Creation of an Isogenic Human iPSC-Based RGC Model of Dominant Optic Atrophy Harboring the Pathogenic Variant c.1861C>T (p.Gln621Ter) in the OPA1 Gene.

Autosomal dominant optic atrophy (ADOA) is a rare progressive disease mainly caused by mutations in OPA1, a nuclear gene encoding for a mitochondrial protein that plays an essential role in mitochondrial dynamics, cell survival, oxidative phosphorylation, and mtDNA maintenance. ADOA is characterized by the degeneration of retinal ganglion cells (RGCs). This causes visual loss, which can lead to legal blindness in many cases. Nowadays, there is no effective treatment for ADOA. In this article, we have established an isogenic human RGC model for ADOA using iPSC technology and the genome editing tool CRISPR/Cas9 from a previously generated iPSC line of an ADOA plus patient harboring the pathogenic variant NM_015560.3: c.1861C>T (p.Gln621Ter) in heterozygosis in OPA1. To this end, a protocol based on supplementing the iPSC culture media with several small molecules and defined factors trying to mimic embryonic development has been employed. Subsequently, the created model was validated, confirming the presence of a defect of intergenomic communication, impaired mitochondrial respiration, and an increase in apoptosis and ROS generation. Finally, we propose the analysis of OPA1 expression by qPCR as an easy read-out method to carry out future drug screening studies using the created RGC model. In summary, this model provides a useful platform for further investigation of the underlying pathophysiological mechanisms of ADOA plus and for testing compounds with potential pharmacological action.

Generation of the First Human In Vitro Model for McArdle Disease Based on iPSC Technology.

McArdle disease is a rare autosomal recessive disorder caused by mutations in the PYGM gene. This gene encodes for the skeletal muscle isoform of glycogen phosphorylase (myophosphorylase), the first enzyme in glycogenolysis. Patients with this disorder are unable to obtain energy from their glycogen stored in skeletal muscle, prompting an exercise intolerance. Currently, there is no treatment for this disease, and the lack of suitable in vitro human models has prevented the search for therapies against it. In this article, we have established the first human iPSC-based model for McArdle disease. For the generation of this model, induced pluripotent stem cells (iPSCs) from a patient with McArdle disease (harbouring the homozygous mutation c.148C>T; p.R50* in the PYGM gene) were differentiated into myogenic cells able to contract spontaneously in the presence of motor neurons and generate calcium transients, a proof of their maturity and functionality. Additionally, an isogenic skeletal muscle model of McArdle disease was created. As a proof-of-concept, we have tested in this model the rescue of PYGM expression by two different read-through compounds (PTC124 and RTC13). The developed model will be very useful as a platform for testing drugs or compounds with potential pharmacological activity.

Correction: Enhanced tumorigenicity by mitochondrial DNA mild mutations.

[This corrects the article DOI: 10.18632/oncotarget.3698.].

The mutation m.13513G>A impairs cardiac function, favoring a neuroectoderm commitment, in a mutant-load dependent way.

Mitochondrial disorders (MDs) arise as a result of a respiratory chain dysfunction. While some MDs can affect a single organ, many involve several organs, the brain being the most affected, followed by heart and/or muscle. Many of these diseases are associated with heteroplasmic mutations in the mitochondrial DNA (mtDNA). The proportion of mutated mtDNA must exceed a critical threshold to produce disease. Therefore, understanding how embryonic development determines the heteroplasmy level in each tissue could explain the organ susceptibility and the clinical heterogeneity observed in these patients. In this report, the dynamics of heteroplasmy and the influence in cardiac commitment of the mutational load of the m.13513G>A mutation has been analyzed. This mutation has been reported as a frequent cause of Leigh syndrome (LS) and is commonly associated with cardiac problems. In this report, induced pluripotent stem cell (iPSc) technology has been used to delve into the molecular mechanisms underlying cardiac disease in LS. When mutation m.13513G>A is above a threshold, iPSc-derived cardiomyocytes (iPSc-CMs) could not be obtained due to an inefficient epithelial-mesenchymal transition. Surprisingly, these cells are redirected toward neuroectodermal lineages that would give rise to the brain. However, when mutation is below that threshold, dysfunctional CM are generated in a mutant-load dependent way. We suggest that distribution of the m.13513G>A mutation during cardiac differentiation is not at random. We propose a possible explanation of why neuropathology is a frequent feature of MD, but cardiac involvement is not always present.

iPSCs: A powerful tool for skeletal muscle tissue engineering.

Both volumetric muscle loss (VML) and muscle degenerative diseases lead to an important decrease in skeletal muscle mass, condition that nowadays lacks an optimal treatment. This issue has driven towards an increasing interest in new strategies in tissue engineering, an emerging field that can offer very promising approaches. In addition, the discovery of induced pluripotent stem cells (iPSCs) has completely revolutionized the actual view of personalized medicine, and their utilization in skeletal muscle tissue engineering could, undoubtedly, add myriad benefits. In this review, we want to provide a general vision of the basic aspects to consider when engineering skeletal muscle tissue using iPSCs. Specifically, we will focus on the three main pillars of tissue engineering: the scaffold designing, the selection of the ideal cell source and the addition of factors that can enhance the resemblance with the native tissue.

Expresión de tuftelina en gérmenes dentales humanos / Expression of tuftelin in human dental germs

La tuftelina es una proteína secretada en la matriz adamantina en desarrollo durante la formación del esmalte. Su función continúa sin esclarecerse, aunque se presume que juega un papel importante en la biomineralización de esmalte y dentina, así como en el desarrollo del órgano dental. Con el presente estudio se identificó su localización en las diferentes estructuras de gérmenes dentales de fetos humanos, conforme a los resultados se observó su expresión en el estadio pre-secretor observándose en el citoplasma de los ameloblastos, retículo estrellado, papila dental, así como en el estrato intermedio; en el secretor se identificó principalmente en la unión amelodentinaria, y en la superficie externa del esmalte, observando una marcada expresión de la proteína en la porción basal del proceso odontoblástico, pero no en la matriz extracelular de la dentina. De acuerdo a los resultados obtenidos se puede considerar que su expresión se presenta tanto en la amelogénesis, como en la odontogénesis en tejidos sin mineralizar.

The tuftelin is a secreted protein in the adamantine matrix in developing during the enamel formation. Its function continues unclarified, although it plays a role in the biomineralization of the dental organ. With the present studio the location was identified in the different structures of dental germs from human fetuses, according to the results it was observed the expression in the pre-secretor stage being observed in the cytoplasm of ameloblasts, stellate reticulum, dental papilla, also in the intermediate stratum; in the secretor it was mainly identified in the amelodentinal junction and in the outer surface of enamel, observing a marked expression of the protein in the basal portion of the odontoblastic process, but not in the extracellular matrix of the dentine. According to the results obtained it can be considered that its expression occurs in both amelogenesis and odontegenesis in unmineralized tissues.

Estudio transversal comparativo de la relación maxilo-mandibular de McNamara aplicadas a sujetos mexicanos / Transversal comparative study of McNamara maxillofacial mandibular ratio applied in Mexican subjects

Las características antropológicas entre diversas razas en las cuales se incluyen etnia, sexo y edad en el mundo varían, es preciso analizar que un estudio establecido para una población no puede ser usado para otra, ya que presenta formas y características diferentes; es por esto que existe la necesidad de realizar estudios en diversas razas comparándolos con las normas establecidas. El objetivo fue comparar la relación maxilo-mandibular de McNamara aplicadas en sujetos mexicanos. Se analizaron 60 radiografías laterales de cráneo con trazados de McNamara. Se identificaron diferencias en los patrones genéticos de crecimiento de los caucásicos y los mexicanos. Nuestros resultados muestran un crecimiento típico de los pacientes clase II esqueletal con maxilar hipotónico posteroanteriormente, una mandíbula disminuida y poca altura vertical, esto como consecuencia de una falta de crecimiento del tercio medio facial por una pobre ventilación aérea.

The anthropological characteristics between different races in which ethnicity, sex and age vary in the world, and it becomes necessary to analyze a set for one population study that cannot be used for another, with different shapes and characteristics. There is a need for studies in different races for comparison with established standards. The objective of the research was to compare the McNamara maxilla-mandibular relationship applied in Mexicans subjects and analyze 60 lateral radiographs of the skulls with McNamara traces. Genetic differences in growth patterns of Caucasians and Mexicans were identified. Our results show typical growth of skeletal class II patients with hypotonic posterior jaw, and jaw with diminished vertical height caused by a lack of midface growth the result of poor air flow.

Reprogramming for Cardiac Regeneration-Strategies for Innovation.

It is well-known that the human myocardium has a low capacity for self-regeneration. This fact is especially important after acute myocardial infarction with subsequent heart failure and adverse tissue remodeling. New potential strategies have recently emerged for treating heart diseases, such as the possibility of generating large quantities of cardiomyocytes through genetic iPSC reprogramming, transdifferentiation for in vitro disease modeling, in vivo therapies or telomerase gene reactivation. Approaches based on these techniques may represent the new horizon in cardiology with an appropriate 180-degree turn perspective. J. Cell. Physiol. 231: 1849-1851, 2016. © 2016 Wiley Periodicals, Inc.

iPSCs-based anti-aging therapies: Recent discoveries and future challenges.

The main biological hallmarks of the aging process include stem cell exhaustion and cellular senescence. Consequently, research efforts to treat age-related diseases as well as anti-aging therapies in general have recently focused on potential 'reprogramming' regenerative therapies. These new approaches are based on induced pluripotent stem cells (iPSCs), including potential in vivo reprogramming for tissue repair. Another possibility is targeting pathways of cellular senescence, e.g., through modulation of p16INK4a signaling and especially inhibition of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). Here, we reviewed and discussed these recent developments together with their possible usefulness for future treatments against sarcopenia, a major age-related condition.

IPSCs, a Promising Tool to Restore Muscle Atrophy.

Induced pluripotent stem cells (iPSCs) are a promising tool for regenerative medicine in chronic conditions associated with muscle atrophy since iPSCs are easier to obtain, pose less ethical limitations and can better capture human genetic diversity compared with human embryonic stem cells. We highlight the potentiality of iPSCs for treating muscle-affecting conditions for which no effective cure is yet available, notably aging sarcopenia and inherited neurometabolic conditions. J. Cell. Physiol. 231: 259-260, 2016. © 2015 Wiley Periodicals, Inc.

Enhanced tumorigenicity by mitochondrial DNA mild mutations.

To understand how mitochondria are involved in malignant transformation we have generated a collection of transmitochondrial cybrid cell lines on the same nuclear background (143B) but with mutant mitochondrial DNA (mtDNA) variants with different degrees of pathogenicity. These include the severe mutation in the tRNALys gene, m.8363G>A, and the three milder yet prevalent Leber's hereditary optic neuropathy (LHON) mutations in the MT-ND1 (m.3460G>A), MT-ND4 (m.11778G>A) and MT-ND6 (m.14484T>C) mitochondrial genes. We found that 143B ρ0 cells devoid of mtDNA and cybrids harboring wild type mtDNA or that causing severe mitochondrial dysfunction do not produce tumors when injected in nude mice. By contrast cybrids containing mild mutant mtDNAs exhibit different tumorigenic capacities, depending on OXPHOS dysfunction.The differences in tumorigenicity correlate with an enhanced resistance to apoptosis and high levels of NOX expression. However, the final capacity of the different cybrid cell lines to generate tumors is most likely a consequence of a complex array of pro-oncogenic and anti-oncogenic factors associated with mitochondrial dysfunction.Our results demonstrate the essential role of mtDNA in tumorigenesis and explain the numerous and varied mtDNA mutations found in human tumors, most of which give rise to mild mitochondrial dysfunction.

The pathogenicity scoring system for mitochondrial tRNA mutations revisited.

Confirming the pathogenicity of mitochondrial tRNA point mutations is one of the classical challenges in the field of mitochondrial medicine. In addition to genetic and functional studies, the evaluation of a genetic change using a pathogenicity scoring system is extremely useful to discriminate between disease-causing mutations from neutral polymorphisms. The pathogenicity scoring system is very robust for confirming pathogenicity, especially of mutations that show impaired activity in functional studies. However, mutations giving normal results using the same functional approaches are disregarded, and this compromises the power of the system to rule out pathogenicity. We propose to include a new criterion in the pathogenicity scoring systems regarding mutations which fail to show any mitochondrial defect in functional studies. To evaluate this proposal we characterized two mutations, m.8296A>G and m.8347A>G, in the mitochondrial tRNA(L) (ys) gene (MT-TK) using trans-mitochondrial cybrid analysis. m.8347A>G mutation severely impairs oxidative phosphorylation, suggesting that it is highly pathogenic. By contrast, the behavior of cybrids homoplasmic for the m.8296A>G mutation is similar to cybrids containing wild-type mitochondrial DNA (mtDNA). The results indicate that including not only positive but also negative outcomes of functional studies in the scoring system is critical for facilitating the diagnosis of this complex group of diseases.

Glutamyl-tRNAGln amidotransferase is essential for mammalian mitochondrial translation in vivo.

Translational accuracy depends on the correct formation of aminoacyl-tRNAs, which, in the majority of cases, are produced by specific aminoacyl-tRNA synthetases that ligate each amino acid to its cognate isoaceptor tRNA. Aminoacylation of tRNAGln, however, is performed by various mechanisms in different systems. Since no mitochondrial glutaminyl-tRNA synthetase has been identified to date in mammalian mitochondria, Gln-tRNAGln has to be formed by an indirect mechanism in the organelle. It has been demonstrated that human mitochondria contain a non-discriminating glutamyl-tRNA synthetase and the heterotrimeric enzyme GatCAB (where Gat is glutamyl-tRNAGln amidotransferase), which are able to catalyse the formation of Gln-tRNAGln in vitro. In the present paper we demonstrate that mgatA (mouse GatA) interference in mouse cells produces a strong defect in mitochondrial translation without affecting the stability of the newly synthesized proteins. As a result, interfered cells present an impairment of the oxidative phosphorylation system and a significant increase in ROS (reactive oxygen species) levels. MS analysis of mitochondrial proteins revealed no glutamic acid found in the position of glutamines, strongly suggesting that misaminoacylated Glu-tRNAGln is rejected from the translational apparatus to maintain the fidelity of mitochondrial protein synthesis in mammals.

The thyroid hormone receptor ß induces DNA damage and premature senescence.

There is increasing evidence that the thyroid hormone (TH) receptors (THRs) can play a role in aging, cancer and degenerative diseases. In this paper, we demonstrate that binding of TH T3 (triiodothyronine) to THRB induces senescence and deoxyribonucleic acid (DNA) damage in cultured cells and in tissues of young hyperthyroid mice. T3 induces a rapid activation of ATM (ataxia telangiectasia mutated)/PRKAA (adenosine monophosphate-activated protein kinase) signal transduction and recruitment of the NRF1 (nuclear respiratory factor 1) and THRB to the promoters of genes with a key role on mitochondrial respiration. Increased respiration leads to production of mitochondrial reactive oxygen species, which in turn causes oxidative stress and DNA double-strand breaks and triggers a DNA damage response that ultimately leads to premature senescence of susceptible cells. Our findings provide a mechanism for integrating metabolic effects of THs with the tumor suppressor activity of THRB, the effect of thyroidal status on longevity, and the occurrence of tissue damage in hyperthyroidism.

Depleción del ácido desoxirribonucleico mitocondrial y mutaciones de POLG en un paciente con neuropatía sensorial atáxica, disartria y oftalmoplejía / Mitochondrial DNA depletion and polg mutations in a patient with sensory ataxia, dysarthria and ophthalmoplegia

Fundamento y objetivo: Un amplio espectro de enfermedades mitocondriales está producido por mutaciones de POLG y se caracterizan por una alteración en la integridad del genoma mitocondrial. La oftalmoplejía progresiva externa suele ser el marcador clínico en los casos de deleciones múltiples, pero no lo es en aquellas enfermedades que cursan con depleción del ácido desoxirribonucleico mitocondrial (ADNmt). En este trabajo presentamos un paciente con la tríada clínica que define el síndrome de neuropatía sensorial atáxica, disartria y oftalmoplejía, las mutaciones del gen POLG y la presencia inusual de una marcada disminución en el contenido del ADNmt en el músculo esquelético.Paciente y método: El paciente presentó un cuadro clínico caracterizado por neuropatía sensorial atáxica, disartria y oftalmoplejía. El diagnóstico se realizó mediante estudios histológicos y análisis molecular del ADNmt y del gen POLG. Resultados: La biopsia del nervio sural detectó una pérdida intensa de las fibras nerviosas mielinizadas gruesas. El estudio molecular reveló mutaciones en el gen POLG, así como deleciones múltiples y una marcada depleción del genoma mitocondrial. Conclusiones: Los pacientes con síndromes atáxicos de origen mitocondrial presentan fenotipos mitocondriales moleculares diferentes, por lo que se aconseja la búsqueda de mutaciones del gen POLG en todos ellos, independientemente de la anomalía que presenten en el genoma mitocondrial (AU)

Background and objetive: A broad spectrum of clinical disorders is produced by mutations in the DNA polymerase gamma mitochondrial (POLG) gene which are associated with altered mitochondrial DNA (mtDNA) integrity. The majority of disorders characterized by multiple mtDNA deletions present with progressive external ophthalmoplegia, though this feature is not usually found in syndromes caused by mtDNA depletion. We report on a patient having the clinical triad of sensory ataxic neuropathy, dysarthria and ophthalmoplegia (SANDO), POLG mutations and reduced muscle mtDNA content. Patient and methods: The patient presented with sensory ataxic neuropathy, dysarthria and ophthalmoplegia. Diagnosis was established by using histological and genetic procedures (nerve biopsy, mtDNA molecular analysis in skeletal muscle and mutation screening in the POLG gene). Results: Sural nerve biopsy showed marked loss of large myelinated fibers. Skeletal muscle analysis revealed multiple mtDNA deletions, a marked decrease in mtDNA copy number and pathogenic mutations in the POLG gene. Conclusions: POLG mutations must be considered in all patients with the cardinal findings of the SANDO phenotype, without taking into account the type of abnormalities encountered in the mitochondrial genome (AU)

[Mitochondrial DNA depletion and POLG mutations in a patient with sensory ataxia, dysarthria and ophthalmoplegia]. / Depleción del ácido desoxirribonucleico mitocondrial y mutaciones de POLG en un paciente con neuropatía sensorial atáxica, disartria y oftalmoplejía.

BACKGROUND AND OBJECTIVE: A broad spectrum of clinical disorders is produced by mutations in the DNA polymerase gamma mitochondrial (POLG) gene which are associated with altered mitochondrial DNA (mtDNA) integrity. The majority of disorders characterized by multiple mtDNA deletions present with progressive external ophthalmoplegia, though this feature is not usually found in syndromes caused by mtDNA depletion. We report on a patient having the clinical triad of sensory ataxic neuropathy, dysarthria and ophthalmoplegia (SANDO), POLG mutations and reduced muscle mtDNA content. PATIENT AND METHODS: The patient presented with sensory ataxic neuropathy, dysarthria and ophthalmoplegia. Diagnosis was established by using histological and genetic procedures (nerve biopsy, mtDNA molecular analysis in skeletal muscle and mutation screening in the POLG gene). RESULTS: Sural nerve biopsy showed marked loss of large myelinated fibers. Skeletal muscle analysis revealed multiple mtDNA deletions, a marked decrease in mtDNA copy number and pathogenic mutations in the POLG gene. CONCLUSIONS: POLG mutations must be considered in all patients with the cardinal findings of the SANDO phenotype, without taking into account the type of abnormalities encountered in the mitochondrial genome.

OPA1 mutations induce mitochondrial DNA instability and optic atrophy 'plus' phenotypes.

Mutations in OPA1, a dynamin-related GTPase involved in mitochondrial fusion, cristae organization and control of apoptosis, have been linked to non-syndromic optic neuropathy transmitted as an autosomal-dominant trait (DOA). We here report on eight patients from six independent families showing that mutations in the OPA1 gene can also be responsible for a syndromic form of DOA associated with sensorineural deafness, ataxia, axonal sensory-motor polyneuropathy, chronic progressive external ophthalmoplegia and mitochondrial myopathy with cytochrome c oxidase negative and Ragged Red Fibres. Most remarkably, we demonstrate that these patients all harboured multiple deletions of mitochondrial DNA (mtDNA) in their skeletal muscle, thus revealing an unrecognized role of the OPA1 protein in mtDNA stability. The five OPA1 mutations associated with these DOA 'plus' phenotypes were all mis-sense point mutations affecting highly conserved amino acid positions and the nuclear genes previously known to induce mtDNA multiple deletions such as POLG1, PEO1 (Twinkle) and SLC25A4 (ANT1) were ruled out. Our results show that certain OPA1 mutations exert a dominant negative effect responsible for multi-systemic disease, closely related to classical mitochondrial cytopathies, by a mechanism involving mtDNA instability.