Gene-Dose-Dependent Reduction Fshr Expression Improves Spatial Memory Deficits in Alzheimer's Mice.

Alzheimer's disease (AD) is a major progressive neurodegenerative disorder of the aging population. High post-menopausal levels of the pituitary gonadotropin follicle-stimulating hormone (FSH) are strongly associated with the onset of AD, and we have shown recently that FSH directly activates the hippocampal Fshr to drive AD-like pathology and memory loss in mice. To establish a role for FSH in memory loss, we used female 3xTg;Fshr+/+, 3xTg;Fshr+/- and 3xTg;Fshr-/- mice that were either left unoperated or underwent sham surgery or ovariectomy at 8 weeks of age. Unoperated and sham-operated 3xTg;Fshr-/- mice were implanted with 17ß-estradiol pellets to normalize estradiol levels. Morris Water Maze and Novel Object Recognition behavioral tests were performed to study deficits in spatial and recognition memory, respectively, and to examine the effects of Fshr depletion. 3xTg;Fshr+/+ mice displayed impaired spatial memory at 5 months of age; both the acquisition and retrieval of the memory were ameliorated in 3xTg;Fshr-/- mice and, to a lesser extent, in 3xTg;Fshr+/- mice- -thus documenting a clear gene-dose-dependent prevention of hippocampal-dependent spatial memory impairment. At 5 and 10 months, sham-operated 3xTg;Fshr-/- mice showed better memory performance during the acquasition and/or retrieval phases, suggesting that Fshr deletion prevented the progression of spatial memory deficits with age. However, this prevention was not seen when mice were ovariectomized, except in the 10-month-old 3xTg;Fshr-/- mice. In the Novel Object Recognition test performed at 10 months, all groups of mice, except ovariectomized 3xTg;Fshr-/- mice showed a loss of recognition memory. Consistent with the neurobehavioral data, there was a gene-dose-dependent reduction mainly in the amyloid ß40 isoform in whole brain extracts. Finally, serum FSH levels < 8 ng/mL in 16-month-old APP/PS1 mice were associated with better retrieval of spatial memory. Collectively, the data provide compelling genetic evidence for a protective effect of inhibiting FSH signaling on the progression of spatial and recognition memory deficits in mice, and lay a firm foundation for the use of an FSH-blocking agent for the early prevention of cognitive decline in postmenopausal women.

Abaloparatide prevents immobilization-induced cortical but not trabecular bone loss after spinal cord injury.

Spinal cord injury (SCI) causes severe and resistant sublesional disuse bone loss. Abaloparatide, a modified parathyroid hormone related peptide, is an FDA approved drug for treatment of severe osteoporosis with potent anabolic activity. The effects of abaloparatide on SCI-induced bone loss remain undefined. Thus, female mice underwent sham or severe contusion thoracic SCI causing hindlimb paralysis. Mice then received subcutaneous injection of vehicle or 20 µg/kg/day abaloparatide for 35 days. Micro-computed tomography (micro-CT) analysis of the distal and midshaft femoral regions of the SCI-vehicle mice revealed reduced trabecular fractional bone volume (56%), thickness (75%), and cortical thickness (80%) compared to sham-vehicle controls. Treatment with abaloparatide did not prevent SCI-induced changes in trabecular or cortical bone. However, histomorphometry evaluation of the SCI-abaloparatide mice demonstrated that abaloparatide treatment increased osteoblast (241%) and osteoclast (247%) numbers and the mineral apposition rate (131%) compared to SCI-vehicle animals. In another independent experiment, treatment with 80 µg/kg/day abaloparatide significantly attenuated SCI-induced loss in cortical bone thickness (93%) when compared to SCI-vehicle mice (79%) but did not prevent SCI-induced trabecular bone loss or elevation in cortical porosity. Biochemical analysis of the bone marrow supernatants of the femurs showed that SCI-abaloparatide animals had 2.3-fold increase in procollagen type I N-terminal propeptide, a bone formation marker than SCI-vehicle animals. SCI groups had 70% higher levels of cross-linked C-telopeptide of type I collagen, a bone resorption marker, than sham-vehicle mice. These findings suggest that abaloparatide protects the cortical bone against the deleterious effects of SCI by promoting bone formation.

Independent Skeletal Actions of Pituitary Hormones.

Over the past years, pituitary hormones and their receptors have been shown to have non-traditional actions that allow them to bypass the hypothalamus-pituitary-effector glands axis. Bone cells-osteoblasts and osteoclasts-express receptors for growth hormone, follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), adrenocorticotrophic hormone (ACTH), prolactin, oxytocin, and vasopressin. Independent skeletal actions of pituitary hormones on bone have been studied using genetically modified mice with haploinsufficiency and by activating or inactivating the receptors pharmacologically, without altering systemic effector hormone levels. On another front, the discovery of a TSH variant (TSH-ßv) in immune cells in the bone marrow and skeletal action of FSHß through tumor necrosis factor α provides new insights underscoring the integrated physiology of bone-immune-endocrine axis. Here we discuss the interaction of each pituitary hormone with bone and the potential it holds in understanding bone physiology and as a therapeutic target.

The role of PDGF-BB in the bone-vascular relationship during aging.

Cardiovascular disease (CVD) and osteoporosis often occur together, suggesting an association between CVD and bone loss. Similarly, the correlation of bone loss, atherosclerosis, and aortic calcification, especially in patients with chronic kidney disease, exemplifies a bone-vessel connection. In this issue of the JCI, Santhanam et al. investigated the role of the angiogenesis factor platelet-derived growth factor-BB (PDGF-BB) in vascular stiffening. Serum levels of bone-derived PDGF-BB differed between young and aged mice, and in mice fed a high-fat diet (HFD) compared with those fed normal chow. Experiments with genetic models led the authors to conclude that bone-derived PDGF-BB mediates the hallmark arterial stiffening of aging and metabolic stress. Notably, excessive preosteoclast-derived PDGF-BB production during aging inhibited osteoblastic bone formation and increased circulating PDGF-BB, which in turn, accelerated vascular stiffness. These findings suggest that modifying circulating PDGF-BB levels may benefit patients with CVD, osteoporosis, and other age-related diseases.

Thyrotropin, Hyperthyroidism, and Bone Mass.

Thyrotropin (TSH), traditionally seen as a pituitary hormone that regulates thyroid glands, has additional roles in physiology including skeletal remodeling. Population-based observations in people with euthyroidism or subclinical hyperthyroidism indicated a negative association between bone mass and low-normal TSH. The findings of correlative studies were supported by small intervention trials using recombinant human TSH (rhTSH) injection, and genetic and case-based evidence. Genetically modified mouse models, which disrupt the reciprocal relationship between TSH and thyroid hormone, have allowed us to examine an independent role of TSH. Since the first description of osteoporotic phenotype in haploinsufficient Tshr +/- mice with normal thyroid hormone levels, the antiosteoclastic effect of TSH has been documented in both in vitro and in vivo studies. Further studies showed that increased osteoclastogenesis in Tshr-deficient mice was mediated by tumor necrosis factor α. Low TSH not only increased osteoclastogenesis, but also decreased osteoblastogenesis in bone marrow-derived primary osteoblast cultures. However, later in vivo studies using small and intermittent doses of rhTSH showed a proanabolic effect, which suggests that its action might be dose and frequency dependent. TSHR was shown to interact with insulin-like growth factor 1 receptor, and vascular endothelial growth factor and Wnt pathway might play a role in TSH's effect on osteoblasts. The expression and direct skeletal effect of a biologically active splice variant of the TSHß subunit (TSHßv) in bone marrow-derived macrophage and other immune cells suggest a local skeletal effect of TSHR. Further studies of how locally secreted TSHßv and systemic TSHß interact in skeletal remodeling through the endocrine, immune, and skeletal systems will help us better understand the hyperthyroidism-induced bone disease.

The hepcidin regulator erythroferrone is a new member of the erythropoiesis-iron-bone circuitry.

Background: Erythroblast erythroferrone (ERFE) secretion inhibits hepcidin expression by sequestering several bone morphogenetic protein (BMP) family members to increase iron availability for erythropoiesis. Methods: To address whether ERFE functions also in bone and whether the mechanism of ERFE action in bone involves BMPs, we utilize the Erfe-/- mouse model as well as ß-thalassemic (Hbbth3/+) mice with systemic loss of ERFE expression. In additional, we employ comprehensive skeletal phenotyping analyses as well as functional assays in vitro to address mechanistically the function of ERFE in bone. Results: We report that ERFE expression in osteoblasts is higher compared with erythroblasts, is independent of erythropoietin, and functional in suppressing hepatocyte hepcidin expression. Erfe-/- mice display low-bone-mass arising from increased bone resorption despite a concomitant increase in bone formation. Consistently, Erfe-/- osteoblasts exhibit enhanced mineralization, Sost and Rankl expression, and BMP-mediated signaling ex vivo. The ERFE effect on osteoclasts is mediated through increased osteoblastic RANKL and sclerostin expression, increasing osteoclastogenesis in Erfe-/- mice. Importantly, Erfe loss in Hbbth3/+mice, a disease model with increased ERFE expression, triggers profound osteoclastic bone resorption and bone loss. Conclusions: Together, ERFE exerts an osteoprotective effect by modulating BMP signaling in osteoblasts, decreasing RANKL production to limit osteoclastogenesis, and prevents excessive bone loss during expanded erythropoiesis in ß-thalassemia. Funding: YZG acknowledges the support of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (R01 DK107670 to YZG and DK095112 to RF, SR, and YZG). MZ acknowledges the support of the National Institute on Aging (U19 AG60917) and NIDDK (R01 DK113627). TY acknowledges the support of the National Institute on Aging (R01 AG71870). SR acknowledges the support of NIDDK (R01 DK090554) and Commonwealth Universal Research Enhancement (CURE) Program Pennsylvania.

Bone resorption goes green.

In this issue of Cell, McDonald et al. show that giant multinucleated, bone-resorbing osteoclasts dissolve into smaller cells, termed "osteopmorhs," which re-form into osteoclasts at distal bone sites (McDonald et al., 2021). These findings overturn the long-standing premise that osteoclasts differentiate solely from hematopoietic precursors and undergo apoptosis after completing resorption.

Serum FSH Is Associated With BMD, Bone Marrow Adiposity, and Body Composition in the AGES-Reykjavik Study of Older Adults.

CONTEXT: Follicle-stimulating hormone (FSH) concentrations increase during the perimenopausal transition and remain high after menopause. Loss of bone mineral density (BMD) and gain of bone marrow adiposity (BMA) and body fat mass also occur during this time. In mice, blocking the action of FSH increases bone mass and decreases fat mass. OBJECTIVE: To investigate the associations between endogenous FSH levels and BMD, BMA, and body composition in older adults, independent of estradiol and testosterone levels. DESIGN, SETTING, AND PARTICIPANTS: Older adults from the AGES-Reykjavik Study, an observational cohort study. MAIN OUTCOME MEASURES: Areal BMD, total body fat, and lean mass were measured with dual-energy x-ray absorptiometry. Lumbar vertebral BMA was measured by 1H-magnetic resonance spectroscopy. Volumetric BMD and visceral and subcutaneous adipose tissue (VAT, SAT) areas were measured with quantitative computed tomography. The least squares means procedure was used to determine sex hormone-adjusted associations between quartiles of serum FSH and BMD, BMA, and body composition. RESULTS: In women (N = 238, mean age 81 years), those in the highest FSH quartile, compared with the lowest quartile, had lower adjusted mean spine integral BMD (-8.6%), lower spine compressive strength index (-34.8%), higher BMA (+8.4%), lower weight (-8.4%), lower VAT (-17.6%), lower lean mass (-6.1%), and lower fat mass (-11.9%) (all P < 0.05). In men, FSH level was not associated with any outcome. CONCLUSIONS: Older postmenopausal women with higher FSH levels have higher BMA, but lower BMD and lower fat and lean mass, independent of estradiol and testosterone levels. Longitudinal studies are needed to better understand the underlying mechanisms.

The NO-cGMP-PKG pathway in skeletal remodeling.

The nitric oxide (NO)-cyclic guanosine monophosphate (cGMP)-protein kinase G (PKG) pathway plays a critical role in skeletal homeostasis. Preclinical data using NO and its donors and genetically modified mice demonstrated that NO was required in bone remodeling and partly mediated the anabolic effects of mechanical stimuli and estrogen. However, the off-target effects and tachyphylaxis of NO limit its long-term use, and previous clinical trials using organic nitrates for osteoporosis have been disappointing. Among the other components in the downstream pathway, targeting cGMP-specific phosphodiesterase to promote the NO-cGMP-PKG signal is a viable option. There are growing in vitro and in vivo data that, among many other PDE families, PDE5A is highly expressed in skeletal tissue, and inhibiting PDE5A using currently available PDE5A inhibitors might increase the osteoanabolic signal and protect the skeleton. These preclinical data open the possibility of repurposing PDE5A inhibitors for treating osteoporosis. Further research is needed to address the primary target bone cell of PDE5A inhibition, the contribution of direct and indirect effects of PDE5A inhibition, and the pathophysiological changes in skeletal PDE5A expression in aging and hypogonadal animal models.

First-in-class humanized FSH blocking antibody targets bone and fat.

Blocking the action of FSH genetically or pharmacologically in mice reduces body fat, lowers serum cholesterol, and increases bone mass, making an anti-FSH agent a potential therapeutic for three global epidemics: obesity, osteoporosis, and hypercholesterolemia. Here, we report the generation, structure, and function of a first-in-class, fully humanized, epitope-specific FSH blocking antibody with a KD of 7 nM. Protein thermal shift, molecular dynamics, and fine mapping of the FSH-FSH receptor interface confirm stable binding of the Fab domain to two of five receptor-interacting residues of the FSHß subunit, which is sufficient to block its interaction with the FSH receptor. In doing so, the humanized antibody profoundly inhibited FSH action in cell-based assays, a prelude to further preclinical and clinical testing.

The Human TSHß Subunit Proteins and Their Binding Sites on the TSH Receptor Using Molecular Dynamics Simulation.

To gain further insight into the binding of the normal and variant human TSHß subunits (TSHß and TSHßv), we modeled the 2 monomeric proteins and studied their interaction with the TSH receptor ectodomain (TSHR-ECD) using molecular dynamics simulation Furthermore, analyzed their bioactivity in vitro using recombinant proteins to confirm that such binding was physiologically relevant. Examining the interaction of TSHß and TSHßv with the TSHR-ECD model using molecular dynamic simulation revealed strong binding of these proteins to the receptor ECD. The specificity of TSHß and TSHßv binding to the TSHR-ECD was examined by analyzing the hydrogen-bonding residues of these subunits to the FSH receptor ECD, indicating the inability of these molecules to bind to the FSH receptors. Furthermore, the modelling suggests that TSHß and TSHßv proteins clasped the concave surface of the leucine rich region of the TSHR ECD in a similar way to the native TSH using dynamic hydrogen bonding. These mutually exclusive stable interactions between the subunits and ECD residues included some high-affinity contact sites corresponding to binding models of native TSH. Furthermore, we cloned TSHß and TSHßv proteins using the entire coding ORF and purified the flag-tagged proteins. The expressed TSHß subunit proteins retained bioactivity both in a coculture system as well as with immune-purified proteins. In summary, we showed that such interactions can result in a functional outcome and may exert physiological or pathophysiological effects in immune cells.

Beyond bone biology: Lessons from team science.

Today, research in biomedicine often requires the knowledge and technologies in diverse fields. Therefore, there is an increasing need for collaborative team science that crosses traditional disciplines. Here, we discuss our own lessons from both interdisciplinary and transdisciplinary teams, which ultimately ushered us to expand our research realm beyond bone biology.

Oxytocin regulates body composition.

The primitive neurohypophyseal nonapeptide oxytocin (OXT) has established functions in parturition, lactation, appetite, and social behavior. We have shown that OXT has direct actions on the mammalian skeleton, stimulating bone formation by osteoblasts and modulating the genesis and function of bone-resorbing osteoclasts. We deleted OXT receptors (OXTRs) selectively in osteoblasts and osteoclasts using Col2.3Cre and Acp5Cre mice, respectively. Both male and female Col2.3Cre+:Oxtrfl/fl mice recapitulate the low-bone mass phenotype of Oxtr+/- mice, suggesting that OXT has a prominent osteoblastic action in vivo. Furthermore, abolishment of the anabolic effect of estrogen in Col2.3Cre+:Oxtrfl/fl mice suggests that osteoblastic OXTRs are necessary for estrogen action. In addition, the high bone mass in Acp5Cre+:Oxtrfl/fl mice indicates a prominent action of OXT in stimulating osteoclastogenesis. In contrast, we found that in pregnant and lactating Col2.3Cre+:Oxtrfl/fl mice, elevated OXT inhibits bone resorption and rescues the bone loss otherwise noted during pregnancy and lactation. However, OXT does not contribute to ovariectomy-induced bone loss. Finally, we show that OXT acts directly on OXTRs on adipocytes to suppress the white-to-beige transition gene program. Despite this direct antibeiging action, injected OXT reduces total body fat, likely through an action on OXT-ergic neurons. Consistent with an antiobesity action of OXT, Oxt-/- and Oxtr-/- mice display increased total body fat. Overall, the actions of OXT on bone mass and body composition provide the framework for future therapies for osteoporosis and obesity.

FSH-metabolic circuitry and menopause.

FSH has a primary function in procreation, wherein it induces estrogen production in females and regulates spermatogenesis in males. However, in line with our discoveries over the past decade of non-unitary functions of pituitary hormones, we and others have described hitherto uncharacterized functions of FSH. Through high-affinity receptors, some of which are variants of the ovarian FSH receptor (FSHR), FSH regulates bone mass, adipose tissue function, energy metabolism, and cholesterol production in both sexes. These newly described actions of FSH may indeed be relevant to the pathogenesis of bone loss, dysregulated energy homeostasis, and disordered lipid metabolism that accompany the menopause in females and aging in both genders. We are therefore excited about the possibility of modulating circulating FSH levels toward a therapeutic benefit for a host of age-associated diseases, including osteoporosis, obesity and dyslipidemia, among other future possibilities.

Cytochrome c oxidase dysfunction enhances phagocytic function and osteoclast formation in macrophages.

The mitochondria-to-nucleus retrograde signaling (MtRS) pathway aids in cellular adaptation to stress. We earlier reported that the Ca2+- and calcineurin-dependent MtRS induces macrophage differentiation to bone-resorbing osteoclasts. However, mechanisms through which macrophages sense and respond to cellular stress remain unclear. Here, we induced mitochondrial stress in macrophages by knockdown (KD) of subunits IVi1 or Vb of cytochrome c oxidase (CcO). Whereas both IVi1 and Vb KD impair CcO activity, IVi1 KD cells produced higher levels of cellular and mitochondrial reactive oxygen species with increased glycolysis. Additionally, IVi1 KD induced the activation of MtRS factors NF-κB, NFAT2, and C/EBPδ as well as inflammatory cytokines, NOS 2, increased phagocytic activity, and a greater osteoclast differentiation potential at suboptimal RANK-L concentrations. The osteoclastogenesis in IVi1 KD cells was reversed fully with an IL-6 inhibitor LMT-28, whereas there was minimal rescue of the enhanced phagocytosis in these cells. In agreement with our findings in cultured macrophages, primary bone marrow-derived macrophages from MPV17-/- mice, a model for mitochondrial dysfunction, also showed higher propensity for osteoclast formation. This is the first report showing that CcO dysfunction affects inflammatory pathways, phagocytic function, and osteoclastogenesis.-Angireddy, R., Kazmi, H. R., Srinivasan, S., Sun, L., Iqbal, J., Fuchs, S. Y., Guha, M., Kijima, T., Yuen, T., Zaidi, M., Avadhani, N. G. Cytochrome c oxidase dysfunction enhances phagocytic function and osteoclast formation in macrophages.

FSH Beyond Fertility.

The traditional view of follicle-stimulating hormone (FSH) as a reproductive hormone is changing. It has been shown that FSH receptors (FSHRs) are expressed in various extra-gonadal tissues and mediate the biological effects of FSH at those sites. Molecular, animal, epidemiologic, and clinical data suggest that elevated serum FSH may play a significant role in the evolution of bone loss and obesity, as well as contributing to cardiovascular and cancer risk. This review summarizes recent data on FSH action beyond reproduction.

FSIP1 regulates autophagy in breast cancer.

Fibrous sheath interacting protein 1 (FSIP1) is a cancer antigen expressed in the majority of breast cancer tissues and is associated with poor prognosis. However, the role of FSIP1 in the progression and drug sensitivity of triple-negative breast cancer (TNBC) has not been explored. Here, we show that FSIP1 deficiency by shRNA-mediated knockdown or CRISPR-Cas9-mediated knockout significantly inhibits the proliferation and invasion of TNBC cells and impairs chemotherapy-induced growth inhibition in vivo. Computational modeling predicted that FSIP1 binds to ULK1, and this was established by coimmunoprecipitation. FSIP1 deficiency promoted autophagy, enhanced AMP-activated protein kinase (AMPK) signaling, and decreased mechanistic target of rapamycin (mTOR) and Wnt/ß-catenin activity. In contrast, knockdown of AMPK or inhibition of autophagy restored the sensitivity to chemotherapy drugs in TNBC cells. Our findings uncover a role of FSIP1 as well as mechanisms underlying FSIP1 action in drug sensitivity and may, therefore, aid in design of TNBC therapies.

FSH, Bone Mass, Body Fat, and Biological Aging.

The Study of Women's Health Across the Nation has taught us that impending ovarian failure during late perimenopause is associated with a sharp rise in serum FSH, which coincides with the most rapid rate of bone loss and the onset of visceral adiposity. At this time in a woman's life, serum estrogen levels are largely unaltered, so the hypothesis that hypoestrogenemia is the sole cause of bone loss and visceral obesity does not offer a full explanation. An alternative explanation, arising from animal models and human data, is that both physiologic aberrations, obesity and osteoporosis, arise at least in part from rising FSH levels. Here, we discuss recent findings on the mechanism through which FSH exerts biological actions on bone and fat and review clinical data that support a role for FSH in causing osteoporosis and obesity. We will also provide a conceptual framework for using a single anti-FSH agent to prevent and treat both osteoporosis and obesity in women across the menopausal transition.

Epitope-specific monoclonal antibodies to FSHß increase bone mass.

Pituitary hormones have long been thought solely to regulate single targets. Challenging this paradigm, we discovered that both anterior and posterior pituitary hormones, including FSH, had other functions in physiology. We have shown that FSH regulates skeletal integrity, and, more recently, find that FSH inhibition reduces body fat and induces thermogenic adipose tissue. A polyclonal antibody raised against a short, receptor-binding epitope of FSHß was found not only to rescue bone loss postovariectomy, but also to display marked antiobesity and probeiging actions. Questioning whether a single agent could be used to treat two medical conditions of public health importance--osteoporosis and obesity--we developed two further monoclonal antibodies, Hf2 and Mf4, against computationally defined receptor-binding epitopes of FSHß. Hf2 has already been shown to reduce body weight and fat mass and cause beiging in mice on a high-fat diet. Here, we show that Hf2, which binds mouse Fsh in immunoprecipitation assays, also increases cortical thickness and trabecular bone volume, and microstructural parameters, in sham-operated and ovariectomized mice, noted on microcomputed tomography. This effect was largely recapitulated with Mf4, which inhibited bone resorption by osteoclasts and stimulated new bone formation by osteoblasts. These effects were exerted in the absence of alterations in serum estrogen in wild-type mice. We also reconfirm the existence of Fshrs in bone by documenting the specific binding of fluorescently labeled FSH, FSH-CH, in vivo. Our study provides the framework for the future development of an FSH-based therapeutic that could potentially target both bone and fat.