In recent decades, a global increase in the incidence of skin cancer, particularly squamous cell carcinoma (SCC), has been observed. To explore the pathogenesis and potential therapeutic approaches for this cancer type, in vivo studies employing various mouse models and ultraviolet (UV) light have been conducted. A comparative study on skin carcinogenesis across four hairless mouse models subjected to UV light exposure was initiated. The mouse strains utilized in this research were: SKH-hr1, SKH-hr2, SKH-hr2+ApoE, and immunodeficient Nude. Based on the various measured parameters, in contrast to the SKH-hr1, SKH-hr2+apoE and SKH-hr2 models were identified as the most appropriate.  The bark extract of Pinus maritima (PBE) was examined for SCC preventive action. It was evaluated in two different experimental animal tumor models induced by ultraviolet radiation (UVR) and combination of UVR with 7,12-dimethylbenz[a]anthracene. A significant decrease in the number of animals bearing tumors, increase in viability and delayed appearance of tumors were observed. Through immunochemical analysis, the expression of P-glycoprotein, multi-drug resistance-associated protein (MRP), and glucose (GLUT-1) transporters in SCC, SCC adjacent area, and normal skin tissues were examined. It was revealed that all assessed transporters were expressed across all skin tissues; however, expression levels were notably higher in tumor and tumor-adjacent areas compared to normal tissues. Male and female hairless SKH-2 mice were exposed for 10 months to cigarette smoke (CS) and/or UV light after administration or not of French maritime pine bark extract (PBE) to study the SCC induction and possible protection by PBE. The results showed that UV and CS were harmful and act synergistically inducing SCC, whereas PBE seems to protect skin against SCC. Type 1 and 2 diabetic, and nondiabetic male mice were exposed to UV radiation for eight months. Remarkably, Type 1 diabetic mice did not develop squamous cell carcinoma or pigmented nevi, contrary to normal and Type 2 diabetic skin. Type 1 diabetic mice showed protection against oxidative stress.

Ageing is a complex process characterised by the gradual deterioration of physiological functions and increased susceptibility to various age-related diseases. Mitochondrial dysfunction is an important factor contributing to ageing. Sirtuin 3 (Sirt3), a mitochondrial protein essential for energy homeostasis, plays a critical role in maintaining mitochondrial function, as loss of Sirt3 reduces energy and impairs cellular repair, which accelerates ageing. The aim of this study was to investigate the role of Sirt3 in male and female mouse embryonic fibroblasts (MEF) exposed to etoposide-induced DNA damage. We employed state-of-the-art genetic, molecular, and imaging technologies as well as metabolomic analyses to provide insights into the molecular mechanisms underlying these responses. We found that the loss of Sirt3 affected metabolic responses differently depending on sex: while male MEF showed minimal damage, possibly due to earlier stress adaptation, female MEF lacking Sirt3 were more vulnerable, suggesting that Sirt3 plays a critical role in enhancing their ability to withstand such challenges. By focusing on Sirt3 and sex-specific signalling pathways it modulates, this study has a potential for developing new strategies to combat diseases associated with DNA damage — a cornerstone of the ageing process.

Ferroptosis, a form of regulated cell death characterized by iron-dependent lipid peroxidation, is linked to neurodegenerative disorders and cold-induced cell death. SLC7A11 (xCT) plays a crucial role in protecting cells against ferroptosis by maintaining intracellular cysteine and glutathione levels. SLC7A11 requires the chaperone protein SLC3A2 for its localization on the plasma membrane to mediate cystine uptake. Arctic ground squirrels (AGS) are known to be protected from cold tissue temperatures and oxidative stress and to resist neuropathology following cerebral ischemia/reperfusion. This study investigated how ferroptosis is influenced by the hibernation season in AGS hippocampus. RNA-Seq, gene expression, and differential gene expression analysis were conducted on hippocampus tissue samples from male and female AGS collected during the summer active season, torpor, and interbout arousal (IBA). Hippocampus was dissected from partially thawed whole brain prior to RNA extraction.  Total RNA samples were used for cDNA library construction and sequencing by BGI Americas Corporation (Cambridge, MA) and analyzed using CLC Genomics Workbench (QIAGEN). Genes were mapped to the Ictidomys tridecemlineatus reference genome and transcript (HiC_Itri_2, GCF_016881025.1). Results show the highest number of differentially expressed genes (4,042) in torpor compared to summer active animals. Notably, SLC7A11 expression was elevated in torpor compared to summer active animals (fold change: 1.80, FDR-p value: 0.0034). Additionally, SLC3A2 was significantly upregulated in torpor compared to IBA (fold change: 1.24; FDR-p value: 0.030). SLC7A11 transports glutamate(out)/cystine(in). Cystine is rapidly converted into cysteine, a limiting reactant for glutathione synthesis, in the presence of NADPH. These findings suggest that SLC7A11 and SLC3A2 may protect AGS from ferroptosis during the hibernation season. This research provides insights into the molecular mechanisms underlying neuroprotection in hibernating AGS and may have implications for understanding and potentially treating neurodegenerative disorders.

Individual complexes of the mitochondrial oxidative phosphorylation system (OXPHOS) are not linked solely by their function; they also share dependencies at the maintenance/assembly level, where one complex depends on the presence of a different individual complex. Despite the relevance of this ‘interdependence’ behavior for mitochondrial diseases, its true nature remains elusive. To understand the mechanism that can explain this phenomenon, we examined the consequences of the aberration of different OXPHOS complexes in human cells. We demonstrate here that complete disruption of each of the OXPHOS complexes resulted in a perturbation in energy deficiency sensing pathways, including the integrated stress response (ISR) pathway. The secondary decrease of complex I (cI) level was triggered by both complex IV and complex V deficiency, and it was independent of ISR signaling. On the other hand, we identified the unifying mechanism behind cI downregulation in the downregulation of mitochondrial ribosomal proteins and, thus, mitochondrial translation. We conclude that the secondary cI defect is due to mitochondrial protein synthesis attenuation, while the responsible signaling pathways could differ based on the origin of the OXPHOS defect.

Changes in carbohydrate metabolism are a key feature of aging which also manifest in the epidermis. Furthermore, the synthesis and distribution of epidermal lipids changes with age. Both these parameters cannot be investigated with immunohistochemistry, as neither serves as useful epitope. We developed a multimodal analytical histocytometry approach combining modalities that localize lipids and enzymatic activities with immunofluorescent imaging of the skin to localize changes that are correlated with appearance of senescent cells. The activities of key metabolic enzymes were determined on tissue sections of aged and juvenile skin with a formazan-based assay. Lipids were localized and quantified using FTICR MALDI - mass spectrometric imaging. We correlated those modalities with immunofluorescent imaging and analyzed the intensities of the respective signals at single cell level, using Strataquest tissue cytometry. We analyzed skin from donors of young (< 30 y) versus advanced (> 67 y) ages and we investigated epidermal equivalent models containing labeled UV-damaged or senescent keratinocytes. Enzymatic activities displayed specific patterns across the stratifying epidermis, and had diverging trajectories in aging, with a marked decrease in suprabasal glucose-6-phosphate dehydrogenase (G6PD) activity. G6PD, the rate limiting enzyme of the pentose phosphate pathway was also identified as a rapid response pathway activated upon UV damage in the epidermis.  The lipid molecular imaging identified differentiation- and age-related changes of polar lipids in skin biopsies and epidermal equivalents, and pro-senescent stress dependent reactive aldehydophospholipid species in the basal epidermal layers.  While these methodologies are still in development, it is evident that correlative analytical imaging – with the aid of AI driven histocytometry – will continue to yield novel insights into skin and epidermal biology by localizing previously undetectable parameters within the epidermis in the context of aging.

Rett syndrome (RTT), a devastating neurodevelopmental disorder, is caused in 95% of the cases by mutations in the X-chromosome-localized MECP2 gene. RTT manifests as a range of multisystem disturbances including altered lipid profile, subclinical inflammation, and overall OxInflammatory status in which mitochondrial dysfunction acts as central player. To decipher the molecular mechanisms underlying the pathophysiological manifestations affecting patients, we investigated whether mitochondria may play a role in the aberrant immune and oxidative responses of RTT. Recent findings from our and other labs unraveled several abnormalities in RTT mitochondria including atypical mitochondrial structure, deregulated expression of genes encoding oxidative phosphorylation factors and mitochondrial organization factors, impaired mitochondrial quality control, depressed energetic profile, and augmented mt-ROS production. In other brain diseases, mitochondrial dysfunction is a vital event during the activation of NLPR3 inflammasome, a multi-protein complex involved in innate immune response, that represents a common denominator in the crosstalk between inflammation and oxidative stress. Interestingly, using primary fibroblasts and lympho-monocytes isolated from RTT patients, we found a constitutive hyperactivation of NLRP3:ASC inflammasome associated with increased levels of nuclear p65 and ASC proteins, and pro-IL-1β mRNA, without the ability to further respond to the LPS + ATP stimuli. Furthermore, increased circulating levels of ASC, interleukin (IL)-18, and 1β were found in RTT individuals, thus corroborating the aforementioned cellular findings. In order to evaluate NLRP3 involvement in the transition from pre-symptomatic to symptomatic phase of RTT, we detected higher serum levels of IL-1β and IL-18 in symptomatic Het mice compared to WT. Of note, increased gene expression of Il-1b, Nlrp3, and ASC was observed in Het brains at the pre-symptomatic stage, suggesting a likely role of NLRP3 impairment in the early stages of the disease. Preliminary data showed that treatment with resveratrol, known to improve mitochondrial function, ameliorated the RTT mouse phenotype by restoring levels of some NLRP3-related components. Furthermore, mitochondrial dysfunction can result in ferroptosis, a form of cell death characterized by iron-dependent lipid peroxidation and accumulation of reactive oxygen species.  After treatment with two ferroptosis inducers, erastin (GPX4 inhibitor) or RSL3 (inhibitor of the cystine/glutamate antiporter), we found changes in GPx and GR activity, alteration in GPX4 protein levels and increased formation of 4HNE protein adducts. Mitochondrial ROS production and lipid peroxidation levels were higher in RTT after ferroptosis induction, while co-treatment with ferrostatin-1, a well-known inhibitor of ferroptosis, significantly prevented these processes. Interestingly, co-treatment with mito-TEMPO, a mitochondria-targeted superoxide dismutase mimetic, mitigated mitochondrial oxidative burden and prevented ferroptosis cell death in RTT cells. Overall, our results demonstrate the decisive role of mitochondrial dysfunction in RTT OxInflammation. Thus, we can speculate that exposure of RTT cells to any condition affecting the already compromised mitochondrial function could not only hyperactivate the inflammatory status but also precipitate ferroptosis cell death. Targeting mitochondria in RTT could represent a strategic coadjuvant therapy to improve the quality of life of the affected patients.

In mammals, a multi-oscillator circadian system generates behavioural, metabolic and physiological ~24h rhythms, with tissue-specific physiological cues enabling local circadian phase adjustments. Emerging work has shown musculoskeletal tissue homeostasis and mechanical responses to be under circadian control. Nuclear factor erythroid 2-related factor 2 (NRF2), a master regulator of the antioxidant response, is a clock-controlled target in several peripheral tissues and also modifies circadian gene expression and rhythmicity. However, the role of NRF2 in mechanical loading-induced changes in musculoskeletal tissues has yet to be elucidated. Wild-type (WT) and Nrf2 KO mice of young (3-6m) or old age (18-20m) harbouring a PER2::luciferase clock reporter were subjected to acute mechanical joint loading of the right leg (peak load 9N, 40 cycles of 10sec) during light phase whilst the contralateral (left) leg served as a non-loaded control. Musculoskeletal tissues were collected for analysis 4 hrs later. Real-time bioluminescence imaging of clock gene reporter activity, protein and mRNA levels of target markers, NRF2/ARE transactivation and genome-wide RNAseq analyses were undertaken. We show that acute mechanical loading in WT mice led to a decrease in gene expression of key members in the negative and auxiliary feedback loops of the molecular clock, associated with the phase-resetting of PER2::luc protein oscillations in the skeletal muscle and a knee joint. This was accompanied by a significant increase in the markers of oxidative burden as well as gene expression and protein abundance levels of antioxidant enzymes. Moreover, acute mechanical loading induced a significant activation of the redox-sensitive energy sensor, AMP-activated kinase (AMPK), known to be involved in molecular clock resetting. We thus examined whether the above acute mechanical responses were dependent on NRF2 activity. Nrf2 KO mice showed an altered response to acute mechanical loading, characterized by blunted circadian resetting and antioxidant responses, and altered AMPK activation. Furthermore, dampened responses to acute mechanical signals were found in ageing WT mouse musculoskeletal tissues, whilst AMPK activator treatments in WT mice induced circadian resetting and antioxidant responses in an NRF2-dependent manner. In conclusion, these data demonstrate that AMPK/NRF2 axis is required for relaying acute mechanical signals to the musculoskeletal system by controlling redox-mediated phase resetting of musculoskeletal clocks and antioxidant protection, which have important implications in understanding biomechanical mechanisms involved in musculoskeletal tissue maintenance in health and with ageing.

The incidence of non-alcoholic fatty liver disease (NAFLD) is gradually increasing with the prevalence of obesity, which is the strongest risk factor for steatosis. Lipid droplet (LD) accumulation in hepatocytes is a hallmark of NAFLD. Seipin protein, which is LD related protein, resides in the endoplasmic reticulum membrane and a shortage of this protein leads to accumulation of abnormal LDs in adipose tissue. Although it has been shown that adipose-specific Seipin deficiency causes increased lipid accumulation in liver and muscle tissue following abnormal LD formation and loss of adipose tissue function, Seipin protein deficiency in liver tissue and its effect on lipid accumulation have not been investigated. Our study aimed to investigate the effect of Seipin deficiency on ER stress and lipophagy in cholesterol-accumulated mouse hepatocyte cells (AML12 cell line). In this direction cholesterol accumulation in mouse hepatocyte cells was established by administrating cholesterol-containing liposome and Seipin levels were reduced using siRNA transfection. Following liposome-cholesterol and siRNA administrations, lipophagy was determined by confocal microscopy, and mRNA levels of GRP78, GRP94, and ATF4 were examined by qRT-PCR. Our findings show that cholesterol-containing liposome administration in hepatocytes increases both Seipin protein and number of large LDs. However, Seipin silencing reduced the increase of cholesterol-mediated large LDs and GRP78 mRNA. Additionally, lysosome-LD colocalization increased only in cells treated with cholesterol-containing liposome, while the siRNA against Seipin did not lead to any significant difference. According to our results, we hypothesise that Seipin silencing in hepatocytes reduced cholesterol-mediated LD maturation as well as GRP78 levels, but not lipophagy.

The in vivo determination of oxidative stress always remains a great challenge. Our approach in Liège CHU consists of simultaneously measuring in blood samples four different kinds of biomarkers: enzymatic and non-enzymatic antioxidants, trace elements, markers of oxidative damage to lipids, and identification of sources leading to increased reactive oxygen species (ROS) production. All these biomarkers (n = 16) have been investigated in patients: 1) with Abdominal Aortic Aneurysm (AAA)¹ or operated for Thoracic Abdominal Dissection (TAD)², 2) suffering from Chronic Obstructive Pulmonary Disease (COPD)³ or FacioScapuloHumeral Myopathy (FSHM)⁴, 3) with COVID-19^5,6 and 4) with delirium⁷. When compared to our internal reference values, depletion in non-enzymatic antioxidants (vitamin C, β-carotene, vitamin C/vitamin E ratio, thiol proteins) and trace elements (zinc, selenium) was observed in the majority of these pathologies. By contrast, increased levels in glutathione peroxidase, copper/zinc ratio, lipid peroxides (ROOH), and myeloperoxidase are common in all these diseases.

Mitochondrial production of O₂^•– and H₂O₂ has been implicated in redox signaling and in the pathogenesis of numerous diseases including cancer, neurodegeneration, and cardiovascular diseases. To understand the exact role of those species, new chemical biology tools for selective and efficient induction of mitochondrial superoxide production are needed. Here, we report the development of a new viologen-based redox cycling agent, mito-diquat (Mito-DQ), capable of inducing targeted mitochondrial O₂^•– production at significantly higher rates as compared to previously reported mito-paraquat (Mito-PQ), a widely used chemical tool to study mitochondria-dependent redox signaling.