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By means of a proteomic approach, we assessed the pathways involved in cerebellar neurodegeneration in a mouse model (Harlequin, Hq) of mitochondrial disorder. A differential proteomic profile study (iTRAQ) was performed in cerebellum homogenates of male Hq and wild-type (WT) mice 8 weeks after the onset of clear symptoms of ataxia in the Hq mice (aged 5.2 ± 0.2 and 5.3 ± 0.1 months for WT and Hq, respectively), followed by a biochemical validation of the most relevant changes. Additional groups of 2-, 3- and 6-month-old WT and Hq mice were analyzed to assess the disease progression on the proteins altered in the proteomic study. The proteomic analysis showed that beyond the expected deregulation of oxidative phosphorylation, the cerebellum of Hq mice showed a marked astroglial activation together with alterations in Ca2+ homeostasis and neurotransmission, with an up- and downregulation of GABAergic and glutamatergic neurotransmission, respectively, and the downregulation of cerebellar "long-term depression", a synaptic plasticity phenomenon that is a major player in the error-driven learning that occurs in the cerebellar cortex. Our study provides novel insights into the mechanisms associated with cerebellar degeneration in the Hq mouse model, including a complex deregulation of neuroinflammation, oxidative phosphorylation and glutamate, GABA and amino acids' metabolism.
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Enfermedades Cerebelosas , Enfermedades Mitocondriales , Enfermedades Neurodegenerativas , Ratones , Masculino , Animales , Proteómica , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Mitocondriales/metabolismo , Cerebelo/metabolismoRESUMEN
BACKGROUND: Successful pregnancy is supported by a healthy maternal-fetal interface (i.e., the decidual tissues) which holds the conceptus and safeguards it against stressors from the beginning of pregnancy. Any disturbance of this interface can presumably lead to the loss of pregnancy. The use of the immunosuppressive drug mycophenolic acid (MPA) should be discontinued in pregnancy given its abortive and embryotoxic effects. Direct teratogenic effects have been observed in mammalian embryos cultured in MPA, but the underlying mechanisms of abortion by MPA are less understood. METHODS: Decidual stromal cells isolated from human placentas are cultured in the presence of clinically relevant doses of MPA. Data regarding the effects of MPA on the proliferation and viability of decidua cultures are first analysed and then, molecular pathways contributing to these effects are unravelled. RESULTS: MPA treatment of decidual stromal cells results in loss of proliferation capacity and a decrease in the viability of decidua cultures. The molecular pathways involved in the effects of MPA on decidual stromal cells are a reduction in pre-rRNA synthesis and subsequent disruption of the nucleolus. The nucleolar stress stabilizes p53, which in turn, leads to a p21-mediated cell cycle arrest in late S and G2 phases, preventing the progression of the decidua cells into the mitosis. Furthermore, MPA does not induce apoptosis but activate mechanisms of autophagy and senescence in decidual stromal cells. CONCLUSION: The irreversible growth arrest of decidua cells, whose role in the maintenance of the pregnancy microenvironment is known, may be one cause of miscarriage in MPA treated pregnant women.
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Aborto Espontáneo/inducido químicamente , Decidua/fisiopatología , Inmunosupresores/efectos adversos , Ácido Micofenólico/efectos adversos , Envejecimiento , Femenino , Humanos , Placenta/fisiopatología , EmbarazoRESUMEN
Mitochondrial disorders (MD) comprise a group of heterogeneous clinical disorders for which non-invasive diagnosis remains a challenge. Two protein biomarkers have so far emerged for MD detection, FGF-21 and GDF-15, but the identification of additional biomarkers capable of improving their diagnostic accuracy is highly relevant. Previous studies identified Gelsolin as a regulator of cell survival adaptations triggered by mitochondrial defects. Gelsolin presents a circulating plasma isoform (pGSN), whose altered levels could be a hallmark of mitochondrial dysfunction. Therefore, we investigated the diagnostic performance of pGSN for MD relative to FGF-21 and GDF-15. Using ELISA assays, we quantified plasma levels of pGSN, FGF-21, and GDF-15 in three age- and gender-matched adult cohorts: 60 genetically diagnosed MD patients, 56 healthy donors, and 41 patients with unrelated neuromuscular pathologies (non-MD). Clinical variables and biomarkers' plasma levels were compared between groups. Discrimination ability was calculated using the area under the ROC curve (AUC). Optimal cut-offs and the following diagnostic parameters were determined: sensitivity, specificity, positive and negative predictive values, positive and negative likelihood ratios, and efficiency. Comprehensive statistical analyses revealed significant discrimination ability for the three biomarkers to classify between MD and healthy individuals, with the best diagnostic performance for the GDF-15/pGSN combination. pGSN and GDF-15 preferentially discriminated between MD and non-MD patients under 50 years, whereas FGF-21 best classified older subjects. Conclusion: pGSN improves the diagnosis accuracy for MD provided by FGF-21 and GDF-15.
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Factores de Crecimiento de Fibroblastos/sangre , Gelsolina/sangre , Factor 15 de Diferenciación de Crecimiento/sangre , Enfermedades Mitocondriales/sangre , Enfermedades Mitocondriales/diagnóstico , Adulto , Biomarcadores/sangre , Estudios de Casos y Controles , Femenino , Humanos , Masculino , Persona de Mediana Edad , FenotipoRESUMEN
Our goal was to analyze postmortem tissues of an adult patient with late-onset thymidine kinase 2 (TK2) deficiency who died of respiratory failure. Compared with control tissues, we found a low mtDNA content in the patient's skeletal muscle, liver, kidney, small intestine, and particularly in the diaphragm, whereas heart and brain tissue showed normal mtDNA levels. mtDNA deletions were present in skeletal muscle and diaphragm. All tissues showed a low content of OXPHOS subunits, and this was especially evident in diaphragm, which also exhibited an abnormal protein profile, expression of non-muscular ß-actin and loss of GAPDH and α-actin. MALDI-TOF/TOF mass spectrometry analysis demonstrated the loss of the enzyme fructose-bisphosphate aldolase, and enrichment for serum albumin in the patient's diaphragm tissue. The TK2-deficient patient's diaphragm showed a more profound loss of OXPHOS proteins, with lower levels of catalase, peroxiredoxin 6, cytosolic superoxide dismutase, p62 and the catalytic subunits of proteasome than diaphragms of ventilated controls. Strong overexpression of TK1 was observed in all tissues of the patient with diaphragm showing the highest levels. TK2 deficiency induces a more profound dysfunction of the diaphragm than of other tissues, which manifests as loss of OXPHOS and glycolytic proteins, sarcomeric components, antioxidants and overactivation of the TK1 salvage pathway that is not attributed to mechanical ventilation.
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ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Diafragma/metabolismo , Mitocondrias/metabolismo , Insuficiencia Respiratoria/metabolismo , Timidina Quinasa/deficiencia , Timidina Quinasa/genética , Actinas/metabolismo , Adulto , Autopsia , Encéfalo/metabolismo , Catalasa/metabolismo , Diafragma/enzimología , Femenino , Fructosa-Bifosfato Aldolasa/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/metabolismo , Humanos , Intestino Delgado/metabolismo , Riñón/metabolismo , Hígado/metabolismo , Espectrometría de Masas , Mitocondrias/enzimología , Mitocondrias/genética , Músculo Esquelético/metabolismo , Fosforilación Oxidativa , Peroxiredoxina VI/metabolismo , Complejo de la Endopetidasa Proteasomal , Proteoma/genética , Proteoma/metabolismo , Insuficiencia Respiratoria/genética , Insuficiencia Respiratoria/mortalidad , Superóxido Dismutasa/metabolismo , Timidina Quinasa/metabolismo , Regulación hacia ArribaRESUMEN
This work introduces NeoMag, a system designed to enhance cell mechanics assays in substrate deformation studies. NeoMag uses multidomain magneto-active materials to mechanically actuate the substrate, transmitting reversible mechanical cues to cells. The system boasts full flexibility in alternating loading substrate deformation modes, seamlessly adapting to both upright and inverted microscopes. The multidomain substrates facilitate mechanobiology assays on 2D and 3D cultures. The integration of the system with nanoindenters allows for precise evaluation of cellular mechanical properties under varying substrate deformation modes. The system is used to study the impact of substrate deformation on astrocytes, simulating mechanical conditions akin to traumatic brain injury and ischemic stroke. The results reveal local heterogeneous changes in astrocyte stiffness, influenced by the orientation of subcellular regions relative to substrate strain. These stiffness variations, exceeding 50% in stiffening and softening, and local deformations significantly alter calcium dynamics. Furthermore, sustained deformations induce actin network reorganization and activate Piezo1 channels, leading to an initial increase followed by a long-term inhibition of calcium events. Conversely, fast and dynamic deformations transiently activate Piezo1 channels and disrupt the actin network, causing long-term cell softening. These findings unveil mechanical and functional alterations in astrocytes during substrate deformation, illustrating the multiple opportunities this technology offers.
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Astrocitos , Astrocitos/metabolismo , Astrocitos/citología , Animales , Calcio/metabolismo , Calcio/química , Fenómenos Biomecánicos , Fenómenos Mecánicos , Actinas/metabolismo , Canales Iónicos/metabolismo , RatonesRESUMEN
We analyzed the effects of apoptosis-inducing factor (AIF) deficiency, as well as those of an exercise training intervention on autophagy across tissues (heart, skeletal muscle, cerebellum and brain), that are primarily affected by mitochondrial diseases, using a preclinical model of these conditions, the Harlequin (Hq) mouse. Autophagy markers were analyzed in: (i) 2, 3 and 6 month-old male wild-type (WT) and Hq mice, and (ii) WT and Hq male mice that were allocated to an exercise training or sedentary group. The exercise training started upon onset of the first symptoms of ataxia in Hq mice and lasted for 8 weeks. Higher content of autophagy markers and free amino acids, and lower levels of sarcomeric proteins were found in the skeletal muscle and heart of Hq mice, suggesting increased protein catabolism. Leupeptin-treatment demonstrated normal autophagic flux in the Hq heart and the absence of mitophagy. In the cerebellum and brain, a lower abundance of Beclin 1 and ATG16L was detected, whereas higher levels of the autophagy substrate p62 and LAMP1 levels were observed in the cerebellum. The exercise intervention did not counteract the autophagy alterations found in any of the analyzed tissues. In conclusion, AIF deficiency induces tissue-specific alteration of autophagy in the Hq mouse, with accumulation of autophagy markers and free amino acids in the heart and skeletal muscle, but lower levels of autophagy-related proteins in the cerebellum and brain. Exercise intervention, at least if starting when muscle atrophy and neurological symptoms are already present, is not sufficient to mitigate autophagy perturbations.
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Combination therapies constitute a powerful tool for cancer treatment. By combining drugs with different mechanisms of action, the limitations of each individual agent can be overcome, while increasing therapeutic benefit. Here, we propose employing tumor-migrating decidua-derived mesenchymal stromal cells as therapeutic agents combining antiangiogenic therapy and chemotherapy. First, a plasmid encoding the antiangiogenic protein endostatin was transfected into these cells by nucleofection, confirming its expression by ELISA and its biological effect in an ex ovo chick embryo model. Second, doxorubicin-loaded mesoporous silica nanoparticles were introduced into the cells, which would act as vehicles for the drug being released. The effect of the drug was evaluated in a coculture in vitro model with mammary cancer cells. Third, the combination of endostatin transfection and doxorubicin-nanoparticle loading was carried out with the decidua mesenchymal stromal cells. This final cell platform was shown to retain its tumor-migration capacity in vitro, and the combined in vitro therapeutic efficacy was confirmed through a 3D spheroid coculture model using both cancer and endothelial cells. The results presented here show great potential for the development of combination therapies based on genetically-engineered cells that can simultaneously act as cellular vehicles for drug-loaded nanoparticles.
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AIM: Cerebellar neurodegeneration is a main phenotypic manifestation of mitochondrial disorders caused by apoptosis-inducing factor (AIF) deficiency. We assessed the effects of an exercise training intervention at the cerebellum and brain level in a mouse model (Harlequin, Hq) of AIF deficiency. METHODS: Male wild-type (WT) and Hq mice were assigned to an exercise (Ex) or control (sedentary [Sed]) group (n = 10-12/group). The intervention (aerobic and resistance exercises) was initiated upon the first symptoms of ataxia in Hq mice (â¼3 months on average) and lasted 8 weeks. Histological and biochemical analyses of the cerebellum were performed at the end of the training program to assess indicators of mitochondrial deficiency, neuronal death, oxidative stress and neuroinflammation. In brain homogenates analysis of enzyme activities and levels of the oxidative phosphorylation system, oxidative stress and neuroinflammation were performed. RESULTS: The mean age of the mice at the end of the intervention period did not differ between groups: 5.2 ± 0.2 (WT-Sed), 5.2 ± 0.1 (WT-Ex), 5.3 ± 0.1 (Hq-Sed), and 5.3 ± 0.1 months (Hq-Ex) (p = 0.489). A significant group effect was found for most variables indicating cerebellar dysfunction in Hq mice compared with WT mice irrespective of training status. However, exercise intervention did not counteract the negative effects of the disease at the cerebellum level (i.e., no differences for Hq-Ex vs. Hq-Sed). On the contrary, in brain, the activity of complex V was higher in both Hq mice groups in comparison with WT animals (p < 0.001), and post hoc analysis also revealed differences between sedentary and trained Hq mice. CONCLUSION: A combined training program initiated when neurological symptoms and neuron death are already apparent is unlikely to promote neuroprotection in the cerebellum of Hq model of mitochondrial disorders, but it induces higher complex V activity in the brain.
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Purpose: Mitochondrial diseases (MD) are among the most prevalent neuromuscular disorders. Unfortunately, no curative treatment is yet available. This study analyzed the effects of exercise training in an animal model of respiratory chain complex I deficiency, the Harlequin (Hq) mouse, which replicates the clinical features of this condition. Methods: Male heterozygous Harlequin (Hq/Y) mice were assigned to an "exercise" (n = 10) or a "sedentary" control group (n = 11), with the former being submitted to an 8 week combined exercise training intervention (aerobic + resistance training performed five times/week). Aerobic fitness, grip strength, and balance were assessed at the beginning and at the end of the intervention period in all the Hq mice. Muscle biochemical analyses (with results expressed as percentage of reference data from age/sex-matched sedentary wild-type mice [n = 12]) were performed at the end of the aforementioned period for the assessment of major molecular signaling pathways involved in muscle anabolism (mTOR activation) and mitochondrial biogenesis (proliferator activated receptor gamma co-activator 1α [PGC-1α] levels), and enzyme activity and levels of respiratory chain complexes, and antioxidant enzyme levels. Results: Exercise training resulted in significant improvements in aerobic fitness (-33 ± 13 m and 83 ± 43 m for the difference post- vs. pre-intervention in total distance covered in the treadmill tests in control and exercise group, respectively, p = 0.014) and muscle strength (2 ± 4 g vs. 17 ± 6 g for the difference post vs. pre-intervention, p = 0.037) compared to the control group. Higher levels of ribosomal protein S6 kinase beta-1 phosphorylated at threonine 389 (156 ± 30% vs. 249 ± 30%, p = 0.028) and PGC-1α (82 ± 7% vs. 126 ± 19% p = 0.032) were observed in the exercise-trained mice compared with the control group. A higher activity of respiratory chain complexes I (75 ± 4% vs. 95 ± 6%, p = 0.019), III (79 ± 5% vs. 97 ± 4%, p = 0.031), and V (77 ± 9% vs. 105 ± 9%, p = 0.024) was also found with exercise training. Exercised mice presented with lower catalase levels (204 ± 22% vs. 141 ± 23%, p = 0.036). Conclusion: In a mouse model of MD, a training intervention combining aerobic and resistance exercise increased aerobic fitness and muscle strength, and mild improvements were found for activated signaling pathways involved in muscle mitochondrial biogenesis and anabolism, OXPHOS complex activity, and redox status in muscle tissue.
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PURPOSE: We determined the effects of an innovative 8-wk exercise intervention (aerobic, resistance, and inspiratory muscle training) for patients with mitochondrial disease. METHODS: Several end points were assessed in 12 patients (19-59 yr, 4 women) at pretraining, posttraining, and after 4-wk detraining: aerobic power, muscle strength/power and maximal inspiratory pressure (main end points), ability to perform activities of daily living, body composition, quality of life, and blood myokines (secondary end points). RESULTS: The program was safe, with patients' adherence being 94% ± 5%. A significant time effect was found for virtually all main end points (P ≤ 0.004), indicating a training improvement. Similar findings (P ≤ 0.003) were found for activities of daily living tests, total/trunk/leg lean mass, total fat mass, femoral fracture risk, and general health perception. No differences were found for blood myokines, except for an acute exertional increase in interleukin 8 at posttraining/detraining (P = 0.002) and in fatty acid binding protein 3 at detraining (P = 0.002). CONCLUSIONS: An intervention including novel exercises for mitochondrial disease patients (e.g., inspiratory muscle training) produced benefits in numerous indicators of physical capacity and induced a previously unreported shift toward a healthier body composition phenotype.