Regulación de la formación de especies reactivas de oxígeno por la cadena respiratoria mitocondrial en células neurales

[ES]Para conocer la función fisiológica de las especies reactivas de oxígeno (ROS) en cerebro, es necesario analizar la contribución de los diferentes tipos de células neurales en la formación de ROS. En la Tesis Doctoral hemos evaluado la capacidad de las neuronas y astrocitos de generar ROS de forma espontánea. Hemos observado que la producción de ROS es mayor (desde 1.5 a 10 veces) en astrocitos que en neuronas, en cultivos primarios de ratas Wistar y ratones C57BL6, con independencia de las condiciones de cultivo y el método de determinación de ROS. Las diferencias en la producción de ROS se confirmó a partir de neuronas y astrocitos diseccionadas de ratones adultos C57BL6, sugiriendo fuertemente que se trata de un fenómeno conservado in vivo. Además, las diferencias en la producción de ROS entre neuronas y astrocitos presenta un origen mitocondrial. Para conocer el mecanismo responsable de las diferencias en la producción mitocondrial de ROS (mROS), hemos analizado el ensamblaje de la cadena respiratoria mitocondrial. Usando electroforesis nativas, análisis proteómicos, y transferencia tipo Western, hemos observado que, en astrocitos, una gran proporción del complejo I se encuentra libre, mientras que en neuronas la mayor parte se encuentra formando parte de supercomplejos. Además, la abundancia de la subunidad del complejo I NDUFS1, en el complejo I libre, es menor en astrocitos que en neuronas. La sobreexpresión de NDUFS1 en astrocitos incrementó la proporción del complejo I en supercomplejos, reduciendo la producción de mROS. Por el contrario, el silenciamiento de NDUFS1 en neuronas disminuyó la proporción del complejo I en supercomplejos, incrementando la producción de mROS. Además, hemos observado que la reducción de los niveles de ROS en astrocitos, tras la incubación con GSH-etil éster, estabilizó al complejo I e incrementó su ensamblaje en supercomplejos. Finalmente, la reducción de mROS en astrocitos, por la expresión de una forma mitocondrial de la catalasa (mitoCatalasa), disminuyó la actividad de NRF2, y la estabilidad de HIF1. Por tanto, estos resultados son los primeros en demostrar como la modulación del complejo I en supercomplejos regula la producción mitocondrial de ROS en un sistema biológico intacto. Además, este mecanismo explica las diferencias intrínsecas en la producción de ROS entre neuronas y astrocitos, posiblemente ejerciendo un papel señalizador sobre las funciones fisiológicas

Abrogating mitochondrial ROS in neurons or astrocytes reveals cell-specific impact on mouse behaviour

Article 101917, (2021)[EN]Cells naturally produce mitochondrial reactive oxygen species (mROS), but the in vivo pathophysiological significance has long remained controversial. Within the brain, astrocyte-derived mROS physiologically regulate behaviour and are produced at one order of magnitude faster than in neurons. However, whether neuronal mROS abundance differentially impacts on behaviour is unknown. To address this, we engineered genetically modified mice to down modulate mROS levels in neurons in vivo. Whilst no alterations in motor coordination were observed by down modulating mROS in neurons under healthy conditions, it prevented the motor discoordination caused by the pro-oxidant neurotoxin, 3-nitropropionic acid (3-NP). In contrast, abrogation of mROS in astrocytes showed no beneficial effect against the 3-NP insult. These data indicate that the impact of modifying mROS production on mouse behaviour critically depends on the specific cell-type where they are generated

Fatty acid oxidation organizes mitochondrial supercomplexes to sustain astrocytic ROS and cognition

Having direct access to brain vasculature, astrocytes can take up available blood nutrients and metabolize them to fulfil their own energy needs and deliver metabolic intermediates to local synapses. These glial cells should be, therefore, metabolically adaptable to swap different substrates. However, in vitro and in vivo studies consistently show that astrocytes are primarily glycolytic, suggesting glucose is their main metabolic precursor. Notably, transcriptomic data and in vitro studies reveal that mouse astrocytes are capable of mitochondrially oxidizing fatty acids and that they can detoxify excess neuronal-derived fatty acids in disease models. Still, the factual metabolic advantage of fatty acid use by astrocytes and its physiological impact on higher-order cerebral functions remain unknown. Here, we show that knockout of carnitine-palmitoyl transferase-1A (CPT1A)—a key enzyme of mitochondrial fatty acid oxidation—in adult mouse astrocytes causes cognitive impairment. Mechanistically, decreased fatty acid oxidation rewired astrocytic pyruvate metabolism to facilitate electron flux through a super-assembled mitochondrial respiratory chain, resulting in attenuation of reactive oxygen species formation. Thus, astrocytes naturally metabolize fatty acids to preserve the mitochondrial respiratory chain in an energetically inefficient disassembled conformation that secures signalling reactive oxygen species and sustains cognitive performance.We acknowledge the technical assistance of M. Resch, M. Carabias-Carrasco, L. Martin and E. Prieto-Garcia, from the University of Salamanca. This work was funded by the European Regional Development Fund, Agencia Estatal de Investigación (grant nos. PID2019-105699RB-I00/AEI/10.13039/501100011033 and RED2018‐102576‐T to J.P.B. and SAF2017-90794-REDT to A.A.), Instituto de Salud Carlos III (grant nos. CB16/10/00282 to J.P.B. and PI18/00285 and RD16/0019/0018 to A.A.), Junta de Castilla y León (grant no. CS/151P20) and Escalera de Excelencia (grant no. CLU-2017-03 to J.P.B. and A.A.)

Essentiality of fatty acid synthase in the 2D to anchorage-independent growth transition in transforming cells

Upregulation of fatty acid synthase (FASN) is a common event in cancer, although its mechanistic and potential therapeutic roles are not completely understood. In this study, we establish a key role of FASN during transformation. FASN is required for eliciting the anaplerotic shift of the Krebs cycle observed in cancer cells. However, its main role is to consume acetyl-CoA, which unlocks isocitrate dehydrogenase (IDH)-dependent reductive carboxylation, producing the reductive power necessary to quench reactive oxygen species (ROS) originated during the switch from two-dimensional (2D) to three-dimensional (3D) growth (a necessary hallmark of cancer). Upregulation of FASN elicits the 2D-to-3D switch; however, FASN's synthetic product palmitate is dispensable for this process since cells satisfy their fatty acid requirements from the media. In vivo, genetic deletion or pharmacologic inhibition of FASN before oncogenic activation prevents tumor development and invasive growth. These results render FASN as a potential target for cancer prevention studies.M.Q.F. is a recipient of the following grants: FIS PI13/00430 and FIS PI16/00354 funded by the Instituto de Salud Carlos III (ISCIII) and co-funded by the European Regional Development Fund (ERDF) and AECC Scientific Foundation (Beca de Retorno 2010). R.C. is a recipient of the following grants: FIS PI11/00832 and FIS PI14/00726 funded by the Instituto de Salud Carlos III (ISCIII) and co-funded by the European Regional Development Fund (ERDF), II14/00009 and PIE15/00068 from the Ministerio de Sanidad, Spain. N.S.C. is a recipient of an NIH grant (5R35CA197532). O.Y.T. is a recipient of the grants BFU2014-57466 from the Ministerio de Economia y Competitividad (MINECO). J.P.B. is funded by MINECO (SAF2016-78114-R), Instituto de Salud Carlos III (RD12/0043/0021), Junta de Castilla y Leon (Escalera de Excelencia CLU-2017-03), Ayudas Equipos Investigacion Biomedicina 2017 Fundacion BBVA, and Fundacion Ramon Areces. This study was partially supported by the generous donations from Fundacion CRIS Contra el Cancer and AVON Spain. We thank Drs. Erwin Wagner and Nabil Djouder for their critical review of the paper.S

Mitochondrial ROS contribute to neuronal ceroid lipofuscinosis pathogenesis

Trabajo presentado al 20th Biennial Meeting of The Society for Free Radical Research International (SFRR-I) del 15 al 18 de marzo de forma virtualNeuronal ceroid lipofuscinoses (NCLs), known as Batten disease, are the most common of the rare neurodegenerative disorders in children. These disorders are grouped together based on clinical similarities and uniform neuropathological features, including accumulation of lipofuscin in lysosomes and widespread gliosis. CLN7 disease is one of these NCLs that present in late infancy and is caused by mutations in the CLN7/MFSD8 gene, which encodes a lysosomal membrane glycoprotein of unknown function, hence the biochemical processes affected by CLN7-loss of function are not understood. Here, we found in the Cln7Δex2 mouse model of CLN7 disease that failure in the autophagy-lysosomal pathway causes aberrant accumulation of reactive oxygen species (ROS)-producing brain mitochondria. Metabolic profile analysis of Cln7Δex2 neurons revealed a decrease in the basal oxygen consumption rate (OCR), ATP-linked and maximal OCR and proton leak, indicating bioenergetically impaired mitochondria. To assess the impact of ROS on CLN7 disease progression, Cln7Δex2 mice were crossed with mice expressing a mitochondrial-tagged form of catalase (mCAT) governed by a neuron-specific promoter (Cln7Δex2-CAMKIIaCre-mCAT). The increased mROS observed in Cln7Δex2 neurons was abolished in Cln7Δex2- CAMKIIaCre-mCAT neurons, verifying the efficacy of this approach. The brain mitochondrial swelling and mitochondrial cristae profile widening observed in Cln7Δex2 mice were abolished in Cln7Δex2-CAMKIIaCre-mCAT mice. Notably, Cln7Δex2 brain accumulation of subunit C-ATPase and lysosomal lipofuscin, as well as gliosis, which are hallmarks of the disease, were ameliorated in Cln7Δex2- CAMKIIaCre-mCAT mice. Altogether, these findings indicate that the generation of ROS by bioenergetically-impaired mitochondria in Cln7Δex2 neurons contributes to the histopathological symptoms of CLN7 disease

Fatty acid oxidation organizes mitochondrial supercomplexes to sustain astrocytic ROS and cognition

[EN]Having direct access to brain vasculature, astrocytes can take up available blood nutrients and metabolize them to fulfil their own energy needs and deliver metabolic intermediates to local synapses1,2. These glial cells should be, therefore, metabolically adaptable to swap different substrates. However, in vitro and in vivo studies consistently show that astrocytes are primarily glycolytic3-7, suggesting glucose is their main metabolic precursor. Notably, transcriptomic data8,9 and in vitro10 studies reveal that mouse astrocytes are capable of mitochondrially oxidizing fatty acids and that they can detoxify excess neuronal-derived fatty acids in disease models11,12. Still, the factual metabolic advantage of fatty acid use by astrocytes and its physiological impact on higher-order cerebral functions remain unknown. Here, we show that knockout of carnitine-palmitoyl transferase-1A (CPT1A)-a key enzyme of mitochondrial fatty acid oxidation-in adult mouse astrocytes causes cognitive impairment. Mechanistically, decreased fatty acid oxidation rewired astrocytic pyruvate metabolism to facilitate electron flux through a super-assembled mitochondrial respiratory chain, resulting in attenuation of reactive oxygen species formation. Thus, astrocytes naturally metabolize fatty acids to preserve the mitochondrial respiratory chain in an energetically inefficient disassembled conformation that secures signalling reactive oxygen species and sustains cognitive performance

Repurposing of tamoxifen ameliorates CLN3 and CLN7 disease phenotype

Batten diseases (BDs) are a group of lysosomal storage disorders characterized by seizure, visual loss, and cognitive and motor deterioration. We discovered increased levels of globotriaosylceramide (Gb3) in cellular and murine models of CLN3 and CLN7 diseases and used fluorescent-conjugated bacterial toxins to label Gb3 to develop a cell-based high content imaging (HCI) screening assay for the repurposing of FDA-approved compounds able to reduce this accumulation within BD cells. We found that tamoxifen reduced the lysosomal accumulation of Gb3 in CLN3 and CLN7 cell models, including neuronal progenitor cells (NPCs) from CLN7 patient-derived induced pluripotent stem cells (iPSC). Here, tamoxifen exerts its action through a mechanism that involves activation of the transcription factor EB (TFEB), a master gene of lysosomal function and autophagy. In vivo administration of tamoxifen to the CLN7Δex2 mouse model reduced the accumulation of Gb3 and SCMAS, decreased neuroinflammation, and improved motor coordination. These data strongly suggest that tamoxifen may be a suitable drug to treat some types of Batten disease.This work was funded by the European Union’s Horizon 2020 research and innovation programme (BATCure, grant No. 666918 to DLM, JPB, SEM, TB and SS). JPB is funded by the Agencia Estatal de Investigación (PID2019-105699RB-I00/ AEI / 10.13039/501100011033 and RED2018-102576-T), Plan Nacional sobre Drogas (2020I028), Junta de Castilla y León (Escalera de Excelencia CLU-2017-03), Ayudas Equipos Investigación Biomedicina 2017 Fundación BBVA and Fundación Ramón Areces. SS was funded by a grant from the Mila’s Miracle Foundation. TB was supported by German Research Council (DFG) grant FOR2625. SM benefits from MRC funding to the MRC Laboratory for Molecular Cell Biology University Unit at UCL (award code MC_U12266B) towards laboratory and office space. We acknowledge Marcella Cesana for providing the TFEB virus. Graphical abstract was created using BioRender.com

Aberrant upregulation of the glycolytic enzyme PFKFB3 in CLN7 neuronal ceroid lipofuscinosis

CLN7 neuronal ceroid lipofuscinosis is an inherited lysosomal storage neurodegenerative disease highly prevalent in children. CLN7/MFSD8 gene encodes a lysosomal membrane glycoprotein, but the biochemical processes affected by CLN7-loss of function are unexplored thus preventing development of potential treatments. Here, we found, in the Cln7∆ex2 mouse model of CLN7 disease, that failure in autophagy causes accumulation of structurally and bioenergetically impaired neuronal mitochondria. In vivo genetic approach reveals elevated mitochondrial reactive oxygen species (mROS) in Cln7∆ex2 neurons that mediates glycolytic enzyme PFKFB3 activation and contributes to CLN7 pathogenesis. Mechanistically, mROS sustains a signaling cascade leading to protein stabilization of PFKFB3, normally unstable in healthy neurons. Administration of the highly selective PFKFB3 inhibitor AZ67 in Cln7∆ex2 mouse brain in vivo and in CLN7 patients-derived cells rectifies key disease hallmarks. Thus, aberrant upregulation of the glycolytic enzyme PFKFB3 in neurons may contribute to CLN7 pathogenesis and targeting PFKFB3 could alleviate this and other lysosomal storage diseases.This work was funded by the European Regional Development Fund, European Union’s Horizon 2020 Research and Innovation Programme (BATCure grant No. 666918 to J.P.B., S.E.M., D.L.M., S.S., and T.R.M.; PANA grant No. 686009 to A.A.), Agencia Estatal de Investigación (PID2019-105699RB-I00/AEI/10.13039/501100011033 and RED2018‐102576‐T to J.P.B.; SAF2017-90794-REDT to A.A.), Instituto de Salud Carlos III (CB16/10/00282 to J.P.B.; PI18/00285; RD16/0019/0018 to A.A.), Junta de Castilla y León (CS/151P20 and Escalera de Excelencia CLU-2017-03 to J.P.B. and A.A.), Ayudas Equipos Investigación Biomedicina 2017 Fundación BBVA (to J.P.B.), and Fundación Ramón Areces (to J.P.B. and A.A.). SM benefits from MRC funding to the MRC Laboratory for Molecular Cell Biology University Unit at UCL (award code MC_U12266B) towards lab and office space. Part of this work was funded by Gero Discovery L.L.C. M.G.M. is an ISCIII-Sara Borrel contract recipient (CD18/00203)

Aberrant upregulation of glycolysis mediates CLN7 neuronal ceroid lipofuscinosis

Resumen del trabajo presentado en el 43rd Annual Meeting of the Spanish Society of Biochemistry & Molecular Biology, celebrado en Barcelona, del 19 al 22 de julio de 2021CLN7 neuronal ceroid lipofuscinosis is an inherited lysosomal storage neurodegenerative disease highly prevalent in children. CLN7/MFSD8 gene encodes a lysosomal membrane glycoprotein, but the biochemical processes affected by CLN7-loss of function are unexplored thus preventing development of potential treatments. Here, we found, in the Cln7∆ex2 mouse model of CLN7 disease, that failure in the autophagy-lysosomal pathway causes accumulation of structurally and bioenergetically impaired neuronal mi- tochondria. In vivo genetic approach revealed elevated mitochondrial reactive oxygen species (mROS) in Cln7∆ex2 neurons that mediates glycolysis activation and contributes to CLN7 pathogenesis. Mechanistically, mROS sustains a signaling cascade leading to protein stabilization of PFK- FB3, a glycolytic-promoting enzyme normally unstable in healthy neurons. Pharmacological inhibition of PFKFB3 in Cln7∆ex2 mouse brain in vivo and in CLN7 patients-derived cells rectified key disease hallmarks. Thus, aberrant upregulation of neuronal glycolysis contributes to CLN7 patho-genesis and targeting PFKFB3 may alleviate this and other lysosomal storage diseasesThis work was funded by Agencia Estatal de Investigación (PID2019-105699RB-I00).Peer reviewe

Regulación de la formación de especies reactivas de oxígeno por la cadena respiratoria mitocondrial en células neurales

Tesis Doctoral presentada por la Licenciada en Biotecnología Dña. Irene López Fabuel, en el Departamento de Bioquímica y Biología Molecular y en el Instituto de Biología Funcional y Genómica, de la Universidad de Salamanca para optar al Título de Doctor Internacional.[ES]: Para conocer la función fisiológica de las especies reactivas de oxígeno (ROS) en cerebro, es necesario analizar la contribución de los diferentes tipos de células neurales en la formación de ROS. En la Tesis Doctoral hemos evaluado la capacidad de las neuronas y astrocitos de generar ROS de forma espontánea. Hemos observado que la producción de ROS es mayor (desde 1.5 a 10 veces) en astrocitos que en neuronas, en cultivos primarios de ratas Wistar y ratones C57BL6, con independencia de las condiciones de cultivo y el método de determinación de ROS. Las diferencias en la producción de ROS se confirmó a partir de neuronas y astrocitos diseccionadas de ratones adultos C57BL6, sugiriendo fuertemente que se trata de un fenómeno conservado in vivo. Además, las diferencias en la producción de ROS entre neuronas y astrocitos presenta un origen mitocondrial. Para conocer el mecanismo responsable de las diferencias en la producción mitocondrial de ROS (mROS), hemos analizado el ensamblaje de la cadena respiratoria mitocondrial. Usando electroforesis nativas, análisis proteómicos, y transferencia tipo Western, hemos observado que, en astrocitos, una gran proporción del complejo I se encuentra libre, mientras que en neuronas la mayor parte se encuentra formando parte de supercomplejos. Además, la abundancia de la subunidad del complejo I NDUFS1, en el complejo I libre, es menor en astrocitos que en neuronas. La sobreexpresión de NDUFS1 en astrocitos incrementó la proporción del complejo I en supercomplejos, reduciendo la producción de mROS. Por el contrario, el silenciamiento de NDUFS1 en neuronas disminuyó la proporción del complejo I en supercomplejos, incrementando la producción de mROS. Además, hemos observado que la reducción de los niveles de ROS en astrocitos, tras la incubación con GSH-etil éster, estabilizó al complejo I e incrementó su ensamblaje en supercomplejos. Finalmente, la reducción de mROS en astrocitos, por la expresión de una forma mitocondrial de la catalasa (mitoCatalasa), disminuyó la actividad de NRF2, y la estabilidad de HIF1. Por tanto, estos resultados son los primeros en demostrar como la modulación del complejo I en supercomplejos regula la producción mitocondrial de ROS en un sistema biológico intacto. Además, este mecanismo explica las diferencias intrínsecas en la producción de ROS entre neuronas y astrocitos, posiblemente ejerciendo un papel señalizador sobre las funciones fisiológicas.[EN]: Understanding the physiological roles of reactive oxygen species (ROS) in the brain requires dissecting out the contribution of different neural cell types to ROS formation. Here, the abilities of neurons and astrocytes to spontaneously generate ROS were characterized. We found that ROS production is higher (from 1.5- to 10-fold) in astrocytes than in neurons, as assessed in primary cultures prepared from Wistar rats and C57Bl6 mice, regardless of the culture medium conditions and methods of ROS assessment. Such difference in ROS production was confirmed in neurons and astrocytes acutely dissociated from adult C57Bl6 mice, strongly suggesting an in vivo phenomenon. Furthermore, higher ROS production was found to take place in mitochondria isolated from cultured astrocytes when compared with those isolated from neurons. To understand the mechanism explaining this different mitochondrial ROS production, we focused on the assembly of the mitochondrial respiratory chain. Using blue-native gel electrophoresis, proteomic analysis, and Western blotting, we found that, in astrocytes, a large proportion of complex I occurs free, whereas in neurons most complex I is embedded into supercomplexes. Furthermore, the abundance of complex I subunit, NDUFS1, in free complex I, was found to be lower in astrocytes than in neurons. Over-expression of NDUFS1 in astrocytes increased the proportion of complex I into supercomplexes and decreased mitochondrial ROS production. Conversely, knockdown of NDUFS1 in neurons decreased the proportion of complex I into supercomplexes and increased mitochondrial ROS production. We also found that, reduction of ROS abundance in astrocytes by incubation with GSH-ethyl ester stabilized complex I and promoted its assembly into supercomplexes. Finally, reduction of mitochondrial ROS in astrocytes by the expression a mitochondrial-tagged form of catalase (mitoCatalase), decreased NRF2 activity and HIF1α stability. Altogether, these results are the first to demonstrate that the modulation of complex I assembly into supercomplexes regulates mitochondrial ROS production in an intact biological system. Moreover, this mechanism explains intrinsic differences in ROS production between neurons and astrocytes, likely playing different cell signalling physiological functions.Peer Reviewe