Osteochondroprogenitor cells and neutrophils expressing p21 and senescence markers modulate fracture repair.

Cells expressing features of senescence, including upregulation of p21 and p16, appear transiently following tissue injury, yet the properties of these cells or how they contrast with age-induced senescent cells remains unclear. Here, we used skeletal injury as a model and identified the rapid appearance following fracture of p21+ cells expressing senescence markers, mainly as osteochondroprogenitors (OCHs) and neutrophils. Targeted genetic clearance of p21+ cells suppressed senescence-associated signatures within the fracture callus and accelerated fracture healing. By contrast, p21+ cell clearance did not alter bone loss due to aging; conversely, p16+ cell clearance, known to alleviate skeletal aging, did not affect fracture healing. Following fracture, p21+ neutrophils were enriched in signaling pathways known to induce paracrine stromal senescence, while p21+ OCHs were highly enriched in senescence-associated secretory phenotype factors known to impair bone formation. Further analysis revealed an injury-specific stem cell-like OCH subset that was p21+ and highly inflammatory, with a similar inflammatory mesenchymal population (fibro-adipogenic progenitors) evident following muscle injury. Thus, intercommunicating senescent-like neutrophils and mesenchymal progenitor cells were key regulators of tissue repair in bone and potentially across tissues. Moreover, our findings established contextual roles of p21+ vs p16+ senescent/senescent-like cells that may be leveraged for therapeutic opportunities.

Paired plasma lipidomics and proteomics analysis in the conversion from mild cognitive impairment to Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is a neurodegenerative condition for which there is currently no available medication that can stop its progression. Previous studies suggest that mild cognitive impairment (MCI) is a phase that precedes the disease. Therefore, a better understanding of the molecular mechanisms behind MCI conversion to AD is needed. METHOD: Here, we propose a machine learning-based approach to detect the key metabolites and proteins involved in MCI progression to AD using data from the European Medical Information Framework for Alzheimer's Disease Multimodal Biomarker Discovery Study. Proteins and metabolites were evaluated separately in multiclass models (controls, MCI and AD) and together in MCI conversion models (MCI stable vs converter). Only features selected as relevant by 3/4 algorithms proposed were kept for downstream analysis. RESULTS: Multiclass models of metabolites highlighted nine features further validated in an independent cohort (0.726 mean balanced accuracy). Among these features, one metabolite, oleamide, was selected by all the algorithms. Further in-vitro experiments in rodents showed that disease-associated microglia excreted oleamide in vesicles. Multiclass models of proteins stood out with nine features, validated in an independent cohort (0.720 mean balanced accuracy). However, none of the proteins was selected by all the algorithms. Besides, to distinguish between MCI stable and converters, 14 key features were selected (0.872 AUC), including tTau, alpha-synuclein (SNCA), junctophilin-3 (JPH3), properdin (CFP) and peptidase inhibitor 15 (PI15) among others. CONCLUSIONS: This omics integration approach highlighted a set of molecules associated with MCI conversion important in neuronal and glia inflammation pathways.

The clinical importance of suspected non-Alzheimer disease pathophysiology.

The development of biomarkers for Alzheimer disease (AD) has led to the origin of suspected non-AD pathophysiology (SNAP) - a heterogeneous biomarker-based concept that describes individuals with normal amyloid and abnormal tau and/or neurodegeneration biomarker status. In this Review, we describe the origins of the SNAP construct, along with its prevalence, diagnostic and prognostic implications, and underlying neuropathology. As we discuss, SNAP can be operationalized using different biomarker modalities, which could affect prevalence estimates and reported characteristics of SNAP in ways that are not yet fully understood. Moreover, the underlying aetiologies that lead to a SNAP biomarker profile, and whether SNAP is the same in people with and without cognitive impairment, remains unclear. Improved insight into the clinical characteristics and pathophysiology of SNAP is of major importance for research and clinical practice, as well as for trial design to optimize care and treatment of individuals with SNAP.

Local patterns of genetic sharing challenge the boundaries between neuropsychiatric and insulin resistance-related conditions.

The co-occurrence of insulin resistance (IR)-related metabolic conditions with neuropsychiatric disorders is a complex public health challenge. Evidence of the genetic links between these phenotypes is emerging, but little is currently known about the genomic regions and biological functions that are involved. To address this, we performed Local Analysis of [co]Variant Association (LAVA) using large-scale (N=9,725-933,970) genome-wide association studies (GWASs) results for three IR-related conditions (type 2 diabetes mellitus, obesity, and metabolic syndrome) and nine neuropsychiatric disorders. Subsequently, positional and expression quantitative trait locus (eQTL)-based gene mapping and downstream functional genomic analyses were performed on the significant loci. Patterns of negative and positive local genetic correlations (|rg|=0.21-1, pFDR<0.05) were identified at 109 unique genomic regions across all phenotype pairs. Local correlations emerged even in the absence of global genetic correlations between IR-related conditions and Alzheimer's disease, bipolar disorder, and Tourette's syndrome. Genes mapped to the correlated regions showed enrichment in biological pathways integral to immune-inflammatory function, vesicle trafficking, insulin signalling, oxygen transport, and lipid metabolism. Colocalisation analyses further prioritised 10 genetically correlated regions for likely harbouring shared causal variants, displaying high deleterious or regulatory potential. These variants were found within or in close proximity to genes, such as SLC39A8 and HLA-DRB1, that can be targeted by supplements and already known drugs, including omega-3/6 fatty acids, immunomodulatory, antihypertensive, and cholesterol-lowering drugs. Overall, our findings underscore the complex genetic landscape of IR-neuropsychiatric multimorbidity, advocating for an integrated disease model and offering novel insights for research and treatment strategies in this domain.

Osteochondroprogenitor cells and neutrophils expressing p21 and senescence markers modulate fracture repair.

Cells expressing features of senescence, including upregulation of p21 and p16, appear transiently following tissue injury, yet the properties of these cells or how they contrast with age-induced senescent cells remains unclear. Here, we used skeletal injury as a model and identified the rapid appearance following fracture of p21+ cells expressing senescence markers, mainly as osteochondroprogenitors (OCHs) and neutrophils. Targeted genetic clearance of p21+ cells suppressed senescence-associated signatures within the fracture callus and accelerated fracture healing. By contrast, p21+ cell clearance did not alter bone loss due to aging; conversely, p16+ cell clearance, known to alleviate skeletal aging, did not affect fracture healing. Following fracture, p21+ neutrophils were enriched in signaling pathways known to induce paracrine stromal senescence, while p21+ OCHs were highly enriched in senescence-associated secretory phenotype factors known to impair bone formation. Further analysis revealed an injury-specific stem cell-like OCH subset that was p21+ and highly inflammatory, with a similar inflammatory mesenchymal population (fibro-adipogenic progenitors) evident following muscle injury. Thus, intercommunicating senescent-like neutrophils and mesenchymal progenitor cells are key regulators of tissue repair in bone and potentially across tissues. Moreover, our findings establish contextual roles of p21+ vs p16+ senescent/senescent-like cells that may be leveraged for therapeutic opportunities.

The association of glucose metabolism measures and diabetes status with Alzheimer's disease biomarkers of amyloid and tau: A systematic review and meta-analysis.

Conflicting evidence exists on the relationship between diabetes mellitus (DM) and Alzheimer's disease (AD) biomarkers. Therefore, we conducted a random-effects meta-analysis to evaluate the correlation of glucose metabolism measures (glycated hemoglobin, fasting blood glucose, insulin resistance indices) and DM status with AD biomarkers of amyloid-ß and tau measured by positron emission tomography or cerebrospinal fluid. We selected 37 studies from PubMed and Embase, including 11,694 individuals. More impaired glucose metabolism and DM status were associated with higher tau biomarkers (r=0.11[0.03-0.18], p=0.008; I2=68%), but were not associated with amyloid-ß biomarkers (r=-0.06[-0.13-0.01], p=0.08; I2=81%). Meta-regression revealed that glucose metabolism and DM were specifically associated with tau biomarkers in population settings (p=0.001). Furthermore, more impaired glucose metabolism and DM status were associated with lower amyloid-ß biomarkers in memory clinic settings (p=0.004), and in studies with a higher prevalence of dementia (p<0.001) or lower cognitive scores (p=0.04). These findings indicate that DM is associated with biomarkers of tau but not with amyloid-ß. This knowledge is valuable for improving dementia and DM diagnostics and treatment.

Contributions of Vascular Burden and Amyloid Abnormality to Cognitive Decline in Memory Clinic Patients.

Background: Alzheimer's disease pathology and vascular burden are highly prevalent and often co-occur in elderly. It remains unclear how both relate to cognitive decline. Objective: To investigate whether amyloid abnormality and vascular burden synergistically contribute to cognitive decline in a memory clinic population. Methods: We included 227 patients from Maastricht and Aachen memory clinics. Amyloid abnormality (A+) was defined by CSF Aß42 using data-driven cut-offs. Vascular burden (V+) was defined as having moderate to severe white matter hyperintensities, or any microbleeds, macrohemorrhage or infarcts on MRI. Longitudinal change in global cognition, memory, processing speed, executive functioning, and verbal fluency was analysed across the A-V-, A-V+, A+V-, A+V+ groups by linear mixed models. Additionally, individual MRI measures, vascular risk and vascular disease were used as V definitions. Results: At baseline, the A+V+ group scored worse on global cognition and verbal fluency compared to all other groups, and showed worse memory compared to A-V+ and A-V- groups. Over time (mean 2.7+ - 1.5 years), A+V+ and A+V- groups showed faster global cognition decline than A-V+ and A-V- groups. Only the A+V- group showed decline on memory and verbal fluency. The A-V+ group did not differ from the A-V- group. Individual MRI vascular measures only indicated an independent association of microbleeds with executive functioning decline. Findings were similar using other V definitions. Conclusions: Our study demonstrates that amyloid abnormality predicts cognitive decline independent from vascular burden in a memory clinic population. Vascular burden shows a minor contribution to cognitive decline in these patients. This has important prognostic implications.

Modest Effects of Osteoclast-Specific ERα Deletion after Skeletal Maturity.

Estrogen regulates bone mass in women and men, but the underlying cellular mechanisms of estrogen action on bone remain unclear. Although both estrogen receptor (ER)α and ERß are expressed in bone cells, ERα is the dominant receptor for skeletal estrogen action. Previous studies using either global or cell-specific ERα deletion provided important insights, but each of these approaches had limitations. Specifically, either high circulating sex steroid levels in global ERα knockout mice or the effects of deletion of ERα during growth and development in constitutive cell-specific knockout mice have made it difficult to clearly define the role of ERα in specific cell types in the adult skeleton. We recently generated and characterized mice with tamoxifen-inducible ERα deletion in osteocytes driven by the 8-kb Dmp1 promoter (ERαΔOcy mice), revealing detrimental effects of osteocyte-specific ERα deletion on trabecular bone volume (-20.1%) and bone formation rate (-18.9%) in female, but not male, mice. Here, we developed and characterized analogous mice with inducible ERα deletion in osteoclasts using the Cathepsin K promoter (ERαΔOcl mice). In a study design identical to that with the previously described ERαΔOcy mice, adult female, but not male, ERαΔOcl mice showed a borderline (-10.2%, p = 0.084) reduction in trabecular bone volume, no change in osteoclast numbers, but a significant increase in serum CTx levels, consistent with increased osteoclast activity. These findings in ERαΔOcl mice differ from previous studies of constitutive osteoclast-specific ERα deletion, which led to clear deficits in trabecular bone and increased osteoclast numbers. Collectively, these data indicate that in adult mice, estrogen action in the osteocyte is likely more important than via the osteoclast and that ERα deletion in osteoclasts from conception onward has more dramatic skeletal effects than inducible osteoclastic ERα deletion in adult mice. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Multivariate GWAS of Alzheimer's disease CSF biomarker profiles implies GRIN2D in synaptic functioning.

BACKGROUND: Genome-wide association studies (GWAS) of Alzheimer's disease (AD) have identified several risk loci, but many remain unknown. Cerebrospinal fluid (CSF) biomarkers may aid in gene discovery and we previously demonstrated that six CSF biomarkers (ß-amyloid, total/phosphorylated tau, NfL, YKL-40, and neurogranin) cluster into five principal components (PC), each representing statistically independent biological processes. Here, we aimed to (1) identify common genetic variants associated with these CSF profiles, (2) assess the role of associated variants in AD pathophysiology, and (3) explore potential sex differences. METHODS: We performed GWAS for each of the five biomarker PCs in two multi-center studies (EMIF-AD and ADNI). In total, 973 participants (n = 205 controls, n = 546 mild cognitive impairment, n = 222 AD) were analyzed for 7,433,949 common SNPs and 19,511 protein-coding genes. Structural equation models tested whether biomarker PCs mediate genetic risk effects on AD, and stratified and interaction models probed for sex-specific effects. RESULTS: Five loci showed genome-wide significant association with CSF profiles, two were novel (rs145791381 [inflammation] and GRIN2D [synaptic functioning]) and three were previously described (APOE, TMEM106B, and CHI3L1). Follow-up analyses of the two novel signals in independent datasets only supported the GRIN2D locus, which contains several functionally interesting candidate genes. Mediation tests indicated that variants in APOE are associated with AD status via processes related to amyloid and tau pathology, while markers in TMEM106B and CHI3L1 are associated with AD only via neuronal injury/inflammation. Additionally, seven loci showed sex-specific associations with AD biomarkers. CONCLUSIONS: These results suggest that pathway and sex-specific analyses can improve our understanding of AD genetics and may contribute to precision medicine.

Multiparametric senescent cell phenotyping reveals targets of senolytic therapy in the aged murine skeleton.

Senescence drives organismal aging, yet the deep characterization of senescent cells in vivo remains incomplete. Here, we apply mass cytometry by time-of-flight using carefully validated antibodies to analyze senescent cells at single-cell resolution. We use multiple criteria to identify senescent mesenchymal cells that are growth-arrested and resistant to apoptosis. These p16 + Ki67-BCL-2+ cells are highly enriched for senescence-associated secretory phenotype and DNA damage markers, are strongly associated with age, and their percentages are increased in late osteoblasts/osteocytes and CD24high osteolineage cells. Moreover, both late osteoblasts/osteocytes and CD24high osteolineage cells are robustly cleared by genetic and pharmacologic senolytic therapies in aged mice. Following isolation, CD24+ skeletal cells exhibit growth arrest, senescence-associated ß-galactosidase positivity, and impaired osteogenesis in vitro. These studies thus provide an approach using multiplexed protein profiling to define senescent mesenchymal cells in vivo and identify specific skeletal cell populations cleared by senolytics.

Multiomics profiling of human plasma and cerebrospinal fluid reveals ATN-derived networks and highlights causal links in Alzheimer's disease.

INTRODUCTION: This study employed an integrative system and causal inference approach to explore molecular signatures in blood and CSF, the amyloid/tau/neurodegeneration [AT(N)] framework, mild cognitive impairment (MCI) conversion to Alzheimer's disease (AD), and genetic risk for AD. METHODS: Using the European Medical Information Framework (EMIF)-AD cohort, we measured 696 proteins in cerebrospinal fluid (n = 371), 4001 proteins in plasma (n = 972), 611 metabolites in plasma (n = 696), and genotyped whole-blood (7,778,465 autosomal single nucleotide epolymorphisms, n = 936). We investigated associations: molecular modules to AT(N), module hubs with AD Polygenic Risk scores and APOE4 genotypes, molecular hubs to MCI conversion and probed for causality with AD using Mendelian randomization (MR). RESULTS: AT(N) framework associated with protein and lipid hubs. In plasma, Proprotein Convertase Subtilisin/Kexin Type 7 showed evidence for causal associations with AD. AD was causally associated with Reticulocalbin 2 and sphingomyelins, an association driven by the APOE isoform. DISCUSSION: This study reveals multi-omics networks associated with AT(N) and causal AD molecular candidates.

The value of maintaining cognition in patients with mild cognitive impairment: The innovation headroom and potential cost-effectiveness of roflumilast.

INTRODUCTION: Early health-technology assessment can support discussing scarce resource allocation among stakeholders. We explored the value of maintaining cognition in patients with mild cognitive impairment (MCI) by estimating: (1) the innovation headroom and (2) the potential cost effectiveness of roflumilast treatment in this population. METHODS: The innovation headroom was operationalized by a fictive 100% efficacious treatment effect, and the roflumilast effect on memory word learning test was assumed to be associated with 7% relative risk reduction of dementia onset. Both were compared to Dutch setting usual care using the adapted International Pharmaco-Economic Collaboration on Alzheimer's Disease (IPECAD) open-source model. RESULTS: The total innovation headroom expressed as net health benefit was 4.2 (95% bootstrap interval: 2.9-5.7) quality-adjusted life years (QALYs). The potential cost effectiveness of roflumilast was k€34 per QALY. DISCUSSION: The innovation headroom in MCI is substantial. Although the potential cost effectiveness of roflumilast treatment is uncertain, further research on its effect on dementia onset is likely valuable.

Local senolysis in aged mice only partially replicates the benefits of systemic senolysis.

Clearance of senescent cells (SnCs) can prevent several age-related pathologies, including bone loss. However, the local versus systemic roles of SnCs in mediating tissue dysfunction remain unclear. Thus, we developed a mouse model (p16-LOX-ATTAC) that allowed for inducible SnC elimination (senolysis) in a cell-specific manner and compared the effects of local versus systemic senolysis during aging using bone as a prototype tissue. Specific removal of Sn osteocytes prevented age-related bone loss at the spine, but not the femur, by improving bone formation without affecting osteoclasts or marrow adipocytes. By contrast, systemic senolysis prevented bone loss at the spine and femur and not only improved bone formation, but also reduced osteoclast and marrow adipocyte numbers. Transplantation of SnCs into the peritoneal cavity of young mice caused bone loss and also induced senescence in distant host osteocytes. Collectively, our findings provide proof-of-concept evidence that local senolysis has health benefits in the context of aging, but, importantly, that local senolysis only partially replicates the benefits of systemic senolysis. Furthermore, we establish that SnCs, through their senescence-associated secretory phenotype (SASP), lead to senescence in distant cells. Therefore, our study indicates that optimizing senolytic drugs may require systemic instead of local SnC targeting to extend healthy aging.

Multiparametric senescent cell phenotyping reveals CD24 osteolineage cells as targets of senolytic therapy in the aged murine skeleton.

Senescence drives organismal aging, yet the deep characterization of senescent cells in vivo remains incomplete. Here, we applied mass cytometry by time-of-flight (CyTOF) using carefully validated antibodies to analyze senescent cells at single-cell resolution. We used multiple criteria to identify senescent mesenchymal cells that were growth arrested and resistant to apoptosis (p16+/Ki67-/BCL-2+; "p16KB" cells). These cells were highly enriched for senescence-associated secretory phenotype (SASP) and DNA damage markers and were strongly associated with age. p16KB cell percentages were also increased in CD24+ osteolineage cells, which exhibited an inflammatory SASP in aged mice and were robustly cleared by both genetic and pharmacologic senolytic therapies. Following isolation, CD24+ skeletal cells exhibited growth arrest, SA-ßgal positivity, and impaired osteogenesis in vitro . These studies thus provide a new approach using multiplexed protein profiling by CyTOF to define senescent mesenchymal cells in vivo and identify a highly inflammatory, senescent CD24+ osteolineage population cleared by senolytics.

Whole-exome rare-variant analysis of Alzheimer's disease and related biomarker traits.

INTRODUCTION: Despite increasing evidence of a role of rare genetic variation in the risk of Alzheimer's disease (AD), limited attention has been paid to its contribution to AD-related biomarker traits indicative of AD-relevant pathophysiological processes. METHODS: We performed whole-exome gene-based rare-variant association studies (RVASs) of 17 AD-related traits on whole-exome sequencing (WES) data generated in the European Medical Information Framework for Alzheimer's Disease Multimodal Biomarker Discovery (EMIF-AD MBD) study (n = 450) and whole-genome sequencing (WGS) data from ADNI (n = 808). RESULTS: Mutation screening revealed a novel probably pathogenic mutation (PSEN1 p.Leu232Phe). Gene-based RVAS revealed the exome-wide significant contribution of rare coding variation in RBKS and OR7A10 to cognitive performance and protection against left hippocampal atrophy, respectively. DISCUSSION: The identification of these novel gene-trait associations offers new perspectives into the role of rare coding variation in the distinct pathophysiological processes culminating in AD, which may lead to identification of novel therapeutic and diagnostic targets.

The Sex-Specific Splicing of Doublesex in Brine Shrimp Artemia franciscana.

The understanding of sex determination and differentiation in animals has recently made remarkable strides through the use of advanced research tools. At the gene level, the Mab-3-related transcription factor (Dmrt) gene family, which encodes for the typical DNA-binding doublesex/Mab-3 (DM) domain in their protein, is known for its contribution to sex determination and differentiation in insects. In this study, DNA-binding DM domain screening has identified eight transcripts from Artemia franciscana transcriptomic that encode proteins containing one conserved DNA-binding DM domain. The genome mapping confirmed that these eight transcripts are transcribed from six different loci on the A. franciscana genome assembly. One of those loci, the Af.dsx-4 locus, is closely related to Doublesex, a gene belonging to the Dmrt gene family. This locus could be transcribed into three alternative transcripts, namely Af.dsx4, Af.dsxF and Af.dsxM. While Af.dsx4 and Af.dsxF could putatively be translated to form an identical Af.dsxF protein of 186 aa long, Af.dsxM translates for an Af.dsxM protein of 289 aa long but shares a DNA-binding DM domain. Interestingly, Af.dsxF and Af.dsxM are confirmed as sex-specific transcripts, Af.dsxF is only present in females, and Af.dsxM is only present in male individuals. The results suggest that the sex-specific splicing mechanism of the doublesex described in insects is also present in A. franciscana. Af.dxs-4 locus can be used in further studies to clarify the sex determination pathways in A. fracnciscana.

The link between cognition and somatic conditions related to insulin resistance in the UK Biobank study cohort: a systematic review.

Clinical and genomic studies have shown an overlap between neuropsychiatric disorders and insulin resistance (IR)-related somatic conditions, including obesity, type 2 diabetes, and cardiovascular diseases. Impaired cognition is often observed among neuropsychiatric disorders, where multiple cognitive domains may be affected. In this review, we aimed to summarise previous evidence on the relationship between IR-related diseases/traits and cognitive performance in the large UK Biobank study cohort. Electronic searches were conducted on PubMed, Scopus, and Web of Science until April 2022. Eighteen articles met the inclusion criteria and were qualitatively reviewed. Overall, there is substantial evidence for an association between IR-related cardio-metabolic diseases/traits and worse performance on various cognitive domains, which is largely independent of possible confoundings. The most consistent findings referred to IR-related associations with poorer verbal and numerical reasoning ability, as well as slower processing speed. The observed associations might be mediated by alterations in immune-inflammation, brain integrity/connectivity, and/or comorbid somatic or psychiatric diseases/traits. Our findings provide impetus for further research into the underlying neurobiology and possible new therapeutic targets.

Temporally ordered associations between type 2 diabetes and brain disorders - a Danish register-based cohort study.

BACKGROUND: Type 2 diabetes mellitus (T2DM) is linked with several neurodegenerative and psychiatric disorders, either as a comorbid condition or as a risk factor. We aimed to expand the evidence by examining associations with a broad range of brain disorders (psychiatric and neurological disorders, excluding late-onset neurodegenerative disorders), while also accounting for the temporal order of T2DM and these brain disorders. METHODS: In a population-based cohort-study of 1,883,198 Danish citizens, born 1955-1984 and followed until end of 2016, we estimated associations between T2DM and 16 brain disorders first diagnosed between childhood and mid-adulthood. We calculated odds ratios (OR) and hazard ratios (HR) with 95% confidence intervals (CI) in temporally ordered analyses (brain disorder diagnosis after T2DM and vice versa), adjusted for sex, age, follow-up, birth year, and parental factors. RESULTS: A total of 67,660 (3.6%) of the study population were identified as T2DM cases after age 30 and by a mean age of 45 years (SD of 8 years). T2DM was associated with most psychiatric disorders. Strongest associations were seen with other (i.e. non-anorectic) eating disorders (OR [95% CI]: 2.64 [2.36-2.94]) and schizophrenia spectrum disorder (2.73 [2.63-2.84]). Among neurological disorders especially inflammatory brain diseases (1.73 [1.57-1.91]) and epilepsy (1.67 [1.60-1.75]) were associated with T2DM. Most associations remained in both directions in the temporally ordered analyses. For most psychiatric disorders, associations were strongest in females. CONCLUSIONS: T2DM was associated with several psychiatric and neurological disorders, and most associations were consistently found for both temporal order of disorders. This suggests a shared etiology of T2DM and those brain disorders. This study can form the starting point for studies directed at further elucidating potential causal links between disorders and shared biological mechanisms.

Skeletal Effects of Inducible ERα Deletion in Osteocytes in Adult Mice.

Estrogen is known to regulate bone metabolism in both women and men, but substantial gaps remain in our knowledge of estrogen and estrogen receptor alpha (ERα) regulation of adult bone metabolism. Studies using global ERα-knockout mice were confounded by high circulating sex-steroid levels, and osteocyte/osteoblast-specific ERα deletion may be confounded by ERα effects on growth versus the adult skeleton. Thus, we developed mice expressing the tamoxifen-inducible CreERT2 in osteocytes using the 8-kilobase (kb) Dmp1 promoter (Dmp1CreERT2 ). These mice were crossed with ERαfl//fl mice to create ERαΔOcy mice, permitting inducible osteocyte-specific ERα deletion in adulthood. After intermittent tamoxifen treatment of adult 4-month-old mice for 1 month, female, but not male, ERαΔOcy mice exhibited reduced spine bone volume fraction (BV/TV (-20.1%, p = 0.004) accompanied by decreased trabecular bone formation rate (-18.9%, p = 0.0496) and serum P1NP levels (-38.9%, p = 0.014). Periosteal (+65.6%, p = 0.004) and endocortical (+64.1%, p = 0.003) expansion were higher in ERαΔOcy mice compared to control (Dmp1CreERT2 ) mice at the tibial diaphysis, reflecting the known effects of estrogen to inhibit periosteal apposition and promote endocortical formation. Increases in Sost (2.1-fold, p = 0.001) messenger RNA (mRNA) levels were observed in trabecular bone at the spine in ERαΔOcy mice, consistent with previous reports that estrogen deficiency is associated with increased circulating sclerostin as well as bone SOST mRNA levels in humans. Further, the biological consequences of increased Sost expression were reflected in significant overall downregulation in panels of osteoblast and Wnt target genes in osteocyte-enriched bones from ERαΔOcy mice. These findings thus establish that osteocytic ERα is critical for estrogen action in female, but not male, adult bone metabolism. Moreover, the reduction in bone formation accompanied by increased Sost, decreased osteoblast, and decreased Wnt target gene expression in ERαΔOcy mice provides a direct link in vivo between ERα and Wnt signaling. © 2022 American Society for Bone and Mineral Research (ASBMR).

Identification of a suitable endogenous control miRNA in bone aging and senescence.

MicroRNAs (miRNAs) are promising tools as biomarkers and therapeutic agents in various chronic diseases such as osteoporosis, cancers, type I and II diabetes, and cardiovascular diseases. Considering the rising interest in the regulatory role of miRNAs in bone metabolism, aging, and cellular senescence, accurate normalization of qPCR-based miRNA expression data using an optimal endogenous control becomes crucial. We used a systematic approach to select candidate endogenous control miRNAs that exhibit high stability with aging from our miRNA sequence data and literature search. Validation of miRNA expression was performed using qPCR and their comprehensive stability was assessed using the RefFinder tool which is based on four statistical algorithms: GeNorm, NormFinder, BestKeeper, and comparative delta CT. The selected endogenous control was then validated for its stability in mice and human bone tissues, and in bone marrow stromal cells (BMSCs) following induction of senescence and senolytic treatment. Finally, the utility of selected endogenous control versus U6 was tested by using each as a normalizer to measure the expression of miR-34a, a miRNA known to increase with age and senescence. Our results show that Let-7f did not change across the groups with aging, senescence or senolytic treatment, and was the most stable miRNA, whereas U6 was the least stable. Moreover, using Let-7f as a normalizer resulted in significantly increased expression of miR-34a with aging and senescence and decreased expression following senolytic treatment. However, the expression pattern for miR-34a reversed for each of these conditions when U6 was used as a normalizer. We show that optimal endogenous control miRNAs, such as Let-7f, are essential for accurate normalization of miRNA expression data to increase the reliability of results and prevent misinterpretation. Moreover, we present a systematic strategy that is transferrable and can easily be used to identify endogenous control miRNAs in other biological systems and conditions.