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OBJECTIVE: Mitochondrial proteostasis is critical for cellular function. The molecular chaperone HSP60 is essential for cell function and dysregulation of HSP60 expression has been implicated in cancer and diabetes. The few reported patients carrying HSP60 gene variants show neurodevelopmental delay and brain hypomyelination. Hsp60 interacts with more than 260 mitochondrial proteins but the mitochondrial proteins and functions affected by HSP60 deficiency are poorly characterized. METHODS: We studied two model systems for HSP60 deficiency: (1) engineered HEK cells carrying an inducible dominant negative HSP60 mutant protein, (2) zebrafish HSP60 knockout larvae. Both systems were analyzed by RNASeq, proteomics, and targeted metabolomics, and several functional assays relevant for the respective model. In addition, skin fibroblasts from patients with disease-associated HSP60 variants were analyzed by proteomics. RESULTS: We show that HSP60 deficiency leads to a differentially downregulated mitochondrial matrix proteome, transcriptional activation of stress responses, and dysregulated cholesterol biosynthesis. This leads to lipid accumulation in zebrafish knockout larvae. CONCLUSIONS: Our data provide a compendium of the effects of HSP60 deficiency on the mitochondrial matrix proteome. We show that HSP60 is a master regulator and modulator of mitochondrial functions and metabolic pathways. HSP60 dysfunction also affects cellular metabolism and disrupts the integrated stress response. The effect on cholesterol synthesis explains the effect of HSP60 dysfunction on myelination observed in patients carrying genetic variants of HSP60.
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The genetic architecture of the QT interval, defined as the period from onset of depolarisation to completion of repolarisation of the ventricular myocardium, is incompletely understood. Only a minor part of the QT interval variation in the general population has been linked to autosomal variant loci. Altered X chromosome dosage in humans, as seen in sex chromosome aneuploidies such as Turner syndrome (TS) and Klinefelter syndrome (KS), is associated with altered QTc interval (heart rate corrected QT), indicating that genes, located in the pseudoautosomal region 1 of the X and Y chromosomes may contribute to QT interval variation. We investigate the dosage effect of the pseudoautosomal gene SLC25A6, encoding the membrane ADP/ATP translocase 3 in the inner mitochondrial membrane, on QTc interval duration. To this end we used human participants and in vivo zebrafish models. Analyses in humans, based on 44 patients with KS, 44 patients with TS, 59 male and 22 females, revealed a significant negative correlation between SLC25A6 expression level and QTc interval duration. Similarly, downregulation of slc25a6 in zebrafish increased QTc interval duration with pharmacological inhibition of KATP channels restoring the systolic duration, whereas overexpression of SLC25A6 shortened QTc, which was normalized by pharmacological activation of KATP channels. Our study demonstrate an inverse relationship between SLC25A6 dosage and QTc interval indicating that SLC25A6 contributes to QT interval variation.
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Síndrome de Klinefelter , Síndrome de QT Prolongado , Síndrome de Turner , Animales , Femenino , Humanos , Masculino , Adenosina Trifosfato , Electrocardiografía , Síndrome de QT Prolongado/genética , Cromosoma X , Pez Cebra/genética , Translocador 3 del Nucleótido AdeninaRESUMEN
Bromodomain containing 1 (BRD1) encodes an epigenetic regulator that controls the expression of genetic networks linked to mental illness. BRD1 is essential for normal brain development and its role in psychopathology has been demonstrated in genetic and preclinical studies. However, the neurobiology that bridges its molecular and neuropathological effects remains poorly explored. Here, using publicly available datasets, we find that BRD1 targets nuclear genes encoding mitochondrial proteins in cell lines and that modulation of BRD1 expression, irrespective of whether it is downregulation or upregulation of one or the other existing BRD1 isoforms (BRD1-L and BRD1-S), leads to distinct shifts in the expression profile of these genes. We further show that the expression of nuclear genes encoding mitochondrial proteins is negatively correlated with the expression of BRD1 mRNA during human brain development. In accordance, we identify the key gate-keeper of mitochondrial metabolism, Peroxisome proliferator-activated receptor (PPAR) among BRD1's co-transcription factors and provide evidence that BRD1 acts as a co-repressor of PPAR-mediated transcription. Lastly, when using quantitative PCR, mitochondria-targeted fluorescent probes, and the Seahorse XFe96 Analyzer, we demonstrate that modulation of BRD1 expression in cell lines alters mitochondrial physiology (mtDNA content and mitochondrial mass), metabolism (reducing power), and bioenergetics (among others, basal, maximal, and spare respiration) in an expression level- and isoform-dependent manner. Collectively, our data suggest that BRD1 is a transcriptional regulator of nuclear-encoded mitochondrial proteins and that disruption of BRD1's genomic actions alters mitochondrial functions. This may be the mechanism underlying the cellular and atrophic changes of neurons previously associated with BRD1 deficiency and suggests that mitochondrial dysfunction may be a possible link between genetic variation in BRD1 and psychopathology in humans.
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Histona Acetiltransferasas , Esquizofrenia , Metabolismo Energético , Histona Acetiltransferasas/fisiología , Humanos , Mitocondrias/metabolismo , Proteínas Mitocondriales , Receptores Activados del Proliferador del Peroxisoma/metabolismo , Isoformas de Proteínas/metabolismo , Esquizofrenia/genéticaRESUMEN
Type 2 diabetes mellitus (T2DM) is associated with impaired skeletal muscle function and degeneration of the skeletal muscles. However, the mechanisms underlying the degeneration are not well described in human skeletal muscle. Here we show that skeletal muscle of T2DM patients exhibit degenerative remodeling of the extracellular matrix that is associated with a selective increase of a subpopulation of fibro-adipogenic progenitors (FAPs) marked by expression of THY1 (CD90)-the FAPCD90+. We identify platelet-derived growth factor (PDGF) as a key FAP regulator, as it promotes proliferation and collagen production at the expense of adipogenesis. FAPsCD90+ display a PDGF-mimetic phenotype, with high proliferative activity, clonogenicity, and production of extracellular matrix. FAPCD90+ proliferation was reduced by in vitro treatment with metformin. Furthermore, metformin treatment reduced FAP content in T2DM patients. These data identify a PDGF-driven conversion of a subpopulation of FAPs as a key event in the fibrosis development in T2DM muscle.
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Diabetes Mellitus Tipo 2 , Enfermedades Musculares , Adipogénesis , Diferenciación Celular , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/metabolismo , Humanos , Músculo Esquelético/metabolismo , Atrofia Muscular/metabolismo , Enfermedades Musculares/metabolismoRESUMEN
High-contrast brightfield (HCBF) microscopy has emerged as a strong tool for noninvasive counting of cells in culture. HCBF imaging delivers precise cell growth data and is completely label free rendering it an attractive alternative to common cell counting procedures that often adversely affect cell growth. With computational image analysis, HCBF achieves efficient high-throughput automated workflows, extremely relevant for drug and chemical screens in pharmaceutical, toxicological, and biomedical research. We demonstrate the applicability of HCBF microscopy to count three common cell types (HEK293, Huh7, and primary human dermal fibroblasts) with diverse morphology challenging the method. The three cell types required different analysis settings, and we identified two parameters of the computational image analysis, which after cell-specific optimization significantly improved the cell counting accuracy, namely the lower size limit and the intensity threshold. Three-dimensional (3D) imaging approaches, which have obtained great attention in recent years, were an interesting prospect to combine with HCBF microscopy. We optimized the analysis of two 3D outputs but found 3D HCBF imaging to be inferior to the optimized single-layer HCBF imaging for cell counting. HCBF cell counts were highly linearly correlated with (R2 > 0.99) and highly similar (<15% difference) to cell counts obtained through Hoechst staining, over a broad range of densities allowing at least this level of accuracy for two to three cell generations in Huh7 cells and fibroblasts. Counts of HEK293 cells correlated somewhat less. In conclusion, the HCBF cell counting method is excellently suited for cell proliferation assays and cytotoxicity assays.
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Técnicas de Cultivo de Célula , Imagenología Tridimensional , Recuento de Células , Células Cultivadas , Humanos , Microscopía FluorescenteRESUMEN
Standardization of the use of next-generation sequencing for the diagnosis of rare neurological disorders has made it possible to detect potential disease-causing genetic variations, including de novo variants. However, the lack of a clear pathogenic relevance of gene variants poses a critical limitation for translating this genetic information into clinical practice, increasing the necessity to perform functional assays. Genetic screening is currently recommended in the guidelines for diagnosis of hypomyelinating leukodystrophies (HLDs). HLDs represent a group of rare heterogeneous disorders that interfere with the myelination of the neurons in the central nervous system. One of the HLD-related genes is HSPD1, encoding the mitochondrial chaperone heat shock protein 60 (HSP60), which functions as folding machinery for the mitochondrial proteins imported into the mitochondrial matrix space. Disease-causing HSPD1 variants have been associated with an autosomal recessive form of fatal hypomyelinating leukodystrophy (HLD4, MitCHAP60 disease; MIM #612233) and an autosomal dominant form of spastic paraplegia, type 13 (SPG13; MIM #605280). In 2018, a de novo HSPD1 variant was reported in a patient with HLD. Here, we present another case carrying the same heterozygous de novo variation in the HSPD1 gene (c.139T > G, p.Leu47Val) associated with an HLD phenotype. Our molecular studies show that the variant HSP60 protein is stably present in the patient's fibroblasts, and functional assays demonstrate that the variant protein lacks in vivo function, thus confirming its disease association. We conclude that de novo variations of the HSPD1 gene should be considered as potentially disease-causing in the diagnosis and pathogenesis of the HLDs.
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Chaperonina 60/genética , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Variación Genética , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/diagnóstico , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/genética , Enfermedades Mitocondriales/diagnóstico , Enfermedades Mitocondriales/genética , Proteínas Mitocondriales/genética , Adulto , Alelos , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Chaperonina 10/genética , Chaperonina 60/química , Niño , Femenino , Estudios de Asociación Genética/métodos , Genotipo , Humanos , Lactante , Imagen por Resonancia Magnética , Masculino , Proteínas Mitocondriales/química , Modelos Moleculares , Mutación , Proteínas Gestacionales/genética , Conformación Proteica , Recurrencia , Relación Estructura-Actividad , Factores Supresores Inmunológicos/genéticaRESUMEN
The HSP60/HSP10 chaperonin assists folding of proteins in the mitochondrial matrix space by enclosing them in its central cavity. The chaperonin forms part of the mitochondrial protein quality control system. It is essential for cellular survival and mutations in its subunits are associated with rare neurological disorders. Here we present the first survey of interactors of the human mitochondrial HSP60/HSP10 chaperonin. Using a protocol involving metabolic labeling of HEK293 cells, cross-linking, and immunoprecipitation of HSP60, we identified 323 interacting proteins. As expected, the vast majority of these proteins are localized to the mitochondrial matrix space. We find that approximately half of the proteins annotated as mitochondrial matrix proteins interact with the HSP60/HSP10 chaperonin. They cover a broad spectrum of functions and metabolic pathways including the mitochondrial protein synthesis apparatus, the respiratory chain, and mitochondrial protein quality control. Many of the genes encoding HSP60 interactors are annotated as disease genes. There is a correlation between relative cellular abundance and relative abundance in the HSP60 immunoprecipitates. Nineteen abundant matrix proteins occupy more than 60% of the HSP60/HSP10 chaperonin capacity. The reported inventory of interactors can form the basis for interrogating which proteins are especially dependent on the chaperonin.
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Chaperonina 10/metabolismo , Chaperonina 60/metabolismo , Proteínas Mitocondriales/metabolismo , Células HEK293 , Humanos , Mitocondrias/metabolismoRESUMEN
Besides providing the majority of ATP production in cells, mitochondria are also involved in many other cellular functions and are central for cellular stress signaling. Mitochondrial dysfunction induces not only inherited mitochondrial disorders but also contributes to neurodegenerative diseases, cancer, diabetes, and metabolic syndrome. The HSP60/HSP10 molecular chaperone complex facilitates folding of mitochondrial proteins and is thus an important factor for many mitochondrial functions. To model different degrees of oxidative stress and mitochondrial dysfunction we here describe a HEK293 derived Flp-In cell system with stable insertion and tunable expression of HSP60 cDNA carrying a dominant negative mutation. When expressed the dominant negative HSP60 mutant is incorporated into endogenously encoded HSP60/HSP10 complexes and impairs chaperone activity of the HSP60/HSP10 complex in a dose dependent manner. Using this system, different levels of oxidative stress and mitochondrial dysfunction challenges can be generated depending on the induction level of the mutant HSP60 cDNA insert. Here we describe our system and pertinent analysis methodology for use in studies of mitochondrial chaperone deficiency and resulting effects of increased production of reactive oxygen species and mitochondrial dysfunction.
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Chaperonina 60/deficiencia , Susceptibilidad a Enfermedades , Mitocondrias/genética , Mitocondrias/metabolismo , Modelos Biológicos , Estrés Oxidativo , Chaperonina 60/química , Chaperonina 60/genética , Chaperonina 60/metabolismo , Citometría de Flujo , Humanos , Espectrometría de Masas , Potencial de la Membrana MitocondrialRESUMEN
Sex differences in brain structure and function are of substantial scientific interest because of sex-related susceptibility to psychiatric and neurological disorders. Neuroinflammation is a common denominator of many of these diseases, and thus microglia, as the brain's immunocompetent cells, have come into focus in sex-specific studies. Here, we show differences in the structure, function, and transcriptomic and proteomic profiles in microglia freshly isolated from male and female mouse brains. We show that male microglia are more frequent in specific brain areas, have a higher antigen-presenting capacity, and appear to have a higher potential to respond to stimuli such as ATP, reflected in higher baseline outward and inward currents and higher protein expression of purinergic receptors. Altogether, we provide a comprehensive resource to generate and validate hypotheses regarding brain sex differences.