Lilian Plotkin

Background: The T riggering R eceptor E xpressed on M yeloid Cells 2 (TREM2) gene is expressed in cells of the hematopoietic lineage, such as microglia and osteoclasts found in the brain and bone, respectively. Previous studies showed that expression of the TREM2 gene variant known as TREM2-R47H leads to altered musculoskeletal phenotype in a sex-dependent manner. Further, gonadectomy ameliorates the skeletal phenotype of gonadal female TREM2 ^R47H/+ mice. We also showed that sex chromosomes play a key role on skeletal mass and bone strength. Due to the sex-dimorphic bone and skeletal muscle phenotype exhibited by mice expressing the TREM2 gene variant, we investigated the role of chromosomal (XX vs. XY) or gonadal (ovaries vs. testes) sex in the Four Core Genotypes (FCG) mice model. Methods: FCG C57Bl/6J mice expressing the TREM2-R47H variant were mated to obtain TREM2 wildtype (TREM2 ^+/+ , WT) and TREM2 ^R47H/+ FCG mice. Four to 5.5-month-old gonadal male (XXT and XYT) and female (XXO and XYO) mice were analyzed. Body weight and bone mineral density were measured at baseline and at the endpoint (5.5 months of age) by DXA/Piximus. Micro-computed tomography, dynamic and static histomorphometry, 3-point bending test (mechanical properties), and bone turnover markers were measured at the endpoint. Two-way ANOVA analyses were performed using Prism 10 to identify the contributions of chromosome sex, the presence of the TREM2-R47H variant, and their interaction, separately for each gonadal sex. Results: Gonadal males : chromosome sex (XX/XY) effects are found for several bone structural parameters in femur and lumbar vertebra 5, whereas there was an interaction between gonadal sex and chromosome sex for other structural measurements in both bones by µCT. This suggests that the effect of the TREM2 variant depends on the presence of the Y chromosome in gonadal males. Mechanical testing shows chromosome sex effects, with higher overall values for XXT mice regardless of the presence of the TREM2 variant. At the cellular level, chromosome sex influences osteoblast, but not osteoclast, number and surface similarly for wild type and TREM2 mutants. Bone formation on the femur cortex and serum markers of formation/resorption were unchanged, suggesting that structural changes result from bone modeling/remodeling at an earlier age. Gonadal females : Chromosome sex affects body weight gain (higher in XYO than XXO mice), but no bone mineral density accrual. Chromosome sex affects the percent change (comparing final and initial values) in total lean mass (XYO > XXO) with chromosome sex x TREM2 genotype interaction and differences in total/%fat mass (TREM2 ^R47H/+ < WT), and %lean mass (TREM2 ^R47H/+ > WT) only for XYO mice. Chromosome sex affects distal femur volumetric bone mass (XYO > XXO), but the TREM2 genotype influences lumbar vertebra trabecular number and separation, which trended higher in TREM2 ^R47H/+ vs. WT mice for sex complement. Chromosome sex influences femur cortical bone, with overall higher values in XXO mice, independent of TREM2 genotype. Mechanical testing parameters were also influenced by chromosome sex with XXO > XYO mice. Femur cortical bone formation is higher on the endocortical but lower on the periosteal surface in XXO vs. XYO (chromosome sex effect). The opposite effects on the bone surfaces might explain the unchanged serum bone formation marker, Procollagen Type 1 N-terminal propeptide (P1NP). Yet, chromosome sex affects the levels of the resorption marker, C-terminal telopeptide of type 1 collagen (CTX-1), which were lower in XXO mice compared to XYO. At the cellular level, there is an interaction between chromosome sex and TREM2 genotype for osteoclast, but not osteoblast, parameters in vertebral trabecular bone. Osteoclast number and surface are higher in TREM2 ^R47H/+ mice only for XYO mice, and, as for CTX-1, are lower in XXO mice compared to XYO only for the mutant mice. Conclusion: Our findings suggest that chromosome sex partially affects the consequences of expression of the TREM2-R47H variant on bone structure in 5.5 month-old mice, whereas the outcomes of the gene variant depend on the mouse gonadal sex. Overall, the findings of the current studies add to our previous publications and overall suggest that the consequences of expression of the R47H variant of the TREM2 gene on the musculoskeletal system depend on the presence of sex hormones and the chromosome sex in a manner that depends on the bone site or skeletal muscle studied. Future studies will determine whether the consequences of the genetic manipulations at the cellular level (both number and activity) result in a stronger bone phenotype at an older age.

Lung cancer is a leading cause of death worldwide and is often accompanied by declines in musculoskeletal health (i.e., cachexia). Despite affecting a majority of lung cancer patients, cachexia remains understudied and currently has no cure. We have previously demonstrated that liver metastases (LMs) exacerbate cachexia in murine models of colorectal cancer, and, while the liver represents a common site of metastases and is associated with poor prognosis in patients with lung cancer, whether LMs heighten musculoskeletal wasting in mice bearing lung cancer is unknown. Here, we aimed to characterize the impact of LMs on musculoskeletal health in a mouse model of lung cancer cachexia. C57BL/6J male mice were injected with LLC tumor cells either subcutaneously or intrasplenically (LMs) to mimic hepatic metastases (n = 6–9/group). Upon sacrifice, skeletal muscle, bone, and plasma were collected for morphological and molecular analyses. Consistently, compared to healthy controls, metastatic tumor hosts displayed greater reductions in muscle weights (~17%), in line with decreased muscle torque (~23%) and reduced muscle cross-sectional area (~10%). On a molecular level, skeletal muscle from mice bearing LMs had elevated levels of pStat3, Murf1, and Atrogin-1, suggesting enhanced protein catabolism. Similar to skeletal muscle, metastatic tumor hosts displayed greater losses in trabecular bone and increased skeletal fragility. Plasma proteomics identified 211 and 131 differentially expressed proteins in metastatic hosts compared to control animals and subcutaneous LLC hosts, respectively. Top regulated pathways in mice bearing LMs included neutrophil degranulation, BAG2 signaling, and cachexia signaling. Overall, our findings demonstrate that LMs are accompanied by accelerated musculoskeletal wasting and weakness in a mouse model of lung cancer cachexia. This work highlights the need for animal models that mimic advanced cancer, thus providing a better understanding of the mechanisms that mediate cachexia.

Background The Triggering Receptor Expressed on Myeloid Cells 2 (TREM2) gene is expressed in cells of the hematopoietic lineage, like microglia and osteoclasts. A TREM2 gene variant known as TREM2-R47H is associated with an increased risk of developing Alzheimer’s disease (AD). Previous studies have shown sex-dimorphic bone and muscle consequences that are associated with the TREM2 variant. Sex chromosomes have also been shown to play a key contributor to skeletal mass and bone strength. Due to the sex-dimorphic bone and skeletal muscle phenotype exhibited by mice expressing the TREM2 gene variant, we investigated the role of chromosomal (XX vs XY) or gonadal (ovaries vs testes) sex. Methods Four Core Genotypes (FCG) C57Bl/6J mice expressing the TREM2-R47H variant were mated to obtain TREM2 wildtype (TREM2 ^+/+ , WT) and TREM2 ^R47H/+ FCG mice. Four to 5.5-month-old gonadal male (XXT and XYT) and female (XXO and XYT) mice were analyzed. Body weight and bone mineral density were initially measured at baseline and endpoint (5.5 months of age) by DXA/Piximus. Micro-computed tomography, dynamic histomorphometry, 3-point bending test (mechanical properties), and bone turnover markers were measured at the endpoint. Two-way ANOVA analyses were performed through Prism 10 to identify the contributions of chromosome sex, the presence of the TREM2-R47H variant, and their interaction, separately for each gonadal sex. Results Gonadal males: chromosome sex (XX/XY) effects are found for several bone structural parameters in femur and lumbar vertebra 5, whereas there was an interaction between gonadal sex and chromosome sex for other structural measurements in both bones by µCT. Overall, values are higher for TREM2 ^R47H/+ than WT for XYT, but not XXT mice, suggesting that the TREM2 genotype effects depend on the presence of the Y chromosome. Mechanical testing shows chromosome sex effects, with higher overall values for XXT mice. Bone formation on the femur cortex and serum formation/resorption markers were unchanged, suggesting that structural changes result from bone modeling/remodeling at an earlier age. Gonadal females: Chromosome sex affects body weight gain (higher in XYO than XXO mice), but no bone mineral density accrual. Chromosome sex affects total lean mass (XYO > XXO) with chromosome sex x TREM2 genotype interaction and differences in total/%fat mass (TREM2 ^R47H/+ WT) only for XYO mice. Chromosome sex affects distal femur volumetric bone mass (XYO > XXO), but the TREM2 genotype influences lumbar vertebra trabecular number and separation, which trended higher in TREM2 ^R47H/+ vs WT mice for sex complement. Chromosome sex influences femur cortical bone, with overall higher values in XXO mice, independent of TREM2 genotype. Mechanical testing parameters also were XXO > XYO mice. Femur cortical bone formation is higher on the endocortical but lower on the periosteal surface in XXO vs XYO (chromosome sex effect). The opposite effects on the bone surfaces might explain the unchanged serum bone formation marker, Procollagen Type 1 N-terminal propeptide (P1NP). Yet, chromosome sex affects the levels of the resorption marker, C-terminal telopeptide of type 1 collagen (CTX-1), which were lower in XXO mice. Conclusion Our findings suggest that chromosome sex partially affects the consequences of expression of the TREM2-R47H variant on bone structure, whereas the outcomes of the gene variant depend on the mouse gonadal sex.

The R47H variant of the Triggering Receptor Expressed on Myeloid Cells 2 (TREM2) is a risk factor for Alzheimer’s disease in humans and leads to lower bone mass accrual in female but not male 12-month-old mice. To determine whether, as with aging, gonadectomy results in sex-specific musculoskeletal effects, gonad removal or SHAM surgery were performed in 4-month-old TREM2^R47H/+ mice and wild type (WT) male and female littermates (n = 10-12/group), with sexes analyzed separately. Body weight was lower in males, but higher in females after gonadectomy, independently of their genotype. Gonadectomy also lead to decreased BMD in males at all sites and in whole body (total) and spine in female mice for both genotypes. Total and femur BMD was lower in gonadectomized male mice 6-weeks post-surgery, independently of the genotype. On the other hand, BMD was only lower in ovariectomized WT but not TREM2^R47H/+ mice in all sites measured at this time point. Bone formation and resorption markers levels were not affected by orchiectomy, whereas CTX was higher 3 weeks after surgery and P1NP showed a tendency towards lower values at the 6 weeks timepoint only in ovariectomized WT mice. μCT analyses showed no differences resulting from gonadectomy in structural parameters in femoral cortical bone for either sex, but lower tissue mineral density in males of either genotype 6 weeks post-surgery. Nevertheless, biomechanical properties were overall lower in gonadectomized males of either genotype, and only for WT ovariectomized mice. Distal femur cancellous bone structure was also affected by gonadectomy in a genotype- and sex-dependent manner, with genotype-independent changes in males, and only in WT female mice. Thus, expression of the TREM2 R47H variant minimally alters the impact of gonadectomy in the musculoskeletal system in males, whereas it partially ameliorates the consequences of ovariectomy in female mice. A link between dementia and loss of bone mass and risk of fractures (osteoporosis) has been suggested, but never demonstrated. Bone loss can be detected before cognitive decline and reduced mobility in Alzheimer’s disease (AD) patients. Since TREM2 gene mutations are associated with AD/dementia and bone fragility, we investigated whether expression of the TREM2-R47H mutated gene alters the response to removal of sex steroids in male and female mice. Expression of TREM2-R47H minimally alters the impact of sex steroid removal in the musculoskeletal system in males, whereas it partially ameliorates the consequences of removing the ovaries in female mice.

Vertebrate sexual dimorphism is ascribed to the presence of testes or ovaries, and, hence, to the secretion of gonad-specific hormones. However, mounting evidence indicates that sex differences in tissues and organs also stem from the presence of sex chromosomes (XX or XY). To tease out the contribution of gonads from sex chromosomes to the musculoskeletal system, we used the Four-Core Genotypes (FCG) mouse model, in which the Sry gene, which dictates testis formation, was either deleted in the Y chromosome, resulting in XY mice with ovaries (XY^-SryO), or overexpressed in XX mice, resulting in XX mice with testes (XXT), together with gonadal males XY^-SryT (Sry deletion and overexpression of the Sry transgene in chromosome 3) and females XXO. The FCG mice are generated by crossing XXO with XY^-SryT mice, all of C57BL/6 J background. We now show that the musculoskeletal phenotype of 2- to 4-month-old FCG mice varies based on both gonads and sex chromosomes, depending on the age and the organ/tissue/cell analyzed. The effect of sex chromosomes on body weight, fat and lean/skeletal muscle mass, and bone mass and structure is minor in 2-/3-month-old mice, soon after sexual maturation. The contribution of sex chromosomes (XX versus XY^-Sry in mice with the same gonads and sex hormones) in several of our measurements becomes apparent in adult 4-month-old mice. Contribution of 1X and 1Y^-Sry versus 2X chromosomes varies among different measurements in gonadal males or females, and mice with XY^-Sry chromosomes might have higher or lower values that XX mice. Our study shows XX versus XY^-Sry chromosome contribution to the musculoskeletal phenotype, which becomes more evident as the animals reach peak bone mass, suggesting that while gonadal sex has a major role, sex chromosomes are a so far unrecognized contributor to musculoskeletal mass and bone strength. Sexual dimorphism, or the morphological differences between males and females, is often attributed to the hormones produced by the testes or ovaries. However, evidence suggests that sex chromosomes (XX or XY) also play a significant role. To separate the effects of these two factors on bone health, this work used a unique mouse model known as the Four-Core Genotypes (FCG). The FCG model allows us to manipulate the expression of the Sry gene, which determines testis development, resulting in four groups: XX mice with ovaries (XXO), XX mice with testes (XXT), XY mice with ovaries (XY^-SryO), and XY mice with testes (XY^-SryT). The results presented in this study revealed that the influence of sex chromosomes on the development of body weight, fat and lean/skeletal muscle mass, and bone mass becomes evident in an age-dependent manner, specifically after sexual maturity. Our evidence suggests that there are distinct differences in musculoskeletal development between mice with XX or XY chromosomes. We conclude that our findings uncovered sex chromosomes as a previously unacknowledged factor contributing to the sexual differences observed in musculoskeletal health.

Mounting evidence indicates that whereas some fundamental aspects of bone cell differentiation and function are similar in females and males, there is a clear contribution of sex/gender on the effects of signaling molecules on bone mass and strength and, consequently, on the effects of pharmacologic approaches to treat skeletal disorders. However, until recently, most studies were designed and performed using only 1 sex, resulting in a scarcity of published information on sexual dimorphism of the musculoskeletal system, including the mandible/masticatory muscles and the axial and appendicular bones and skeletal muscles. Further, it is now recognized that scientific rigor requires the study of both males and females. Therefore, there is an increasing need to understand the molecular and cellular basis for the differential outcomes of genetic manipulations and therapeutic agent administration depending on the sex of the experimental animals. Studies have shown higher muscle mass, cancellous bone mass, and long bone width in males compared with females as well as different traits in the pelvis and the skull, which are usually used for gender identification in forensic anthropology. Yet, most reports focus on the role of sex hormones, in particular, the consequences of estrogen deficiency with menopause in humans and in ovariectomized animal models. In addition, emerging data is starting to unveil the effects of gender-affirming hormonal therapy on the musculoskeletal system. We summarize here the current knowledge on the sex/gender-dependent phenotypic characteristics of the bone and skeletal muscles in humans and rodents, highlighting studies in which side by side comparisons were made.

Background: Yerba mate (YM, Ilex paraguariensis) consumption beneficially affects the bones. However, whether YM components exert their effect on bone cells directly remains elusive.Methods: We evaluated how main YM components affect osteoblastic (MC3T3-E1) and osteocytic (MLO-Y4) cells in vitro when administered separately or in an aqueous extract. MC3T3-E1 and MLO-Y4 cells were exposed to three different experimental conditions: (1) Caffeine, chlorogenic acid, and their combinations; (2) Caffeine, rutin, and their combinations; (3) Aqueous YM extract.Results: All polyphenol and caffeine concentrations as well as that of their tested combinations significantly increased MC3T3-E1 cell viability from 16.6% to 34.8% compared to the control. In MLO-Y4 cells, the lowest rutin and the two highest caffeine concentrations significantly increased cell viability by 11.9, 14.9, and 13.7%, respectively. While rutin and caffeine combinations tended to increase MLO-Y4 cell viability, different chlorogenic acid and caffeine combinations did not affect it. Finally, the aqueous YM extract significantly increased MLO-Y4, MC3T3-E1, and differentiated MC3T3-E1 cell viability compared to the control without treatment.Conclusions: YM components (rutin, chlorogenic acid, and caffeine) positively affected bone cells, mainly pre-osteoblast cells. Moreover, the aqueous YM extract significantly increased MLO-Y4, MC3T3-E1, and differentiated MC3T3-E1 cell viabilities indicating an additional relevant nutritional property of YM infusion. Further studies would be required to elucidate the underlying effector mechanism of YM on the bones and its relationship with previously described in vivo positive effects.

Low levels of microRNA (miR) 21 may explain the higher osteocyte apoptosis with Cx43-deficient and aged female mice. However, miR21 exerts a sex-divergent role in osteocytes, regulating bone mass and architecture through non-cell autonomous effects on osteoblasts and osteoclasts, via sex-specific regulation of osteocyte cytokine production. miR21 deficiency improves bone strength in females, and, to a higher extent, in male miR21-deficient mice. To understand the molecular basis for the effects of miR21 deletion, mRNA was isolated from miR21^fl/fl (controls) or miR21-deficient (by deletion in cells expressing Cre recombinase under the control of the 8 kb fragment of the DMP1 promoter: miR21^ΔOt mice). miR21 was 50% lower in miR21^ΔOt whole calvaria bone compared to control mice of the corresponding sex. RNAseq was performed in 4 samples/sex and genotype. There were 152 genes with <.05 P-value and >1 absolute log2 fold change in the male data analysis, and expression of most genes was higher in the miR21^fl/fl group. Two of the genes, Actn3 and Myh4, had a false discovery rate < 0.1. Gene enrichment analysis of significant genes on both KEGG pathways and gene ontology (GO) gene sets shows that the significant genes were enriched in muscle contraction. Some muscle-related genes like Actn3 were included in multiple significant pathways. For females, only 65 genes had P-value <.05 and >1 absolute log2 fold change. Yet, no significant KEGG or GO pathways, including ≥5 significant genes, were seen, and no overlap of significant genes was found between male and female samples. Therefore, deletion of miR21 has a stronger effect on male transcriptome in calvaria, compared to females. Further, no enrichment of any pathway was detected in female samples. Thus, either there are no differences between 2 groups in female or the effect size is small, and a larger sample size is needed to uncover miR21-dependent differences. Small RNAs, name microRNAs or miRs, modulate the expression of genes by targeting messenger RNA leading to its degradation and the suppression of protein synthesis. We have previously shown that removal of miR21from murine osteocytes, the cells embedded in the mineralized bone matrix, results in changes in bone at the structural and biomechanical level, as well as in the number and activity of bone cells. Importantly, these effects differ between males and females. We now show that the genes regulated by the removal of the miR are also different in bones from males and females at 4 mo of age. As expected, the expression of most genes was higher in the control mice expressing miR21, compared to miR21-deficient animals. However, although 152 genes were significantly different in bones from male mice, only 65 were different in females, and there was no overlap between the genes changed in males and females. This study adds to the evidence that sexual dimorphism plays a role in skeletal development and growth.

Purpose of Review: This review examines the linked pathophysiology of Alzheimer’s disease/related dementia (AD/ADRD) and bone disorders like osteoporosis. The emphasis is on “inflammaging”—a low-level inflammation common to both, and its implications in an aging population. Recent Findings: Aging intensifies both ADRD and bone deterioration. Notably, ADRD patients have a heightened fracture risk, impacting morbidity and mortality, though it is uncertain if fractures worsen ADRD. Therapeutically, agents targeting inflammation pathways, especially Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) and TNF-α, appear beneficial for both conditions. Additionally, treatments like Sirtuin 1 (SIRT-1), known for anti-inflammatory and neuroprotective properties, are gaining attention. Summary: The interconnectedness of AD/ADRD and bone health necessitates a unified treatment approach. By addressing shared mechanisms, we can potentially transform therapeutic strategies, enriching our understanding and refining care in our aging society. This review article is part of a series of multiple manuscripts designed to determine the utility of using artificial intelligence for writing scientific reviews.