SMDT1 variants impair EMRE-mediated mitochondrial calcium uptake in patients with muscle involvement.

Ionic calcium (Ca2+) is a key messenger in signal transduction and its mitochondrial uptake plays an important role in cell physiology. This uptake is mediated by the mitochondrial Ca2+ uniporter (MCU), which is regulated by EMRE (essential MCU regulator) encoded by the SMDT1 (single-pass membrane protein with aspartate rich tail 1) gene. This work presents the genetic, clinical and cellular characterization of two patients harbouring SMDT1 variants and presenting with muscle problems. Analysis of patient fibroblasts and complementation experiments demonstrated that these variants lead to absence of EMRE protein, induce MCU subcomplex formation and impair mitochondrial Ca2+ uptake. However, the activity of oxidative phosphorylation enzymes, mitochondrial morphology and membrane potential, as well as routine/ATP-linked respiration were not affected. We hypothesize that the muscle-related symptoms in the SMDT1 patients result from aberrant mitochondrial Ca2+ uptake.

Stress-dependent macromolecular crowding in the mitochondrial matrix.

Macromolecules of various sizes induce crowding of the cellular environment. This crowding impacts on biochemical reactions by increasing solvent viscosity, decreasing the water-accessible volume and altering protein shape, function, and interactions. Although mitochondria represent highly protein-rich organelles, most of these proteins are somehow immobilized. Therefore, whether the mitochondrial matrix solvent exhibits macromolecular crowding is still unclear. Here, we demonstrate that fluorescent protein fusion peptides (AcGFP1 concatemers) in the mitochondrial matrix of HeLa cells display an elongated molecular structure and that their diffusion constant decreases with increasing molecular weight in a manner typical of macromolecular crowding. Chloramphenicol (CAP) treatment impaired mitochondrial function and reduced the number of cristae without triggering mitochondrial orthodox-to-condensed transition or a mitochondrial unfolded protein response. CAP-treated cells displayed progressive concatemer immobilization with increasing molecular weight and an eightfold matrix viscosity increase, compatible with increased macromolecular crowding. These results establish that the matrix solvent exhibits macromolecular crowding in functional and dysfunctional mitochondria. Therefore, changes in matrix crowding likely affect matrix biochemical reactions in a manner depending on the molecular weight of the involved crowders and reactants.

The decylTPP mitochondria-targeting moiety lowers electron transport chain supercomplex levels in primary human skin fibroblasts.

Attachment of cargo molecules to lipophilic triphenylphosphonium (TPP+) cations is a widely applied strategy for mitochondrial targeting. We previously demonstrated that the vitamin E-derived antioxidant Trolox increases the levels of active mitochondrial complex I (CI), the first complex of the electron transport chain (ETC), in primary human skin fibroblasts (PHSFs) of Leigh Syndrome (LS) patients with isolated CI deficiency. Primed by this finding, we here studied the cellular effects of mitochondria-targeted Trolox (MitoE10), mitochondria-targeted ubiquinone (MitoQ10) and their mitochondria-targeting moiety decylTPP (C10-TPP+). Chronic treatment (96 h) with these molecules of PHSFs from a healthy subject and an LS patient with isolated CI deficiency (NDUFS7-V122M mutation) did not greatly affect cell number. Unexpectedly, this treatment reduced CI levels/activity, lowered the amount of ETC supercomplexes, inhibited mitochondrial oxygen consumption, increased extracellular acidification, altered mitochondrial morphology and stimulated hydroethidine oxidation. We conclude that the mitochondria-targeting decylTPP moiety is responsible for the observed effects and advocate that every study employing alkylTPP-mediated mitochondrial targeting should routinely include control experiments with the corresponding alkylTPP moiety.

Stimulation of cholesterol biosynthesis in mitochondrial complex I-deficiency lowers reductive stress and improves motor function and survival in mice.

The majority of cellular energy is produced by the mitochondrial oxidative phosphorylation (OXPHOS) system. Failure of the first OXPHOS enzyme complex, NADH:ubiquinone oxidoreductase or complex I (CI), is associated with multiple signs and symptoms presenting at variable ages of onset. There is no approved drug treatment yet to slow or reverse the progression of CI-deficient disorders. Here, we present a comprehensive human metabolic network model of genetically characterized CI-deficient patient-derived fibroblasts. Model calculations predicted that increased cholesterol production, export, and utilization can counterbalance the surplus of reducing equivalents in patient-derived fibroblasts, as these pathways consume considerable amounts of NAD(P)H. We show that fibrates attenuated increased NAD(P)H levels and improved CI-deficient fibroblast growth by stimulating the production of cholesterol via enhancement of its cellular efflux. In CI-deficient (Ndufs4-/-) mice, fibrate treatment resulted in prolonged survival and improved motor function, which was accompanied by an increased cholesterol efflux from peritoneal macrophages. Our results shine a new light on the use of compensatory biological pathways in mitochondrial dysfunction, which may lead to novel therapeutic interventions for mitochondrial diseases for which currently no cure exists.

Mitochondria-targeted phenolic antioxidants induce ROS-protective pathways in primary human skin fibroblasts.

Phytochemical antioxidants like gallic and caffeic acid are constituents of the normal human diet that display beneficial health effects, potentially via activating stress response pathways. Using primary human skin fibroblasts (PHSFs) as a model, we here investigated whether such pathways were induced by novel mitochondria-targeted variants of gallic acid (AntiOxBEN2) and caffeic acid (AntiOxCIN4). Both molecules reduced cell viability with similar kinetics and potency (72 h incubation, IC50 ~23 µM). At a relatively high but non-toxic concentration (12.5 µM), AntiOxBEN2 and AntiOxCIN4 increased ROS levels (at 24 h), followed by a decline (at 72 h). Further analysis at the 72 h timepoint demonstrated that AntiOxBEN2 and AntiOxCIN4 did not alter mitochondrial membrane potential (Δψ), but increased cellular glutathione (GSH) levels, mitochondrial NAD(P)H autofluorescence, and mitochondrial superoxide dismutase 2 (SOD2) protein levels. In contrast, cytosolic SOD1 protein levels were not affected. AntiOxBEN2 and AntiOxCIN4 both stimulated the gene expression of Nuclear factor erythroid 2-related factor 2 (NRF2; a master regulator of the cellular antioxidant response toward oxidative stress). AntiOxBEN2 and ANtiOxCIN4 differentially affected the gene expression of the antioxidants Heme oxygenase 1 (HMOX1) and NAD(P)H dehydrogenase (quinone) 1 (NQO1). Both antioxidants did not protect from cell death induced by GSH depletion and AntiOxBEN2 (but not AntiOxCIN4) antagonized hydrogen peroxide-induced cell death. We conclude that AntiOxBEN2 and AntiOxCIN4 increase ROS levels, which stimulates NRF2 expression and, as a consequence, SOD2 and GSH levels. This highlights that AntiOxBEN2 and AntiOxCIN4 can act as prooxidants thereby activating endogenous ROS-protective pathways.

Long-Term and Acute Benefits of Reduced Sitting on Vascular Flow and Function.

PURPOSE: Sedentary behavior increases the risk for cardiovascular and cerebrovascular disease. To understand potential benefits and underlying mechanisms, we examined the acute and long-term effect of reduced sitting intervention on vascular and cerebrovascular function. METHODS: This prospective study included 24 individuals with increased cardiovascular risk (65 ± 5 yr, 29.8 ± 3.9 kg·m-2). Before and after 16-wk reduced sitting, using a mobile health device with vibrotactile feedback, we examined (i) vascular function (flow-mediated dilation [FMD]), (ii) cerebral blood flow velocity (CBFv, transcranial Doppler), and (iii) cerebrovascular function (cerebral autoregulation [CA] and cerebral vasomotor reactivity [CVMR]). To better understand potential underlying mechanisms, before and after intervention, we evaluated the effects of 3 h sitting with and without light-intensity physical activity breaks (every 30 min). RESULTS: The first wave of participants showed no change in sedentary time (n = 9, 10.3 ± 0.5 to 10.2 ± 0.5 h·d-1, P = 0.87). Upon intervention optimization by participants' feedback, the subsequent participants (n = 15) decreased sedentary time (10.2 ± 0.4 to 9.2 ± 0.3 h·d-1, P < 0.01). This resulted in significant increases in FMD (3.1% ± 0.3% to 3.8% ± 0.4%, P = 0.02) and CBFv (48.4 ± 2.6 to 51.4. ±2.6 cm·s-1, P = 0.02), without altering CA or CVMR. Before and after the 16-wk intervention, 3-h exposure to uninterrupted sitting decreased FMD and CBFv, whereas physical activity breaks prevented a decrease (both P < 0.05). CA and CVMR did not change (P > 0.20). CONCLUSION: Long-term reduction in sedentary behavior improves peripheral vascular function and cerebral blood flow and acutely prevents impaired vascular function and decreased cerebral blood flow. These results highlight the potential benefits of reducing sedentary behavior to acutely and chronically improve cardio- or cerebrovascular risk.

NDUFS4 deletion triggers loss of NDUFA12 in Ndufs4-/- mice and Leigh syndrome patients: A stabilizing role for NDUFAF2.

Mutations in NDUFS4, which encodes an accessory subunit of mitochondrial oxidative phosphorylation (OXPHOS) complex I (CI), induce Leigh syndrome (LS). LS is a poorly understood pediatric disorder featuring brain-specific anomalies and early death. To study the LS pathomechanism, we here compared OXPHOS proteomes between various Ndufs4-/- mouse tissues. Ndufs4-/- animals displayed significantly lower CI subunit levels in brain/diaphragm relative to other tissues (liver/heart/kidney/skeletal muscle), whereas other OXPHOS subunit levels were not reduced. Absence of NDUFS4 induced near complete absence of the NDUFA12 accessory subunit, a 50% reduction in other CI subunit levels, and an increase in specific CI assembly factors. Among the latter, NDUFAF2 was most highly increased. Regarding NDUFS4, NDUFA12 and NDUFAF2, identical results were obtained in Ndufs4-/- mouse embryonic fibroblasts (MEFs) and NDUFS4-mutated LS patient cells. Ndufs4-/- MEFs contained active CI in situ but blue-native-PAGE highlighted that NDUFAF2 attached to an inactive CI subcomplex (CI-830) and inactive assemblies of higher MW. In NDUFA12-mutated LS patient cells, NDUFA12 absence did not reduce NDUFS4 levels but triggered NDUFAF2 association to active CI. BN-PAGE revealed no such association in LS patient fibroblasts with mutations in other CI subunit-encoding genes where NDUFAF2 was attached to CI-830 (NDUFS1, NDUFV1 mutation) or not detected (NDUFS7 mutation). Supported by enzymological and CI in silico structural analysis, we conclude that absence of NDUFS4 induces near complete absence of NDUFA12 but not vice versa, and that NDUFAF2 stabilizes active CI in Ndufs4-/- mice and LS patient cells, perhaps in concert with mitochondrial inner membrane lipids.

Homozygous damaging SOD2 variant causes lethal neonatal dilated cardiomyopathy.

BACKGROUND: Idiopathic dilated cardiomyopathy (DCM) is recognised to be a heritable disorder, yet clinical genetic testing does not produce a diagnosis in >50% of paediatric patients. Identifying a genetic cause is crucial because this knowledge can affect management options, cardiac surveillance in relatives and reproductive decision-making. In this study, we sought to identify the underlying genetic defect in a patient born to consanguineous parents with rapidly progressive DCM that led to death in early infancy. METHODS AND RESULTS: Exome sequencing revealed a potentially pathogenic, homozygous missense variant, c.542G>T, p.(Gly181Val), in SOD2. This gene encodes superoxide dismutase 2 (SOD2) or manganese-superoxide dismutase, a mitochondrial matrix protein that scavenges oxygen radicals produced by oxidation-reduction and electron transport reactions occurring in mitochondria via conversion of superoxide anion (O2-·) into H2O2. Measurement of hydroethidine oxidation showed a significant increase in O2-· levels in the patient's skin fibroblasts, as compared with controls, and this was paralleled by reduced catalytic activity of SOD2 in patient fibroblasts and muscle. Lentiviral complementation experiments demonstrated that mitochondrial SOD2 activity could be completely restored on transduction with wild type SOD2. CONCLUSION: Our results provide evidence that defective SOD2 may lead to toxic increases in the levels of damaging oxygen radicals in the neonatal heart, which can result in rapidly developing heart failure and death. We propose SOD2 as a novel nuclear-encoded mitochondrial protein involved in severe human neonatal cardiomyopathy, thus expanding the wide range of genetic factors involved in paediatric cardiomyopathies.

Sixteen-Week Physical Activity Intervention in Subjects With Increased Cardiometabolic Risk Shifts Innate Immune Function Towards a Less Proinflammatory State.

Background Low-grade inflammation, largely mediated by monocyte-derived macrophages, contributes to atherosclerosis. Sedentary behavior is associated with atherosclerosis and cardiovascular diseases (CVD). We examined whether reducing sedentary behavior and improving walking time improves monocyte inflammatory phenotype in subjects with increased cardiovascular risk. Methods and Results Across 2 waves, 16 individuals with increased cardiovascular risk performed a 16-week intervention study (age 64±6 years, body mass index 29.9±4.3 kg/m2), using a device with vibration feedback to promote physical activity. Before and after intervention, we objectively examined physical activity (ActivPAL), cytokine production capacity after ex vivo stimulation in peripheral blood mononuclear cells, metabolism of peripheral blood mononuclear cells, circulating cytokine concentrations, and monocyte immunophenotype. Overall, no significant increase in walking time was found (1.9±0.7 to 2.2±1.2 h/day, P=0.07). However, strong, inverse correlations were observed between the change in walking time and the change in production of interleukin (IL)-1ß, IL-6, IL-8, and IL-10 after lipopolysaccharide stimulation (rs=-0.655, -0.844, -0.672, and -0.781, respectively, all P<0.05). After intervention optimization based on feedback from wave 1, participants in wave 2 (n=8) showed an increase in walking time (2.2±0.8 to 3.0±1.3 h/day, P=0.001) and attenuated cytokine production of IL-6, IL-8, and IL-10 (all P<0.05). Glycolysis (P=0.08) and maximal OXPHOS (P=0.04) of peripheral blood mononuclear cells decreased after intervention. Lower IL-6 concentrations (P=0.06) and monocyte percentages (P<0.05), but no changes in monocyte subsets were found. Conclusions Successfully improving walking time shifts innate immune function towards a less proinflammatory state, characterized by a lower capacity to produce inflammatory cytokines, in individuals with increased cardiovascular risk. Clinical Trial Registration Information URL: http://www.trialregister.nl. Unique identifier: NTR6387.

Octa-arginine boosts the penetration of elastin-like polypeptide nanoparticles in 3D cancer models.

Elastin-like polypeptide (ELP) nanoparticles are a versatile platform for targeted drug delivery. As for any type of nanocarrier system, an important challenge remains the ability of deep (tumor) tissue penetration. In this study, ELP particles with controlled surface density of the cell-penetrating peptide (CPP) octa-arginine (R8) were created by temperature-induced co-assembly. ELPs formed micellar nanoparticles with a diameter of around 60 nm. Cellular uptake in human skin fibroblasts was directly dependent on the surface density of R8 as confirmed by flow cytometry and confocal laser scanning microscopy. Remarkably, next to promoting cellular uptake, the presence of the CPP also enhanced penetration into spheroids generated from human glioblastoma U-87 cells. After 24 h, uptake into cells was observed in multiple layers towards the spheroid core. ELP particles not carrying any CPP did not penetrate. Clearly, a high CPP density exerted a dual benefit on cellular uptake and tissue penetration. At low nanoparticle concentration, there was evidence of a binding site barrier as observed for the penetration of molecules binding with high affinity to cell surface receptors. In conclusion, R8-functionalized ELP nanoparticles form an excellent delivery vehicle that combines tunability of surface characteristics with small and well-defined size.

Mitochondrial Morphofunction in Mammalian Cells.

Significance: In addition to their classical role in cellular ATP production, mitochondria are of key relevance in various (patho)physiological mechanisms including second messenger signaling, neuro-transduction, immune responses and death induction. Recent Advances: Within cells, mitochondria are motile and display temporal changes in internal and external structure ("mitochondrial dynamics"). During the last decade, substantial empirical and in silico evidence was presented demonstrating that mitochondrial dynamics impacts on mitochondrial function and vice versa. Critical Issues: However, a comprehensive and quantitative understanding of the bidirectional links between mitochondrial external shape, internal structure and function ("morphofunction") is still lacking. The latter particularly hampers our understanding of the functional properties and behavior of individual mitochondrial within single living cells. Future Directions: In this review we discuss the concept of mitochondrial morphofunction in mammalian cells, primarily using experimental evidence obtained within the last decade. The topic is introduced by briefly presenting the central role of mitochondria in cell physiology and the importance of the mitochondrial electron transport chain (ETC) therein. Next, we summarize in detail how mitochondrial (ultra)structure is controlled and discuss empirical evidence regarding the equivalence of mitochondrial (ultra)structure and function. Finally, we provide a brief summary of how mitochondrial morphofunction can be quantified at the level of single cells and mitochondria, how mitochondrial ultrastructure/volume impacts on mitochondrial bioreactions and intramitochondrial protein diffusion, and how mitochondrial morphofunction can be targeted by small molecules.

Rescue from galactose-induced death of Leigh Syndrome patient cells by pyruvate and NAD.

Cell models of mitochondrial complex I (CI) deficiency display activation of glycolysis to compensate for the loss in mitochondrial ATP production. This adaptation can mask other relevant deficiency-induced aberrations in cell physiology. Here we investigated the viability, mitochondrial morphofunction, ROS levels and ATP homeostasis of primary skin fibroblasts from Leigh Syndrome (LS) patients with isolated CI deficiency. These cell lines harbored mutations in nuclear DNA (nDNA)-encoded CI genes (NDUFS7, NDUFS8, NDUFV1) and, to prevent glycolysis upregulation, were cultured in a pyruvate-free medium in which glucose was replaced by galactose. Following optimization of the cell culture protocol, LS fibroblasts died in the galactose medium, whereas control cells did not. LS cell death was dose-dependently inhibited by pyruvate, malate, oxaloacetate, α-ketoglutarate, aspartate, and exogenous NAD+ (eNAD), but not by lactate, succinate, α-ketobutyrate, and uridine. Pyruvate and eNAD increased the cellular NAD+ content in galactose-treated LS cells to a different extent and co-incubation studies revealed that pyruvate-induced rescue was not primarily mediated by NAD+. Functionally, in LS cells glucose-by-galactose replacement increased mitochondrial fragmentation and mass, depolarized the mitochondrial membrane potential (Δψ), increased H2DCFDA-oxidizing ROS levels, increased mitochondrial ATP generation, and reduced the total cellular ATP content. These aberrations were differentially rescued by pyruvate and eNAD, supporting the conclusion that these compounds rescue galactose-induced LS cell death via different mechanisms. These findings establish a cell-based strategy for intervention testing and enhance our understanding of CI deficiency pathophysiology.

Extracellular acidification induces ROS- and mPTP-mediated death in HEK293 cells.

The extracellular pH (pHe) is a key determinant of the cellular (micro)environment and needs to be maintained within strict boundaries to allow normal cell function. Here we used HEK293 cells to study the effects of pHe acidification (24h), induced by mitochondrial inhibitors (rotenone, antimycin A) and/or extracellular HCl addition. Lowering pHe from 7.2 to 5.8 reduced cell viability by 70% and was paralleled by a decrease in cytosolic pH (pHc), hyperpolarization of the mitochondrial membrane potential (Δψ), increased levels of hydroethidine-oxidizing ROS and stimulation of protein carbonylation. Co-treatment with the antioxidant α-tocopherol, the mitochondrial permeability transition pore (mPTP) desensitizer cyclosporin A and Necrostatin-1, a combined inhibitor of Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and Indoleamine 2,3-dioxygenase (IDO), prevented acidification-induced cell death. In contrast, the caspase inhibitor zVAD.fmk and the ferroptosis inhibitor Ferrostatin-1 were ineffective. We conclude that extracellular acidification induces necroptotic cell death in HEK293 cells and that the latter involves intracellular acidification, mitochondrial functional impairment, increased ROS levels, mPTP opening and protein carbonylation. These findings suggest that acidosis of the extracellular environment (as observed in mitochondrial disorders, ischemia, acute inflammation and cancer) can induce cell death via a ROS- and mPTP opening-mediated pathogenic mechanism.

Modulation of oxidative phosphorylation and redox homeostasis in mitochondrial NDUFS4 deficiency via mesenchymal stem cells.

BACKGROUND: Disorders of the oxidative phosphorylation (OXPHOS) system represent a large group among the inborn errors of metabolism. The most frequently observed biochemical defect is isolated deficiency of mitochondrial complex I (CI). No effective treatment strategies for CI deficiency are so far available. The purpose of this study was to investigate whether and how mesenchymal stem cells (MSCs) are able to modulate metabolic function in fibroblast cell models of CI deficiency. METHODS: We used human and murine fibroblasts with a defect in the nuclear DNA encoded NDUFS4 subunit of CI. Fibroblasts were co-cultured with MSCs under different stress conditions and intercellular mitochondrial transfer was assessed by flow cytometry and fluorescence microscopy. Reactive oxygen species (ROS) levels were measured using MitoSOX-Red. Protein levels of CI were analysed by blue native polyacrylamide gel electrophoresis (BN-PAGE). RESULTS: Direct cellular interactions and mitochondrial transfer between MSCs and human as well as mouse fibroblast cell lines were demonstrated. Mitochondrial transfer was visible in 13.2% and 6% of fibroblasts (e.g. fibroblasts containing MSC mitochondria) for human and mouse cell lines, respectively. The transfer rate could be further stimulated via treatment of cells with TNF-α. MSCs effectively lowered cellular ROS production in NDUFS4-deficient fibroblast cell lines (either directly via co-culture or indirectly via incubation of cell lines with cell-free MSC supernatant). However, CI protein expression and activity were not rescued by MSC treatment. CONCLUSION: This study demonstrates the interplay between MSCs and fibroblast cell models of isolated CI deficiency including transfer of mitochondria as well as modulation of cellular ROS levels. Further exploration of these cellular interactions might help to develop MSC-based treatment strategies for human CI deficiency.

Controlling T-Cell Activation with Synthetic Dendritic Cells Using the Multivalency Effect.

Artificial antigen-presenting cells (aAPCs) have recently gained a lot of attention. They efficiently activate T cells and serve as powerful replacements for dendritic cells in cancer immunotherapy. Focusing on a specific class of polymer-based aAPCs, so-called synthetic dendritic cells (sDCs), we have investigated the importance of multivalent binding on T-cell activation. Using antibody-functionalized sDCs, we have tested the influence of polymer length and antibody density. Increasing the multivalent character of the antibody-functionalized polymer lowered the effective concentration required for T-cell activation. This was evidenced for both early and late stages of activation. The most important effect observed was the significantly prolonged activation of the stimulated T cells, indicating that multivalent sDCs sustain T-cell signaling. Our results highlight the importance of multivalency for the design of aAPCs and will ultimately allow for better mimics of natural dendritic cells that can be used as vaccines in cancer treatment.

Acute stimulation of glucose influx upon mitoenergetic dysfunction requires LKB1, AMPK, Sirt2 and mTOR-RAPTOR.

Mitochondria play a central role in cellular energy production, and their dysfunction can trigger a compensatory increase in glycolytic flux to sustain cellular ATP levels. Here, we studied the mechanism of this homeostatic phenomenon in C2C12 myoblasts. Acute (30 min) mitoenergetic dysfunction induced by the mitochondrial inhibitors piericidin A and antimycin A stimulated Glut1-mediated glucose uptake without altering Glut1 (also known as SLC2A1) mRNA or plasma membrane levels. The serine/threonine liver kinase B1 (LKB1; also known as STK11) and AMP-activated protein kinase (AMPK) played a central role in this stimulation. In contrast, ataxia-telangiectasia mutated (ATM; a potential AMPK kinase) and hydroethidium (HEt)-oxidizing reactive oxygen species (ROS; increased in piericidin-A- and antimycin-A-treated cells) appeared not to be involved in the stimulation of glucose uptake. Treatment with mitochondrial inhibitors increased NAD+ and NADH levels (associated with a lower NAD+:NADH ratio) but did not affect the level of Glut1 acetylation. Stimulation of glucose uptake was greatly reduced by chemical inhibition of Sirt2 or mTOR-RAPTOR. We propose that mitochondrial dysfunction triggers LKB1-mediated AMPK activation, which stimulates Sirt2 phosphorylation, leading to activation of mTOR-RAPTOR and Glut1-mediated glucose uptake.

Multiplexed high-content analysis of mitochondrial morphofunction using live-cell microscopy.

Mitochondria have a central role in cellular (patho)physiology, and they display a highly variable morphology that is probably coupled to their functional state. Here we present a protocol that allows unbiased and automated quantification of mitochondrial 'morphofunction' (i.e., morphology and membrane potential), cellular parameters (size, confluence) and nuclear parameters (number, morphology) in intact living primary human skin fibroblasts (PHSFs). Cells are cultured in 96-well plates and stained with tetramethyl rhodamine methyl ester (TMRM), calcein-AM (acetoxy-methyl ester) and Hoechst 33258. Next, multispectral fluorescence images are acquired using automated microscopy and processed to extract 44 descriptors. Subsequently, the descriptor data are subjected to a quality control (QC) algorithm based upon principal component analysis (PCA) and interpreted using univariate, bivariate and multivariate analysis. The protocol requires a time investment of ∼4 h distributed over 2 d. Although it is specifically developed for PHSFs, which are widely used in preclinical research, the protocol is portable to other cell types and can be scaled up for implementation in high-content screening.