (αS60) that run around 60 kDa on SDS-PAGE, but also point at the dynamic nature of cellular αS states. Standardization of αS cross-linking methods will facilitate efforts to study the effects of genetic, pharmacological, and environmental factors on αS conformation. Here, we present detailed protocols for cross-linking cellular αS multimers in cultured cells and brain tissues. These protocols will benefit future studies aimed at characterizing αS conformation in its cellular environment, both at steady state and upon perturbation, be it chronic or acute.


Assuntos
Modelos Moleculares , Conformação Proteica , alfa-Sinucleína/química , Células Cultivadas , Mutação , Doença de Parkinson/metabolismo , Multimerização Proteica , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo
16.
Sci Rep ; 8(1): 8023, 2018 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-29795181

RESUMO

Public concerns over the use of synthetic pesticides are growing since many studies have shown their impact on human health. A new environmental movement in occidental countries promoting an organic agriculture favours the rebirth of botanical pesticides. These products confer an effective alternative to chemical pesticides such as glyphosate. Among the biopesticides, the α-terthienyls found in the roots of Tagetes species, are powerful broad-spectrum pesticides. We found that an α-terthienyl analogue with herbicidal properties, called A6, triggers resistant SDS oligomers of the pathogenic prion protein PrPSc (rSDS-PrPSc) in cells. Our main question is to determine if we can induce those rSDS-PrPSc oligomers in vitro and in vivo, and their impact on prion aggregation and propagation. Using wild-type mice challenged with prions, we showed that A6 accelerates or slows down prion disease depending on the concentration used. At 5 mg/kg, A6 is worsening the pathology with a faster accumulation of PrPSc, reminiscent to soluble toxic rSDS-PrPSc oligomers. In contrast, at 10 and 20 mg/kg of A6, prion disease occurred later, with less PrPSc deposits and with rSDS-PrPSc oligomers in the brain reminiscent to non-toxic aggregates. Our results are bringing new openings regarding the impact of biopesticides in prion and prion-like diseases.


Assuntos
Encéfalo/patologia , Neuroblastoma/tratamento farmacológico , Praguicidas/farmacologia , Proteínas PrPC/química , Doenças Priônicas/prevenção & controle , Pirimidinas/química , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Relação Dose-Resposta a Droga , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Praguicidas/química , Proteínas PrPC/efeitos dos fármacos , Proteínas PrPC/metabolismo , Doenças Priônicas/metabolismo , Doenças Priônicas/patologia , Células Tumorais Cultivadas
17.
Mol Neurodegener ; 11: 11, 2016 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-26809712

RESUMO

BACKGROUND: Prion diseases are characterized by the accumulation in the central nervous system of an abnormally folded isoform of the prion protein, named PrP(Sc). Aggregation of PrP(Sc) into oligomers and fibrils is critically involved in the pathogenesis of prion diseases. Oligomers are supposed to be the key neurotoxic agents in prion disease, so modulation of prion aggregation pathways with small molecules can be a valuable strategy for studying prion pathogenicity and for developing new diagnostic and therapeutic approaches. We previously identified thienyl pyrimidine compounds that induce SDS-resistant PrP(Sc) (rSDS-PrP(Sc)) oligomers in prion-infected samples. RESULTS: Due to the low effective doses of the thienyl pyrimidine hits, we synthesized a quaterthiophene-bis-triazine compound, called MR100 to better evaluate their diagnostic and therapeutic potentials. This molecule exhibits a powerful activity inducing rSDS-PrP(Sc) oligomers at nanomolar concentrations in prion-infected cells. Fluorescence interaction studies of MR100 with mouse PrP fibrils showed substantial modification of the spectrum, and the interaction was confirmed in vitro by production of rSDS-oligomer species upon incubation of MR100 with fibrils in SDS-PAGE gel. We further explored whether MR100 compound has a potential to be used in the diagnosis of prion diseases. Our results showed that: (i) MR100 can detect rSDS-oligomers in prion-infected brain homogenates of various species, including human samples from CJD patients; (ii) A protocol, called "Rapid Centrifugation Assay" (RCA), was developed based on MR100 property of inducing rSDS-PrP(Sc) oligomers only in prion-infected samples, and avoiding the protease digestion step. RCA allows the detection of both PK-sensitive and PK-resistant PrP(Sc) species in rodents samples but also from patients with different CJD forms (sporadic and new variant); (iii) A correlation could be established between the amount of rSDS-PrP(Sc) oligomers revealed by MR100 and the duration of the symptomatic phase of the disease in CJD patients; and (iv) Bioassay experiments showed that MR100 can trap prion infectivity more efficiently than P30 drug. CONCLUSIONS: MR100 is a powerful tool not only for studying the prion aggregation pathways regarding oligomeric and sPrP(Sc) species, but also for developing alternative methods for the detection of prion-infected samples. Considering our bioassay results, MR100 is a promising molecule for the development of prion decontamination approaches.


Assuntos
Encéfalo/metabolismo , Proteínas PrPC/metabolismo , Doenças Priônicas/diagnóstico , Doenças Priônicas/metabolismo , Príons , Pirimidinas/metabolismo , Animais , Western Blotting/métodos , Modelos Animais de Doenças , Corantes Fluorescentes/metabolismo , Humanos , Camundongos
18.
PLoS One ; 9(12): e113331, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25436888

RESUMO

Prion protein is involved in severe neurodegenerative disorders but its physiological role is still in debate due to an absence of major developmental defects in knockout mice. Previous reports in zebrafish indicate that the two prion genes, PrP1 and PrP2, are both involved in several steps of embryonic development thus providing a unique route to discover prion protein function. Here we investigate the role of PrP2 during development of a mechano-sensory system, the posterior lateral line, using morpholino knockdown and PrP2 targeted inactivation. We confirm the efficiency of the translation blocking morpholino at the protein level. Development of the posterior lateral line is altered in PrP2 morphants, including nerve axonal outgrowth and primordium migration defects. Reduced neuromast deposition was observed in PrP2 morphants as well as in PrP2-/- mutants. Rosette formation defects were observed in PrP2 morphants, strongly suggesting an abnormal primordium organization and reflecting loss of cell cohesion during migration of the primordium. In addition, the adherens junction proteins, E-cadherin and ß-catenin, were mis-localized after reduction of PrP2 expression and thus contribute to the primordium disorganization. Consequently, hair cell differentiation and number were affected and this resulted in reduced functional neuromasts. At later developmental stages, myelination of the posterior lateral line nerve was altered. Altogether, our study reports an essential role of PrP2 in collective migration process of the primordium and in neuromast formation, further implicating a role for prion protein in cell adhesion.


Assuntos
Movimento Celular , Mecanorreceptores/citologia , Príons/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Junções Aderentes/metabolismo , Animais , Axônios/metabolismo , Adesão Celular , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Inativação Gênica , Células Ciliadas Auditivas/citologia , Humanos , Mecanorreceptores/metabolismo , Camundongos , Príons/genética , Células de Schwann/citologia , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genética
19.
Int J Cell Biol ; 2013: 975832, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24222767

RESUMO

Prion diseases are fatal neurodegenerative sporadic, inherited, or acquired disorders. In humans, Creutzfeldt-Jakob disease is the most studied prion disease. In animals, the most frequent prion diseases are scrapie in sheep and goat, bovine spongiform encephalopathy in cattle, and the emerging chronic wasting disease in wild and captive deer in North America. The hallmark of prion diseases is the deposition in the brain of PrP(Sc), an abnormal ß -sheet-rich form of the cellular prion protein (PrP(C)) (Prusiner 1982). According to the prion hypothesis, PrP(Sc) can trigger the autocatalytic conversion of PrP(C) into PrP(Sc), presumably in the presence of cofactors (lipids and small RNAs) that have been recently identified. In this review, we will come back to the original works that led to the discovery of prions and to the protein-only hypothesis proposed by Dr. Prusiner. We will then describe the recent reports on mammalian synthetic prions and recombinant prions that strongly support the protein-only hypothesis. The new concept of "deformed templating" regarding a new mechanism of PrP(Sc) formation and replication will be exposed. The review will end with a chapter on the prion-like propagation of other neurodegenerative disorders, such as Alzheimer's and Parkinson's disease and tauopathies.

20.
Curr Top Med Chem ; 13(19): 2477-83, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24059332

RESUMO

Transmissible spongiform encephalopathies (TSEs), also called prion diseases, are fatal, infectious, genetic or sporadic neurodegenerative disorders of humans and animals. In humans, TSEs are represented by Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker syndrome, Fatal Familial Insomnia and Kuru. In animals, the most prominent prion diseases are scrapie of sheep and goats, bovine spongiform encephalopathy (BSE) of cattle and chronic wasting disease (CWD) of deer and elk. A critical event in prion diseases is the accumulation in the central nervous system (CNS) of the abnormally folded PrP(Sc) protein that is the protease-resistant isoform of a normal cellular protein encoded by the host and called PrP(C). PrP(Sc) (also known as rPrP(Sc) or PrP27-30) represents the main marker of prion diseases and is routinely used in the reference method for the diagnosis of prion diseases. Most of the therapeutic strategies developed so far aimed at identifying compounds that diminish the levels of PrP(Sc), with variable success when tested in vivo. In this review, we present an alternative approach in which small molecules that induce PrP(Sc) oligomers are identified. By using virtual and cellular screenings, we found several thienyl pyrimidine compounds that trigger PrP(Sc) oligomerization and trap prion infectivity.


Assuntos
Proteínas PrPSc/agonistas , Doenças Priônicas/tratamento farmacológico , Dobramento de Proteína/efeitos dos fármacos , Pirimidinas/química , Pirimidinas/farmacologia , Animais , Avaliação Pré-Clínica de Medicamentos , Humanos , Modelos Moleculares , Estrutura Molecular , Proteínas PrPSc/metabolismo , Doenças Priônicas/diagnóstico , Pirimidinas/uso terapêutico
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA