RESUMEN

Activating transcription factor-5 (ATF5) is a stress-response transcription factor induced upon different cell stressors like fasting, amino-acid limitation, cadmium or arsenite. ATF5 is also induced, and promotes transcription of anti-apoptotic target genes like MCL1, during the unfolded protein response (UPR) triggered by endoplasmic reticulum stress. In the brain, high ATF5 levels are found in gliomas and also in neural progenitor cells, which need to decrease their ATF5 levels for differentiation into mature neurons or glia. This initially led to believe that ATF5 is not expressed in adult neurons. More recently, we reported basal neuronal ATF5 expression in adult mouse brain and its neuroprotective induction during UPR in a mouse model of status epilepticus. Here we aimed to explore whether ATF5 is also expressed by neurons in human brain both in basal conditions and in Huntington's disease (HD), where UPR has been described to be partially impaired due to defective ATF6 processing. Apart from confirming that ATF5 is present in human adult neurons, here we report accumulation of ATF5 within the characteristic polyglutamine-containing neuronal nuclear inclusions in brains of HD patients and mice. This correlates with decreased levels of soluble ATF5 and of its antiapoptotic target MCL1. We then confirmed the deleterious effect of ATF5 deficiency in a Caenorhabditis elegans model of polyglutamine-induced toxicity. Finally, ATF5 overexpression attenuated polyglutamine-induced apoptosis in a cell model of HD. These results reflect that decreased ATF5 in HD-probably secondary to sequestration into inclusions-renders neurons more vulnerable to mutant huntingtin-induced apoptosis and that ATF5-increasing interventions might have therapeutic potential for HD.

Asunto(s)

Factores de Transcripción Activadores/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Enfermedad de Huntington/metabolismo , Cuerpos de Inclusión/metabolismo , Neuronas/metabolismo , Péptidos/metabolismo , Animales , Apoptosis , Caenorhabditis elegans , Línea Celular Tumoral , Modelos Animales de Enfermedad , Estrés del Retículo Endoplásmico/fisiología , Humanos , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Enfermedad de Huntington/patología , Cuerpos de Inclusión/patología , Ratones Transgénicos , Neuronas/patología , Neuroprotección/fisiología

RESUMEN

Lewy bodies and neurites are the pathological hallmark of Parkinson's disease. These structures are composed of fibrillized and ubiquitinated alpha-synuclein suggesting that impaired protein clearance is an important event in aggregate formation. The A30P mutation is known for its fast oligomerization, but slow fibrillization rate. Despite its toxicity to neurons, mechanisms involved in either clearance or conversion of A30P alpha-synuclein from its soluble state into insoluble fibrils and their effects in vivo are poorly understood. Synphilin-1 is present in Lewy bodies, interacting with alpha-synuclein in vivo and in vitro and promotes its sequestration into aggresomes, which are thought to act as cytoprotective agents facilitating protein degradation. We therefore crossed animals overexpressing A30P alpha-synuclein with synphilin-1 transgenic mice to analyze its impact on aggregation, protein clearance and phenotype progression. We observed that co-expression of synphilin-1 mildly delayed the motor phenotype caused by A30P alpha-synuclein. Additionally, the presence of N- and C-terminal truncated alpha-synuclein species and fibrils were strongly reduced in double-transgenic mice when compared with single-transgenic A30P mice. Insolubility of mutant A30P and formation of aggresomes was still detectable in aged double-transgenic mice, paralleled by an increase of ubiquitinated proteins and high autophagic activity. Hence, this study supports the notion that co-expression of synphilin-1 promotes formation of autophagic-susceptible aggresomes and consecutively the degradation of human A30P alpha-synuclein. Notably, although synphilin-1 overexpression significantly reduced formation of fibrils and astrogliosis in aged animals, a similar phenotype is present in single- and double-transgenic mice suggesting additional neurotoxic processes in disease progression.

Asunto(s)

Proteínas Portadoras/genética , Proteínas del Tejido Nervioso/genética , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Envejecimiento , Animales , Autofagia/fisiología , Benzotiazoles , Encéfalo/metabolismo , Encéfalo/patología , Proteínas Portadoras/metabolismo , Expresión Génica , Humanos , Ratones , Ratones Transgénicos , Mutación , Proteínas del Tejido Nervioso/metabolismo , Enfermedad de Parkinson/genética , Pliegue de Proteína , Solubilidad , Tiazoles/metabolismo , Ubiquitina/metabolismo

RESUMEN

Huntington's disease is caused by an expanded polyglutamine repeat in the huntingtin protein (HTT), but the pathophysiological sequence of events that trigger synaptic failure and neuronal loss are not fully understood. Alterations in N-methyl-D-aspartate (NMDA)-type glutamate receptors (NMDARs) have been implicated. Yet, it remains unclear how the HTT mutation affects NMDAR function, and direct evidence for a causative role is missing. Here we show that mutant HTT redirects an intracellular store of juvenile NMDARs containing GluN3A subunits to the surface of striatal neurons by sequestering and disrupting the subcellular localization of the endocytic adaptor PACSIN1, which is specific for GluN3A. Overexpressing GluN3A in wild-type mouse striatum mimicked the synapse loss observed in Huntington's disease mouse models, whereas genetic deletion of GluN3A prevented synapse degeneration, ameliorated motor and cognitive decline and reduced striatal atrophy and neuronal loss in the YAC128 Huntington's disease mouse model. Furthermore, GluN3A deletion corrected the abnormally enhanced NMDAR currents, which have been linked to cell death in Huntington's disease and other neurodegenerative conditions. Our findings reveal an early pathogenic role of GluN3A dysregulation in Huntington's disease and suggest that therapies targeting GluN3A or pathogenic HTT-PACSIN1 interactions might prevent or delay disease progression.

Asunto(s)

Conducta Animal , Enfermedad de Huntington/metabolismo , Enfermedad de Huntington/patología , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapsis/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas Portadoras/metabolismo , Muerte Celular/efectos de los fármacos , Proteínas del Citoesqueleto , Modelos Animales de Enfermedad , Eliminación de Gen , Células HEK293 , Humanos , Enfermedad de Huntington/fisiopatología , Inmunoprecipitación , Péptidos y Proteínas de Señalización Intracelular , Ratones , Actividad Motora/efectos de los fármacos , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Proteínas Mutantes/toxicidad , Neostriado/metabolismo , Neostriado/patología , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Neuropéptidos/metabolismo , Fosfoproteínas/metabolismo , Unión Proteica/efectos de los fármacos , Estructura Cuaternaria de Proteína , Prueba de Desempeño de Rotación con Aceleración Constante , Sinapsis/efectos de los fármacos , Sinapsis/ultraestructura

RESUMEN

Activating transcription factor 5 (ATF5) is a basic-leucine-zipper transcription factor of the ATF/CREB family. The Atf5 gene generates two transcripts, Atf5α and Atf5ß, of which Atf5α is known to be selectively translated upon endoplasmic reticulum stress response in non-neuronal cells. ATF5 is highly expressed in the developing brain where it modulates proliferation of neural progenitor cells. These cells show a high level of ATF5 that has to decrease to allow them to differentiate into mature neurons or glial cells. This has led to the extended notion that differentiated neural cells do not express ATF5 unless they undergo tumourigenic transformation. However, no systematic analysis of the distribution of ATF5 in adult brain or of its potential role in neuronal endoplasmic reticulum stress response has been reported. By immunostaining here we confirm highest ATF5 levels in neuroprogenitor cells of the embryonic and adult subventricular zone but also found ATF5 in a large variety of neurons in adult mouse brain. By combining Atf5 in situ hybridization and immunohistochemistry for the neuronal marker NeuN we further confirmed Atf5 messenger RNA in adult mouse neurons. Quantitative reverse transcriptase polymerase chain reaction demonstrated that Atf5α is the most abundant transcript in adult mouse encephalon and injection of the endoplasmic reticulum stress inducer tunicamycin into adult mouse brain increased neuronal ATF5 levels. Accordingly, ATF5 levels increased in hippocampal neurons of a mouse model of status epilepticus triggered by intra-amygdala injection of kainic acid, which leads to abnormal hippocampal neuronal activity and endoplasmic reticulum stress. Interestingly, ATF5 upregulation occurred mainly in hippocampal neuronal fields that do not undergo apoptosis in this status epilepticus model such as CA1 and dentate gyrus, thus suggesting a neuroprotective role. This was confirmed in a primary neuronal culture model in which ATF5 overexpression resulted in decreased endoplasmic reticulum stress-induced apoptosis and the opposite result was achieved by Atf5 RNA interference. Furthermore, in vivo administration of the eIF2α phosphatase inhibitor salubrinal resulted in increased ATF5 hippocampal levels and attenuated status epilepticus-induced neuronal death in the vulnerable CA3 subfield. In good agreement with the neuroprotective effect of increased ATF5, we found that apoptosis-resistant epileptogenic foci from patients with temporal lobe epilepsy also showed increased levels of ATF5. Thus, our results demonstrate that adult neurons express ATF5 and that they increase its levels upon endoplasmic reticulum stress as a pro-survival mechanism, thus opening a new field for neuroprotective strategies focused on ATF5 modulation.

Asunto(s)

Factores de Transcripción Activadores/biosíntesis , Estrés del Retículo Endoplásmico/fisiología , Neuronas/metabolismo , Fármacos Neuroprotectores/metabolismo , Estado Epiléptico/metabolismo , Estado Epiléptico/patología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Cinamatos/administración & dosificación , Cinamatos/farmacología , Modelos Animales de Enfermedad , Estrés del Retículo Endoplásmico/efectos de los fármacos , Humanos , Ratones , Ratones Endogámicos C57BL , Neuronas/efectos de los fármacos , Neuronas/patología , Estado Epiléptico/tratamiento farmacológico , Tiourea/administración & dosificación , Tiourea/análogos & derivados , Tiourea/farmacología

RESUMEN

Brain-derived neurotrophic factor (BDNF) is a key player in learning and memory processes. However, little is known about brain area-specific functions of this neurotrophin. Here we investigated whether BDNF could differently affect motor neocortical and hippocampal-related cognitive and plastic morphologic changes in young (12-week-old) and middle-aged (30-week-old) BDNF heterozygous (BDNF⁺/⁻) and wild type (wt) mice. We found that at 30 weeks of age, BDNF⁺/⁻ mice showed impaired performance in accelerating rotarod and grasping tests while preserved spatial learning in a T-maze and recognition memory in an object recognition task compared with wt mice suggesting a specific neocortical dysfunction. Accordingly, a significant reduction of synaptic markers (PSD-95 and GluR1) and corresponding puncta was observed in motor neocortex but not in hippocampus of BDNF⁺/⁻ mice. Interestingly, 30-week-old BDNF⁺/⁻ mice displayed increased TrkB levels in the hippocampus but not in the motor neocortex, which suggests specific hippocampal compensatory mechanisms as a consequence of BDNF decrease. In conclusion, our data indicates that BDNF could differentially regulate the neuronal micro-structures and cognition in a region-specific and in an age-dependent manner.

Asunto(s)

Envejecimiento/fisiología , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Encéfalo/metabolismo , Guanilato-Quinasas/biosíntesis , Proteínas de la Membrana/biosíntesis , Receptor trkB/biosíntesis , Animales , Western Blotting , Factor Neurotrófico Derivado del Encéfalo/genética , Espinas Dendríticas/metabolismo , Homólogo 4 de la Proteína Discs Large , Ensayo de Inmunoadsorción Enzimática , Heterocigoto , Aprendizaje/fisiología , Memoria/fisiología , Ratones , Microscopía Confocal , Plasticidad Neuronal/fisiología

RESUMEN

We study the striatal susceptibility to NMDA receptor (NMDAR)-mediated injury of two Huntington's disease (HD) transgenic mice: R6/1 and R6/1:BDNF(+/-). We found that R6/1:BDNF(+/-) mice--which express reduced levels of BDNF--were more resistant than R6/1 mice to intrastriatal injection of quinolinate. This increased resistance is related to a differential reduction in expression of NMDAR scaffolding proteins, MAGUKs (PSD-95, PSD-93, SAP-102 and SAP-97) but not to altered levels or synaptic location of NMDAR. A robust reorganization of postsynaptic density (PSD) was detected in HD transgenic mice, shown by a switch of PSD-93 by PSD-95 in PSD. Furthermore, NMDAR signaling pathways were affected by different BDNF levels in HD mice; we found a reduction of synaptic alpha CaMKII (but not of nNOS) in R6/1:BDNF(+/-) compared to R6/1 mice. The specific regulation of MAGUKs and alpha CaMKII in striatal neurons may reflect a protective mechanism against expression of mutant huntingtin exon-1.

Asunto(s)

Cuerpo Estriado/fisiología , Mutación/fisiología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/genética , Receptores de Glutamato/fisiología , Receptores de N-Metil-D-Aspartato/fisiología , Transmisión Sináptica/genética , Adulto , Anciano , Animales , Cuerpo Estriado/patología , Humanos , Proteína Huntingtina , Masculino , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/fisiología , Proteínas Nucleares/fisiología , Receptores de N-Metil-D-Aspartato/biosíntesis , Receptores de N-Metil-D-Aspartato/genética , Transducción de Señal/fisiología , Sinapsis/genética , Sinapsis/metabolismo , Sinapsis/patología

RESUMEN

Accumulating evidence has suggested that neurotrophins participate in the pathophysiology of mood disorders. We have developed transgenic mice overexpressing the full-length neurotrophin-3 receptor TrkC (TgNTRK3) in the central nervous system. TgNTRK3 mice show increased anxiety-like behavior and enhancement of panic reaction in the mouse defense test battery, along with an increase in the number and density of catecholaminergic (tyrosine hydroxylase positive) neurons in locus coeruleus and substantia nigra. Furthermore, treatment of TgNTRK3 mice with diazepam significantly attenuated the anxiety-like behaviors in the plus maze. These results provide evidence for the involvement of TrkC in the development of noradrenergic neurons in the central nervous system with consequences on anxiety-like behavior and panic reaction. Thus, changes in TrkC expression levels could contribute to the phenotypic expression of panic disorder through a trophic effect on noradrenergic neurons in the locus coeruleus. Our results demonstrate that the elevated NT3-TrkC tone via overexpression of TrkC in the brain may constitute a molecular mechanism for the expression of anxiety and anxiety.

Asunto(s)

Trastornos de Ansiedad/metabolismo , Encéfalo/metabolismo , Encéfalo/fisiopatología , Catecolaminas/metabolismo , Neuronas/metabolismo , Trastorno de Pánico/metabolismo , Receptor trkC/metabolismo , Animales , Trastornos de Ansiedad/genética , Trastornos de Ansiedad/fisiopatología , Enfermedades del Sistema Nervioso Autónomo/genética , Enfermedades del Sistema Nervioso Autónomo/metabolismo , Enfermedades del Sistema Nervioso Autónomo/fisiopatología , Conducta Animal/fisiología , Encéfalo/patología , Recuento de Células , Proliferación Celular , Modelos Animales de Enfermedad , Femenino , Predisposición Genética a la Enfermedad/genética , Locus Coeruleus/metabolismo , Locus Coeruleus/patología , Locus Coeruleus/fisiopatología , Masculino , Ratones , Ratones Transgénicos , Vías Nerviosas/metabolismo , Vías Nerviosas/patología , Vías Nerviosas/fisiopatología , Pruebas Neuropsicológicas , Norepinefrina/metabolismo , Trastorno de Pánico/genética , Trastorno de Pánico/fisiopatología , Receptor trkC/genética , Sustancia Negra/metabolismo , Sustancia Negra/patología , Sustancia Negra/fisiopatología , Regulación hacia Arriba/genética

RESUMEN

The primary mechanism responsible for Huntington's disease remains unknown. Postulated early pathogenic events include the following: impaired protein folding, altered protein degradation, mitochondrial dysfunction, and transcriptional dysregulation. Although related therapies can delay disease progression in mouse models, they target downstream and probably indirect effects of mutant-huntingtin expression. Accordingly, in case they prove beneficial in humans, they might only palliate some aspects of disease. Our previous studies in the Tet/HD94 conditional model and the recently reported efficacy of RNA interference against mutant huntingtin in another mouse model support silencing mutant-huntingtin expression as a valid therapeutic approach that has the advantage of targeting toxicity at its root. Here, we address whether gene silencing can still be beneficial in the late stages of disease with detectable striatal neuron loss. Stereological analysis was applied to determine an age at which Tet/HD94 mice show a decrease in the number of striatal neurons. Then, progression of neuropathology and motor phenotype were analyzed in mice that were allowed to continue expressing mutant huntingtin and in mice that no longer expressed it. Neuronal loss did not revert in gene-off mice, but the additional loss that takes place in gene-on mice was prevented. The total number of huntingtin-containing inclusions dramatically reverted, but a small fraction of inclusions positive for the amyloid dye thioflavin-S remained. Interestingly, despite a 20% decrease in striatal neurons and the presence of amyloid-like irreversible inclusions, gene-off mice fully recover from their motor deficit, thus ruling out amyloid-like huntingtin inclusions as the main toxic species and suggesting that gene-silencing therapies might work in late stages of disease.

Asunto(s)

Amiloide/metabolismo , Cuerpo Estriado/patología , Modelos Animales de Enfermedad , Enfermedad de Huntington/patología , Destreza Motora , Neuronas/patología , Amiloide/genética , Amiloide/fisiología , Animales , Recuento de Células/métodos , Muerte Celular/fisiología , Cuerpo Estriado/fisiología , Enfermedad de Huntington/genética , Ratones , Ratones Transgénicos , Destreza Motora/fisiología , Recuperación de la Función/fisiología

RESUMEN

The mechanism that controls the selective vulnerability of striatal neurons in Huntington's disease is unclear. Brain-derived neurotrophic factor (BDNF) protects striatal neurons and is regulated by Huntingtin through the interaction with the neuron-restrictive silencer factor. Here, we demonstrate that the downregulation of BDNF by mutant Huntingtin depends on the length and levels of expression of the CAG repeats in cell cultures. To analyze the functional effects of these changes in BDNF in Huntington's disease, we disrupted the expression of bdnf in a transgenic mouse model by cross-mating bdnf(+/ -) mice with R6/1 mice. Thus, we compared transgenic mice for mutant Huntingtin with different levels of BDNF. Using this double mutant mouse line, we show that the deficit of endogenous BDNF modulates the pathology of Huntington's disease. The decreased levels of this neurotrophin advance the onset of motor dysfunctions and produce more severe uncoordinated movements. This behavioral pathology correlates with the loss of striatal dopamine and cAMP-regulated phosphoprotein-32-positive projection neurons. In particular, the insufficient levels of BDNF cause specific degeneration of the enkephalinergic striatal projection neurons, which are the most affected cells in Huntington's disease. This neuronal dysfunction can specifically be restored by administration of exogenous BDNF. Therefore, the decrease in BDNF levels plays a key role in the specific pathology observed in Huntington's disease by inducing dysfunction of striatal enkephalinergic neurons that produce severe motor dysfunctions. Hence, administration of exogenous BDNF may delay or stop illness progression.

Asunto(s)

Factor Neurotrófico Derivado del Encéfalo/fisiología , Encefalinas/deficiencia , Enfermedad de Huntington/patología , Proteínas del Tejido Nervioso/fisiología , Proteínas Nucleares/fisiología , Edad de Inicio , Animales , Ataxia/genética , Factor Neurotrófico Derivado del Encéfalo/deficiencia , Factor Neurotrófico Derivado del Encéfalo/uso terapéutico , Muerte Celular , Línea Celular Transformada , Corea/genética , Cuerpo Estriado/citología , Cruzamientos Genéticos , Endocitosis , Encefalinas/biosíntesis , Regulación de la Expresión Génica , Proteína Huntingtina , Enfermedad de Huntington/metabolismo , Ratones , Ratones Noqueados , Ratones Transgénicos , Trastornos del Movimiento/genética , Degeneración Nerviosa , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Neuronas/citología , Neuronas/metabolismo , Proteínas Nucleares/deficiencia , Proteínas Nucleares/genética , Fenotipo , Células Madre/citología , Transfección , Repeticiones de Trinucleótidos