Antioxidants and PPARγ agonists protect the mitochondria of hippocampal neurons from neurodegenerative processes

Las mitocondrias y su función metabólica cambian con el envejecimiento y pueden ser el factor más importante en el desarrollo de diferentes enfermedades neurodegenerativas relacionadas con la edad, incluida la enfermedad de Alzheimer (EA). En esta condición patológica, las mitocondrias muestran un potencial de membrana reducido, aumento de la permeabilidad, dishomeóstasis del calcio con una producción excesiva de especies reactivas de oxigeno (ROS), pudiendo producir un daño importante en las proteínas, lípidos y ácidos nucleicos de la células. En la EA, la sobreproducción de la proteína precursora del amiloide (APP) y del péptido β-amiloide (Aβ) por el procesamiento proteolítico de la APP, junto a los ovillos neurofibrilares producto de la hiperfosforilación de la proteína Tau, pueden afectar el equilibrio dinámico mitocondrial (fusión/fisión), aumentando la fisión. En particular, el Aβ es capaz de interactuar con las membranas y proteínas mitocondriales, contribuyendo a la fisiopatología de la neurodegeneración. En esta tesis doctoral nos hemos centrado en tratamientos dirigidos a mejorar el metabolismo de las mitocondrias con estrategias como es el uso de antioxidantes que regulen la producción de ROS en las neuronas, pero que también contribuyan a la biogénesis mitocondrial. El uso de la quercetina un antioxidante presente normalmente en los frutos rojos y plantas del género Allium, con bajo nivel de actividad antioxidante se ha visualizado como una terapia eficaz, porque es capaz de proteger a la neuronas del daño producido tanto por el estrés oxidativo como por el péptido Aβ, pero además puede activar vías de señalización relacionadas con la defensa celular antioxidante, como es la activación del factor de transcripcion erytheroid-derived- 2-like 2 (Nrf2), resultando en el aumento de los niveles de catalasa y peroxidasa en la célula. Por otra parte, agonistas de los receptores de activadores de proliferación peroxisomal del tipo gamma (RAPPgamma), usados en la actualidad en el tratamiento de la diabetes tipo 2 han demostrado también una actividad anti-inflamatoria. Estas moléculas pueden ser utilizados para inducir la biogénesis mitocondrial, reestablecer la homeostasis del calcio y proteger a las neuronas del daño oxidativo. Es en este sentido que la búsqueda de moléculas que ayuden a restablecer la homeostasis celular y proteger del daño oxidativo a las neuronas son importantes para el tratamiento y/o prevención de las enfermedades neurodegenerativas.Mitochondria and their metabolic function change with aging and these events may be the most important factor in the development of different neurodegenerative diseases related to age, including Alzheimer's disease (AD). In this pathological condition, the mitochondria show a reduced membrane potential, increased permeability, dishomeostasis of calcium with an excessive production of reactive oxygen species (ROS), and may cause significant damage to the proteins, lipids and nucleic acids of the cells. In AD, the overproduction of amyloid precursor protein (APP) and β-amyloid peptide (Aβ) by the proteolytic processing of APP, together with the neurofibrillary tangles resulting from the hyperphosphorylation of the Tau protein, can affect the mitochondrial dynamic equilibrium (fusion/fission), increasing fission. In particular, Aβ is able to interact with mitochondrial membranes and proteins, contributing to the pathophysiology of neurodegeneration. In this doctoral thesis, we have focused on treatments aimed at improving the metabolism of mitochondria by strategies such as the use of antioxidants that regulate ROS production in neurons, but also contribute to mitochondrial biogenesis. The use of quercetin, an antioxidant normally present in red fruits and plants of the genus Allium, with a low level of antioxidant activity has been seen as an effective therapy, because it is able to protect the neurons from the damage caused by both oxidative stress and it can also activate signaling pathways related to antioxidant cellular defense, such as the activation of the transcription factor erytheroid-derived- 2-like 2 (Nrf2), resulting in increased levels of catalase and peroxidase in the cell. On the other hand, agonists of peroxisomal proliferation activator receptors of the gamma type (PPARgamma), currently used in the treatment of type 2 diabetes have also demonstrated an anti-inflammatory activity. These molecules can be used to induce mitochondrial biogenesis, reestablish calcium homeostasis and protect neurons from oxidative damage. It is in this sense that the search for molecules that help to restore cellular homeostasis and protect from oxidative damage to neurons are important for the treatment and/or prevention of neurodegenerative diseases

Antioxidants and PPARγ agonists protect the mitochondria of hippocampal neurons from neurodegenerative processes

Las mitocondrias y su función metabólica cambian con el envejecimiento y pueden ser el factor más importante en el desarrollo de diferentes enfermedades neurodegenerativas relacionadas con la edad, incluida la enfermedad de Alzheimer (EA). En esta condición patológica, las mitocondrias muestran un potencial de membrana reducido, aumento de la permeabilidad, dishomeóstasis del calcio con una producción excesiva de especies reactivas de oxigeno (ROS), pudiendo producir un daño importante en las proteínas, lípidos y ácidos nucleicos de la células. En la EA, la sobreproducción de la proteína precursora del amiloide (APP) y del péptido β-amiloide (Aβ) por el procesamiento proteolítico de la APP, junto a los ovillos neurofibrilares producto de la hiperfosforilación de la proteína Tau, pueden afectar el equilibrio dinámico mitocondrial (fusión/fisión), aumentando la fisión. En particular, el Aβ es capaz de interactuar con las membranas y proteínas mitocondriales, contribuyendo a la fisiopatología de la neurodegeneración. En esta tesis doctoral nos hemos centrado en tratamientos dirigidos a mejorar el metabolismo de las mitocondrias con estrategias como es el uso de antioxidantes que regulen la producción de ROS en las neuronas, pero que también contribuyan a la biogénesis mitocondrial. El uso de la quercetina un antioxidante presente normalmente en los frutos rojos y plantas del género Allium, con bajo nivel de actividad antioxidante se ha visualizado como una terapia eficaz, porque es capaz de proteger a la neuronas del daño producido tanto por el estrés oxidativo como por el péptido Aβ, pero además puede activar vías de señalización relacionadas con la defensa celular antioxidante, como es la activación del factor de transcripcion erytheroid-derived- 2-like 2 (Nrf2), resultando en el aumento de los niveles de catalasa y peroxidasa en la célula. Por otra parte, agonistas de los receptores de activadores de proliferación peroxisomal del tipo gamma (RAPPgamma), usados en la actualidad en el tratamiento de la diabetes tipo 2 han demostrado también una actividad anti-inflamatoria. Estas moléculas pueden ser utilizados para inducir la biogénesis mitocondrial, reestablecer la homeostasis del calcio y proteger a las neuronas del daño oxidativo. Es en este sentido que la búsqueda de moléculas que ayuden a restablecer la homeostasis celular y proteger del daño oxidativo a las neuronas son importantes para el tratamiento y/o prevención de las enfermedades neurodegenerativas.Mitochondria and their metabolic function change with aging and these events may be the most important factor in the development of different neurodegenerative diseases related to age, including Alzheimer's disease (AD). In this pathological condition, the mitochondria show a reduced membrane potential, increased permeability, dishomeostasis of calcium with an excessive production of reactive oxygen species (ROS), and may cause significant damage to the proteins, lipids and nucleic acids of the cells. In AD, the overproduction of amyloid precursor protein (APP) and β-amyloid peptide (Aβ) by the proteolytic processing of APP, together with the neurofibrillary tangles resulting from the hyperphosphorylation of the Tau protein, can affect the mitochondrial dynamic equilibrium (fusion/fission), increasing fission. In particular, Aβ is able to interact with mitochondrial membranes and proteins, contributing to the pathophysiology of neurodegeneration. In this doctoral thesis, we have focused on treatments aimed at improving the metabolism of mitochondria by strategies such as the use of antioxidants that regulate ROS production in neurons, but also contribute to mitochondrial biogenesis. The use of quercetin, an antioxidant normally present in red fruits and plants of the genus Allium, with a low level of antioxidant activity has been seen as an effective therapy, because it is able to protect the neurons from the damage caused by both oxidative stress and it can also activate signaling pathways related to antioxidant cellular defense, such as the activation of the transcription factor erytheroid-derived- 2-like 2 (Nrf2), resulting in increased levels of catalase and peroxidase in the cell. On the other hand, agonists of peroxisomal proliferation activator receptors of the gamma type (PPARgamma), currently used in the treatment of type 2 diabetes have also demonstrated an anti-inflammatory activity. These molecules can be used to induce mitochondrial biogenesis, reestablish calcium homeostasis and protect neurons from oxidative damage. It is in this sense that the search for molecules that help to restore cellular homeostasis and protect from oxidative damage to neurons are important for the treatment and/or prevention of neurodegenerative diseases

Wnt5a inhibits K(+) currents in hippocampal synapses through nitric oxide production

Hippocampal synapses play a key role in memory and learning processes by inducing long-term potentiation and depression. Wnt signaling is essential in the development and maintenance of synapses via several mechanisms. We have previously found that Wnt5a induces the production of nitric oxide (NO), which modulates NMDA receptor expression in the postsynaptic regions of hippocampal neurons. Here, we report that Wnt5a selectively inhibits a voltage-gated K(+) current (Kv current) and increases synaptic activity in hippocampal slices. Further supporting a specific role for Wnt5a, the soluble Frizzled receptor protein (sFRP-2; a functional Wnt antagonist) fully inhibits the effects of Wnt5a. We additionally show that these responses to Wnt5a are mediated by activation of a ROR2 receptor and increased NO production because they are suppressed by the shRNA-mediated knockdown of ROR2 and by 7-nitroindazole, a specific inhibitor of neuronal NOS. Together, our results show that Wnt5a increases NO production by acting on ROR2 receptors, which in turn inhibit Kv currents. These results reveal a novel mechanism by which Wnt5a may regulate the excitability of hippocampal neurons.This work was supported by grants from Fondecyt no. 1120156 and from the Basal Centre for Excellence in Science and Technology (Conicyt-PFB 12/2007) to N.C.I; Fondecyt no. 11121206 to WC; the Plan Estatal de I + D + i 2013–2016 and ISCIII-Subdirección General de Evaluación y Fomento de la Investigación (Grant PI13/00408) and FEDER to F.J.M.; grants from the Fundación Ciencia y Vida (CONICYT PFB16/ 2007) and FONDECYT no. 1131137 to I.E.A. and M.V-G

Wnt5a inhibits K(+) currents in hippocampal synapses through nitric oxide production

Hippocampal synapses play a key role in memory and learning processes by inducing long-term potentiation and depression. Wnt signaling is essential in the development and maintenance of synapses via several mechanisms. We have previously found that Wnt5a induces the production of nitric oxide (NO), which modulates NMDA receptor expression in the postsynaptic regions of hippocampal neurons. Here, we report that Wnt5a selectively inhibits a voltage-gated K(+) current (Kv current) and increases synaptic activity in hippocampal slices. Further supporting a specific role for Wnt5a, the soluble Frizzled receptor protein (sFRP-2; a functional Wnt antagonist) fully inhibits the effects of Wnt5a. We additionally show that these responses to Wnt5a are mediated by activation of a ROR2 receptor and increased NO production because they are suppressed by the shRNA-mediated knockdown of ROR2 and by 7-nitroindazole, a specific inhibitor of neuronal NOS. Together, our results show that Wnt5a increases NO production by acting on ROR2 receptors, which in turn inhibit Kv currents. These results reveal a novel mechanism by which Wnt5a may regulate the excitability of hippocampal neurons.This work was supported by grants from Fondecyt no. 1120156 and from the Basal Centre for Excellence in Science and Technology (Conicyt-PFB 12/2007) to N.C.I; Fondecyt no. 11121206 to WC; the Plan Estatal de I + D + i 2013–2016 and ISCIII-Subdirección General de Evaluación y Fomento de la Investigación (Grant PI13/00408) and FEDER to F.J.M.; grants from the Fundación Ciencia y Vida (CONICYT PFB16/ 2007) and FONDECYT no. 1131137 to I.E.A. and M.V-G

Mitostasis, calcium and free radicals in health, aging and neurodegeneration

Mitochondria play key roles in ATP supply, calcium homeostasis, redox balance control and apoptosis, which in neurons are fundamental for neurotransmission and to allow synaptic plasticity. Their functional integrity is maintained by mitostasis, a process that involves mitochondrial transport, anchoring, fusion and fission processes regulated by different signaling pathways but mainly by the peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α). PGC-1α also favors Ca2+ homeostasis, reduces oxidative stress, modulates inflammatory processes and mobilizes mitochondria to where they are needed. To achieve their functions, mitochondria are tightly connected to the endoplasmic reticulum (ER) through specialized structures of the ER termed mitochondria-associated membranes (MAMs), which facilitate the communication between these two organelles mainly to aim Ca2+ buffering. Alterations in mitochondrial activity enhance reactive oxygen species (ROS) production, disturbing the physiological metabolism and causing cell damage. Furthermore, cytosolic Ca2+ overload results in an increase in mitochondrial Ca2+, resulting in mitochondrial dysfunction and the induction of mitochondrial permeability transition pore (mPTP) opening, leading to mitochondrial swelling and cell death through apoptosis as demonstrated in several neuropathologies. In summary, mitochondrial homeostasis is critical to maintain neuronal function; in fact, their regulation aims to improve neuronal viability and to protect against aging and neurodegenerative diseases.This work was supported by the Spanish Ministry of Science and Innovation and FEDER Funds through grants SAF2017-83372-R (FJM); RTI2018-094809-B-I00 (JMFF); PID2019-106755RB-I00 (RV); and through the “María de Maeztu” Programme for Units of Excellence in R&D (award CEX2018-000792-M)

Amyloid-β peptide nitrotyrosination stabilizes oligomers and enhances NMDAR-mediated toxicity

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the pathological aggregation of the amyloid-β peptide (Aβ). Monomeric soluble Aβ can switch from helicoidal to β-sheet conformation, promoting its assembly into oligomers and subsequently to amyloid fibrils. Oligomers are highly toxic to neurons and have been reported to induce synaptic transmission impairments. The progression from oligomers to fibrils forming senile plaques is currently considered a protective mechanism to avoid the presence of the highly toxic oligomers. Protein nitration is a frequent post-translational modification under AD nitrative stress conditions. Aβ can be nitrated at tyrosine 10 (Y10) by peroxynitrite. Based on our analysis of ThT binding, Western blot and electron and atomic force microscopy, we report that Aβ nitration stabilizes soluble, highly toxic oligomers and impairs the formation of fibrils. We propose a mechanism by which fibril elongation is interrupted upon Y10 nitration: Nitration disrupts fibril-forming folds by preventing H14-mediated bridging, as shown with an Aβ analog containing a single residue (H to E) replacement that mimics the behavior of nitrated Aβ related to fibril formation and neuronal toxicity. The pathophysiological role of our findings in AD was highlighted by the study of these nitrated oligomers on mouse hippocampal neurons, where an increased NMDAR-dependent toxicity of nitrated Aβ oligomers was observed. Our results show that Aβ nitrotyrosination is a post-translational modification that increases Aβ synaptotoxicity. SIGNIFICANCE STATEMENT: We report that nitration (i.e., the irreversible addition of a nitro group) of the Alzheimer-related peptide amyloid-β (Aβ) favors the stabilization of highly toxic oligomers and inhibits the formation of Aβ fibrils. The nitrated Aβ oligomers are more toxic to neurons due to increased cytosolic calcium levels throughout their action on NMDA receptors. Sustained elevated calcium levels trigger excitotoxicity, a characteristic event in Alzheimer's disease.This work was supported by Spanish Plan Estatal de I+D+I 2013-2016 and the ISCIII-Subdirección General de Evaluación y Fomento de la Investigación Grants PI13/00408 and Red HERACLES RD12/0042/0014 and FEDER Funds, Spanish Ministerio de Economía y Competitividad BIO2014-57518-R, BIO2014-52872-R, and AGL2014-52395-C2-2-R and FEDER Funds, the Generalitat de Catalunya (Spain) 2014-SGR-938, and Chilean Funds for Science CONICYTPFB 12/2007 and FONDECYT N° 1160724. A.P.-M. was the recipient of the Universitat Autònoma de Barcelona-Programa Banco de Santander Fellowshi

Amyloid-β peptide nitrotyrosination stabilizes oligomers and enhances NMDAR-mediated toxicity

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the pathological aggregation of the amyloid-β peptide (Aβ). Monomeric soluble Aβ can switch from helicoidal to β-sheet conformation, promoting its assembly into oligomers and subsequently to amyloid fibrils. Oligomers are highly toxic to neurons and have been reported to induce synaptic transmission impairments. The progression from oligomers to fibrils forming senile plaques is currently considered a protective mechanism to avoid the presence of the highly toxic oligomers. Protein nitration is a frequent post-translational modification under AD nitrative stress conditions. Aβ can be nitrated at tyrosine 10 (Y10) by peroxynitrite. Based on our analysis of ThT binding, Western blot and electron and atomic force microscopy, we report that Aβ nitration stabilizes soluble, highly toxic oligomers and impairs the formation of fibrils. We propose a mechanism by which fibril elongation is interrupted upon Y10 nitration: Nitration disrupts fibril-forming folds by preventing H14-mediated bridging, as shown with an Aβ analog containing a single residue (H to E) replacement that mimics the behavior of nitrated Aβ related to fibril formation and neuronal toxicity. The pathophysiological role of our findings in AD was highlighted by the study of these nitrated oligomers on mouse hippocampal neurons, where an increased NMDAR-dependent toxicity of nitrated Aβ oligomers was observed. Our results show that Aβ nitrotyrosination is a post-translational modification that increases Aβ synaptotoxicity. SIGNIFICANCE STATEMENT: We report that nitration (i.e., the irreversible addition of a nitro group) of the Alzheimer-related peptide amyloid-β (Aβ) favors the stabilization of highly toxic oligomers and inhibits the formation of Aβ fibrils. The nitrated Aβ oligomers are more toxic to neurons due to increased cytosolic calcium levels throughout their action on NMDA receptors. Sustained elevated calcium levels trigger excitotoxicity, a characteristic event in Alzheimer's disease.This work was supported by Spanish Plan Estatal de I+D+I 2013-2016 and the ISCIII-Subdirección General de Evaluación y Fomento de la Investigación Grants PI13/00408 and Red HERACLES RD12/0042/0014 and FEDER Funds, Spanish Ministerio de Economía y Competitividad BIO2014-57518-R, BIO2014-52872-R, and AGL2014-52395-C2-2-R and FEDER Funds, the Generalitat de Catalunya (Spain) 2014-SGR-938, and Chilean Funds for Science CONICYTPFB 12/2007 and FONDECYT N° 1160724. A.P.-M. was the recipient of the Universitat Autònoma de Barcelona-Programa Banco de Santander Fellowshi