RESUMEN
Ubiquitin-immunoreactive neuronal inclusions composed of TAR DNA binding protein of 43 kDa (TDP-43) are a major pathological feature of frontotemporal lobar degeneration (FTLD-TDP). In vivo studies with TDP-43 knockout mice have suggested that TDP-43 plays a critical, although undefined role in development. In the current report, we generated transgenic mice that conditionally express wild-type human TDP-43 (hTDP-43) in the forebrain and established a paradigm to examine the sensitivity of neurons to TDP-43 overexpression at different developmental stages. Continuous TDP-43 expression during early neuronal development produced a complex phenotype, including aggregation of phospho-TDP-43, increased ubiquitin immunoreactivity, mitochondrial abnormalities, neurodegeneration and early lethality. In contrast, later induction of hTDP-43 in the forebrain of weaned mice prevented early death and mitochondrial abnormalities while yielding salient features of FTLD-TDP, including progressive neurodegeneration and ubiquitinated, phospho-TDP-43 neuronal cytoplasmic inclusions. These results suggest that neurons in the developing forebrain are extremely sensitive to TDP-43 overexpression and that timing of TDP-43 overexpression in transgenic mice must be considered when distinguishing normal roles of TDP-43, particularly as they relate to development, from its pathogenic role in FTLD-TDP and other TDP-43 proteinopathies. Finally, our adult induction of hTDP-43 strategy provides a mouse model that develops critical pathological features that are directly relevant for human TDP-43 proteinopathies.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Neuronas/metabolismo , Proteinopatías TDP-43/metabolismo , Animales , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Humanos , Ratones , Ratones de la Cepa 129 , Ratones Transgénicos , Mitocondrias/genética , Mitocondrias/metabolismo , Neuronas/citología , Proteinopatías TDP-43/genética , Factores de Tiempo , Ubiquitina/metabolismoRESUMEN
The zinc finger protein EVI1 is causally associated with acute myeloid leukemogenesis, and inhibition of its function with a small molecule therapeutic may provide effective therapy for EVI1-expressing leukemias. In this paper we describe the development of a pyrrole-imidazole polyamide to specifically block EVI1 binding to DNA. We first identify essential domains for leukemogenesis through structure-function studies on both EVI1 and the t(3;21)(q26;q22)-derived RUNX1-MDS1-EVI1 (RME) protein, which revealed that DNA binding to the cognate motif GACAAGATA via the first of two zinc finger domains (ZF1, encompassing fingers 1-7) is essential transforming activity. To inhibit DNA binding via ZF1, we synthesized a pyrrole-imidazole polyamide 1, designed to bind to a subsite within the GACAAGATA motif and thereby block EVI1 binding. DNase I footprinting and electromobility shift assays revealed a specific and high affinity interaction between polyamide 1 and the GACAAGATA motif. In an in vivo CAT reporter assay using NIH-3T3-derived cell line with a chromosome-embedded tet-inducible EVI1-VP16 as well as an EVI1-responsive reporter, polyamide 1 completely blocked EVI1-responsive reporter activity. Growth of a leukemic cell line bearing overexpressed EVI1 was also inhibited by treatment with polyamide 1, while a control cell line lacking EVI1 was not. Finally, colony formation by RME was attenuated by polyamide 1 in a serial replating assay. These studies provide evidence that a cell permeable small molecule may effectively block the activity of a leukemogenic transcription factor and provide a valuable tool to dissect critical functions of EVI1 in leukemogenesis.
Asunto(s)
Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/química , Inhibidores de Crecimiento/farmacología , Imidazoles/farmacología , Nylons/farmacología , Pirroles/farmacología , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular Transformada , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Sistemas de Liberación de Medicamentos/métodos , Inhibidores de Crecimiento/química , Inhibidores de Crecimiento/metabolismo , Humanos , Imidazoles/química , Imidazoles/metabolismo , Proteína del Locus del Complejo MDS1 y EV11 , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Células Mieloides/efectos de los fármacos , Células Mieloides/metabolismo , Células Mieloides/patología , Nylons/química , Nylons/metabolismo , Unión Proteica/genética , Proto-Oncogenes/genética , Pirroles/química , Pirroles/metabolismo , Ratas , Retroviridae/genética , Factores de Transcripción/genéticaRESUMEN
The Mds1 and Evi1 complex locus (Mecom) gives rise to several alternative transcripts implicated in leukemogenesis. However, the contribution that Mecom-derived gene products make to normal hematopoiesis remains largely unexplored. To investigate the role of the upstream transcription start site of Mecom in adult hematopoiesis, we created a mouse model with a lacZ knock-in at this site, termed ME(m1), which eliminates Mds1-Evi1 (ME), the longer, PR-domain-containing isoform produced by the gene (also known as PRDM3). ß-galactosidase-marking studies revealed that, within hematopoietic cells, ME is exclusively expressed in the stem cell compartment. ME deficiency leads to a reduction in the number of HSCs and a complete loss of long-term repopulation capacity, whereas the stem cell compartment is shifted from quiescence to active cycling. Genetic exploration of the relative roles of endogenous ME and EVI1 isoforms revealed that ME preferentially rescues long-term HSC defects. RNA-seq analysis in Lin(-)Sca-1(+)c-Kit(+) cells (LSKs) of ME(m1) documents near complete silencing of Cdkn1c, encoding negative cell-cycle regulator p57-Kip2. Reintroduction of ME into ME(m1) LSKs leads to normalization of both p57-Kip2 expression and growth control. Our results clearly demonstrate a critical role of PR-domain-containing ME in linking p57-kip2 regulation to long-term HSC function.
Asunto(s)
Hematopoyesis , Células Madre Hematopoyéticas/citología , Animales , Inhibidor p57 de las Quinasas Dependientes de la Ciclina/genética , Exones , Regulación del Desarrollo de la Expresión Génica , Técnicas de Sustitución del Gen , Técnicas de Inactivación de Genes , Células Madre Hematopoyéticas/metabolismo , Operón Lac , Leucemia/genética , Leucocitosis/genética , Ratones , Ratones Endogámicos C57BL , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína , Trombocitopenia/genéticaRESUMEN
Progranulin (PGRN) is involved in wound repair, inflammation, and tumor formation, but its function in the central nervous system is unknown. Roles in development, sexual differentiation, and long-term neuronal survival have been suggested. Mutations in the GRN gene resulting in partial loss of the encoded PGRN protein cause frontotemporal lobar degeneration with ubiquitin immunoreactive inclusions. We sought to understand the neuropathological consequences of loss of PGRN function throughout the lifespan of GRN-deficient ((-/+) and (-/-)) mice. An aged series of GRN-deficient and wild-type mice were compared by histology, immunohistochemistry, and electron microscopy. Although GRN-deficient mice were viable, GRN(-/-) mice were produced at lower than predicted frequency. Neuropathologically, GRN(-/+) were indistinguishable from controls; however, GRN(-/-) mice developed age-associated, abnormal intraneuronal ubiquitin-positive autofluorescent lipofuscin. Lipofuscin was noted in aged GRN(+/+) mice at levels comparable with those of young GRN(-/-) mice. GRN(-/-) mice developed microgliosis, astrogliosis, and tissue vacuolation, with focal neuronal loss and severe gliosis apparent in the oldest GRN(-/-) mice. Although no overt frontotemporal lobar degeneration with ubiquitin immunoreactive inclusions type- or TAR DNA binding protein-43-positive lesions were observed, robust lipofuscinosis and ubiquitination in GRN(-/-) mice is strikingly similar to changes associated with aging and cellular decline in humans and animal models. Our data suggests that PGRN plays a key role in maintaining neuronal function during aging and supports the notion that PGRN is a trophic factor essential for long-term neuronal survival.
Asunto(s)
Envejecimiento/fisiología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Ratones Noqueados , Lipofuscinosis Ceroideas Neuronales/metabolismo , Animales , Encéfalo/citología , Encéfalo/metabolismo , Encéfalo/patología , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Ratones , Lipofuscinosis Ceroideas Neuronales/genética , Lipofuscinosis Ceroideas Neuronales/patología , Neuronas/citología , Neuronas/metabolismo , Progranulinas , Ubiquitina/metabolismo , UbiquitinaciónRESUMEN
A unifying characteristic of prion diseases is the conversion of a normal cellular protein (PrP(c)) to an abnormal pathogenic conformation, designated PrP(sc). Antibodies directed against PrP(c), when added to scrapie-infected cell cultures or passively administered in vivo, can result in elimination of PrP(sc) or prevent its replication, respectively. In our efforts to develop an approach with potential prophylactic utility we employed a recombinant adeno-associated vector type 2 (rAAV2) viral vector platform to express PrP(c)-specific single-chain fragment variable (scFv) antibodies within the central nervous system (CNS) of susceptible mice that were subsequently inoculated peripherally with infectious prions. Vector expressed scFvs delayed onset of prion pathogenesis as evidenced by improvements in clinical signs and rotarod performance, in extended incubation periods, and in decreased PrP(sc) burden in the CNS. This novel antibody delivery platform enables the in vivo translation of prion prophylactics to other species afflicted by transmissible spongiform encephalopathies (TSEs) and which also has relevance to the development of therapeutics for other protein-misfolding diseases such as Alzheimer's or Parkinson's disease.
Asunto(s)
Anticuerpos/genética , Sistema Nervioso Central/metabolismo , Dependovirus/genética , Proteínas PrPC/inmunología , Enfermedades por Prión/prevención & control , Secuencia de Aminoácidos , Animales , Anticuerpos/inmunología , Western Blotting , Línea Celular , Densitometría , Endopeptidasa K/metabolismo , Ensayo de Inmunoadsorción Enzimática , Vectores Genéticos/genética , Humanos , Immunoblotting , Inmunohistoquímica , Inmunoprecipitación , Ratones , Datos de Secuencia Molecular , Enfermedades por Prión/terapia , Homología de Secuencia de Aminoácido , Resonancia por Plasmón de Superficie , Transducción GenéticaRESUMEN
Parkinson's disease (PD) is a common neurodegenerative disease of unknown etiology. Evidence suggests a role for protein misfolding in disease pathogenesis. One pathologic feature observed in dopaminergic neurons is the intracytoplasmic eosinophilic inclusions known as Lewy bodies. One component of Lewy bodies, the presynaptic protein, alpha-synuclein forms oligomers and higher order aggregates and is proposed to be involved in dopaminergic neuronal death. In an effort to discriminate between alpha-synuclein conformational forms as well as design potential disruptors of pathogenic misfolding we panned a human phage antibody library for anti-synuclein single chain antibodies (scFvs). We identified six scFvs which recognize different conformers of alpha-synuclein in both an ELISA and Western blot analysis. These scFvs may further our understanding of alpha-synuclein's role in PD.
Asunto(s)
Región Variable de Inmunoglobulina/química , Región Variable de Inmunoglobulina/inmunología , alfa-Sinucleína/química , alfa-Sinucleína/inmunología , Humanos , Región Variable de Inmunoglobulina/análisis , alfa-Sinucleína/análisisRESUMEN
Strategies that employ HSV amplicon vectors in the prevention and/or amelioration of pathogenic states afflicting the central nervous system (CNS) have been extensively documented in preclinical disease models. The versatility of the HSV amplicon platform allows for the implementation of therapeutic approaches that require expression of genes exhibiting neuroprotective or neuroplastic activities, or even applications that necessitate the elaboration of antigen-specific immune responses to pathogenic proteins/structures harbored within the CNS. This discourse highlights the successes and challenges encountered using HSV amplicon vectors as tools for the dissection of neural network function and as therapeutics directed against a variety of neurologic disorders.