RESUMEN
The aggregation of α-synuclein (aSyn) leading to the formation of Lewy bodies is the defining pathological hallmark of Parkinson's disease (PD). Rare familial PD-associated mutations in aSyn render it aggregation-prone; however, PD patients carrying wild type (WT) aSyn also have aggregated aSyn in Lewy bodies. The mechanisms by which WT aSyn aggregates are unclear. Here, we report that inflammation can play a role in causing the aggregation of WT aSyn. We show that activation of the inflammasome with known stimuli results in the aggregation of aSyn in a neuronal cell model of PD. The insoluble aggregates are enriched with truncated aSyn as found in Lewy bodies of the PD brain. Inhibition of the inflammasome enzyme caspase-1 by chemical inhibition or genetic knockdown with shRNA abated aSyn truncation. In vitro characterization confirmed that caspase-1 directly cleaves aSyn, generating a highly aggregation-prone species. The truncation-induced aggregation of aSyn is toxic to neuronal culture, and inhibition of caspase-1 by shRNA or a specific chemical inhibitor improved the survival of a neuronal PD cell model. This study provides a molecular link for the role of inflammation in aSyn aggregation, and perhaps in the pathogenesis of sporadic PD as well.
Asunto(s)
Caspasa 1/genética , Inflamasomas/metabolismo , Cuerpos de Lewy/metabolismo , Neuronas/metabolismo , Agregado de Proteínas/genética , alfa-Sinucleína/genética , Compuestos de Alumbre/farmacología , Caspasa 1/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Dipéptidos/farmacología , Regulación de la Expresión Génica , Humanos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Cuerpos de Lewy/efectos de los fármacos , Cuerpos de Lewy/patología , Lipopolisacáridos/farmacología , Neuronas/efectos de los fármacos , Neuronas/patología , Nigericina/farmacología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Vitamina K 3/farmacología , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , para-Aminobenzoatos/farmacologíaRESUMEN
Over 30% of patients with amyotrophic lateral sclerosis (ALS) exhibit cognitive deficits indicative of frontotemporal dementia (FTD), suggesting a common pathogenesis for both diseases. Consistent with this hypothesis, neuronal and glial inclusions rich in TDP43, an essential RNA-binding protein, are found in the majority of those with ALS and FTD, and mutations in TDP43 and a related RNA-binding protein, FUS, cause familial ALS and FTD. TDP43 and FUS affect the splicing of thousands of transcripts, in some cases triggering nonsense-mediated mRNA decay (NMD), a highly conserved RNA degradation pathway. Here, we take advantage of a faithful primary neuronal model of ALS and FTD to investigate and characterize the role of human up-frameshift protein 1 (hUPF1), an RNA helicase and master regulator of NMD, in these disorders. We show that hUPF1 significantly protects mammalian neurons from both TDP43- and FUS-related toxicity. Expression of hUPF2, another essential component of NMD, also improves survival, whereas inhibiting NMD prevents rescue by hUPF1, suggesting that hUPF1 acts through NMD to enhance survival. These studies emphasize the importance of RNA metabolism in ALS and FTD, and identify a uniquely effective therapeutic strategy for these disorders.
Asunto(s)
Esclerosis Amiotrófica Lateral/fisiopatología , Modelos Biológicos , Neuronas/efectos de los fármacos , Transactivadores/fisiología , Supervivencia Celular , Humanos , Fármacos Neuroprotectores/farmacología , Degradación de ARNm Mediada por Codón sin Sentido , ARN HelicasasRESUMEN
FUS/TLS is a nucleic acid binding protein that, when mutated, can cause a subset of familial amyotrophic lateral sclerosis (fALS). Although FUS/TLS is normally located predominantly in the nucleus, the pathogenic mutant forms of FUS/TLS traffic to, and form inclusions in, the cytoplasm of affected spinal motor neurons or glia. Here we report a yeast model of human FUS/TLS expression that recapitulates multiple salient features of the pathology of the disease-causing mutant proteins, including nuclear to cytoplasmic translocation, inclusion formation, and cytotoxicity. Protein domain analysis indicates that the carboxyl-terminus of FUS/TLS, where most of the ALS-associated mutations are clustered, is required but not sufficient for the toxicity of the protein. A genome-wide genetic screen using a yeast over-expression library identified five yeast DNA/RNA binding proteins, encoded by the yeast genes ECM32, NAM8, SBP1, SKO1, and VHR1, that rescue the toxicity of human FUS/TLS without changing its expression level, cytoplasmic translocation, or inclusion formation. Furthermore, hUPF1, a human homologue of ECM32, also rescues the toxicity of FUS/TLS in this model, validating the yeast model and implicating a possible insufficiency in RNA processing or the RNA quality control machinery in the mechanism of FUS/TLS mediated toxicity. Examination of the effect of FUS/TLS expression on the decay of selected mRNAs in yeast indicates that the nonsense-mediated decay pathway is probably not the major determinant of either toxicity or suppression.
Asunto(s)
ADN Helicasas/metabolismo , Proteína FUS de Unión a ARN/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Transactivadores/metabolismo , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Núcleo Celular/genética , Citoplasma/genética , Citoplasma/metabolismo , ADN Helicasas/genética , Regulación de la Expresión Génica , Mutación , Neuronas/metabolismo , ARN Helicasas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteína FUS de Unión a ARN/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
A heterologously expressed form of the human Parkinson disease-associated protein α-synuclein with a 10-residue N-terminal extension is shown to form a stable tetramer in the absence of lipid bilayers or micelles. Sequential NMR assignments, intramonomer nuclear Overhauser effects, and circular dichroism spectra are consistent with transient formation of α-helices in the first 100 N-terminal residues of the 140-residue α-synuclein sequence. Total phosphorus analysis indicates that phospholipids are not associated with the tetramer as isolated, and chemical cross-linking experiments confirm that the tetramer is the highest-order oligomer present at NMR sample concentrations. Image reconstruction from electron micrographs indicates that a symmetric oligomer is present, with three- or fourfold symmetry. Thermal unfolding experiments indicate that a hydrophobic core is present in the tetramer. A dynamic model for the tetramer structure is proposed, based on expected close association of the amphipathic central helices observed in the previously described micelle-associated "hairpin" structure of α-synuclein.
Asunto(s)
Modelos Moleculares , Polímeros/química , Estructura Secundaria de Proteína , alfa-Sinucleína/química , Dicroismo Circular , Humanos , Microscopía Electrónica , Resonancia Magnética Nuclear Biomolecular , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The full complement of molecular pathways contributing to the pathogenesis of Parkinson disease (PD) remains unknown. Here we address this issue by taking a broad approach, beginning by using functional MRI to identify brainstem regions differentially affected and resistant to the disease. Relying on these imaging findings, we then profiled gene expression levels from postmortem brainstem regions, identifying a disease-related decrease in the expression of the catabolic polyamine enzyme spermidine/spermine N1-acetyltransferase 1 (SAT1). Next, a range of studies were completed to support the pathogenicity of this finding. First, to test for a causal link between polyamines and α-synuclein toxicity, we investigated a yeast model expressing α-synuclein. Polyamines were found to enhance the toxicity of α-synuclein, and an unbiased genome-wide screen for modifiers of α-synuclein toxicity identified Tpo4, a member of a family of proteins responsible for polyamine transport. Second, to test for a causal link between SAT1 activity and PD histopathology, we investigated a mouse model expressing α-synuclein. DENSPM (N1, N11-diethylnorspermine), a polyamine analog that increases SAT1 activity, was found to reduce PD histopathology, whereas Berenil (diminazene aceturate), a pharmacological agent that reduces SAT1 activity, worsened the histopathology. Third, to test for a genetic link, we sequenced the SAT1 gene and a rare but unique disease-associated variant was identified. Taken together, the findings from human patients, yeast, and a mouse model implicate the polyamine pathway in PD pathogenesis.
Asunto(s)
Acetiltransferasas/metabolismo , Tronco Encefálico/metabolismo , Enfermedad de Parkinson/metabolismo , Poliaminas/metabolismo , alfa-Sinucleína/metabolismo , Acetiltransferasas/genética , Animales , Tronco Encefálico/patología , Diminazeno/análogos & derivados , Diminazeno/farmacología , Variación Genética , Humanos , Imagen por Resonancia Magnética , Ratones , Ratones Transgénicos , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología , Pemolina/análogos & derivados , Pemolina/farmacologíaRESUMEN
FUS is a nucleic acid binding protein that, when mutated, cause a subset of familial amyotrophic lateral sclerosis (ALS). Expression of FUS in yeast recapitulates several pathological features of the disease-causing mutant proteins, including nuclear to cytoplasmic translocation, formation of cytoplasmic inclusions, and cytotoxicity. Genetic screens using the yeast model of FUS have identified yeast genes and their corresponding human homologs suppressing FUS induced toxicity in yeast, neurons and animal models. To expand the search for human suppressor genes of FUS induced toxicity, we carried out a genome-scale genetic screen using a newly constructed library containing 13570 human genes cloned in an inducible yeast-expression vector. Through multiple rounds of verification, we found 37 human genes that, when overexpressed, suppress FUS induced toxicity in yeast. Human genes with DNA or RNA binding functions are overrepresented among the identified suppressor genes, supporting that perturbations of RNA metabolism is a key underlying mechanism of FUS toxicity.
Asunto(s)
Esclerosis Amiotrófica Lateral , Proteína FUS de Unión a ARN , Animales , Citoplasma , Humanos , Cuerpos de Inclusión , Mutación , Proteína FUS de Unión a ARN/genética , Saccharomyces cerevisiae/genéticaRESUMEN
The identification of hot spots, i.e., binding regions that contribute substantially to the free energy of ligand binding, is a critical step for structure-based drug design. Here we present the application of two fragment-based methods to the detection of hot spots for DJ-1 and glucocerebrosidase (GCase), targets for the development of therapeutics for Parkinson's and Gaucher's diseases, respectively. While the structures of these two proteins are known, binding information is lacking. In this study we employ the experimental multiple solvent crystal structures (MSCS) method and computational fragment mapping (FTMap) to identify regions suitable for the development of pharmacological chaperones for DJ-1 and GCase. Comparison of data derived via MSCS and FTMap also shows that FTMap, a computational method for the identification of fragment binding hot spots, is an accurate and robust alternative to the performance of expensive and difficult crystallographic experiments.
Asunto(s)
Descubrimiento de Drogas , Glucosilceramidasa/química , Péptidos y Proteínas de Señalización Intracelular/química , Proteínas Oncogénicas/química , Bibliotecas de Moléculas Pequeñas/química , Sitios de Unión , Cristalografía por Rayos X , Enfermedad de Gaucher/tratamiento farmacológico , Humanos , Ligandos , Proteínas de la Membrana/química , Terapia Molecular Dirigida , Enfermedad de Parkinson/tratamiento farmacológico , Unión Proteica , Conformación Proteica , Proteína Desglicasa DJ-1 , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Solventes/química , Propiedades de SuperficieRESUMEN
Budding yeast has been widely used as a model in studying proteins associated with human diseases. Genome-wide genetic screening is a powerful tool commonly used in yeast studies. The expression of a number of neurodegenerative disease-associated proteins in yeast causes cytotoxicity and aggregate formation, recapitulating findings seen in patients with these disorders. Here, we describe a method for screening a yeast model of the Amyotrophic Lateral Sclerosis-associated protein FUS for modifiers of its toxicity. Instead of using transformation, this new screening platform relies on the mating of yeast to introduce an arrayed library of plasmids into the yeast model. The mating method has two clear advantages: first, it is highly efficient; second, the pre-transformed arrayed library of plasmids can be stored for long-term as a glycerol stock, and quickly applied to other screens without the labor-intensive step of transformation into the yeast model each time. We demonstrate how this method can successfully be used to screen for genes that modify the toxicity of FUS.
Asunto(s)
Biblioteca de Genes , Enfermedades Neurodegenerativas/diagnóstico , Proteínas/metabolismo , Deficiencias en la Proteostasis/diagnóstico , Saccharomyces cerevisiae/patogenicidad , HumanosRESUMEN
Latrepirdine (Dimebon), an anti-histamine, has shown some benefits in trials of neurodegenerative diseases characterized by accumulation of aggregated or misfolded protein such as Alzheimer's disease (AD) and has been shown to promote the removal of α-synuclein protein aggregates in vivo. An important pathway for removal of aggregated or misfolded proteins is the autophagy-lysosomal pathway, which has been implicated in AD pathogenesis, and enhancing this pathway has been shown to have therapeutic potential in AD and other proteinopathies. Here we use a yeast model, Saccharomyces cerevisiae, to investigate whether latrepirdine can enhance autophagy and reduce levels of amyloid-ß (Aß)42 aggregates. Latrepirdine was shown to upregulate yeast vacuolar (lysosomal) activity and promote transport of the autophagic marker (Atg8) to the vacuole. Using an in vitro green fluorescent protein (GFP) tagged Aß yeast expression system, we investigated whether latrepirdine-enhanced autophagy was associated with a reduction in levels of intracellular GFP-Aß42. GFP-Aß42 was localized into punctate patterns compared to the diffuse cytosolic pattern of GFP and the GFP-Aß42 (19:34), which does not aggregate. In the autophagy deficient mutant (Atg8Δ), GFP-Aß42 showed a more diffuse cytosolic localization, reflecting the inability of this mutant to sequester GFP-Aß42. Similar to rapamycin, we observed that latrepirdine significantly reduced GFP-Aß42 in wild-type compared to the Atg8Δ mutant. Further, latrepirdine treatment attenuated Aß42-induced toxicity in wild-type cells but not in the Atg8Δ mutant. Together, our findings provide evidence for a novel mechanism of action for latrepirdine in inducing autophagy and reducing intracellular levels of GFP-Aß42.
Asunto(s)
Péptidos beta-Amiloides/antagonistas & inhibidores , Autofagia/fisiología , Proteínas Fluorescentes Verdes/metabolismo , Indoles/farmacología , Líquido Intracelular/metabolismo , Fragmentos de Péptidos/antagonistas & inhibidores , Saccharomyces cerevisiae/metabolismo , Péptidos beta-Amiloides/metabolismo , Autofagia/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Proteínas Fluorescentes Verdes/antagonistas & inhibidores , Humanos , Líquido Intracelular/efectos de los fármacos , Fragmentos de Péptidos/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Studies have shown that the inositol biosynthetic pathway and the enzyme glycogen synthase kinase-3 (GSK-3) are targets of the mood-stabilizing drugs lithium and valproate. However, a relationship between these targets has not been previously described. We hypothesized that GSK-3 may play a role in inositol synthesis, and that loss of GSK-3 may lead to inositol depletion, thus providing a mechanistic link between the two drug targets. Utilizing a yeast Saccharomyces cerevisiae gsk-3Delta quadruple-null mutant, in which all four genes encoding homologues of mammalian GSK-3 are disrupted, we tested the hypothesis that GSK-3 is required for de novo inositol biosynthesis. The gsk-3Delta mutant exhibited multiple features of inositol depletion, including defective growth in inositol-lacking medium, decreased intracellular inositol, increased INO1 and ITR1 expression, and decreased levels of phosphatidylinositol. Treatment of wild-type cells with a highly specific GSK-3 inhibitor led to a significant increase in INO1 expression. Supplementation with inositol alleviated the temperature sensitivity of gsk-3Delta. Activity of myo-inositol-3 phosphate synthase, the rate-limiting enzyme in inositol de novo biosynthesis, was decreased in gsk-3Delta. These results demonstrate for the first time that GSK-3 is required for optimal myo-inositol-3 phosphate synthase activity and de novo inositol biosynthesis, and that loss of GSK-3 activity causes inositol depletion.
Asunto(s)
Glucógeno Sintasa Quinasa 3/metabolismo , Inositol/metabolismo , Inhibidores Enzimáticos/farmacología , Regulación Fúngica de la Expresión Génica , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/genética , Inositol/farmacología , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Monosacáridos , Mutación , Mio-Inositol-1-Fosfato Sintasa/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Temperatura , Tiadiazoles/farmacologíaRESUMEN
In a genetic screen for Saccharomyces cerevisiae mutants hypersensitive to the inositol-depleting drugs lithium and valproate, a loss of function allele of TPI1 was identified. The TPI1 gene encodes triose phosphate isomerase, which catalyzes the interconversion of dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate. A single mutation (N65K) in tpi1 completely abolished Tpi1p enzyme activity and led to a 30-fold increase in the intracellular DHAP concentration. The tpi1 mutant was unable to grow in the absence of inositol and exhibited the "inositol-less death" phenotype. Similarly, the pgk1 mutant, which accumulates DHAP as a result of defective conversion of 3-phosphoglyceroyl phosphate to 3-phosphoglycerate, exhibited inositol auxotrophy. DHAP as well as glyceraldehyde 3-phosphate and oxaloacetate inhibited activity of both yeast and human myo-inositol-3 phosphate synthase, the rate-limiting enzyme in de novo inositol biosynthesis. Implications for the pathology associated with TPI deficiency and responsiveness to inositol-depleting anti-bipolar drugs are discussed. This study is the first to establish a connection between perturbation of glycolysis and inhibition of de novo inositol biosynthesis.
Asunto(s)
Inositol/biosíntesis , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Cartilla de ADN , Glucólisis , Litio/farmacología , Datos de Secuencia Molecular , Mutación , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , Triosa-Fosfato Isomerasa/química , Triosa-Fosfato Isomerasa/genética , Triosa-Fosfato Isomerasa/metabolismo , Ácido Valproico/farmacologíaRESUMEN
Valproate (VPA) is one of the two drugs approved by the Food and Drug Administration (FDA) for the treatment of bipolar disorder. The therapeutic mechanism of VPA has not been established. We have shown previously that growth of the yeast Saccharomyces cerevisiae in the presence of VPA causes a decrease in intracellular inositol and inositol-1-P, and a dramatic increase in expression of INO1, which encodes the rate limiting enzyme for de novo inositol biosynthesis. To understand the underlying mechanism of action of VPA, INO1, CHO1 and INO2 expression, intracellular inositol and phospholipid biosynthesis were studied as a function of acute and chronic exposure of growing cells to the drug. A decrease in intracellular inositol was apparent immediately after addition of VPA. Surprisingly, expression of genes that are usually derepressed during inositol depletion, including INO1, CHO1 and INO2 (that contain inositol-responsive UASINO sequences) decreased several fold during the first hour, after which expression began to increase. Incorporation of 32Pi into total phospholipids was significantly decreased. Pulse labelling of CDP-DG and PG, shown previously to increase during inositol depletion, increased within 30 min. However, pulse labelling of PS, which normally increases during inositol depletion, was decreased within 30 min. PS synthase activity in cell extracts decreased with time, although VPA did not directly inhibit PS synthase enzyme activity. Thus, in contrast to the effect of chronic VPA treatment, short-term exposure to VPA abrogated the normal response to inositol depletion of inositol responsive genes and led to aberrant synthesis of phospholipids.
Asunto(s)
Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Inositol/metabolismo , Fosfolípidos/biosíntesis , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Ácido Valproico/farmacología , Regiones no Traducidas 5'/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Northern Blotting , CDPdiacilglicerol-Serina O-Fosfatidiltransferasa/metabolismo , División Celular/efectos de los fármacos , Electroforesis en Gel de Agar , Mio-Inositol-1-Fosfato Sintasa/genética , Mio-Inositol-1-Fosfato Sintasa/metabolismo , Hibridación de Ácido Nucleico , Fosfolípidos/genética , Proteínas Represoras/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/genéticaRESUMEN
We have cloned, sequenced, and expressed a human cDNA encoding 1-d-myo-inositol-3-phosphate (MIP) synthase (hINO1). The encoded 62-kDa human enzyme converted d-glucose 6-phosphate to 1-d-myo-inositol 3-phosphate, the rate-limiting step for de novo inositol biosynthesis. Activity of the recombinant human MIP synthase purified from Escherichia coli was optimal at pH 8.0 at 37 degrees C and exhibited K(m) values of 0.57 mm and 8 microm for glucose 6-phosphate and NAD(+), respectively. NH(4)(+) and K(+) were better activators than other cations tested (Na(+), Li(+), Mg(2+), Mn(2+)), and Zn(2+) strongly inhibited activity. Expression of the protein in the yeast ino1Delta mutant lacking MIP synthase (ino1Delta/hINO1) complemented the inositol auxotrophy of the mutant and led to inositol excretion. MIP synthase activity and intracellular inositol were decreased about 35 and 25%, respectively, when ino1Delta/hINO1 was grown in the presence of a therapeutically relevant concentration of the anti-bipolar drug valproate (0.6 mm). However, in vitro activity of purified MIP synthase was not inhibited by valproate at this concentration, suggesting that inhibition by the drug is indirect. Because inositol metabolism may play a key role in the etiology and treatment of bipolar illness, functional conservation of the key enzyme in inositol biosynthesis underscores the power of the yeast model in studies of this disorder.