Caloric restriction reduces trabecular bone loss during aging and improves bone marrow adipocyte endocrine function in male mice.

Introduction: Caloric restriction (CR) is a nutritional intervention that increases life expectancy while lowering the risk for cardio-metabolic disease. Its effects on bone health, however, remain controversial. For instance, CR has been linked to increased accumulation of bone marrow adipose tissue (BMAT) in long bones, a process thought to elicit detrimental effects on bone. Qualitative differences have been reported in BMAT in relation to its specific anatomical localization, subdividing it into physiological and potentially pathological BMAT. We here examine the local impact of CR on bone composition, microstructure and its endocrine profile in the context of aging. Methods: Young and aged male C57Bl6J mice were subjected to CR for 8 weeks and were compared to age-matched littermates with free food access. We assessed bone microstructure and BMAT by micro-CT, bone fatty acid and transcriptomic profiles, and bone healing. Results: CR increased tibial BMAT accumulation and adipogenic gene expression. CR also resulted in elevated fatty acid desaturation in the proximal and mid-shaft regions of the tibia, thus more closely resembling the biochemical lipid profile of the distally located, physiological BMAT. In aged mice, CR attenuated trabecular bone loss, suggesting that CR may revert some aspects of age-related bone dysfunction. Cortical bone, however, was decreased in young mice on CR and remained reduced in aged mice, irrespective of dietary intervention. No negative effects of CR on bone regeneration were evident in either young or aged mice. Discussion: Our findings indicate that the timing of CR is critical and may exert detrimental effects on bone biology if administered during a phase of active skeletal growth. Conversely, CR exerts positive effects on trabecular bone structure in the context of aging, which occurs despite substantial accumulation of BMAT. These data suggest that the endocrine profile of BMAT, rather than its fatty acid composition, contributes to healthy bone maintenance in aged mice.

Short-term consumption of highly processed diets varying in macronutrient content impair the sense of smell and brain metabolism in mice.

OBJECTIVE: Food processing greatly contributed to increased food safety, diversity, and accessibility. However, the prevalence of highly palatable and highly processed food in our modern diet has exacerbated obesity rates and contributed to a global health crisis. While accumulating evidence suggests that chronic consumption of such foods is detrimental to sensory and neural physiology, it is unclear whether its short-term intake has adverse effects. Here, we assessed how short-term consumption (<2 months) of three diets varying in composition and macronutrient content influence olfaction and brain metabolism in mice. METHODS: The diets tested included a grain-based standard chow diet (CHOW; 54% carbohydrate, 32% protein, 14% fat; #8604 Teklad Rodent diet , Envigo Inc.), a highly processed control diet (hpCTR; 70% carbohydrate, 20% protein, 10% fat; #D12450B, Research Diets Inc.), and a highly processed high-fat diet (hpHFD; 20% carbohydrate, 20% protein, 60% fat; #D12492, Research Diets Inc.). We performed behavioral and metabolic phenotyping, electro-olfactogram (EOG) recordings, brain glucose metabolism imaging, and mitochondrial respirometry in different brain regions. We also performed RNA-sequencing (RNA-seq) in the nose and across several brain regions, and conducted differential expression analysis, gene ontology, and network analysis. RESULTS: We show that short-term consumption of the two highly processed diets, but not the grain-based diet, regardless of macronutrient content, adversely affects odor-guided behaviors, physiological responses to odorants, transcriptional profiles in the olfactory mucosa and brain regions, and brain glucose metabolism and mitochondrial respiration. CONCLUSIONS: Even short periods of highly processed food consumption are sufficient to cause early olfactory and brain abnormalities, which has the potential to alter food choices and influence the risk of developing metabolic disease.

Genome-Wide RNA Tomography in the Mouse Whole Olfactory Mucosa.

Spatial transcriptomics allows for the genome-wide profiling of topographic gene expression patterns within a tissue of interest. Here, we describe our methodology to generate high-quality RNA-seq libraries from cryosections from fresh frozen mouse whole olfactory mucosae. This methodology can be extended to virtually any vertebrate organ or tissue sample.

A 3D transcriptomics atlas of the mouse nose sheds light on the anatomical logic of smell.

The sense of smell helps us navigate the environment, but its molecular architecture and underlying logic remain understudied. The spatial location of odorant receptor genes (Olfrs) in the nose is thought to be independent of the structural diversity of the odorants they detect. Using spatial transcriptomics, we create a genome-wide 3D atlas of the mouse olfactory mucosa (OM). Topographic maps of genes differentially expressed in space reveal that both Olfrs and non-Olfrs are distributed in a continuous and overlapping fashion over at least five broad zones in the OM. The spatial locations of Olfrs correlate with the mucus solubility of the odorants they recognize, providing direct evidence for the chromatographic theory of olfaction. This resource resolves the molecular architecture of the mouse OM and will inform future studies on mechanisms underlying Olfr gene choice, axonal pathfinding, patterning of the nervous system, and basic logic for the peripheral representation of smell.

Congenital iRHOM2 deficiency causes ADAM17 dysfunction and environmentally directed immunodysregulatory disease.

We report a pleiotropic disease due to loss-of-function mutations in RHBDF2, the gene encoding iRHOM2, in two kindreds with recurrent infections in different organs. One patient had recurrent pneumonia but no colon involvement, another had recurrent infectious hemorrhagic colitis but no lung involvement and the other two experienced recurrent respiratory infections. Loss of iRHOM2, a rhomboid superfamily member that regulates the ADAM17 metalloproteinase, caused defective ADAM17-dependent cleavage and release of cytokines, including tumor-necrosis factor and amphiregulin. To understand the diverse clinical phenotypes, we challenged Rhbdf2-/- mice with Pseudomonas aeruginosa by nasal gavage and observed more severe pneumonia, whereas infection with Citrobacter rodentium caused worse inflammatory colitis than in wild-type mice. The fecal microbiota in the colitis patient had characteristic oral species that can predispose to colitis. Thus, a human immunodeficiency arising from iRHOM2 deficiency causes divergent disease phenotypes that can involve the local microbial environment.

Deconstructing the mouse olfactory percept through an ethological atlas.

Odor perception in non-humans is poorly understood. Here, we generated the most comprehensive mouse olfactory ethological atlas to date, consisting of behavioral responses to a diverse panel of 73 odorants, including 12 at multiple concentrations. These data revealed that mouse behavior is incredibly diverse and changes in response to odorant identity and concentration. Using only behavioral responses observed in other mice, we could predict which of two odorants was presented to a held-out mouse 82% of the time. Considering all 73 possible odorants, we could uniquely identify the target odorant from behavior on the first try 20% of the time and 46% within five attempts. Although mouse behavior is difficult to predict from human perception, they share three fundamental properties: first, odor valence parameters explained the highest variance of olfactory perception. Second, physicochemical properties of odorants can be used to predict the olfactory percept. Third, odorant concentration quantitatively and qualitatively impacts olfactory perception. These results increase our understanding of mouse olfactory behavior and how it compares to human odor perception and provide a template for future comparative studies of olfactory percepts among species.

Differential regulation of the immune system in a brain-liver-fats organ network during short-term fasting.

OBJECTIVE: Fasting regimens can promote health, mitigate chronic immunological disorders, and improve age-related pathophysiological parameters in animals and humans. Several ongoing clinical trials are using fasting as a potential therapy for various conditions. Fasting alters metabolism by acting as a reset for energy homeostasis, but the molecular mechanisms underlying the beneficial effects of short-term fasting (STF) are not well understood, particularly at the systems or multiorgan level. METHODS: We performed RNA-sequencing in nine organs from mice fed ad libitum (0 h) or subjected to fasting five times (2-22 h). We applied a combination of multivariate analysis, differential expression analysis, gene ontology, and network analysis for an in-depth understanding of the multiorgan transcriptome. We used literature mining solutions, LitLab™ and Gene Retriever™, to identify the biological and biochemical terms significantly associated with our experimental gene set, which provided additional support and meaning to the experimentally derived gene and inferred protein data. RESULTS: We cataloged the transcriptional dynamics within and between organs during STF and discovered differential temporal effects of STF among organs. Using gene ontology enrichment analysis, we identified an organ network sharing 37 common biological pathways perturbed by STF. This network incorporates the brain, liver, interscapular brown adipose tissue, and posterior-subcutaneous white adipose tissue; hence, we named it the brain-liver-fats organ network. Using Reactome pathways analysis, we identified the immune system, dominated by T cell regulation processes, as a central and prominent target of systemic modulations during STF in this organ network. The changes we identified in specific immune components point to the priming of adaptive immunity and parallel the fine-tuning of innate immune signaling. CONCLUSIONS: Our study provides a comprehensive multiorgan transcriptomic profiling of mice subjected to multiple periods of STF and provides new insights into the molecular modulators involved in the systemic immunotranscriptomic changes that occur during short-term energy loss.

A transcriptomic atlas of mammalian olfactory mucosae reveals an evolutionary influence on food odor detection in humans.

The mammalian olfactory system displays species-specific adaptations to different ecological niches. To investigate the evolutionary dynamics of olfactory sensory neuron (OSN) subtypes across mammalian evolution, we applied RNA sequencing of whole olfactory mucosa samples from mouse, rat, dog, marmoset, macaque, and human. We find that OSN subtypes, representative of all known mouse chemosensory receptor gene families, are present in all analyzed species. Further, we show that OSN subtypes expressing canonical olfactory receptors are distributed across a large dynamic range and that homologous subtypes can be either highly abundant across all species or species/order specific. Highly abundant mouse and human OSN subtypes detect odorants with similar sensory profiles and sense ecologically relevant odorants, such as mouse semiochemicals or human key food odorants. Together, our results allow for a better understanding of the evolution of mammalian olfaction in mammals and provide insights into the possible functions of highly abundant OSN subtypes.

Interspecific variation of olfactory preferences in flies, mice, and humans.

Aiming to unravel interspecific differences in olfactory preferences, we performed comparative studies of odor valence in flies, mice, and humans. Our analysis suggests a model where flies and mice share similar olfactory preferences, but neither species share odor preferences with humans. This model contrasts with a previous study by Mandairon et al., which suggested that the olfactory preferences of mice and humans are similar. A probabilistic examination revealed that underpowered studies can result in spurious significant correlations, which can account for the differences between both studies. Future analyses aimed at dissecting the olfactory preferences across species need to test large numbers of odorants to stress-test the model proposed here and identify robust associations.

Profiling olfactory stem cells from living patients identifies miRNAs relevant for autism pathophysiology.

BACKGROUND: Autism spectrum disorders (ASD) are a group of neurodevelopmental disorders caused by the interaction between genetic vulnerability and environmental factors. MicroRNAs (miRNAs) are key posttranscriptional regulators involved in multiple aspects of brain development and function. Previous studies have investigated miRNAs expression in ASD using non-neural cells like lymphoblastoid cell lines (LCL) or postmortem tissues. However, the relevance of LCLs is questionable in the context of a neurodevelopmental disorder, and the impact of the cause of death and/or post-death handling of tissue likely contributes to the variations observed between studies on brain samples. METHODS: miRNA profiling using TLDA high-throughput real-time qPCR was performed on miRNAs extracted from olfactory mucosal stem cells (OMSCs) biopsied from eight patients and six controls. This tissue is considered as a closer tissue to neural stem cells that could be sampled in living patients and was never investigated for such a purpose before. Real-time PCR was used to validate a set of differentially expressed miRNAs, and bioinformatics analysis determined common pathways and gene targets. Luciferase assays and real-time PCR analysis were used to evaluate the effect of miRNAs misregulation on the expression and translation of several autism-related transcripts. Viral vector-mediated expression was used to evaluate the impact of miRNAs deregulation on neuronal or glial cells functions. RESULTS: We identified a signature of four miRNAs (miR-146a, miR-221, miR-654-5p, and miR-656) commonly deregulated in ASD. This signature is conserved in primary skin fibroblasts and may allow discriminating between ASD and intellectual disability samples. Putative target genes of the differentially expressed miRNAs were enriched for pathways previously associated to ASD, and altered levels of neuronal transcripts targeted by miR-146a, miR-221, and miR-656 were observed in patients' cells. In the mouse brain, miR-146a, and miR-221 display strong neuronal expression in regions important for high cognitive functions, and we demonstrated that reproducing abnormal miR-146a expression in mouse primary cell cultures leads to impaired neuronal dendritic arborization and increased astrocyte glutamate uptake capacities. CONCLUSIONS: While independent replication experiments are needed to clarify whether these four miRNAS could serve as early biomarkers of ASD, these findings may have important diagnostic implications. They also provide mechanistic connection between miRNA dysregulation and ASD pathophysiology and may open up new opportunities for therapeutic.

Erosion of X Chromosome Inactivation in Human Pluripotent Cells Initiates with XACT Coating and Depends on a Specific Heterochromatin Landscape.

Human pluripotent stem cells (hPSCs) display extensive epigenetic instability, particularly on the X chromosome. In this study, we show that, in hPSCs, the inactive X chromosome has a specific heterochromatin landscape that predisposes it to erosion of X chromosome inactivation (XCI), a process that occurs spontaneously in hPSCs. Heterochromatin remodeling and gene reactivation occur in a non-random fashion and are confined to specific H3K27me3-enriched domains, leaving H3K9me3-marked regions unaffected. Using single-cell monitoring of XCI erosion, we show that this instability only occurs in pluripotent cells. We also provide evidence that loss of XIST expression is not the primary cause of XCI instability and that gene reactivation from the inactive X (Xi) precedes loss of XIST coating. Notably, expression and coating by the long non-coding RNA XACT are early events in XCI erosion and, therefore, may play a role in mediating this process.

A prominent and conserved role for YY1 in Xist transcriptional activation.

Accumulation of the noncoding RNA Xist on one X chromosome in female cells is a hallmark of X-chromosome inactivation (XCI) in eutherians. Here we uncover an essential function for the ubiquitous autosomal transcription factor Yin-Yang 1 (YY1) in the transcriptional activation of Xist in both human and mouse. We show that loss of YY1 prevents Xist upregulation during the initiation and maintenance of X-inactivation, and that YY1 binds directly the Xist 5' region to trigger the activity of the Xist promoter. Binding of YY1 to the Xist 5' region before XCI competes with the Xist repressor REX1, whereas DNA methylation controls mono-allelic fixation of YY1 to Xist at the onset of XCI. YY1 is thus the first autosomal activating factor involved in a fundamental and conserved pathway of Xist regulation that ensures the asymmetric transcriptional upregulation of the master regulator of XCI.

Linking X chromosome inactivation to pluripotency: Necessity or fate?

Silencing one X chromosome is essential for the development of female mammals, but the regulation of this process appears to vary between species. In the mouse, which has thus far been the leading model system in the field, X chromosome inactivation (XCI) is tightly coupled to pluripotency and the underlying mechanisms have just begun to be deciphered. However, mechanistic aspects of XCI regulation in other species have yet to be thoroughly investigated. Here we review current knowledge of the developmental regulation of XCI in mice and humans and discuss the extent to which the intimate link between XCI and pluripotency extends beyond rodents.

Partial reversal of the methylation pattern of the X-linked gene HUMARA during hematopoietic differentiation of human embryonic stem cells.

Human embryonic stem cells (hESCs) can be induced to differentiate towards hematopoiesis with high efficiency. In this work, we analyzed the methylation status of the X-linked HUMARA (human androgen receptor) gene in hematopoietic cells derived from hESC line H9 before and after induction of hematopoietic differentiation. All passages of H9 and H9-derived hematopoietic cells displayed homogenous methylation pattern with disappearance of the same allele upon HpaII digestion. This pattern persisted in the great majority of different hematopoietic progenitors derived from H9, except in 11 of 86 individually plucked colonies in which an equal digestion of the HUMARA alleles has been found, suggesting that a methylation change occurring at this locus during differentiation. Interestingly, quantification of X inactive-specific transcript (XIST) RNA in undifferentiated H9 cell line and day 14 embryoid bodies (EB) by RT-PCR did not show any evidence of XIST expression either before or after differentiation. Thus, during self-renewal conditions and after induction of commitment towards the formation of EB, the methylation pattern of the HUMARA locus appears locked with the same unmethylated allele. However, hematopoietic differentiation seems to be permissive to the reversal of methylation status of HUMARA in some terminally differentiated progenitors. These data suggest that monitoring methylation of HUMARA gene during induced differentiation could be of use for studying hESC-derived hematopoiesis.