HSP60 chaperone deficiency disrupts the mitochondrial matrix proteome and dysregulates cholesterol synthesis.

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.

Advancing Clinical Trial Design for Non-Muscle Invasive Bladder Cancer.

BACKGROUND: Despite recent drug development for non-muscle invasive bladder cancer (NMIBC), few therapies have been approved by the US Food and Drug Administration (FDA), and there remains an unmet clinical need. Bacillus Calmette-Guerin (BCG) supply issues underscore the importance of developing safe and effective drugs for NMIBC. OBJECTIVE: On November 18-19, 2021, the FDA held a public virtual workshop to discuss NMIBC research needs and potential trial designs for future development of effective therapies. METHODS: Representatives from various disciplines including urologists, oncologists, pathologists, statisticians, basic and translational scientists, and the patient advocacy community participated. The workshop format included invited lectures, panel discussions, and opportunity for audience discussion and comment. RESULTS: In a pre-workshop survey, 92% of urologists surveyed considered the development of alternatives to BCG as a high drug development priority for BCG-naïve high-risk patients. Key topics discussed included definitions of disease states; trial design for BCG-naïve NMIBC, BCG-unresponsive carcinoma in situ, and BCG-unresponsive papillary carcinoma; strengths and limitations of single-arm trial designs; assessing patient-reported outcomes; and considerations for assessing avoidance of cystectomy as an efficacy measure. CONCLUSIONS: The workshop discussed several important opportunities for trial design refinement in NMIBC. FDA encourages sponsors to meet with the appropriate review division to discuss trial design proposals for NMIBC early in drug development.

A genome-wide CRISPR-Cas9 knockout screen identifies novel PARP inhibitor resistance genes in prostate cancer.

DNA repair gene mutations are frequent in castration-resistant prostate cancer (CRPC), suggesting eligibility for poly(ADP-ribose) polymerase inhibitor (PARPi) treatment. However, therapy resistance is a major clinical challenge and genes contributing to PARPi resistance are poorly understood. Using a genome-wide CRISPR-Cas9 knockout screen, this study aimed at identifying genes involved in PARPi resistance in CRPC. Based on the screen, we identified PARP1, and six novel candidates associated with olaparib resistance upon knockout. For validation, we generated multiple knockout populations/clones per gene in C4 and/or LNCaP CRPC cells, which confirmed that loss of PARP1, ARH3, YWHAE, or UBR5 caused olaparib resistance. PARP1 or ARH3 knockout caused cross-resistance to other PARPis (veliparib and niraparib). Furthermore, PARP1 or ARH3 knockout led to reduced autophagy, while pharmacological induction of autophagy partially reverted their PARPi resistant phenotype. Tumor RNA sequencing of 126 prostate cancer patients identified low ARH3 expression as an independent predictor of recurrence. Our results advance the understanding of PARPi response by identifying four novel genes that contribute to PARPi sensitivity in CRPC and suggest a new model of PARPi resistance through decreased autophagy.

The psychiatric risk gene BRD1 modulates mitochondrial bioenergetics by transcriptional regulation.

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.

Clinical characteristics and symptom duration among outpatients with COVID-19.

BACKGROUND: Approximately 80% of people with COVID-19 do not require hospitalization. Studies examining the outpatient experience have not tracked symptoms to resolution leading to unknown expected symptom duration. Our objectives were to (1) determine symptom duration among patients with COVID-19 who do not require hospitalization and (2) identify potential risk factors associated with prolonged symptom duration. DESIGN: This is a retrospective cohort study conducted across an academic healthcare system including adult patients with laboratory-confirmed SARS-CoV-2 infection between March 18th and April 28th, 2020 who were not hospitalized. Symptom duration encompassed time from patient-reported symptom onset as documented in the chart until documented symptom resolution. We calculated the median symptom duration and tested if demographics, comorbidities, or reported symptoms were associated with symptom duration. KEY RESULTS: Of 294 patients meeting inclusion criteria, 178 (60.5%) had documented symptom resolution. The median [interquartile range (IQR)] symptom duration for included patients was 15 (8-24) days. No associations were found between comorbidities and symptom duration. Factors associated with prolonged symptom duration were presence vs lack of lower respiratory symptoms [median (IQR) 16.5 (10.75-33.5) vs 14.5 (7-21.75) days respectively, P < .001] and neurologic symptoms [median (IQR) 17 (9-28) vs 9.5 (4-17) days, P < .001] at disease onset. CONCLUSIONS: The median symptom duration in outpatients is 15 days and over 25% of patients have symptoms longer than 21 days.

Paediatric Strategy Forum for medicinal product development of chimeric antigen receptor T-cells in children and adolescents with cancer: ACCELERATE in collaboration with the European Medicines Agency with participation of the Food and Drug Administration.

The seventh multi-stakeholder Paediatric Strategy Forum focused on chimeric antigen receptor (CAR) T-cells for children and adolescents with cancer. The development of CAR T-cells for patients with haematological malignancies, especially B-cell precursor acute lymphoblastic leukaemia (BCP-ALL), has been spectacular. However, currently, there are scientific, clinical and logistical challenges for use of CAR T-cells in BCP-ALL and other paediatric malignancies, particularly in acute myeloid leukaemia (AML), lymphomas and solid tumours. The aims of the Forum were to summarise the current landscape of CAR T-cell therapy development in paediatrics, too identify current challenges and future directions, with consideration of other immune effector modalities and ascertain the best strategies to accelerate their development and availability to children. Although the effect is of limited duration in about half of the patients, anti-CD19 CAR T-cells produce high response rates in relapsed/refractory BCP-ALL and this has highlighted previously unknown mechanisms of relapse. CAR T-cell treatment as first- or second-line therapy could also potentially benefit patients whose disease has high-risk features associated with relapse and failure of conventional therapies. Identifying patients with very early and early relapse in whom CAR T-cell therapy may replace haematopoietic stem cell transplantation and be definitive therapy versus those in whom it provides a more effective bridge to haematopoietic stem cell transplantation is a very high priority. Development of approaches to improve persistence, either by improving T cell fitness or using more humanised/fully humanised products and co-targeting of multiple antigens to prevent antigen escape, could potentially further optimise therapy. Many differences exist between paediatric B-cell non-Hodgkin lymphomas (B-NHL) and BCP-ALL. In view of the very small patient numbers with relapsed lymphoma, careful prioritisation is needed to evaluate CAR T-cells in children with Burkitt lymphoma, primary mediastinal B cell lymphoma and other NHL subtypes. Combination trials of alternative targets to CD19 (CD20 or CD22) should also be explored as a priority to improve efficacy in this population. Development of CD30 CAR T-cell immunotherapy strategies in patients with relapsed/refractory Hodgkin lymphoma will likely be most efficiently accomplished by joint paediatric and adult trials. CAR T-cell approaches are early in development for AML and T-ALL, given the unique challenges of successful immunotherapy actualisation in these diseases. At this time, CD33 and CD123 appear to be the most universal targets in AML and CD7 in T-ALL. The results of ongoing or planned first-in-human studies are required to facilitate further understanding. There are promising early results in solid tumours, particularly with GD2 targeting cell therapies in neuroblastoma and central nervous system gliomas that represent significant unmet clinical needs. Further understanding of biology is critical to success. The comparative benefits of autologous versus allogeneic CAR T-cells, T-cells engineered with T cell receptors T-cells engineered with T cell receptor fusion constructs, CAR Natural Killer (NK)-cell products, bispecific T-cell engager antibodies and antibody-drug conjugates require evaluation in paediatric malignancies. Early and proactive academia and multi-company engagement are mandatory to advance cellular immunotherapies in paediatric oncology. Regulatory advice should be sought very early in the design and preparation of clinical trials of innovative medicines, for which regulatory approval may ultimately be sought. Aligning strategic, scientific, regulatory, health technology and funding requirements from the inception of a clinical trial is especially important as these are very expensive therapies. The model for drug development for cell therapy in paediatric oncology could also involve a 'later stage handoff' to industry after early development in academic hands. Finally, and very importantly, strategies must evolve to ensure appropriate ease of access for children who need and could potentially benefit from these therapies.

Human skeletal muscle CD90+ fibro-adipogenic progenitors are associated with muscle degeneration in type 2 diabetic patients.

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.

Electron transfer flavoprotein and its role in mitochondrial energy metabolism in health and disease.

Electron transfer flavoprotein (ETF) is an enzyme with orthologs from bacteria to humans. Human ETF is nuclear encoded by two separate genes, ETFA and ETFB, respectively. After translation, the two subunits are imported to the mitochondrial matrix space and assemble into a heterodimer containing one FAD and one AMP as cofactors. ETF functions as a hub taking up electrons from at least 14 flavoenzymes, feeding them into the respiratory chain. This represents a major source of reducing power for the electron transport chain from fatty acid oxidation and amino acid degradation. Transfer of electrons from the donor enzymes to ETF occurs by direct transfer between the enzyme bound flavins, a process that is tightly regulated by the polypeptide chain and by protein:protein interactions. ETF, in turn relays electrons to the iron sulfur cluster of the inner membrane protein ETF:QO, from where they travel via the FAD in ETF:QO to ubiquinone, entering the respiratory chain at the level of complex III. ETF recognizes its dehydrogenase partners via a recognition loop that anchors the protein on its partner followed by dynamic movements of the ETF flavin domain that bring redox cofactors in close proximity, thus promoting electron transfer. Genetic mutations in the ETFA or ETFB genes cause the Mendelian disorder multiple acyl-CoA dehydrogenase deficiency (MADD; OMIM #231680). We here review the knowledge on human ETF and investigations of the effects of disease-associated missense mutations in this protein that have promoted the understanding of the essential role that ETF plays in cellular metabolism and human disease.

Optimized High-Contrast Brightfield Microscopy Application for Noninvasive Proliferation Assays of Human Cell Cultures.

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.

A recurrent de novo HSPD1 variant is associated with hypomyelinating leukodystrophy.

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.

Riboflavin Deficiency-Implications for General Human Health and Inborn Errors of Metabolism.

As an essential vitamin, the role of riboflavin in human diet and health is increasingly being highlighted. Insufficient dietary intake of riboflavin is often reported in nutritional surveys and population studies, even in non-developing countries with abundant sources of riboflavin-rich dietary products. A latent subclinical riboflavin deficiency can result in a significant clinical phenotype when combined with inborn genetic disturbances or environmental and physiological factors like infections, exercise, diet, aging and pregnancy. Riboflavin, and more importantly its derivatives, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), play a crucial role in essential cellular processes including mitochondrial energy metabolism, stress responses, vitamin and cofactor biogenesis, where they function as cofactors to ensure the catalytic activity and folding/stability of flavoenzymes. Numerous inborn errors of flavin metabolism and flavoenzyme function have been described, and supplementation with riboflavin has in many cases been shown to be lifesaving or to mitigate symptoms. This review discusses the environmental, physiological and genetic factors that affect cellular riboflavin status. We describe the crucial role of riboflavin for general human health, and the clear benefits of riboflavin treatment in patients with inborn errors of metabolism.

An inventory of interactors of the human HSP60/HSP10 chaperonin in the mitochondrial matrix space.

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.

Potential complementation effects of two disease-associated mutations in tetrameric glutaryl-CoA dehydrogenase is due to inter subunit stability-activity counterbalance.

Glutaric Aciduria Type I (GA-I), is an autosomal recessive neurometabolic disease caused by mutations in the GCDH gene that encodes for glutaryl-CoA dehydrogenase (GCDH), a flavoprotein involved in the metabolism of tryptophan, lysine and hydroxylysine. Although over 200 disease mutations have been reported a clear correlation between genotype and phenotype has been difficult to establish. To contribute to a better molecular understanding of GA-I we undertook a detailed molecular study on two GCDH disease-related variants, GCDH-p.Arg227Pro and GCDH-p.Val400Met. Heterozygous patients harbouring these two mutations have increased residual enzymatic activity in relation to homozygous patients with only one of the mutations, suggesting a complementation effect between the two. Combining biochemical, biophysical and structural methods we here establish the effects of these mutations on protein folding, stability and catalytic activity. We show that both variants retain the overall protein fold, but with compromised enzymatic activities. Detailed enzyme kinetic studies reveal that GCDH-p.Arg227Pro has impaired function due to deficient substrate affinity as evidenced by its higher Km, and that the lower activity in GCDH-p.Val400Met results from weaker interactions with its physiological redox partner (electron transfer flavoprotein). Moreover, the GCDH-p.Val400Met variant has a significantly lower thermal stability (ΔTm ≈ 9 °C), and impaired binding of the FAD cofactor in relation to wild-type protein. On these grounds, we provide a rational for the possible interallelic complementation observed in heterozygous patients based on the fact that in GCDH, the low active p.Arg227Pro variant contributes to stabilize the tetramer while the structurally unstable p.Val400Met variant compensates for enzyme activity.

A Cell Model for HSP60 Deficiencies: Modeling Different Levels of Chaperonopathies Leading to Oxidative Stress and Mitochondrial Dysfunction.

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.

Deficiency of the mitochondrial sulfide regulator ETHE1 disturbs cell growth, glutathione level and causes proteome alterations outside mitochondria.

The mitochondrial enzyme ETHE1 is a persulfide dioxygenase essential for cellular sulfide detoxification, and its deficiency causes the severe and complex inherited metabolic disorder ethylmalonic encephalopathy (EE). In spite of well-described clinical symptoms of the disease, detailed cellular and molecular characterization is still ambiguous. Cellular redox regulation has been described to be influenced in ETHE1 deficient cells, and to clarify this further we applied image cytometry and detected decreased levels of reduced glutathione (GSH) in cultivated EE patient fibroblast cells. Cell growth initiation of the EE patient cells was impaired, whereas cell cycle regulation was not. Furthermore, Seahorse metabolic analyzes revealed decreased extracellular acidification, i. e. decreased lactate formation from glycolysis, in the EE patient cells. TMT-based large-scale proteomics was subsequently performed to broadly elucidate cellular consequences of the ETHE1 deficiency. More than 130 proteins were differentially regulated, of which the majority were non-mitochondrial. The proteomics data revealed a link between ETHE1-deficiency and down-regulation of several ribosomal proteins and LIM domain proteins important for cellular maintenance, and up-regulation of cell surface glycoproteins. Furthermore, several proteins of endoplasmic reticulum (ER) were perturbed including proteins influencing disulfide bond formation (e.g. protein disulfide isomerases and peroxiredoxin 4) and calcium-regulated proteins. The results indicate that decreased level of reduced GSH and alterations in proteins of ribosomes, ER and of cell adhesion lie behind the disrupted cell growth of the EE patient cells.

APD-Containing Cyclolipodepsipeptides Target Mitochondrial Function in Hypoxic Cancer Cells.

The natural product family of macrocyclic lipodepsipeptides containing the 4-amido-2,4-pentadienoate functionality possesses intriguing cytotoxic selectivity toward hypoxic cancer cells. These subpopulations of cancer cells display increased metastatic potential and resistance to chemo- and radiotherapy. In this paper, we present studies on the mechanism of action of these natural products in hypoxic cancer cells and show that this involves rapid and hypoxia-selective collapse of mitochondrial integrity and function. These events drive a regulated cell death process that potentially could function as a powerful tool in the fight against chemo- and radiotherapy-resistant cancer cells. Toward that end, we demonstrate activity in two different mouse tumor models.

The clinical outcome of LMNA missense mutations can be associated with the amount of mutated protein in the nuclear envelope.

AIMS: Lamin A/C mutations are generally believed to be associated with a severe prognosis. The aim of this study was to investigate disease expression in three affected families carrying different LMNA missense mutations. Furthermore, the potential molecular disease mechanisms of the mutations were investigated in fibroblasts obtained from mutation carriers. METHODS AND RESULTS: A LMNA-p.Arg216Cys missense mutation was identified in a large family with 36 mutation carriers. Disease expression was unusual with a late onset and a favourable prognosis. Two smaller families with severe disease expression were shown to carry a LMNA-p.Arg471Cys and LMNA-p.Arg471His mutation, respectively. LMNA gene and protein expression was investigated in eight different mutation carriers by quantitative reverse transcriptase polymerase chain reaction, Western blotting, immunohistochemistry, and protein mass spectrometry. The results showed that all mutation carriers incorporated mutated lamin protein into the nuclear envelope. Interestingly, the ratio of mutated to wild-type protein was only 30:70 in LMNA-p.Arg216Cys carriers with a favourable prognosis while LMNA-p.Arg471Cys and LMNA-p.Arg471His carriers with a more severe outcome expressed significantly more of the mutated protein by a ratio of 50:50. CONCLUSION: The clinical findings indicated that some LMNA mutations may be associated with a favourable prognosis and a low risk of sudden death. Protein expression studies suggested that a severe outcome was associated with the expression of high amounts of mutated protein. These findings may prove to be helpful in counselling and risk assessment of LMNA families.

Metformin targets brown adipose tissue in vivo and reduces oxygen consumption in vitro.

AIMS: To test the hypothesis that brown adipose tissue (BAT) is a metformin target tissue by investigating in vivo uptake of [11 C]-metformin tracer in mice and studying in vitro effects of metformin on cultured human brown adipocytes. MATERIALS AND METHODS: Tissue-specific uptake of metformin was assessed in mice by PET/CT imaging after injection of [11 C]-metformin. Human brown adipose tissue was obtained from elective neck surgery and metformin transporter expression levels in human and murine BAT were determined by qPCR. Oxygen consumption in metformin-treated human brown adipocyte cell models was assessed by Seahorse XF technology. RESULTS: Injected [11 C]-metformin showed avid uptake in the murine interscapular BAT depot. Metformin exposure in BAT was similar to hepatic exposure. Non-specific inhibition of the organic cation transporter (OCT) protein by cimetidine administration eliminated BAT exposure to metformin, demonstrating OCT-mediated uptake. Gene expression profiles of OCTs in BAT revealed ample OCT3 expression in both human and mouse BAT. Incubation of a human brown adipocyte cell models with metformin reduced cellular oxygen consumption in a dose-dependent manner. CONCLUSION: These results support BAT as a putative metformin target.

Proteomics of human mitochondria.

Proteomics have passed through a tremendous development in the recent years by the development of ever more sensitive, fast and precise mass spectrometry methods. The dramatically increased research in the biology of mitochondria and their prominent involvement in all kinds of diseases and ageing has benefitted from mitochondrial proteomics. We here review substantial findings and progress of proteomic analyses of human cells and tissues in the recent past. One challenge for investigations of human samples is the ethically and medically founded limited access to human material. The increased sensitivity of mass spectrometry technology aids in lowering this hurdle and new approaches like generation of induced pluripotent cells from somatic cells allow to produce patient-specific cellular disease models with great potential. We describe which human sample types are accessible, review the status of the catalog of human mitochondrial proteins and discuss proteins with dual localization in mitochondria and other cellular compartments. We describe the status and developments of pertinent mass spectrometric strategies, and the use of databases and bioinformatics. Using selected illustrative examples, we draw a picture of the role of proteomic analyses for the many disease contexts from inherited disorders caused by mutation in mitochondrial proteins to complex diseases like cancer, type 2 diabetes and neurodegenerative diseases. Finally, we speculate on the future role of proteomics in research on human mitochondria and pinpoint fields where the evolving technologies will be exploited.

Mitochondrial Spare Respiratory Capacity Is Negatively Correlated with Nuclear Reprogramming Efficiency.

Nuclear reprogramming efficiency has been shown to be highly variable among different types of somatic cells and different individuals, yet the underlying mechanism remains largely unknown. Several studies have shown that reprogramming of fibroblasts into induced pluripotent stem cells (iPSCs) requires remodeling of mitochondria and a metabolic shift from an oxidative state to a more glycolytic state. In this study, we evaluated the nuclear reprogramming efficiency in relation to mitochondrial bioenergetic parameters of fibroblasts from seven different human individuals. Using the Seahorse extracellular energy flux analyzer, we measured oxygen consumption rate (OCR) profiles of the cells, along with their nuclear reprogramming efficiency into iPSCs. Our results showed that fibroblasts with the lowest mitochondrial spare respiratory capacity (SRC) had the highest nuclear reprogramming efficiency, opposed to fibroblasts with the highest mitochondrial SRC, which showed lowest reprogramming efficiency. Furthermore, we found that targeted fluorescent tagging of endogenous genes (MYH6 and COL2A1) by CRISPR/Cas9-mediated homologous recombination was accompanied by an increase in the SRC level of the modified fibroblasts and impaired reprogramming efficiency. Our findings indicate a negative correlation between high mitochondrial SRC in somatic cells and low reprogramming efficiencies. This type of analysis potentially allows screening and predicting reprogramming efficiency before reprogramming, and further suggests that nuclear reprogramming might be improved by approaches that modulate the SRC.