Search

Background: Oculopharyngeal muscular dystrophy (OPMD) is a rare, late-onset, slowly progressive neuromuscular disorder characterized by ptosis, dysphagia, and proximal limb weakness. Emerging clinical trials require rapidly accessible and sensitive biomarkers to evaluate OPMD disease progression and potential response to future treatments., Objective: This cross-sectional study was designed to identify candidate circulating protein and imaging biomarkers of OPMD severity for future use in clinical trials., Methods: Twenty-five individuals with OPMD (age 63.3 ± 10.5 years; GCN copy number of 13 in PABPN1 ) were assessed using the 7k SOMAScan assay to profile the plasma proteome, and MRI to quantify replacement of muscle by fat. OPMD severity was first categorized using the clinical presence/absence of limb weakness, and protein signals were considered distinguishing if they differed by more than 30% between subgroups and had statistically significant P -values after correcting for multiple comparisons. Distinguishing proteins were contrasted with age-matched controls ( n  = 10). OPMD severity was also treated as a continuous variable using fat fraction of the soleus muscle, and proteins were considered distinguishing if the slope of relationship between protein signal and soleus fat fraction differed significantly from zero after correcting for multiple comparisons. Pathway analyses were conducted using Metascape and the Database for Annotation, Visualization, and Integrated Discovery webtools., Results: Eighteen plasma proteins distinguished OPMD on both indicators of severity. Pathway analyses identified skeletal muscle tissue, phagocytosis/engulfment, and extracellular matrix organization as enriched ontology clusters in OPMD with limb weakness. The most distinguishing plasma protein signals (ACTN2, MYOM2, CA3, APOBEC2, MYL3, and PDLIM3) were over 200% higher in OPMD with limb weakness than OPMD without limb weakness as well as controls, and correlated strongly with percent of fatty replacement of soleus ( r  = 0.89 ± 0.04)., Conclusions: The candidate biomarkers identified contribute to the ongoing search for sensitive and accessible biomarkers of OPMD progression, prognosis, and monitoring., Competing Interests: Declaration of conflicting interestsDr Hanns Lochmuller is an Editorial Board Member of this journal, but was not involved in the peer-review process nor had access to any information regarding its peer-review.

Mitochondrial function is typically assessed by measuring oxygen consumption at a given time point. However, this approach cannot monitor respiratory changes that occur over time. Here, we present a protocol to measure mitochondrial respiration in freshly isolated muscle stem cells, primary skeletal muscle, and immortalized C2C12 myoblasts in real time using the Resipher platform. We describe steps for preparing and plating cells, performing media changes, setting up the software and device, and analyzing data. This method can be adapted to other cell types. For complete details on the use and execution of this protocol, please refer to Triolo et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Mitochondrial dynamics orchestrate many essential cellular functions, including metabolism, which is instrumental in promoting cancer growth and metastatic progression. However, how mitochondrial dynamics influences metastatic progression remains poorly understood. Here, we show that breast cancer cells with low metastatic potential exhibit a more fused mitochondrial network compared to highly metastatic cells. To study the impact of mitochondrial dynamics on metastasis, we promoted mitochondrial elongation in metastatic breast cancer cells by individual genetic deletion of three key regulators of mitochondrial fission (Drp1, Fis1, Mff) or by pharmacological intervention with leflunomide. Omics analyses revealed that mitochondrial elongation causes substantial alterations in metabolic pathways and processes related to cell adhesion. In vivo, enhanced mitochondrial elongation by loss of mitochondrial fission mediators or treatment with leflunomide notably reduced metastasis formation. Furthermore, the transcriptomic signature associated with elongated mitochondria correlated with improved clinical outcome in patients with breast cancer. Overall, our findings highlight mitochondrial dynamics as a potential therapeutic target in breast cancer.

From quiescence to activation and myogenic differentiation, muscle stem cells (MuSCs) experience drastic alterations in their signaling activity and metabolism. Through balanced cycles of fission and fusion, mitochondria alter their morphology and metabolism, allowing them to affect their decisive role in modulating MuSC activity and fate decisions. This tightly regulated process contributes to MuSC regulation by mediating changes in redox signaling pathways, cell cycle progression, and cell fate decisions. In this review, we discuss the role of mitochondrial dynamics as an integral modulator of MuSC activity, fate, and maintenance. Understanding the influence of mitochondrial dynamics in MuSCs in health and disease will further the development of therapeutics that support MuSC integrity and thus may aid in restoring the regenerative capacity of skeletal muscle.

Impaired mitochondrial function is a hallmark of aging and a major contributor to neurodegenerative diseases. We have shown that disrupted mitochondrial dynamics typically found in aging alters the fate of neural stem cells (NSCs) leading to impairments in learning and memory. At present, little is known regarding the mechanisms by which neural stem and progenitor cells survive and adapt to mitochondrial dysfunction. Using Opa1-inducible knockout as a model of aging and neurodegeneration, we identify a decline in neurogenesis due to impaired stem cell activation and progenitor proliferation, which can be rescued by the mitigation of oxidative stress through hypoxia. Through sc-RNA-seq, we identify the ATF4 pathway as a critical mechanism underlying cellular adaptation to metabolic stress. ATF4 knockdown in Opa1-deficient NSCs accelerates cell death, while the increased expression of ATF4 enhances proliferation and survival. Using a Slc7a11 mutant, an ATF4 target, we show that ATF4-mediated glutathione production plays a critical role in maintaining NSC survival and function under stress conditions. Together, we show that the activation of the integrated stress response (ISR) pathway enables NSCs to adapt to metabolic stress due to mitochondrial dysfunction and metabolic stress and may serve as a therapeutic target to enhance NSC survival and function in aging and neurodegeneration., (© 2024 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

Maintenance of mitochondrial function plays a crucial role in the regulation of muscle stem cell (MuSC), but the underlying mechanisms remain ill defined. In this study, we monitored mitophagy in MuSCS under various myogenic states and examined the role of PINK1 in maintaining regenerative capacity. Results indicate that quiescent MuSCs actively express mitophagy genes and exhibit a measurable mitophagy flux and prominent mitochondrial localization to autophagolysosomes, which become rapidly decreased during activation. Genetic disruption of Pink1 in mice reduces PARKIN recruitment to mitochondria and mitophagy in quiescent MuSCs, which is accompanied by premature activation/commitment at the expense of self-renewal and progressive loss of muscle regeneration, but unhindered proliferation and differentiation capacity. Results also show that impaired fate decisions in PINK1-deficient MuSCs can be restored by scavenging excess mitochondrial ROS. These data shed light on the regulation of mitophagy in MuSCs and position PINK1 as an important regulator of their mitochondrial properties and fate decisions., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Myogenic differentiation is integral for the regeneration of skeletal muscle following tissue damage. Though high-energy post-mitotic muscle relies predominantly on mitochondrial respiration, the importance of mitochondrial remodeling in enabling muscle differentiation and the players involved are not fully known. Here we show that the mitochondrial fusion protein OPA1 is essential for muscle differentiation. Our study demonstrates that OPA1 loss or inhibition, through genetic and pharmacological means, abolishes in vivo muscle regeneration and in vitro myotube formation. We show that both the inhibition and genetic deletion of OPA1 prevent the early onset metabolic switch required to drive myoblast differentiation. In addition, we observe an OPA1-dependent upregulation of the supercomplex assembly factor, SCAF1, at the onset of differentiation. Importantly, preventing the upregulation of SCAF1, through OPA1 loss or siRNA-mediated SCAF1 knockdown, impairs metabolic reprogramming and muscle differentiation. These findings reveal the integral role of OPA1 and mitochondrial reprogramming at the onset of myogenic differentiation., Competing Interests: The authors declare no competing interests., (© 2024 The Author(s).)

Since changes in mitochondrial morphology regulate key functions of stem cells, it is important to assess their structure under physiological and pathophysiological conditions. Here, we present techniques optimized in rare adult muscle stem cells (MuSCs). For evaluating mitochondrial length and volume within a compact cytoplasmic area in MuSCs on intact myofibers, we describe steps for mitochondrial staining, imaging, and quantification. For evaluating mitochondrial ultrastructure in small cell numbers, we describe steps for agarose embedding and quantification by TEM. For complete details on generation and use of this protocol, please refer to Baker et al. (2022). 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

During the inflammatory response, macrophage phenotypes can be broadly classified as pro-inflammatory/classically activated "M1", or pro-resolving/alternatively "M2" macrophages. Although the classification of macrophages is general and assumes there are distinct phenotypes, in reality macrophages exist across a spectrum and must transform from a pro-inflammatory state to a proresolving state following an inflammatory insult. To adapt to changing metabolic needs of the cell, mitochondria undergo fusion and fission, which have important implications for cell fate and function. We hypothesized that mitochondrial fission and fusion directly contribute to macrophage function during the pro-inflammatory and proresolving phases. In the present study, we find that mitochondrial length directly contributes to macrophage phenotype, primarily during the transition from a pro-inflammatory to a proresolving state. Phenocopying the elongated mitochondrial network (by disabling the fission machinery using siRNA) leads to a baseline reduction in the inflammatory marker IL-1β, but a normal inflammatory response to LPS, similar to control macrophages. In contrast, in macrophages with a phenocopied fragmented phenotype (by disabling the fusion machinery using siRNA) there is a heightened inflammatory response to LPS and increased signaling through the ATF4/c-Jun transcriptional axis compared to control macrophages. Importantly, macrophages with a fragmented mitochondrial phenotype show increased expression of proresolving mediator arginase 1 and increased phagocytic capacity. Promoting mitochondrial fragmentation caused an increase in cellular lactate, and an increase in histone lactylation which caused an increase in arginase 1 expression. These studies demonstrate that a fragmented mitochondrial phenotype is critical for the proresolving response in macrophages and specifically drive epigenetic changes via lactylation of histones following an inflammatory insult.

Quiescence regulation is essential for adult stem cell maintenance and sustained regeneration. Our studies uncovered that physiological changes in mitochondrial shape regulate the quiescent state of adult muscle stem cells (MuSCs). We show that MuSC mitochondria rapidly fragment upon an activation stimulus, via systemic HGF/mTOR, to drive the exit from deep quiescence. Deletion of the mitochondrial fusion protein OPA1 and mitochondrial fragmentation transitions MuSCs into G-alert quiescence, causing premature activation and depletion upon a stimulus. OPA1 loss activates a glutathione (GSH)-redox signaling pathway promoting cell-cycle progression, myogenic gene expression, and commitment. MuSCs with chronic OPA1 loss, leading to mitochondrial dysfunction, continue to reside in G-alert but acquire severe cell-cycle defects. Additionally, we provide evidence that OPA1 decline and impaired mitochondrial dynamics contribute to age-related MuSC dysfunction. These findings reveal a fundamental role for OPA1 and mitochondrial dynamics in establishing the quiescent state and activation potential of adult stem cells., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Mitochondria are considered the metabolic hubs within a cell. These organelles are highly dynamic and continuously undergo cycles of fission and fusion events. The balance in the dynamic state of mitochondria is critical for maintaining key physiological events within cells. Here we discuss the emerging role of mitochondrial dynamics in regulating stem cell function and highlight the crosstalk between mitochondrial shape and intracellular signaling cascades within the context of stem cells., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Mitochondria are dynamic organelles that rely on a balance of opposing fission and fusion events to sustain mitochondrial function and efficiently meet the energy demands of a cell. As high-energy demanding cells, neurons rely heavily on optimally functional mitochondria with balanced mitochondrial dynamics, to ensure a sufficient energy supply required to maintain cell survival, establish membrane excitability and partake in processes of neurotransmission and plasticity. As such, many neurodegenerative diseases (e.g., Alzheimer's disease, Parkinson's disease) and stress conditions (e.g., stroke) leading to neuronal dysfunction or death are often associated with impaired mitochondrial function and dynamics, characterized by excessive mitochondrial fragmentation. For this reason, the assessment of mitochondrial morphology in neurons and within the brain can provide valuable information. The dynamic nature of mitochondria is not only observed in shape changes, but also changes in mitochondrial network connectivity and in cristae architecture. In this chapter, we will describe how mitochondrial morphology can be examined in vitro using hippocampal neuronal cultures and in vivo using mouse brain sections by immunocytochemistry, immunohistochemistry, and electron microscopy techniques., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Mitochondrial reactive oxygen species (mtROS) and redox regulation play an important role in stem cell maintenance and cell fate decisions. Although changes in mtROS and redox homeostasis represent a physiological mechanism to drive stem cell commitment and differentiation, dysregulation of this system can lead to defects in stem cell maintenance and regenerative capacity. This chapter explains the methods used to assess mitochondrial superoxide levels and redox regulation in stem cell populations.

The fundamental importance of functional mitochondria in the survival of most eukaryotic cells, through regulation of bioenergetics, cell death, calcium dynamics and reactive oxygen species (ROS) generation, is undisputed. However, with new avenues of research in stem cell biology these organelles have now emerged as signaling entities, actively involved in many aspects of stem cell functions, including self-renewal, commitment and differentiation. With this recent knowledge, it becomes evident that regulatory pathways that would ensure the maintenance of mitochondria with state-specific characteristics and the selective removal of organelles with sub-optimal functions must play a pivotal role in stem cells. As such, mitophagy, as an essential mitochondrial quality control mechanism, is beginning to gain appreciation within the stem cell field. Here we review and discuss recent advances in our knowledge pertaining to the roles of mitophagy in stem cell functions and the potential contributions of this specific quality control process on to the progression of aging and diseases.

Polyphosphates (polyPs) are long chains of inorganic phosphates linked by phosphoanhydride bonds. They are found in all kingdoms of life, playing roles in cell growth, infection, and blood coagulation. Unlike in bacteria and lower eukaryotes, the mammalian enzymes responsible for polyP metabolism are largely unexplored. We use RNA sequencing (RNA-seq) and mass spectrometry to define a broad impact of polyP produced inside of mammalian cells via ectopic expression of the E. coli polyP synthetase PPK. We find that multiple cellular compartments can support accumulation of polyP to high levels. Overproduction of polyP is associated with reprogramming of both the transcriptome and proteome, including activation of the ERK1/2-EGR1 signaling axis. Finally, fractionation analysis shows that polyP accumulation results in relocalization of nuclear/cytoskeleton proteins, including targets of non-enzymatic lysine polyphosphorylation. Our work demonstrates that internally produced polyP can activate diverse signaling pathways in human cells., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Mitochondria play a crucial role in neuronal survival through efficient energy metabolism. In pathological conditions, mitochondrial stress leads to neuronal death, which is regulated by the anti-apoptotic BCL-2 family of proteins. MCL-1 is an anti-apoptotic BCL-2 protein localized to mitochondria either in the outer membrane (OM) or inner membrane (Matrix), which have distinct roles in inhibiting apoptosis and promoting bioenergetics, respectively. While the anti-apoptotic role for Mcl1 is well characterized, the protective function of MCL-1 Matrix remains poorly understood. Here, we show MCL-1 OM and MCL-1 Matrix prevent neuronal death through distinct mechanisms. We report that MCL-1 Matrix functions to preserve mitochondrial energy transduction and improves respiratory chain capacity by modulating mitochondrial oxygen consumption in response to mitochondrial stress. We show that MCL-1 Matrix protects neurons from stress by enhancing respiratory function, and by inhibiting mitochondrial permeability transition pore opening. Taken together, our results provide novel insight into how MCL-1 Matrix may confer neuroprotection under stress conditions involving loss of mitochondrial function.

The dynamic and fluid nature of mitochondria allows for modifications in mitochondrial shape, connectivity and cristae architecture. The precise balance of mitochondrial dynamics is among the most critical features in the control of mitochondrial function. In the past few years, mitochondrial shape has emerged as a key regulatory factor in the determination of the bioenergetic capacity of cells. This is mostly due to the recent discoveries linking changes in cristae organization with supercomplex assembly of the electron transport chain (ETC), also defined as the formation of respirosomes. Here we will review the most current advances demonstrating the impact of mitochondrial dynamics and cristae shape on oxidative metabolism, respiratory efficiency, and redox state. Furthermore, we will discuss the implications of mitochondrial dynamics and supercomplex assembly under physiological and pathological conditions., (Copyright © 2019 Elsevier B.V. and Mitochondria Research Society. All rights reserved.)

Emerging evidence now indicates that mitochondria are central regulators of neural stem cell (NSC) fate decisions and are crucial for both neurodevelopment and adult neurogenesis, which in turn contribute to cognitive processes in the mature brain. Inherited mutations and accumulated damage to mitochondria over the course of ageing serve as key factors underlying cognitive defects in neurodevelopmental disorders and neurodegenerative diseases, respectively. In this Review, we explore the recent findings that implicate mitochondria as crucial regulators of NSC function and cognition. In this respect, mitochondria may serve as targets for stem-cell-based therapies and interventions for cognitive defects.

Significance: Recent discoveries in mitochondrial biology have transformed and further solidified the importance of mitochondria in development, aging, and disease. Within the realm of regenerative and stem cell research, these recent advances have brought forth new concepts that revolutionize our understanding of metabolic and redox states in the establishment of cellular identity and fate decisions. Recent Advances: Mitochondrial metabolism, morphology, and cellular redox states are dynamic characteristics that undergo shifts during stem cell differentiation. Although it was once thought that this was solely because of changing metabolic needs of differentiating cells, it is now clear that these events are driving forces in the regulation of stem cell identity and fate decisions. Critical Issues: Although recent discoveries have placed mitochondrial function and physiological reactive oxygen species (ROS) at the forefront for the regulation of stem cell self-renewal, how this may impact tissue homeostasis and regenerative capacity is poorly understood. In addition, the role of mitochondria and ROS on the maintenance of a stem cell population in many degenerative diseases and during aging is not clear, despite the fact that mitochondrial dysfunction and elevated ROS levels are commonly observed in these conditions. Future Directions: Given the newly established role for mitochondria and ROS in stem cell self-renewal capacity, special attention should now be directed in understanding how this would impact the development and progression of aging and diseases, whereby mitochondrial and ROS defects are a prominent factor. Antioxid. Redox Signal. 28, 1090-1101.

Mitochondria are classically known to be the cellular energy producers, but a renewed appreciation for these organelles has developed with the accumulating discoveries of additional functions. The importance of mitochondria within the brain has been long known, particularly given the high-energy demanding nature of neurons. The energy demands imposed by neurons require the well-orchestrated morphological adaptation and distribution of mitochondria. Recent studies now reveal the importance of mitochondrial dynamics not only in mature neurons but also during neural development, particularly during the process of neurogenesis and neural stem cell fate decisions. In this review, we will highlight the recent findings that illustrate the importance of mitochondrial dynamics in neurodevelopment and neural stem cell function. Developmental Dynamics 247:47-53, 2018. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

Mitochondria are classically known as the essential energy producers in cells. As such, the activation of mitochondrial metabolism upon cellular differentiation was deemed a necessity to fuel the high metabolic needs of differentiated cells. However, recent studies have revealed a direct role for mitochondrial activity in the regulation of stem cell fate and differentiation. Several components of mitochondrial metabolism and respiration have now been shown to regulate different aspects of stem cell differentiation through signaling, transcriptional, proteomic and epigenetic modulations. In light of these findings mitochondrial metabolism is no longer considered a consequence of cellular differentiation, but rather a key regulatory mechanism of this process. This review will focus on recent progress that defines mitochondria as the epicenters for the regulation of stem cell fate decisions., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Mitochondrial dysfunction is a common feature of many genetic disorders that target the brain and cognition. However, the exact role these organelles play in the etiology of such disorders is not understood. Here, we show that mitochondrial dysfunction impairs brain development, depletes the adult neural stem cell (NSC) pool and impacts embryonic and adult neurogenesis. Using deletion of the mitochondrial oxidoreductase AIF as a genetic model of mitochondrial and neurodegenerative diseases revealed the importance of mitochondria in multiple steps of the neurogenic process. Developmentally, impaired mitochondrial function causes defects in NSC self-renewal, neural progenitor cell proliferation and cell cycle exit, as well as neuronal differentiation. Sustained mitochondrial dysfunction into adulthood leads to NSC depletion, loss of adult neurogenesis and manifests as a decline in brain function and cognitive impairment. These data demonstrate that mitochondrial dysfunction, as observed in genetic mitochondrial and neurodegenerative diseases, underlies the decline of brain function and cognition due to impaired stem cell maintenance and neurogenesis., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The amyloid state of protein organization is typically associated with debilitating human neuropathies and is seldom observed in physiology. Here, we uncover a systemic program that leverages the amyloidogenic propensity of proteins to regulate cell adaptation to stressors. On stimulus, cells assemble the amyloid bodies (A-bodies), nuclear foci containing heterogeneous proteins with amyloid-like biophysical properties. A discrete peptidic sequence, termed the amyloid-converting motif (ACM), is capable of targeting proteins to the A-bodies by interacting with ribosomal intergenic noncoding RNA (rIGSRNA). The pathological β-amyloid peptide, involved in Alzheimer's disease, displays ACM-like activity and undergoes stimuli-mediated amyloidogenesis in vivo. Upon signal termination, elements of the heat-shock chaperone pathway disaggregate the A-bodies. Physiological amyloidogenesis enables cells to store large quantities of proteins and enter a dormant state in response to stressors. We suggest that cells have evolved a post-translational pathway that rapidly and reversibly converts native-fold proteins to an amyloid-like solid phase., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Regulated mechanisms of stem cell maintenance are key to preventing stem cell depletion and aging. While mitochondrial morphology plays a fundamental role in tissue development and homeostasis, its role in stem cells remains unknown. Here, we uncover that mitochondrial dynamics regulates stem cell identity, self-renewal, and fate decisions by orchestrating a transcriptional program. Manipulation of mitochondrial structure, through OPA1 or MFN1/2 deletion, impaired neural stem cell (NSC) self-renewal, with consequent age-dependent depletion, neurogenesis defects, and cognitive impairments. Gene expression profiling revealed ectopic expression of the Notch self-renewal inhibitor Botch and premature induction of transcription factors that promote differentiation. Changes in mitochondrial dynamics regulate stem cell fate decisions by driving a physiological reactive oxygen species (ROS)-mediated process, which triggers a dual program to suppress self-renewal and promote differentiation via NRF2-mediated retrograde signaling. These findings reveal mitochondrial dynamics as an upstream regulator of essential mechanisms governing stem cell self-renewal and fate decisions through transcriptional programming., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Cristae, the organized invaginations of the mitochondrial inner membrane, respond structurally to the energetic demands of the cell. The mechanism by which these dynamic changes are regulated and the consequences thereof are largely unknown. Optic atrophy 1 (OPA1) is the mitochondrial GTPase responsible for inner membrane fusion and maintenance of cristae structure. Here, we report that OPA1 responds dynamically to changes in energetic conditions to regulate cristae structure. This cristae regulation is independent of OPA1's role in mitochondrial fusion, since an OPA1 mutant that can still oligomerize but has no fusion activity was able to maintain cristae structure. Importantly, OPA1 was required for resistance to starvation-induced cell death, for mitochondrial respiration, for growth in galactose media and for maintenance of ATP synthase assembly, independently of its fusion activity. We identified mitochondrial solute carriers (SLC25A) as OPA1 interactors and show that their pharmacological and genetic blockade inhibited OPA1 oligomerization and function. Thus, we propose a novel way in which OPA1 senses energy substrate availability, which modulates its function in the regulation of mitochondrial architecture in a SLC25A protein-dependent manner., (© 2014 The Authors.)

Sustained cellular function and viability of high-energy demanding post-mitotic cells rely on the continuous supply of ATP. The utilization of mitochondrial oxidative phosphorylation for efficient ATP generation is a function of oxygen levels. As such, oxygen deprivation, in physiological or pathological settings, has profound effects on cell metabolism and survival. Here we show that mild extracellular acidosis, a physiological consequence of anaerobic metabolism, can reprogramme the mitochondrial metabolic pathway to preserve efficient ATP production regardless of oxygen levels. Acidosis initiates a rapid and reversible homeostatic programme that restructures mitochondria, by regulating mitochondrial dynamics and cristae architecture, to reconfigure mitochondrial efficiency, maintain mitochondrial function and cell survival. Preventing mitochondrial remodelling results in mitochondrial dysfunction, fragmentation and cell death. Our findings challenge the notion that oxygen availability is a key limiting factor in oxidative metabolism and brings forth the concept that mitochondrial morphology can dictate the bioenergetic status of post-mitotic cells.

Mitochondrial dysfunction plays an important role in the etiology of neurodegenerative diseases. However, the progressive nature of neuronal loss in genetic models of mitochondrial dysfunction suggests the presence of compensatory mechanisms promoting neuronal survival under these conditions. Here, we identified the energy metabolism kinase LKB1 as a key regulator of the compensatory mechanisms activated in neurons, following mitochondrial dysfunction. To accomplish this, we have created an in vivo neurodegenerative model based on the deletion of the mitochondrial protein apoptosis-inducing factor (AIF) in postmitotic neurons. Loss of mitochondrial function caused by AIF deletion induced several adaptive mechanisms, including increased glycolysis and mitochondrial biogenesis. Importantly, the activation of these adaptive mechanisms was abrogated by the deletion of one allele of LKB1, resulting in impaired neuronal survival. Because loss of mitochondrial function is a central mechanism implicated in neurodegenerative diseases, modulation of LKB1-dependent pathways may represent an important strategy to preserve neuronal survival and function.

The von Hippel-Lindau (VHL) tumor suppressor protein, the recognition component of an E3 ubiquitin ligase complex, recruits the alpha-subunit of the hypoxia-inducible factor (HIFalpha) for oxygen-dependent degradation. The ability of VHL to mediate efficient degradation of HIFalpha is also dependent on its oxygen/pH-regulated subcellular trafficking. Under aerobic conditions, VHL engages in nuclear-cytoplasmic trafficking that requires ongoing transcription and is mediated by a novel nuclear export motif, the transcription-dependent nuclear export motif (TD-NEM). Disease-causing mutations targeting TD-NEM restrain VHL from mediating efficient oxygen-dependent degradation of HIFalpha by altering its subcellular dynamics. In addition, decreasing the extracellular pH, during anaerobic metabolism, stabilizes HIFalpha by triggering the relocalization and static detention of VHL to nucleoli. Together, these recent findings support the critical role of subcellular trafficking and dynamic properties for the function of VHL in promoting HIF regulation and tumor suppression.

The cytoplasmic translation factor eEF1A has been implicated in the nuclear export of tRNA species in lower eukaryotes. Here we demonstrate that eEF1A plays a central role in nuclear export of proteins in mammalian cells. TD-NEM (transcription-dependent nuclear export motif), a newly characterized nuclear export signal, mediates efficient nuclear export of several proteins including the von Hippel-Lindau (VHL) tumor suppressor and the poly(A)-binding protein (PABP1) in a manner that is dependent on ongoing RNA polymerase II (RNA PolII)-dependent transcription. eEF1A interacts specifically with TD-NEM of VHL and PABP1 and disrupting this interaction, by point mutations of key TD-NEM residues or treatment with actinomycin D, an inhibitor of RNA PolII-dependent transcription, prevents assembly and nuclear export. siRNA-induced knockdown or antibody-mediated depletion of eEF1A prevents in vivo and in vitro nuclear export of TD-NEM-containing proteins. Nuclear retention experiments and inhibition of the Exportin-5 pathway suggest that eEF1A stimulates nuclear export of proteins from the cytoplasmic side of the nuclear envelope, without entering the nucleus. Together, these data identify a role for eEF1A, a cytoplasmic mediator of tRNA export in yeast, in the nuclear export of proteins in mammalian cells. These results also provide a link between the translational apparatus and subcellular trafficking machinery demonstrating that these two central pathways in basic metabolism can act cooperatively.

It is thought that degradation of nuclear proteins by the ubiquitylation system requires nuclear-cytoplasmic trafficking of E3 ubiquitin ligases. The von Hippel-Lindau (VHL) tumor suppressor protein is the substrate recognition component of a Cullin-2-containing E3 ubiquitin ligase that recruits hypoxia-inducible factor (HIF) for oxygen-dependent degradation. We demonstrated that VHL engages in nuclear-cytoplasmic trafficking that requires ongoing transcription to promote efficient HIF degradation. Here, we report the identification of a discreet motif, DXGX(2)DX(2)L, that directs transcription-dependent nuclear export of VHL and which is targeted by naturally occurring mutations associated with renal carcinoma and polycythemia in humans. The DXGX(2)DX(2)L motif is also found in other proteins, including poly(A)-binding protein 1, to direct its transcription-dependent nuclear export. We define DXGX(2)DX(2)L as TD-NEM (transcription-dependent nuclear export motif), since inhibition of transcription by actinomycin D or 5,6-dichlorobenzimidazole abrogates its nuclear export activity. Disease-causing mutations of key residues of TD-NEM restrain the ability of VHL to efficiently mediate oxygen-dependent degradation of HIF by altering its nuclear export dynamics without affecting interaction with its substrate. These results identify a novel nuclear export motif, further highlight the role of nuclear-cytoplasmic shuttling of E3 ligases in degradation of nuclear substrates, and provide evidence that disease-causing mutations can target subcellular trafficking.

Proteins share peptidic sequences, such as a nuclear localization signal (NLS), which guide them to particular membrane-bound compartments. Similarities have also been observed within different classes of signals that target proteins to membrane-less subnuclear compartments. Common localization signals affect spatial and temporal subcellular organization and are thought to allow the coordinated response of different molecular networks to a given signaling cue. Here we identify a higher-order and predictive code, {[RR(I/L)X(3)r]((n, n > or = 1))+[L(phi/N)(V/L)]((n,n>1))}, that establishes high-affinity interactions between a group of proteins and the nucleolus in response to a specific signal. This position-independent code is referred to as a nucleolar detention signal regulated by H(+) (NoDS(H+)) and the class of proteins includes the cIAP2 apoptotic regulator, VHL ubiquitylation factor, HSC70 heat shock protein and RNF8 transcription regulator. By identifying a common subnuclear targeting consensus sequence, our work reveals rules governing the dynamics of subnuclear organization and ascribes new modes of regulation to several proteins with diverse steady-state distributions and dynamic properties.

Biological evolution abides by an unbending rule obligating organisms to maintain energy equilibrium. Hypoxia reduces cellular energy supply and is thus thought to be deleterious. We report that cells have evolved pH-sensitive mechanisms to maintain energy equilibrium by lowering energy demand. We found that fermentation-induced acidosis allows hypoxic cells to maintain energy equilibrium and viability under hypoxia by restricting ribosomal biogenesis, the most energy-demanding cellular process. Acidosis triggers nucleolar condensation, decreases precursor rRNA synthesis, reduces the dynamic profile of the RNA polymerase I preinitiation factor UBF1 and its interaction with the promoter of rRNA genes (rDNA). These changes require the pH-dependent interaction of the statically detained von Hippel-Lindau tumor suppressor protein (VHL) with rDNA. This phenomenon is promoted by, but does not require, activation of the hypoxia-inducible factor (HIF), a transcription factor implicated in extracellular acidification, energy production and oxygen homeostasis. Abrogating this program by silencing VHL expression, competing rDNA-VHL interaction or preventing environmental acidification triggers energy starvation and cell death under hypoxia. Our data suggest that oxygen-starved cells maintain energy equilibrium by gauging the environmental concentration of H(+) to statically detain VHL to nucleolar rDNA and restrict ribosome production. These findings also provide an explanation for the protective effect of acidosis in ischemic settings such as development, stroke and cancer.

Cellular pathways relay information through dynamic protein interactions. We have assessed the kinetic properties of the murine double minute protein (MDM2) and von Hippel-Lindau (VHL) ubiquitin ligases in living cells under physiological conditions that alter the stability of their respective p53 and hypoxia-inducible factor substrates. Photobleaching experiments reveal that MDM2 and VHL are highly mobile proteins in settings where their substrates are efficiently degraded. The nucleolar architecture converts MDM2 and VHL to a static state in response to regulatory cues that are associated with substrate stability. After signal termination, the nucleolus is able to rapidly release these proteins from static detention, thereby restoring their high mobility profiles. A protein surface region of VHL's beta-sheet domain was identified as a discrete [H+]-responsive nucleolar detention signal that targets the VHL/Cullin-2 ubiquitin ligase complex to nucleoli in response to physiological fluctuations in environmental pH. Data shown here provide the first evidence that cells have evolved a mechanism to regulate molecular networks by reversibly switching proteins between a mobile and static state.

Hypoxia and acidosis are common features of several physiological and pathological situations, including cancer and stroke. The HIF (hypoxia-inducible factor) transcription factor plays a seminal role in orchestrating cellular responses to alterations in oxygen availability. HIF is degraded in normal oxygen tension by the VHL (von Hippel-Lindau) tumor suppressor protein but stabilized by hypoxia to activate an array of genes implicated in oxygen homeostasis including vascular endothelial growth factor. Cells respond to a comparatively mild decline in oxygen tension by converting to an anaerobic state of respiration and secreting lactic acid. We recently reported that a decrease in environmental pH triggers sequestration of VHL into the nucleolus neutralizing its ability to degrade HIF. This implies that cells have evolved a parallel mechanism of HIF activation that responds to changes in oxygen levels by sensing extracellular [H+]. Here we discuss the implications of this new VHL regulatory mechanism on oxygen homeostasis and hypoxic cell memory.