RESUMO
Small-molecule high-throughput screening (HTS) campaigns have frequently been used to identify lead molecules that can alter expression of disease-relevant proteins in cell-based assays. However, most cell-based HTS assays require short compound exposure periods to avoid toxicity and ensure that compounds are stable in media for the duration of the exposure. This limits the ability of HTS assays to detect inhibitors of the synthesis of target proteins with long half-lives, which can often exceed the exposure times utilized in most HTS campaigns. One such target is alpha-synuclein (α-syn)-a protein well-known for its pathological aggregation in Parkinson's Disease (PD) and other forms of neurodegeneration known collectively as synucleinopathies. Here, we report the development of an HTS assay using a CRISPR-engineered neuroblastoma cell line expressing a destabilized luciferase reporter inserted at the end of the coding region of the SNCA locus. The resultant destabilized fusion protein exhibited a significant reduction in half-life compared to the endogenous, unmodified α-syn protein, and accurately reported reductions in α-syn levels due to known protein translation inhibitors and specific α-syn siRNAs. The robustness and utility of this approach was shown by using the resulting cell line (dsLuc-Syn) to screen a focused library of 3,192 compounds for reduction of α-syn. These data demonstrate the general utility of converting endogenous loci into destabilized reporter genes capable of identifying inhibitors of gene expression of highly stable proteins even in short-term assays.
Assuntos
Doença de Parkinson , alfa-Sinucleína , Linhagem Celular , Expressão Gênica , Ensaios de Triagem em Larga Escala/métodos , Humanos , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMO
Lafora disease (LD), a progressive form of inherited epilepsy, is associated with widespread neurodegeneration and the formation of polyglucosan bodies in the neurons. Laforin, a protein phosphatase, and malin, an E3 ubiquitin ligase, are two of the proteins that are defective in LD. We have shown recently that laforin and malin (referred together as LD proteins) are recruited to aggresome upon proteasomal blockade, possibly to clear misfolded proteins through the ubiquitin-proteasome system (UPS). Here we test this possibility using a variety of cytotoxic misfolded proteins, including the expanded polyglutamine protein, as potential substrates. Laforin and malin, together with Hsp70 as a functional complex, suppress the cellular toxicity of misfolded proteins, and all the three members of this complex are required for this function. Laforin and malin interact with misfolded proteins and promote their degradation through the UPS. LD proteins are recruited to the polyglutamine aggregates and reduce the frequency of aggregate-positive cells. Taken together, our results suggest that the malin-laforin complex is a novel player in the neuronal response to misfolded proteins and could be potential therapeutic targets for neurodegenerative disorders associated with cytotoxic proteins.
Assuntos
Proteínas de Transporte/metabolismo , Doença de Lafora/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Dobramento de Proteína , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Ubiquitina/metabolismo , Animais , Células COS , Proteínas de Transporte/genética , Chlorocebus aethiops , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Doença de Lafora/genética , Camundongos , Camundongos Transgênicos , Complexo de Endopeptidases do Proteassoma/genética , Ligação Proteica , Proteínas Tirosina Fosfatases não Receptoras/genética , Ubiquitina/genética , Ubiquitina-Proteína LigasesRESUMO
Correction to: J. Biosci. 40(5), December 2015, 863-871 https://doi.org/10.1007/s12038-015-9570-0 The image of anti-Myc blot of figure 2C (third panel; Malin-Myc [C26S]) was inadvertently used once again for the c-tubulin loading control of figure 2B. The revised figure 2B with the correct image of the c-tubulin loading control is given below. The interpretation and conclusion provided in the article do not change because of the correction.
RESUMO
Copy number mutations implicate excess production of α-synuclein as a possibly causative factor in Parkinson's disease (PD). Using an unbiased screen targeting endogenous gene expression, we discovered that the ß2-adrenoreceptor (ß2AR) is a regulator of the α-synuclein gene (SNCA). ß2AR ligands modulate SNCA transcription through histone 3 lysine 27 acetylation of its promoter and enhancers. Over 11 years of follow-up in 4 million Norwegians, the ß2AR agonist salbutamol, a brain-penetrant asthma medication, was associated with reduced risk of developing PD (rate ratio, 0.66; 95% confidence interval, 0.58 to 0.76). Conversely, a ß2AR antagonist correlated with increased risk. ß2AR activation protected model mice and patient-derived cells. Thus, ß2AR is linked to transcription of α-synuclein and risk of PD in a ligand-specific fashion and constitutes a potential target for therapies.
Assuntos
Regulação da Expressão Gênica , Doença de Parkinson/etnologia , Doença de Parkinson/genética , Receptores Adrenérgicos beta 2/metabolismo , alfa-Sinucleína/genética , Acetilação , Agonistas de Receptores Adrenérgicos beta 1/farmacologia , Antagonistas Adrenérgicos beta/farmacologia , Antagonistas Adrenérgicos beta/uso terapêutico , Albuterol/farmacologia , Albuterol/uso terapêutico , Animais , Linhagem Celular Tumoral , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica/efeitos dos fármacos , Histonas/metabolismo , Humanos , Ligantes , Camundongos , Fármacos Neuroprotetores/farmacologia , Noruega/etnologia , Doença de Parkinson/tratamento farmacológico , Regiões Promotoras Genéticas , Propranolol/farmacologia , Propranolol/uso terapêutico , Receptores Adrenérgicos beta 2/genética , Risco , Substância Negra/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
Peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) is a central regulator of cellular and mitochondrial metabolism. Cellular bioenergetics are critically important in "energy-guzzling" neurons, but the components and wiring of the transcriptional circuit through which PGC-1α regulates the neuronal electron transport chain have not been established. This information may be vital for restoring neuronal bioenergetics gene expression that is compromised during incipient Parkinson's neuropathology and in aging-dependent brain diseases. Here we delineate a neuronal transcriptional circuit controlled by endogenous PGC-1α. We show that a feed-forward circuit of endogenous neuronal PGC-1α and the orphan nuclear estrogen-related receptor α (ERRα) activates the nuclear-encoded mitochondrial electron transport chain. PGC-1α not only trans-activated expression of ERRα, but also coactivated ERRα target genes in complexes I, II, IV, and V of the neuronal electron transport chain via association with evolutionary conserved ERRα promoter binding motifs. Chemical activation of this transcriptional program induced transcription of the neuronal electron transport chain. These data highlight a neuronal transcriptional circuit regulated by PGC-1α that can be therapeutically targeted for Parkinson's and other neurodegenerative diseases.
RESUMO
Lafora's progressive myoclonus epilepsy (Lafora disease: LD) is caused by mutations in the EPM2A or NHLRC1 gene, but cellular mechanisms of the pathogenesis remain unclear. In an attempt to understand and elucidate the disease pathway, we have investigated the global gene expression profile in a mouse model for LD that developed a phenotype similar to that observed in human patients, including presence of Lafora bodies, neurodegeneration and profound neurological disturbances. We found 62 differentially expressed genes in the Epm2a knockout mice brains. These genes encode factors involved in protein catabolism, phosphatase, transcription factors, and molecules involved in protein translation, and homeostasis. The two largest functional groups of mRNAs that showed altered expression were predicted to be involved in post-translational modification of proteins and transcriptional regulation, suggesting that defects in protein activity and/or turnover may be the key trigger in the pathophysiology of LD. Furthermore we show that changes in gene expression are not limited to brain and are seen in other organs that develop Lafora bodies. Our study may provide valuable insights into the pathophysiology of LD and may aid in developing potential therapeutic targets.
Assuntos
Química Encefálica/genética , Encéfalo/metabolismo , Regulação da Expressão Gênica/fisiologia , Doença de Lafora/genética , Degeneração Neural/genética , Proteínas do Tecido Nervoso/genética , Animais , Encéfalo/patologia , Encéfalo/fisiopatologia , Modelos Animais de Doenças , Fosfatases de Especificidade Dupla , Feminino , Perfilação da Expressão Gênica , Genes Reguladores/genética , Doença de Lafora/metabolismo , Doença de Lafora/fisiopatologia , Masculino , Camundongos , Camundongos Knockout , Mutação/genética , Degeneração Neural/metabolismo , Degeneração Neural/fisiopatologia , Proteínas do Tecido Nervoso/metabolismo , Processamento de Proteína Pós-Traducional/genética , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases não Receptoras , RNA Mensageiro/análise , RNA Mensageiro/genéticaRESUMO
Lafora disease (LD), an autosomal recessive and fatal form of neurodegenerative disorder, is characterized by the presence of polyglucosan inclusions in the affected tissues including the brain. LD can be caused by defects either in the EPM2A gene coding for the laforin protein phosphatase or the NHLRC1 gene coding for the malin ubiquitin ligase. Since the clinical symptoms of LD patients representing the two genetic groups are very similar and since malin is known to interact with laforin, we were curious to examine the possibility that the two proteins regulate each other's function. Using cell biological assays we demonstrate here that (i) malin promotes its own degradation via autoubiquitination, (ii) laforin prevents the auto-degradation of malin by presenting itself as a substrate and (iii) malin preferentially degrades the phosphatase-inactive laforin monomer. Our results that laforin and malin regulate each other's stability and activity offers a novel and attractive model to explain the molecular basis of locus heterogeneity observed in LD.
Assuntos
Proteínas de Transporte/metabolismo , Doença de Lafora/metabolismo , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Animais , Células COS , Proteínas de Transporte/genética , Chlorocebus aethiops , Humanos , Doença de Lafora/genética , Estabilidade Proteica , Proteínas Tirosina Fosfatases não Receptoras/genética , Proteólise , Ubiquitina-Proteína Ligases , UbiquitinaçãoRESUMO
Neurodegenerative disorders are a group of hereditary and sporadic conditions that are characterized by progressive nervous system dysfunctions. These disorders are often associated with neuronal atrophy and are characterized by the presence of intra- or extra-neuronal inclusions in the central or peripheral nervous system. The emerging understanding on these apparently diverse set of disorders suggest that they share a few key pathomechanisms, one of which could be the abnormality in the protein quality control pathways. Recent studies have shown that either an overload on the proteolytic pathways - the ubiquitin-proteasome system and the autophagosome-lysosome system - or defects in the critical components of these pathways might underlie the neuropathology. Here, we review the recent advances in our understanding on the role of protein quality control systems in the pathomechanisms of neurodegenerative disorders, highlight the interdependence between the two pathways and their involvement in neuronal survival.
Assuntos
Encéfalo/patologia , Doenças Neurodegenerativas/patologia , Neurônios/patologia , Encéfalo/metabolismo , Humanos , Doenças Neurodegenerativas/metabolismo , Neurônios/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Superóxido Dismutase/metabolismo , Ubiquitina/metabolismoRESUMO
Lafora disease (LD), an autosomal recessive and fatal form of neurodegenerative disorder, is characterized by the presence of polyglucosan inclusions in the affected tissues including the brain. LD can be caused by defects either in the EPM2A gene coding for the laforin protein phosphatase or the NHLRC1 gene coding for the malin ubiquitin ligase. Since the clinical symptoms of LD patients representing the two genetic groups are very similar and since malin is known to interact with laforin, we were curious to examine the possibility that the two proteins regulate each other’s function. Using cell biological assays we demonstrate here that (i) malin promotes its own degradation via autoubiquitination, (ii) laforin prevents the auto-degradation of malin by presenting itself as a substrate and (iii) malin preferentially degrades the phosphatase-inactive laforin monomer. Our results that laforin and malin regulate each other’s stability and activity offers a novel and attractive model to explain the molecular basis of locus heterogeneity observed in LD.
RESUMO
Lafora disease (LD), an autosomal recessive neurodegenerative disorder, is characterized by the presence of cytoplasmic polyglucosan inclusions known as Lafora bodies in several tissues including the brain. Laforin, a protein phosphatase, and malin, an ubiquitin ligase, are two of the proteins that are known to be defective in LD. Malin interacts with laforin and promotes its polyubiquitination and degradation. Here we show that malin and laforin co-localize in endoplasmic reticulum (ER) and that they form centrosomal aggregates when treated with proteasomal inhibitors in both neuronal and non-neuronal cells. Laforin/malin aggregates co-localize with gamma-tubulin and cause redistribution of alpha-tubulin. These aggregates are also immunoreactive to ubiquitin, ubiquitin-conjugating enzyme, ER chaperone and proteasome subunits, demonstrating their aggresome-like properties. Furthermore, we show that the centrosomal aggregation of laforin and malin is dependent on the functional microtubule network. Laforin and malin form aggresome when expressed together or otherwise, suggesting that the two proteins are recruited to the centrosome independent of each other. Taken together, our results suggest that the centrosomal accumulation of malin, possibly with the help of laforin, may enhance the ubiquitination of its substrates and facilitate their efficient degradation by proteasome. Defects in malin or laforin may thus lead to increased levels of misfolded and/or target proteins, which may eventually affect the physiological processes of the neuron. Thus, defects in protein degradation and clearance are likely to be the primary trigger in the physiopathology of LD.
Assuntos
Proteínas de Transporte/metabolismo , Doença de Lafora/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Acetilcisteína/análogos & derivados , Acetilcisteína/farmacologia , Animais , Células COS , Proteínas de Transporte/genética , Linhagem Celular , Linhagem Celular Tumoral , Centrossomo/metabolismo , Chlorocebus aethiops , Inibidores de Cisteína Proteinase/farmacologia , Retículo Endoplasmático/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Immunoblotting , Imuno-Histoquímica , Corpos de Inclusão/metabolismo , Leupeptinas/farmacologia , Microscopia de Fluorescência , Ligação Proteica/efeitos dos fármacos , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases não Receptoras , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Tubulina (Proteína)/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína LigasesRESUMO
Lafora's disease (LD) is an autosomal recessive and fatal form of progressive myoclonus epilepsy with onset in late childhood or adolescence. LD is characterised by the presence of intracellular polyglucosan inclusions, called Lafora bodies, in tissues including the brain, liver and skin. Patients have progressive neurologic deterioration, leading to death within 10 years of onset. No preventive or curative treatment is available for LD. At least three genes underlie LD, of which two have been isolated and mutations characterised: EPM2A and NHLRC1. The EPM2A gene product laforin is a protein phosphatase while the NHLRC1 gene product malin is an E3 ubiquitin ligase that ubiquitinates and promotes the degradation of laforin. Analyses of the structure and function of these gene products suggest defects in post-translational modification of proteins as the common mechanism that leads to the formation of Lafora inclusion bodies, neurodegeneration and the epileptic phenotype of LD. In this review, we summarise the available information on the genetic basis of LD, and correlate these advances with the rapidly expanding information about the mechanisms of LD gained from studies on both cell biological and animal models. Finally, we also discuss a possible mechanism to explain the locus heterogeneity observed in LD.