Clinical and biological characterization of 20 patients with TANGO2 deficiency indicates novel triggers of metabolic crises and no primary energetic defect.

TANGO2 disease is a severe inherited disorder associating multiple symptoms such as metabolic crises, encephalopathy, cardiac arrhythmias, and hypothyroidism. The mechanism of action of TANGO2 is currently unknown. Here, we describe a cohort of 20 French patients bearing mutations in the TANGO2 gene. We found that the main clinical presentation was the association of neurodevelopmental delay (n = 17), acute metabolic crises (n = 17) and hypothyroidism (n = 12), with a large intrafamilial clinical variability. Metabolic crises included rhabdomyolysis (15/17), neurological symptoms (14/17), and cardiac features (12/17; long QT (n = 10), Brugada pattern (n = 2), cardiac arrhythmia (n = 6)) that required intensive care. We show previously uncharacterized triggers of metabolic crises in TANGO2 patients, such as some anesthetics and possibly l-carnitine. Unexpectedly, plasma acylcarnitines, plasma FGF-21, muscle histology, and mitochondrial spectrometry were mostly normal. Moreover, in patients' primary myoblasts, palmitate and glutamine oxidation rates, and the mitochondrial network were also normal. Finally, we found variable mitochondrial respiration and defective clearance of oxidized DNA upon cycles of starvation and refeeding. We conclude that TANGO2 disease is a life-threatening disease that needs specific cardiac management and anesthesia protocol. Mechanistically, TANGO2 disease is unlikely to originate from a primary mitochondrial defect. Rather, we suggest that mitochondrial defects are secondary to strong extrinsic triggers in TANGO2 deficient patients.

A thermolabile aldolase A mutant causes fever-induced recurrent rhabdomyolysis without hemolytic anemia.

Aldolase A deficiency has been reported as a rare cause of hemolytic anemia occasionally associated with myopathy. We identified a deleterious homozygous mutation in the ALDOA gene in 3 siblings with episodic rhabdomyolysis without hemolytic anemia. Myoglobinuria was always triggered by febrile illnesses. We show that the underlying mechanism involves an exacerbation of aldolase A deficiency at high temperatures that affected myoblasts but not erythrocytes. The aldolase A deficiency was rescued by arginine supplementation in vitro but not by glycerol, betaine or benzylhydantoin, three other known chaperones, suggesting that arginine-mediated rescue operated by a mechanism other than protein chaperoning. Lipid droplets accumulated in patient myoblasts relative to control and this was increased by cytokines, and reduced by dexamethasone. Our results expand the clinical spectrum of aldolase A deficiency to isolated temperature-dependent rhabdomyolysis, and suggest that thermolability may be tissue specific. We also propose a treatment for this severe disease.

FDX2 and ISCU Gene Variations Lead to Rhabdomyolysis With Distinct Severity and Iron Regulation.

BACKGROUND AND OBJECTIVES: To determine common clinical and biological traits in 2 individuals with variants in ISCU and FDX2, displaying severe and recurrent rhabdomyolyses and lactic acidosis. METHODS: We performed a clinical characterization of 2 distinct individuals with biallelic ISCU or FDX2 variants from 2 separate families and a biological characterization with muscle and cells from those patients. RESULTS: The individual with FDX2 variants was clinically more affected than the individual with ISCU variants. Affected FDX2 individual fibroblasts and myoblasts showed reduced oxygen consumption rates and mitochondrial complex I and PDHc activities, associated with high levels of blood FGF21. ISCU individual fibroblasts showed no oxidative phosphorylation deficiency and moderate increase of blood FGF21 levels relative to controls. The severity of the FDX2 individual was not due to dysfunctional autophagy. Iron was excessively accumulated in ISCU-deficient skeletal muscle, which was accompanied by a downregulation of IRP1 and mitoferrin2 genes and an upregulation of frataxin (FXN) gene expression. This excessive iron accumulation was absent from FDX2 affected muscle and could not be correlated with variable gene expression in muscle cells. DISCUSSION: We conclude that FDX2 and ISCU variants result in a similar muscle phenotype, that differ in severity and skeletal muscle iron accumulation. ISCU and FDX2 are not involved in mitochondrial iron influx contrary to frataxin.

Compromised mitochondrial quality control triggers lipin1-related rhabdomyolysis.

LPIN1 mutations are responsible for inherited recurrent rhabdomyolysis, a life-threatening condition with no efficient therapeutic intervention. Here, we conduct a bedside-to-bench-and-back investigation to study the pathophysiology of lipin1 deficiency. We find that lipin1-deficient myoblasts exhibit a reduction in phosphatidylinositol-3-phosphate close to autophagosomes and late endosomes that prevents the recruitment of the GTPase Armus, locks Rab7 in the active state, inhibits vesicle clearance by fusion with lysosomes, and alters their positioning and function. Oxidized mitochondrial DNA accumulates in late endosomes, where it activates Toll-like receptor 9 (TLR9) and triggers inflammatory signaling and caspase-dependent myolysis. Hydroxychloroquine blocks TLR9 activation by mitochondrial DNA in vitro and may attenuate flares of rhabdomyolysis in 6 patients treated. We suggest a critical role for defective clearance of oxidized mitochondrial DNA that activates TLR9-restricted inflammation in lipin1-related rhabdomyolysis. Interventions blocking TLR9 activation or inflammation can improve patient care in vivo.

Derivation and Characterization of Immortalized Human Muscle Satellite Cell Clones from Muscular Dystrophy Patients and Healthy Individuals.

In Duchenne muscular dystrophy (DMD) patients, absence of dystrophin causes muscle wasting by impacting both the myofiber integrity and the properties of muscle stem cells (MuSCs). Investigation of DMD encompasses the use of MuSCs issued from human skeletal muscle. However, DMD-derived MuSC usage is restricted by the limited number of divisions that human MuSCs can undertake in vitro before losing their myogenic characteristics and by the scarcity of human material available from DMD muscle. To overcome these limitations, immortalization of MuSCs appears as a strategy. Here, we used CDK4/hTERT expression in primary MuSCs and we derived MuSC clones from a series of clinically and genetically characterized patients, including eight DMD patients with various mutations, four congenital muscular dystrophies and three age-matched control muscles. Immortalized cultures were sorted into single cells and expanded as clones into homogeneous populations. Myogenic characteristics and differentiation potential were tested for each clone. Finally, we screened various promoters to identify the preferred gene regulatory unit that should be used to ensure stable expression in the human MuSC clones. The 38 clonal immortalized myogenic cell clones provide a large collection of controls and DMD clones with various genetic defects and are available to the academic community.

TUBG1 missense variants underlying cortical malformations disrupt neuronal locomotion and microtubule dynamics but not neurogenesis.

De novo heterozygous missense variants in the γ-tubulin gene TUBG1 have been linked to human malformations of cortical development associated with intellectual disability and epilepsy. Here, we investigated through in-utero electroporation and in-vivo studies, how four of these variants affect cortical development. We show that TUBG1 mutants affect neuronal positioning, disrupting the locomotion of new-born neurons but without affecting progenitors' proliferation. We further demonstrate that pathogenic TUBG1 variants are linked to reduced microtubule dynamics but without major structural nor functional centrosome defects in subject-derived fibroblasts. Additionally, we developed a knock-in Tubg1Y92C/+ mouse model and assessed consequences of the mutation. Although centrosomal positioning in bipolar neurons is correct, they fail to initiate locomotion. Furthermore, Tubg1Y92C/+ animals show neuroanatomical and behavioral defects and increased epileptic cortical activity. We show that Tubg1Y92C/+ mice partially mimic the human phenotype and therefore represent a relevant model for further investigations of the physiopathology of cortical malformations.

Myogenic Progenitor Cells Exhibit Type I Interferon-Driven Proangiogenic Properties and Molecular Signature During Juvenile Dermatomyositis.

OBJECTIVE: Juvenile dermatomyositis (JDM) is an inflammatory pediatric myopathy characterized by focal capillary loss in muscle, followed by progressive recovery upon adequate treatment with immunomodulating drugs, although some patients remain refractory to treatment. While the underlying mechanism of capillary depletion remains uncertain, recent studies have identified an up-regulation of type I interferon (IFN) expression specific to JDM. Given that myogenic precursor cells (MPCs) exert proangiogenic activity during normal skeletal muscle regeneration, we hypothesized that they may also modulate vascular remodeling/angiogenesis during JDM. The aim of this study was to investigate that hypothesis. METHODS: Human cell cocultures were used to analyze angiogenic properties in patients with JDM, patients with Duchenne's muscular dystrophy (DMD) (control patients for vascular remodeling), and healthy control subjects. Transcriptome analysis was used to examine muscle-derived MPCs. Histologic analysis of type I IFN in muscle biopsy samples was also performed. RESULTS: Using human cell cocultures, we showed highly angiogenic properties of MPCs from JDM patients in association with the expression of an angiogenic molecular signature. Transcriptome analysis of MPCs freshly isolated from muscle samples revealed type I IFN as the master regulator of the most up-regulated genes in JDM-derived MPCs. Functionally, treatment of normal MPCs with type I IFN recapitulated the molecular pattern and the proangiogenic functions of JDM-derived MPCs. In vivo histologic investigation showed that MPCs synthesized type I IFN and major proangiogenic molecules in JDM muscle. Moreover, MPCs derived from JDM muscles that were characterized by strong vasculopathy produced higher levels of type I IFN, confirming MPCs as a cellular source of type I IFN during JDM, and this correlated with the severity of the disease. CONCLUSION: These results demonstrate a new type I IFN pathway in JDM that activates the production of angiogenic effectors by MPCs, triggering their proangiogenic function to promote vessel recovery and muscle reconstruction.

Dual mTORC1/2 inhibition induces anti-proliferative effect in NF1-associated plexiform neurofibroma and malignant peripheral nerve sheath tumor cells.

Approximately 30-50% of individuals with Neurofibromatosis type 1 develop benign peripheral nerve sheath tumors, called plexiform neurofibromas (PNFs). PNFs can undergo malignant transformation to highly metastatic malignant peripheral nerve sheath tumors (MPNSTs) in 5-10% of NF1 patients, with poor prognosis. No effective systemic therapy is currently available for unresectable tumors. In tumors, the NF1 gene deficiency leads to Ras hyperactivation causing the subsequent activation of the AKT/mTOR and Raf/MEK/ERK pathways and inducing multiple cellular responses including cell proliferation. In this study, three NF1-null MPNST-derived cell lines (90-8, 88-14 and 96-2), STS26T sporadic MPNST cell line and PNF-derived primary Schwann cells were used to test responses to AZD8055, an ATP-competitive "active-site" mTOR inhibitor. In contrast to rapamycin treatment which only partially affected mTORC1 signaling, AZD8055 induced a strong inhibition of mTORC1 and mTORC2 signaling in MPNST-derived cell lines and PNF-derived Schwann cells. AZD8055 induced full blockade of mTORC1 leading to an efficient decrease of global protein synthesis. A higher cytotoxic effect was observed with AZD8055 compared to rapamycin in the NF1-null MPNST-derived cell lines with IC50 ranging from 70 to 140 nM and antiproliferative effect was confirmed in PNF-derived Schwann cells. Cell migration was impaired by AZD8055 treatment and cell cycle analysis showed a G0/G1 arrest. Combined effects of AZD8055 and PD0325901 MEK inhibitor as well as BRD4 (BromoDomain-containing protein 4) inhibitors showed a synergistic antiproliferative effect. These data suggest that NF1-associated peripheral nerve sheath tumors are an ideal target for AZD8055 as a single molecule or in combined therapies.

Apoptosis-induced proteinase 3 membrane expression is independent from degranulation.

Proteinase 3 (PR3) and human neutrophil elastase (HNE) are serine proteinases stored in the azurophilic granules of neutrophils. In contrast to HNE, PR3 is the target of antineutrophil cytoplasm antibodies (ANCA) in Wegener's granulomatosis. The mechanisms leading to the membrane expression of PR3 and HNE are still unclear and appear to be critical to understand the pathophysiological role of ANCA. Stably transfected rat basophilic cell lines (RBL) with PR3 or HNE were used to analyze the PR3 and HNE secretion mechanisms and differentiate between them. RBL cells were lacking endogenous PR3 and HNE. They were stably transfected with HNE or PR3 or an inactive mutant of PR3 (PR3S203A). Using the calcium ionophore A23187 as a secretagogue, higher serine proteinase activity was secreted in the supernatant of RBL/HNE than in RBL/PR3. It is interesting that PR3 and PR3/S203A were also expressed at the plasma membrane, thus demonstrating that serine protease activity was not required for plasma membrane expression. In contrast, no expression of plasma membrane HNE could be detected in RBL/HNE. Apoptosis induced by etoposide was evaluated by DNA fragmentation, the presence of cytoplasmic histone-associated DNA fragments, and annexin V labeling. No membrane HNE was detected in RBL/HNE. In contrast, in RBL/PR3 and in RBL/PR3S203A, the membrane expression of PR3 and PR3S203A increased with etoposide concentrations and appeared closely related to annexin V labeling. Our data suggest that membrane PR3 originates from two distinct pools, the granular pool mobilized following degranulation or a plasma membrane pool mobilized upon apoptosis.

Dp412e: a novel human embryonic dystrophin isoform induced by BMP4 in early differentiated cells.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a devastating X-linked recessive genetic myopathy. DMD physiopathology is still not fully understood and a prenatal onset is suspected but difficult to address. METHODS: The bone morphogenetic protein 4 (BMP4) is a critical signaling molecule involved in mesoderm commitment. Human induced pluripotent stem cells (hiPSCs) from DMD and healthy individuals and human embryonic stem cells (hESCs) treated with BMP4 allowed us to model the early steps of myogenesis in normal and DMD contexts. RESULTS: Unexpectedly, 72h following BMP4 treatment, a new long DMD transcript was detected in all tested hiPSCs and hESCs, at levels similar to that found in adult skeletal muscle. This novel transcript named "Dp412e" has a specific untranslated first exon which is conserved only in a sub-group of anthropoids including human. The corresponding novel dystrophin protein of 412-kiloDalton (kDa), characterized by an N-terminal-truncated actin-binding domain, was detected in normal BMP4-treated hiPSCs/hESCs and in embryoid bodies. Finally, using a phosphorodiamidate morpholino oligomer (PMO) targeting the DMD exon 53, we demonstrated the feasibility of exon skipping validation with this BMP4-inducible hiPSCs model. CONCLUSIONS: In this study, the use of hiPSCs to analyze early phases of human development in normal and DMD contexts has led to the discovery of an embryonic 412 kDa dystrophin isoform. Deciphering the regulation process(es) and the function(s) associated to this new isoform can contribute to a better understanding of the DMD physiopathology and potential developmental defects. Moreover, the simple and robust BMP4-inducible model highlighted here, providing large amount of a long DMD transcript and the corresponding protein in only 3 days, is already well-adapted to high-throughput and high-content screening approaches. Therefore, availability of this powerful cell platform can accelerate the development, validation and improvement of DMD genetic therapies.

Cell cloning-based transcriptome analysis in cyclin-dependent kinase-like 5 mutation patients with severe epileptic encephalopathy.

Mutations in the human CDKL5 gene have been shown to cause infantile spasms, as well as Rett syndrome-like phenotype. Because CDKL5 is subjected to X chromosome inactivation (XCI), individual cells from CDKL5 mutation girls either express the wild-type or mutant allele, likely resulting in different consequences at both the cellular and molecular levels. To identify these consequences, we carried out gene expression profiling on clonal populations derived from primary cultures of three patients' fibroblasts either expressing the wild-type or mutant allele. A total of 16 up-regulated and 20 down-regulated genes were identified. The differentially expressed gene products, mostly involved in differentiation and oxidative stress may be related to a mechanism underlying mental retardation and epilepsy. Among these, the apoptosis signal-regulated kinase MAP3K5 expression was found to be altered in non-neuronal, but also in neuronal CDKL5-deficient cells. Due to the fact that MAP3K5 activates MAP kinase pathway, which mediates signals leading to both differentiation and survival in neuronal cells, we suggest that a CDKL5 deficit may induce changes in synaptic plasticity in the patient's brain.

The cleavage of neutrophil leukosialin (CD43) by cathepsin G releases its extracellular domain and triggers its intramembrane proteolysis by presenilin/gamma-secretase.

The highly negatively charged membrane sialoglycoprotein leukosialin, CD43, is shed during neutrophil activation. This is generally thought to enhance cell adhesion. We here describe two novel consequences of this shedding, during neutrophil activation by phorbol esters or by chemoattractants after TNF-alpha priming. CD43 proteolysis was investigated by Western blotting, using a polyclonal antibody to CD43 intracellular domain. Our data emphasize the importance of a juxtamembranous cleavage of about 50% of membrane CD43 molecules by cathepsin G. Indeed, it is inhibited by alpha1-antichymotrypsin and cathepsin G inhibitor I and is reproduced by exogenous purified cathepsin G. The resulting membrane-anchored C-terminal fragment, CD43-CTF, becomes susceptible to presenilin/gamma-secretase, which releases CD43 intracytoplasmic domain: preincubation with three different gamma-secretase inhibitors, before PMN treatment by agonists or by purified cathepsin G, results in the accumulation of CD43-CTF. Because CD43 binds E-selectin, we also investigated the effect of the soluble extracellular domain CD43s, released by cathepsin G juxtamembranous cleavage, on neutrophil adhesion to endothelial cells. A recombinant CD43s-Fc fusion protein inhibited neutrophil E selectindependent adhesion to endothelial cells under flow conditions, while it had no effect on neutrophil static adhesion. We thus propose that, in addition to its potential pro-adhesive role, CD43 proteolysis results in: (i) the release, by cathepsin G, of CD43 extracellular domain, able to inhibit the adhesion of flowing neutrophils on endothelial cells and thus to participate to the natural control of inflammation; (ii) the release and/or the clearance, by presenilin/gamma-secretase, of CD43 intracellular domain, thereby regulating CD43-mediated signaling.

Distinct signaling pathways are involved in leukosialin (CD43) down-regulation, membrane blebbing, and phospholipid scrambling during neutrophil apoptosis.

Although leukosialin (CD43) membrane expression decreases during neutrophil apoptosis, the CD43 molecule, unexpectedly, is neither proteolyzed nor internalized. We thus wondered whether it could be shed on bleb-derived membrane vesicles. Membrane blebbing is a transient event, hardly appreciated during the asynchronous, spontaneous apoptosis of neutrophils. Cell pre-synchronization at 15 degrees C made it possible to observe numerous blebbing neutrophils for a short 1-h period at 37 degrees C. CD43 down-regulation co-occurred with the blebbing stage and phosphatidylserine externalization, shortly after mitochondria depolarization and before nuclear condensation. Blebs detaching from the cell body were observed by time lapse fluorescence microscopy, and the release of bleb-derived vesicles was followed by flow cytometry. Phosphatidylserine externalization required caspases and protein kinase C (PKC) but not the myosin light chain kinase (MLCK). By contrast, bleb formation and release was caspase- and PKC-independent but required an active MLCK, whereas CD43 down-regulation involved caspases but neither PKC nor MLCK. Furthermore, CD43 appeared mostly excluded from membrane blebs by electron microscopy. Thus, CD43 down-regulation does not result from the release of bleb-derived vesicles. Ultracentrifugation of apoptotic cell supernatants made it possible to recover <1 microM microparticles, which contained the entire CD43 molecule. These microparticles expressed neutrophil membrane markers such as CD11b, CD66b, and CD63, together with CD43. In conclusion, we show that the three early membrane events of apoptosis, namely blebbing, phosphatidylserine externalization, and CD43 down-regulation, result from different signaling pathways and can occur independently from one another. CD43 down-regulation results from the shedding of microparticles released during apoptosis but unrelated to the blebbing.

Rapamycin inhibits human renal epithelial cell proliferation: effect on cyclin D3 mRNA expression and stability.

BACKGROUND: Recent data have suggested that rapamycin use during the initial period after transplantation is associated with prolonged delayed graft function (DGF). Because of the known effects of rapamycin in other cell types, we speculated that this action may be secondary to human renal epithelial cells (HRECs) inhibition of proliferation. METHODS: Primary cultures of HRECs were incubated with various concentrations of rapamycin. Cell proliferation was evaluated by cytotoxicity assays. The cell cycle was analyzed by flow cytometry. Protein expression levels were assessed by Western blot. Cyclin D3 mRNA levels were measured by quantitative real-time polymerase chain reaction (PCR). The transcriptional activity of the cyclin D3 gene was evaluated using transient transfection. RESULTS: Rapamycin exerted a significant concentration-dependent antiproliferative effect on growing HRECs by inhibiting the G(1) to S transition. The p70(S6) kinase pathway leading to cell cycle progression was found to be active, and low concentrations of rapamycin dramatically reduced p70(S6) kinase phosphorylation. Rapamycin completely inhibited the increase in cyclin D3 protein expression and mRNA accumulation induced by fetal calf serum, but did not affect cyclin E or cdk-inhibitor expression levels. This regulation of cyclin D3 protein expression is mainly due to a destabilization of its mRNA. Rapamycin reduced the mRNA half-life by 26% (4.8 +/- 1.3 hours vs. 6.5 +/- 1.0 hours, P < 0.001). CONCLUSION: Rapamycin inhibits the proliferative response of HRECs to mitogenic stimuli, and causes cell cycle arrest in the early G(1) phase, not only by a nonspecific process due to inhibition of the p70(S6k) pathway, but also by a direct effect on cyclin D3 mRNA stability.

Beta4 integrin and laminin 5 are aberrantly expressed in polycystic kidney disease: role in increased cell adhesion and migration.

Extracellular matrix alterations have been suggested to be part of the early events occurring in Autosomal Dominant Polycystic Kidney Disease (ADPKD), a disease characterized by formation of renal cysts and progressive renal failure. Here we report that cDNA array analysis identified beta(4) integrin aberrant expression in ADPKD cells. Furthermore, laminin 5 (Ln-5), the main alpha(6)beta(4) integrin ligand, was also found to be abnormally expressed in ADPKD. Studies performed with ADPKD cyst-lining epithelial cells (CC) by comparison with normal tubular cells indicate that integrin alpha(6)beta(4)-Ln-5 interactions are involved in cellular events of potential importance for cystogenesis: 1) laminin 5 is a preferential adhesion substrate for CC, mainly through alpha(6)beta(4) interaction, 2) CC increased haptotactic and chemotactic motility depends on the presence of Ln-5 and requires integrin alpha(3)beta(1) cooperation, and 3) CC haptotactic or chemotactic migration is specifically increased by mAb-mediated beta(4) integrin ligation, through an alpha(3)beta(1) integrin-dependent and independent pathway, respectively. These results highlight the role of Ln-5 and alpha(6)beta(4) integrin in adhesive and motility properties of cyst-lining epithelial cells, and further suggest that integrins and extracellular matrix modifications may be of general relevance to kidney epithelial cell cyst formation.