9 research outputs found

    Connecting mitochondrial dynamics and life-or-death events via Bcl-2 family proteins

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    International audienceThe morphology of a population of mitochondria is the result of several interacting dynamical phenomena, including fission, fusion, movement, elimination and biogenesis. Each of these phenomena is controlled by underlying molecular machinery, and when defective can cause disease. New understanding of the relationships between form and function of mitochondria in health and disease is beginning to be unraveled on several fronts. Studies in mammals and model organisms have revealed that mitochondrial morphology, dynamics and function appear to be subject to regulation by the same proteins that regulate apoptotic cell death. One protein family that influences mitochondrial dynamics in both healthy and dying cells is the Bcl-2 protein family. Connecting mitochondrial dynamics with life-death pathway forks may arise from the intersection of Bcl-2 family proteins with the proteins and lipids that determine mitochondrial shape and function. Bcl-2 family proteins also have multifaceted influences on cells and mitochondria, including calcium handling, autophagy and energetics, as well as the subcellular localization of mitochondrial organelles to neuronal synapses. The remarkable range of physical or functional interactions by Bcl-2 family proteins is challenging to assimilate into a cohesive understanding. Most of their effects may be distinct from their direct roles in apoptotic cell death and are particularly apparent in the nervous system. Dual roles in mitochondrial dynamics and cell death extend beyond BCL-2 family proteins. In this review, we discuss many processes that govern mitochondrial structure and function in health and disease, and how Bcl-2 family proteins integrate into some of these processes

    Regulation of Skeletal Muscle Mitochondrial, Quality Control by the Transcription Factors P53 and ATF4

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    Well-appreciated for its role in locomotion and posture, the metabolic profile of muscle has extended implications for mobility, along with the onset of disease. It is well-documented that endurance exercise promotes enhanced aerobic capacity, while prolonged disuse results in a diminished respiratory function, accompanied by fiber atrophy and weakness. A unique, and natural, feature of aging is the progressive loss of muscle mass, which develops from molecular changes within muscle that include diminished aerobic capacity and muscle strength. Thus, understanding the molecular mechanisms that promote muscle health or contribute to muscle decline are of considerable importance to identify therapeutic strategies that can preserve muscle function. Mitochondria are the culpable organelles in the maintenance of skeletal muscle metabolic health, and are therefore important regulators of a variety of factors contributing to muscle dysfunction, such as oxidative stress and antioxidants, apoptosis, inflammation, and Ca2+ handling, in addition to their role as the energy hubs of the cell. Mitochondrial quality control (MQC) involves multiple processes that coordinately regulate organellar biogenesis, turnover, and proteostasis (mitochondrial unfolded protein response) to maintain an optimal mitochondrial pool. While recent research has elucidated the importance of this synchronized control, science has yet characterized a single regulator of broad MQC pathways. The transcription factors p53 and ATF4 are two candidates that, in muscle, respond to various stressors, and are positioned at the nexus of these processes. Therefore, we explored the necessity of p53 and ATF4 in mediating mitochondrial adaptations. p53 muscle-specific knockout mice (mKO) had dysregulated signaling for MQC following 1-day of denervation- induced disuse, with further decrements in organelle function and MQC regulation after 7 days, especially with respect to the autophagy-lysosome system. Additionally, through ATF4 overexpression (OE) and knockdown (KD) in C2C12 myotubes, we found ATF4 to be an import mediator of MQC pathways, contributing to an enhanced mitochondrial network with augmented function, following C2C12 myotube differentiation, as well as acute and chronic contractile activity. Our data identify both p53 and ATF4 as two transcriptional regulators that each exhibit multifaceted control of mitochondrial health in muscle during both the pro- and mal-adaptive stimuli of exercise and disuse

    Redox remodelling in diaphragm muscle adaptation to chronic sustained hypoxia

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    Chronic sustained hypoxia (CH) induces functional weakness, atrophy, and mitochondrial remodelling in the diaphragm muscle. Animal models of CH present with changes similar to patients with respiratory-related disease, thus, elucidating the molecular mechanisms driving these adaptations is clinically important. We hypothesize that ROS are pivotal in diaphragm muscle adaptation to CH. C57BL6/J mice were exposed to CH (FiO2=0.1) for one, three, and six weeks. Sternohyoid (upper airway dilator), extensor digitorum longus (EDL), and soleus were studied as reference muscles as well as the diaphragm. The diaphragm was profiled using a redox proteomics approach followed by mass spectrometry. Following this, redox-modified metabolic enzyme activities and atrophy signalling were assessed using spectrophotometric assays and ELISA. Diaphragm isotonic performance was assessed after six weeks of CH ± chronic antioxidant supplementation. Protein carbonyl and free thiol content in the diaphragm were increased and decreased respectively after six weeks of CH – indicative of protein oxidation. These changes were temporally modulated and muscle specific. Extensive remodelling of metabolic proteins occurred and the stress reached the cross-bridge. Metabolic enzyme activities in the diaphragm were, for the most part, decreased by CH and differential muscle responses were observed. Redox sensitive chymotrypsin-like proteasome activity of the diaphragm was increased and atrophy signalling was observed through decreased phospho-FOXO3a and phospho-mTOR. Phospho-p38 MAPK content was increased and this was attenuated by antioxidant treatment. Hypoxia decreased power generating capacity of the diaphragm and this was restored by N-acetyl-cysteine (NAC) but not by tempol. Redox remodelling is pivotal for diaphragm adaptation to chronic sustained hypoxia. Muscle changes are dependent on duration of the hypoxia stimulus, activity profile of the muscle, and molecular composition of the muscle. The working respiratory muscles and slow oxidative fibres are particularly susceptible. NAC (antioxidant) may be useful as an adjunct therapy in respiratory-related diseases characterised by hypoxic stress

    Psr1p interacts with SUN/sad1p and EB1/mal3p to establish the bipolar spindle

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    Regular Abstracts - Sunday Poster Presentations: no. 382During mitosis, interpolar microtubules from two spindle pole bodies (SPBs) interdigitate to create an antiparallel microtubule array for accommodating numerous regulatory proteins. Among these proteins, the kinesin-5 cut7p/Eg5 is the key player responsible for sliding apart antiparallel microtubules and thus helps in establishing the bipolar spindle. At the onset of mitosis, two SPBs are adjacent to one another with most microtubules running nearly parallel toward the nuclear envelope, creating an unfavorable microtubule configuration for the kinesin-5 kinesins. Therefore, how the cell organizes the antiparallel microtubule array in the first place at mitotic onset remains enigmatic. Here, we show that a novel protein psrp1p localizes to the SPB and plays a key role in organizing the antiparallel microtubule array. The absence of psr1+ leads to a transient monopolar spindle and massive chromosome loss. Further functional characterization demonstrates that psr1p is recruited to the SPB through interaction with the conserved SUN protein sad1p and that psr1p physically interacts with the conserved microtubule plus tip protein mal3p/EB1. These results suggest a model that psr1p serves as a linking protein between sad1p/SUN and mal3p/EB1 to allow microtubule plus ends to be coupled to the SPBs for organization of an antiparallel microtubule array. Thus, we conclude that psr1p is involved in organizing the antiparallel microtubule array in the first place at mitosis onset by interaction with SUN/sad1p and EB1/mal3p, thereby establishing the bipolar spindle.postprin

    Removal of antagonistic spindle forces can rescue metaphase spindle length and reduce chromosome segregation defects

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    Regular Abstracts - Tuesday Poster Presentations: no. 1925Metaphase describes a phase of mitosis where chromosomes are attached and oriented on the bipolar spindle for subsequent segregation at anaphase. In diverse cell types, the metaphase spindle is maintained at a relatively constant length. Metaphase spindle length is proposed to be regulated by a balance of pushing and pulling forces generated by distinct sets of spindle microtubules and their interactions with motors and microtubule-associated proteins (MAPs). Spindle length appears important for chromosome segregation fidelity, as cells with shorter or longer than normal metaphase spindles, generated through deletion or inhibition of individual mitotic motors or MAPs, showed chromosome segregation defects. To test the force balance model of spindle length control and its effect on chromosome segregation, we applied fast microfluidic temperature-control with live-cell imaging to monitor the effect of switching off different combinations of antagonistic forces in the fission yeast metaphase spindle. We show that spindle midzone proteins kinesin-5 cut7p and microtubule bundler ase1p contribute to outward pushing forces, and spindle kinetochore proteins kinesin-8 klp5/6p and dam1p contribute to inward pulling forces. Removing these proteins individually led to aberrant metaphase spindle length and chromosome segregation defects. Removing these proteins in antagonistic combination rescued the defective spindle length and, in some combinations, also partially rescued chromosome segregation defects. Our results stress the importance of proper chromosome-to-microtubule attachment over spindle length regulation for proper chromosome segregation.postprin

    NF-κB activation in skeletal muscle during ageing: role in development of sarcopenia.

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    Sarcopenia is the loss of muscle mass and function in older age. An increase in pro-inflammatory cytokines is associated with many age-related conditions including sarcopenia. A chronic activation of nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) has been shown in muscles from old wild type (WT) and this has also been shown in a model of accelerated loss of muscle mass and function, mice lacking CuZn superoxide dismutase (SOD1KO mice). The consequence of such a chronic activation of NF-κB is unclear, but muscle is known to be an endocrine organ, with changes in the release of cytokines and chemokines, particularly following contractions where pro inflammatory cytokines can be released. In advancing age there is a notable decrease in muscle mass, with substantial loss of protein, suggesting that the balance between protein synthesis and degradation is net negative. Activation of NF-κB is also associated with activation of protein degradation processes. The aims of is thesis were to: 1) Quantify the changes in mass, structure and function of muscles of old WT and adult SOD1KO mice compared with adult WT mice; 2) Determine the localisation of nuclei with increase activation of the canonical pathway of NF-κB in muscles of old WT and adult SOD1KO compared with adult WT mice; 3) Identify whether any changes in cytokine and chemokine levels in the plasma of old WT and adult SOD1KO mice are associated with increased cytokine production by muscle and 4) to determine whether activation or inhibition of protein turnover pathways are associated with changes in NF-κB activity in muscles of old WT mice when compared with adult WT mice. Morphological and functional characteristics of muscle were examined using the tissue cell geometry plugin on ImageJ and MyoVision. Components of the NF-κB pathway were measured through qPCR and western blotting of lysates from gastrocnemius muscles and by immunohistochemistry analysis of EDL muscles. The levels of cytokines and chemokines were determined in muscle lysates and plasma and in media derived from isolated muscle fibres from adult WT, old WT and adult SOD1KO mice. The association of nuclear localisation of p65 with the regenerative stage of the muscle fibre was also examined in a model of regenerating extensor digitorum longus (EDL) muscles from adult WT, old WT and SOD1KO mice following injury induced via BaCl2 injection into the EDL muscle. Finally, fractional synthesis rates of individual proteins were determined in muscles of adult WT and old WT mice using heavy water SILAM where mice and unlabelled proteomics was also performed on these mice. The datasets from these experiments were compared with an RNA sequencing dataset to determine whether transcription was the driving factor in differences observed in fractional synthesis rates. There was an increase in inflammatory cytokines/chemokines in the plasma of old WT mice and an increase in specific chemokines in muscle homogenates or released from isolated muscle fibres from old WT muscles, suggesting that muscle of old WT mice may be a source of specific cytokines/chemokines, some of which add to the plasma pool and others acting more locally. The levels and patterns of cytokines/chemokines in plasma and muscle of adult SOD1KO mice were very different, suggesting that these mice are a poor model of inflammaging. Higher levels of p65 (the major transcription factor involved with canonical NF-κB activation) were observed in muscle fibres containing a centrally positioned nucleus, seen particularly in muscles of SOD1KO mice and during regeneration following chemically induced muscle damage but it is unlikely that this activation results in a substantial increase in release of cytokines/chemokines by these muscle fibres. Bioinformatic analysis of the unlabelled proteomics dataset revealed there was a clear upregulation of protein degradation pathways and protein misfolding pathways in the muscles of old WT mice. There was little evidence for changes in overall protein synthesis rates suggesting that increased protein degradation is the main driver of muscle protein loss with age. Increased activation of NF-κB may play a role in increased activation of degenerative pathways in muscles of old WT mice, but further analyses including inhibitor studies are required to confirm the role of NF-κB
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