Marc Catalán-García

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.

Pyruvate kinase M2 and the mitochondrial ATPase Inhibitory Factor 1 provide novel biomarkers of dermatomyositis: a metabolic link to oncogenesis

BackgroundMetabolic alterations play a role in the development of inflammatory myopathies (IMs). Herein, we have investigated through a multiplex assay whether proteins of energy metabolism could provide biomarkers of IMs.MethodsA cohort of thirty-two muscle biopsies and forty plasma samples comprising polymyositis (PM), dermatomyositis (DM) and sporadic inclusion body myositis (sIBM) and control donors was interrogated with monoclonal antibodies against proteins of energy metabolism using reverse phase protein microarrays (RPPA).ResultsWhen compared to controls the expression of the proteins is not significantly affected in the muscle of PM patients. However, the expression of β-actin is significantly increased in DM and sIBM in consistence with muscle and fiber regeneration. Concurrently, the expression of some proteins involved in glucose metabolism displayed a significant reduction in muscle of sIBM suggesting a repression of glycolytic metabolism in these patients. In contrasts to these findings, the expression of the glycolytic pyruvate kinase isoform M2 (PKM2) and of the mitochondrial ATPase Inhibitor Factor 1 (IF1) and Hsp60 were significantly augmented in DM when compared to other IMs in accordance with a metabolic shift prone to cancer development. PKM2 alone or in combination with other biomarkers allowed the discrimination of control and IMs with very high (>95%) sensitivity and specificity. Unfortunately, plasma levels of PKM2 were not significantly altered in DM patients to recommend its use as a non-invasive biomarker of the disease.ConclusionsExpression of proteins of energy metabolism in muscle enabled discrimination of patients with IMs. RPPA identified the glycolysis promoting PKM2 and IF1 proteins as specific biomarkers of dermatomyositis, providing a biochemical link of this IM with oncogenesis.

Mitochondrial and apoptotic in vitro modelling of differential HIV-1 progression and antiretroviral toxicity

OBJECTIVES Ex vivo analysis of mitochondrial function may reveal HIV progression and the impact of ART. We propose a mitochondrial and apoptotic in vitro model using Jurkat T cells incubated with plasma. The objectives of this study were to evaluate mitochondrial and apoptotic lesions in this model in relation to HIV progression, and to assess the effect of >1 year of standard non-thymidine-containing therapy. METHODS This was a cross-sectional comparison among three age- and gender-matched groups (n = 19 × 3): healthy non-HIV-infected participants, HIV-infected long-term non-progressors (LTNPs) and standard antiretroviral-naive chronically infected patients [standard progressors (Sps)], longitudinally evaluated before (Sp1) and after (Sp2) >1 year of efavirenz + tenofovir + emtricitabine therapy. We analysed mitochondrial DNA content by RT-PCR, mitochondrial function by spectrophotometry, mitochondrial protein synthesis by western blot analysis, mitochondrial dynamics by western blot analysis (MFN2), apoptotic transition pore formation by western blot analysis (VDAC-1) and mitochondrial membrane potential and annexin V/propidium iodide fluorescence by flow cytometry. RESULTS There was a decreasing non-significant trend towards lower mitochondrial parameters for HIV-infected values with respect to uninfected control reference values. HIV progression (LTNP versus Sp1) was associated with decreased mitochondrial genetic, functional and translational parameters, which partially recovered after treatment intervention (Sp2). Mitochondrial fusion showed a trend to decrease non-significantly in Sp patients compared with LTNP patients, especially after therapy. All apoptotic parameters showed a trend to increase in Sp1 with respect to LTNP, followed by recovery in Sp2. CONCLUSIONS We proposed an in vitro model for mitochondrial and apoptotic assessment to test the effects of HIV infection and its therapy, resembling in vivo conditions. This model could be useful for clinical research purposes.

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.

Placental Mitochondrial Toxicity, Oxidative Stress, Apoptosis, and Adverse Perinatal Outcomes in Hiv Pregnancies Under Antiretroviral Treatment Containing Zidovudine

Objective: To determine whether mitochondrial, oxidative, and apoptotic abnormalities in placenta derived from HIV and combined antiretroviral therapy (cART) containing zidovudine (AZT) could be associated with adverse perinatal outcome. Design: Cross-sectional, controlled, observational study. Methods: We studied obstetric results and mitochondrial, oxidative, and apoptotic state in placenta of 24 treated HIV-infected and 32 -uninfected pregnant women. We measured mitochondrial DNA (mtDNA) content by quantitative reverse transcriptase–polymerase chain reaction (mtND2/n18SrRNA), oxidative stress by the spectrophotometric quantification of lipid peroxidation and apoptosis by Western blot analysis of active caspase-3 respect to &bgr;-actin content and analysis of the terminal deoxynucleotidyl transferase dUTP nick end labeling. Results: Global adverse perinatal outcome (defined as preterm delivery or/and small newborns for gestational age) was significantly increased in HIV pregnancies [or 6.7 (1.3–33.2); P < 0.05]. mtDNA content in HIV-infected women was significantly depleted (39.20% ± 2.78%) with respect to controls (0.59 ± 0.03 vs. 0.97 ± 0.07; P < 0.001). A significant 29.50% ± 9.14% increase in oxidative stress was found in placentas of HIV-infected women (23.23 ± 1.64 vs. 17.94 ± 1.03; P < 0.01). A trend toward 41.18% ± 29.41% increased apoptosis active caspase-3/&bgr;-actin was found in HIV patients (0.48 ± 0.10 vs. 0.34 ± 0.05; P = not significant), confirmed by transferase dUTP nick end labeling assay. Adverse perinatal outcome did not correlate mitochondrial, oxidative, or apoptotic findings. Conclusions: Placentas of HIV-infected pregnant women under AZT cART showed evidence of mtDNA depletion, increased oxidative stress levels, and apoptosis suggestive of secondary mitochondrial failure, potential base of associated adverse perinatal outcome. Despite the fact that further demonstration of causality would need new approaches and bigger sample sizes, AZT-sparing cART should be considered in the context of pregnancy.

Exhaustion of mitochondrial and autophagic reserve may contribute to the development of LRRK2 G2019S -Parkinson’s disease

BackgroundMutations in leucine rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson’s disease (PD). Mitochondrial and autophagic dysfunction has been described as etiologic factors in different experimental models of PD. We aimed to study the role of mitochondria and autophagy in LRRK2G2019S-mutation, and its relationship with the presence of PD-symptoms.MethodsFibroblasts from six non-manifesting LRRK2G2019S-carriers (NM-LRRK2G2019S) and seven patients with LRRK2G2019S-associated PD (PD-LRRK2G2019S) were compared to eight healthy controls (C). An exhaustive assessment of mitochondrial performance and autophagy was performed after 24-h exposure to standard (glucose) or mitochondrial-challenging environment (galactose), where mitochondrial and autophagy impairment may be heightened.ResultsA similar mitochondrial phenotype of NM-LRRK2G2019S and controls, except for an early mitochondrial depolarization (54.14% increased, p = 0.04), was shown in glucose. In response to galactose, mitochondrial dynamics of NM-LRRK2G2019S improved (− 17.54% circularity, p = 0.002 and + 42.53% form factor, p = 0.051), probably to maintain ATP levels over controls. A compromised bioenergetic function was suggested in PD-LRRK2G2019S when compared to controls in glucose media. An inefficient response to galactose and worsened mitochondrial dynamics (− 37.7% mitochondrial elongation, p = 0.053) was shown, leading to increased oxidative stress. Autophagy initiation (SQTSM/P62) was upregulated in NM-LRRK2G2019S when compared to controls (glucose + 118.4%, p = 0.014; galactose + 114.44%, p = 0.009,) and autophagosome formation increased in glucose media. Despite of elevated SQSTM1/P62 levels of PD-NMG2019S when compared to controls (glucose + 226.14%, p = 0.04; galactose + 78.5%, p = 0.02), autophagosome formation was deficient in PD-LRRK2G2019S when compared to NM-LRRK2G2019S (− 71.26%, p = 0.022).ConclusionsEnhanced mitochondrial performance of NM-LRRK2G2019S in mitochondrial-challenging conditions and upregulation of autophagy suggests that an exhaustion of mitochondrial bioenergetic and autophagic reserve, may contribute to the development of PD in LRRK2G2019S mutation carriers.