373 research outputs found
Identification of the HetR Recognition Sequence Upstream of hetZ in Anabaena sp Strain PCC 7120
No full textHetR is the master regulator of heterocyst differentiation in Anabaena sp. strain PCC 7120 and has been found to specifically bind to an inverted-repeat-containing region upstream of hetP, a heterocyst differentiation gene. However, no such inverted-repeat sequence can be found in promoters of other genes in the genome. hetZ is a gene involved in early heterocyst differentiation. As shown with the gfp reporter gene, transcription from P-hetZ was correlated to the expression level of hetR and inhibition by RGSGR, the pentapeptide derived from the C terminus of PatS. As detected by electrophoretic mobility shift assay, a recombinant HetR showed specific binding to the region upstream of hetZ, and the binding was inhibited by RGSGR. Tests of a series of the upstream fragments delimited the HetR-binding site to a 40-bp region that shows similarity to that upstream of hetP. The introduction of substitutions of bases conserved in the two HetR-binding sites showed that at least 12 bases are required for recognition by HetR. Deletion of a 51-bp region containing the HetR-binding site completely eliminated the transcription activity of P-hetZ. Based on the HetR recognition sequence of hetZ, those upstream of hetR and patA are proposed.HetR is the master regulator of heterocyst differentiation in Anabaena sp. strain PCC 7120 and has been found to specifically bind to an inverted-repeat-containing region upstream of hetP, a heterocyst differentiation gene. However, no such inverted-repeat sequence can be found in promoters of other genes in the genome. hetZ is a gene involved in early heterocyst differentiation. As shown with the gfp reporter gene, transcription from P-hetZ was correlated to the expression level of hetR and inhibition by RGSGR, the pentapeptide derived from the C terminus of PatS. As detected by electrophoretic mobility shift assay, a recombinant HetR showed specific binding to the region upstream of hetZ, and the binding was inhibited by RGSGR. Tests of a series of the upstream fragments delimited the HetR-binding site to a 40-bp region that shows similarity to that upstream of hetP. The introduction of substitutions of bases conserved in the two HetR-binding sites showed that at least 12 bases are required for recognition by HetR. Deletion of a 51-bp region containing the HetR-binding site completely eliminated the transcription activity of P-hetZ. Based on the HetR recognition sequence of hetZ, those upstream of hetR and patA are proposed
Optimized adeno-associated vector to deliver the unfolded protein response transcription factor XBP1s into the hippocampus ameliorates Alzheimer’s disease features in mouse models
No full textProteostasis impairment at the level of the endoplasmic reticulum (ER) is a salient feature of Alzheimer’s disease (AD). The unfolded protein response (UPR) is the main adaptive pathway to cope with protein folding stress at the ER, where the expression of the transcription factor X-Box binding protein 1 (XBP1) is central to establish repair programs. To artificially enforce the adaptive capacity of the UPR in the AD brain, we recently reported the protective effects of overexpressing active XBP1 in the nervous system through the local delivery into the hippocampus of an AD mice using adeno-associated vectors (AAVs). Here we have generated a next generation vector suitable for clinical trials by (i) expressing codon optimized human XBP1s without tags, (ii) the use the synapsin promoter to restrict expression to neurons, and (iii) a novel variant of AAV2 that has greater spreading capacity (AAV-TT) in the brain because of reduced affinity to heparin (termed AAV-TT-Syn-hXBP1s or Proteostaser-1). Treatment of 5xFAD mice with AAV-TT-Syn-hXBP1s improved long-term potentiation, memory performance, and reduced amyloid plaques deposition. In addition, we validated the protective effects of AAV-TT-Syn-hXBP1s on a model of sporadic AD based on the intracerebral injection of amyloid beta oligomers. Our results further support the therapeutic potential of targeting UPR-dependent gene expression programs as a strategy to ameliorate AD features and sustain synaptic function
Artificial enforcement of the unfolded protein response (UPR) reduces disease features in multiple preclinical models of ALS/FTD.
No full textData used for Research Article submission to Molecular Therapy Journal for review Amyotrophic lateral sclerosis (ALS) and fronto-temporal dementia (FTD) are part of a spectrum of diseases that share several causative genes, resulting on a combinatory of motor and cognitive symptoms and abnormal protein aggregation. Multiple unbiased studies have revealed that proteostasis impairment at the level of the endoplasmic reticulum (ER) is a transversal pathogenic feature of ALS/FTD. The transcription factor XBP1s is a master regulator of the unfolded protein response (UPR), the main adaptive pathway to cope with ER stress. Here we provide evidence of suboptimal activation of the UPR in ALS/FTD models under experimental ER stress. To artificially engage the UPR, we intracerebroventricularly administrated adeno-associated viruses (AAV) to express the active form of XBP1 (XBP1s) in the nervous system of ALS/FTD models. XBP1s expression improved motor performance and extended life span of mutant SOD1 mice, associated with reduced protein aggregation. AAV-XBP1s administration also attenuated disease progression in models of TDP-43 and C9orf72 pathogenesis. Proteomic profiling of spinal cord tissue revealed that XBP1s overexpression improved proteostasis and modulated the expression of a cluster of synaptic and cell morphology proteins. Our results suggest that strategies to improve ER proteostasis may serve as a pan-therapeutic strategy to treat ALS/FTD
Expression of a protein disulfide isomerase A3 variant associated to amyotrophic lateral sclerosis triggers disease features in mice
No full textAmyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by loss of motoneurons and compromised proteostasis. Dysfunction of the endoplasmic reticulum (ER) has been identified as a transversal pathogenic mechanism associated to motoneurons vulnerability in ALS. Protein disulfide isomerases (PDIs) are key enzymes catalyzing protein folding at the ER that are altered in the disease, involving both biochemical and genetic perturbations. We previously identified mutations in the gene encoding PDIA3 (also known as Grp58 or ERp57) in ALS cases, which were associated with altered neurite outgrowth in cell culture and abnormal motoneuron connectivity in zebrafish. Here we report the generation of transgenic mice expressing the ALS-associated PDIA3Q481K variant. Moderate PDIA3Q481K overexpression resulted in altered motor capacity accompanied by decreased motoneurons number and induction of ER stress in the spinal cord. The adverse effects of PDIA3Q481K were associated with altered electromyogram without evident morphological changes in neuromuscular junctions. Our results suggest that the PDIA3Q481K variant is pathogenic and its overexpression in mice recapitulate some ALS features, further supporting the concept that altered proteostasis due to PDI dysfunction constitute a risk factor to develop the disease
ER stress and the unfolded protein response in neurodegeneration
No full textThe clinical manifestation of neurodegenerative diseases is initiated by the selective alteration in the functionality of distinct neuronal populations. The pathology of many neurodegenerative diseases includes accumulation of misfolded proteins in the brain. In physiological conditions, the proteostasis network maintains normal protein folding, trafficking and degradation; alterations in this network - particularly disturbances to the function of endoplasmic reticulum (ER) - are thought to contribute to abnormal protein aggregation. ER stress triggers a signalling reaction known as the unfolded protein response (UPR), which induces adaptive programmes that improve protein folding and promote quality control mechanisms and degradative pathways or can activate apoptosis when damage is irreversible. In this Review, we discuss the latest advances in defining the functional contribution of ER stress to brain diseases, including novel evidence that relates the UPR to synaptic function, which has implications for cognition and memory. A complex concept is emerging wherein the consequences of ER stress can differ drastically depending on the disease context and the UPR signalling pathway that is altered. Strategies to target specific components of the UPR using small molecules and gene therapy are in development, and promise interesting avenues for future interventions to delay or stop neurodegeneration.FONDAP
15150012
US Office of Naval Research-Global (ONR-G)
N62909-16-1-2003
Millennium Institute
P09-015-F
FONDEF
ID16I10223
D11E1007
US Air Force Office of Scientific Research
FA9550-16-1-0384
CONICYT-Brazil
441921/2016-7
ALS Therapy Alliance
2014-F-059
Muscular Dystrophy Association
382453
Michael J Fox Foundation for Parkinson's Research - Target Validation
9277
FONDECYT
1140549
ALSRP Therapeutic Idea Award
AL150111
Synapsis Foundation
Stiftung UNISCIENTIA
Frick foundation for ALS research
Swiss National Science Foundation
European Research Council
72582
The unfolded protein response transcription factor XBP1s ameliorates Alzheimer’s disease by improving synaptic function and proteostasis
No full textAlteration in the buffering capacity of the proteostasis network is an emerging feature of Alzheimer´s disease (AD), highlighting the occurrence of endoplasmic reticulum (ER) stress. The unfolded protein response (UPR) is the main signaling pathway emerging from the ER to cope with protein folding stress. Inositol requiring enzyme-1 (IRE1) is an ER-located kinase and endoribonuclease that operates as a central ER stress sensor, enabling the establishment of adaptive programs through the control of the expression of the transcription factor X-Box binding protein 1 (XBP1). A polymorphism in the XBP1 promoter was suggested as a risk factor to develop AD. To artificially enforce the adaptive capacity of the UPR, here we developed strategies to express the active form of XBP1 in neurons on a preclinical model of AD. The overexpression of XBP1s in the nervous system using transgenic mice significantly reduced the load of amyloid deposition in cerebral cortex and hippocampus, in addition to preserve synaptic and cognitive function. Moreover, the local delivery of XBP1s in the hippocampus of AD mice using viral vectors improved long-term potentiation, memory performance and the density of dendritic spines. Quantitative proteomics of hippocampus indicated that XBP1 expression restores the levels of many synaptic protein and factors involved in actin cytoskeleton regulation and axonal growth. Our results illustrate the therapeutic potential of XBP1s-deppedent responses as a strategy to ameliorate AD features and sustain synaptic function
Bax inhibitor-1: regula negativamente al sensor de estrés IRE1a
No full textThe endoplasmic reticulum (ER) is an essential organelle for protein
folding and secretion. Different conditions alter its functioning promoting the
accumulation of misfolded proteins at its lumen, a condition referred as "ER
stress". Adaptation to ER stress is mediated by the activat¡on of a complex
signal transduction pathway known as the unfolded protein response (UPR).
One of the most conserved ER stress sensors is lRElc¿, which controls the
expression of the transcription factor X-Box binding protein-1 (XBP-1), an
essential component for adaptive processes. When ER stress becomes
irreversible and UPR is not enough to re-establish homeostasis, prolonged
ER stress leads to apoptosis by mechanisms not well understood.
Bax inhibitor -1 (Bl-1) is an evolutionary conserved ER-resident
protein, capable of suppress cell death. ln mammals Bl-1 regulates apoptosis
initiated by a variety of intrinsic death stimuli. Recent studies suggest a
hyperactivation of the UPR, under ischemic and reperfusion conditions in Bl- 'l deficient mice. Nonetheless the mechanism which explains these
observations is unknown. ln this thesis we investigated the role of Bl-1 in
UPR. Bl-1 deficient cells show hyperactivation of lRElc¿, producing an
increase of the downstream targets, such as the expression of XBP-1 and
over-expression of XBP-1-dependent target genes. This phenotype can be
explained by a marked delay of lREl signalling deactivation observed in
absence of Bl-1. The inhibitory effects of Bl-1 in UPR are assoc¡ated with the
formation of a stable protein complex with IRE-1 cytosolic domain, which is
dependent of C-terminal region of Bl-1 a domain previously linked to its antiapoptotic
activity. Phylogenetic analysis of Bl-1 sequence revealed that this
domain is conserved in Drosophila Melanogaster and Mus musculus among
other species. With the aim of validating our results in vivo, we analyzed the
levels of activation of lRElo in Bl-1 deficient m¡ce or in a D- Melanogaster
strain that over-express Bl-1. ln both models, it was possible to observe that
Bl-1 suppresses lRElcr activity, when these animals where under ER stress
conditions. These results suggest a new role for Bl-1 in adaptive processes
aga¡nst ER stress, a function that contrasts with its inhibitory effect of
apoptosis,El retículo endoplásmico (RE) es un organelo esencial para el
plegamiento y secreción de proteínas. Diferentes condiciones alteran el
funcionamiento del RE, promoviendo la acumulación de proteínas mal
plegadas en su lumen, un estado llamado "estrés de RE". La adaptación
celular frente al estrés del RE esta mediada por la activación de una
compleja vía de transducción de señales conocida como respuesta a
proteínas mal plegadas (UPR). Uno de los sensores de estrés de RE mas
conservado es lRElo, el cual controla la expresión del factor de trascripc¡ón
"X-Box binding protein-l" (XBP-1), componente esencial para gatillar
pro@sos adaptativos. Cuando el estrés de RE daña irreversiblemente a la
célula y la UPR no es suficiente para reestablecer la homeostasis, esta via
desencadena apoptosis por mecanismos aun no bien conocidos.
"Bax inhibitor-l" (Bl-1) es una proteína residente del RE, conservada
en la evolución, capaz de suprimir la muerte celular en diferentes especies.
En mamíferos Bl-1 regula la apoptosis mediada por diferentes estímulos
intrínsecos. Estudios recientes sugieren una sobre-activación de la UPR en
ratones deficientes para Bl-'t bajo condiciones de isquemia y reperfusión. Sin
embargo, el mecanismo que explica estas observaciones aun se desconoce.
En esta tesis investigamos el posible rol de Bl-l en la UPR. Células
deficientes en Bl-1 muestran hiperactivación de lRElc¿, asociado a un
aumento en los niveles de sus blancos río abajo, como es la expresión de
XBP-I y una sobre-expresión de los genes blanco dependientes de XBP-1.
Este fenotipo puede ser explicado por un marcado retraso en la
desactivación de la señalización de lRElcr, observada en la ausencia de BlI . LoE efectos inhibitorios de Bl-1 en la UPR están asociados con la
formación de un complejo proteico estable con el dominio citosólico de
lRElc¿, el cual es dependiente de la región C-terminal de Bl-1, un dom¡n¡o
previamente asociado a su actividad anti-apoptótica. Análisis filogenéticos
revelaron que este dominio se encuentra conservado en Drosophila
melanogaster y Mus musculus, entre otras especies. Con el objeto de validar
nuestros resultados in vivo, analizamos los niveles de activación de lRElcr
en ratones deficientes para Bl-1 o en una cepa de D. melanogasfer que
sobreexpresa Bl-l . En ambos modelos se observó que Bl-1 suprime la
actividad de lREl cr, al ser sometidos estos animales a estrés de RE
experimental. Nuestros resultados sugieren un nuevo papel para Bl-1 en
procesos adaptativos contra el estrés de RE, función que contrasta con su
conocido efecto inhibidor de la apoptosis gatillada por diversos estlmulos de
muerte celular intrínsecos
Caracterización de modelos de pérdida y ganancia de función para la foldasa ERP57 en el sistema nervioso central
No full textERp57 es una proteína miembro de la familia de las proteínas Disulfuro Isomerasas y una foldasa del retículo endoplasmico que participa en el plegamiento de glicoproteinas, siendo parte del ciclo calnexina/calreticulina. ERp57 ha sido relacionada con diversas enfermedades, como cáncer y hepatitis, en las cuales su contribución en enfermedades neurogenerativas, como Alzheimer y Esclerosis Lateral Amiotrofica aun no ha sido dilucidado
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