Creatine protects against rotenone induced cell death of cerebellar granule neurons

Tese de mestrado, Ciências Biofarmacêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2012Parkinson´s Disease (PD) is the second most prevalent neurodegenerative brain disorder worldwide. Nevertheless, there is lack of certainty on the pathophysiology of the neurodegenerative mechanisms underlying PD. Several neurotoxins, rotenone among them, have been shown to induce parkinsonism-like brain degeneration and are widely used in cellular and animal models of PD. In spite of an extensive association of PD with dopaminergic neuron degeneration of the substantia nigra pars compacta, other brain areas like the cerebellum have been more recently implicated in the pathology of the disease. Therefore, we used a rotenone/cerebellar granule neurons (CGN) model to study not only the potential of creatine (as ergogenic compound), epicatechin and kaempferol (as antioxidant compounds) to afford neuroprotection against rotenone neurotoxicity but also to better understand the cellular mechanisms underlying this neurotoxicity. Our results revealed a strong protection by creatine against rotenone-induced CGN death, while kaempferol did not afford a significant protection and epicatechin elicited at most a very weak protection. On the contrary, kaempferol also antagonized the protective effect of creatine. These results lend support to the potential use of creatine in PD therapeutics, and alert for the avoidance of the consumption of foods or infusions with high content in kaempferol. Furthermore, we noted that rotenone triggered an energetic failure in CGN as a primary event, supported not only by the protection afforded by creatine but also by a deregulation in calcium homeostasis thought voltage operate calcium channels type L and N-Methyl-D-aspartate receptors stimulation and store-operated calcim entry inhibition, by the elevation of AMP-kinase active levels and by mitochondrial membrane depolarization. Rotenone also promoted an enhanced production of reactive oxygen species and a weak nitrosative stress, but only as a later event in the development of CGN death. In conclusion, our results support a role for creatine in affording neuroprotection against rotenone neurotoxicity, through a mechanim that prevent an energetic failure.A doença de Parkinson é a segunda doença neurodegenerativa mais prevalente a nível mundial. Apesar disso, são ainda pouco conhecidos os mecanismos neurodegenerativos que estão por detrás desta doença. Varias neurotoxinas, nas quais se inclui a rotenona, têm vindo a ser demonstradas como indutoras de degenerescência cerebral de tipo-Parkinsonismo e são, por isso, vastamente utilizadas em modelos celulares e animais da doença de Parkinson. Apesar da extensiva associação entra a doença de Parkinson e a degenerescência dos neurónios dopaminérgicos da substantia nigra pars compacta, mais recentemente, outras áreas cerebrais, nomeadamente o cerebelo, têm sido implicadas na patogénese da doença. Portanto, neste trabalho, foi utilizado um modelo celular de neurónios granulares do cerebelo expostos à neurotoxina rotenona. Este modelo foi utilizado, não só para estudar a capacidade da creatina (como composto ergogénico) e de epicatequina e kaempferol (como antioxidantes) em proteger contra a neurotoxicidade despoletada pela rotenona, mas também para melhor entender os mecanismos subjacentes a esta neurotoxicidade. Os resultados obtidos revelaram que a creatina apresenta uma elevada protecção contra a morte celular, induzida pela rotenona nos neurónios granulares do cerebelo, enquanto o kaempferol não ofereceu qualquer protecção e a epicatequina, por sua vez, promoveu uma protecção demasiado fraca. Pelo contrário, o kaempferol demostrou um efeito antagónico relativamente à creatina. Os resultados suportam, assim, o potencial uso da creatina na terapêutica da doença de Parkinson e alertam para que se evite o consumo de comidas e infusões com um elevado conteúdo em kaempferol nesta mesma terapêutica. Para além disso, os nossos resultados revelaram que a rotenona como evento primário conduz a uma falência energética nos neurónios granulares do cerebelo. Esta conclusão é suportada, não só pela protecção exercida pela creatina, mas também pela observação de uma desregulação nos níveis citosólicos de cálcio através de uma estimulação dos canais de cálcio do tipo L operados por voltagem e dos receptores N-Metil-D-aspartato e pela inibição da entrada capacitativa de cálcio; pelo aumento dos níveis da AMP-quinase activa e ainda pela despolarização da membrana mitocondrial. A rotenona promoveu, por fim, a produção de espécies reactivas de oxigénio e um stresse nitrosativo fraco, no decurso da morte dos neurónios granulares do cerebelo. Em suma, os resultados suportam a utilização da creatina como composto neuroprotector contra a neurotoxicidade exercida pela rotenona, através da prevenção da ocorrência de uma falência energética.Funded by ERASMUS fellowship (29206-IC-1-2007-1-PT-ERASMUS-EUCX-1, registo nº 38/SMP/2011) and grant GR10092 of Junta de Extremadura with FEDER cofinanciatitio

Methyl–cyclodextrin impairs the phosphorylation of the 2 subunit of L-type calcium channels and cytosolic calcium homeostasis in mature cerebellar granule neurons

La activación de los canales de calcio de tipo L (LTCC) impide que las neuronas de gránulos cerebelosos (CGN) entren en la apoptosis inducida por bajo K+. En trabajos anteriores, demostramos que los LTCC están en gran medida asociados con balsas de lípidos ricos en caveolina-1 en la membrana plasmática de las CGN. En este trabajo, mostramos que la proteína cinasa A (PKA) y la proteína cinasa dependiente de calmodulina II (CaMK-II) están asociadas con las balsas de lípidos ricos en caveolina-1 de los CGN maduros, y mostramos además que el tratamiento con el agente atrapador de colesterol y disruptor de balsa de lípidos metil- β -ciclodextrina disminuye el nivel de fosforilación de la subunidad β2 del CCLT y la concentración de calcio en estado estable en los somas neuronales ([Ca2+]i) a valores cercanos a los medidos en condiciones proapoptóticas de 5 mM KCl. Estos efectos se correlacionan con los efectos producidos por un tratamiento corto (15 min) de CGN con inhibidores H-89 y KN-93 de PKA y CaMK-II, respectivamente, en un medio de 25 mM KCl. Además, sólo una incubación de 15 min de CGN con H-89 produce alrededor de un 90% de inhibición de la entrada de calcio que normalmente ocurriría a través de los LTCC para aumentar [Ca2+]i al elevar el K+ extracelular de 5 a 25 mM, es decir, de condiciones proapoptóticas a condiciones de supervivencia. En conclusión, los resultados de este trabajo sugieren que las balsas de lípidos ricos en caveolina-1 desempeñan un papel importante en el control del nivel de fosforilación inducida por la PKA y el CaMK-II de la subunidad β2 del CCLT, evitando así que los CGN entren en apoptosis.The activation of L-type calcium channels (LTCCs) prevents cerebellar granule neurons (CGNs) from entering low-K+-induced apoptosis. In previous works, we showed that LTCCs are largely associated with caveolin-1-rich lipid rafts in the CGN plasma membrane. In this work, we show that protein kinase A (PKA) and calmodulin-dependent protein kinase II (CaMK-II) are associated with caveolin-1-rich lipid rafts of mature CGNs, and we further show that treatment with the cholesterol-trapping and lipid raft-disrupting agent methyl- β -cyclodextrin decreases the phosphorylation level of the LTCC β2 subunit and the steady-state calcium concentration in neuronal somas ([Ca2+]i) to values close to those measured in 5 mM KCl proapoptotic conditions. These effects correlate with the effects produced by a short (15 min) treatment of CGNs with H-89 and KN-93—inhibitors of PKA and CaMK-II, respectively—in 25 mM KCl medium. Moreover, only a 15 min incubation of CGNs with H-89 produces about a 90% inhibition of the calcium entry that would normally occur through LTCCs to increase [Ca2+]i upon raising the extracellular K+ from 5 to 25 mM, i.e., from proapoptotic to survival conditions. In conclusion, the results of this work suggest that caveolin-1-rich lipid rafts play a major role in the control of the PKA- and CaMK-II-induced phosphorylation level of the LTCC β2 subunit, thus preventing CGNs from entering apoptosis.• Ministerio de Economía, y Competitividad Fondos FEDER. Beca BFU2014-53641-P (I+D+i) • Junta de Junta de Extremadura y Fondos FEDER. Ayuda BBB008 (Plan Regional de I+D+I) • Fundação para a Ciência e Tecnologia (Portugal). Beca predoctoral SFRH/BD/84543/2012, para Sofia Isabel Almeida FortalezaspeerReviewe

Cytochrome c stimulates the superoxide anion production by cytochrome b5 reductase in neuronal synaptic plasma membrane vesicles

info:eu-repo/grantAgreement/FCT/5876/147258/PT Work financed by Grants FCT/MEC (UID/Multi/04378/2013). POCI-01-0145-FEDER-007728 and BFU2014-53641-P of the Spanish Ministerio de Economia y Competitividad co-financed by FEDER.authorsversionpublishe

Chemical characterization and bioactivity of phytochemicals from Iberian endemic Santolina semidentata and strategies for ex situ propagation

Asteraceae family members are well-known for their medicinal potential, comprising several properties that make them unique among plants. Here we focus on Santolina semidentata, an endemic plant from the Iberian Peninsula, not yet described for its medicinal properties. Phytochemical characterization of S. semidentata was performed, concerning total phenol content, flavonoid content, antioxidant capacity, HPLC-DAD profile, acetylcholinesterase inhibitory capacity, cytotoxicity and neuroprotective effect in a human neurodegeneration cell model. Moreover, essential oil composition and antifungal activity were also analised. This oil might be useful for therapeutical purposes, particularly in the treatment of dermatophytosis. S. semidentata potential for neuroprotection was revealed by acetylcholinesterase inhibitory capacity and also by an effective protective effect in human neuronal cells. Furthermore, different seed conservation protocols, as well as successful in vitro propagation were established which may be useful when integrated in a broad strategy for the conservation of these endemic plants and their sustainable use for potential biotechnological applications. The results presented here greatly contribute to value this species regarding its potential as a source of phytochemicals with prospective neuroprotective health benefits, either as alternative neuroprotective drugs or as leads for synthetizing more effective molecules.The authors wish to thank to “Fundo EDP para a Biodiversidade” for financial support. This work was also supported by “Fundação para a Ciência e a Tecnologia” through grant PEst-OE/EQB/LA0004/2011, BGCT/33418/2008, Green-it: UID/Multi/04551/2013, iNOVA4Health: UID/Multi/04462/2013 and financial support to CNS (IF/01097/20132), RP (SFRH/BD/63615/2009), IF (SFRH/BD/86584/2012) and AG (SFRH/BD/103155/2014).info:eu-repo/semantics/publishedVersio

Funciones de los nanodominios asociados a las balsas lipídicas de la membrana plasmática de neuronas en la regulación y coordinación de la señalización por calcio y por especies reactivas del oxígeno y sus implicaciones para la excitabilidad y supervivencia neuronal

Una alteración sostenida de la homeostasis intracelular del calcio y un incremento sostenido del estrés oxidativo celular son puntos convergentes en los procesos neurodegenerativos prevalentes actualmente. En esta tesis se ha puesto de manifiesto la relevancia para el control de la homeostasis del calcio citosólico en las neuronas granulares del cerebelo de rata (CGN) de los sistemas de transporte del calcio asociados a los “rafts” lipídicos utilizando metil-β-ciclodextrina (MβCD). El tratamiento con MβCD de las CGN maduras produjo una disminución de los niveles de fosforilación de la subunidad β de los canales de calcio de tipo L (LTCC) y su inactivación. Un efecto similar se alcanzó con el tratamiento con inhibidores de dos proteínas quinasas asociadas con los “rafts”, concluyéndose que su disrupción genera LTCC disfuncionales. Adicionalmente, se ha demostrado que estos nanodominios experimentan un notable incremento durante la maduración de las CGN y contribuyen significativamente a la producción total de ROS/RNS en los cultivos primarios de CGN “in vitro”, con una importante contribución de la isoforma 3 de la citocromo b₅ reductasa al control de la producción total de ROS en CGN maduras. Se ha mostrado la implicación de los sistemas de transporte del calcio asociados a estos nanodominios en la elevación sostenida de la concentración del calcio citosólico inducida por concentraciones neurotóxicas de la rotenona y que la creatina protege contra la desregulación de la homeostasis del calcio citosólico inducida por rotenona, que precede a la despolarización mitocondrial y al incremento del estrés oxidativo en este proceso neurotóxico.A sustained alteration of intracellular calcium homeostasis and a sustained increase in cellular oxidative stress are convergent points in the neurodegenerative processes prevalent nowadays. In this Ph.D. work it has been shown the relevance for the control of cytosolic calcium homeostasis in rat cerebellar granule neurons (CGN) of calcium transport systems associated with lipid rafts using methyl-β-cyclodextrin (MβCD). MβCD-induced rafts disruption of mature CGN lowered the levels of LTCC´s β-subunit phosphorylation and these calcium channels were inactivated. A similar result was obtained with mature CGN treatment with inhibitors of two protein kinases associated with these rafts, leading to the conclusion that lipid rafts disruption produce dysfunctional LTCCs. Additionally, it is demonstrated that during CGN maturation these nanodomains increase more than two-fold and afford a significant contribution to the overall ROS/RNS production in primary cultures of CGN “in vitro”, with the isoform 3 of cytochrome b₅ reductase playing a major role in the control of the overall ROS production in mature CGN. It is also shown the involvement of calcium transport systems associated with these nanodomains in the sustained raise of cytosolic calcium induced by neurotoxic concentrations of rotenone, and that creatine protects against rotenone-induced dysregulation of cytosolic calcium homeostasis. The results also show that the sustained raise of cytosolic calcium concentration precedes the mitochondrial membrane depolarization and the raise of ROS that generates the oxidative stress in rotenone neurotoxicity.Gobierno de España. Plan Nacional de I+D+I: Ayudas BFU2014-53641-P y BFU2017-85723-P · Junta de Extremadura, cofinanciado con fondos FEDER: Ayuda GR15139 al grupo de investigación BBB008 · Fundação para a Ciência e a Tecnologia (FCT): Beca predoctoral (SFRH/BD/84543/2012

The critical role of lipid rafts nanodomains in the cross-talk between calcium and reactive oxygen and nitrogen species in cerebellar granule neurons apoptosis by extracellular potassium deprivation

Sofia Fortalezas has been supported by a predoctoral fellowship of the Portuguese Fundação para a Ciência e a Tecnologia (FCT). Alejandro K. Samhan-Arias is supported by a Post-doctoral Fellowship SFRH/BPD/100069/2014 of the Fundação para a Ciência e Tecnologia, Portugal.The apoptosis of cerebellar granule neurons (CGN) induced by low-potassium in serum free medium in vitro has become a widely used model for neuronal apoptosis during in vivo brain development. In this review we shall summarize first the basic features of this model for neuronal apoptosis. Next, we shall focus on the L-type calcium channels (LTCC) inactivation as the primary pro-apoptotic signal in low K+-induced CGN death. This apoptotic process can be split into two major and sequential cellular signaling phases: one reversible phase that offers a temporal window for therapeutic interventions to prevent neuronal death, and an irreversible later phase. Therefore, we shall comment next the critical role of reactive oxygen species (ROS) production and major ROS sources triggering the entry of CGN in the irreversible stages of low K+-induced apoptosis. Then, we shall present the experimental evidences showing clustering of LTCC and ROS producing enzymes in plasma membrane lipid rafts of CGN matured in vitro, which have opened new perspectives for cell signaling in the early and reversible phase of this apoptosis. The role of lipid rafts nanodomains as fast response calcium/nitric oxide transducers of the switch of CGN to low K+ medium will be discussed next. The two major conclusions drawn from this review are: (1) deregulation of the pool of cytochrome b5 reductase associated to plasma membrane-lipid rafts, at least in part due to overexpression of cytochrome b5, can account for the critical superoxide anion overshot which triggers the entry in the irreversible phase of low K+ apoptosis of CGN, and (2) LTCC inactivation is rapidly transduced by lipid rafts nanodomains into a large drop of cytosolic calcium, a switch-off of nitric oxide production and subsequent inactivation of survival signaling pathways dependent on the activity of CaMKII, PKA and Akt/PKB kinases.publishersversionpublishe

Cytochrome b5 reductase is the component from neuronal synaptic plasma membrane vesicles that generates superoxide anion upon stimulation by cytochrome c

In this work, we measured the effect of cytochrome c on the NADH-dependent superoxide anion production by synaptic plasma membrane vesicles from rat brain. In these membranes, the cytochrome c stimulated NADH-dependent superoxide anion production was inhibited by antibodies against cytochrome b5 reductase linking the production to this enzyme. Measurement of the superoxide anion radical generated by purified recombinant soluble and membrane cytochrome b5 reductase corroborates the production of the radical by different enzyme isoforms. In the presence of cytochrome c, a burst of superoxide anion as well as the reduction of cytochrome c by cytochrome b5 reductase was measured. Complex formation between both proteins suggests that cytochrome b5 reductase is one of the major partners of cytochrome c upon its release from mitochondria to the cytosol during apoptosis. Superoxide anion production and cytochrome c reduction are the consequences of the stimulated NADH consumption by cytochrome b5 reductase upon complex formation with cytochrome c and suggest a major role of this enzyme as an anti-apoptotic protein during cell death