RESUMEN

Injecting proteins into the central nervous system that stimulate neuronal growth can lead to beneficial effects in animal models of disease. In particular, glial cell line-derived neurotrophic factor (GDNF) has shown promise in animal and cell models of Parkinson's disease, Huntington's disease and amyotrophic lateral sclerosis (ALS). Here, systemic AAV9-GDNF was delivered via tail vein injections to young rats to determine whether this could be a safe and functional strategy to treat the SOD1G93A rat model of ALS and, therefore, translated to a therapy for ALS patients. We found that GDNF administration in this manner resulted in modest functional improvement, whereby grip strength was maintained for longer and the onset of forelimb paralysis was delayed compared to non-treated rats. This did not, however, translate into an extension in survival. In addition, ALS rats receiving GDNF exhibited slower weight gain, reduced activity levels and decreased working memory. Collectively, these results confirm that caution should be applied when applying growth factors such as GDNF systemically to multiple tissues.

Asunto(s)

Esclerosis Amiotrófica Lateral/terapia , Sistema Nervioso Central/fisiopatología , Factor Neurotrófico Derivado de la Línea Celular Glial/uso terapéutico , Neuronas Motoras/patología , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/fisiopatología , Animales , Sistema Nervioso Central/efectos de los fármacos , Dependovirus/genética , Modelos Animales de Enfermedad , Terapia Genética , Vectores Genéticos/genética , Vectores Genéticos/uso terapéutico , Factor Neurotrófico Derivado de la Línea Celular Glial/efectos adversos , Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Fuerza de la Mano/fisiología , Humanos , Neuronas Motoras/metabolismo , Ratas , Médula Espinal/efectos de los fármacos , Médula Espinal/fisiopatología , Superóxido Dismutasa/genética

RESUMEN

Expansions of a hexanucleotide repeat (GGGGCC) in the noncoding region of the C9orf72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. Decreased expression of C9orf72 is seen in expansion carriers, suggesting that loss of function may play a role in disease. We found that two independent mouse lines lacking the C9orf72 ortholog (3110043O21Rik) in all tissues developed normally and aged without motor neuron disease. Instead, C9orf72 null mice developed progressive splenomegaly and lymphadenopathy with accumulation of engorged macrophage-like cells. C9orf72 expression was highest in myeloid cells, and the loss of C9orf72 led to lysosomal accumulation and altered immune responses in macrophages and microglia, with age-related neuroinflammation similar to C9orf72 ALS but not sporadic ALS human patient tissue. Thus, C9orf72 is required for the normal function of myeloid cells, and altered microglial function may contribute to neurodegeneration in C9orf72 expansion carriers.

Asunto(s)

Esclerosis Amiotrófica Lateral/inmunología , Demencia Frontotemporal/inmunología , Factores de Intercambio de Guanina Nucleótido/fisiología , Macrófagos/inmunología , Microglía/inmunología , Células Mieloides/inmunología , Proteínas/fisiología , Envejecimiento/inmunología , Esclerosis Amiotrófica Lateral/genética , Animales , Proteína C9orf72 , Demencia Frontotemporal/genética , Técnicas de Silenciamiento del Gen , Factores de Intercambio de Guanina Nucleótido/genética , Heterocigoto , Humanos , Enfermedades Linfáticas/genética , Enfermedades Linfáticas/inmunología , Ratones , Ratones Noqueados , Proteínas/genética , Ratas , Esplenomegalia/genética , Esplenomegalia/inmunología

RESUMEN

The sensation commonly referred to as 'pain' has two components. The first is the sensory-discriminative component and provides information on location, modality and intensity of stimuli. The second is the affective-motivational component and refers to the emotional responses (fear, distress etc.) and the urge to respond evoked by the somatic sensation, and at the cortical level these two components appear to be located in different regions. The cortex probably influences pain by two different mechanisms. There is good evidence that the cortex can reduce pain by interrupting the transmission of noxious information from the spinal cord level by activating descending pain modulatory systems located in the brainstem. Less well established is the idea that modulation can also occur at the cortical level to change the affective-motivational aspects of nociception so that pain is perceived but looses its emotional and aversive component.

Asunto(s)

Corteza Cerebral/fisiopatología , Nociceptores/fisiología , Dolor/fisiopatología , Animales , Corteza Cerebral/diagnóstico por imagen , Estimulación Eléctrica , Humanos , Radiografía

RESUMEN

Caspase-8 plays an essential role in apoptosis induced by Fas activation. Moreover, caspase-8 can be processed also in response to exposure to genotoxic agents. To decipher the role of caspase-8 in DNA damaging agent (DDA)-induced apoptosis as well as the pathway(s) leading to its activation in response to genotoxic stress, we investigated caspase-8 processing induced by ionizing radiation (IR) or mitomycin C (MMC) treatment in human B-lymphoblasts. Altogether, our observations establish that caspase-8 is actively processed in both receptor-mediated and DDA-induced cell death. However, while Fas-dependent apoptosis absolutely required caspase-8 activity, it is not necessary for completion of the apoptotic program induced by IR and MMC. Experiments performed to understand the molecular pathway(s) of the caspase-8 activation after DDA demonstrated that for both IR and MMC, the Fas/Fas-L interaction is dispensable. Data obtained from caspase inhibitors and from lymphoblasts carrying mutations in ATM and FANCC proteins, involved in DDA response, clearly showed that distinct mechanisms are responsible for caspase-8 activation by IR and MMC in B-lymphoblasts. IR-dependent processing of caspase-8 involves ATM, mitochondrial collapse, FANCC, and caspase-3 activation. Caspase-8 activation by MMC evokes the mitochondrial pathways involving FANCC but not ATM. Collectively, our data indicate that caspase-8 activation is essentially a bystander effect and not a major determinant of the behavior of DDA-exposed cells.

Asunto(s)

Apoptosis/fisiología , Linfocitos B/metabolismo , Caspasas/metabolismo , Proteínas de Ciclo Celular , Daño del ADN/fisiología , Proteínas de Unión al ADN , Mutágenos/farmacología , Células Progenitoras Mieloides/metabolismo , Proteínas Nucleares , Receptor fas/metabolismo , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Proteínas de la Ataxia Telangiectasia Mutada , Linfocitos B/efectos de los fármacos , Linfocitos B/efectos de la radiación , Caspasa 3 , Caspasa 8 , Caspasa 9 , Caspasas/efectos de los fármacos , Caspasas/efectos de la radiación , Células Cultivadas/efectos de los fármacos , Células Cultivadas/metabolismo , Células Cultivadas/efectos de la radiación , Daño del ADN/efectos de los fármacos , Daño del ADN/efectos de la radiación , Inhibidores Enzimáticos/farmacología , Proteína del Grupo de Complementación C de la Anemia de Fanconi , Proteínas del Grupo de Complementación de la Anemia de Fanconi , Proteína Ligando Fas , Humanos , Inmunohistoquímica , Glicoproteínas de Membrana/efectos de los fármacos , Glicoproteínas de Membrana/metabolismo , Mitomicina/farmacología , Células Progenitoras Mieloides/efectos de los fármacos , Células Progenitoras Mieloides/efectos de la radiación , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Proteínas Serina-Treonina Quinasas/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/efectos de la radiación , Proteínas/efectos de los fármacos , Proteínas/metabolismo , Proteínas/efectos de la radiación , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Transducción de Señal/efectos de la radiación , Proteínas Supresoras de Tumor , Receptor fas/efectos de los fármacos , Receptor fas/efectos de la radiación

RESUMEN

PURPOSE: To define the role of the ataxia telangiectasia (A-T) mutated gene (ATM) in activation and progress of apoptosis. MATERIAL AND METHODS: Three normal and three A-T EBV-transformed cell lines were studied. Following irradiation (IR), Fas activation or ceramide exposure, viability and apoptosis were measured by trypan blue dye exclusion assay and as sub-G1 cell fraction by flow cytometric analysis of propidium iodide stained cultures, respectively. Activation of caspase-3 was evaluated by immunoblot and by an in vitro activity assay on cytosolic cell extracts. To assess changes in mitochondrial potential and reactive oxygen species, cells were stained by 3,3'-dihexyloxacarbocynine iodide or hydroethidine, respectively, and scored by flow cytometry. RESULTS: The observations establish that A-T cells are equipped with a proficient apoptotic machinery, as demonstrated by their ability to undergo mitochondrial collapse and caspase-3 activation after Fas activation or ceramide treatment. Both treatments have a similar cytotoxic effect on normal and A-T cells. In contrast, in spite of the stronger cytotoxicity induced by IR exposure, irradiated A-T cells are unable to undergo mitochondrial collapse and caspase-3 activation. CONCLUSIONS: The data indicate that ATM is necessary in the initiation of molecular pathway(s) leading to IR-induced apoptosis, and suggest that increased radiosensitivity of A-T cells is more likely a direct consequence of necrotic cell death.

Asunto(s)

Apoptosis/fisiología , Apoptosis/efectos de la radiación , Ataxia Telangiectasia/metabolismo , Mitocondrias/metabolismo , Mitocondrias/efectos de la radiación , Proteínas Serina-Treonina Quinasas/metabolismo , Apoptosis/genética , Ataxia Telangiectasia/genética , Ataxia Telangiectasia/patología , Proteínas de la Ataxia Telangiectasia Mutada , Caspasa 3 , Caspasas/metabolismo , Proteínas de Ciclo Celular , Línea Celular , Línea Celular Transformada , Proteínas de Unión al ADN , Activación Enzimática/efectos de la radiación , Humanos , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Supresoras de Tumor

RESUMEN

Fanconi anemia (FA) is a human genetic disease featuring cancer predisposition, genetic instability and DNA damage hypersensitivity. Although abnormalities in DNA repair and cell cycle checkpoint have been proposed as the underlying defect in this syndrome, these hypotheses did not provide full explanations of the complex phenotype. Although not exclusive of such possibilities, alterations in the control of apoptosis might account for the pleiotropic phenotype of this syndrome. We and others have previously reported a deregulation of the apoptotic response to mitomycin C, suggesting that the products of the Fanconi anemia group C protein (FANCC) contribute to the regulation of apoptosis. To explore the functional importance of the apoptotic alterations in FA we analyzed biochemical steps of the execution phase of apoptosis stimulated by another DNA damaging agent, the gamma-ray using FA cell lines derived from complementation group C (FA-C) independent patients. It is shown that the poly(ADP-ribose) polymerase, a target of caspase-3, is not cleaved in FA-C after ionizing radiation (IR). Moreover, caspase-3 is not processed in its active form and, its activity is not increased by IR in FA-C cells compared to normal cells. Altogether, these results demonstrate that loss of the FANCC activity results in a deficiency of the IR-induced apoptosis which is due to an inability to activate caspase-3. Our work suggests that apoptosis signaling induced by mitomycin C and IR is subject to common regulation involving the FANCC protein.

Asunto(s)

Caspasas/metabolismo , Proteínas de Ciclo Celular , Proteínas de Unión al ADN , Anemia de Fanconi/metabolismo , Anemia de Fanconi/radioterapia , Proteínas Nucleares , Proteínas/metabolismo , Apoptosis/efectos de la radiación , Caspasa 3 , Inhibidores de Caspasas , Caspasas/efectos de la radiación , Muerte Celular/efectos de la radiación , Línea Celular , Cumarinas/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Activación Enzimática/efectos de la radiación , Anemia de Fanconi/genética , Proteína del Grupo de Complementación C de la Anemia de Fanconi , Proteínas del Grupo de Complementación de la Anemia de Fanconi , Rayos gamma , Humanos , Oligopéptidos/metabolismo , Oligopéptidos/farmacología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Poli(ADP-Ribosa) Polimerasas/efectos de la radiación , Proteínas/genética , Proteínas/efectos de la radiación , Radiación Ionizante

RESUMEN

As an approach to integrate the chicken genetic and cytogenetic maps, bacterial artificial chromosome (BAC) and P1-derived artificial chromosome (PAC) clones were localized by fluorescence in situ hybridization (FISH) on chromosomes and by genetic mapping on the East Lansing and Compton reference families. Some of the clones used in this study were previously selected for the presence of potentially polymorphic (CA)n repeats and a microsatellite marker was developed when possible for genetic mapping. For other clones, a single strand conformational polymorphism (SSCP) was developed and used for this purpose. Between the two approaches, 18 markers linking the cytogenetic and genetic maps, seven on macrochromosomes and 11 on microchromosomes, were generated. Our results enabled the assignment and orientation of a linkage group to chromosome 3, together with the assignment of linkage groups to eight different microchromosomes, a fraction of the genome lacking mapping data and for which the degree of coverage by the genetic map was not well estimated previously.

Asunto(s)

Pollos/genética , Mapeo Cromosómico , Marcadores Genéticos , Animales , Secuencia de Bases , Clonación Molecular , Citogenética , Cartilla de ADN/genética , Ligamiento Genético , Biblioteca Genómica , Hibridación Fluorescente in Situ , Repeticiones de Microsatélite , Polimorfismo Genético , Polimorfismo Conformacional Retorcido-Simple