Miostatina inhibe la vía de señalización IGF-1/PI3K/PLCγ/NFATc3 a través del receptor Activina tipo IB (ActRIB)

Tesis (Magíster en Biotecnología)Miostatina, un miembro de la familia TGF-β, es una proteína expresada principalmente en el músculo esquelético que cumple un rol inhibidor en la proliferación y diferenciación de células musculares. Miostatina ejerce su acción a través de un complejo de receptores Activina, los cuales median la actividad de los factores de transcripción Smads encargados de regular negativamente el proceso miogénico. Sin embargo, en los últimos años han surgido antecedentes que demuestran que la inhibición del proceso de diferenciación de células musculares por miostatina puede ser regulado de manera independiente a la actividad de los factores de transcripción Smads. Estudios realizados por nuestro grupo de laboratorio han mostrado que IGF-1 es capaz de inducir de manera transitoria la liberación de calcio desde reservorios intracelulares a través de un mecanismo que involucra la actividad de la PI3K y la PLC. Complementariamente determinamos que la preincubación con miostatina inhibió significativamente la liberación de calcio inducida por IGF-1. Esta observación nos lleva a formular la pregunta acerca de qué mecanismos median la inhibición de liberación de calcio inducida por IGF-1, surgiendo como un candidato el receptor Activina tipo IB. Si bien no existen antecedentes reportados de una interacción entre el receptor Activina tipo IB y componentes de la vía de IGF-1, sí existen reportes de la modulación de la actividad PI3K por parte de receptores de la familia TGF-En base a esto se postuló la siguiente hipótesis de trabajo “El receptor para miostatina Activina tipo IB modula negativamente la vía de señalización IGF-1/PI3K/PLC/NFATc3”. Para comprobar esta hipótesis se determinó el efecto de miostatina en la vía para IGF-1 usando las herramientas de western blot y vector reportero en mioblastos. Para demostrar la participación directa del receptor ActRIB, se realizaron ensayos de knock-down con siRNA para receptores de Activina tipo IB y finalmente se evaluó la interacción mediante inmunoprecipitación de ActRIB y componentes de la vía de IGF-1. Los resultados obtenidos sugieren que el receptor para miostatina se une a la subunidad p85 de la PI3K modulando su actividad, así como la regulación de los demás elementos de la vía de IGF-1. La información obtenida de este proyecto revela aspectos desconocidos de la vía de señalización de miostatina.Myostatin, a member of the TGF-β family is a protein expressed mainly in the skeletal muscle that plays an inhibitory role in the proliferation and differentiation of muscle cells. Myostatin exerts its actions through a complex of activin receptors, which mediate the activity of Smads transcription factors responsible for down regulation of myogenic processes. However, in recent years there have been studies that show that the inhibition of the differentiation process of muscle cells by myostatin can be regulated independently of the activity of the Smads transcription factors. Studies by our group have shown that IGF-1 induces in a transient manner the release of calcium from intracellular stores via a mechanism that involves PI3K and PLC activity. In addition, we determined that preincubation with myostatin significantly inhibited the release of calcium induced by IGF-1. This observation leads us to ask which mechanism mediates the inhibition of calcium release induced by IGF-1 emerging as a candidate the activin receptor type IB. While there are no reports about the interaction between the activin receptor type IB and components of the IGF-1 signaling pathway, there are reports regarding the modulation of PI3K activity by receptors of the TGF-β family. Based on these observations we proposed the following hypothesis “The myostatin receptor, activin type IB negatively modulates the IGF-1/PI3K/PLC/NFATc3 signaling pathway”. To test this hypothesis, we studied the effect of myostatin on the IGF-1 pathway by western blotting and luciferase reporter vector in skeletal myoblasts. To demonstrate a direct participation of the activin receptor type IB, siRNA knock-down was tested for the activin type IB receptor. Finally, the interaction between the activin type IB receptor and components of the IGF-1 pathway was determined by immunoprecipitation. The information obtained in this project reveals unknown aspects of the myostatin signaling pathwa

Insulin-like growth factor 2 (IGF2) protects against Huntington's disease through the extracellular disposal of protein aggregates

Impaired neuronal proteostasis is a salient feature of many neurodegenerative diseases, highlighting alterations in the function of the endoplasmic reticulum (ER). We previously reported that targeting the transcription factor XBP1, a key mediator of the ER stress response, delays disease progression and reduces protein aggregation in various models of neurodegeneration. To identify disease modifier genes that may explain the neuroprotective effects of XBP1 deficiency, we performed gene expression profiling of brain cortex and striatum of these animals and uncovered insulin-like growth factor 2 (Igf2) as the major upregulated gene. Here, we studied the impact of IGF2 signaling on protein aggregation in models of Huntington's disease (HD) as proof of concept. Cell culture studies revealed that IGF2 treatment decreases the load of intracellular aggregates of mutant huntingtin and a polyglutamine peptide. These results were validated using induced pluripotent stem cells (iPSC)-derived medium spiny neurons from HD patients and spinocerebellar ataxia cases. The reduction in the levels of mutant huntingtin was associated with a decrease in the half-life of the intracellular protein. The decrease in the levels of abnormal protein aggregation triggered by IGF2 was independent of the activity of autophagy and the proteasome pathways, the two main routes for mutant huntingtin clearance. Conversely, IGF2 signaling enhanced the secretion of soluble mutant huntingtin species through exosomes and microvesicles involving changes in actin dynamics. Administration of IGF2 into the brain of HD mice using gene therapy led to a significant decrease in the levels of mutant huntingtin in three different animal models. Moreover, analysis of human postmortem brain tissue and blood samples from HD patients showed a reduction in IGF2 level. This study identifies IGF2 as a relevant factor deregulated in HD, operating as a disease modifier that buffers the accumulation of abnormal protein species

Rôle de la mécanique des cavéoles dans la dynamique des vésicules extracellulaires impliquées dans la progression tumorale

Extracellular vesicles (EVs) are lipid-enclosed vesicles that are released by all cells studied to date and present in all human bodily fluids. EVs contain genetic material and proteins that are able to be transferred to and generate an effect in other cells. Caveolin-1 (Cav1) is a key component of the small invagination of the plasma membrane called caveolae, where it functions as mechano-sensors and membrane tension buffering device. Cav1 facilitates the migration and invasion of tumor cells and its expression is often increased in the late stages of cancer. Interestingly, high levels of Cav1 have been found in EVs of patients with advanced cancer. Given the importance of mechanical forces in the microenvironment of cancer cells, we hypothesized that caveolae and/or Cav1 may represent key players in the regulation of EV biology and cancer progression under mechanical strain. To test this hypothesis, we subjected different cancer cell lines, having (WT) or deleted for Cav1 expression (KO) to 2D or 3D systems of mechanical stress. EVs were purified from these cells and analyzed by nanoparticle tracking analysis. We found a striking increase in the release of EVs after mechanical stress both in 2D and 3D models. This increase was strictly dependent on the presence of Cav1 and correlated with enhanced fusion of multivesicular bodies to the plasma membrane, indicating that the population of EVs increased upon mechanical stress correspond to exosomes. We observed that Cav1 was enriched in EVs after mechanical strain. The increase of EVs observed after mechanical stress was drastically reduced upon downregulation of the endosomal sorting complex ESCRT-0, further confirming their exosomal nature. Lipidomic analysis revealed differences in the lipid composition of EVs in Cav1KO cells and after mechanical stress, suggesting a difference in the properties of these EVs. Finally, EVs from mechanically stressed cells were shown to promote a Cav1-dependent enhanced migration and invasion phenotype in triple negative breast cancer cells. These data allows us to conclude that mechanical stress is associated with increased release of EVs and the acquisition of metastatic traits in receiving cells in vitro, with Cav1 being a key player of this process. In parallel, we also studied the involvement of Cav1 in the uptake of EVs, a key process in the EV mediated cellular communication context. We used different protein markers to follow and visualize the uptake of EVs. EV uptake was strongly decreased in Cav1 KO cells as compared to WT receiving cells. We also made the observation that Cav1 but not caveolae was required for this uptake of EVs by cells and that the uptake efficiency was correlated with the level of Cav1 expression in receiving cells.. Overall, our work has revealed a new role for Cav1 in cell-to-cell communication and the propagation of metastatic phenotypes through the mechanical control of EV production and dynamics.Les vésicules extracellulaires (VE) sont des vésicules lipidiques qui sont libérées par toutes les cellules étudiées à ce jour et présentes dans tous les fluides corporels humains. Les VE contiennent du matériel génétique et des protéines qui peuvent être transférés à d'autres cellules et produire un effet sur celles-ci. La cavéoline-1 (Cav1) est un composant clé des petites invagination de la membrane plasmique appelées cavéoles, où elles fonctionnent comme mécano-capteurs et régulateur de la tension membranaire. La Cav1 facilite la migration et l'invasion des cellules tumorales et son expression est souvent augmentée dans les stades avancés du cancer. Des niveaux élevés de Cav1 ont ainsi été rapportés dans les VE de patients atteints d'un cancer avancé. Étant donné l'importance des forces mécaniques dans le micro-environnement des cellules cancéreuses, nous avons émis l'hypothèse que les cavéoles et/ou Cav1 pourraient être des acteurs clés dans la régulation de la biologie des VE et la progression du cancer sous contraintes mécaniques. Pour tester cette hypothèse, nous avons soumis différentes lignées cancéreuses exprimant (WT) ou délétées pour l’expression de Cav1 (KO) à des systèmes de stress mécanique en 2D et 3D. Les VE ont été purifiées et analysées par suivi des nanoparticules (NTA). Nous avons trouvé une augmentation conséquente de la libération des VE après un stress mécanique à la fois dans les modèles 2D et 3D. Cette augmentation était strictement dépendante de la présence de Cav1 et corrélée à une fusion accrue des corps multivésiculaires avec la membrane plasmique, indiquant que la population de VE augmentée lors d’un stress mécanique est constituée d'exosomes. Nous avons observé que Cav1 était enrichie dans les VE après une contrainte mécanique. L’augmentation des VE suite à un stress mécanique est considérablement réduite suite à la régulation négative du complexe de tri endosomal ESCRT-0, confirmant la nature exosomale de ces VE. L'analyse lipidomique a révélé des différences dans la composition lipidique des VE provenant de cellules Cav1KO et après un stress mécanique, suggèrant une différence dans les propriétés de ces VE. Enfin, il a été démontré que les EVs provenant de cellules soumises à un stress mécanique favorisent un phénotype de migration et d'invasion accru, dépendant de Cav1, dans les cellules cancéreuses. Ces résultats nous permettent de conclure que le stress mécanique est associé à la libération accrue de VE et à l'acquisition de traits métastatiques dans les cellules réceptrices in vitro, Cav1 étant un acteur clé de ce processus. En parallèle, nous avons également étudié l'implication de Cav1 dans l’endocytose des EVs, un processus clé dans le contexte de la communication cellulaire médiée par les EVs. Nous avons utilisé différents marqueurs protéiques pour suivre et visualiser la capture des VE dans les cellules réceptrices. La capture des VE était fortement diminuée dans les cellules réceptrices Cav1 KO par rapport aux cellules WT. Nous avons également fait l’observation que Cav1, et non les cavéoles, était nécessaire à la capture des VE par les cellules, et que l’efficacité de capture était corrélée au niveau d’expression de Cav1 dans les cellules réceptrices. Dans l'ensemble, nos travaux ont révélé un rôle nouveau de Cav1 dans la communication intercellulaire médiée par les VE et la propagation des phénotypes métastatiques par le contrôle mécanique de la sécrétion et la dynamique des VE

Rôle de la mécanique des cavéoles dans la dynamique des vésicules extracellulaires impliquées dans la progression tumorale

Les vésicules extracellulaires (VE) sont des vésicules lipidiques qui sont libérées par toutes les cellules étudiées à ce jour et présentes dans tous les fluides corporels humains. Les VE contiennent du matériel génétique et des protéines qui peuvent être transférés à d'autres cellules et produire un effet sur celles-ci. La cavéoline-1 (Cav1) est un composant clé des petites invagination de la membrane plasmique appelées cavéoles, où elles fonctionnent comme mécano-capteurs et régulateur de la tension membranaire. La Cav1 facilite la migration et l'invasion des cellules tumorales et son expression est souvent augmentée dans les stades avancés du cancer. Des niveaux élevés de Cav1 ont ainsi été rapportés dans les VE de patients atteints d'un cancer avancé. Étant donné l'importance des forces mécaniques dans le micro-environnement des cellules cancéreuses, nous avons émis l'hypothèse que les cavéoles et/ou Cav1 pourraient être des acteurs clés dans la régulation de la biologie des VE et la progression du cancer sous contraintes mécaniques. Pour tester cette hypothèse, nous avons soumis différentes lignées cancéreuses exprimant (WT) ou délétées pour l’expression de Cav1 (KO) à des systèmes de stress mécanique en 2D et 3D. Les VE ont été purifiées et analysées par suivi des nanoparticules (NTA). Nous avons trouvé une augmentation conséquente de la libération des VE après un stress mécanique à la fois dans les modèles 2D et 3D. Cette augmentation était strictement dépendante de la présence de Cav1 et corrélée à une fusion accrue des corps multivésiculaires avec la membrane plasmique, indiquant que la population de VE augmentée lors d’un stress mécanique est constituée d'exosomes. Nous avons observé que Cav1 était enrichie dans les VE après une contrainte mécanique. L’augmentation des VE suite à un stress mécanique est considérablement réduite suite à la régulation négative du complexe de tri endosomal ESCRT-0, confirmant la nature exosomale de ces VE. L'analyse lipidomique a révélé des différences dans la composition lipidique des VE provenant de cellules Cav1KO et après un stress mécanique, suggèrant une différence dans les propriétés de ces VE. Enfin, il a été démontré que les EVs provenant de cellules soumises à un stress mécanique favorisent un phénotype de migration et d'invasion accru, dépendant de Cav1, dans les cellules cancéreuses. Ces résultats nous permettent de conclure que le stress mécanique est associé à la libération accrue de VE et à l'acquisition de traits métastatiques dans les cellules réceptrices in vitro, Cav1 étant un acteur clé de ce processus. En parallèle, nous avons également étudié l'implication de Cav1 dans l’endocytose des EVs, un processus clé dans le contexte de la communication cellulaire médiée par les EVs. Nous avons utilisé différents marqueurs protéiques pour suivre et visualiser la capture des VE dans les cellules réceptrices. La capture des VE était fortement diminuée dans les cellules réceptrices Cav1 KO par rapport aux cellules WT. Nous avons également fait l’observation que Cav1, et non les cavéoles, était nécessaire à la capture des VE par les cellules, et que l’efficacité de capture était corrélée au niveau d’expression de Cav1 dans les cellules réceptrices. Dans l'ensemble, nos travaux ont révélé un rôle nouveau de Cav1 dans la communication intercellulaire médiée par les VE et la propagation des phénotypes métastatiques par le contrôle mécanique de la sécrétion et la dynamique des VE.Extracellular vesicles (EVs) are lipid-enclosed vesicles that are released by all cells studied to date and present in all human bodily fluids. EVs contain genetic material and proteins that are able to be transferred to and generate an effect in other cells. Caveolin-1 (Cav1) is a key component of the small invagination of the plasma membrane called caveolae, where it functions as mechano-sensors and membrane tension buffering device. Cav1 facilitates the migration and invasion of tumor cells and its expression is often increased in the late stages of cancer. Interestingly, high levels of Cav1 have been found in EVs of patients with advanced cancer. Given the importance of mechanical forces in the microenvironment of cancer cells, we hypothesized that caveolae and/or Cav1 may represent key players in the regulation of EV biology and cancer progression under mechanical strain. To test this hypothesis, we subjected different cancer cell lines, having (WT) or deleted for Cav1 expression (KO) to 2D or 3D systems of mechanical stress. EVs were purified from these cells and analyzed by nanoparticle tracking analysis. We found a striking increase in the release of EVs after mechanical stress both in 2D and 3D models. This increase was strictly dependent on the presence of Cav1 and correlated with enhanced fusion of multivesicular bodies to the plasma membrane, indicating that the population of EVs increased upon mechanical stress correspond to exosomes. We observed that Cav1 was enriched in EVs after mechanical strain. The increase of EVs observed after mechanical stress was drastically reduced upon downregulation of the endosomal sorting complex ESCRT-0, further confirming their exosomal nature. Lipidomic analysis revealed differences in the lipid composition of EVs in Cav1KO cells and after mechanical stress, suggesting a difference in the properties of these EVs. Finally, EVs from mechanically stressed cells were shown to promote a Cav1-dependent enhanced migration and invasion phenotype in triple negative breast cancer cells. These data allows us to conclude that mechanical stress is associated with increased release of EVs and the acquisition of metastatic traits in receiving cells in vitro, with Cav1 being a key player of this process. In parallel, we also studied the involvement of Cav1 in the uptake of EVs, a key process in the EV mediated cellular communication context. We used different protein markers to follow and visualize the uptake of EVs. EV uptake was strongly decreased in Cav1 KO cells as compared to WT receiving cells. We also made the observation that Cav1 but not caveolae was required for this uptake of EVs by cells and that the uptake efficiency was correlated with the level of Cav1 expression in receiving cells.. Overall, our work has revealed a new role for Cav1 in cell-to-cell communication and the propagation of metastatic phenotypes through the mechanical control of EV production and dynamics

Axonal degeneration is mediated by necroptosis activation Necroptosis mediates axonal degeneration

Axonal degeneration contributes to functional impairment in several disorders of the nervous system, constituting an important target for neuroprotection. Several individual factors and subcellular events have been implicated in axonal degeneration, but the identification of an integrative signaling pathway activating this self-destructive process has remained elusive. Through pharmacological and genetic approaches, we tested whether necroptosis, a regulated cell death mechanism, implicated in the pathogenesis of several neurodegenerative diseases, is involved in axonal degeneration. Pharmacological inhibition of the necroptotic kinase RIPK1 using necrostatin-1 strongly delayed axonal degeneration in the peripheral and central nervous system of wild-type mice of either sex and protected in vitro sensory axons from degeneration after mechanical and toxic insults. These effects were also observed after genetic knock down of RIPK3, a second key regulator of necroptosis, and the downstream effector, MLKL RIPK1 inhibition prevented mitochondrial fragmentation in vitro and in vivo, a typical feature of necrotic death, and inhibition of mitochondrial fission by Mdivi also resulted in reduced axonal loss in damaged nerves. Furthermore, electrophysiological analysis demonstrated that inhibition of necroptosis delays not only the morphological degeneration of axons but also the loss of their electrophysiological function after nerve injury. Activation of the necroptotic pathway early during injury-induced axonal degeneration was evidenced by increased phosphorylation of the downstream effector MLKL. Our results demonstrate that axonal degeneration proceeds by necroptosis, defining a novel mechanistic framework in the axonal degenerative cascade for therapeutic interventions in a wide variety of conditions that lead to neuronal loss and functional impairment.SIGNIFICANCE STATEMENTWe show that axonal degeneration triggered by diverse stimuli is mediated by the activation of the necroptotic programmed cell death program by a cell-autonomous mechanism. We believe that this work represents a critical advance for the field since it identifies a defined degenerative pathway involved in axonal degeneration in both PNS and CNS, a process that has been proposed as an early event in several neurodegenerative conditions and a major contributor of neuronal death. The identification of necroptosis as a key mechanism for axonal degeneration, is an important step to develop novel therapeutic strategies for nervous system disorders, particularly those related to chemotherapy-induced peripheral neuropathies or CNS diseases in which axonal degeneration is a common factor

The necroptosis machinery mediates axonal degeneration in a model of Parkinson disease

Geroscience Center for Brain Health and Metabolis

The necroptosis machinery mediates axonal degeneration in a model of Parkinson disease.

Parkinson's disease (PD) is the second most common neurodegenerative condition, characterized by motor impairment due to the progressive degeneration of dopaminergic neurons in the substantia nigra and depletion of dopamine release in the striatum. Accumulating evidence suggest that degeneration of axons is an early event in the disease, involving destruction programs that are independent of the survival of the cell soma. Necroptosis, a programmed cell death process, is emerging as a mediator of neuronal loss in models of neurodegenerative diseases. Here, we demonstrate activation of necroptosis in postmortem brain tissue from PD patients and in a toxin-based mouse model of the disease. Inhibition of key components of the necroptotic pathway resulted in a significant delay of 6-hydroxydopamine-dependent axonal degeneration of dopaminergic and cortical neurons in vitro. Genetic ablation of necroptosis mediators MLKL and RIPK3, as well as pharmacological inhibition of RIPK1 in preclinical models of PD, decreased dopaminergic neuron degeneration, improving motor performance. Together, these findings suggest that axonal degeneration in PD is mediated by the necroptosis machinery, a process here referred to as necroaxoptosis, a druggable pathway to target dopaminergic neuronal loss

Table_1_Neuronal activity-dependent ATP enhances the pro-growth effect of repair Schwann cell extracellular vesicles by increasing their miRNA-21 loading.xlsx

Functional recovery after peripheral nerve injuries is critically dependent on axonal regeneration. Several autonomous and non-cell autonomous processes regulate axonal regeneration, including the activation of a growth-associated transcriptional program in neurons and the reprogramming of differentiated Schwann cells (dSCs) into repair SCs (rSCs), triggering the secretion of neurotrophic factors and the activation of an inflammatory response. Repair Schwann cells also release pro-regenerative extracellular vesicles (EVs), but is still unknown whether EV secretion is regulated non-cell autonomously by the regenerating neuron. Interestingly, it has been described that nerve activity enhances axonal regeneration by increasing the secretion of neurotrophic factors by rSC, but whether this activity modulates pro-regenerative EV secretion by rSC has not yet been explored. Here, we demonstrate that neuronal activity enhances the release of rSC-derived EVs and their transfer to neurons. This effect is mediated by activation of P2Y receptors in SCs after activity-dependent ATP release from sensory neurons. Importantly, activation of P2Y in rSCs also increases the amount of miRNA-21 present in rSC-EVs. Taken together, our results demonstrate that neuron to glia communication by ATP-P2Y signaling regulates the content of SC-derived EVs and their transfer to axons, modulating axonal elongation in a non-cell autonomous manner.</p

Image_3_Neuronal activity-dependent ATP enhances the pro-growth effect of repair Schwann cell extracellular vesicles by increasing their miRNA-21 loading.tiff

Functional recovery after peripheral nerve injuries is critically dependent on axonal regeneration. Several autonomous and non-cell autonomous processes regulate axonal regeneration, including the activation of a growth-associated transcriptional program in neurons and the reprogramming of differentiated Schwann cells (dSCs) into repair SCs (rSCs), triggering the secretion of neurotrophic factors and the activation of an inflammatory response. Repair Schwann cells also release pro-regenerative extracellular vesicles (EVs), but is still unknown whether EV secretion is regulated non-cell autonomously by the regenerating neuron. Interestingly, it has been described that nerve activity enhances axonal regeneration by increasing the secretion of neurotrophic factors by rSC, but whether this activity modulates pro-regenerative EV secretion by rSC has not yet been explored. Here, we demonstrate that neuronal activity enhances the release of rSC-derived EVs and their transfer to neurons. This effect is mediated by activation of P2Y receptors in SCs after activity-dependent ATP release from sensory neurons. Importantly, activation of P2Y in rSCs also increases the amount of miRNA-21 present in rSC-EVs. Taken together, our results demonstrate that neuron to glia communication by ATP-P2Y signaling regulates the content of SC-derived EVs and their transfer to axons, modulating axonal elongation in a non-cell autonomous manner.</p

Image_1_Neuronal activity-dependent ATP enhances the pro-growth effect of repair Schwann cell extracellular vesicles by increasing their miRNA-21 loading.tiff

Functional recovery after peripheral nerve injuries is critically dependent on axonal regeneration. Several autonomous and non-cell autonomous processes regulate axonal regeneration, including the activation of a growth-associated transcriptional program in neurons and the reprogramming of differentiated Schwann cells (dSCs) into repair SCs (rSCs), triggering the secretion of neurotrophic factors and the activation of an inflammatory response. Repair Schwann cells also release pro-regenerative extracellular vesicles (EVs), but is still unknown whether EV secretion is regulated non-cell autonomously by the regenerating neuron. Interestingly, it has been described that nerve activity enhances axonal regeneration by increasing the secretion of neurotrophic factors by rSC, but whether this activity modulates pro-regenerative EV secretion by rSC has not yet been explored. Here, we demonstrate that neuronal activity enhances the release of rSC-derived EVs and their transfer to neurons. This effect is mediated by activation of P2Y receptors in SCs after activity-dependent ATP release from sensory neurons. Importantly, activation of P2Y in rSCs also increases the amount of miRNA-21 present in rSC-EVs. Taken together, our results demonstrate that neuron to glia communication by ATP-P2Y signaling regulates the content of SC-derived EVs and their transfer to axons, modulating axonal elongation in a non-cell autonomous manner.</p