RESUMO
Inclusions comprised of microtubule-associated protein tau (tau) are implicated in a group of neurodegenerative diseases, collectively known as tauopathies, that include Alzheimer's disease (AD). The spreading of misfolded tau "seeds" along neuronal networks is thought to play a crucial role in the progression of tau pathology. Consequently, restricting the release or uptake of tau seeds may inhibit the spread of tau pathology and potentially halt the advancement of the disease. Previous studies have demonstrated that the Mammalian Suppressor of Tauopathy 2 (MSUT2), an RNA binding protein, modulates tau pathogenesis in a transgenic mouse model. In this study, we investigated the impact of MSUT2 on tau pathogenesis using tau seeding models. Our findings indicate that the loss of MSUT2 mitigates human tau seed-induced pathology in neuron cultures and mouse models. In addition, MSUT2 regulates many gene transcripts, including the Adenosine Receptor 1 (A1AR), and we show that down regulation or inhibition of A1AR modulates the activity of the "ArfGAP with SH3 Domain, Ankyrin Repeat, and PH Domain 1 protein" (ASAP1), thereby influencing the internalization of pathogenic tau seeds into neurons resulting in reduction of tau pathology.
Assuntos
Doença de Alzheimer , Tauopatias , Camundongos , Humanos , Animais , Encéfalo/patologia , Proteínas tau/metabolismo , Tauopatias/patologia , Doença de Alzheimer/patologia , Neurônios/patologia , Camundongos Transgênicos , Mamíferos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismoRESUMO
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with no known cure. Aggregates of the nuclear protein TDP-43 have been recognized as a hallmark of proteinopathy in both familial and sporadic cases of ALS. Post-translational modifications of this protein, include hyperphosphorylation, cause disruption of TDP-43 homeostasis and as a consequence, promotion of its neurotoxicity. Among the kinases involved in these changes, cell division cycle kinase 7 (CDC7) plays an important role by directly phosphorylating TDP-43. In the present manuscript the discovery, synthesis, and optimization of a new family of selective and ATP-competitive CDC7 inhibitors based on 6-mercaptopurine scaffold are described. Moreover, we demonstrate the ability of these inhibitors to reduce TDP-43 phosphorylation in both cell cultures and transgenic animal models such as C. elegans and Prp-hTDP43 (A315T) mice. Altogether, the compounds described here may be useful as versatile tools to explore the role of CDC7 in TDP-43 phosphorylation and also as new drug candidates for the future development of ALS therapies.
Assuntos
Esclerose Lateral Amiotrófica/tratamento farmacológico , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ligação a DNA/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Esclerose Lateral Amiotrófica/metabolismo , Animais , Comportamento Animal/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta a Droga , Humanos , Camundongos , Camundongos Transgênicos , Simulação de Acoplamento Molecular , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Amyotrophic lateral sclerosis (ALS), a common motor neuron disease affecting two per 100,000 people worldwide, encompasses at least five distinct pathological subtypes, including, ALS-SOD1, ALS-C9orf72, ALS-TDP-43, ALS-FUS and Guam-ALS. The etiology of a major subset of ALS involves toxicity of the TAR DNA-binding protein-43 (TDP-43). A second RNA/DNA binding protein, fused in sarcoma/translocated in liposarcoma (FUS/TLS) has been subsequently associated with about 1% of ALS patients. While mutations in TDP-43 and FUS have been linked to ALS, the key contributing molecular mechanism(s) leading to cell death are still unclear. One unique feature of TDP-43 and FUS pathogenesis in ALS is their nuclear clearance and simultaneous cytoplasmic aggregation in affected motor neurons. Since the discoveries in the last decade implicating TDP-43 and FUS toxicity in ALS, a majority of studies have focused on their cytoplasmic aggregation and disruption of their RNA-binding functions. However, TDP-43 and FUS also bind to DNA, although the significance of their DNA binding in disease-affected neurons has been less investigated. A recent observation of accumulated genomic damage in TDP-43 and FUS-linked ALS and association of FUS with neuronal DNA damage repair pathways indicate a possible role of deregulated DNA binding function of TDP-43 and FUS in ALS. In this review, we discuss the different ALS disease subtypes, crosstalk of etiopathologies in disease progression, available animal models and their limitations, and recent advances in understanding the specific involvement of RNA/DNA binding proteins, TDP-43 and FUS, in motor neuron diseases.
Assuntos
Esclerose Lateral Amiotrófica/genética , Proteínas de Ligação a DNA/genética , Proteína FUS de Ligação a RNA/genética , HumanosRESUMO
OBJECTIVE: Kinase hyperactivity occurs in both neurodegenerative disease and cancer. Lesions containing hyperphosphorylated aggregated TDP-43 characterize amyotrophic lateral sclerosis and frontotemporal lobar degeneration with TDP-43 inclusions. Dual phosphorylation of TDP-43 at serines 409/410 (S409/410) drives neurotoxicity in disease models; therefore, TDP-43-specific kinases are candidate targets for intervention. METHODS: To find therapeutic targets for the prevention of TDP-43 phosphorylation, we assembled and screened a comprehensive RNA interference library targeting kinases in TDP-43 transgenic Caenorhabditis elegans. RESULTS: We show CDC7 robustly phosphorylates TDP-43 at pathological residues S409/410 in C. elegans, in vitro, and in human cell culture. In frontotemporal lobar degeneration (FTLD)-TDP cases, CDC7 immunostaining overlaps with the phospho-TDP-43 pathology found in frontal cortex. Furthermore, PHA767491, a small molecule inhibitor of CDC7, reduces TDP-43 phosphorylation and prevents TDP-43-dependent neurodegeneration in TDP-43-transgenic animals. INTERPRETATION: Taken together, these data support CDC7 as a novel therapeutic target for TDP-43 proteinopathies, including FTLD-TDP and amyotrophic lateral sclerosis.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Doenças Neurodegenerativas/etiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteinopatias TDP-43/terapia , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Linhagem Celular Transformada , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Lobo Frontal/metabolismo , Lobo Frontal/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Humanos , Movimento/fisiologia , Mutação/genética , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Fosforilação , Piperidonas/farmacologia , Pirróis/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Serina/metabolismo , Proteinopatias TDP-43/complicações , Proteinopatias TDP-43/tratamento farmacológico , Proteinopatias TDP-43/genética , TransfecçãoRESUMO
Proteolytic cleavage of tau at glutamic acid 391 (E391) is linked to the pathogenesis of Alzheimer disease (AD). This C-terminal-truncated tau species exists in neurofibrillary tangles and abnormal neurites in the brains of AD patients and may potentiate tau polymerization. We generated a mouse model that expresses human tau truncated at E391 to begin to elucidate the role of this C-terminal-truncated tau species in the development of tau pathology. Our results show that truncated but otherwise wild-type human tau is sufficient to drive pretangle pathologic changes in tau, including accumulation of insoluble tau, somatodendritic redistribution, formation of pathologic conformations, and dual phosphorylation of tau at sites associated with AD pathology. In addition, these mice exhibit atypical neuritic tau immunoreactivity, including abnormal neuritic processes and dystrophic neurites. These results suggest that changes in tau proteolysis can initiate tauopathy.
Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Encéfalo/patologia , Ácido Glutâmico/genética , Mutação/genética , Proteínas tau/genética , Adenosina Trifosfatases/metabolismo , Animais , Encéfalo/metabolismo , Proteínas de Transporte de Cátions/metabolismo , ATPases Transportadoras de Cobre , Modelos Animais de Doenças , Regulação da Expressão Gênica/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/metabolismo , Neurônios/patologia , Fosforilação , Placa Amiloide/metabolismo , Placa Amiloide/patologia , Proteínas tau/metabolismoRESUMO
The neuropathological hallmark of the majority of amyotrophic lateral sclerosis (ALS) and a class of frontotemporal lobar degeneration is ubiquitinated cytoplasmic aggregates composed of transactive response DNA binding protein 43 kDa (TDP-43). Genetic manipulation of TDP-43 in animal models has been used to study the protein's role in pathogenesis. Transgenic rodents for TDP-43 have recapitulated key aspects of ALS such as paralysis, loss of spinal motor neurons and muscle atrophy. Viral vectors are an alternate approach to express pathological proteins in animals. Use of the recombinant adeno-associated virus vector serotype 9 has permitted widespread transgene expression throughout the central nervous system after intravenous administration. Expressing TDP-43 in rats with this method produced a phenotype that was consistent with and similar to TDP-43 transgenic lines. Increased levels of TDP-43 in the nucleus are toxic to neurons and sufficient to produce ALS-like symptoms. Animal models based on TDP-43 will address the relationships between TDP-43 expression levels, pathology, neuronal loss, muscle atrophy, motor function and causative mechanisms of disease. New targets that modify TDP-43 function, or targets from previous ALS models and other models of spinal cord diseases, could be tested for efficacy in the recent rodent models of ALS based on TDP-43. The vector approach could be an important therapeutic channel because the entire spinal cord can be affected from a one-time peripheral administration.
Assuntos
Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/terapia , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Terapia Genética/métodos , Roedores , Esclerose Lateral Amiotrófica/patologia , Esclerose Lateral Amiotrófica/fisiopatologia , Animais , Animais Geneticamente Modificados , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Vetores Genéticos/uso terapêutico , Células Germinativas/fisiologia , Humanos , Medula Espinal/patologia , Medula Espinal/fisiologia , Medula Espinal/fisiopatologiaRESUMO
Lesions containing aggregated and hyperphosphorylated tau protein are characteristic of neurodegenerative tauopathies. We have developed a cellular model of pathological tau deposition and clearance by overexpressing wild type human tau in HEK293 cells. When proteasome activity is inhibited, HEK293/tau cells accumulate tau protein in structures that bear many of the hallmarks of aggresomes. These include recruitment of tau into large spherical inclusions, accumulation of the retrograde motor protein dynein at the centrosome, formation of an intermediate filament cage around inclusions, and clustering of mitochondria at the aggresome. Tau aggresomes form rapidly and can be cleared upon relief of proteasome inhibition. We observe recruitment of pathological misfolded phospho-tau species to aggresomes. Immunoblotting reveals accumulation of detergent insoluble aggregated tau species. Knockdown of histone deacetylase 6, a protein known to interact with tau, reveals a requirement for HDAC6 activity in tau aggresome formation. Direct observation of the accumulation and clearance of abnormal tau species will allow us to dissect the cellular and molecular mechanisms at work in clearing aggresomal tau and its similarity to disease relevant pathological tau clearance mechanisms.