RESUMO
Amyotrophic lateral sclerosis (ALS) is a heterogenous neurodegenerative disorder that affects motor neurons and voluntary muscle control1. ALS heterogeneity includes the age of manifestation, the rate of progression and the anatomical sites of symptom onset. Disease-causing mutations in specific genes have been identified and define different subtypes of ALS1. Although several ALS-associated genes have been shown to affect immune functions2, whether specific immune features account for ALS heterogeneity is poorly understood. Amyotrophic lateral sclerosis-4 (ALS4) is characterized by juvenile onset and slow progression3. Patients with ALS4 show motor difficulties by the time that they are in their thirties, and most of them require devices to assist with walking by their fifties. ALS4 is caused by mutations in the senataxin gene (SETX). Here, using Setx knock-in mice that carry the ALS4-causative L389S mutation, we describe an immunological signature that consists of clonally expanded, terminally differentiated effector memory (TEMRA) CD8 T cells in the central nervous system and the blood of knock-in mice. Increased frequencies of antigen-specific CD8 T cells in knock-in mice mirror the progression of motor neuron disease and correlate with anti-glioma immunity. Furthermore, bone marrow transplantation experiments indicate that the immune system has a key role in ALS4 neurodegeneration. In patients with ALS4, clonally expanded TEMRA CD8 T cells circulate in the peripheral blood. Our results provide evidence of an antigen-specific CD8 T cell response in ALS4, which could be used to unravel disease mechanisms and as a potential biomarker of disease state.
Assuntos
Esclerose Lateral Amiotrófica , Linfócitos T CD8-Positivos , Células Clonais , Esclerose Lateral Amiotrófica/imunologia , Esclerose Lateral Amiotrófica/patologia , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Células Clonais/patologia , DNA Helicases/genética , DNA Helicases/metabolismo , Técnicas de Introdução de Genes , Camundongos , Neurônios Motores/patologia , Enzimas Multifuncionais/genética , Enzimas Multifuncionais/metabolismo , Mutação , RNA Helicases/genética , RNA Helicases/metabolismoRESUMO
Amyotrophic lateral sclerosis (ALS) is a rapidly progressing, highly heterogeneous neurodegenerative disease, underscoring the importance of obtaining information to personalize clinical decisions quickly after diagnosis. Here, we investigated whether ALS-relevant signatures can be detected directly from biopsied patient fibroblasts. We profiled familial ALS (fALS) fibroblasts, representing a range of mutations in the fused in sarcoma (FUS) gene and ages of onset. To differentiate FUS fALS and healthy control fibroblasts, machine-learning classifiers were trained separately on high-content imaging and transcriptional profiles. "Molecular ALS phenotype" scores, derived from these classifiers, captured a spectrum from disease to health. Interestingly, these scores negatively correlated with age of onset, identified several pre-symptomatic individuals and sporadic ALS (sALS) patients with FUS-like fibroblasts, and quantified "movement" of FUS fALS and "FUS-like" sALS toward health upon FUS ASO treatment. Taken together, these findings provide evidence that non-neuronal patient fibroblasts can be used for rapid, personalized assessment in ALS.
Assuntos
Esclerose Lateral Amiotrófica , Fibroblastos , Proteína FUS de Ligação a RNA , Humanos , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Proteína FUS de Ligação a RNA/metabolismo , Proteína FUS de Ligação a RNA/genética , Mutação/genética , Masculino , Feminino , Pele/patologia , Pele/metabolismo , Aprendizado de Máquina , Pessoa de Meia-IdadeRESUMO
Fused in sarcoma (FUS) is an RNA-binding protein that is genetically and pathologically associated with rare and aggressive forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). To explore the mechanisms by which mutant FUS causes neurodegeneration in ALS-FTD, we generated a series of FUS knock-in mouse lines that express the equivalent of ALS-associated mutant FUSP525L and FUSΔEX14 protein. In FUS mutant mice, we show progressive, age-dependent motor neuron loss as a consequence of a dose-dependent gain of toxic function, associated with the insolubility of FUS and related RNA-binding proteins. In this disease-relevant mouse model of ALS-FUS, we show that ION363, a non-allele-specific FUS antisense oligonucleotide, efficiently silences Fus and reduces postnatal levels of FUS protein in the brain and spinal cord, delaying motor neuron degeneration. In a patient with ALS with a FUSP525L mutation, we provide preliminary evidence that repeated intrathecal infusions of ION363 lower wild-type and mutant FUS levels in the central nervous system, resulting in a marked reduction in the burden of FUS aggregates that are a pathological hallmark of disease. In mouse genetic and human clinical studies, we provide evidence in support of FUS silencing as a therapeutic strategy in FUS-dependent ALS and FTD.
Assuntos
Esclerose Lateral Amiotrófica/terapia , Inativação Gênica/efeitos dos fármacos , Oligonucleotídeos Antissenso/farmacologia , Proteína FUS de Ligação a RNA/genética , Esclerose Lateral Amiotrófica/genética , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Transgênicos , MutaçãoRESUMO
PURPOSE: For many tumors, signaling exchanges between cancer cells and other cells in their microenvironment influence overall tumor signaling. Some of these exchanges depend on expression of the primary cilium on nontransformed cell populations, as extracellular ligands including Sonic Hedgehog (SHH), PDGFRα, and others function through receptors spatially localized to cilia. Cell ciliation is regulated by proteins that are themselves therapeutic targets. We investigated whether kinase inhibitors of clinical interest influence ciliation and signaling by proteins with ciliary receptors in cancer and other cilia-relevant disorders, such as polycystic kidney disease (PKD). EXPERIMENTAL DESIGN: We screened a library of clinical and preclinical kinase inhibitors, identifying drugs that either prevented or induced ciliary disassembly. Specific bioactive protein targets of the drugs were identified by mRNA depletion. Mechanism of action was defined, and activity of select compounds investigated. RESULTS: We identified multiple kinase inhibitors not previously linked to control of ciliation, including sunitinib, erlotinib, and an inhibitor of the innate immune pathway kinase, IRAK4. For all compounds, activity was mediated through regulation of Aurora-A (AURKA) activity. Drugs targeting cilia influenced proximal cellular responses to SHH and PDGFRα. In vivo, sunitinib durably limited ciliation and cilia-related biological activities in renal cells, renal carcinoma cells, and PKD cysts. Extended analysis of IRAK4 defined a subset of innate immune signaling effectors potently affecting ciliation. CONCLUSIONS: These results suggest a paradigm by which targeted drugs may have unexpected off-target effects in heterogeneous cell populations in vivo via control of a physical platform for receipt of extracellular ligands.
Assuntos
Cílios/efeitos dos fármacos , Cílios/metabolismo , Descoberta de Drogas , Animais , Biomarcadores , Linhagem Celular , Suscetibilidade a Doenças , Cloridrato de Erlotinib/farmacologia , Proteínas Hedgehog/metabolismo , Humanos , Doenças Renais Císticas/etiologia , Doenças Renais Císticas/metabolismo , Doenças Renais Císticas/patologia , Camundongos , Modelos Biológicos , Comunicação Parácrina/efeitos dos fármacos , Fator de Crescimento Derivado de Plaquetas/metabolismo , Rim Policístico Autossômico Dominante/metabolismo , Inibidores de Proteínas Quinases/farmacologia , RNA Interferente Pequeno/genética , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas , Sunitinibe/farmacologiaRESUMO
Gemini surfactants with hexadecyl tails and hydroxyethylated head groups bridged with tetramethylene (G4), hexamethylene (G6) and dodecamethylene (G12) spacers were shown to self-assemble at the lower critical micelle concentration compared to their conventional m-s-m analogs. The lipoplex formation and the plasmid DNA transfer into different kinds of host cells were studied. In the case of eukaryotic cells, high transfection efficacy has been demonstrated for DNA-gemini complexes, which increased as follows: G6Assuntos
Técnicas de Transferência de Genes
, Compostos de Amônio Quaternário/química
, Tensoativos/química
, Transfecção/métodos
, DNA/química
, DNA/genética
, Eletroporação
, Proteínas de Fluorescência Verde/genética
, Proteínas de Fluorescência Verde/metabolismo
, Células HEK293
, Humanos
, Microscopia de Força Atômica
, Estrutura Molecular
, Conformação de Ácido Nucleico
, Plasmídeos/química
, Plasmídeos/genética
, Reprodutibilidade dos Testes
, Relação Estrutura-Atividade
, Termodinâmica
RESUMO
Anti-Müllerian hormone (AMH) and its type II receptor AMHR2, both previously thought to primarily function in gonadal tissue, were unexpectedly identified as potent regulators of transforming growth factor (TGF-ß)/bone morphogenetic protein (BMP) signaling and epithelial-mesenchymal transition (EMT) in lung cancer. AMH is a TGF-ß/BMP superfamily member, and AMHR2 heterodimerizes with type I receptors (ALK2, ALK3) also used by the type II receptor for BMP (BMPR2). AMH signaling regulates expression of BMPR2, ALK2, and ALK3, supports protein kinase B-nuclear factor κB (AKT-NF-κB) and SMAD survival signaling, and influences BMP-dependent signaling in non-small cell lung cancer (NSCLC). AMH and AMHR2 are selectively expressed in epithelial versus mesenchymal cells, and loss of AMH/AMHR2 induces EMT. Independent induction of EMT reduces expression of AMH and AMHR2. Importantly, EMT associated with depletion of AMH or AMHR2 results in chemoresistance but sensitizes cells to the heat shock protein 90 (HSP90) inhibitor ganetespib. Recognition of this AMH/AMHR2 axis helps to further elucidate TGF-ß/BMP resistance-associated signaling and suggests new strategies for therapeutic targeting of EMT.