GBA mutation promotes early mitochondrial dysfunction in 3D neurosphere models

Glucocerebrosidase (GBA) mutations are the most important genetic risk factor for the development of Parkinson disease (PD). GBA encodes the lysosomal enzyme glucocerebrosidase (GCase). Loss-of-GCase activity in cellular models has implicated lysosomal and mitochondrial dysfunction in PD disease pathogenesis, although the exact mechanisms remain unclear. We hypothesize that GBA mutations impair mitochondria quality control in a neurosphere model.We have characterized mitochondrial content, mitochondrial function and macroautophagy flux in 3D-neurosphere-model derived from neural crest stem cells containing heterozygous and homozygous N370SGBA mutations, under carbonyl cyanide-m-chlorophenyl-hydrazine (CCCP)- induced mitophagy.Our findings on mitochondrial markers and ATP levels indicate that mitochondrial accumulation occurs in mutant N370SGBA neurospheres under basal conditions, and clearance of depolarised mitochondria is impaired following CCCP-treatment. A significant increase in TFEB-mRNA levels, the master regulator of lysosomal and autophagy genes, may explain an unchanged macroautophagy flux in N370SGBA neurospheres. PGC1α-mRNA levels were also significantly increased following CCCP-treatment in heterozygote, but not homozygote neurospheres, and might contribute to the increased mitochondrial content seen in cells with this genotype, probably as a compensatory mechanism that is absent in homozygous lines.Mitochondrial impairment occurs early in the development of GCase-deficient neurons. Furthermore, impaired turnover of depolarised mitochondria is associated with early mitochondrial dysfunction.In summary, the presence of GBA mutation may be associated with higher levels of mitochondrial content in homozygous lines and lower clearance of damaged mitochondria in our neurosphere model

HIV-1 promonocytic and lymphoid cell lines: an in vitro model of in vivo mitochondrial and apoptotic lesion.

To characterize mitochondrial/apoptotic parameters in chronically human immunodeficiency virus (HIV-1)-infected promonocytic and lymphoid cells which could be further used as therapeutic targets to test pro-mitochondrial or anti-apoptotic strategies as in vitro cell platforms to deal with HIV-infection. Mitochondrial/apoptotic parameters of U1 promonocytic and ACH2 lymphoid cell lines were compared to those of their uninfected U937 and CEM counterparts. Mitochondrial DNA (mtDNA) was quantified by rt-PCR while mitochondrial complex IV (CIV) function was measured by spectrophotometry. Mitochondrial-nuclear encoded subunits II-IV of cytochrome-c-oxidase (COXII-COXIV), respectively, as well as mitochondrial apoptotic events [voltage-dependent-anion-channel-1(VDAC-1)-content and caspase-9 levels] were quantified by western blot, with mitochondrial mass being assessed by spectrophotometry (citrate synthase) and flow cytometry (mitotracker green assay). Mitochondrial membrane potential (JC1-assay) and advanced apoptotic/necrotic events (AnexinV/propidium iodide) were measured by flow cytometry. Significant mtDNA depletion spanning 57.67% (P < 0.01) was found in the U1 promonocytic cells further reflected by a significant 77.43% decrease of mitochondrial CIV activity (P < 0.01). These changes were not significant for the ACH2 lymphoid cell line. COXII and COXIV subunits as well as VDAC-1 and caspase-9 content were sharply decreased in both chronic HIV-1-infected promonocytic and lymphoid cell lines (<0.005 in most cases). In addition, U1 and ACH2 cells showed a trend (moderate in case of ACH2), albeit not significant, to lower levels of depolarized mitochondrial membranes. The present in vitro lymphoid and especially promonocytic HIV model show marked mitochondrial lesion but apoptotic resistance phenotype that has been only partially demonstrated in patients. This model may provide a platform for the characterization of HIV-chronicity, to test novel therapeutic options or to study HIV reservoirs

Mitochondrial DNA disturbances and deregulated expression of oxidative phosphorylation and mitochondrial fusion proteins in sporadic inclusion body myositis

Sporadic inclusion body myositis (sIBM) is one of the most common myopathies in elderly people. Mitochondrial abnormalities at the histological level are present in these patients. We hypothesize that mitochondrial dysfunction may play a role in disease aetiology. We took the following measurements of muscle and peripheral blood mononuclear cells (PBMCs) from 30 sIBM patients and 38 age-and gender-paired controls: mitochondrial DNA (mtDNA) deletions, amount of mtDNA and mtRNA, mitochondrial protein synthesis, mitochondrial respiratory chain (MRC) complex I and IV enzymatic activity, mitochondrial mass, oxidative stress and mitochondrial dynamics (mitofusin 2 and optic atrophy 1 levels). Depletion of mtDNA was present in muscle from sIBM patients and PBMCs showed deregulated expression of mitochondrial proteins in oxidative phosphorylation. MRC complex IV/citrate synthase activity was significantly decreased in both tissues and mitochondrial dynamics were affected in muscle. Depletion of mtDNA was significantly more severe in patients with mtDNA deletions, which also presented deregulation of mitochondrial fusion proteins. Imbalance in mitochondrial dynamics in muscle was associated with increased mitochondrial genetic disturbances (both depletion and deletions), demonstrating that proper mitochondrial turnover is essential for mitochondrial homoeostasis and muscle function in these patients

Comment on Yeste et al.: Polyphenols and IUGR Pregnancies: Intrauterine Growth Restriction and Hydroxytyrosol Affect the Development and Neurotransmitter Profile of the Hippocampus in a Pig Model

Intrauterine growth restriction (IUGR) affects 5-10% of newborns and increases the risks of intrauterine demise, neonatal morbidity, and death. In their recent publication, Yeste et al. found the benefits of hydroxytyrosol supplementation on brain remodeling from an IUGR pig model. Additionally, we found a significant decrease in phenolic alcohol (tyrosol and hydroxytyrosol) intake in IUGR pregnant women. Altogether, these findings support the notion that dietetic interventions, through supplementation but mostly via a balanced diet, can ameliorate IUGR complications. Furthermore, diet intervention combined with early biomarkers may allow clinicians to eventually anticipate IUGR diagnosis and help avoid one of the most frequent causes of newborn mortality and morbidity

Mitochondrial toxicity and caspase activation in HIV pregnant women.

To assess the impact of HIV-infection and highly active anti-retroviral treatment in mitochondria and apoptotic activation of caspases during pregnancy and their association with adverse perinatal outcome. Changes of mitochondrial parameters and apoptotic caspase activation in maternal peripheral blood mononuclear cells were compared at first trimester of pregnancy and delivery in 27 HIV-infected and -treated pregnant women versus 24 uninfected pregnant controls. We correlated immunovirological, therapeutic and perinatal outcome with experimental findings: mitochondrial DNA (mtDNA) content, mitochondrial protein synthesis, mitochondrial function and apoptotic caspase activation. The HIV pregnancies showed increased adverse perinatal outcome (OR: 4.81 [1.14-20.16]; P < 0.05) and decreased mtDNA content (42.66 ± 5.94%, P < 0.01) compared to controls, even higher in naïve participants. This depletion caused a correlated decrease in mitochondrial protein synthesis (12.82 ± 5.73%, P < 0.01) and function (20.50 ± 10.14%, P < 0.001), not observed in controls. Along pregnancy, apoptotic caspase-3 activation increased 63.64 ± 45.45% in controls (P < 0.001) and 100.00 ± 47.37% in HIV-pregnancies (P < 0.001), in correlation with longer exposure to nucleoside analogues. HIV-infected women showed increased obstetric problems and declined genetic and functional mitochondrial parameters during pregnancy, especially those firstly exposed to anti-retrovirals. The apoptotic activation of caspases along pregnancy is emphasized in HIV pregnancies promoted by nucleoside analogues. However, we could not demonstrate direct mitochondrial or apoptotic implication in adverse obstetric outcome probably because of the reduced sample size

Cardiac and placental mitochondrial characterization in a rabbit model of intrauterine growth restriction

BACKGROUND: Intrauterine growth restriction (IUGR) is associated with cardiovascular remodeling persisting into adulthood. Mitochondrial bioenergetics, essential for embryonic development and cardiovascular function, are regulated by nuclear effectors as sirtuins. A rabbit model of IUGR and cardiovascular remodeling was generated, in which heart mitochondrial alterations were observed by microscopic and transcriptomic analysis. We aimed to evaluate if such alterations are translated at a functional mitochondrial level to establish the etiopathology and potential therapeutic targets for this obstetric complication. METHODS: Hearts and placentas from 16 IUGR-offspring and 14 controls were included to characterize mitochondrial function. RESULTS: Enzymatic activities of complexes II, IV and II + III in IUGR-hearts (-11.96 ± 3.16%; -15.58 ± 5.32%; -14.73 ± 4.37%; p < 0.05) and II and II + III in IUGR-placentas (-17.22 ± 3.46%; p < 0.005 and -29.64 ± 4.43%; p < 0.001) significantly decreased. This was accompanied by a not significant reduction in CI-stimulated oxygen consumption and significantly decreased complex II SDHB subunit expression in placenta (-44.12 ± 5.88%; p < 0.001). Levels of mitochondrial content, Coenzyme Q and cellular ATP were conserved. Lipid peroxidation significantly decreased in IUGR-hearts (-39.02 ± 4.35%; p < 0.001), but not significantly increased in IUGR-placentas. Sirtuin3 protein expression significantly increased in IUGR-hearts (84.21 ± 31.58%; p < 0.05) despite conserved anti-oxidant SOD2 protein expression and activity in both tissues. CONCLUSIONS: IUGR is associated with cardiac and placental mitochondrial CII dysfunction. Up-regulated expression of Sirtuin3 may explain attenuation of cardiac oxidative damage and preserved ATP levels under CII deficiency. GENERAL SIGNIFICANCE: These findings may allow the design of dietary interventions to modulate Sirtuin3 expression and consequent regulation of mitochondrial imbalance associated with IUGR and derived cardiovascular remodeling

Mitochondrial and autophagic alterations in skin fibroblasts from Parkinson disease patients with Parkin mutations.

PRKN encodes an E3-ubiquitin-ligase involved in multiple cell processes including mitochondrial homeostasis and autophagy. Previous studies reported alterations of mitochondrial function in fibroblasts from patients with PRKN mutation-associated Parkinson's disease (PRKN-PD) but have been only conducted in glycolytic conditions, potentially masking mitochondrial alterations. Additionally, autophagy flux studies in this cell model are missing.We analyzed mitochondrial function and autophagy in PRKN-PD skin-fibroblasts (n=7) and controls (n=13) in standard (glucose) and mitochondrial-challenging (galactose) conditions.In glucose, PRKN-PD fibroblasts showed preserved mitochondrial bioenergetics with trends to abnormally enhanced mitochondrial respiration that, accompanied by decreased CI, may account for the increased oxidative stress. In galactose, PRKN-PD fibroblasts exhibited decreased basal/maximal respiration vs. controls and reduced mitochondrial CIV and oxidative stress compared to glucose, suggesting an inefficient mitochondrial oxidative capacity to meet an extra metabolic requirement. PRKN-PD fibroblasts presented decreased autophagic flux with reduction of autophagy substrate and autophagosome synthesis in both conditions.The alterations exhibited under neuron-like oxidative environment (galactose), may be relevant to the disease pathogenesis potentially explaining the increased susceptibility of dopaminergic neurons to undergo degeneration. Abnormal PRKN-PD phenotype supports the usefulness of fibroblasts to model disease and the view of PD as a systemic disease where molecular alterations are present in peripheral tissues

Unraveling Inclusion Body Myositis Using a Patient-derived Fibroblast Model

Background: Inclusion body myositis (IBM) is an inflammatory myopathy clinically characterized by proximal and distal muscle weakness, with inflammatory infiltrates, rimmed vacuoles and mitochondrial changes in muscle histopathology. There is scarce knowledge on IBM aetiology, and non-established biomarkers or effective treatments are available, partly due to the lack of validated disease models. Methods: We have performed transcriptomics and functional validation of IBM muscle pathological hallmarks in fibroblasts from IBM patients (n = 14) and healthy controls (n = 12), paired by age and sex. The results comprise an mRNA-seq, together with functional inflammatory, autophagy, mitochondrial and metabolic changes between patients and controls. Results: Gene expression profile of IBM vs control fibroblasts revealed 778 differentially expressed genes (P-value adj < 0.05) related to inflammation, mitochondria, cell cycle regulation and metabolism. Functionally, an increased inflammatory profile was observed in IBM fibroblasts with higher supernatant cytokine secretion (three-fold increase). Autophagy was reduced considering basal protein mediators (18.4% reduced), time-course autophagosome formation (LC3BII 39% reduced, P-value < 0.05), and autophagosome microscopic evaluation. Mitochondria displayed reduced genetic content (by 33.9%, P-value < 0.05) and function (30.2%-decrease in respiration, 45.6%-decline in enzymatic activity (P-value < 0.001), 14.3%-higher oxidative stress, 135.2%-increased antioxidant defence (P-value < 0.05), 11.6%-reduced mitochondrial membrane potential (P-value < 0.05) and 42.8%-reduced mitochondrial elongation (P-value < 0.05)). In accordance, at the metabolite level, organic acid showed a 1.8-fold change increase, with conserved amino acid profile. Correlating to disease evolution, oxidative stress and inflammation emerge as potential markers of prognosis. Conclusions: These findings confirm the presence of molecular disturbances in peripheral tissues from IBM patients and prompt patients’ derived fibroblasts as a promising disease model, which may eventually be exported to other neuromuscular disorders. We additionally identify new molecular players in IBM associated with disease progression, setting the path to deepen in disease aetiology, in the identification of novel biomarkers or in the standardization of biomimetic platforms to assay new therapeutic strategies for preclinical studies

Disrupted mitochondrial and metabolic plasticity underlie comorbidity between age-Related and degenerative disorders as parkinson disease and type 2 diabetes mellitus.

Idiopathic Parkinson's disease (iPD) and type 2 diabetes mellitus (T2DM) are chronic, multisystemic, and degenerative diseases associated with aging, with eventual epidemiological co-morbidity and overlap in molecular basis. This study aims to explore if metabolic and mitochondrial alterations underlie the previously reported epidemiologic and clinical co-morbidity from a molecular level. To evaluate the adaptation of iPD to a simulated pre-diabetogenic state, we exposed primary cultured fibroblasts from iPD patients and controls to standard (5 mM) and high (25 mM) glucose concentrations to further characterize metabolic and mitochondrial resilience. iPD fibroblasts showed increased organic and amino acid levels related to mitochondrial metabolism with respect to controls, and these differences were enhanced in high glucose conditions (citric, suberic, and sebacic acids levels increased, as well as alanine, glutamate, aspartate, arginine, and ornithine amino acids; p-values between 0.001 and 0.05). The accumulation of metabolites in iPD fibroblasts was associated with (and probably due to) the concomitant mitochondrial dysfunction observed at enzymatic, oxidative, respiratory, and morphologic level. Metabolic and mitochondrial plasticity of controls was not observed in iPD fibroblasts, which were unable to adapt to different glucose conditions. Impaired metabolism and mitochondrial activity in iPD may limit energy supply for cell survival. Moreover, reduced capacity to adapt to disrupted glucose balance characteristic of T2DM may underlay the co-morbidity between both diseases. Conclusions: Fibroblasts from iPD patients showed mitochondrial impairment, resulting in the accumulation of organic and amino acids related to mitochondrial metabolism, especially when exposed to high glucose. Mitochondrial and metabolic defects down warding cell plasticity to adapt to changing glucose bioavailability may explain the comorbidity between iPD and T2DM

CIBERER : Spanish national network for research on rare diseases: A highly productive collaborative initiative

Altres ajuts: Instituto de Salud Carlos III (ISCIII); Ministerio de Ciencia e Innovación.CIBER (Center for Biomedical Network Research; Centro de Investigación Biomédica En Red) is a public national consortium created in 2006 under the umbrella of the Spanish National Institute of Health Carlos III (ISCIII). This innovative research structure comprises 11 different specific areas dedicated to the main public health priorities in the National Health System. CIBERER, the thematic area of CIBER focused on rare diseases (RDs) currently consists of 75 research groups belonging to universities, research centers, and hospitals of the entire country. CIBERER's mission is to be a center prioritizing and favoring collaboration and cooperation between biomedical and clinical research groups, with special emphasis on the aspects of genetic, molecular, biochemical, and cellular research of RDs. This research is the basis for providing new tools for the diagnosis and therapy of low-prevalence diseases, in line with the International Rare Diseases Research Consortium (IRDiRC) objectives, thus favoring translational research between the scientific environment of the laboratory and the clinical setting of health centers. In this article, we intend to review CIBERER's 15-year journey and summarize the main results obtained in terms of internationalization, scientific production, contributions toward the discovery of new therapies and novel genes associated to diseases, cooperation with patients' associations and many other topics related to RD research