Targeting Mitochondria to Control Ageing and Senescence

Ageing is accompanied by a progressive impairment of cellular function and a systemic deterioration of tissues and organs, resulting in increased vulnerability to multiple diseases. Here, we review the interplay between two hallmarks of ageing, namely, mitochondrial dysfunction and cellular senescence. The targeting of specific mitochondrial features in senescent cells has the potential of delaying or even reverting the ageing process. A deeper and more comprehensive understanding of mitochondrial biology in senescent cells is necessary to effectively face this challenge. Here, we discuss the main alterations in mitochondrial functions and structure in both ageing and cellular senescence, highlighting the differences and similarities between the two processes. Moreover, we describe the treatments available to target these pathways and speculate on possible future directions of anti-ageing and anti-senescence therapies targeting mitochondria

Remodelling of the Mitochondrial Bioenergetic Pathways in Human Cultured Fibroblasts with Carbohydrates

Mitochondrial oxidative phosphorylation defects underlie many neurological and neuromuscular diseases. Patients’ primary dermal fibroblasts are one of the most commonly used in vitro models to study mitochondrial pathologies. However, fibroblasts tend to rely more on glycolysis than oxidative phosphorylation for their energy when cultivated in standard high-glucose medium, rendering it difficult to expose mitochondrial dysfunctions. This study aimed to systematically investigate to which extent the use of galactose- or fructose-based medium switches the fibroblasts’ energy metabolism to a more oxidative state. Highly proliferative cells depend more on glycolysis than less proliferative cells. Therefore, we investigated two primary dermal fibroblast cultures from healthy subjects: a highly proliferative neonatal culture and a slower-growing adult culture. Cells were cultured with 25 mM glucose, galactose or fructose, and 4 mM glutamine as carbon sources. Compared to glucose, both galactose and fructose reduce the cellular proliferation rate, but the galactose-induced drop in proliferation is much more profound than the one observed in cells cultivated in fructose. Both galactose and fructose result in a modest increase in mitochondrial content, including mitochondrial DNA, and a disproportionate increase in protein levels, assembly, and activity of the oxidative phosphorylation enzyme complexes. Galactose- and fructose-based media induce a switch of the prevalent biochemical pathway in cultured fibroblasts, enhancing aerobic metabolism when compared to glucose-based medium. While both galactose and fructose stimulate oxidative phosphorylation to a comparable degree, galactose decreases the cellular proliferation rate more than fructose, suggesting that a fructose-based medium is a better choice when studying partial oxidative phosphorylation defects in patients’ fibroblasts

Mitochondrial Structure and Bioenergetics in Normal and Disease Conditions.

Mitochondria are ubiquitous intracellular organelles found in almost all eukaryotes and involved in various aspects of cellular life, with a primary role in energy production. The interest in this organelle has grown stronger with the discovery of their link to various pathologies, including cancer, aging and neurodegenerative diseases. Indeed, dysfunctional mitochondria cannot provide the required energy to tissues with a high-energy demand, such as heart, brain and muscles, leading to a large spectrum of clinical phenotypes. Mitochondrial defects are at the origin of a group of clinically heterogeneous pathologies, called mitochondrial diseases, with an incidence of 1 in 5000 live births. Primary mitochondrial diseases are associated with genetic mutations both in nuclear and mitochondrial DNA (mtDNA), affecting genes involved in every aspect of the organelle function. As a consequence, it is difficult to find a common cause for mitochondrial diseases and, subsequently, to offer a precise clinical definition of the pathology. Moreover, the complexity of this condition makes it challenging to identify possible therapies or drug targets

Interplay between Mitochondrial Protein Import and Respiratory Complexes Assembly in Neuronal Health and Degeneration.

The fact that >99% of mitochondrial proteins are encoded by the nuclear genome and synthesised in the cytosol renders the process of mitochondrial protein import fundamental for normal organelle physiology. In addition to this, the nuclear genome comprises most of the proteins required for respiratory complex assembly and function. This means that without fully functional protein import, mitochondrial respiration will be defective, and the major cellular ATP source depleted. When mitochondrial protein import is impaired, a number of stress response pathways are activated in order to overcome the dysfunction and restore mitochondrial and cellular proteostasis. However, prolonged impaired mitochondrial protein import and subsequent defective respiratory chain function contributes to a number of diseases including primary mitochondrial diseases and neurodegeneration. This review focuses on how the processes of mitochondrial protein translocation and respiratory complex assembly and function are interlinked, how they are regulated, and their importance in health and disease

Novel compound heterozygous pathogenic variants in nucleotide-binding protein like protein (NUBPL) cause leukoencephalopathy with multi-systemic involvement.

NUBPL (Nucleotide-binding protein like) protein encodes a member of the Mrp/NBP35 ATP-binding proteins family, deemed to be involved in mammalian complex I (CI) assembly process. Exome sequencing of a patient presenting with infantile-onset hepatopathy, renal tubular acidosis, developmental delay, short stature, leukoencephalopathy with minimal cerebellar involvement and multiple OXPHOS deficiencies revealed the presence of two novel pathogenic compound heterozygous variants in NUBPL (p.Phe242Leu/p.Leu104Pro). We investigated patient's and control immortalised fibroblasts and demonstrated that both the peripheral and the membrane arms of complex I are undetectable in mutant NUBPL cells, resulting in virtually absent CI holocomplex and loss of enzyme activity. In addition, complex III stability was moderately affected as well. Lentiviral-mediated expression of the wild-type NUBPL cDNA rescued both CI and CIII assembly defects, confirming the pathogenicity of the variants. In conclusion, this is the first report describing a complex multisystemic disorder due to NUBPL defect. In addition, we confirmed the role of NUBPL in Complex I assembly associated with secondary effect on Complex III stability and we demonstrated a defect of mtDNA-related translation which suggests a potential additional role of NUBPL in mtDNA expression

AMPK-dependent phosphorylation of MTFR1L regulates mitochondrial morphology

Mitochondria are dynamic organelles that undergo membrane remodeling events in response to metabolic alterations to generate an adequate mitochondrial network. Here, we investigated the function of mitochondrial fission regulator 1-like protein (MTFR1L), an uncharacterized protein that has been identified in phosphoproteomic screens as a potential AMP-activated protein kinase (AMPK) substrate. We showed that MTFR1L is an outer mitochondrial membrane-localized protein modulating mitochondrial morphology. Loss of MTFR1L led to mitochondrial elongation associated with increased mitochondrial fusion events and levels of the mitochondrial fusion protein, optic atrophy 1. Mechanistically, we show that MTFR1L is phosphorylated by AMPK, which thereby controls the function of MTFR1L in regulating mitochondrial morphology both in mammalian cell lines and in murine cortical neurons in vivo. Furthermore, we demonstrate that MTFR1L is required for stress-induced AMPK-dependent mitochondrial fragmentation. Together, these findings identify MTFR1L as a critical mitochondrial protein transducing AMPK-dependent metabolic changes through regulation of mitochondrial dynamics.</p

Identification and functional characterisation of different inner mitochondrial membrane supercomplexes

The inner mitochondria membrane (IMM) is densely packed with proteins necessary for mitochondrial activity, including oxidative phosphorylation (OXPHOS) complexes and transporters. Mitochondrial respiratory chain (MRC) complexes, which generate ATP by OXPHOS, associate in higher-order assemblies, known as supercomplexes (SC), that are structurally interdependent. Numerous patients carrying mutations in a single complex, indeed, present with combined enzyme deficiencies and, in particular, the destabilisation of complex I (CI) has been often described in the absence of complex III (CIII). To clarify the structural and functional relationships between complexes, we have analysed a MTCYB-deficient human cell line, unable to assemble CIII. Our results showed that in this line, CI biogenesis was blocked by preventing the incorporation of the NADH module, the last step of CI assembly, rather than decreasing its stability. Moreover, complex IV (CIV) biogenesis was impaired as well, and CIV subunits appeared sequestered within CIII subassemblies. Therefore, we propose that CIII is central not only for the formation of SC but also for the maturation of the other electron transport chain complexes. These results challenge the previous SC model that described the formation of fully assembled individual complexes before the association in SC. In contrast, they support a cooperative-assembly model in which the main role of CIII in SC is to provide a structural and functional platform for the completion of overall MRC biogenesis. Next, we identified and characterised the interaction between CIV and the mitochondrial calcium uniporter complex (MCUC), responsible for mitochondrial calcium uptake and homeostasis. Our data, indeed, showed a specific physical interaction between this ETC enzyme and various subunits of MCUC in first and second dimension blue native PAGE, SILAC labelling/pulldown, and BioID proteomic-based methods. We then investigated the effects of this association with CIV, measuring enzyme activity and mitochondrial respiration, but also on MCUC and CIV distribution in the IMM, by N-structured illuminated super-resolution microscopy (N-SIM). Our results showed a specific reduction in CIV activity in the absence of MICU1, the main regulator of the uniporter, but surprisingly no effects were observed after MCU downregulation or pharmacological inhibition of mitochondrial Ca2+ entry. Instead, the lack of CIV seems to have an impact on MCU-containing complexes formation and induces a re-localisation of MCU from the cristae membrane to the inner boundary membrane. Further experiments will be performed to shed light on the physiological relevance of this interaction.MR

Mitochondrial Structure and Bioenergetics in Normal and Disease Conditions

Mitochondria are ubiquitous intracellular organelles found in almost all eukaryotes and involved in various aspects of cellular life, with a primary role in energy production. The interest in this organelle has grown stronger with the discovery of their link to various pathologies, including cancer, aging and neurodegenerative diseases. Indeed, dysfunctional mitochondria cannot provide the required energy to tissues with a high-energy demand, such as heart, brain and muscles, leading to a large spectrum of clinical phenotypes. Mitochondrial defects are at the origin of a group of clinically heterogeneous pathologies, called mitochondrial diseases, with an incidence of 1 in 5000 live births. Primary mitochondrial diseases are associated with genetic mutations both in nuclear and mitochondrial DNA (mtDNA), affecting genes involved in every aspect of the organelle function. As a consequence, it is difficult to find a common cause for mitochondrial diseases and, subsequently, to offer a precise clinical definition of the pathology. Moreover, the complexity of this condition makes it challenging to identify possible therapies or drug targets

Respiratory supercomplexes act as a platform for complex III-mediated maturation of human mitochondrial complexes I and IV

Mitochondrial respiratory chain (MRC) enzymes associate in supercomplexes (SCs) that are structurally interdependent. This may explain why defects in a single component often produce combined enzyme deficiencies in patients. A case in point is the alleged destabilization of complex I in the absence of complex III. To clarify the structural and functional relationships between complexes, we have used comprehensive proteomic, functional, and biogenetical approaches to analyze a MT-CYB-deficient human cell line. We show that the absence of complex III blocks complex I biogenesis by preventing the incorporation of the NADH module rather than decreasing its stability. In addition, complex IV subunits appeared sequestered within complex III subassemblies, leading to defective complex IV assembly as well. Therefore, we propose that complex III is central for MRC maturation and SC formation. Our results challenge the notion that SC biogenesis requires the pre-formation of fully assembled individual complexes. In contrast, they support a cooperative-assembly model in which the main role of complex III in SCs is to provide a structural and functional platform for the completion of overall MRC biogenesis