Conference Agenda

The intestinal mucosa (IM) is an immunological and selectively permeable barrier within the gastrointestinal tract that is responsible for nutrient absorption and waste secretion. Despite interconnectivity along the small intestine, duodenal, jejunal, and ileal segments have distinct roles in digestion, nutrient transit, and absorption. More recently, segment-specific roles of IM in metabolism and neuroendocrine signaling have been revealed, indicating that segments of the small intestine may function more like independent organs systems with discrete molecular architectures. Mitochondrial function plays a pivotal role in intestinal homeostasis. However, the segment specific regulation of IM mitochondria remains entirely unknown. The purpose of this study was to investigate the bioenergetic functions of IM derived from distinct segments of the small intestine. To address this, whole intestine was harvested from male wildtype C57BL/6J mice ± diet-induced obesity (DIO) and duodenal ileal interposition, as well as human organ donors on life support. Intestine was flushed with ice cold saline, segmented by the duodenum, jejunum, and ileum, and fileted to expose IM. IM were collected by scraping the brush border membrane from the tissue. Respiratory capacity was determined by high-resolution respirometry. Cryopreserved IM were used for determination of citrate synthase activity (CSA), RNA isolation, and bulk transcriptomic analyses across segments. Gene set enrichment analysis was conducted between segments using MitoCarta3.0. IM mitochondrial respiratory activity was highest in the duodenum and decreased in a stepwise manner distally along the small intestine in mice and humans. CSA was lowest in the duodenum and increased in a stepwise manner distally across the small intestine in mice and humans. Diet-induced obesity and ileal interposition surgery did not alter IM mitochondrial capacity, indicating minimal bioenergetic plasticity between segments. Whole transcriptome analyses revealed discrete segmental signatures characterized by differential regulation of genes mediating bioenergetic function, ultrastructure, and morphology. Mitochondrially-targeted transcriptomic analysis revealed differential regulation of OXPHOS subunits and complex assembly, with higher enrichment in the duodenum and jejunum compared to the ileum. Taken together, IM from discrete segments of the small intestine have distinct bioenergetic features related to intrinsic cues of the organ to support substrate oxidation and respiratory capacity. Importantly, the innate segmental differences were similar in mice and humans, indicative of a conserved feature of mammalian biology.

Triple Negative Breast Cancer (TNBC) is an aggressive subtype of breast cancer that does not express any of the receptors commonly found in breast cancer, namely the estrogen receptor (ER), the progesterone receptor (PR) and human epidermal growth factor (HER2) receptor. As these receptors are widely used as therapeutic targets for patients with non-TNBC, novel therapeutic targets need to be found for patients with TNBC. In an endeavour to discover novel therapeutic targets we compared phenotypic, metabolic and bioenergetics profiles a parental TNBC cell line (Hs578T) and a more invasive subtype (Hs578Ts(i)₈) derived from that cell line (isogenic). Our study confirmed greater migratory and invasive potential in Hs578Ts(i)₈ cells compared to the Hs578T isogenic parental cells, as determined by in vitro transwell migration/ invasion analysis. In addition, Hs578Ts(i)₈ cells exhibit a ~3-fold greater cellular/ mitochondrial oxygen consumption rate when compared to their isogenic parental counterparts, as determined by the Seahorse Flux Analyzer. Glycolytic capacity is also greater (~2.5-fold) in Hs578Ts(i)₈ cells.

It was also observed that Hs578Ts(i)₈ cell survival, in Dulbecco Modified Eagles Medium, is more sensitive to glutamine deprivation and glutaminase inhibition than the isogenic parental cells. Additionally, supplementation of medium with dimethyl-ɑ-ketoglutarate or glutamate effectively rescues Hs578Ts(i)₈ cell death from glutamine deficiency. Subsequent GCMS analysis demonstrated that Hs578Ts(i)₈ cells have greater glutaminolysis and pentose phosphate pathway flux when compared to the parental cell line (Hs578T). The differential pentose phosphate pathway flux was mirrored by increased transcripts for several enzymes in that pathway, as determined by comparative transcriptomic analysis (RNA-Seq).

Our findings demonstrate significant metabolic differences between parental the TNBC cell line (Hs578T) and its more invasive derived isogenic subtype (Hs578Ts(i)₈). Future work will determine whether, and if so how, these metabolic differences are linked to the differential migratory and invasive phenotypes.

It is widely accepted that neurons undergo a metabolic switch during their differentiation from nonpolarized cells. While proliferating stem cells are mainly rely on glycolysis, differentiated neurons are mainly dependent on oxidative phosphorylation (OXPHOS) as a source of ATP. In adult neurons, it is assumed that mitochondria in different sub-compartments also differ with respect to their metabolism. Using hiPSC-derived neurons and a portfolio of different biosensors, we visualized how different bioenergetic parameters change during neuronal differentiation. We also analyzed mitochondrial function and OXPHOS spatially resolved an in soma, axon and growth cone by high-resolution imaging. Surprisingly, we found no significant differences in mitochondrial physiology between perinuclear and peripherical mitochondria at one time point of neuronal differentiation but significant differences during the development indicating a metabolic switch from glycolytic to OXPHOS conditions. In particular, we implemented local pH-sensors in the matrix and intra-cristae space. This allowed us to determine pH changes in addition to modulations in DY_m as determinants of the proton motive force. Moreover, we employed a supercomplex sensor for N-respirasomes [1]. Preliminary results indicate a trend toward more supercomplexes in more mature neurons. In conclusion, using an image-based approach, we were able to demonstrate the temporal shift in neuronal metabolism, but found no significant heterogeneity between mitochondria in different subcompartments (spatial analysis).

[1] B. Rieger, D.N. Shalaeva, A.C. Sohnel, W. Kohl, P. Duwe, A.Y. Mulkidjanian, K.B. Busch, Lifetime imaging of GFP at CoxVIIIa reports respiratory supercomplex assembly in live cells, Sci Rep 7 (2017) 46055.

Mitochondrial dysfunction plays a central role in the development of ischemia/reperfusion (I/R) brain injury, but the mechanism of damage is not completely understood. Here, we utilized in vivo and in vitro models of brain I/R and measured mitochondrial function, content of flavoproteins, performed proteomics/metabolomics analyses, and assessed neurological deficit.

Brain ischemia in vivo decreased the activity of mitochondrial complex I (primary energy failure), which was partially recovered at 15 minutes of reperfusion, but then irreversibly declined after 1 hour, indicating secondary energy failure. Other respiratory complexes were not affected. Irreversible complex I inactivation was associated with a decrease in the content of enzyme-bound flavin mononucleotide (FMN), the first redox-center of complex I. As a result, the FMN-deficient complex I is not able to catalyze physiological NADH oxidation and ROS generation. Metabolomics analysis of the brain samples after in vivo I/R demonstrated a dramatic accumulation of succinate and glycerol-3-phosphate in the brain during ischemia. Upon reoxygenation, these substrates are oxidized leading to reverse electron transfer (RET), when a fraction of electrons from quinol pool is directed toward mitochondrial complex I. RET over-reduced complex I causing FMNH₂ to dissociate from the active center of the enzyme. Most likely, released FMNH₂ is rapidly autoxidized and either partially rebinds to complex I or is dephosphorylated to riboflavin. Importantly, we also found that administration of FMN precursor riboflavin (vitamin B2) during in vivo brain I/R decreased infarct size, preserved complex I activity, and attenuated neurological deficit.

In vitro, the rate of FMN release during RET was not affected by the oxygen level up to 5-10 µM O₂ and by amount of endogenously produced ROS. No effect of RET on complex I subunits integrity or respiratory supercomplex assembly was found by complexome profiling. We suggest that the RET-induced flavin dissociation is a brain-specific phenomenon, due to the presence of a complex I tissue-specific long isoform of the NDUFV3 subunit located close to the FMN-binding site. Unlike the canonical short version, the long isoform of NDUFV3 can reach toward the FMN-binding pocket and affect the nucleotide affinity to the apoenzyme.

Our results suggest a central role of complex I in the development of bioenergetic failure at the early phase of brain I/R injury. Dissociation of FMN from mitochondrial complex I represents a previously undescribed mechanism of enzyme inactivation leading to brain mitochondrial impairment in I/R.

Leigh syndrome (LS) is a pediatric mitochondrial disorder that affects multiple tissues and organs during early development and causes death in infancy. Since there are no adequate models for understanding the rapid fatality associated with LS, we have established patient-specific human-induced pluripotent stem cells (hiPSC) for understanding the pathologies and for drug testing and development. Our results demonstrate altered mitochondrial morphologies, bioenergetic deficiencies and aberrant differentiation potential in neurons and cardiomyocytes during early development. Our ongoing efforts will better aid in creating customized diagnostic and therapeutic platforms thus benefiting the mitochondrial disease community.

[1] Meshrkey F, Cabrera Ayuso A, Rao RR, Iyer S. Quantitative analysis of mitochondrial morphologies in human induced pluripotent stem cells for Leigh syndrome. Stem Cell Research: 57:102572.

[2] Meshrkey F, Scheulin KM, Littlejohn CM, Stabach J, Saikia B, Thorat V, Huang Y, LaFramboise T, Lesnefsky EJ, Rao RR, West FD, Iyer S. Induced pluripotent stem cells derived from patients carrying mitochondrial mutations exhibit altered bioenergetics and aberrant differentiation potential. Stem Cell Research and Therapy, 14(1):320.

VDAC1 (Voltage-Dependent Anion-selective Channel 1), the most abundant protein of the outer mitochondrial membrane (OMM), is responsible for the high permeability of the OMM where it forms channels for the entry/exit into/from the mitochondrion of ions and small metabolites, including ATP/ADP, NAD+/NADH and Krebs cycle intermediates [1]. In ALS1, the genetic form of amyotrophic lateral sclerosis (ALS) caused by mutations in the gene encoding the antioxidant enzyme Cu/Zn Superoxide Dismutase (SOD1), VDAC1 represents the preferred docking site for selective recruitment to the spinal mitochondria of misfolded SOD1 aggregates. In particular, mitochondrial accumulation of SOD1 mutants, as observed for SOD1 G93A, triggers organelle dysfunction by affecting VDAC1 conductance and the ADP/ATP exchanges, resulting in reduced oxygen consumption rates and inhibition of mitophagy [2]. Indeed, downregulation of VDAC1 in SOD1 transgenic rats accelerates the onset of ALS symptoms and shortens the animals' lifespan.

We show that stable and enhanced expression of VDAC1, obtained by neonatal intraspinal injection of a recombinant AVV vector, recovers the mitochondrial respiratory profile of pre-symptomatic mice to that of wild-types. High-resolution respirometry analyses show a recovery of OXPHOS respiration, ATP-bound flux and maximum electron transport chain (ETC) capacity. Interestingly, this correlates with increased expression and activity of key regulators of mitochondrial function (ETC complex I, NAD⁺-dependent sirtuin deacetylases as Sirt3 and Sirt5) and the receptor subunit of the TOM complex, the main gateway for the import of mitochondrial pre-proteins into the organelle. Thus, the upregulation of VDAC1 appears to promote the activation of a TOM complex-dependent mitochondrial quality control pathway that stabilizes the Complex I-Sirt3 axis. Indeed, a more efficient exchange of NAD+/NADH increases the availability and utilization of NADH by Complex I and the derived NAD+ acts as an activator of Sirt3 [3]. Overall, our data suggests that a gene therapy based on VDAC1 could be a promising pharmacological tool for ALS.

[1] V. Shoshan-Barmatz et al. Mol Aspects Med. 2010 Jun;31(3):227-85
[2] A. Magrì et al. Cell Death Dis, 2023, 14: 122

[3] A. Magrì et al. Cell Death Discov. 2024 Apr 16;10(1):178.

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