Conference Agenda

AGC1 deficiency is a severe infantile encephalopathy caused by mutations of SLC25A12/AGC1 the isoform 1 of the mitochondrial aspartate/glutamate carrier catalysing the unidirectional exchange of mitochondrial aspartate with cytosolic glutamate. AGC1 is a key component of the malate-aspartate NADH shuttle (MAS) and plays a crucial role in energy metabolism, cellular redox state balancing, and myelin synthesis in the CNS [1]. AGC1 deficiency manifests with brain atrophy, hypotonia, epilepsy, and hypomyelination. To gain further insights into the pathogenesis of AGC1 deficiency, neuronal progenitors (NPs) were obtained from induced Pluripotent Stem Cells (iPSCs) of a patient expressing the R353Q AGC1 mutant that retains 15% of WT AGC1 activity [2], and of a patient carrying two compound heterozygous mutations (c.225del;p(Glu76Serfs*17) and c.1747C>A;p.(=)) that completely prevented AGC1 expression due to altered splicing. Compared to three unrelated healthy controls, all patient NPs revealed a proliferation deficit when grown deprived of glutamine, along with an increased cell death. Consistent with impaired MAS activity, NPs from both patients displayed higher glycolysis compensating for a dramatic decrease of mitochondrial respiration in the presence of glucose, pyruvate and lactate, also associated with a significant reduction of complex I activity in the mitochondrial respiratory chain. Since ketogenic diet improves clinical outcomes in patients with AGC1 deficiency, including resumed myelination [3], we evaluated the impact of ketone bodies acetoacetate and beta-OH-butyrate on NPs mitochondrial respiration. Ketone bodies significantly enhanced oxygen consumption rates in NPs with impaired AGC1, particularly in the absence of glucose and when combined with glutamine. Our data suggest that NPs generated from AGC1 deficiency patients benefit from the administration of alternative metabolites generating acetyl-CoA in the mitochondria and feeding TCA cycle, bypassing the limited mitochondrial oxidation of pyruvate derived from glycolysis.

[1] Wibom R, et al., N Engl J Med. 2009;361:489-95.

[2] Falk MJ, et al., JIMD Rep. 2014;14:77-85.

[3] Dahlin M, et al. Epilepsia. 2015;56(11): e176-81.

Paediatric Leigh syndrome (LS) is an early-onset and fatal neurodegenerative disorder, for which there is no treatment. LS is frequently caused by mutations in the NDUFS4 gene, which encodes an accessory subunit of mitochondrial complex I (CI), the first complex of the oxidative phosphorylation (OXPHOS) system. Whole-body Ndufs4 knock-out (KO) mice (WB-KO mice) are widely used to study isolated CI deficiency, LS pathology and interventions. These animals develop a brain-specific phenotype via an incompletely understood pathomechanism. To better understand the latter, we here performed a quantitative analysis of the sub-brain proteome in six-weeks old WB-KO mice. Tissues consisted of a brain slice (BS), cerebellum (CB), cerebral cortex (­­CC), hippocampus (HC), inferior colliculus (IC), and superior colliculus (SC). Proteome analysis demonstrated similarities between CC/HC, and between IC/SC, whereas BS and CB differed from these two groups and from each other. All brain tissues displayed greatly reduced levels of two CI structural subunits (NDUFS4, NDUFA12) and an increased level of the CI assembly factor NDUFAF2. The level of CI-Q module subunits was significantly more reduced in IC/SC than in BS/CB/CC/HC, whereas other OXPHOS complex levels were not reduced. Gene ontology (GO) term and pathway enrichment analysis demonstrated tissue-specific and common proteome changes between brain tissues. Across brain tissues, upregulation of cold shock-associated proteins, mitochondrial fatty acid (FA) degradation and synthesis (mtFAS) were the most prominent. FA-related pathways were predominantly upregulated in CB and HS. We argue that stimulation of these pathways constitutes part of a futile and pro-pathological adaptation to CI deficiency in LS.

Leigh syndrome (LS) is a fatal neurometabolic disease characterized by progressive loss of central nervous system function. There are over 100 genetic mutations that can cause LS, most of which involve proteins associated with the mitochondrial electron transport chain (ETC). Currently no therapeutics are approved for the treatment of patients with LS. Because LS involves dysfunctional ETC activity and cellular redox stress, we sought to determine the effect of the novel compound SBT-589 in a cell model of LS. We have previously shown that SBT-589 restored bioenergetics by bypassing Complex I (CI) of the ETC and ameliorated iron-mediated cell death. SBT-589 comes from a novel class of molecules that cross the blood-brain-barrier and have homogenous biodistribution throughout regions of the rodent brain. In this study, we examined the efficacy of SBT-589 in the LS patient-derived fibroblast line GM03672. Following redox stress induced by RAS-selective lethal 3 (RSL3) administration, SBT-589 dose-dependently attenuated cell death and preserved intracellular ATP levels. Fibroblasts stressed with the glutathione synthesis inhibitor erastin were also protected with SBT-589 treatment. To assess the impact of SBT-589 on lipid peroxidation, the metabolic by-product 15-HETE was measured in the supernatant of cultured LS fibroblasts. Co-treatment of erastin or RSL3-injured fibroblasts with 1 μM SBT-589 led to a reduction in 15-HETE compared to untreated cells. Furthermore, SBT-589 (1 μM) restored mitochondrial membrane potential and integrity to near baseline in cells that were treated with RSL3. The impact of SBT-589 on bioenergetics in the presence of a CI inhibitor was evaluated using high-resolution respirometry. After chemical inhibition of CI, treatment with SBT-589 restored oxygen consumption rates to near basal levels. In parallel studies using isolated mitochondria, SBT-589 reduced mitochondrial emission of reactive oxygen species (ROS). These data highlight the therapeutic potential of SBT-589 as a disease-modifying treatment for individuals living with LS.

Majority of nuclear-encoded defects of ATP synthase are caused by mutations in TMEM70, an assembly factor that facilitates the incorporation of c-ring into the membrane domain of the enzyme. TMEM70 deficiency in patients leads to a prominent decrease of assembled ATP synthase, manifesting as severe neonatal encephalo-cardio-myopathy accompanied by lactic acidosis and 3-methlyglutaconinc aciduria. Currently, there are no curative treatments for patients with TMEM70 defects. To explore potential treatment interventions, we established relevant TMEM70 rodent models – inducible Tmem70 knockout mice (iTmem70) and SHR-Tmem70^ko/ko,tg/0 rats. At the organ level, liver and skeletal muscle are primarily affected in iTmem70 mice, while heart pathology is most prominent in Tmem70^ko/ko,tg/0 rats. At a molecular level, both models display a pronounced decrease in the content of fully assembled F₁F_o-ATP synthase and accumulation of F₁ subcomplex, copying the phenotype of examined patient’s tissues. Since lipid infusions are known to be beneficial for TMEM70 patients during metabolic crises, we performed dietary intervention to evaluate the beneficial effect of lipids on the iTmem70 mice model.

A ketogenic diet was administered ad libitum and in iTmem70 model drastically prolonged overall survival. Also, it stabilized body weight loss and altered the profile of enzymes involved in fatty acid oxidation. Interestingly, BNE/CNE analyses show increased content of subunit c accompanied by the upregulation of assembled ATP synthase and a drop in the content of the free F₁ domain. Both mouse and rat models on the ketogenic diet have significantly higher respiratory control ratios and respiratory rates for complex I. A ketogenic diet on TMEM70 deficient mice lead to the upregulation of most inner mitochondrial membrane proteins and phospholipids (PC and PE) in the liver, indicating improvement in mitochondrial biogenesis and ultrastructure. iTmem70 animals show disturbances in the urea cycle, that were partially corrected by the ketogenic diet. In conclusion, the ketogenic diet significantly attenuates Tmem70 pathology in rodent models. It can be considered a treatment strategy for TMEM70 patients.

Funded by Czech Medical Research Council (NU21-07-00550).

Major depressive disorder (MDD) impairs the cognitive, affective and somatic functioning of affected individuals. While the clinical diagnosis depends on the symptoms and their severity, clinically-applicable biomarkers of MDD remain missing. This gap limits predictive and preventive approaches as well as biologically-embedded clinical treatment modalities for MDD that - so far - rather follow reactive medical care strategies. Now, Omics sciences allow for the hypothesis-free identification of new biomarker candidates and their characteristic signatures in biosamples. Using an untargeted proteomics approach with hair samples, we intended to identify a correlative crosslink to previously reported impaired mitochondrial bioenergetics in peripheral blood mononuclear cells (PBMC).

Female individuals with and without MDD (N=44) were recruited at the AMEOS Psychiatry Clinic in Hildesheim, Germany. The Beck Depression Inventory (BDI-II; self-report) and the Montgomery Asberg Depression Rating Scale (MADRS) were used to assess the clinical severity of depressive symptoms. Whole blood was collected to measure mitochondrial respiration in Ficoll-isolated and cryopreserved PBMC. Thawed samples were prepared to assess oxygen consumption rates using high-resolution respirometry (HRR) with the O2k Oxygraph. In addition, hair samples were collected from the posterior vertex of the scalp, and Orbitrap mass spectrometry was performed with 3cm long hair strands to analyze and characterize the hair proteome.

Mitochondrial respiration in PBMC from depressed individuals was significantly lower for characteristic profiles compared to the MDD-free control group. In addition, these impairments correlated significantly with the clinical severity of depressive symptoms. Next, the analysis of hair revealed a total of N=1,143 different proteins. Here, proteins directly linked to cellular bioenergetics including subunits of Complex V (ATP synthase F0) again showed (i) a significant difference between depressed and non-depressed individuals, (ii) a significant correlation with the clinical severity of MDD symptoms, and (iii) a significant correlation with mitochondrial functioning characteristics measured by HRR.

Together with mitochondrial bioenergetics, Omics sciences provide access to new concepts, methods and biological information highly relevant to catalyze MDD biomarker research. Here, we provide the first data on hair proteomics and report a correlation with mitochondrial bioenergetics that synergistically stimulate current perspectives about the etiology and manifestation of MDD. As a result, these advances may lead to the identification of new biomarkers of MDD. Even though the feasibility could be demonstrated, given limitations (e.g. sex and age distribution, cohort size, and medication) have to be considered in future replication studies.