Mitochondrial Response Research Articles

AML-282 Venetoclax Upregulates Mitochondrial Stress Response to Overcome the Resistance to Arsenic Trioxide in Acute Promyelocytic Leukemia

AML-282 Venetoclax Upregulates Mitochondrial Stress Response to Overcome the Resistance to Arsenic Trioxide in Acute Promyelocytic Leukemia

POSTER: AML-282 Venetoclax Upregulates Mitochondrial Stress Response to Overcome the Resistance to Arsenic Trioxide in Acute Promyelocytic Leukemia

POSTER: AML-282 Venetoclax Upregulates Mitochondrial Stress Response to Overcome the Resistance to Arsenic Trioxide in Acute Promyelocytic Leukemia

Mitochondrial reactivity following acute exposure to experimental pain testing in people with HIV and chronic pain.

Background: Physical stressors can cause a physiological response that can contribute to an increase in mitochondrial dysfunction and Mitochondrial DNA damage (mtDNA damage). People living with HIV (PWH) are more likely to suffer from chronic pain and may be more susceptible to mitochondrial dysfunction following exposure to a stressor. We used Quantitative Sensory Testing (QST) as an acute painful stressor in order to investigate whether PWH with/without chronic pain show differential mitochondrial physiological responses. Methods: The current study included PWH with (n = 26), and without (n = 29), chronic pain. Participants completed a single session that lasted approximately 180min, including QST. Blood was taken prior to and following the QST battery for assays measuring mtDNA damage, mtDNA copy number, and mtDNA damage-associated molecular pattern (DAMP) levels (i.e., ND1 and ND6). Results: We examined differences between those with and without pain on various indicators of mitochondrial reactivity following exposure to QST. However, only ND6 and mtDNA damage were shown to be statistically significant between pain groups. Conclusion: PWH with chronic pain showed greater mitochondrial reactivity to laboratory stressors. Consequently, PWH and chronic pain may be more susceptible to conditions in which mitochondrial damage/dysfunction play a central role, such as cognitive decline.

Long-read direct RNA sequencing of the mitochondrial transcriptome of Saccharomyces cerevisiae reveals condition-dependent intron abundance.

Mitochondria fulfil many essential roles and have their own genome, which is expressed as polycistronic transcripts that undergo co- or posttranscriptional processing and splicing. Due to the inherent complexity and limited technical accessibility of the mitochondrial transcriptome, fundamental questions regarding mitochondrial gene expression and splicing remain unresolved, even in the model eukaryote Saccharomyces cerevisiae. Long-read sequencing could address these fundamental questions. Therefore, a method for the enrichment of mitochondrial RNA and sequencing using Nanopore technology was developed, enabling the resolution of splicing of polycistronic genes and the quantification of spliced RNA. This method successfully captured the full mitochondrial transcriptome and resolved RNA splicing patterns with single-base resolution and was applied to explore the transcriptome of S. cerevisiae grown with glucose or ethanol as the sole carbon source, revealing the impact of growth conditions on mitochondrial RNA expression and splicing. This study uncovered a remarkable difference in the turnover of Group II introns between yeast grown in either mostly fermentative or fully respiratory conditions. Whether this accumulation of introns in glucose medium has an impact on mitochondrial functions remains to be explored. Combined with the high tractability of the model yeast S. cerevisiae, the developed method enables to monitor mitochondrial transcriptome responses in a broad range of relevant contexts, including oxidative stress, apoptosis and mitochondrial diseases.

Inhibition of mitochondrial fusion via SIRT1/PDK2/PARL axis breaks mitochondrial metabolic plasticity and sensitizes cancer cells to glucose restriction therapy

Mitochondria dynamically change their morphology via fusion and fission, a process called mitochondrial dynamics. Dysregulated mitochondrial dynamics respond rapidly to metabolic cues, and are linked to the initiation and progression of diverse human cancers. Metabolic adaptations significantly contribute to tumor development and escape from tissue homeostatic defenses. In this work, we identified oroxylin A (OA), a dual GLUT1/mitochondrial fusion inhibitor, which restricted glucose catabolism of hepatocellular carcinoma cells and simultaneously inhibited mitochondrial fusion by disturbing SIRT1/PDK2/PARL axis. Based the dual action of OA in metabolic regulation and mitochondrial dynamics, further results revealed that mitochondrial functional status and spare respiratory capacity (SRC) of cancer cells had a close correlation with mitochondrial metabolic plasticity, and played important roles in the susceptibility to cancer therapy aiming at glucose restriction. Cancer cells with healthy mitochondria and high SRC exhibit greater metabolic flexibility and higher resistance to GLUT1 inhibitors. This phenomenon is attributed to the fact that high SRC cells fuse mitochondria in response to glucose restriction, enhancing tolerance to energy deficiency, but undergo less mitochondrial oxidative stress compared to low SRC cells. Thus, inhibiting mitochondrial fusion breaks mitochondrial metabolic plasticity and increases cancer cell susceptibility to glucose restriction therapy. Collectively, these finding indicate that combining a GLUT1 inhibitor with a mitochondrial fusion inhibitor can work synergistically in cancer therapy and, more broadly, suggest that the incorporations of mitochondrial dynamics and metabolic regulation may become the targetable vulnerabilities bypassing the genotypic heterogeneity of multiple malignancies.

The neurobehavioral impacts of typical antibiotics toward zebrafish larvae

Due to the widely usage in livestock, aquaculture and clinics, antibiotic residues are existed in aqueous environments and their potential toxicity to aquatic organisms is concerning. Here, we used zebrafish as the model to investigate the neurotoxicity and involved mechanism of seven antibiotics that were frequently detected in surface waters. The results revealed that the short-term exposure to clarithromycin (CLA), chlortetracycline (CTC) and roxithromycin (ROX) induced behavioral effects, with effective concentration of 1 μg/L (CTC and ROX) and 100 μg/L (CLA, CTC and ROX) respectively. A significant decrease in the travel distance and velocity as well as an increase in turn angle was measured. TUNEL assay identified increased cell apoptosis in brain sections of larvae exposed to three neurotoxic antibiotics, which raised the possibility that the behavioral symptoms were associated with neural damage. Transcriptome sequencing showed that the three antibiotics could affect the nervous system of zebrafish including the alteration of synaptogenesis and neurotransmission. Additionally, ROX and CTC affected pathways involved in mitochondrial stress response and endocrine system in zebrafish larvae. Besides, BDNF, ASCL1, and CREBBP are potential upstream regulatory factors that mediated these impacts. These findings indicated that exposure of CTC, ROX and CLA may cause abnormal behavior toward zebrafish larvae under environmental relevant concentration and revealed the potential role of neural cell apoptosis and synaptogenesis signaling in mediating this effect.

Recent progress in understanding mitokines as diagnostic and therapeutic targets in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most prevalent tumors worldwide and the leading contributor to cancer-related deaths. The progression and metastasis of HCC are closely associated with altered mitochondrial metabolism, including mitochondrial stress response. Mitokines, soluble proteins produced and secreted in response to mitochondrial stress, play an essential immunomodulatory role. Immunotherapy has emerged as a crucial treatment option for HCC. However, a positive response to therapy is typically dependent on the interaction of tumor cells with immune regulation within the tumor microenvironment. Therefore, exploring the specific immunomodulatory mechanisms of mitokines in HCC is essential for improving the efficacy of immunotherapy. This study provides a comprehensive overview of the association between HCC and the immune microenvironment and highlights recent progress in understanding the involvement of mitochondrial function in preserving liver function. In addition, a systematic review of mitokines-mediated immunomodulation in HCC is presented. Finally, the potential diagnostic and therapeutic roles of mitokines in HCC are prospected and summarized. Recent progress in mitokine research represents a new prospect for mitochondrial therapy. Considering the potential of mitokines to regulate immune function, investigating them as a relevant molecular target holds great promise for the diagnosis and treatment of HCC.

Chronic Thermal Acclimation Effects on Critical Thermal Maxima (CTmax) and Oxidative Stress Differences in White Epaxial Muscle between Surface and Cave Morphotypes of the Mexican Cavefish (Astyanax mexicanus).

AbstractIn the face of increasing environmental temperatures, operative differences between mitochondrial function and whole-animal phenotypic response to the environment are underrepresented in research, especially in subtemperate ectothermic vertebrates. A novel approach to exploring this connection is to examine model species that are genetically similar but that have different whole-animal phenotypes, each of which inhabits different environments. The blind Mexican cavefish (Astyanax mexicanus) has the following two morphotypes: a surface form found in aboveground rivers and an obligate cave-dwelling form. Each morphotype inhabits vastly different thermal and oxygen environments. Whole-animal and mitochondrial responses to thermal acclimation and oxidative stress, with respect to increasing temperatures, have not been previously determined in either morphotype of this species. Here, we chronically acclimated both morphotypes to three temperatures (14°C, 25°C, and 31°C) to establish potential for acclimation and critical thermal maxima (CTmax) for each morphotype of this species. After measuring CTmax in six cohorts, we additionally measured enzymatic antioxidant capacity (catalase, superoxide dismutase, and glutathione peroxidase activities), peroxyl scavenging capacity, and lipid peroxidation damage in white epaxial muscle for each individual. We found a significant effect of acclimation temperature on CTmax (, ) but no effect of morphotype on CTmax (, ). Additionally, we found that morphotype had a significant effect on glutathione peroxidase activity, with the surface morphotype having increased glutathione peroxidase activity compared with the cave morphotype (, ). No other oxidative stress variable demonstrated significant differences. Increases in CTmax with chronic thermal acclimation to higher temperatures suggests that there is some degree of phenotypic plasticity in this species that nominally occupies thermally stable environments. The decreased glutathione peroxidase activity in the cave morphotype may be related to decreased environmental oxygen concentration and decreased metabolic rate in this environmentally constrained morphotype compared to in its surface-living counterparts.

Abstract B031: Effect of macrophage inflammatory response and VHL mutation status on the development of clear cell renal carcinoma

Abstract Kidney[A1] cancer is a serious health care issue problem around the worldworldwide. It is symptomless asymptomatic in the early stage and is refractory to most [A2] of the cancer treatment modalities. The most prevalent form of the kidney cancer, renal cell carcinoma of the clear-cell type (ccRCC), is mainly caused by loss-of-function of the von Hippel-Lindau tumor suppressor gene (VHL). VHL protein is an E3 ubiquitin ligase and. iIts most prominentmain degradation target is the alpha subunit of the hypoxia-inducible factor (HIF). However, the etiology of ccRCC formation development remains unclear. Recent animal model studies in our laboratory have indicatedWe recently observed that tissue inflammation and fibrosis are the early events in ccRCC development is tissue inflammation and fibrosis. This interesting result provides important supports for the concept notion that chronic inflammation is can induce tumorigenesisic and. It is also consistent with the epidemiological evidence linking inflammatory kidney disease and kidney cancer. In the current present study, we explored the mechanistic detailsm by which changes within the VHL mutant cells orchestrateeffect[A3] a systemic inflammatory response[A4], and found that the metabolic abnormalities caused by VHL gene mutation lead to unresolved chronic endoplasmic reticular stress (ER stress), which in turn induces activation of JNK and NFκB, two major components of pro-inflammatory signaling pathways components[A5]. On the other hand, our previous study also demonstrated that LCN2[A6] sensitizes the inflammatory response of VHL mutant HK-2 cells in vhl mutant condition and the chemotactic abilityies of macrophage RAW 264.7 cells. [A7] However, the wider implication of this tumorigenic process [A8] that of RAW 264.7 cells is stillremains unclear and then warrants further investigationswill be discussed. Accumulating evidence indicated that Lon protease (Lon) is a multifunctional protein that mediatesinvolved in protein quality control and oxidative stress responses in mitochondria. However, the molecular mechanisms underlying the effects of Lon on the progression of ccRCC progression and inflammatory response inof the vhl VHL mutant condition cells remain largely unknown. Therefore, in this study, we aimed to elucidate the inflammatory effect role of inflammatory RAW 264.7 macrophages on the development of ccRCC development inusing cultured vhl VHL mutant renal tubular cells and RAW264.7 cells. Moreover, d Decoding how the action involved signaling pathways drive or suppress signals cellular and inflammatory responses or preventing it is a major research challenge. Once elucidated, it will for future research and will require the definition ofprovide the rationale for drug design and application on anti-ccRCC formation therapeutic strategies based onvia targeting macrophages infiltration. Citation Format: Chan-Yen Kuo. Effect of macrophage inflammatory response and VHL mutation status on the development of clear cell renal carcinoma [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr B031.

Metabolic Disruption Induced by mTOR Signaling Pathway Inhibition in Regulatory T-Cell Expansion for Clinical Application.

Regulatory T cell (Treg) therapy is considered an alternative approach to induce tolerance in transplantation. If successful, this therapy may have implications on immunosuppression minimization/withdrawal to reduce drug-induced toxicity in patients. The aim of this study was to assess the efficacy of the mTORC1/C2 inhibitor, AZD8055, in the manufacturing of clinically competent Treg cells and compare the effects with those induced by rapamycin (RAPA), another mTOR inhibitor commonly used in Treg expansion protocols. Primary human Treg cells were isolated from leukapheresis product. Cell viability, expansion rates, suppressive function, autophagy, mitochondrial unfolded protein response (mitoUPR), and cell metabolic profile were assessed. We observed a stronger inhibition of the mTORC2 signaling pathway and downstream events triggered by Interleukin 2 (IL2)-receptor in AZD8055-treated cells compared with those treated with RAPA. AZD8055 induced progressive metabolic changes in mitochondrial respiration and glycolytic pathways that disrupted the long-term expansion and suppressive function of Tregs. Unlike RAPA, AZD8055 treatment impaired autophagy and enhanced the mitoUPR cell stress response pathway. A distinct pattern of mTOR inhibition by AZD, compared with RAPA, induced mitochondrial stress response and dysfunction, impaired autophagy, and disrupted cellular bioenergetics, resulting in the loss of proliferative potential and suppressive function of Treg cells.

Propionic acid induces alterations in mitochondrial morphology and dynamics in SH-SY5Y cells

Propionic acid (PPA) is used to study the role of mitochondrial dysfunction in neurodevelopmental conditions like autism spectrum disorders. PPA is known to disrupt mitochondrial biogenesis, metabolism, and turnover. However, the effect of PPA on mitochondrial dynamics, fission, and fusion remains challenging to study due to the complex temporal nature of these mechanisms. Here, we use complementary quantitative visualization techniques to examine how PPA influences mitochondrial ultrastructure, morphology, and dynamics in neuronal-like SH-SY5Y cells. PPA (5 mM) induced a significant decrease in mitochondrial area (p < 0.01), Feret's diameter and perimeter (p < 0.05), and in area2 (p < 0.01). Mitochondrial event localiser analysis demonstrated a significant increase in fission and fusion events (p < 0.05) that preserved mitochondrial network integrity under stress. Moreover, mRNA expression of cMYC (p < 0.0001), NRF1 (p < 0.01), TFAM (p < 0.05), STOML2 (p < 0.0001), and OPA1 (p < 0.01) was significantly decreased. This illustrates a remodeling of mitochondrial morphology, biogenesis, and dynamics to preserve function under stress. Our data provide new insights into the influence of PPA on mitochondrial dynamics and highlight the utility of visualization techniques to study the complex regulatory mechanisms involved in the mitochondrial stress response.

Mitochondrial integrated stress response controls lung epithelial cell fate.

Alveolar epithelial type 1 (AT1) cells are necessary to transfer oxygen and carbon dioxide between the blood and air. Alveolar epithelial type 2 (AT2) cells serve as a partially committed stem cell population, producing AT1 cells during postnatal alveolar development and repair after influenza A and SARS-CoV-2 pneumonia1-6. Little is known about the metabolic regulation of the fate of lung epithelial cells. Here we report that deleting the mitochondrial electron transport chain complex I subunit Ndufs2 in lung epithelial cells during mouse gestation led to death during postnatal alveolar development. Affected mice displayed hypertrophic cells with AT2 and AT1 cell features, known as transitional cells. Mammalian mitochondrial complex I, comprising 45 subunits, regenerates NAD+ and pumps protons. Conditional expression of yeast NADH dehydrogenase (NDI1) protein that regenerates NAD+ without proton pumping7,8 was sufficient to correct abnormal alveolar development and avert lethality. Single-cell RNA sequencing revealed enrichment of integrated stress response (ISR) genes in transitional cells. Administering an ISR inhibitor9,10 or NAD+ precursor reduced ISR gene signatures in epithelial cells and partially rescued lethality in the absence of mitochondrial complex I function. Notably, lung epithelial-specific loss of mitochondrial electron transport chain complex II subunit Sdhd, which maintains NAD+ regeneration, did not trigger high ISR activation or lethality. These findings highlight an unanticipated requirement for mitochondrial complex I-dependent NAD+ regeneration in directing cell fate during postnatal alveolar development by preventing pathological ISR induction.

Unravelling the Therapeutic Potential of Antibiotics in Hypoxia in a Breast Cancer MCF-7 Cell Line Model.

Antibiotics inhibit breast cancer stem cells (CSCs) by suppressing mitochondrial biogenesis. However, the effectiveness of antibiotics in clinical settings is inconsistent. This inconsistency raises the question of whether the tumor microenvironment, particularly hypoxia, plays a role in the response to antibiotics. Therefore, the goal of this study was to evaluate the effectiveness of five commonly used antibiotics for inhibiting CSCs under hypoxia using an MCF-7 cell line model. We assessed the number of CSCs through the mammosphere formation assay and aldehyde dehydrogenase (ALDH)-bright cell count. Additionally, we examined the impact of antibiotics on the mitochondrial stress response and membrane potential. Furthermore, we analyzed the levels of proteins associated with therapeutic resistance. There was no significant difference in the number of CSCs between cells cultured under normoxic and hypoxic conditions. However, hypoxia did affect the rate of CSC inhibition by antibiotics. Specifically, azithromycin was unable to inhibit sphere formation in hypoxia. Erythromycin and doxycycline did not reduce the ratio of ALDH-bright cells, despite decreasing the number of mammospheres. Furthermore, treatment with chloramphenicol, doxycycline, and tetracycline led to the overexpression of the breast cancer resistance protein. Our findings suggest that hypoxia may weaken the inhibitory effects of antibiotics on the breast cancer model.

Kojic acid induces oxidative stress and anti-inflammatory effects in human hepatocellular carcinoma (HepG2) cells

The cosmetic industry makes extensive use of kojic acid (KA); however, the toxicity of KA in humans is not well known. By monitoring oxidative stress, mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NFκB) signalling in human hepatoma (HepG2) cells after a 24 h exposure, this study aimed to identify the toxicity of KA. KA toxicity [4.22, 8.02 and 12.67 mM] was assessed using mitochondrial output, antioxidant responses, macromolecule damage, MAPK signalling, inflammation, and cell death markers, using spectrophotometry, luminometry, Western blot and qPCR. Apoptosis was confirmed by reduced cell viability and increased caspases −9 (p < 0.0001), −8 (p = 0.0003), and −3/7 (p < 0.0001) activities at 4.22 mM and 8.02 mM. LDH leakage was present at 12.67 mM, providing significant evidence of necrosis. Malondialdehyde (MDA) levels significantly increased at 4.22 mM (p < 0.0001). There was an increase of phosphorylated nuclear factor erythroid-2 factor-2 (p-Nrf2) at 4.22 mM and 8.02 mM, whilst at 12.67 mM decreased p-Nrf2 (p < 0.0001) was observed. KA increased p38 expression (p = 0.0011). The findings point to significant suppression of the NFκB inflammatory pathway at 8.02 mM (p < 0.0001). This study showed that KA initiated MAPK signalling due to oxidative stress and suppressed inflammation. HepG2 cells showed minimal toxicity to KA.

Mitochondria: at the crossroads between mechanobiology and cell metabolism.

Metabolism and mechanics are two key facets of structural and functional processes in cells, such as growth, proliferation, homeostasis and regeneration. Their reciprocal regulation has been increasingly acknowledged in recent years: external physical and mechanical cues entail metabolic changes, which in return regulate cell mechanosensing and mechanotransduction. Since mitochondria are pivotal regulators of metabolism, we review here the reciprocal links between mitochondrial morphodynamics, mechanics and metabolism. Mitochondria are highly dynamic organelles which sense and integrate mechanical, physical and metabolic cues to adapt their morphology, the organization of their network and their metabolic functions. While some of the links between mitochondrial morphodynamics, mechanics and metabolism are already well established, others are still poorly documented and open new fields of research. First, cell metabolism is known to correlate with mitochondrial morphodynamics. For instance, mitochondrial fission, fusion and cristae remodeling allow the cell to fine-tune its energy production through the contribution of mitochondrial oxidative phosphorylation and cytosolic glycolysis. Second, mechanical cues and alterations in mitochondrial mechanical properties reshape and reorganize the mitochondrial network. Mitochondrial membrane tension emerges as a decisive physical property which regulates mitochondrial morphodynamics. However, the converse link hypothesizing a contribution of morphodynamics to mitochondria mechanics and/or mechanosensitivity has not yet been demonstrated. Third, we highlight that mitochondrial mechanics and metabolism are reciprocally regulated, although little is known about the mechanical adaptation of mitochondria in response to metabolic cues. Deciphering the links between mitochondrial morphodynamics, mechanics and metabolism still presents significant technical and conceptual challenges but is crucial both for a better understanding of mechanobiology and for potential novel therapeutic approaches in diseases such as cancer. This article is protected by copyright. All rights reserved.

Chronic exposure to dim artificial light disrupts the daily rhythm in mitochondrial respiration in mouse suprachiasmatic nucleus

ABSTRACT Circadian rhythms of physiology, behavior, and metabolism have an endogenous 24 h period that synchronizes with environmental cycles of light/dark and food availability. Alterations in light cycles are stressful and disrupt such diurnal oscillations. Recently, we witnessed a sudden rise in studies describing the mechanisms behind the interaction between the key characteristics of mitochondrial functions, peripheral clocks, and stress responses. To our knowledge, there is no study in the suprachiasmatic nuclei (SCN) describing the dysregulated mitochondrial bioenergetics under abnormal lighting conditions, which is common in today’s modern world. Thus, we aimed to investigate the existence of daily changes in mitochondrial bioenergetics (respiratory control rate, RCR), mitochondrial abundance (mtDNA/nDNA), plasma corticosterone, and to test whether disturbances in the lighting conditions might influence such rhythms. To confirm this, mice were sacrificed, mitochondria were isolated from the suprachiasmatic nuclei in the brain and blood was collected, every 3 h at various time points zeitgeber time/circadian time, (0, 3, 6, 9, 12, 15, 18, 21, and 24 h) under 12:12 h light-dark (LD, 150 lux L: 0 lux D) cycle and chronic artificial dim lighting (LL, 5 lux: 5lux) conditions, of a 24 h period, respectively. Our results demonstrate the existence of robust daily rhythmicity in RCR, mtDNA/nDNA and plasma CORT under a normal LD cycle. However, these rhythms were significantly disrupted and clock genes expressions were dysregulated under chronic dim LL. Furthermore, mitochondrial abundance was significantly reduced during LL compared to their numbers under LD cycle. Our data demonstrate that the circadian clock regulates mitochondrial functions (RCR, number), essential for accomplishing daily energy demands and supply by the SCN neurons. Abnormal light exposure dysregulates mitochondrial functions in the SCN and may alter metabolism, resulting in obesity, diabetes, and other metabolic disorders. Therefore, properly designing lighting conditions in workplaces is essential to mitigate the adverse consequences of light on humans.

T lymphocytes expressing the switchable chimeric Fc receptor CD64 exhibit augmented persistence and antitumor activity

Chimeric antigen receptor (CAR) Tcell therapy lacks persistent efficacy with "on-target, off-tumor" toxicities for treating solid tumors. Thus, an antibody-guided switchable CAR vector, the chimeric Fc receptor CD64 (CFR64), composed of a CD64 extracellular domain, is designed. Tcells expressing CFR64 exert more robust cytotoxicity against cancer cells than CFR Tcells with high-affinity CD16 variant (CD16v) or CD32A as their extracellular domains. CFR64 Tcells also exhibit better long-term cytotoxicity and resistance to Tcell exhaustion compared with conventional CAR Tcells. With trastuzumab, the immunological synapse (IS) established by CFR64 is more stable with lower intensity induction of downstream signaling than anti-HER2 CAR Tcells. Moreover, CFR64 Tcells exhibit fused mitochondria in response to stimulation, while CARH2 Tcells contain predominantly punctate mitochondria. These results show that CFR64 Tcells may serve as a controllable engineered Tcell therapy with prolonged persistence and long-term antitumor activity.

Long-Term Photobiomodulation of Cellular and Mitochondrial Activities in IMR-32 Cells Using an 810 nm Light-Emitting Diode

In photobiomodulation (PBM), biological tissues are stimulated using low-energy light. Mitochondria are thought to be integral to the process, and PBM effects vary with light parameters. Biphasic curves are commonly used to describe the dose response of PBM but do not fit in some cases. Dose-dependent PBM effects, especially mitochondrial responses, must be understood to optimize clinical therapies. However, most previous studies have reported short-term mitochondrial activity changes, neglecting long-term effects or dose-dependent changes in whole transcriptomes. We studied the effects of 3-day PBM with an 810 nm circular LED beam on human neuroblastoma IMR-32 cells. Two light doses with opposite effects on mitochondrial DNA copy number were chosen to analyze total RNA, protein content and function of mitochondrial respiratory complexes, and mitochondrial morphology. Daily 15-minute 810 nm light treatment enhanced drug resistance to the mitochondrial inhibitors and uncoupling agent via two distinct mechanisms: 1.40 mW/cm <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sup> light increased cell proliferation and the proportion of cells in the cell cycle S phase; 1.95 mW/cm <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sup> light enhanced oxidative phosphorylation at the RNA, protein, and functional levels and the proportion of network-like mitochondria. Our findings reveal the complexity of PBM dose responses and provide insights into light-cell interactions.

P-393 The mitochondrial targeted antioxidant Mitoquinol increases survival of isolated human preantral follicles in vitro

Abstract Study question Does Mitoquinol have a protective effect on survival and mitochondrial oxidative stress in cultured human preantral follicles? Summary answer Mitoquinol significantly increased survival of human preantral follicles cultured for 8 days and significantly decreased expression of genes related to the mitochondrial oxidative stress response. What is known already Culturing human ovarian follicles is a promising new source of mature oocytes for fertility preservation, as well as a key model system to investigate fundamental biology. Nevertheless, only a handful of studies have yielded mature human oocytes from preantral stage follicles and optimizing culture systems is challenging due to the scarcity of human material. Mitoquinol is an antioxidant that contains a form of Coenzyme Q10 called ubiquinone that has been modified to selectively target and protect mitochondria from oxidative damage. In human oocytes, the presence of Mitoquinol in the IVM media increased nuclear maturation and protected against chromosomal misalignment. Study design, size, duration Human preantral follicles (n = 213; mean diameter: 71 µm; range: 26-189 µm) were isolated from ovarian medulla tissue. Preantral follicles were mechanically and enzymatically isolated, encapsulated in 0.5% alginate and cultured for 8 days in three experimental groups: Control (n = 68); 50nM Mitoquinol (n = 71), 250nM Mitoquinol (n = 74). The primary endpoints were follicular growth and survival. Secondary endpoints included follicular gene expression and hormone analysis of the spent culture media. Participants/materials, setting, methods Ovarian medulla tissue was donated by 5 patients (aged 30-37 years) undergoing unilateral oophorectomy for ovarian tissue cryopreservation. Follicular growth and survival were assessed every second day during culture by microscopy. For follicles bigger than 95 µm, AMH and Estradiol concentrations were measured by ELISA in the spent media on day 8. Finally surviving follicles were snap-frozen and gene expression was analysed by qPCR; mitochondrial oxidative stress genes: HSP-60, TFAM and apoptosis genes: BAX/BCL-2. Main results and the role of chance After 8 days in culture, the follicular survival rate in the 50nM Mitoquinol group (68%; n = 51/74) was significantly higher than the control group (49%; n = 32/68; p = 0.0344) and the 250nM Mitoquinol (54%; n = 39/71; p = 0.0443). The protective effect of 50nM Mitoquinol was more pronounced in the subset of small follicles (Day0 &amp;lt; 70 µm, n = 135) in which survival rate of the 50nM Mitoquinol group (65%; n = 31/47) was significantly higher than the control (41%; n = 18/43; p = 0.005) and the 250nM group (44%; n = 20/4; p = 0.019). The average growth of surviving follicles was similar in the three experimental groups (Control: 40.7 ± 17.4 µm; Mitoquinol 50nM: 36.9 ± 19 μm; Mitoquinol 250nM: 36.4 ±16.5 μm). A significant positive association between the diameter of the follicle and the concentration of AMH and Estradiol was observed (AMH: p = 0.0215, R = 0.43 and Estradiol: p = 0.0413, R = 0.38) but no differences were found between the experimental groups. The relative expression of genes involved in mitochondrial oxidative stress response were significantly lower in the 50nM Mitoquinol group compared to the 250mM Mitoquinol group (HSP-60 p = 0.018; TFAM p = 0.013) and similar to the control group (HSP-60 p = 0.056; TFAM p = 0.071). Moreover, the gene expression of the ratio BAX/BCL2 was similar in the three groups. Limitations, reasons for caution A larger sample size is required to confirm the statistical analysis and to extrapolate the results to primordial, primary, and secondary stage follicles. Furthermore, long-term culture studies are required to determine the impact of Mitoquinol on antral follicle development and oocyte maturation Wider implications of the findings Our findings show that Mitoquinol can be used as an antioxidant in the culture of human preantral follicles since it increased follicle survival and reduced gene expression of genes related to mitochondrial oxidative stress response. Trial registration number Not appliclable

P-628 Prenatally androgenized PCOS-like mice present with dysregulated endometrium during window of implantation (WOI) and decidualization

Abstract Study question How do prenatal androgenization and excessive body weight impact endometrial preparation for implantation? Summary answer Prenatal androgenization rather than excessive body weight causes abnormal uterine function and gene expression during artificially induced window of implantation and decidualization events in vivo. What is known already Polycystic ovary syndrome (PCOS) is a common endocrine disorder in women characterized by reproductive defects including irregular menstruation and pregnancy complications, but also by metabolic derangements such as obesity, insulin resistance and cardiovascular diseases. It is hypothesized that PCOS originates from prenatal androgenization (PNA), hence PNA mice models mimicking PCOS features have been generated. PNA has been shown to disrupt reproductive cycles, ovarian functioning and normal embryonic development; however, endometrial function in PNA mice is currently understudied. Therefore, this project aimed to study endometrial function during endometrial preparation for implantation in a PNA PCOS-like mouse model. Study design, size, duration Female PNA mice received a high-fat high-sugar (HFHS) diet to capture both reproductive and metabolic phenotypes of PCOS. Uteri were collected for histological and transcriptome analysis from 12-week-old ovariectomized PNA mice undergoing hormonal treatments to induce window of implantation (WOI) or decidualization states. Sample collection was done from minimum 6 animals per group from 4 different study groups depending on PNA status (PNA versus control) and diet (HFHS versus normal diet). Participants/materials, setting, methods PNA mice were generated by administrating 250 µg dihydrotestosterone daily to pregnant C57BL/6J dams during E16.5-E18.5. The female pups received a HFHS diet to induce obesity. Estrous cyclicity and anogenital distance were assessed to confirm PCOS phenotype. After ovariectomy, estrogen and progesterone injections were used to artificially induce WOI or decidualization states (intra-uterine injection of 25 µl oil was used for decidual trauma). The collected uteri were analyzed using RNA sequencing (RNAseq) and immunohistochemistry. Main results and the role of chance PNA mice showed increased anogenital distance and disrupted estrous cycles, confirming the PCOS-like phenotype. PNA, but not HFHS diet caused defective uterine luminal closure. PNA mice had altered gene expression during the WOI, including genes important for endometrial receptivity, genes associated with human PCOS, and genes related to reproduction failure. The HFHS diet induced a milder phenotype, i.e., enriched pathways related to mitochondrial functions, cell cycle and DNA damage response. Analysis of artificially decidualized uteri showed a severe morphological decidualization defect in PNA groups, as demonstrated by the reduced weights of the decidualized uterine horns, whereas HFHS diet did not have a significant additive effect. RNA sequencing data showed that PNA decidualized horns clustered together with non-decidualized control and PNA horns, irrespective of diet. Pathways enriched in PNA were associated with changes in extracellular matrix and ion homeostasis. HFHS diet with PNA further aggravated endometrial dysfunction, as shown by reduced prolactin expression, a classical marker for endometrial decidualization. Role of chance: while drastic effects of PNA on endometrial function are detectable, the sample size might be too small to detect more subtle morphological effects caused by the HFHS diet. Limitations, reasons for caution PNA mouse models display selective features of PCOS but cannot mirror the comprehensive PCOS phenotype due to the complexity and heterogeneity of the disorder. Nevertheless, this model provides insights into the impact of PNA and excessive body weight on endometrial physiology. Wider implications of the findings This in-dept investigation of PNA and overweight mice demonstrates the utility of the model for studying PCOS-related endometrial dysfunction and provides cues towards its pathophysiology. This report supports the hypothesis of prenatal androgen exposure as the major factor in the aetiology of PCOS-related subfertility, rather than metabolic status. Trial registration number not applicable