RESUMEN
Genetic defects in DNA repair are increasingly recognized as being able to cause degenerative ataxia syndromes. It remains a mystery, however, why disruption of a process fundamental to proliferating cells can be selectively toxic to postmitotic neurons. Recent studies now reveal that an ataxia gene, tyrosyl phosphodiesterase 1 (TDP1), repairs single-stranded DNA breaks in nondividing cells. Here we review the implications of this and other findings for a growing list of hereditary ataxias.
Asunto(s)
Reparación del ADN/fisiología , Degeneraciones Espinocerebelosas/genética , Animales , ADN de Cadena Simple/fisiología , Humanos , Hidrolasas Diéster Fosfóricas/genética , Degeneraciones Espinocerebelosas/fisiopatologíaRESUMEN
Analyses of the biologic effects of mutations in the BRI2 (ITM2b) and the amyloid beta precursor protein (APP) genes support the hypothesis that cerebral accumulation of amyloidogenic peptides in familial British and familial Danish dementias and Alzheimer's disease (AD) is associated with neurodegeneration. We have used somatic brain transgenic technology to express the BRI2 and BRI2-Abeta1-40 transgenes in APP mouse models. Expression of BRI2-Abeta1-40 mimics the suppressive effect previously observed using conventional transgenic methods, further validating the somatic brain transgenic methodology. Unexpectedly, we also find that expression of wild-type human BRI2 reduces cerebral Abeta deposition in an AD mouse model. Additional data indicate that the 23 aa peptide, Bri23, released from BRI2 by normal processing, is present in human CSF, inhibits Abeta aggregation in vitro and mediates its anti-amyloidogenic effect in vivo. These studies demonstrate that BRI2 is a novel mediator of Abeta deposition in vivo.
Asunto(s)
Péptidos beta-Amiloides/antagonistas & inhibidores , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/antagonistas & inhibidores , Precursor de Proteína beta-Amiloide/metabolismo , Amiloide/fisiología , Encéfalo/metabolismo , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Amiloide/genética , Péptidos beta-Amiloides/administración & dosificación , Péptidos beta-Amiloides/genética , Precursor de Proteína beta-Amiloide/administración & dosificación , Precursor de Proteína beta-Amiloide/genética , Animales , Encéfalo/patología , Pollos , Cricetinae , Dependovirus/genética , Femenino , Técnicas de Transferencia de Gen , Humanos , Masculino , Glicoproteínas de Membrana , Proteínas de la Membrana , Ratones , Ratones Transgénicos , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/genética , FenotipoRESUMEN
Huntington's disease (HD) and other polyglutamine (polyQ) neurodegenerative diseases are characterized by neuronal accumulation of the disease protein, suggesting that the cellular ability to handle abnormal proteins is compromised. As both a cochaperone and ubiquitin ligase, the C-terminal Hsp70 (heat shock protein 70)-interacting protein (CHIP) links the two major arms of protein quality control, molecular chaperones, and the ubiquitin-proteasome system. Here, we demonstrate that CHIP suppresses polyQ aggregation and toxicity in transfected cell lines, primary neurons, and a novel zebrafish model of disease. Suppression by CHIP requires its cochaperone function, suggesting that CHIP acts to facilitate the solubility of mutant polyQ proteins through its interactions with chaperones. Conversely, HD transgenic mice that are haploinsufficient for CHIP display a markedly accelerated disease phenotype. We conclude that CHIP is a critical mediator of the neuronal response to misfolded polyQ protein and represents a potential therapeutic target in this important class of neurodegenerative diseases.
Asunto(s)
Inhibición Neural/efectos de los fármacos , Neuronas/efectos de los fármacos , Péptidos/metabolismo , Ubiquitina-Proteína Ligasas/farmacología , Animales , Western Blotting/métodos , Células Cultivadas , Corteza Cerebral/citología , Chlorocebus aethiops , Modelos Animales de Enfermedad , Embrión de Mamíferos , Embrión no Mamífero , Técnica del Anticuerpo Fluorescente/métodos , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/metabolismo , Enfermedad de Huntington/tratamiento farmacológico , Técnicas In Vitro , Ratones , Ratones Transgénicos , Proteínas Asociadas a Microtúbulos/metabolismo , Mutación , Neuronas/citología , Péptidos/genética , Radioinmunoensayo/métodos , Ratas , Transfección/métodos , Pez CebraRESUMEN
Tau and amyloid precursor protein (APP) are key proteins in the pathogenesis of sporadic and inherited Alzheimer's disease. Thus, developing ways to inhibit production of these proteins is of great research and therapeutic interest. The selective silencing of mutant alleles, moreover, represents an attractive strategy for treating inherited dementias and other dominantly inherited disorders. Here, using tau and APP as model targets, we describe an efficient method for producing small interfering RNA (siRNA) against essentially any targeted region of a gene. We then use this approach to develop siRNAs that display optimal allele-specific silencing against a well-characterized tau mutation (V337M) and the most widely studied APP mutation (APPsw). The allele-specific RNA duplexes identified by this method then served as templates for constructing short hairpin RNA (shRNA) plasmids that successfully silenced mutant tau or APP alleles. These plasmids should prove useful in experimental and therapeutic studies of Alzheimer's disease. Our results suggest guiding principles for the production of allele-specific siRNA, and the general method described here should facilitate the production of gene-specific siRNAs.
Asunto(s)
Alelos , Enfermedad de Alzheimer/genética , Mutación/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animales , Secuencia de Bases , Células COS , ARN Polimerasas Dirigidas por ADN/metabolismo , Genes Reporteros/genética , Células HeLa , Humanos , Especificidad por Sustrato , Proteínas Virales , Proteínas tau/genéticaRESUMEN
BACKGROUND: Alzheimer's disease (AD) and a host of other neurodegenerative central nervous system (CNS) proteinopathies are characterized by the accumulation of misfolded protein aggregates. Simplistically, these aggregates can be divided into smaller, soluble, oligomeric and larger, less-soluble or insoluble, fibrillar forms. Perhaps the major ongoing debate in the neurodegenerative disease field is whether the smaller oligomeric or larger fibrillar aggregates are the primary neurotoxin. Herein, we propose an integrative hypothesis that provides new insights into how a variety of misfolded protein aggregates can result in neurodegeneration. RESULTS: We introduce the concept that a wide range of highly stable misfolded protein aggregates in AD and other neurodegenerative proteinopathies are recognized as non-self and chronically activate the innate immune system. This pro-inflammatory state leads to physiological senescence of CNS cells. Once CNS cells undergo physiological senescence, they secrete a variety of pro-inflammatory molecules. Thus, the senescence of cells, which was initially triggered by inflammatory stimuli, becomes a self-reinforcing stimulus for further inflammation and senescence. Ultimately, senescent CNS cells become functionally impaired and eventually die, and this neurodegeneration leads to brain organ failure. CONCLUSION: This integrative hypothesis, which we will refer to as the proteinopathy-induced senescent cell hypothesis of AD and other neurodegenerative diseases, links CNS proteinopathies to inflammation, physiological senescence, cellular dysfunction, and ultimately neurodegeneration. Future studies characterizing the senescent phenotype of CNS cells in AD and other neurodegenerative diseases will test the validity of this hypothesis. The implications of CNS senescence as a contributing factor to the neurodegenerative cascade and its implications for therapy are discussed.
RESUMEN
In SCF (Skp1/Cullin/F-box protein) ubiquitin ligases, substrate specificity is conferred by a diverse array of F-box proteins. Only in fully assembled SCF complexes, it is believed, can substrates bound to F-box proteins become ubiquitinated. Here we show that Fbx2, a brain-enriched F-box protein implicated in the ubiquitination of glycoproteins discarded from the endoplasmic reticulum, binds the co-chaperone/ubiquitin ligase CHIP (C terminus of Hsc-70-interacting protein) through a unique N-terminal PEST domain in Fbx2. CHIP facilitates the ubiquitination and degradation of Fbx2-bound glycoproteins, including unassembled NMDA receptor subunits. These findings indicate that CHIP acts with Fbx2 in a novel ubiquitination pathway that links CHIP to glycoprotein quality control in neurons. In addition, they expand the repertoire of pathways by which F-box proteins can regulate ubiquitination and suggest a new role for PEST domains as a protein interaction motif.
Asunto(s)
Proteínas de Drosophila/metabolismo , Glicoproteínas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Ligasas SKP Cullina F-box/metabolismo , Ubiquitina/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Células COS , Chlorocebus aethiops , Cartilla de ADN , Proteínas de Drosophila/genética , Glicoproteínas/genética , Glicosilación , Humanos , Cinética , Datos de Secuencia Molecular , Neuronas/fisiología , Proteínas Nucleares/genética , Proteínas Recombinantes de Fusión/metabolismo , Transfección , Ubiquitina/genéticaRESUMEN
1.RNA interference (RNAi) is a recently discovered biological pathway that mediates post-transcriptional gene silencing. The process of RNAi is orchestrated by an increasingly well-understood cellular machinery. 2. The common entry point for both natural and engineered RNAi are double stranded RNA molecules known as short interfering RNAs (siRNAs), that mediate the sequence-specific identification and degradation of the targeted messenger RNA (mRNA). The study and manipulation of these siRNAs has recently revolutionized biomedical research. 3. In this review, we first provide a brief overview of the process of RNAi, focusing on its potential role in brain function and involvement in neurological disease. We then describe the methods developed to manipulate RNAi in the laboratory and its applications to neuroscience. Finally, we focus on the potential therapeutic application of RNAi to neurological disease.
Asunto(s)
Biología Molecular/tendencias , Neurociencias/tendencias , Interferencia de ARN/fisiología , ARN Interferente Pequeño/genética , Animales , Química Encefálica/genética , Silenciador del Gen/fisiología , Terapia Genética/métodos , Terapia Genética/tendencias , Humanos , Biología Molecular/métodos , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Neurociencias/métodos , ARN Mensajero/genéticaRESUMEN
Protein misfolding and aggregation are central features of the polyglutamine neurodegenerative disorders, but the dynamic properties of expanded polyglutamine proteins are poorly understood. Here, we use fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) with green fluorescent protein fusion proteins to study polyglutamine protein kinetics in living cells. Our results reveal markedly divergent mobility states for an expanded polyglutamine protein, ataxin-3, and establish that nuclear inclusions formed by this protein are aggregates. Additional studies of green fluorescent protein-tagged cAMP response element binding protein coexpressed with either of two mutant polyglutamine proteins, ataxin-3 and huntingtin, support a model of disease in which coaggregation of transcriptional components contributes to pathogenesis. Finally, studies of a third polyglutamine disease protein, ataxin-1, reveal unexpected heterogeneity in the dynamics of inclusions formed by different disease proteins, a finding which may help explain disease-specific elements of pathogenesis in these neurodegenerative disorders.
Asunto(s)
Trastornos Heredodegenerativos del Sistema Nervioso/genética , Trastornos Heredodegenerativos del Sistema Nervioso/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Péptidos/genética , Péptidos/metabolismo , Animales , Ataxina-3 , Células COS , Proteína de Unión a CREB , Proteínas Fluorescentes Verdes , Trastornos Heredodegenerativos del Sistema Nervioso/etiología , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Modelos Neurológicos , Mutación , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras , Transactivadores/metabolismo , Transfección , Repeticiones de TrinucleótidosRESUMEN
A three-nucleotide (GAG) deletion in the TOR1A gene is the most common cause of inherited dystonia, DYT1. Because the mutant protein, TorsinA (TA), is thought to act in a dominant manner to cause disease, inhibiting expression from the mutant gene represents a potentially powerful therapeutic strategy. In an effort to develop therapy for this disease, we tested whether small interfering RNA (siRNA) could selectively silence expression of mutant TA. Exploiting the three-base pair difference between wild-type and mutant alleles, we designed siRNAs to silence expression of mutant, wild-type, or both forms of TA. In transfected cells, siRNA successfully suppressed wild-type or mutant TA in an allele-specific manner: for example, mutant-specific siRNA reduced the levels of mutant TA to less than 1% of controls with minimal effect on wild-type TA expression. In cells expressing both alleles, thus simulating the heterozygous state, siRNA-mediated suppression remained robust and allele specific. Our siRNA studies demonstrate allele-specific targeting of a dominant neurogenetic disease gene and suggest the broad therapeutic potential of siRNA for DYT1 dystonia and other dominantly inherited neurological diseases.
Asunto(s)
Alelos , Proteínas Portadoras/genética , Distonía/genética , Distonía/terapia , Silenciador del Gen , Terapia Genética/métodos , Chaperonas Moleculares , Mutación Puntual/genética , ARN Interferente Pequeño/genética , Western Blotting , Técnicas de Cultivo de Célula , Eliminación de Gen , Humanos , Microscopía Fluorescente , Plásmidos/genética , Transfección/métodosRESUMEN
Small interfering RNA (siRNA) holds therapeutic promise for silencing dominantly acting disease genes, particularly if mutant alleles can be targeted selectively. In mammalian cell models we demonstrate that allele-specific silencing of disease genes with siRNA can be achieved by targeting either a linked single-nucleotide polymorphism (SNP) or the disease mutation directly. For a polyglutamine neurodegenerative disorder in which we first determined that selective targeting of the disease-causing CAG repeat is not possible, we took advantage of an associated SNP to generate siRNA that exclusively silenced the mutant Machado-Joseph disease/spinocerebellar ataxia type 3 allele while sparing expression of the WT allele. Allele-specific suppression was accomplished with all three approaches currently used to deliver siRNA: in vitro-synthesized duplexes as well as plasmid and viral expression of short hairpin RNA. We further optimized siRNA to specifically target a missense Tau mutation, V337M, that causes frontotemporal dementia. These studies establish that siRNA can be engineered to silence disease genes differing by a single nucleotide and highlight a key role for SNPs in extending the utility of siRNA in dominantly inherited disorders.