Mone Zaidi - US grants

During bone resorption, an osteoclast generates and, as a consequence, becomes exposed to millimolar Ca2+ levels. The goal of the present study is to define pathways through which changes in extracellular Ca2+, and corresponding changes in intracellular Ca2+, are transduced first into changes in intranuclear Ca2+, and then into alterations in gene expression. Notably, the osteoclast plasma membrane represents a unique site for the location of a ryanodine receptor. Ryanodine receptors are Ca2+-permeable channels that normally reside in microsomal membranes. However, in the osteoclast, a plasma membrane-resident, type II, ryanodine receptor isoform serves to sense changes in extracellular Ca2+, hence the term Ca2+ sensor. It has been shown recently that ryanodine receptors are also located in nuclear membranes at which site they gate the flux of Ca2_+ into the nucleoplasm. By making separate measurement of nuclear envelope and nucleoplasmic Ca2+ levels, as well as by performing confocal microscopic studies using epitope-specific antisera, we first propose to determine whether Ca2+ transport across the osteoclast nuclear membrane occurs through ryanodine receptor-gated Ca2+ channels. Such studies are particularly relevant to recent observations showing that nuclear Ca2+ levels regulate gene expression directly. Thus, by applying the in situ reverse transcriptase polymerase chain reaction (RT=PCR) to isolated single osteoclasts, we propose to investigate whether Ca2+ modulates expression of the gene for the osteoclast cytokine, interleukin-6, as well as that of its receptor. Finally, we will also assess whether the secreted interleukin-6 attenuates Ca2+ sensing, a phenomenon that, we believe, should allow a resorbing osteoclast 'escape' (or recover) from Ca2+-induced inhibition. Important therapeutic implications should follow from a better understanding of mechanisms that underlie extracellular Ca2+ sensing by, and Ca2+ homeostasis in, the osteoclast.

An osteoclast is unique in being exposed locally to high millimolar Ca2+ levels resulting from mineral dissolution. We recently discovered that high ambient Ca2+ activates a ryanodine receptor-gated Ca2+ channel located, quite unusually, in the cell's plasma membrane. Classically, however, ryanodine receptors function as Ca2+ release channels at their microsomal, and more recently discovered, nuclear membrane locations. They are gated primarily by Ca2+ and the second messenger, cyclic adenosine diphosphate-ribose (cADPr). The latter is formed from NAD+ following its cyclization by the enzyme, CD38 (an ADP-ribosyl cyclase). We provide compelling preliminary data demonstrating that: (a) CD38 mRNA is expressed in the osteoclast; (b) immunoreactive CD38 is localized to the cell's plasma membrane; (c) when activated, CD38 triggers a cytosolic Ca2+ signal likely via cADPr generation from NAD+; and (d) CD38-induced Ca2+ signaling is associated with resorption inhibition and enhanced interleukin-6 secretion. Our goal is to examine whether CD38, by converting NAD+ to cADPr, regulates osteoclast Ca2+ homeostasis and hence, bone resorption and cytokine gene expression. Specifically, we will first examine whether cADPr, generated from NAD+ through CD38 catalysis, triggers cytosolic and nucleoplasmic Ca2+ transients via ryanodine receptor activation at the plasma, microsomal, and nuclear membranes. For this, we will use 'functional' CD38 antibodies and cADPr inhibitors together with state-of-the-art single cell and nuclear Ca2+ microfluorimetry, patch clamp electrophysiology, and VOXEL-assisted confocal microscopy. Next, using the pit (resorption) assay, together with in situ RT-PCR cytoimaging and the RNase protection assay, we propose to investigate the mechanism through which CD38 inhibits bone resorption, but paradoxically enhances interelukin-6 expression. Finally, we shall study any possible feedback regulation of CD38 gene expression by interleukin-6 and Ca2+ again utilizing in situ RT-PCR cytoimaging and RNase protection assays. To determine whether effects are transcriptional, and having cloned the full-length CD38 cDNA, we shall soon be poised to measure activity of the CD38 gene promoter following its cloning and characterization from a rabbit genomic library. Taken together, the studies should provide mechanistic insights into the role of the NAD+/CD38/cADPr/Ca2+ system in osteoclast control.

CD38 (ADP-ribosyl cyclase) catalyses the cyclization of NAD+ to cyclic ADP-ribose (cADPr), a second messenger that releases Ca2+ from ryanodine receptor-gated Ca2+ stores. During the current funding period, we have made several key observations. Firstly, we showed that CD38 is expressed in abundance in the osteoclast, and its stimulation by an activating antibody triggers Ca2+ release via ryanodine receptors resulting resorption inhibition. Second, we found that the CD38-/- mouse displays profound osteoporosis characterized by excessive bone loss due to increased osteoclast formation and resorptive function. Finally, to study the topological requirements for NAD+-induced Ca2+ signaling, we made several mutated CD38 constructs that did not localize the plasma membrane. We demonstrated that microsomal membrane, rather that the plasma membrane CD38 is necessary for NAD+-induced Ca2+ release in NIH3T3 cells. Our hypothesis is that CD38 negatively regulates osteoclast formation and function by acting as an intracellular ?NAD? receptor? that couples intermediary metabolism for Ca2+ signaling. We will explore this hypothesis in two specific aims. Specific aim 1 will focus on the function of CD38 as a negative regulation of osteoclast formation. We will further characterize the bone phenotype of CD38-/- mice using densitometry, 3-dimensional pQCT imaging, histomorphometry, and biomechanical testing. We will also examine ex vivo osteoclast formation in CD38-/- mice, and more importantly, determine whether the cellular phenotype (a) is mediated via supporting osteoblasts, and (b) can be rescued by adenoviral CD38 transfer. Specific aim 2 will investigate whether CD38, as a putative NAD+ regulator, negatively regulates the resorptive function of mature osteoclasts. We will first examine the localization and function of endogenous and recombinant full-length CD38 and each CD38 mutant in the osteoclast by confocal microscopy, immunoblotting and cyclase assays. We will next characterize NAD+-induced cytosolic Ca2+ responses in osteoclasts infected with full length CD38 or its mutated constructed. We will also investigated whether CD38 inhibits osteoclastic bone resorption in the pit assay by sensitizing both microsomal and plasma membrane ryanodine receptors. Finally, we will determine whether CD38 over-expression in transgenic mice results in an osteopetrotic phenotype and dysfunctional osteoclasts.

[unreadable] DESCRIPTION (provided by applicant): Calcineurin is a Ca2+/calmodulin activated phosphatase that couples Ca 2+ pulses to gene transcription. Importantly, it is the target for two of the most widely used immunosuppressants, cyclosporin A and FK506, both of which inhibit bone formation and cause skeletal loss. We now find that deletion of the alpha isoform of calcineurin A impairs, by -50%, the ability of hematopoetic stem cell precursors to form osteoclasts in response to RANK-L, a key osteoclastogenic cytokine. This effect is mimicked by both cyclosporin A and FK506, suggesting that calcineurin is downstream of RANK-L. We also find that RANK-L stimulates the expression and nuclear import of calcineurin's primary substrate, NFATc1. Likewise, Ca2+ triggers NFATc1 import, stimulates osteoclast formation, and inhibits the transcription of an anti-osteoclastogenic gene, CD38. Dominant-negative NFAT attenuates, while constitutively active NFAT enhances osteoclast precursor differentiation. However, apart from NFATc1, calcineurin also binds to and dephosphorylates the transcription inhibitor, lkB, likely as a means to prevent unrestricted osteoclastogenesis. Thus, our central hypothesis is that calcineurin and NFATc1 are both required for the osteoclastogenesis induced by RANKL and Ca2+ that is in turn regulated by the concomitant dephosphorylation of IkB. Specific Aim 1 will determine whether calcineurin is necessary for RANK-L-induced osteoclast formation through gain- and loss-of-function experiments involving (a) transduction of constitutively active and dominant negative calcineurin mutants as TAT fusion proteins, (b) stably transfecting osteoclast precursors with isoform-specific calcineurin snRNAs, and (c) rescuing the defective osteoclastogenesis in Aa-/- stem cells. Specific Aim 2 will examine the relative contributions of NFATc1 and IkBa in modulating RANK-L-induced osteoclastogenesis. Specific Aim 3 will investigate whether the effects of Ca2+ on osteoclast formation and target gene expression require calcineurin and NFATc1. For both the latter aims, we will combine cross-linking, co-immunoprecipitation, dephosphorylation, and nuclear translocation assays with constitutively active and dominant-negative NFAT transduction in vitro. In Specific Aim 2, the two mutants will also be over-expressed in osteoclasts in transgenic mice and effects on the skeleton examined by bone densitometry, micro-computerized tomography, histomorphometry, and remodeling marker measurements. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Calcineurin is a Ca2+/calmodulin activated phosphatase that couples Ca 2+ pulses to gene transcription. Importantly, it is the target for two of the most widely used immunosuppressants, cyclosporin A and FK506, both of which inhibit bone formation and cause skeletal loss. We now find that deletion of the alpha isoform of calcineurin A impairs, by -50%, the ability of hematopoetic stem cell precursors to form osteoclasts in response to RANK-L, a key osteoclastogenic cytokine. This effect is mimicked by both cyclosporin A and FK506, suggesting that calcineurin is downstream of RANK-L. We also find that RANK-L stimulates the expression and nuclear import of calcineurin's primary substrate, NFATc1. Likewise, Ca2+ triggers NFATc1 import, stimulates osteoclast formation, and inhibits the transcription of an anti-osteoclastogenic gene, CD38. Dominant-negative NFAT attenuates, while constitutively active NFAT enhances osteoclast precursor differentiation. However, apart from NFATc1, calcineurin also binds to and dephosphorylates the transcription inhibitor, lkB, likely as a means to prevent unrestricted osteoclastogenesis. Thus, our central hypothesis is that calcineurin and NFATc1 are both required for the osteoclastogenesis induced by RANKL and Ca2+ that is in turn regulated by the concomitant dephosphorylation of IkB. Specific Aim 1 will determine whether calcineurin is necessary for RANK-L-induced osteoclast formation through gain- and loss-of-function experiments involving (a) transduction of constitutively active and dominant negative calcineurin mutants as TAT fusion proteins, (b) stably transfecting osteoclast precursors with isoform-specific calcineurin snRNAs, and (c) rescuing the defective osteoclastogenesis in Aa-/- stem cells. Specific Aim 2 will examine the relative contributions of NFATc1 and IkBa in modulating RANK-L-induced osteoclastogenesis. Specific Aim 3 will investigate whether the effects of Ca2+ on osteoclast formation and target gene expression require calcineurin and NFATc1. For both the latter aims, we will combine cross-linking, co-immunoprecipitation, dephosphorylation, and nuclear translocation assays with constitutively active and dominant-negative NFAT transduction in vitro. In Specific Aim 2, the two mutants will also be over-expressed in osteoclasts in transgenic mice and effects on the skeleton examined by bone densitometry, micro-computerized tomography, histomorphometry, and remodeling marker measurements. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The New York Academy of Sciences (NYAS) is planning a conference titled, "Skeletal Development and Remodeling in Health, Disease and Aging" to be held from May 18 to March 21, 2005 in New York City. The conference is being organized by Mone Zaidi, MD, PhD, FRCP, Professor of Medicine and Director, Mount Sinai Bone Program, Mount Sinai School of Medicine. It is an ideal time for a conference on this subject, as the past decade has seen an enormous expansion in our understanding of how the skeleton is remodeled and repaired. Using state-of-the-art molecular technologies including Tran genesis, gene knockout, and gene array, scientists are gaining a better understanding of both the precise pathways through which osteoblasts lay down new bone and how osteoclasts remove old bone. These studies have helped clarify basic biological properties of the skeleton, how it responds to hormonal, cytokine and mechanical stimulation, as well as clarified path physiology. The eventual goal of such discoveries is to lay down a firm scientific foundation for the identification of novel cellular and molecular targets for future drug development. The conference will be organized along four themes: Skeletal Development and Repair, Molecular Endocrinology of Bone, Bone Cell Biology and Conservation of Skeletal Integrity, while enabling participants to engage in a highly focused discussion within each category. The final day will specifically focus on current and emerging therapies for osteoporosis. The conference will draw wide spread participation among high powered contributors in bone and cartilage biology research, as well as junior faculty member and senior postdoctoral research fellows working in the field. We also expect the meeting to be attended also by biologists, chemists, and medical doctors whose research and/or patients stand to benefit from the information exchanged at the conference. A local forum, which will provide the non-scientific public an informal opportunity to learn about the latest research and therapies emerging in the field, is also planned.

[unreadable] DESCRIPTION (provided by applicant): The New York Academy of Sciences (NYAS) is planning a major 4-day conference entitled "Skeletal Biology and Medicine" to be held April 25-28, 2007 at the New York Academy of Sciences and the Mount Sinai Medical Center, New York, NY. The conference is being organized by Mone Zaidi, MD, PhD, FRCP, Professor of Medicine and Physiology, Director Mount Sinai Bone Program. New insights into the mechanisms of bone development including cellular and mechanical factors, receptors, and signaling pathways have contributed to our understanding of both normative and pathologic states of bone and the skeleton. Bone cells undergo dynamic processes whereby they are influenced by numerous genetic factors and cellular mediators. Recent findings are shaping therapeutic directions to focus on multiple modes of intervention involving anti-resorptive treatments and anabolic agents. Also significant is the role that other physiological systems or disease states such as the immune system, inflammation, infection and cancer have on bone and musculoskeletal health. A better understanding of the fundamentals of skeletal biology, the pathophysiology associated with skeletal disease and the molecular and genetic basis for some of these disorders will impact on the ability to find effective treatments. This conference will combine basic, clinical and translational research in a forum designed to provide the most current information on aspects of skeletal development and its relationship to bone disease and its treatment. Specific objectives of this conference are to: 1) examine the physiology of bone development; 2) explore the pathophysiology, of skeletal diseases; 3) discuss improved treatment paradigms for bone disease and 4) disseminate the proceedings by print and electronic means so that a wider audience can benefit from the insights shared. [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Post-menopausal osteoporosis, a global public health problem, has for decades been attributed solely to declining estrogen levels, and although follicle stimulating hormone (FSH) levels rise sharply in parallel, a direct effect of FSH on the skeleton has never been explored. The only ascribed function of FSH is ovarian estrogen secretion. We speculate that, in addition to declining estrogen, FSH drives the decreases in bone mass during the early menopause by stimulating the osteoclast, the cell that resorbs bone. Mice devoid of FSH or its receptor do not display hyper-resorption or bone loss despite being severely hypogonadal. That FSH is pro-resorptive is supported by in vitro evidence for a G-protein coupled FSH receptor (FSHR) on the osteoclast. FSH also enhances the release of the osteoclastogenic cytokine TNFa from osteoclast precursors, and additionally, promotes osteoclast survival. The hypothesis emerging from our study, supported by the tight correlations between bone mass and serum FSH in humans, is that circulating FSH directly stimulates osteoclastic bone resorption. We will therefore investigate in Specific Aim 1 whether FSH causes bone loss in vivo independently of lowered estrogen. For this, we will administer or over-express FSH in mice lacking the two estrogen receptors, ER??-/-, as well as GnRH-deficient hpg mice. We will also examine whether selective FSHR deletion in the osteoclast will prevent ovariectomy-induced bone loss, and whether transgenic reconstitution of the FSHR in FSHR-/- osteoclasts will restore resorptive activity. In Specific Aim 2 we determine the mechanism of the FSH effect. We will first study the mechanism of FSH-induced TNFa expression, and then, using TNFa-/- mice, elucidate if the entire effect of FSH is TNFa-dependent. Finally, using Akt-deficient cells, we will also determine whether the pro-survival action of FSH is Akt-mediated. If FSH is proven to be pro-resorptive in vivo, we envisage attenuating FSH in humans to a skeletal advantage without compromising ovarian function, for example by a monoclonal antibody. The latter premise arises from our observation that FSH haploinsufficiency in mice increases bone mass, while sparing the ovaries. The significance of this work thus lies not only in our challenging an archetypal paradigm, estrogen deficiency, as being the full explanation for menopausal bone loss, but also in establishing that pituitary hormones, such as FSH, act beyond traditional target endocrine organs.

DESCRIPTION (provided by applicant): The New York Skeletal Biology and Medicine (NYSBM) is the third international conference in a series of biennial meetings that began in 2005, initially under the joint auspices of the New York Academy of Sciences and Mount Sinai School of Medicine. The target audience for this conference comprises of basic scientists, clinical investigators and clinicians with interests in diverse disciplines of endocrinology, physiology, cell biology, pathology, genetics, molecular biology, rheumatology, radiology, orthopedics and oncology as they relate to bone development, homeostasis and health. The previous conferences have had up to 200 participants, of which up to 80 experts in various areas of basic and clinical bone disease were invited to speak or chair sessions. The past years'meetings have been cherished by the participants and attendees from all parts of the world. The past meeting, in particular, featured amongst other plenary and invited lectures, Dr. Aaron Ciechanover, Nobel Laureate in Chemistry (2004) from Haifa, Israel. It also featured the Rosalyn Yalow Lecture given by John Potts (Harvard Medical School) and the Gideon Rodan Memorial Lecture given by Gerard Karsenty (Columbia). The conference now commands the reputation as being a premiere meeting in skeletal metabolism, and has therefore become an imperative. The conference provides vital insights into the mechanisms of bone development and restructuring, including cellular and mechanical triggers, receptors and signaling pathways. The past two meetings have contributed significantly to our understanding of both normative and pathologic states of bone and the skeleton. Bone cells undergo dynamic processes, where by they are influenced by genetic factors and cellular mediators. Recent findings are shaping therapeutic directions to focus on multiple modes of intervention involving anti-resorptive and importantly anabolic agents. Also significant is the role that other physiological systems or disease states such as the immune system, inflammation, diabetes, infection and cancer have on bone and musculoskeletal health. A better understanding of the fundamentals of skeletal biology, the pathophysiology associated with skeletal disease and the molecular and genetic basis of these disorders will impact on our ability to find effective treatments. The 2009 NYSMB will combine basic, clinical and translational research in a forum designed to provide the most current information on aspects of skeletal development and its relationship to bone disease and its treatment.

DESCRIPTION (provided by applicant): Thyrotoxic osteoporosis, which is accompanied by a high fracture risk, is known to arise from pro-resorptive effects of high thyroid hormone. We reported that TSH-signaling deficient mice lacking the TSH receptor (TSHR) display severe osteoporosis, suggesting that low TSH levels also contribute to thyrotoxic bone loss. That haploinsufficient euthyroid TSHR mice had an equally profound phenotype suggested that the effects of TSH were independent of thyroid hormones. Nonetheless, it remains unclear whether TSHR activation by stimulating antibodies in Graves'disease reduces the hyperthyroid bone loss that is due to high thyroid hormones and low TSH. We further showed that TSHR activation inhibits osteoclast formation, function and survival, as well as the production of TNFa. When TNFa is ablated from TSHR-/- osteoclasts the enhanced osteoclastogenesis and osteopenia are both rescued, suggesting that TNFa plays a key role in thyrotoxic bone disease. Recently, we observed that TSH, when injected intermittently as far apart as once every two weeks, prevented and restored ovariectomy-induced bone loss by inhibiting bone resorption and stimulating bone formation. We hypothesize that TSH preserves the skeleton through potent anti-resorptive and anabolic actions, and that a loss of these actions contributes to the bone loss of hyperthyroidism. We will use genetically modified mice and state-of-the-art molecular approaches to understand the role of TSH in hyperthyroid bone loss. We will first attempt to rescue the TSHR-/- phenotype by deleting TNFa or its receptors, p55 or p75, in double mutants, or by transgenically reconstituting TSHRs in TSHR-/- osteoclasts or osteoblasts. Next, we will determine whether stimulating anti-TSHR antibodies given by injection or produced in vivo by adeno-TSHR immunization attenuate hyperthyroid bone loss. Finally, using mice in which TSHRs are restored cell-selectively in osteoclasts or osteoblasts on a TSHR-/- background, we will examine which cell contributes to the prevention and restoration of post-ovariectomy bone loss by TSH. These foundation studies should allow us to consider skeletal protection by TSH in post-menopausal women whose TSH levels are suppressed by thyroxine therapy for non-cancer causes. PUBLIC HEALTH RELEVANCE: Hyperthyroidism affects one in 1000 American women and is accompanied by osteoporosis and a high fracture risk. We showed that decrements in the pituitary hormone TSH accompany the high thyroid hormone levels, both of which contribute to the bone loss. This proposal examines the molecular mechanism through which TSH acts directly on the skeleton.

DESCRIPTION (provided by applicant): The 2011 New York Skeletal Biology and Medicine (NYSBM) conference is the fourth international conference in a series of biennial meetings that began in 2005, initially under the joint auspices of the New York Academy of Sciences and Mount Sinai School of Medicine. The target audience for this conference comprises of basic scientists, clinical investigators and clinicians with interests in diverse disciplines of endocrinology, physiology, cell biology, pathology, genetics, molecular biology, rheumatology, radiology, orthopedics and oncology as they relate to bone development, homeostasis and health. The previous conferences have had up to 250 participants, of which up to 85 experts in various areas of basic and clinical bone disease were invited to speak or chair sessions. The past years'meetings have been cherished by the participants and attendees from all parts of the world. Featured amongst other plenary lectures in 2009, were the Diane Wolf Memorial Inaugural Lecture given by Dr. Victor Dzau (Duke University), the 2nd Rosalyn Yalow Lecture given by Sir Michael Berridge (University of Cambridge) and the 2nd Gideon Rodan Memorial Lecture given by Roland Baron (Harvard). The conference now commands the reputation as being a premiere reference meeting in skeletal biology. It provides vital insights into the mechanisms of bone development and restructuring, including cellular and mechanical triggers, receptors and signaling pathways. It also touches upon the significant role that other physiological systems or disease states such as the immune system, inflammation, diabetes, infection and cancer play in musculoskeletal health. Recent findings are also shaping therapeutic directions to focus on multiple modes of intervention involving anti-resorptive and importantly anabolic agents. A better understanding of the fundamentals of skeletal biology, the pathophysiology associated with skeletal disease and the molecular and genetic basis of these disorders is thus likely to impact on our ability to find effective treatments. The 2011 NYSMB will combine basic, clinical and translational research in a forum designed to provide the most current information on aspects of skeletal development and its relationship to bone disease and its treatment.

DESCRIPTION (provided by applicant): We reported recently that the posterior pituitary hormone oxytocin (OT) thought primarily to regulate lactation and social bonding is anabolic to the skeleton. Heterozygote mice with circulating OT reduced to half that of wild type mice showed no lactation defect, but instead displayed severe osteopenia and reduced bone formation. Bone resorption remained unaffected, likely due to the opposing actions of OT on osteoclast formation and function. Together the data suggest that the bone forming action of OT is dominant, and perhaps more ancient than its effect on the breast. Expectedly, OT injected into wild type mice increased bone mass by enhancing osteoblastogenesis, whereas in stromal cell cultures, it stimulated mineralized colony formation. Furthermore, we found recently that bone marrow osteoblasts not only possess abundant OT receptors (Oxtrs), but also produce OT. This means that an autocrine OT circuit in marrow could potentially amplify the bone forming action of injected OT. We hypothesize that OT is an anabolic bone hormone, and that its action is mediated through an osteoblast Oxtr, which when stimulated by OT, produces OT locally in an autocrine loop. In Specific Aim 1, we will investigate whether injected OT can restore the lost bone in aging and hypogonadal mice. In Specific Aim 2, we will elucidate, through cell-selective genetic ablation of the Oxtr, whether osteoblasts, osteoclasts or both cells participate in the action of OT. In Specific Aim 3, we will determine whether marrow OT is required for the bone forming action of injected OT using OT-/- mice and bone marrow transplantation. Our studies should help establish OT and Oxtrs as potential targets for treating human osteoporosis.

DESCRIPTION (provided by applicant): Gaucher Disease is a debilitating lysosomal storage disorder characterized by striking visceral enlargement and a high risk of crippling fractures. It s caused by mutations in the glucocerebrosidase (GBA1) gene that impair -glycosidase, an enzyme required for sphingolipid catabolism. While enzyme replacement therapy (imiglucerase) is effective, its effects on fracture risk are not fully understood. To identify new therapeutic targets for non-neuronopathic type 1 GD (GD1), attempts have been made to knock in mutations and delete Gba1 in mice. We have successfully deleted Gba1 in cells of the hematopoietic and mesenchymal cell lineage using an Mx1 promoter. Our Mx1-Cre:GD1 mouse phenocopies human GD1 almost in its entirety, displaying severe hepatosplenomegaly, cytopenia, and osteoporosis. The mouse also displays Th1 and Th2 hypercytokinemia and immune cell defects, which might contribute not only to the increased risk of lymphoproliferative malignancy, but also to the bone disease. Our data further show that the osteoporosis is due to a defect in osteoblastic bone formation, not osteoclastic bone resorption. We find that reduced osteoblast viability noted in stromal cell cultures from Mx1-Cre:GD1 mice is recapitulated by exposure to sphingosine, a sphingolipid that accumulates in GD1. We hypothesize that, despite the immune cell dysfunction that may affect bone, the osteopenia noted in Mx1-Cre:GD1 mice arises mainly from the direct action of sphingosine on the osteoblast, thus lowering bone formation. Therefore, in Specific Aim 1, we will determine whether the bone formation defect is autonomous, and if so, which bone cell - osteoblast, osteocyte, or osteoclast - drives it. For this we will delete Gba1 in the three cell types, respectively, using Col2.3-Cre, Dmp1-Cre and CathK-Cre mice. In Specific Aim 2, we will lower sphingolipid levels by inhibiting or deleting glucosylceramide synthase (Gcs), an enzyme upstream of Gba1. For this, we will inject Mx1-Cre:GD1 mice with eliglustat tartrate, a Gcs inhibitor, and, in parallel, generate mice lacking Gba1 and Gcs in the same cells. Of note, we find that eliglustat tartrate lowers serum GL-1 and reverses the visceromegaly and cytopenia in GD1 patients. Finally, in Specific Aim 3, to hone in on the specific lipid that causes osteoblast inhibition, we will lower sphingosine, but not LysoGL-1 levels. We hypothesize that, as the extralysosomal enzyme Gba2 converts LysoGL-1 to sphingosine, Gba2 deletion or its inhibition by AMP-DNM should reverse the osteopenia in Mx1-Cre:GD1 mice. To further examine the action of sphingosine and other sphingolipids on the osteoblast, we will study differentiation, cell cycling and apoptosis in vitro. Our investigations should not only define the target cell and responsible molecule for the osteopenia in GD1, but also identify new therapeutic targets, both upstream (Gcs) and downstream (Gba2) of Gba1, for GD1-associated and other common types of osteoporosis.

DESCRIPTION (provided by applicant): Cigarette smokers suffer from severe osteoporosis, and are therefore at an exceptionally high risk of skeletal fracture. However, the mechanism through which smoke causes bone loss remains unclear. We find that BaP (benzo[a]pyrene) and TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin), two of over 200 known chemicals found in cigarette smoke, trigger the aryl hydrocarbon receptor (Ahr) and the cytochrome P450 (Cyp1) enzymes to stimulate bone removal. Despite these observations, several gaps in our understanding remain. First, we are uncertain which bone cell, osteoclast, osteoblast or osteocyte, primarily mediates this action. Therefore, in Specific Aim 1, we will study the bone phenotype of mice in which the Ahr gene is deleted selectively in each cell type, and then examine the effect of the Ahr agonists BaP and TCDD on skeletal mass and remodeling. Second, we are unclear whether the osteoclast-stimulatory action of Ahr agonists involves the activation of mitochondrial or microsomal Cyp1s, and whether the reactive oxygen species (ROS) so produced mediate this action. Therefore, in Specific Aim 2, we will administer BaP and/or TCDD to knock-in mice expressing Cyp1 proteins either in mitochondria or in microsomes. We will phenotype their skeletons and study ROS production in isolated bone marrow cells. Our previous studies have further shown that ROS activate mitochondria-to-nucleus signals to generate a pro-osteoclastogenic footprint comprising CREB, C/EBP?, NF-?B, NFAT2, and hnRNPA2. In Specific Aim 3, we will determine whether this transcriptional footprint is activated by Ahr agonists. If so, we will utilize siRNA and/or chemical inhibitors to validate the role of each molecule, as well as promoter-reporter assays and ChIP to confirm that the footprint transactivates osteoclast gene expression. These studies should establish the Ahr as a therapeutic target for osteoporosis and unravel, at least to an extent, the molecular basis underlying the osteoporosis noted in smokers.

PROJECT SUMMARY In 2003, we showed that the anterior pituitary hormone thyrotropin (a.k.a. TSH), hitherto known to promote thyroid hormone secretion, is a potent direct regulator of bone mass (Abe et al, Cell, 2003, PMID: 14567913)1. This finding underscored a potential role for low circulating TSH levels in causing the bone loss that has been recognized in patients with hyperthyroidism for over a century2, and, by tradition, has been attributed solely to thyroid hormone excess. We found instead that Tsh receptor-deficient Tshr-/- mice had profound osteoporosis, even when rendered euthyroid1. Importantly, we showed more recently that bone loss in Tshr-/- mice rendered hyperthyroid significantly exceeded that in wild type hyperthyroid mice (Baliram et al, J Clin Invest, 2012, PMID: 22996689)3 ? this finding not only confirmed a direct permissive action of Tshr deficiency on bone, but also buttressed multiple clinical studies showing a tight and highly reproducible correlation between low TSH levels, bone loss, and a high fracture risk in cohorts of hyperthyroid patients worldwide4-24. Furthermore, we found that the osteoclastogenic cytokine, Tnf?, was grossly elevated in Tshr-/- mice, and that its genetic deletion rescued the skeletal phenotype of Tshr deficiency (Hase et al, PNAS, 2006, PMID: 16908863; Sun et al, PNAS, 2013, PMID: 23716650)25,26. This led to the question: which cell ? osteoblast or osteoclast ? drives the effect, and which of the two Tnf receptors, Tnfrsf1a or Tnfrsf1b, mediate the action of Tnf? in Tshr deficiency? Specific Aim 1 will study mice in which the Tshr is deleted selectively in osteoblasts or osteoclasts, as well as double mutants in which both the Tshr and either Tnfrsf1a or Tnfrsf1b are deleted. Complementary co-culture experiments will determine if osteoblastic Tnf? mediates the hyper-resorption in Tshr-/- mice. A second corpus of data, confirmed by other groups27-33, showed that Tsh displays both anti- resorptive and anabolic actions1,34-37. For example, intermittent low dose Tsh injections restored the lost bone 7 months post-ovariectomy, importantly without elevating T4 levels (Sun et al, PNAS, 2008, PMID: 18332426)37. A follow-up question thus arises: is the Tshr a druggable target? Towards finding an answer, we will utilize both genetic and pharmacological approaches. In Specific Aim 2, we will examine whether high Tsh levels are anabolic using mice in which the expression of dominant-negative Tr??337 in the thyrotrope clamps Tsh at ~30-fold higher circulating levels. In Specific Aim 3, we will study the effects of a small molecule activator of the Tshr, MS438, which, we have found, binds Tshrs selectively and with a nanomolar affinity (Latif et al, Thyroid, 2015, PMID: 25333622)38. We also find that MS438 displays pro-osteoblastic and anti-osteoclastic actions in vitro, and does not elevate serum T4. We will thus inject mice with MS438 immediately (?prevention?) or 7-months following (?restoration?) ovariectomy to determine if it can prevent bone loss and/or restore the lost bone. Together, these studies should not only allow an in-depth understanding of Tsh action on bone, but also provide proof-of-concept for a new approach that targets the skeletal Tshr.

PROGRAM SUMMARY Obesity and osteoporosis are global public health hazards that commonly affect older individuals and often co-exist in postmenopausal women. While a restricted armamentarium of therapies is available for osteoporosis, the five approved agents for obesity are limited by poor efficacy and unacceptable side effects. Hence, new approaches to treat these two chronic conditions of aging require a collaborative and rigorous integrative program between independent, but fully interactive laboratories. This U19 builds on a firm foundation of rigorous and transparent research, born from a longstanding collaboration between Drs. Mone Zaidi and Clifford Rosen, the results of which were published last year (Nature, 2017, PMID: 28538730). We identified FSH as a unique target to prevent both obesity and osteoporosis. We raised a polyclonal antibody to Fsh?, which, by blocking its access to the Fsh receptor (Fshr), prevented high-fat-diet-induced obesity and ovariectomy-induced osteoporosis. In addition, our Fsh antibody triggered the appearance of energy- producing ?beige? adipocytes in white adipose tissue. Based on these studies and others, we now postulate that FSH may also be a critical aging hormone. We therefore propose to undertake a comprehensive, multipronged and interdisciplinary study of the effects of blocking Fsh signaling, either pharmacologically using our monoclonal anti-Fsh antibodies or genetically in Fshr-/- mice, on lifespan, fat gain, bone marrow adiposity, and skeletal health in mice. We will also study the mechanism of Fsh action on fat cells using ThermoMice that report ?beiging,? AdipoChaser mice that measure de novo adipogenesis, and state-of-the-art technologies for transcriptome, lipidome and bioenergetic profiling. To buttress our preclinical observations and, with a view of testing our monoclonal antibodies in people, we propose an epidemiological study of older women and men in the AGES-Reykjavik Cohort. We will examine whether serum FSH can be used as a surrogate marker for bone loss, visceral fat gain, bone marrow adiposity, and ultimately, fracture risk. To provide necessary resources across the four investigative sites?Icahn School of Medicine at Mount Sinai, Maine Medical Center Research Institute, University of Texas Southwestern Medical Center and the University of California at San Francisco?we propose three overarching multifunctional cores: a Skeletal and Metabolic Phenotyping Core, an Antibody Production and Testing Core, and an Administrative and Biostatistics Support Core. In sum, our U19 proposal should allow us to break new ground in our understanding of two prevalent disorders of aging, in addition to opening new avenues for therapeutic interventions for our increasing numbers of older adults.

The record of safety and efficacy of the four FDA?approved drugs for erectile dysfunction, namely tadalafil, vardenafil, sildenafil and avanafil, is predicated on their ability to potently inhibit the cellular enzyme, phosphodiesterase 5A (PDE5A). PDE5A hydrolyzes cyclic guanosine monophosphate (cGMP), so that PDE5A inhibitors stimulate the nitric oxide?cGMP?protein kinase G (PKG) pathway. In 1991, we documented for the first time that nitric oxide regulates the osteoclast, the cell that resorbs bone (PMID: 1849281). Multiple studies have since established robust effects of modulating this pathway on both components of bone remodeling ? bone resorption by osteoclasts and bone formation by osteoblasts. Prompted by observations that erectile dysfunction and osteoporosis track together in older men, in men with diabetes, and in men receiving androgen?deprivation therapy for prostate cancer, we sought to investigate the action of tadalafil and vardenafil on bone. The overarching hypothesis was that PDE5A inhibitors could be repurposed for the co?therapy of erectile dysfunction and osteoporosis in men and, even perhaps, solely for osteoporosis in women. We found in mouse studies that tadalafil and vardenafil increased bone mass, importantly in female mice, by stimulating bone formation and inhibiting bone resorption (Kim et al, PNAS, In press). Despite net bone gain, the anabolic action of the drugs was antagonized by a unique sympathetic relay signature originating from central PDE5A?positive neurons in the locus coeruleus, raphe pallidus and hypothalamic paraventricular nucleus. Noting that most osteoporosis drugs are either anti?resorptive or anabolic, any dual?acting agent will have unique value particularly with oral use. Therefore, towards the potential for repurposing PDE5A inhibitors for osteoporosis, our current goal is to understand precisely how the drugs work on bone and to evaluate preclinical efficacy in models of bone loss. In Specific Aim 1, using global and cell?selective knock out mice, we will determine whether the drugs inhibit PDE5A to activate the NO?cGMP?PKG2 pathway in bone, and if so, which cell is the primary target. In Specific Aim 2, we will comprehensively map the distribution of PDE5A in brain at the single transcript level by RNAscope, and interrogate PDE5A?positive nodes through AAV?mediated Pde5a overexpression or knock down. In Specific Aim 3, we will study the ability of tadalafil, vardenafil, sildenafil and/or avanafil to trigger bone gain in 1?year?old aging mice; to prevent hypogonadal bone loss in rats and mice; and to restore bone that is already lost 28 weeks following ovariectomy in rats. Together, our mechanistic and efficacy studies should provide a firm foundation for future clinical trials towards repurposing PDE5A inhibitors for the prevention and treatment of osteoporosis.

PROJECT SUMMARY Alzheimer?s disease (AD) stands out as notable in two respects??in not having a cure and in affecting women more than men. While declining estrogen has been thought to underpin post?menopausal AD, there is a clear clinical correlation of AD with rising levels of follicle?stimulating hormone (FSH). Most notably, there is a ?spike? in cognitive decline in women in the early years of the menopausal transition, when serum estrogen is normal and FSH levels begin to rise. Collaborative studies between the Mount Sinai and Emory groups have identified FSH as a potential driver for AD?and suggest that rising FSH levels may contribute to the disproportionate increase of AD in aging women. Notably, we find that FSH receptors (FSHRs) are expressed in both mouse and human brain, and that the injection of recombinant FSH or ovariectomy (that elevates serum FSH) aggravates AD pathology and cognitive decline in 3xTg mice. Inhibiting the action of FSH in 3xTg or APP/PS1 mice by an FSH?blocking antibody or downregulating Fshr expression in the hippocampus prevents onset of the AD phenotype. The Emory group also provides strong preliminary evidence that FSH upregulates C/EBP?, which activates asparagine endopeptidase (AEP), a ??secretase that cleaves amyloid precursor protein (APP) and Tau??resulting in neuritic plaques and neurofibrillary tangles, respectively. The goal of the transdisciplinary collaboration between the disciplines of endocrinology and neuroscience is to fully understand the mechanism of FSH action on AD?vulnerable brain regions. Thus, in Specific Aim 1, we will map the distribution and cellular localization of the FSHR and its signaling partners CEBPB and LGMN in human and mouse brain using single?transcript technologies. In Specific Aim 2, we will examine the function of the brain FSHR in driving AD pathology and cognitive decline. For this, we will downregulate or overexpress the Fshr in specific brain areas of 3xTg mice by stereotaxically injecting AAV expressing siFshr or Fshr. We will also study the effect of high FSH in 3xTg mice rendered haploinsufficient in Cebpb, and delineate the transcriptomic architecture of FSH?treated human neuronal cells by RNA?seq. In Specific Aim 3, we will determine whether deleting the Fshr or inhibiting FSH action by our murine FSH blocking antibody, Hf2, injected over the lifespan of 3xTg mice can prevent the onset of cognitive decline. To contemporaneously replicate our data, the Emory group will study the effect of treating established cognitive impairment with Hf2 in 18?month?old APP knock?in (KI) mice. In all, our proof?of?concept studies??conducted using our Good Laboratory Practices (GLP) Platform??should not only establish a role for high FSH in driving AD, but also provide a framework for the future testing of our humanized FSH?blocking antibody, Hu6, in aging women.

PROJECT SUMMARY Alzheimer?s disease (AD) stands out as notable in not having a cure among many diseases that affect elderly men and women??in essence, creating an urgent need for a new therapy. It is also unclear why menopausal women have a preponderance of AD, and, while declining estrogen bas been implicated, there is clear clinical correlation with rising levels of follicle stimulating hormone (FSH). We have identified FSH as a target for several aging disorders??osteoporosis, obesity, hypercholesteremia??and now, AD. Inhibiting the action of FSH using blocking antibodies reduces body fat, increases bone mass, lowers serum cholesterol, and from our newest and most exciting results, prevents AD in two mouse models. We have designed a novel humanized monoclonal antibody, Hu6, that binds to a small epitope within the receptor?binding domain of FSH?, thus blocking its action on the FSH receptor (FSHR). Our aspirational goal is to use this lead therapeutic for the therapy and prevention of all four disorders??or, at the very least, AD. Selected from a pool of 30 newly synthesized humanized antibodies, Hu6 displays high?affinity binding to FSH (KD ~7 nM) and thus prevents its action on hippocampal FSHRs to improve cognition in AD mice. These observations, together with its optimal pharmacokinetic profile, lay the groundwork for Hu6 to enter early stage development. In Specific Aim 1, we propose to scale up production of research?grade Hu6; create an optimal formulation; test its physicochemical properties; study the structure of the FSH:Hu6 complex; and manufacture a master cell bank for cGMP?grade Hu6. In Specific Aim 2, we will perform pharmacokinetic studies in Tg32 mice ?humanized? for antibody clearance, and in African green vervet monkeys; determine minimum effective dose(s) in preventing and/or treating AD in 3xTg mice; examine efficacy and safety in young and aged 3xTg mice; and document safety in vervet monkeys. The work will be conducted using Good Laboratory Practice (GLP) standards established at Mount Sinai, Emory, Wake Forest and San Antonio. We have also created cross?functional research teams that will be supported by a distinguished panel of advisors, comprising science and medicine experts, business leaders, and entrepreneurs in biotechnology. Definitive information on dosage, route and frequency, together with early proof of safety should propel us into late stage development and first?in?human studies.