Intégration de la régulation post-transcriptionnelle et des interactions mitochondries/cytosquelette dans les voies de contrôle du métabolisme mitochondrial

Mitochondrion provides energy for cell metabolism through the mechanism of oxidative phosphorylation. This function requires a coordinated expression of nuclear and mitochondrial genomes provided by the family of transcriptional coactivators PGC -1 (peroxisome proliferator-activated receptor γ Coactivator -1), responding to endogenous and/or environmental signals. A fine regulation of the oxidative phosphorylation by miRNAs is now suspected. To specify these regulatory pathways in cellular models of human follicular thyroid carcinomas, we have explored the PRC-related (PGC- related coactivator) pathway and specific microRNAs, in models presenting various mitochondrial abundance and differences in PRC and PGC- 1α expression levels. We have highlighted the role of miR-218 as a key regulatory factor of mitochondrial functions. An adequate energy supply also requires close connections between cytoskeleton and mitochondria to ensure an optimal distribution of mitochondria within the cell. Peptides derived from the light neurofilament subunit, as NFL - TBS.40 -63, are able to specifically enter into human glioblastoma cells and destabilize the microtubule network, leading to cell death by apoptosis. To study the impact of this peptide on mitochondrial network and oxidative phosphorylation, we have treated the T98G human glioblastoma cells by different concentrations of NFL- TBS.40 -63. Our work showed disturbance in mitochondrial network and reduction in mitochondrial respiration rate in the treated cells. All these results should allow the development of therapy targeting the mitochondrial function.La mitochondrie fournit l'énergie nécessaire au fonctionnement cellulaire, grâce au mécanisme de phosphorylation oxydative. Cette fonction nécessite une expression coordonnée des génomes nucléaires et mitochondriaux assurée par la famille de coactivateurs transcriptionnels PGC-1 (Peroxisome proliferator-activated receptor γ Coactivator-1), sensibles aux signaux endogènes et/ou environnementaux. Une régulation plus fine de la phosphorylation oxydative par des miRNAs est maintenant soupçonnée. Afin de préciser ces différents modes de régulation dans des modèles cellulaires de carcinomes thyroïdiens, nous avons exploré la voie PRC-dépendante (PGC-related coactivator) et les miRNAs spécifiquement exprimés dans ces modèles présentant une richesse en mitochondries et des niveaux de PRC et de PGC-1α différents. Ce travail a permis de mettre en évidence miR-218 comme marqueur clé de régulation de la fonction mitochondriale. Au-delà de la régulation de l'expression génique, une fourniture énergétique adéquate nécessite également une répartition optimale des mitochondries au sein de la cellule, grâce à d'étroites connexions entre le cytosquelette et la mitochondrie. Des peptides issus de la sous-unité légère des neurofilaments, dont le NFL-TBS.40-63, sont capables d'entrer spécifiquement dans les cellules de glioblastomes humains et d'y déstabiliser le réseau microtubulaire, conduisant à la mort cellulaire par apoptose. Pour étudier l'impact de ce peptide sur le réseau de mitochondries et leurs fonctions, nous avons traité le modèle cellulaire de glioblastomes humains T98G, par différentes concentrations de NFL-TBS.40-63. Ce travail révèle une perturbation du réseau de mitochondries et une diminution de la respiration mitochondriale dans les cellules exposées. L'ensemble de ces travaux doit permettre le développement de traitements ciblés de la fonction mitochondriale

The NFL-TBS.40-63 anti-glioblastoma peptide disrupts microtubule and mitochondrial networks in the T98G glioma cell line.

Despite aggressive therapies, including combinations of surgery, radiotherapy and chemotherapy, glioblastoma remains a highly aggressive brain cancer with the worst prognosis of any central nervous system disease. We have previously identified a neurofilament-derived cell-penetrating peptide, NFL-TBS.40-63, that specifically enters by endocytosis in glioblastoma cells, where it induces microtubule destruction and inhibits cell proliferation. Here, we explore the impact of NFL-TBS.40-63 peptide on the mitochondrial network and its functions by using global cell respiration, quantitative PCR analysis of the main actors directing mitochondrial biogenesis, western blot analysis of the oxidative phosphorylation (OXPHOS) subunits and confocal microscopy. We show that the internalized peptide disturbs mitochondrial and microtubule networks, interferes with mitochondrial dynamics and induces a rapid depletion of global cell respiration. This effect may be related to reduced expression of the NRF-1 transcription factor and of specific miRNAs, which may impact mitochondrial biogenesis, in regard to default mitochondrial mobility

Quantitative PCR analysis of mitochondrial fission/fusion actors and relevant differentially-expressed miRNA-mRNA complexes in human T98G glioblastoma cells.

<p>5A: Quantitative PCR analysis of mitochondrial fission/fusion actors (FIS1 and MFN2) in T98G cells. The data are expressed in units (mRNA expression of a specific gene normalized to β-globin mRNA expression) that are relative to the control, which was assigned a unit value. 5B: Quantitative PCR analysis of relevant differentially expressed miRNA in T98G cells. The data are expressed in units (miRNA expression relative to U5 snRNA) that are relative to the control, which was assigned a unit value. 5C: Quantitative PCR analysis of PTEN and NAIP mRNA, directly targeted by miR-21 and miR-221, respectively. FGFR3 expression was used as negative control of miR-100, which expression level was unchanged by peptide treatment. The data are expressed in units (mRNA expression of a specific gene normalized to β-globin mRNA expression) that are relative to the control, which was assigned a unit value. The values are the average ± SD for three separate determinations (<i>N</i> = 3). *: <i>P</i><0.05 versus control.</p

The NFL-TBS.40-63 peptide reorganizes mitochondrial networks in human T98G glioblastoma cells.

<p>The white arrow indicates a mitochondrial network (bottom left) superposed with a microtubule network (bottom right). The yellow arrow indicates a mitochondrial network (bottom left) superposed with long peptide sequences (top right). The microtubules were detected using an Alexa647-labeled, anti-α-tubulin antibody (purple); biotinylated NFL-TBS.40-63 peptide was labeled with streptavidin Alexa488 (green), the nuclei with diamidino ph?nylindole (DAPI; blue) and the mitochondria with a mitotracker Red CMX ROS). The cells were examined with a Nikon A1RSI confocal microscope and the images were analyzed with Nikon NIS-element software. The red bars are the measuring scale.</p

Effects of 10 µM of NFL-TBS.40-63 peptide on mitochondrial and microtubule networks in human T98G glioblastoma cells.

<p>3A: NFL peptide accumulates within the cell in a polarized manner, limiting the density of the microtubules and mitochondrial networks. 3B: NFL peptide accumulates at the basis of the midbody and excludes the microtubule network. 3C: The peptide surrounds the microtubules' tips and limits filopodia formation. Microtubules were detected using an Alexa647-labeled, anti-α-tubulin antibody (purple); biotinylated NFL-TBS.40-63 peptide was labeled with streptavidin Alexa488 (green), the nuclei with diamidino phénylindole (DAPI; blue) and mitochondria with a mitotracker (RedCMX ROS). The cells were examined with a Nikon A1RSI confocal microscope and the images were analyzed with Nikon NIS-element software. The <i>red bars</i> are the measuring scale.</p

Action of the NFL-TBS.40-63 peptide on mitochondrial number and functions in T98G cells and NIH 3T3 control cells.

<p>1A: The relative oxygen consumption was measured using a mitostress kit and a Seahorse XF-24 apparatus (from Seahorse Bioscience, North Billerica, MA, USA). The oligomycin-insensitive fraction represents non-phosphorylating respiration, which was recorded after the inhibition of ATP synthase with oligomycin. The oligomycin-sensitive fraction represents the phosphorylating respiration, i.e., the fraction used for ATP synthesis. Results are expressed relative to oxygen consumption of scramble treated cells used as control (pmol/min/mg protein). 1B: The protein expression of mitochondrial subunit IV of complex IV (COX4, MS408, Mitosciences) and subunit Ip of complex II (SDHB, MS203, Mitosciences) were measured by Western blot analysis after a 6-hour exposure to 10 µM NFL-TBS.40-63 peptide and normalized to the α-tubulin level (65 KDa; Abcam, Cambridge, UK). The protein expression for the peptide-treated samples was expressed relative to that of the scramble-treated samples. S: Scramble; P: NFL-TBS.40-63 peptide. Results are expressed relative to protein expression ratio of scramble treated cells used as control. The values represent the average ± SD for three separate determinations (<i>N</i> = 3). *: <i>P</i><0.05 versus control.</p

Expression analysis of the genes involved in the control of mitochondrial biogenesis in T98G cells treated by 10 µM of NFL-TBS.40-63 peptide.

<p>2A: Quantitative PCR analysis: PRC (PPRC1) and PGC-1α (PPARGC1A) coactivators, NRF-1 transcription factor, mitochondrial transcription factor TFAM and a component of the respiratory chain, Cytochrome c (CYCS), were measured. The data are expressed in relative units (mRNA expression of a specific gene normalized to β-globin mRNA expression) and expressed relative to the control, which was assigned a unit value. The values are the average ± SD for three separate determinations (<i>N</i> = 3). *: <i>P</i><0.05 versus control. 2B: Western blot analysis: The protein expression of PGC-1α (105KDa, Ab- 54481, Abcam) and NRF-1 (54 KDa, Ab-86516, Abcam) were measured by Western blot analysis after a 6-hours exposure to 10 µM NFL-TBS.40-63 peptide and normalized to the β-actin level (45 KDa; Abcam). Results are expressed relative to protein expression ratio of scramble treated cells which was assigned a unit value. The values are the average ± SD (<i>N</i> = 3). *: <i>P</i><0.05 versus control.</p