Neurotoxicity of prion peptides mimicking the central domain of the cellular prion protein

The physiological functions of PrP(C) remain enigmatic, but the central domain, comprising highly conserved regions of the protein may play an important role. Indeed, a large number of studies indicate that synthetic peptides containing residues 106-126 (CR) located in the central domain (CD, 95-133) of PrP(C) are neurotoxic. The central domain comprises two chemically distinct subdomains, the charge cluster (CC, 95-110) and a hydrophobic region (HR, 112-133). The aim of the present study was to establish the individual cytotoxicity of CC, HR and CD. Our results show that only the CD peptide is neurotoxic. Biochemical, Transmission Electron Microscopy and Atomic Force Microscopy experiments demonstrated that the CD peptide is able to activate caspase-3 and disrupt the cell membrane, leading to cell death

Reelin expression in Creutzfeldt-Jakob disease and experimental models of transmissible spongiform encephalopathies

Reelin is an extracellular glycoprotein involved in key cellular processes in developing and adult nervous system, including regulation of neuronal migration, synapse formation, and plasticity. Most of these roles are mediated by the intracellular phosphorylation of disabled-1 (Dab1), an intracellular adaptor molecule, in turn mediated by binding Reelin to its receptors. Altered expression and glycosylation patterns of Reelin in cerebrospinal and cortical extracts have been reported in Alzheimer's disease. However, putative changes in Reelin are not described in natural prionopathies or experimental models of prion infection or toxicity. With this is mind, in the present study, we determined that Reelin protein and mRNA levels increased in CJD human samples and in mouse models of human prion disease in contrast to murine models of prion infection. However, changes in Reelin expression appeared only at late terminal stages of the disease, which prevent their use as an efficient diagnostic biomarker. In addition, increased Reelin in CJD and in in vitro models does not correlate with Dab1 phosphorylation, indicating failure in its intracellular signaling. Overall, these findings widen our understanding of the putative changes of Reelin in neurodegeneration

Sistema de secreción de tipo III en "Aeromonas" mesófilas

[spa] Aeromonas hydrophila es un bacilo gram-negativo, patógeno oportunista de animales y humanos. La patogénesis de A. hydrophila es multifactorial. Con objeto de identificar genes implicados en la virulencia de la cepa PPD134/91 de A. hydrophila, se realizaron experimentos de sustracción génica, que llevaron a la detección de 22 fragmentos de ADN que codificaban 19 putativos factores de virulencia, incluyendo un gen que codificaba una proteína de sistema de secreción de tipo III (T3SS). La creciente importancia del T3SS en la patogénesis de diversas bacterias, llevó a identificar y analizar la agrupación génica del T3SS de las cepas AH-1 y AH-3 de A. hydrophila, así como también el efector AexT en la cepa AH-3. La inactivación de los genes de T3SS aopB y aopD en A. hydrophila AH-1, y ascV en A. hydrophila AH-3 conlleva una disminución de la citotoxicidad, un incremento de la fagocitosis, y una reducción de la virulencia en diferentes modelos animales, demostrando estos resultados que el T3SS es necesario para la patogenicidad del género Aeromonas. El mutante en el gen aexT de la cepa AH-3 de A. hydrophila muestra una ligera reducción de la virulencia, ensayada con diferentes métodos. Por otro lado, la proteína AexT presentó actividad ADP-ribosiltransferasa, y se demostró su translocación vía T3SS al interior de células eucariotas, sistema que a su vez parece ser inducible in vitro en condiciones de depleción de calcio. Mediante el uso de diferentes sondas de ADN, se determinó la presencia del T3SS en cepas tanto clínicas como ambientales de diferentes especies del género Aeromonas: A. hydrophila, A. veronii, y A. caviae, y la codistribución de esta agrupación génica y del gen aexT. Finalmente, con el fin de analizar la activación transcripcional de la agrupación génica de T3SS y del efector AexT en A. hydrophila AH-3, los promotores predichos del operón aopN-aopD y el gen aexT se fusionaron con el gen reporter gfp (Green Fluorescence Protein). Además, se demostró que la expresión de ambos promotores depende de diferentes componentes bacterianos, como por ejemplo el sistema de dos componentes PhoP/PhoQ, el sistema de quórum sensing AhyI/AhyR, el sistema flagelar, el lipopolisacárido, la oxidoreductasa DsbA o el complejo enzimático piruvato deshidrogenasa.[eng] Aeromonas hydrophila is a gram-negative opportunistic pathogen of animals and humans and its pathogenesis is multifactorial. Genomic subtraction was used to identify putative virulence genes in A. hydrophila PPD134/91 and led to the identification of 22 unique DNA fragments encoding 19 putative virulence factors, including a gene encoding a type III secretion system (T3SS) protein. The increasing importance of the T3SS in the pathogenesis of several bacteria, led us to identify and further analyse the T3SS gene clusters in A. hydrophila AH-1 and AH-3. Insertional inactivation of the T3SS genes aopB and aopD in A. hydrophila AH-1 and ascV in A. hydrophila AH-3 led to decreased cytotoxicity, increased phagocytosis, and reduced virulence in several animal models. These results show that the T3SS is required for A. hydrophila pathogenicity. We also cloned and sequenced an ADP-ribosylating toxin (AexT) in A. hydrophila AH-3, and showed that this toxin is translocated via the T3SS, which also seems to be inducible in vitro under calcium depleted conditions. The A. hydrophila AH-3 aexT mutant showed a slight reduction in virulence assayed by several methods. By using several gene probes, we also determined the presence of the T3SS in clinical and environmental strains of different Aeromonas species: A. hydrophila, A. veronii, and A. caviae, and the co-distribution of this gene cluster and the gene encoding the toxin AexT. Finally, to study the transcriptional regulation of the T3SS gene cluster and the effector AexT in A. hydrophila AH-3, we isolated the predicted promoter regions of both operon aopN-aopD and gene aexT, which were fused with a green fluorescence protein reporter gene. Furthermore, we established that expression of the T3SS gene cluster and the effector AexT in A. hydrophila AH-3 depends on the expression of several bacterial components, such as the lipopolysaccharide, the polar flagellum, the two component regulatory system PhoP/PhoQ, the quorum sensing system based on AhyI/AhyR proteins, the pyruvate dehydrogenase complex and the periplasmic oxidoreductase DsbA

Sistema de secreción de tipo III en Aeromonas mesòfilas

Aeromonas hydrophila és un bacil gram-negatiu, patogen oportunista d’animal i humans. La patogènesi d’A. Hydrophila és multifactorial. A fi d'identificar gens implicats en la virulència de la soca PPD134/91 d’A. hydrophila, vam realitzar experiments de substracció gènica, que van dur a la detecció de 22 fragments d’ADN que codificaven 19 potencials factors de virulencia, incloent un gen que codificava una proteïna de sistema de secreció de tipus III (T3SS). La importància creixent del T3SS en la patogènesi de diversos bacteris, ens va dur a identificar i analitzar l'agrupació gènica del T3SS de les soques AH-1 i AH-3 d’A. hydrophila. La inactivació dels gens de T3SS aopB i aopD d’A. hydrophila AH-1, i ascV d’A. hydrophila AH-3, comporta una disminució de la citotoxicitat, un increment de la fagocitosi, i una reducció de la virulència en diferents models animals. Aquests resultats demostren que el T3SS és necessari per a la patogenicitat. També vam clonar i seqüenciar una ADP-ribosiltransferasa (AexT) a la soca AH-3 d’A. hydrophila, i vam demostrar que aquesta toxina és translocada via el T3SS, sistema que al seu torn sembla ser induïble in vitro en condicions de depleció de calci. El mutant en el gen aexT de la soca AH-3 d’A. hydrophila va mostrar una lleugera reducció de la virulència, assajada amb diferents mètodes. Mitjançant l'ús de diferents sondes d’ADN, vam determinar la presència del T3SS en soques tant clíniques com ambientals de diferents espècies del gènere Aeromonas: A. hydrophila, A. veronii, i A. caviae, i la codistribució d'aquesta agrupació gènica i el gen aexT. Finalment, amb la finalitat d'estudiar la regulació transcripcional de l'agrupació gènica de T3SS i de l’efector AexT A. hydrophila AH-3, vam aïllar els promotors predits per l’operó aopN-aopD i el gen aexT, i els vam fusionar amb el gen reporter gfp (Green Fluorescence Protein). A més, vam demostrar que l'expressió d'ambdós promotors depèn de diferents components bacterians, com per exemple el sistema de dos components PhoP/PhoQ, el sistema de quorum sensing AhyI/AhyR, o el complex piruvat deshidrogenasa.Aeromonas hydrophila is a gram-negative opportunistic pathogen of animals and humans. The pathogenesis is multifactorial. Genomic subtraction was used to identify putative virulence genes in A. hydrophila PPD134/91 and led to the identification of 22 unique DNA fragments encoding 19 putative virulence factors, including a gene encoding a type III secretion system (T3SS) protein. The increasing importance of the T3SS in the pathogenesis of several bacteria, led us to identify and further analyse the T3SS gene clusters in A. hydrophila AH-1 and AH-3. Insertional inactivation of the T3SS genes aopB and aopD in A. hydrophila AH-1 and ascV in A. hydrophila AH-3 led to decreased cytotoxicity, increased phagocytosis, and reduced virulence in several animal models. These results show that a T3SS is required for A. Hydrophila pathogenicity. We also cloned and sequenced an ADP-ribosylating toxin (AexT) in A. hydrophila AH-3, and showed that this toxin is translocated via the T3SS, which seems to be inducible in vitro under calcium depleted conditions. The A. hydrophila AH-3 aexT mutant showed a slight reduction in virulence assayed by several methods. By using several gene probes, we also determined the presence of the T3SS in clinical and environmental strains of different Aeromonas species: A. hydrophila, A. veronii, and A. caviae, and the co-distribution of this gene cluster and the gene encoding the toxin AexT. Finally, to study the transcriptional regulation of the T3SS gene cluster and the effector AexT in A. hydrophila AH-3, we isolated the predicted promoters region of both operon aopN-aopD and gene aexT, which were fused with a green fluorescence protein reporter gene. Furthermore, we established that expression of the T3SS gene cluster and the effector AexT in A. hydrophila AH-3 depends on the expression of several bacterial components, such as the two component regulatory system PhoP/PhoQ, the quorum sensing system based on AhyI/AhyR proteins, en the pyruvate dehydrogenase complex

Reelin expression in Creutzfeldt-Jakob disease and experimental models of transmissible spongiform encephalopathies

Reelin is an extracellular glycoprotein involved in key cellular processes in developing and adult nervous system, including regulation of neuronal migration, synapse formation, and plasticity. Most of these roles are mediated by the intracellular phosphorylation of disabled-1 (Dab1), an intracellular adaptor molecule, in turn mediated by binding Reelin to its receptors. Altered expression and glycosylation patterns of Reelin in cerebrospinal and cortical extracts have been reported in Alzheimer's disease. However, putative changes in Reelin are not described in natural prionopathies or experimental models of prion infection or toxicity. With this is mind, in the present study, we determined that Reelin protein and mRNA levels increased in CJD human samples and in mouse models of human prion disease in contrast to murine models of prion infection. However, changes in Reelin expression appeared only at late terminal stages of the disease, which prevent their use as an efficient diagnostic biomarker. In addition, increased Reelin in CJD and in in vitro models does not correlate with Dab1 phosphorylation, indicating failure in its intracellular signaling. Overall, these findings widen our understanding of the putative changes of Reelin in neurodegeneration

Reelin expression in Creutzfeldt-Jakob disease and experimental models of transmissible spongiform encephalopathies

Reelin is an extracellular glycoprotein involved in key cellular processes in developing and adult nervous system, including regulation of neuronal migration, synapse formation, and plasticity. Most of these roles are mediated by the intracellular phosphorylation of disabled-1 (Dab1), an intracellular adaptor molecule, in turn mediated by binding Reelin to its receptors. Altered expression and glycosylation patterns of Reelin in cerebrospinal and cortical extracts have been reported in Alzheimer's disease. However, putative changes in Reelin are not described in natural prionopathies or experimental models of prion infection or toxicity. With this is mind, in the present study, we determined that Reelin protein and mRNA levels increased in CJD human samples and in mouse models of human prion disease in contrast to murine models of prion infection. However, changes in Reelin expression appeared only at late terminal stages of the disease, which prevent their use as an efficient diagnostic biomarker. In addition, increased Reelin in CJD and in in vitro models does not correlate with Dab1 phosphorylation, indicating failure in its intracellular signaling. Overall, these findings widen our understanding of the putative changes of Reelin in neurodegeneration

Reelin expression in Creutzfeldt-Jakob disease and experimental models of transmissible spongiform encephalopathies

Reelin is an extracellular glycoprotein involved in key cellular processes in developing and adult nervous system, including regulation of neuronal migration, synapse formation, and plasticity. Most of these roles are mediated by the intracellular phosphorylation of disabled-1 (Dab1), an intracellular adaptor molecule, in turn mediated by binding Reelin to its receptors. Altered expression and glycosylation patterns of Reelin in cerebrospinal and cortical extracts have been reported in Alzheimer's disease. However, putative changes in Reelin are not described in natural prionopathies or experimental models of prion infection or toxicity. With this is mind, in the present study, we determined that Reelin protein and mRNA levels increased in CJD human samples and in mouse models of human prion disease in contrast to murine models of prion infection. However, changes in Reelin expression appeared only at late terminal stages of the disease, which prevent their use as an efficient diagnostic biomarker. In addition, increased Reelin in CJD and in in vitro models does not correlate with Dab1 phosphorylation, indicating failure in its intracellular signaling. Overall, these findings widen our understanding of the putative changes of Reelin in neurodegeneration

Reelin expression in Creutzfeldt-Jakob disease and experimental models of transmissible spongiform encephalopathies

Reelin is an extracellular glycoprotein involved in key cellular processes in developing and adult nervous system, including regulation of neuronal migration, synapse formation, and plasticity. Most of these roles are mediated by the intracellular phosphorylation of disabled-1 (Dab1), an intracellular adaptor molecule, in turn mediated by binding Reelin to its receptors. Altered expression and glycosylation patterns of Reelin in cerebrospinal and cortical extracts have been reported in Alzheimer's disease. However, putative changes in Reelin are not described in natural prionopathies or experimental models of prion infection or toxicity. With this is mind, in the present study, we determined that Reelin protein and mRNA levels increased in CJD human samples and in mouse models of human prion disease in contrast to murine models of prion infection. However, changes in Reelin expression appeared only at late terminal stages of the disease, which prevent their use as an efficient diagnostic biomarker. In addition, increased Reelin in CJD and in in vitro models does not correlate with Dab1 phosphorylation, indicating failure in its intracellular signaling. Overall, these findings widen our understanding of the putative changes of Reelin in neurodegeneration