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Housing in metabolic cages can induce a pronounced stress response. Metabolic cage systems imply housing mice on metal wire mesh for the collection of urine and feces in addition to monitoring food and water intake. Moreover, mice are single-housed, and no nesting, bedding, or enrichment material is provided, which is often argued to have a not negligible impact on animal welfare due to cold stress. We therefore attempted to reduce stress during metabolic cage housing for mice by comparing an innovative metabolic cage (IMC) with a commercially available metabolic cage from Tecniplast GmbH (TMC) and a control cage. Substantial refinement measures were incorporated into the IMC cage design. In the frame of a multifactorial approach for severity assessment, parameters such as body weight, body composition, food intake, cage and body surface temperature (thermal imaging), mRNA expression of uncoupling protein 1 (Ucp1) in brown adipose tissue (BAT), fur score, and fecal corticosterone metabolites (CMs) were included. Female and male C57BL/6J mice were single-housed for 24 h in either conventional Macrolon cages (control), IMC, or TMC for two sessions. Body weight decreased less in the IMC (females—1st restraint: −6.94%; 2nd restraint: −6.89%; males—1st restraint: −8.08%; 2nd restraint: −5.82%) compared to the TMC (females—1st restraint: −13.2%; 2nd restraint: −15.0%; males—1st restraint: −13.1%; 2nd restraint: −14.9%) and the IMC possessed a higher cage temperature (females—1st restraint: 23.7 °C; 2nd restraint: 23.5 °C; males—1st restraint: 23.3 °C; 2nd restraint: 23.5 °C) compared with the TMC (females—1st restraint: 22.4 °C; 2nd restraint: 22.5 °C; males—1st restraint: 22.6 °C; 2nd restraint: 22.4 °C). The concentration of fecal corticosterone metabolites in the TMC (females—1st restraint: 1376 ng/g dry weight (DW); 2nd restraint: 2098 ng/g DW; males—1st restraint: 1030 ng/g DW; 2nd restraint: 1163 ng/g DW) was higher compared to control cage housing (females—1st restraint: 640 ng/g DW; 2nd restraint: 941 ng/g DW; males—1st restraint: 504 ng/g DW; 2nd restraint: 537 ng/g DW). Our results show the stress potential induced by metabolic cage restraint that is markedly influenced by the lower housing temperature. The IMC represents a first attempt to target cold stress reduction during metabolic cage application thereby producing more animal welfare friendlydata.
Sulfur mustard (SM) and its derivatives are potent genotoxic agents, which have been shown to trigger the activation of poly (ADP-ribose) polymerases (PARPs) and the depletion of their substrate, nicotinamide adenine dinucleotide (NAD+). NAD+ is an essential molecule involved in numerous cellular pathways, including genome integrity and DNA repair, and thus, NAD+ supplementation might be beneficial for mitigating mustard-induced (geno)toxicity. In this study, the role of NAD+ depletion and elevation in the genotoxic stress response to SM derivatives, i.e., the monofunctional agent 2-chloroethyl-ethyl sulfide (CEES) and the crosslinking agent mechlorethamine (HN2), was investigated with the use of NAD+ booster nicotinamide riboside (NR) and NAD+ synthesis inhibitor FK866. The effects were analyzed in immortalized human keratinocytes (HaCaT) or monocyte-like cell line THP-1. In HaCaT cells, NR supplementation, increased NAD+ levels, and elevated PAR response, however, did not affect ATP levels or DNA damage repair, nor did it attenuate long- and short-term cytotoxicities. On the other hand, the depletion of cellular NAD+ via FK866 sensitized HaCaT cells to genotoxic stress, particularly CEES exposure, whereas NR supplementation, by increasing cellular NAD+ levels, rescued the sensitizing FK866 effect. Intriguingly, in THP-1 cells, the NR-induced elevation of cellular NAD+ levels did attenuate toxicity of the mustard compounds, especially upon CEES exposure. Together, our results reveal that NAD+ is an important molecule in the pathomechanism of SM derivatives, exhibiting compound-specificity. Moreover, the cell line-dependent protective effects of NR are indicative of system-specificity of the application of this NAD+ booster.
The plasma membrane of mammalian cells links transmembrane receptors, various structural components, and membrane-binding proteins to subcellular processes, allowing inter- and intracellular communication. Therefore, membrane-binding proteins, together with structural components such as actin filaments, modulate the cell membrane in their flexibility, stiffness, and curvature. Investigating membrane components and curvature in cells remains challenging due to the diffraction limit in light microscopy. Preparation of 5–15-nm-thin plasma membrane sheets and subsequent inspection by metal replica transmission electron microscopy (TEM) reveal detailed information about the cellular membrane topology, including the structure and curvature. However, electron microscopy cannot identify proteins associated with specific plasma membrane domains. Here, we describe a novel adaptation of correlative super-resolution light microscopy and platinum replica TEM (CLEM-PREM), allowing the analysis of plasma membrane sheets with respect to their structural details, curvature, and associated protein composition. We suggest a number of shortcuts and troubleshooting solutions to contemporary PREM protocols. Thus, implementation of super-resolution stimulated emission depletion (STED) microscopy offers significant reduction in sample preparation time and reduced technical challenges for imaging and analysis. Additionally, highly technical challenges associated with replica preparation and transfer on a TEM grid can be overcome by scanning electron microscopy (SEM) imaging. The combination of STED microscopy and platinum replica SEM or TEM provides the highest spatial resolution of plasma membrane proteins and their underlying membrane and is, therefore, a suitable method to study cellular events like endocytosis, membrane trafficking, or membrane tension adaptations.
Caenorhabditis elegans (C. elegans) is gaining recognition and importance as an organismic model for toxicity testing in line with the 3Rs principle (replace, reduce, refine). In this study, we explored the use of C. elegans to examine the toxicities of alkylating sulphur mustard analogues, specifically the monofunctional agent 2-chloroethyl-ethyl sulphide (CEES) and the bifunctional, crosslinking agent mechlorethamine (HN2). We exposed wild-type worms at different life cycle stages (from larvae L1 to adulthood day 10) to CEES or HN2 and scored their viability 24 h later. The susceptibility of C. elegans to CEES and HN2 paralleled that of human cells, with HN2 exhibiting higher toxicity than CEES, reflected in LC50 values in the high µM to low mM range. Importantly, the effects were dependent on the worms’ developmental stage as well as organismic age: the highest susceptibility was observed in L1, whereas the lowest was observed in L4 worms. In adult worms, susceptibility to alkylating agents increased with advanced age, especially to HN2. To examine reproductive effects, L4 worms were exposed to CEES and HN2, and both the offspring and the percentage of unhatched eggs were assessed. Moreover, germline apoptosis was assessed by using ced-1p::GFP (MD701) worms. In contrast to concentrations that elicited low toxicities to L4 worms, CEES and HN2 were highly toxic to germline cells, manifesting as increased germline apoptosis as well as reduced offspring number and percentage of eggs hatched. Again, HN2 exhibited stronger effects than CEES. Compound specificity was also evident in toxicities to dopaminergic neurons–HN2 exposure affected expression of dopamine transporter DAT-1 (strain BY200) at lower concentrations than CEES, suggesting a higher neurotoxic effect. Mechanistically, nicotinamide adenine dinucleotide (NAD+) has been linked to mustard agent toxicities. Therefore, the NAD+-dependent system was investigated in the response to CEES and HN2 treatment. Overall NAD+ levels in worm extracts were revealed to be largely resistant to mustard exposure except for high concentrations, which lowered the NAD+ levels in L4 worms 24 h post-treatment. Interestingly, however, mutant worms lacking components of NAD+-dependent pathways involved in genome maintenance, namely pme-2, parg-2, and sirt-2.1 showed a higher and compound-specific susceptibility, indicating an active role of NAD+ in genotoxic stress response. In conclusion, the present results demonstrate that C. elegans represents an attractive model to study the toxicology of alkylating agents, which supports its use in mechanistic as well as intervention studies with major strength in the possibility to analyze toxicities at different life cycle stages.
BACKGROUND: Patients with diabetes exhibit an increased prevalence for emotional disorders compared with healthy humans, partially due to a shared pathogenesis including hormone resistance and inflammation, which is also linked to intestinal dysbiosis. The preventive intake of probiotic lactobacilli has been shown to improve dysbiosis along with mood and metabolism. Yet, a potential role of Lactobacillus rhamnosus (Lacticaseibacillus rhamnosus 0030) (LR) in improving emotional behavior in established obesity and the underlying mechanisms are unknown.
METHODS: Female and male C57BL/6N mice were fed a low-fat diet (10% kcal from fat) or high-fat diet (HFD) (45% kcal from fat) for 6 weeks, followed by daily oral gavage of vehicle or 1 3 10 8 colony-forming units of LR, and assessment of anxiety- and depressive-like behavior. Cecal microbiota composition was analyzed using 16S ribosomal RNA sequencing, plasma and cerebrospinal fluid were collected for metabolomic analysis, and gene expression of different brain areas was assessed using reverse transcriptase quantitative polymerase chain reaction.
RESULTS: We observed that 12 weeks of HFD feeding induced hyperinsulinemia, which was attenuated by LR application only in female mice. On the contrary, HFD-fed male mice exhibited increased anxiety- and depressive-like behavior, where the latter was specifically attenuated by LR application, which was independent of metabolic changes. Furthermore, LR application restored the HFD-induced decrease of tyrosine hydroxylase, along with normalizing cholecystokinin gene expression in dopaminergic brain regions; both tyrosine hydroxylase and cholecystokinin are involved in signaling pathways impacting emotional disorders.
CONCLUSIONS: Our data show that LR attenuates depressive-like behavior after established obesity, with changes in the dopaminergic system in male mice, and mitigates hyperinsulinemia in obese female mice.
Aging is a complex process characterized by several factors, including loss of genetic and epigenetic information, accumulation of chronic oxidative stress, protein damage and aggregates and it is becoming an emergent drug target. Therefore, it is the utmost importance to study aging and agerelated diseases, to provide treatments to develop a healthy aging process. Skeletal muscle is one of the earliest tissues affected by age-related changes with progressive loss of muscle mass and function from 30 years old, effect known as sarcopenia. Several studies have shown the accumulation of protein aggregates in different animal models, as well as in humans, suggesting impaired proteostasis, a hallmark of aging, especially regarding degradation systems. Thus, different publications have explored the role of the main proteolytic systems in skeletal muscle from rodents and humans, like ubiquitin proteasomal system (UPS) and autophagy lysosomal system (ALS), however with contradictory results. Yet, most of the published studies are performed in muscles that comprise more than one fiber type, that means, muscles composed by slow and fast fibers. These fiber types, exhibit different metabolism and contraction speed; the slow fibers or type I display an oxidative metabolism, while fast fibers function towards a glycolytic metabolism ranging from fast oxidative to fast glycolytic fibers. To this extent, the aim of this thesis sought to understand on how aging impacts both fiber types not only regarding proteostasis but also at a metabolome and transcriptome network levels. Therefore, the first part of this thesis, presents the differences between slow oxidative (from Soleus muscle) and fast glycolytic fibers (Extensor digitorum longus, EDL) in terms of degradation systems and how they cope with oxidative stress during aging, while the second part explores the differences between young and old EDL muscle transcriptome and metabolome, unraveling molecular features. More specifically, the results from the present work show that slow oxidative muscle performs better at maintaining the function of UPS and ALS during aging than EDL muscle, which is clearly affected, accounting for the decline in the catalytic activity rates and accumulation of autophagy-related proteins. Strinkingly, transcriptome and metabolome analyses reveal that fast glycolytic muscle evidences significant downregulation of mitochondrial related processes and damaged mitochondria morphology during aging, despite of having a lower oxidative metabolism compared to oxidative fibers. Moreover, predictive analyses reveal a negative association between aged EDL gene signature and lifespan extending interventions such as caloric restriction (CR). Although, CR intervention does not alter the levels of mitochondrial markers in aged EDL muscle, it can reverse the higher mRNA levels of muscle damage markers. Together, the results from this thesis give new insights about how different metabolic muscle fibers cope with age-related changes and why fast glycolytic fibers are more susceptible to aging than slow oxidative fibers.