c-Abl Phosphorylates MFN2 to Regulate Mitochondrial Morphology in Cells under Endoplasmic Reticulum and Oxidative Stress, Impacting Cell Survival and Neurodegeneration.

The endoplasmic reticulum is a subcellular organelle key in the control of synthesis, folding, and sorting of proteins. Under endoplasmic reticulum stress, an adaptative unfolded protein response is activated; however, if this activation is prolonged, cells can undergo cell death, in part due to oxidative stress and mitochondrial fragmentation. Here, we report that endoplasmic reticulum stress activates c-Abl tyrosine kinase, inducing its translocation to mitochondria. We found that endoplasmic reticulum stress-activated c-Abl interacts with and phosphorylates the mitochondrial fusion protein MFN2, resulting in mitochondrial fragmentation and apoptosis. Moreover, the pharmacological or genetic inhibition of c-Abl prevents MFN2 phosphorylation, mitochondrial fragmentation, and apoptosis in cells under endoplasmic reticulum stress. Finally, in the amyotrophic lateral sclerosis mouse model, where endoplasmic reticulum and oxidative stress has been linked to neuronal cell death, we demonstrated that the administration of c-Abl inhibitor neurotinib delays the onset of symptoms. Our results uncovered a function of c-Abl in the crosstalk between endoplasmic reticulum stress and mitochondrial dynamics via MFN2 phosphorylation.

c-Abl Tyrosine Kinase Is Required for BDNF-Induced Dendritic Branching and Growth.

Brain-derived neurotrophic factor (BDNF) induces activation of the TrkB receptor and several downstream pathways (MAPK, PI3K, PLC-γ), leading to neuronal survival, growth, and plasticity. It has been well established that TrkB signaling regulation is required for neurite formation and dendritic arborization, but the specific mechanism is not fully understood. The non-receptor tyrosine kinase c-Abl is a possible candidate regulator of this process, as it has been implicated in tyrosine kinase receptors' signaling and trafficking, as well as regulation of neuronal morphogenesis. To assess the role of c-Abl in BDNF-induced dendritic arborization, wild-type and c-Abl-KO neurons were stimulated with BDNF, and diverse strategies were employed to probe the function of c-Abl, including the use of pharmacological inhibitors, an allosteric c-Abl activator, and shRNA to downregulates c-Abl expression. Surprisingly, BDNF promoted c-Abl activation and interaction with TrkB receptors. Furthermore, pharmacological c-Abl inhibition and genetic ablation abolished BDNF-induced dendritic arborization and increased the availability of TrkB in the cell membrane. Interestingly, inhibition or genetic ablation of c-Abl had no effect on the classic TrkB downstream pathways. Together, our results suggest that BDNF/TrkB-dependent c-Abl activation is a novel and essential mechanism in TrkB signaling.

c-Abl Inhibition Activates TFEB and Promotes Cellular Clearance in a Lysosomal Disorder.

The transcription factor EB (TFEB) has emerged as a master regulator of lysosomal biogenesis, exocytosis, and autophagy, promoting the clearance of substrates stored in cells. c-Abl is a tyrosine kinase that participates in cellular signaling in physiological and pathophysiological conditions. In this study, we explored the connection between c-Abl and TFEB. Here, we show that under pharmacological and genetic c-Abl inhibition, TFEB translocates into the nucleus promoting the expression of its target genes independently of its well-known regulator, mammalian target of rapamycin complex 1. Active c-Abl induces TFEB phosphorylation on tyrosine and the inhibition of this kinase promotes lysosomal biogenesis, autophagy, and exocytosis. c-Abl inhibition in Niemann-Pick type C (NPC) models, a neurodegenerative disease characterized by cholesterol accumulation in lysosomes, promotes a cholesterol-lowering effect in a TFEB-dependent manner. Thus, c-Abl is a TFEB regulator that mediates its tyrosine phosphorylation, and the inhibition of c-Abl activates TFEB promoting cholesterol clearance in NPC models.

c-Abl Deficiency Provides Synaptic Resiliency Against Aß-Oligomers.

Spine pathology has been implicated in the early onset of Alzheimer's disease (AD), where Aß-Oligomers (AßOs) cause synaptic dysfunction and loss. Previously, we described that pharmacological inhibition of c-Abl prevents AßOs-induced synaptic alterations. Hence, this kinase seems to be a key element in AD progression. Here, we studied the role of c-Abl on dendritic spine morphological changes induced by AßOs using c-Abl null neurons (c-Abl-KO). First, we characterized the effect of c-Abl deficiency on dendritic spine density and found that its absence increases dendritic spine density. While AßOs-treatment reduces the spine number in both wild-type (WT) and c-Abl-KO neurons, AßOs-driven spine density loss was not affected by c-Abl. We then characterized AßOs-induced morphological changes in dendritic spines of c-Abl-KO neurons. AßOs induced a decrease in the number of mushroom spines in c-Abl-KO neurons while preserving the populations of immature stubby, thin, and filopodia spines. Furthermore, synaptic contacts evaluated by PSD95/Piccolo clustering and cell viability were preserved in AßOs-exposed c-Abl-KO neurons. In conclusion, our results indicate that in the presence of AßOs c-Abl participates in synaptic contact removal, increasing susceptibility to AßOs damage. Its deficiency increases the immature spine population reducing AßOs-induced synapse elimination. Therefore, c-Abl signaling could be a relevant actor in the early stages of AD.

A Survey of the Regulatory Requirements for BCS-Based Biowaivers for Solid Oral Dosage Forms by Participating Regulators and Organisations of the International Generic Drug Regulators Programme.

PURPOSE: The Biopharmaceutics Classification System (BCS) based biowaiver is a scientific model which enables the substitution of in vivo bioequivalence studies with in vitro data as evidence of therapeutic equivalence subject to certain conditions. Despite being based on the same principles, BCS-based biowaivers are interpreted and regulated differently among international regulatory agencies. In this survey, the Bioequivalence Working Group (BEWG) of the International Generic Drug Regulators Programme (IGDRP) compared the criteria for BCS-based biowaivers applied by the participating regulators and organisations. METHODS: Differences and similarities regarding solubility, permeability, dissolution, excipients and fixed-dose combination products, were identified and compared in a detailed survey of each participant's criteria for BCS-based biowaivers. These criteria were determined based upon the participants' respective regulatory guidance documents, policies and practices. RESULTS: This review has, with the exception of two participants who do not accept BCS-based biowaivers, revealed that most IGDRP participants interpret the BCS principles and conditions similarly but notable differences exist in the application of these principles.  Conclusion: Although many similarities exist, this review identifies several opportunities for greater convergence of regulatory requirements amongst the surveyed jurisdictions. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Reduction of Blood Amyloid-ß Oligomers in Alzheimer's Disease Transgenic Mice by c-Abl Kinase Inhibition.

One of the pathological hallmarks of Alzheimer's disease (AD) is the presence of amyloid plaques, which are deposits of misfolded and aggregated amyloid-beta peptide (Aß). The role of the c-Abl tyrosine kinase in Aß-mediated neurodegeneration has been previously reported. Here, we investigated the therapeutic potential of inhibiting c-Abl using imatinib. We developed a novel method, based on a technique used to detect prions (PMCA), to measure minute amounts of misfolded-Aß in the blood of AD transgenic mice. We found that imatinib reduces Aß-oligomers in plasma, which correlates with a reduction of AD brain features such as plaques and oligomers accumulation, neuroinflammation, and cognitive deficits. Cells exposed to imatinib and c-Abl KO mice display decreased levels of ß-CTF fragments, suggesting that an altered processing of the amyloid-beta protein precursor is the most probable mechanism behind imatinib effects. Our findings support the role of c-Abl in Aß accumulation and AD, and propose AD-PMCA as a new tool to evaluate AD progression and screening for drug candidates.

Terminaciones voluntarias de embarazo de causa genética en Mayabeque / Voluntary endings of pregnancies of genetic cause in Mayabeque

Introducción: el asesoramiento genético que se ofrece a la embarazada durante su seguimiento prenatal, garantiza la realización de una serie de estudios de diagnóstico para la detección de defectos congénitos y enfermedades genéticas. Una vez detectada la presencia de un defecto congénito incompatible con la vida o de pronóstico severo para la calidad de vida del producto de la concepción, la gestante y su pareja tienen el derecho de decidir sobre la continuidad del embarazo.Objetivo: conocer el comportamiento de las terminaciones voluntarias de embarazos de causa genética en la provincia Mayabeque. Métodos: se realizó un estudio retrospectivo de tipo observacional, en el Centro Provincial de Genética Médica de Mayabeque donde se estudiaron diferentes variables de las pacientes que solicitaron terminaciones voluntarias de embarazo de causa genética, en un periodo de tres años.Resultados: las terminaciones voluntarias de embarazo fueron más frecuentes en mujeres con edad materna entre 20-35 años 72,90 por ciento con menos de 20 semanas 43,23 por ciento y con defectos congénitos aislados 84,52 por ciento principalmente de los sistemas nerviosos centrales y cardiovasculares. Dentro de los defectos congénitos predominaron las cardiopatías congénitas, defectos de cierre del tubo neural y cromosomopatías. El ultrasonido fue el medio diagnóstico de mayor utilidad para confirmar el defecto congénito. Conclusiones: la oportuna decisión de la terminación voluntaria de embarazo una vez diagnosticado un defecto congénito incompatible con la vida ha tenido un impacto directo en la reducción de la tasa de mortalidad infantil en la provincia Mayabeque(AU)

Introduction: the genetic advising that is offered to the pregnant woman during her prenatal follow up, guarantees the performance of several diagnostic tests for the detection of congenital defects and genetic diseases. Once detected a congenital defect incompatible with life or of severe prognosis for the life quality of the product of conception, the pregnant woman and her couple have the right to decide about the continuity of the pregnancy. Objective: to know the behavior of the voluntary endings of pregnancies of genetic cause in Mayabeque province Methods: a retrospective observational study was carried out at the Medical Genetic Center from Mayabeque province in which different variables were studied in patients who seek voluntary endings of pregnancies of genetic cause in a period of three years. Results: the voluntary endings of pregnancies were more frequent in women with maternal age of 20-35 years 72,90 percent with less than 20 weeks 43,23 percent and with isolated congenital defects 84,52 percent mainly in the cardiovascular and central nervous system. Within the congenital defects, congenital cardiopathies, defects of the closure of the neural tube and cromosomopathies prevailed, Ultrasound was the diagnostic test of highest quality to confirm the congenital defect. Conclusions: the pertinent decision of voluntary endings of pregnancies of genetic cause, once it was diagnosed a genetic defect incompatible with life, has had a direct impact in the reduction of children mortality rate in Mayabeque province(AU)

EphA4 activation of c-Abl mediates synaptic loss and LTP blockade caused by amyloid-ß oligomers.

The early stages of Alzheimer's disease are characterised by impaired synaptic plasticity and synapse loss. Here, we show that amyloid-ß oligomers (AßOs) activate the c-Abl kinase in dendritic spines of cultured hippocampal neurons and that c-Abl kinase activity is required for AßOs-induced synaptic loss. We also show that the EphA4 receptor tyrosine kinase is upstream of c-Abl activation by AßOs. EphA4 tyrosine phosphorylation (activation) is increased in cultured neurons and synaptoneurosomes exposed to AßOs, and in Alzheimer-transgenic mice brain. We do not detect c-Abl activation in EphA4-knockout neurons exposed to AßOs. More interestingly, we demonstrate EphA4/c-Abl activation is a key-signalling event that mediates the synaptic damage induced by AßOs. According to this results, the EphA4 antagonistic peptide KYL and c-Abl inhibitor STI prevented i) dendritic spine reduction, ii) the blocking of LTP induction and iii) neuronal apoptosis caused by AßOs. Moreover, EphA4-/- neurons or sh-EphA4-transfected neurons showed reduced synaptotoxicity by AßOs. Our results are consistent with EphA4 being a novel receptor that mediates synaptic damage induced by AßOs. EphA4/c-Abl signalling could be a relevant pathway involved in the early cognitive decline observed in Alzheimer's disease patients.

Lysosomal vitamin E accumulation in Niemann-Pick type C disease.

Niemann-Pick C disease (NPC) is a neuro-visceral lysosomal storage disorder mainly caused by genetic defects in the NPC1 gene. As a result of loss of NPC1 function large quantities of free cholesterol and other lipids accumulate within late endosomes and lysosomes. In NPC livers and brains, the buildup of lipids correlates with oxidative damage; however the molecular mechanisms that trigger it remain unknown. Here we study potential alterations in vitamin E (α-tocopherol, α-TOH), the most potent endogenous antioxidant, in liver tissue and neurons from NPC1 mice. We found increased levels of α-TOH in NPC cells. We observed accumulation and entrapment of α-TOH in NPC neurons, mainly in the late endocytic pathway. Accordingly, α-TOH levels were increased in cerebellum of NPC1 mice. Also, we found decreased mRNA levels of the α-TOH transporter, α-Tocopherol Transfer Protein (α-TTP), in the cerebellum of NPC1 mice. Finally, by subcellular fractionation studies we detected a significant increase in the hepatic α-TOH content in purified lysosomes from NPC1 mice. In conclusion, these results suggest that NPC cells cannot transport vitamin E correctly leading to α-TOH buildup in the endosomal/lysosomal system. This may result in a decreased bioavailability and impaired antioxidant function of vitamin E in NPC, contributing to the disease pathogenesis.

Caracterización del climaterio y menopausia en la mujer venezolana / Characterization of climatery and menopause in venezuelan woman

Se realizó un estudio descriptivo prospectivo, de corte transversal, en el consultorio médico Orinoco, municipio Independencia, Parroquia Soledad, Estado Anzoátegui, Venezuela, durante el año 2006 con el objetivo de realizar una caracterización del proceso de climaterio y menopausia en la población femenina de 40 a 59 años. El universo de la investigación estuvo constituido por 65 mujeres y la muestra la conformaron 45, al excluirse del estudio 20 pacientes histerectomizadas. Se estudiaron aspectos como: edad de aparición de la menopausia, principales trastornos genitourinarios, circulatorios, psicológicos y generales asociados a la etapa. Los resultados reflejaron que la mayor parte de las mujeres se encontraban en la perimenopausia y la edad promedio fue de 49 años. La perimenopausia fue el momento de mayor incidencia de los síntomas vasomotores, psicológicos y generales. Se recomendó realizar estudios sobre los factores biopsicosociales de esta etapa en las diferentes áreas de salud y crear consultas de climaterio y menopausia (AU)

It was performed a descriptive, prospective and transversal study, in the Orinoco doctor's office, Independence municipality, Soledad Parish, Anzoategui State, Venezuela, in 2006 with the objective of performing a characterization of the Climatery and menopause process in the female population 40 to 59 years. The research universe consisted of 65 women and the sample consisted of 45, because 20 patients were excluded of the study for having undergone hysterectomy. It was studied aspects like: age of menopause onset, main genitourinary, circulatory, psychological and general disorders related to the stage. The results showed that the majority of women were in perimenopause and the average age was 49 years. Perimenopause was the moment of greater incidence of vasomotor, psychological and general symptoms. It was recommended to develop studies on the biopsychosocial factors at this stage in different areas of health and creating climacteric and menopause consultation (AU)

High-frequency field stimulation of primary neurons enhances ryanodine receptor-mediated Ca2+ release and generates hydrogen peroxide, which jointly stimulate NF-κB activity.

Neuronal electrical activity increases intracellular Ca(2+) concentration and generates reactive oxygen species. Here, we show that high frequency field stimulation of primary hippocampal neurons generated Ca(2+) signals with an early and a late component, and promoted hydrogen peroxide generation via a neuronal NADPH oxidase. Hydrogen peroxide generation required both Ca(2+) entry through N-methyl-D-aspartate receptors and Ca(2+) release mediated by ryanodine receptors (RyR). Field stimulation also enhanced nuclear translocation of the NF-κB p65 protein and NF-κB -dependent transcription, and increased c-fos mRNA and type-2 RyR protein content. Preincubation with inhibitory ryanodine or with the antioxidant N-acetyl L-cysteine abolished the increase in hydrogen peroxide generation and the late Ca(2+) signal component induced by electrical stimulation. Primary cortical cells behaved similarly as primary hippocampal cells. Exogenous hydrogen peroxide also activated NF-κB-dependent transcription in hippocampal neurons; inhibitory ryanodine prevented this effect. Selective inhibition of the NADPH oxidase or N-acetyl L-cysteine also prevented the enhanced translocation of p65 in hippocampal cells, while N-acetyl L-cysteine abolished the increase in RyR2 protein content induced by high frequency stimulation. In conclusion, the present results show that electrical stimulation induced reciprocal activation of ryanodine receptor-mediated Ca(2+) signals and hydrogen peroxide generation, which stimulated jointly NF-κB activity.

Increased hippocampal expression of the divalent metal transporter 1 (DMT1) mRNA variants 1B and +IRE and DMT1 protein after NMDA-receptor stimulation or spatial memory training.

Iron is essential for crucial neuronal functions but is also highly toxic in excess. Neurons acquire iron through transferrin receptor-mediated endocytosis and via the divalent metal transporter 1 (DMT1). The N-terminus (1A, 1B) and C-terminus (+IRE, -IRE) splice variants of DMT1 originate four protein isoforms, all of which supply iron to cells. Diverse physiological or pathological conditions induce differential DMT1 variant expression, which are cell-type dependent. Hence, it becomes relevant to ascertain if activation of neuronal plasticity processes that require functional N-methyl D: -aspartate (NMDA) receptors, including in vitro stimulation of NMDA receptor-mediated signaling and spatial memory training, selectively modify DMT1 variant expression. Here, we report for the first time that brief (5 min) exposure of primary hippocampal cultures to NMDA (50 muM) increased 24 h later the expression of DMT1-1B and DMT1+IRE, but not of DMT1-IRE mRNA. In contrast, endogenous DMT1 mRNA levels remained unaffected following 6 h incubation with brain-derived nerve factor. NMDA (25-50 muM) also enhanced DMT1 protein expression 24-48 h later; this enhancement was abolished by the transcription inhibitor actinomycin D and by the NMDA receptor antagonist MK-801, implicating NMDA receptors in de novo DMT1 expression. Additionally, spatial memory training enhanced DMT1-1B and DMT1+IRE expression and increased DMT1 protein content in rat hippocampus, where the exon1A variant was not found. These results suggest that NMDA receptor-dependent plasticity processes stimulate expression of the iron transporter DMT1-1B+IRE isoform, which presumably plays a significant role in hippocampal spatial memory formation.

Ecocardiografía Doppler en las patologías obstétricas / Doppler echocardiography in obstetric pathologies

Se realizó una revisión bibliográfica sobre la hemodinámica feto placentaria y sus variantes fisiológicas normales, así como los distintos parámetros de la ecocardiografía Doppler y su aplicación para el diagnóstico y seguimientos de fetos con patologías asociadas como Hipertensión Arterial, Crecimiento Intrauterino Retardado, Diabetes mellitus, anemia fetal y discordancia intergemelar. Se constató la importancia de este método no invasivo en la medicina materno fetal lo que permite recomendar el adiestramiento e incorporación de esta práctica en la atención prenatal a los diferentes niveles. Para realizar este trabajo se consultaron bibliografías en la base de datos electrónica accesible en el momento del estudio, libros de textos en formato digital y conferencias impartidas en el diplomado de ecocardiografía fetal 2008 en el Cardiocentro William Soler (AU)

It was conducted a review of the literature on placental and fetal hemodynamics and its normal physiologic variants, as well as the various parameters of Doppler echocardiography and its application for the diagnosis and monitoring of fetuses with associated pathologies like hypertension, retarded intrauterine growth, diabetes mellitus, fetal anemia and intertwin disparity. It was confirmed the importance of this noninvasive method in maternal fetal medicine, which allows to recommend the training and incorporation of this practice in prenatal care at different levels. To make this work it was consulted bibliography in the electronic database accessible at the time of the study, books in digital format and lectures shared at fetal echocardiography diploma course in William Cardiocenter in 2008 (AU)

STI571 prevents apoptosis, tau phosphorylation and behavioural impairments induced by Alzheimer's beta-amyloid deposits.

There is evidence that amyloid beta-protein (Abeta) deposits or Abeta intermediates trigger pathogenic factors in Alzheimer's disease patients. We have previously reported that c-Abl kinase activation involved in cell signalling regulates the neuronal death response to Abeta fibrils (Abeta(f)). In the present study we investigated the therapeutic potential of the selective c-Abl inhibitor STI571 on both the intrahippocampal injection of Abeta(f) and APPsw/PSEN1DeltaE9 transgenic mice Alzheimer's disease models. Injection of Abeta(f) induced an increase in the numbers of p73 and c-Abl immunoreactive cells in the hippocampal area near to the lesion. Chronic intraperitoneal administration of STI571 reduced the rat behavioural deficit induced by Abeta(f), as well as apoptosis and tau phosphorylation. Our in vitro studies suggest that inhibition of the c-Abl/p73 signalling pathway is the mechanism underlying of the effects of STI571 on Abeta-induced apoptosis for the following reasons: (i) Abeta(f) induces p73 phosphorylation, the TAp73 isoform levels increase so as to enhance its proapoptotic function, and all these effects where reduced by STI571; (ii) c-Abl kinase activity is required for neuronal apoptosis and (iii) STI571 prevents the Abeta-induced increase in the expression of apoptotic genes. Furthermore, in the Abeta-injected area there was a huge increase in phosphorylated p73 and a larger number of TAp73-positive cells, with these changes being prevented by STI571 coinjection. Moreover, the intraperitoneal administration of STI571 rescued the cognitive decline in APPsw/PSEN1DeltaE9 mice, p73 phosphorylation, tau phosphorylation and caspase-3 activation in neurons around Abeta deposits. Besides, we observed a decrease in the number and size of Abeta deposits in the APPsw/PSEN1DeltaE9-STI571-treated mice. These results are consistent with the role of the c-Abl/p73 signalling pathway in Abeta neurodegeneration, and suggest that STI571-like compounds would be effective in therapeutic treatments of Alzheimer disease.

NFAT activation by membrane potential follows a calcium pathway distinct from other activity-related transcription factors in skeletal muscle cells.

Depolarization of skeletal muscle cells triggers intracellular Ca2+ signals mediated by ryanodine and inositol 1,4,5-trisphosphate (IP3) receptors. Previously, we have reported that K+-induced depolarization activates transcriptional regulators ERK, cAMP response element-binding protein, c-fos, c-jun, and egr-1 through IP3-dependent Ca2+ release, whereas NF-kappa B activation is elicited by both ryanodine and IP3 receptor-mediated Ca2+ signals. We have further shown that field stimulation with electrical pulses results in an NF-kappa B activation increase dependent of the amount of pulses and independent of their frequency. In this work, we report the results obtained for nuclear factor of activated T cells (NFAT)-mediated transcription and translocation generated by both K+ and electrical stimulation protocols in primary skeletal muscle cells and C2C12 cells. The Ca2+ source for NFAT activation is through release by ryanodine receptors and extracellular Ca2+ entry. We found this activation to be independent of the number of pulses within a physiological range of stimulus frequency and enhanced by long-lasting low-frequency stimulation. Therefore, activation of the NFAT signaling pathway differs from that of NF-kappa B and other transcription factors. Calcineurin enzyme activity correlated well with the relative activation of NFAT translocation and transcription using different stimulation protocols. Furthermore, both K+-induced depolarization and electrical stimulation increased mRNA levels of the type 1 IP3 receptor mediated by calcineurin activity, which suggests that depolarization may regulate IP3 receptor transcription. These results confirm the presence of at least two independent pathways for excitation-transcription coupling in skeletal muscle cells, both dependent on Ca2+ release and triggered by the same voltage sensor but activating different intracellular release channels.

Activation of the neuronal c-Abl tyrosine kinase by amyloid-beta-peptide and reactive oxygen species.

The deposition and accumulation of amyloid-beta-peptide (Abeta) in the brain are considered a sine qua non for Alzheimer's disease. The experimental delivery of fibrilized Abeta serves as a cellular model for several facets of the disease including the induction of synaptic dysfunction and apoptosis. c-Abl kinase is involved in the regulation of apoptosis and its pro-apoptotic function is in part mediated by its interaction with p73, a p53 homologue. We found that c-Abl activation is involved in cell signals that regulate neuronal death response to Abeta fibrils. Abeta peptide fibrils induced an increase of the c-Abl activity in rat hippocampal neurons as well as an increase in nuclear p73 protein levels and the p73-c-Abl complex. The neuronal cell death induced by Abeta fibrils was prevented by the inhibition of c-Abl with imatinib mesylate (Gleevec or STI571) and by the inhibition c-Abl expression by RNAi. These results directly point to a therapeutic strategy for the treatment of Alzheimer's disease.