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Biology subjects

Enriquez, J. A.

Publications and source records attributed to Enriquez, J. A..

At least 19 recordsLinked to original sources

Butyrate extends health and lifespan in mice with mitochondrial deficiency

Mitochondrial diseases progressively lead to multisystemic failure with treatment options remaining extremely limited. To investigate novel strategies that alleviate mitochondrial dysfunction, we have generated an ubiquitous and tamoxifen-inducible knockout mouse model of mitochondrial transcription factor A (TFAM), a nuclear-encoded protein involved in mitochondrial DNA (mtDNA) maintenance -- Tfamfl/flUbCre-ERT2 (iTfamKO) mice. Systemic TFAM deficiency triggers mitochondrial decline in a myriad of tissues in adult mice. Consequently, iTfamKO mice manifest multiorgan dysfunction including lipodystrophy, sarcopenia, metabolic alterations, kidney failure, neurodegeneration, and locomotor dysregulation, which result in the premature death of these mice. Interestingly, iTfamKO mice display intestinal barrier disruption and gut dysbiosis, with diminished levels of microbiota-derived short-fatty acids (SCFAs), such as butyrate. Mice with a deficient proof-reading version of the mtDNA polymerase gamma (mtDNA-mutator mice) phenocopy the dysfunction of the intestinal barrier and bacterial dysbiosis with reduced levels of butyrate, suggesting that different mouse models of mitochondrial dysfunction share deficient generation of butyrate. Transfer of microbiota from healthy control mice or administration of tributyrin, a butyrate precursor, delay multiple signs of multimorbidity extending lifespan in iTfamKO mice. Mechanistically, butyrate supplementation recovers epigenetic histone acylation marks that are lost in the intestine of Tfam deficient mice. Overall, our findings highlight the relevance of preserving host-microbiota symbiosis in disorders related to mitochondrial dysfunction.

molecular biology↗

Astrocytic glycolysis attenuates mitochondrial efficiency to preserve cognition

Astrocytic glycolysis is tightly coupled to neurotransmission and thought to be essential for neurological health. However, the metabolic adaptations that enable astrocytes to maintain a durable glycolytic profile without compromising viability are elusive. Here, using in vivo approaches including cell-specific gene expression disruption, metabolic flux analyses and behavioral tests in mice, we addressed this issue. We found that Pfkfb3 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3) is instrumental in maintaining the astrocytic glycolytic phenotype. Importantly, astrocytic glycolysis sustained by Pfkfb3 is required for normal cognitive performance. Mechanistically, ATP generated through glycolysis is consumed by mitochondria, via the reverse mode of ATP synthase, to conserve the proton gradient across the inner mitochondrial membrane. This enables mitochondria to attenuate pyruvate decarboxylation, tricarboxylic acid cycle and electron transport chain activity, thereby preserving pyruvate for conversion into lactate and delivery to neurons. These findings reveal that astrocytes sacrifice mitochondrial bioenergetic efficiency as a previously underappreciated strategy to support cognition.

cell biology↗

AXL governs axolotl cardiac regeneration and directs mammalian cardiomyocyte dedifferentiation

Cardiac injury outcomes vary widely across species, from complete regeneration to irreversible scarring. Using single-nucleus multiomics and spatial transcriptomics, we generated a spatially resolved atlas of axolotl heart regeneration following injury, identifying a distinct border-zone cardiomyocyte population with a pro-regenerative transcriptional program. Ligand-receptor analysis of the border zone niche identified enrichment of the receptor tyrosine kinase AXL in injury-responsive cardiomyocytes and its ligand Gas6 in endothelial cells. Functional perturbation demonstrated AXL requirement for border-zone cardiomyocyte activation and axolotl heart regeneration. In murine cardiomyocytes, AXL overexpression induced sarcomere disassembly, metabolic rewiring, and immature gene expression without triggering proliferation. These findings show that AXL signaling induces cardiomyocyte dedifferentiation and uncouples dedifferentiation from cell cycle re-entry, providing mechanistic insight into cellular plasticity during heart regeneration.

cell biology↗

Mitochondrial genetics defines anti-tumour immunity through mitochondrial ROS and PD-1 signalling

As central players in cell metabolism, mitochondria influence numerous aspects of health and disease, including the initiation and progression of cancer. Although mitochondrial DNA (mtDNA) mutations have been extensively documented in human cancers for decades, the functional impact of mitochondrial haplogroups on tumour biology remains largely unexplored. Here, we investigate the role of mitochondrial variability in tumour biology using conplastic mouse strains, which are animal models with identical nuclear genomes but different mtDNA haplotypes. We showed that the physiologically relevant variation in mitochondrial ROS (mROS) generation, associated with specific clusters of mtDNA single nucleotide polymorphisms (SNPs), modulated immune responses within the tumour microenvironment and altered tumour growth. We observed strain-dependent differences in the abundance of multiple immune subsets and in PD-1 expression in tumour-infiltrating lymphocytes (TILs). In addition, mtDNA haplotypes influenced cancer progression by modulating tumour angiogenesis through an mROS-independent mechanism. These findings connect nucleo-mitochondrial genetic variability to tumour progression, de novo vessel formation and anti-tumour immunity. Tumour immunotherapies should incorporate the spatial and temporal dynamics of cancer evolution and consider mitochondrial genetics as a targetable layer influencing treatment efficacy.

cancer biology↗

SARS-CoV-2 ORF7a Drives Mitochondrial Dysfunction via PDK4 Activation and Complex I Inhibition

SARS-CoV-2 reprograms host metabolism to promote viral replication and evade immune responses. While infection is known to impair mitochondrial function and enhance glycolysis, the role of viral accessory proteins in these alterations remains unclear. Here, we investigate the metabolic impact of the accessory protein ORF7a. Lentiviral expression of ORF7a in human lung epithelial (A549) and monocytic (THP1) cells, coupled with integrated transcriptomic, proteomic, and metabolomic analyses, revealed profound dysregulation of glucose and lipid metabolism. ORF7a impaired oxidative phosphorylation, reducing basal and maximal respiration, inducing mitochondrial depolarization, and increasing reactive oxygen species. Mechanistically, ORF7a upregulated pyruvate dehydrogenase kinase 4 (PDK4), promoting pyruvate dehydrogenase (PDH) complex phosphorylation and suppressing pyruvate oxidation. However, pharmacological PDK4 inhibition did not restore respiration. High-resolution respirometry in frozen samples revealed impaired complex I function, while Blue Native-PAGE demonstrated defective respiratory supercomplex assembly. By linking enzymatic inhibition with structural destabilization, our study uncovers a functional vulnerability of the mitochondrial respiratory chain to viral manipulation. These findings establish ORF7a as a key modulator of host metabolic reprogramming and highlight mitochondrial pathways as potential therapeutic targets in COVID-19.

immunology↗

Disrupted astrocyte-endothelial crosstalk drives hemangioblastoma lesions in VHL disease

Hemangioblastomas (HBs) are highly vascularized central nervous system (CNS) tumours that can become life-threatening, especially in the context of Von Hippel-Lindau (VHL) disease, caused by the loss of VHL function. The limited pharmacological options targeting VHL-HBs stem from an incomplete understanding of their cellular origin, development, and molecular pathogenesis. Here we use advanced mouse genetics to show that mosaic deletion of Vhl in Apln+ cells leads to the formation of precursor tumour-like lesions, composed by clusters of Vhl-knockout (VhlKO) astrocytes and surrounding Vhl-wild-type (VhlWT) vessels that become malformed, resembling early-stage HBs linked to VHL disease. VhlKO astrocytes morphologically and transcriptomically resembled the reactive astrocytes characteristic of ischemic CNS injury. They exhibited metabolic rewirement towards glycolysis and upregulation of cell growth pathways. They also expressed several secreted proangiogenic molecules that activate and prevent the normal maturation of neighbouring vessels, leading to VHL-HBs. Temporal conditional genetic analysis revealed that Vhl loss need to happen during postnatal development for HBs to form, and that lesions become quiescent in early adulthood. HIF-2 deletion, or MTORC1 inhibition with rapamycin, efficiently inhibited VHL-HBs growth and the associated vascular malformations. Our work shows that the loss of Vhl in single astrocytes induces their growth and pathogenic crosstalk with neighbouring endothelial cells, driving hemangioblastoma development in VHL disease. Our new somatic mosaic mouse models will also enable testing of novel drugs against this disease.

cancer biology↗

MIF-induced CD74+ microglia/macrophages are clinically relevant disease-associated subpopulations in brain metastasis and other CNS disorders

The upregulation of CD74, a chaperone involved in MHC-II antigen processing 1,2, has been broadly reported in virtually all brain disorders analyzed by single-cell RNA sequencing 3-6. However, its expression is usually interpreted as indicative of antigen presentation. In parallel, CD74 expression has also been described in cancer cells across multiple tumor types, but interestingly in glioma its expression has been mainly identified in the microenvironment. However, the functional contribution of CD74 to disease progression in the brain, and specifically in secondary brain tumors, has not been directly addressed. Here we described that, in contrast to what it has been assumed, the presence of CD74+ microglia/macrophages, which is induced by increased levels of interferon gamma in the brain affected by metastases, does not relate to its canonical pathway. Instead, CD74s alternative function as cytokine receptor is pivotal. Rewired by increasing levels of its ligand MIF, produced by proliferating cancer cells, the CD74 receptor, upon binding to this ligand, translocates to the nucleus activating a NF-{kappa}B-dependent program promoting metastasis progression. A brain metastasis-associated CD74 signature involves a more aggressive progression of the local disease in patients, while it has no clinical correlation with the matched primary tumor. Furthermore, we identified the CD74+ myeloid population in additional brain disorders including Alzheimers disease and multiple sclerosis, which shared a pan-disease non-canonical signature with clinical relevance. The brain-penetrant drug ibudilast, which prevents the binding of MIF to CD74, decreases brain metastases in experimental models in vivo and in patient-derived organotypic cultures ex vivo in a primary tumor-agnostic manner. Our findings suggest that MIF/CD74-induced reprogramming of myeloid cells in brain disorders is a novel vulnerability that could be exploited therapeutically against brain metastases, and possibly other brain disorders, guided by a non-invasive molecular strategy.

cancer biology↗

A Quantitative Approach to Mapping Mitochondrial Specialization and Plasticity

Mitochondria are a diverse family of organelles with highly specialized functions. While they share common features, their molecular and functional diversity remains underexplored. Here, we introduce a quantitative pipeline to define the degree of molecular specialization among different mitochondrial phenotypes, or mitotypes. By distilling hundreds of validated mitochondrial genes into 149 biologically interpretable MitoPathway scores, this streamlined mitotyping framework enables investigators to quantify and interpret mitochondrial diversity and plasticity from transcriptomic data across a variety of natural and experimental contexts. Using this approach, we show that mouse and human multi-organ mitotypes segregate along two main axes of mitochondrial specialization, characterize robust longitudinal and perturbation-induced metabolic plasticity in cultured human fibroblasts, and resolve cell-type and single-cell mitochondrial recalibrations across the human brain and sperm developmental trajectory. Together, this framework provides a practical and extensible approach for analyzing mitochondrial specialization in complex biological systems.

cell biology↗

Integrative multi-layer workflow for quantitative analysis of post-translational modifications

Novel algorithms based on ultratolerant database searching have paved the way for comprehensive analysis of all possible post-translational modifications (PTM) that can be detected by mass spectrometry-based proteomics, obviating their prior knowledge. These tools together with novel quantitative statistical models allow hypothesis-free approaches to study the role and impact of PTM on biological systems. However, interpretation of this information from a pathophysiological perspective is challenging due to the huge amounts of PTM data, the existence of chemical, structural, and statistical artifacts and the lack of dedicated tools for their analysis. Here we propose a novel integrative workflow that automatically captures several layers of PTM-related information, including variations in trypsin efficiency, zonal changes, specific PTM changes and hypermodified regions, allowing advanced control of artefacts and coherent and comprehensive interpretation of PTM data. We show the performance of the new workflow by reanalyzing proteomics data from animal models of mitochondrial heteroplasmy and ischemia/reperfusion, revealing relevant PTM information not previously detectable, including consistent detection of novel oxidative modifications in Met and Cys residues from raw proteomics data. The workflow is available through the application PTM-compass.

bioinformatics↗

Uncoupling de novo pyrimidine biosynthesis from mitochondrial electron transport by ectopic expression of cytosolic DHODH

Dihydroorotate dehydrogenase (DHODH) is an enzyme involved in the biosynthesis of pyrimidine nucleotides. In most eukaryotes, this enzyme is bound to the inner mitochondrial membrane, where it couples the synthesis of orotate with the reduction of ubiquinone. As ubiquinone must be regenerated by respiratory complex III, pyrimidine biosynthesis and cellular respiration are tightly coupled. Consequently, inhibition of respiration leads to cessation of DNA synthesis and impairs cell proliferation. We show that expression of Saccharomyces cerevisiae URA1 gene (ScURA) in mammalian cells uncouples biosynthesis of pyrimidines from mitochondrial electron transport. ScURA forms a homodimer in the cytosol that uses fumarate instead of ubiquinone as the electron acceptor, enabling oxygen-independent pyrimidine biosynthesis. Cells expressing ScURA are resistant to drugs that inhibit complex III and the mitochondrial ribosome. ScURA enables the growth of mtDNA-lacking {rho}0 cells in uridine-deficient medium and ameliorates the phenotype of cellular models of mitochondrial diseases. This genetic tool uncovers the contribution of pyrimidine biosynthesis to the phenotypes arising from electron transport chain defects.

molecular biology↗

Time-resolved mitochondrial-focused screening identifies regulatory components of oxidative metabolism

Defects in mitochondrial oxidative metabolism contribute to various genetic inherited disorders, termed mitochondrial diseases, with limited treatment options. Given the lack of functional annotation for numerous mitochondrial proteins, there is a necessity for an extensive gene inventory related to mitochondrial function, with special interest in Oxidative Phosphorylation (OXPHOS). To address this gap, we developed a CRISPR/Cas9 loss-of-function library targeting nuclear-encoded mitochondrial genes and conducted galactose-based screenings at various time points to uncover novel regulators of mitochondrial function. Our study resulted in a gene catalog essential for mitochondrial oxidative metabolism, and constructed a dynamic timeline mapping a broad network of mitochondrial pathways, with a particular focus on the OXPHOS complexes. Computational analysis pinpointed RTN4IP1 and ECHS1 as key genes strongly associated with OXPHOS and whose mutations are associated with mitochondrial diseases in humans. RTN4IP1 was found to be crucial for mitochondrial respiration, with complexome profiling revealing its role as an assembly factor required for the complete assembly of complex I. Furthermore, we discovered that ECHS1 controls oxidative metabolism independently of its canonical function in fatty acid oxidation. Deletion of ECHS1 leads to reduced catabolism of branched-chain amino acids (BCAAs), which impairs the activity of lipoic acid-dependent enzymes such as pyruvate dehydrogenase (PDH). This deleterious phenotype can be rescued by restricting valine intake or catabolism in ECHS1-deficient cells.

molecular biology↗

TET3 regulates cellular terminal differentiation at the metabolic level

TET-family members play an essential role in cell fate commitment and their dysfunctions result in arrested differentiation. TET3 is ubiquitously expressed in differentiated cells and essential in postnatal development due to yet unknown reasons. To define TET3 function in cell differentiation, we profiled the intestinal epithelium at the single-cell level from wild-type and Tet3 knockout mice. Here we show that, in the absence of TET3, enterocytes exhibit an aberrant differentiation trajectory and do not acquire a physiological cell identity due to an impairment in oxidative phosphorylation, specifically due to an ATP synthase assembly deficiency. Furthermore, our analysis demonstrates that the loss of TET3 compromises mitochondrial metabolic maturation and leads to a metabolic profile enriched in glycolysis-dependent anabolic pathways similar to those observed in undifferentiated cells. Collectively, our study has revealed the molecular mechanism by which TET3 regulates terminal differentiation at the metabolic level.

developmental biology↗

An ATF4-centric regulatory network is required for the assembly and function of the OXPHOS system

Identifying the factors that determine mammalian cell viability when oxidative phosphorylation (OXPHOS) function is impaired poses challenges due to the diverse cellular responses and limited clinical material availability. Moreover, animal models often fail to replicate human phenotypes. To address these challenges, this study conducted comprehensive analyses involving multiple defects and species by comparing the RNA-Seq expression profiles of human and murine cell lines with distinct nuclear backgrounds, representing both normal and OXPHOS-deficient models. To minimize species-specific variation, the study employed clustering techniques to group murine genes affected by OXPHOS dysfunction and identified crucial regulators like ATF4, UCP1, and SYVN1. ATF4 consistently displayed activation in response to OXPHOS defects, not only in murine but also in human cells, confirming its pivotal role in the cellular response to mitochondrial dysfunction. By integrating human and murine data, the study unveiled a conserved regulatory network encompassing genes related to the mTOR pathway and folate metabolism. Remarkably, the study uncovered an unexpected finding: the depletion of ATF4 in both mouse and human cells impairs OXPHOS assembly and supercomplex organization. This impairment primarily stems from a severe disruption in complex I assembly in the absence of ATF4, even under non-stress conditions.

molecular biology↗

Na+/H+ antiporter activity by respiratory complex I controls mitochondrial membrane potential and is impaired in LHON disease

The mitochondrial electron transport chain (mETC) converts the energy of substrate oxidation into a H+ electrochemical gradient ({Delta}p), which is composed by an inner mitochondrial membrane (IMM) potential ({Delta}{Psi}mt) and a pH gradient ({Delta}pH). So far, {Delta}{Psi}mt has been assumed to be composed exclusively by H+. Mitochondrial Ca2+ and Na+ homeostasis, which are essential for cellular function, are controlled by exchangers and antiporters in the inner mitochondrial membrane (IMM). In the last few years, some of them have been identified, except for the Na+-specific mitochondrial Na+/H+ exchanger (mNHE). Here, using a rainbow of mitochondrial and nuclear genetic models, we have identified the P-module of complex I (CI) as the major mNHE. In turn, its activity creates a Na+ gradient across the IMM, parallel to {Delta}pH, which accounts for half of the {Delta}{Psi}mt in coupled respiring mitochondria. We have also found that a deregulation of this mNHE function in CI, without affecting its enzymatic activity, occurs in Leber hereditary optic neuropathy (LHON), which has profound consequences in {Delta}{Psi}mt and mitochondrial Ca2+ homeostasis and explains the previously unknown molecular pathogenesis of this neurodegenerative disease.

biochemistry↗

A novel Alex3/Gαq protein complex regulating mitochondrial dynamics, dendritic complexity, and neuronal survival

In neurons, mitochondrial dynamics and trafficking are essential to provide the energy required for neurotransmission and neuronal activity. Recent studies point to GPCR and G proteins as important regulators of mitochondrial dynamics and energy metabolism. Here we show that activation of Gq negatively regulates mitochondrial dynamics and trafficking in neurons. Gq interacts with the mitochondrial trafficking protein Alex3. By generating a CNS-specific armcx3 knock-out mouse line, we demonstrate that Alex3 is required for Gq effects on mitochondrial dynamics and trafficking, and dendritic growth. Armcx3-deficient mice present decreased OXPHOS complex and ER stress response protein levels, which correlate with increased neuronal death, motor neuron and neuromuscular synaptic loss, and severe motor alterations. Finally, we show that Alex3 disassembles from the Miro1/Gq complex upon calcium rise. These data uncover a novel Alex3/Gq complex that regulates neuronal mitochondrial dynamics and neuronal death and allows the control of mitochondrial functions by GPCRs.

cell biology↗

Variant pathogenic prediction by locus variability, the importance of the last picture of evolution.

Accurate pathogenic detection for single nucleotide variants (SNVs) is a key problem to perform variant ranking in whole exome sequencing studies. Several in silico tools have been developed to identify deleterious variants. Locus variability, computed as Shannon entropy from gnomAD/helixMTdb variant allele frequencies can be used as pathogenic variants predictor. In this study we evaluate the use of Shannon entropy in non-coding mitochondrial DNA and also in coding regions with an additional selective pressure other than that imposed by the genetic code, as are splice-sites. To benchmark this functionality in non-coding mitochondrial variants, Shannon entropy was compared with HmtVar disease score, outperforming it in non-coding SNVs (AUCH=0.99 in ROC curve and PR-AUCH=1.00 in Precision-recall curve). In the same way, for splice-sites variants, Shannon entropy was compared against two state-of-the-art ensemble predictors ada score and rf score, matching their overall performance both in ROC curves (AUCH=0.95) and Precision-recall curves (PR-AUC=0.97). Therefore, locus variability could aid in variant ranking process for these specific types of SNVs. Contactjlcabreraa@cnic.es; fscabo@cnic.es

bioinformatics↗

Functional role of respiratory supercomplexes in mice: segmentation of the Qpool and SCAF1

Mitochondrial respiratory complexes assemble into different forms of supercomplexes (SC). In particular, SC III2+IV require the SCAF1 protein. However, the structural role of this factor in the formation of the respirasome (I+III2+IV) and the physiological role of SCs are controversial. Here, we study C57BL/6J mice harbouring either non-functional SCAF1, the full knock-out for SCAF1 or the wild-type version of the protein and found a growth and exercise phenotype due to the lack of functional SCAF1. By combining quantitative data-independent proteomics, high resolution 2D Blue Native Gel Electrophoresis and functional analysis of enriched respirasome fractions, we show that SCAF1 confers structural attachment between III2 and IV within the respirasome, increases NADH-dependent respiration and reduces ROS production. Furthermore, through the expression of AOX in cells and mice we confirm that CI-CIII superassembly segments the CoQ in two pools and modulates CI-NADH oxidative capacity. These data demonstrate that SC assembly, regulated by SCAF1, modulates the functionality of the electron transport chain.

molecular biology↗

Respiratory supercomplexes provide metabolic efficiency in zebrafish

The oxidative phosphorylation (OXPHOS) system is a dynamic system in which the respiratory complexes coexist with super-assembled quaternary structures called supercomplexes (SCs). The physiological role of SCs is still disputed. Here we used zebrafish to study the relevance of respiratory SCs. We combined immunodetection analysis and deep data-independent proteomics to characterize these structures and found similar SCs to those described in mice, as well as novel SCs including III2+IV2, I+IV and I+III2+IV2. To study the physiological role of SCs, we generated two null allele zebrafish lines for supercomplex assembly factor 1 (SCAF1). SCAF1-/- fish displayed altered OXPHOS activity due to the disrupted interaction of complex III and IV. SCAF1-/- fish were smaller in size, and showed abnormal fat deposition and decreased female fertility. These physiological phenotypes were rescued by doubling the food supply, which correlated with improved bioenergetics and alterations in the metabolic gene expression program. These results reveal that SC assembly by SCAF1 modulates OXPHOS efficiency and allows for the optimization of metabolic resources.

physiology↗