TAU is a low affinity receptor of glucocorticoid and needed for glucocorticoid-caused bone loss

Abstract

Provided are methods for prophylaxis or treatment of inflammatory arthritis or disorders that are associated with glucocorticoid administration. The methods comprise administering an agent that inhibits phosphorylation of Tau. The method can include administration of a glucocorticoid. The methods include treating glucocorticoid-induced osteoporosis (GIO), dexamethasone mediated osteoclastogenesis, and inflammatory arthritis.

Description

[0001] TAU IS A LOW AFFINITY RECEPTOR OF GLUCOCORTICOID AND NEEDED FOR GLUCOCORTICOID-CAUSED BONE LOSS

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application No. 63 / 602,896, filed November 27, 2023, the entire disclosure of which is incorporated herein by reference.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0005] This invention was made with government support under grant numbers R01AR062207 and R01AR061484 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] FIELD

[0007] The present disclosure relates generally to prophylaxis or treatment of glucocorticoid- caused bone loss and inflammatory arthritis using a combination of a glucocorticoid and a Tau kinase inhibitor.

[0008] RELATED INFORMATION

[0009] The discovery of glucocorticoids (GCs) therapeutic effects against rheumatoid arthritis by Hench and colleagues led to the Nobel Prize in Physiology and Medicine in 1950 (7) and has revolutionized clinical practice for treating various inflammatory, autoimmune, and neoplastic diseases owing to GC’s invaluable anti-inflammatory and immunosuppressive actions (2, 3). Since then, synthetic GCs, such as dexamethasone and prednisolone, represent the most widely prescribed drugs in the world (- / , 5). Very recently, high dose dexamethasone was a crucial treatment to people with advanced COVID-19 (6). Although attractive, pharmacologic GCs treatment, particularly with high dosage and prolonged usage, can cause a wide range of side effects, including iatrogenic insulin resistance, high blood glucose, disorders of lipid metabolism, myopathy, increased risk of infection, and glucocorticoid induced osteoporosis (GIO), the most common cause of secondary osteoporosis (3, 7).

[0010] The concomitance of therapeutic and deleterious impacts of GCs drives the research focus in two directions: one is to unravel the mechanisms underlying GCs therapeutic antiinflammatory and immunosuppressive actions, and the second is to search for the knowledge behind the side effects of prolonged exposure to pharmacological concentrations of GCs, including GIO. It is well known that GCs bind to the canonical glucocorticoid receptor (GR) (S) with high affinity, which is at the apex of the regulatory network responsible for GCs’ anti-inflammatory and immunosuppressive actions (2, 4, 9). Selective GC actions or ligand- selective GR activators to maintain its anti-inflammatory while limit its skeletal action was the therapeutic goal for the past over 60 years (70). But there has been no previous breakthrough to date. Therefore, there is an urgent, unmet need to explore the mechanisms behind adverse effects associated with the high dosage and prolonged usage of GCs, and to develop approaches to alleviating the adverse effects. The present disclosure is pertinent to this need.

[0011] SUMMARY

[0012] In one aspect the present disclosure relates to prophylaxis or therapy for disorders associated with GC administration. The method involves administering to an individual in need thereof an agent that inhibits phosphorylation of Tau. The individual in need may be experiencing or at risk of developing one or more adverse effects of GC administration. In another aspect, the individual may have or be at risk for developing an inflammatory condition. In a non-limiting example, the agent that inhibits phosphorylation of Tau is TRxO237.

[0013] In examples, the GC is dexamethasone. In examples, the agent inhibits phosphorylation of Tau at Tau amino acid residue Ser422. In examples, the phosphorylation is by a kinase that is tau tubulin kinase 1 (TTBK1). In one approach, the disclosure comprises administering an effective amount of TRxO237 to an individual who is already undergoing GC treatment. The disclosure also comprises administering a combination of TRxO237 and a GC to an individual in need thereof. In various examples, the individual has or is at risk for developing glucocorticoid-induced osteoporosis (GIO). In examples, the individual has or is at risk for developing GC mediated osteoclastogenesis. In examples, the individual has or is at risk for developing inflammatory arthritis.

[0014] BRIEF DESCRIPTION OF FIGURES

[0015] FIGS. 1A-1Q. High dose dexamethasone binds to Tau. (1A) The schematic diagram of human proteome microarray, which contains over 20,000 individual proteins printed in duplicate, to identify binding partners of high dose and low dose dexamethasone, respectively. (IB, 1C) Human proteome microarray analysis reveals the proteins (in blue) which bind to both low and high dose dexamethasone, and proteins (in green) which selectively bind to high dose dexamethasone. (ID) Principle of DARTS assay for the isolation of proteins protected from degradation by dexamethasone. (IE) Dexamethasone protects two groups of protein bands (highlighted in black rectangle) from degradation in DARTS using whole cell lysate from dexamethasone treated Raw264.7 cells coupled with Coomassie blue staining. (IF) Molecular weight (MW) plot of putative high dose dexamethasone binding proteins identified by human proteome microarray analysis. (1G) The protective effects of serial doses of dexamethasone on Tau and GR from digestion by protease are evaluated by DARTS coupled with immunoblotting. GAPDH is resistant to protease under the condition and serves as a loading indicator. (1H) Quantification of Tau and GR stability treated with serial dosage of dexamethasone assayed by DARTS (n = 3). (II) The interactions between dexamethasone and Tau, assayed by solid phase biding. 10 mM Tau was coated to the plate, and a serial dilution of biotin-labeled dexamethasone was added, followed by HRP-labeled Streptavidin and its substrate (n = 3). Inset shows the Scatchard plot analysis for KD value calculation. (1J-1P) One-step kinetic SPR assay for binding of Tau to different glucocorticoids, as indicated. (IQ) qRT-PCR analysis of Tau mRNA levels in different tissues, as indicated (n = 3). BM, bone marrow. Data are mean ± SD in H, I, Q.

[0016] FIGS. 2A-2L. Tau deficiency abolishes high dose dexamethasone induced osteoporosis in mouse CIA model. (2A) Scheme of experimental strategy to analyze the effects of Tau deficiency on dexamethasone induced bone loss in inflammatory arthritis model. (2B, 2C) Clinical arthritis scores (2B) and images of paw (2C) in WT, Tau- / - and GR- / - male mice with CIA treated with or without dexamethasone for 5 weeks (n = 6 mice for each group). (2D, 2E) Representative images of H&E staining (2D), and quantification of histomorphometric analysis of synovial inflammation (2E) of ankle joints from indicated mice (n = 6 mice for each group). (2F) Whole body bone mineral density in WT, Tau- / - and GR- / - male mice with CIA treated with or without dexamethasone for 5 weeks, measured by DEXA scanning (n = 8 mice for each group). (2G, 2H) Representative reconstructed 3D micro-CT images (2G), and quantification of BV / TV, Tb. Th and Tb. N (H) of femur and tibia trabecular bone of WT, Tau- / - and GR- / - male CIA mice with indicated treatment (n = 6 mice for each group). Scale bar = 250 pm in g. (21, 2J) Representative TRAP staining image (21), and quantification of TRAP+ osteoclast surface per bone surface (Oc.S. / B.S.) (2J) of femur distal metaphysis of WT, Tau- / - and GR- / - male mice with CIA in the same experiment (n = 6 mice for each group). Scale bar = 50 pm in I. (2K) Quantification of osteoblast surface per bone surface (Ob.S. / B.S.) in the indicated mice. (2L) Representative H&E stained images of femur showing osteoblasts (yellow arrows) on the trabecular bone. Scale bar = 20 pm. Data are mean ± SD, P values are calculated by two-tailed unpaired Student’ s / -test. FIGS. 3A-3Q. Tau is required for high dose dexamethasone mediated enhancement of osteoclastogenesis. (3 A) Representative bright-field images of TRAP- positive bone marrow macrophage derived osteoclasts in vitro. Bone marrow derived macrophages isolated from WT, Tau- / - and GR- / - mice are differentiated with 20 ng / ml M- CSF and 50 ng / ml RANKL supplemented with low (10 nM) or high (10 pM) dose of dexamethasone for 7 days. Scale bar = 100 pm. (3B) Quantification of number of TRAP+ multinuclear osteoclast shown in a (n = 20, number per field of view, from three mice per age group). (3C) Quantification of number of nuclei per osteoclast shown in a (n = 25 per group). (3D-3F) Representative microscopic images of resorption pit (3D), quantification of the resorption pit areas (3E) and fluorescence intensity released into media (3F), assayed with the osteoclasts derived from WT, Tau- / - and GR- / - mouse bone marrow macrophage cultured with 20 ng / ml M-CSF and 50 ng / ml RANKL supplemented with low or high dose of dexamethasone for 7 days (n = 3). (3G-3J) Gene expression levels of osteoclast differentiation markers NFATcl (following 3 days of differentiation), TRAP, CTSK and CTR (following 5 days of differentiation) determined by qRT-PCR. Bone marrow derived macrophage isolated from WT, Tau- / - and GR- / - mice are differentiated with 20 ng / ml M- CSF and 50 ng / ml RANKL supplemented with low or high dose of dexamethasone (n = 4). (3K, 3L) Tau knockout and GR knockout Raw264.7 macrophage are generated by using CRISPR-Cas9 technique. The knockout efficiency of Tau (3K) and GR (3L) is determined using immunoblotting. (3M, 3N) Representative bright-field images (3M) and corresponding quantification (3N) of TRAP-positive multinuclear osteoclasts differentiated from Raw264.7 macrophages treated with 50 ng / ml RANKL in the presence of 10 pM dexamethasone for 5 days (n = 3). Scale bar = 100pm. (30) Immunoblotting to demonstrate knockout efficiency in Tau- / -, GR- / - and Tau- / -;GR- / - THP-1 human monocytes generated by CRISPR-Cas9. (3P, 3Q) Representative bright-field images (3P) and corresponding quantification (3Q) of TRAP- positive multinuclear osteoclast differentiated from THP-1 derived macrophages with 50ng / ml RANKL in the presence of lOpM dexamethasone for 14 days (n = 3). Scale bar = 100 pm. Data are mean ± SD, except B, C, which show box- and whisker plots with center line as median, box extending from 25thto 75thpercentile and whiskers extending from minimum to maximum values. P values are calculated by one way ANOVA with Bonferroni post-hoc test (3B, 3C, 3E-3J) and two-tailed unpaired Student’s / -test (3N, 3Q). ns, not significant; * P < 0.05; ** P < 0.01.

[0017] FIGS. 4A-4Q. Combinatorial treatment with dexamethasone and TRxO237 enables anti-inflammatory action while prevents dexamethasone induced bone loss in murine CIA. (4A) Clinical arthritis scores of WT CIA mice treated with or without dexamethasone in the presence or absence of TRxO237, as indicated, for 5 weeks (n = 6 mice for each group). (4B, 4C) Representative images of H&E staining (4B), and quantification of histomorphometric analysis of synovial inflammation (4C) of ankle joints from indicated mice (n = 6 mice for each group). Scale bar = 100 pm in B. (4D) Whole body bone mineral density in WT male mice with CIA treated with or without dexamethasone in the presence or absence of TRxO237, as indicated, for 5 weeks, measured by DEXA scanning (n = 8 mice for each group). (4E, 4F) Representative reconstructed 3D micro-CT images, of trabecular bone of femur (4E) and tibia (4F) of WT male CIA mice with indicated treatment. Scale bar = 250 pm. (4G-4I) Quantification of femur and tibia trabecular BV / TV (G), Tb. Th (4H) and Tb. N (41) in the same experiment (n = 6 mice for each group). (4J, 4K) Representative TRAP staining image (4 J), and quantification of TRAP+ osteoclast surface per bone surface (Oc.S. / B.S.) (4K) of the femur distal metaphysis of WT male mice with CIA in the same experiment (n = 6 mice for each group). Scale bar = 50 pm. (4L, 4M) Representative H&E stained images of femur showing osteoblasts (yellow arrows) on the trabecular bone (4L). Scale bar = 20 pm. Quantification of osteoblast surface per bone surface (Ob.S. / B.S.) in the indicated mice (4M). (4N, 40) H&E and immunohistochemistry staining (4N), and corresponding quantification (40) of p-Tau S422 in femur of WT male mice with CIA in the same experiment (n = 6 mice for each group). The areas defined by black rectangle on each representative image are enlarged as insets. Scale bar = 20pm. (4P, 4Q) H&E and immunohistochemistry staining (4P), and corresponding quantification (4Q) of p-Tau Ser422 in human bone tissue of healthy controls (n = 5) and osteoporosis patients (n = 6). The areas defined by black rectangle on each representative image are enlarged as insets. Scale bar = 100 pm. Data are mean ± SD, P values are calculated by one way ANOVA with Bonferroni post-hoc test (4C, 4D, 4G-4I, 4K, 4M, 40) and two-tailed unpaired Student’s / -test (4Q).

[0018] FIGS. 5A-5J. Threonine at position 212 (Thr212) in Tan is critical for dexamethasone binding. (5A) Scheme of constructs encoding full length (FL) and serial deletion mutants of Tau. (5B) Expressions of Flag-tagged full length (FL) and deletion mutants of Tau. HEK293T cells transfected with Flag-tagged FL and deletion mutants of Tau are lyzed and immunoblotted with anti-Flag antibody. (5C) DARTS assay with FL and mutants of Tau. Cell lysates of FL and deletion mutants of Tau transfected HEK293T cells are incubated with lOpM dexamethasone and subjected to proteinase digestion, followed by immunoblotting with anti-Flag antibody. (5D, 5E) Overview of the binding position of dexamethasone (violet) and prednisolone (green) in Tau protein. The Tau protein is depicted as cyan ribbons, and the important interacting residues are shown as sticks. Hydrogen bonds are represented by dotted yellow lines. (5F, 5G) The 2D ligand-receptor interaction diagram of dexamethasone (5F) and prednisolone (5G) with important interactions shown. The amino acids within 4 A are shown as colored bubbles, where green indicates hydrophobic residues, cyan indicates polar residues, red indicates negatively charged residues, and violet indicates positively charged residues. The magenta arrows show the hydrogen bonds. (5H) Docking score and interacting residues in induced-fit docking predicted docking complexes of Tau with dexamethasone and prednisolone. (51) Expressions of Tau in WT and Tau knockout Raw 264.7 cells transfected with FL or indicated point mutations of Tau, determined by immunoblotting with anti-Tau antibody. (5J) DARTS assay with serial point mutations of Tau. Lysates of Tau knockout Raw264.7 cells transfected with FL or various point mutations of Tau, as indicated, are incubated with lOpM dexamethasone and subjected to proteinase digestion, followed by immunoblotting with anti-Flag antibody. Data (5B, 5C, 51, 5J) are representative of 3 experiments.

[0019] FIGS. 6A-6M. Tau is required for GIO. (6A) Representative reconstructed 3D micro-CT images of femoral and tibia trabecular bone from WT, Tau- / - and GR- / - male mice with or without GIO. Scale bar = 250 pm. (6B) Quantification of trabecular parameters including bone volume / total volume (BV / TV), trabecular thickness (Tb. Th) and trabecular number (Tb. N) in WT, Tau- / - and GR- / - male mice without or with GIO (n = 6 mice for each group). (6C, 6D) Representative TRAP staining image (6C), and quantification of TRAP+ osteoclast surface per bone surface (Oc.S. / B.S.) (6D) of femoral distal metaphysis of WT, Tau- / - and GR- / - male mice in the same experiment (n = 6 mice for each group). Scale bar = 50 pm. (6E-6G) The expressions of osteoclastogenesis related genes in WT (6E), Tau- / - (6F) and GR- / - (6G) femur and tibia after treated with dexamethasone for 5 weeks (n = 6 mice for each group). (6H) Representative H&E stained images of femur showing osteoblasts (yellow arrows) on the trabecular bone. Scale bar = 20 pm. (61) Quantification of osteoblast surface per bone surface (Ob.S. / B.S.) in the indicated mice. (6J) Bone formation rate (BFR) determined by calcein labeling in WT, Tau- / -, GR- / - male mice after treated with or without dexamethasone for 5 weeks (n = 6 mice for each group). (6K) Serum levels of RANKL, OPG, RANKL / OPG, CTX-1, and PINP in the indicated male mice, assayed with ELISA (n = 8 mice for each group). (6L, 6M) Representative reconstructed 3D micro-CT images (6L) and quantification (6M) of trabecular bone of the LI and L2 vertebra of WT, Tau- / - and GR- / - male mice treated with or without dexamethasone for 5 weeks (n = 6 mice for each group). Scale bar = 1 mm in 1. Data are mean ± SD, P values are calculated by two-tailed unpaired

[0020] Student’s / -test.

[0021] FIGS. 7A-7H. Tau Deficiency in female mice recapitulates its effect in male GIO model. (7A) Representative reconstructed 3D micro-CT images of femur and tibia trabecular bone of WT, Tau- / - and GR- / - female mice with or without GIO. Scale bar = 250 pm. (7B) Quantification of trabecular parameters including BV / TV, Tb. Th and Tb. N in WT, Tau- / - and GR- / - female mice with or without GIO (n = 6 mice for each group). (7C) Whole body bone mineral density in WT, Tau- / - and GR- / - female mice treated with or without dexamethasone for 5 weeks, measured by DEXA scanning (n = 8 mice for each group). (7D, 7E) Quantification of TRAP+ osteoclast surface per bone surface (Oc.S. / B.S.) (D) and representative TRAP staining image (7E), of the femur distal metaphysis of WT, Tau- / - and GR- / - female mice in the same experiment (n = 6 mice for each group). Scale bar = 20 pm. (7F) Quantification of osteoblast surface per bone surface (Ob.S. / B.S.) in the indicated mice. (7G) Representative H&E stained images of femur showing osteoblasts (yellow arrows) on the trabecular bone. Scale bar = 50 pm. (7H) Serum levels of RANKL, OPG, RANKL / OPG, CTX-1, and PINP in the indicated female mice, assayed with ELISA (n = 8 mice for each group). Data are mean ± SD, P values are calculated by two-tailed unpaired Student’s / -test.

[0022] FIGS. 8A-8H. The effects of high dose dexamethasone on apoptosis and osteoblastogenesis of bone marrow-derived mesenchymal stem cells in vitro. (8A) Apoptosis in bone marrow derived macrophage isolated from WT, Tau- / - and GR -I- mice, cultured with 20 ng / ml M-CSF along with or without 50 ng / ml RANKL and / or different dose of dexamethasone for 5 days, evaluated by TUNEL assay (n = 3). (8B) Apoptosis in bone marrow cells isolated from WT, Tau- / - and GR- / - mice, stimulated without or with osteo- induction media (OIM) in the presence of different dose of dexamethasone for 5 days, evaluated by TUNEL assay (n = 3). (8C, 8D) In vitro osteoblastogenesis of primary WT, Tau- / - and GR- / - bone marrow cells after osteogenic induction with OIM and different dose of dexamethasone for 21 days, determined with Alizarin Red staining. Representative sating (8C) and quantification of osteoblastogenesis (8D) (n = 4). (8E-8H) The expressions of osteogenesis-related genes Runx2 (8E) (1 day of differentiation), and OCN (8F), BSP (8G) and Collet (8H) (5 days of differentiation), determined with qRT-PCR (n = 3). Data are mean ± SD, P values are calculated by one way ANOVA with Bonferroni post-hoc test.

[0023] FIGS. 9A-9P. Tau Ser422 phosphorylation by high dose dexamethasone through TTBK1 is required for dexamethasone enhanced osteoclastogenesis. (9 A) Immunoblotting of pTau S422, S396 and S202 / T205 in Raw264.7 cells treated with low (L, 10 nM) or high (H, 10 pM) dose of dexamethasone for indicated time. GAPDH is used as a loading control. (9B) Immunoblotting of Tau in Tau knockout Raw264.7 cells transfected with empty vector, full length (FL) or serial point mutations of Tau. (9C, 9D) Representative bright-field images (9C) and corresponding quantification (9D) of TRAP -positive multinuclear osteoclasts differentiated from FL or serial point mutations of Tau transfected Tau- / - Raw264.7 macrophage treated with 50 ng / ml RANKL and 10 pM dexamethasone for 5 days. Scale bar = 100 pm. (9E) Confocal images of Raw264.7 cells labeled with antibodies for Tau and tubulin (left panel), and corresponding fluorescence signal intensity plots of Tau (red) and tubulin (green) vesicles (right panel). Cells are treated with or without 50 ng / ml RANKL and 10 pM dexamethasone. Arrows indicate regions in each cell where fluorescent signal intensity plots are obtained. (9F) Confocal images of Raw264.7 cells labeled with antibodies for p-Tau Ser422 and tubulin (left panel), and corresponding fluorescence signal intensity plots of p-Tau Ser422 (red) and tubulin (green) vesicles (right panel). Cells are treated with or without 50 ng / ml RANKL and 10 pM dexamethasone. Arrows indicate regions in each cell where fluorescent signal intensity plots are obtained. (9G) Experimental design and principle used to identify the potential kinase that are involved in dexamethasone dependent p-Tau Ser422. (9H) Summary of the potential kinases responsible for dexamethasone dependent activation of Tau. (91) Knockdown efficiency of TTBK1 in Raw264.7 cells, measured by Western blotting. (9J) Immunoblotting of dexamethasone activated p-Tau Ser422 in Raw264.7 cells transfected with TTBK1 siRNA or treated with different kinase inhibitors, as indicated. (9K) Densitometry analysis of immunoblotting results shown in j. (9L, 9M) Representative bright-field images (9L) and corresponding quantification (9M) of TRAP-positive multinuclear osteoclast differentiated from Raw264.7 macrophage, which are transfected with TTBK1 siRNA or treated with different kinase inhibitor, followed by stimulating with 50 ng / ml RANKL and lOpM dexamethasone for 5 days. Scale bar = 100 pm. (9N) Confocal images of vehicle or lOpM dexamethasone treated Raw264.7 cells labeled with antibodies for Tau and TTBK1 (left panel), and corresponding fluorescence signal intensity plots of Tau (red) and TTBK1 (green) vesicles (right panel). Arrows indicate regions in each cell where fluorescent signal intensity plots are obtained. (90) Immunoprecipitation from lOpM dexamethasone treated Raw264.7 cells with anti-Tau antibody, and detection of TTBK1 by immunoblotting. (9P) Immunoprecipitation from lOpM dexamethasone treated Raw264.7 cells transfected with Flag-tagged FL or serial deletion mutations of Tau with anti-Flag M2 resins, and detection of TTBK1 by immunoblotting, n = 3 biological replicates. Data are mean ± SD, P values are calculated by one way ANOVA with Bonferroni post-hoc test.

[0024] FIGS. 10A-100. The transcription factor p!05 / p50 is a downstream mediator of phosphorylated Tan in mediation of high dose dexamethasone enhanced osteoclastogenesis. (10A) Schematic for application of biochemical co-purification and mass spectrometry approaches to screen the transcriptional factors recruited to activated Tau by high dose dexamethasone. Raw264.7 cells transfected with Flag or Flag-tagged Tau are treated with or without lOpM dexamethasone for 30 min, followed by precipitation with antiFlag M2 resins. (10B) Summary of the identified transcriptional factors recruited to Tau in 10 pM dexamethasone treated Raw264.7 cells. (10C) Knockdown efficiency of CTCF in Raw264.7 cells, measured by immunoblotting. (10D-10G) Representative bright-field images (10D, 10E) and corresponding quantification (10F, 10G) of TRAP -positive multinuclear osteoclast differentiated from Raw264.7 macrophages treated with 50 ng / ml RANKL and 10 pM dexamethasone for 5 days. Raw264.7 cells are treated with andrographolide (AGL, p50 inhibitor) or transfected with CTCF siRNA before differentiating into osteoclasts. Scale bar = 100pm. (10H) Immunoprecipitation from 10 pM dexamethasone treated Raw264.7 cells with anti-Tau antibody, and detection of pl05 / p50 by immunoblotting. (101) Immunoprecipitation from 10 pM dexamethasone treated Raw264.7 cells transfected with Flag-tagged FL or serial deletion mutations of Tau with anti-Flag M2 resins, and detection of pl05 / p50 by immunoblotting. (10J, 10K) Immunoblotting analysis (10J) and quantification (10K) of relative levels of pl05 and p50 in control, Tau- / - and GR- / - Raw264.7 cells after 10 pM dexamethasone stimulation. (10L, 10M) Immunoblotting analysis (10L) and quantification (10M) of relative levels of pl05 and p50 in TTBK1 siRNA transfected Raw264.7 cells after 10 pM dexamethasone stimulation. (10N, 100) Confocal images of Raw264.7 cells labeled with antibodies for plO5 / p5O (10N), and relative fluorescence intensity of plO5 / p5O in cytoplasm and nuclear are shown (100). n = 3 biological replicates. Data are mean ± SD, P values are calculated by one way ANOVA with Bonferroni post-hoc test (10F, 10G, 10K, 10M) and two way ANOVA with Bonferroni post-hoc test (100).

[0025] FIGS. 11A-11M. FDA-approved drug library screen leads to the identification of TRxO237 as a drug that inhibits dexamethasone induced p-Tau S422 and osteoclastogenesis. (11 A) Development of ELISA-based high-throughput screening assay. Immobilized 12.5 ng Tau antibody binds to a serial dilution of biotinylated antiphosphorylated Tau Ser422 antibody (0 - 400 pg / ml) (n = 3). (11B) Heatmap illustration of effects of drugs on 10 pM dexamethasone stimulation of Tau phosphorylation at Ser422, represented by normalized OD450 fold change relative to no drug control. The colors indicate drug activity, and color intensity represents potency. (11C) Identities of two candidate drugs that show inhibition of dexamethasone induced p-Tau S422 from ELISA based screen. (HD, HE) Immunoblotting analysis (HD) and quantification (HE) of p-Tau S422 in lOpM dexamethasone stimulated 1 pM drug pretreated THP-1 cells (n = 3). (HF) Representative bright-field images of TRAP-positive bone marrow macrophage derived osteoclasts in vitro. Primary WT bone marrow derived macrophages are differentiated with 20 ng / ml M-CSF and 50 ng / ml RANKL supplemented with 10 pM dexamethasone and 1 pM indicated drug for 7 days. Scale bar = 100 pm. (11G) Number of TRAP+ multinuclear osteoclasts (n = 20, number per field of view, from three mice per group) and nuclei per osteoclast a (n = 25 per group) shown in f. (11H) Representative microscopic images of TRAP staining of bone marrow macrophage derived osteoclasts. Bone marrow derived macrophages isolated from WT, Tau- / - and GR- / - mice are differentiated with 20 ng / ml M-CSF and 50 ng / ml RANKL supplemented with high dose of dexamethasone and 1 pM TRxO237 or methylene blue (MB) for 7 days. Scale bar = 100 pm. (HI, 11 J) Quantification of number of TRAP+ multinuclear osteoclast (n = 20, number per field of view, from three mice per age group) (HI) and nuclei per osteoclast (n = 25 per group) (HJ). (UK) Representative microscopic images of resorption activity of osteoclast grown on OsteoAssay plates in vitro in the same experiment. (HL) Quantification of the resorption pit areas shown in 11K. (11M) quantification of fluorescence intensity released into media in osteoclasts derived from WT, Tau- / - and GR- / - mouse bone marrow macrophages cultured with 20 ng / ml M-CSF and 50 ng / ml RANKL supplemented with 10 pM dexamethasone and 1 pM TRxO237 or methylene blue (MB) (n = 3) for 7 days. Data are mean ± SD, P values are calculated by one way ANOVA with Bonferroni post-hoc test.

[0026] FIGS. 12A-12H. TRxO237 protects against GIO in a Tan-dependent manner.

[0027] (12A) Whole body bone mineral density in WT, Tau- / - and GR- / - male mice treated with or without 10 mg / kg body weight dexamethasone and 4 mg / kg body weight TRxO237 for 5 weeks, measured by DEXA scanning (n = 8 mice for each group). (12B) Representative reconstructed 3D micro-CT images of femoral and tibia trabecular bone from WT, Tau- / - and GR- / - male with indicated treatment (n = 6 mice for each group). Scale bar = 250 pm. (12C- 12E) Quantification of trabecular BV / TV, Tb. Th and Tb. N in WT (12C), Tau- / - (12D) and GR- / - (12E) male mice in the same experiment (n = 6 mice for each group). (12F, 12G) Representative TRAP staining image (12F) and quantification of TRAP+ osteoclast surface per bone surface (Oc.S. / B.S.) (12G) of distal metaphysis of the femur from WT, Tau- / - and GR- / - male mice with indicate treatment (n = 6 mice for each group). Sale bar = 50 pm. (12H) Quantification of osteoblast surface per bone surface (Ob.S. / B.S.) in the indicated mice. Data are mean ± SD, P values are calculated by one way ANOVA with Bonferroni post-hoc test, except k which is calculated by student Z-Test.

[0028] FIGS. 13A-13L. TRxO237 is therapeutic against glucocorticoid-induced osteoporosis through inhibition of p-Tau Ser422. (13A-13E) Serum levels of RANKL (13A), OPG (13B), RANKL / OPG (13C), CTX-1 (13D), and PINP (13E) in WT, Tau- / - and GR- / - male mice treated with or without 10 mg / kg body weight dexamethasone and 4 mg / kg body weight TRxO237 for 5 weeks, assayed by ELISA (n = 8 mice for each group). (13F) Quantification of pTauSer422 positive cells in femur of indicated mice in the same experiment. (13G) Representative immunohistochemistry staining of pTauSer422 in femur of indicated mice (n = 6 mice for each group). Scale bar = 50 pm. (13H, 131) Representative reconstructed 3D micro-CT images (13H) and quantification (131) of trabecular bone of the LI and L2 vertebra from WT, Tau- / - and GR- / - male mice treated with or without dexamethasone in the presence or absence of TRxO237, as indicated, for 5 weeks (n = 6 mice for each group). Scale bar = 1 mm in 13H. (13J, 13K) Representative TRAP staining image (13J) and corresponding quantification of TRAP+ Oc.S. / B.S. (13K) of the lumbar vertebrae of indicated mice (n = 6 mice for each group). Sale bar = 50 pm. (13L) Quantification of osteoblast surface per bone surface (Ob.S. / B.S.) in the lumbar vertebrae of the indicated mice. Data are mean ± SD, P values are calculated by one way ANOVA with Bonferroni post-hoc test.

[0029] FIGS. 14A-14J. TRxO237 efficacy to protect against bone loss in GIO model. (14A) Whole body bone mineral density in WT male mice treated with or without dexamethasone and serial doses of TRxO237 for 5 weeks, measured by DEXA scanning (n = 8 mice for each group). (14B) Representative reconstructed 3D micro-CT images of trabecular bone of femur and tibia of WT male mice treated with dexamethasone and difference dosage of TRxO237 (n = 6 mice for each group). (14C-14E) Quantification of trabecular, BV / TV (C), Tb. Th (D) and Tb. N (E) in WT male mice treated without or with dexamethasone along with different dose of TRxO237 for 5 weeks (n = 6 mice for each group). (14F) Serum levels of RANKL, OPG, RANKL / OPG, CTX-1, and PINP in the indicated male mice, assayed by ELISA (n = 8 mice for each group). (14G, 14H) Representative TRAP staining image (G) and quantification of TRAP+ Oc.S. / B.S. (H) of distal metaphysis of the femur of WT male mice in the same experiment (n = 6 mice for each group). Images in lower panel (scale bar = 20 pm) are high-resolution versions of the boxed regions in upper panel images (scale bar = 100 pm). (141, 14 J) H&E and immunohistochemistry staining (I), and corresponding quantification (J) of pTau S422 in femur of WT male mice in the same experiment (n = 6 mice for each group). Scale bar = 20 pm. Data are mean ± SD, P values are calculated by one way ANOVA with Bonferroni post- hoc test.

[0030] FIG. 15. A model to compare glucocorticoid / GR mediated anti-inflammatory and glucocorticoid / Tau mediated osteoclastogenesis / bone resorption pathways. Upon binding to low dose glucocorticoid, GR is translocated into the nucleus and binds to the canonical glucocorticoid response element or tethers with NF-KB to activate antiinflammatory or repress pro-inflammatory gene expression, respectively. Alternatively, high dose glucocorticoid directly binds to Tau, and then recruits TTBK1 kinase to Tau, leading to the phosphorylation of Tau at Ser422. Activated p-Tau Ser422 facilitates the processing of NF-KB 1 pl05 into p50 and the nuclear translocation of NF-KB in RANKL primed macrophages, resulting in enhanced osteoclastogenesis, and eventually causing GIO.

[0031] DETAILED DESCRIPTION

[0032] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0033] Unless specified to the contrary, it is intended that every maximum numerical limitation given throughout this description includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification includes every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0034] The singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise. The terms “a” (or “an”), as well as the terms “one or more,” and “at least one” can be used interchangeably herein. Furthermore, “and / or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other.

[0035] The present disclosure relates to treatment or prophylaxis of GC-induced bone loss, or inflammatory arthritis, or a combination thereof. The disclosure demonstrates, among other aspects, that development of GC-induced GIO can be inhibited by an agent that can be considered a kinase inhibitor (and may also function as a tau protein aggregation inhibitor). Non-limiting examples of these aspects are illustrated by the described use of TRxO237. The structure of TRxO237 is known in the art. TRxO237 is commercially available such as from SELLECKCHEM Catalog# S7762. Without intending to be bound by any particular theory, it is considered that the kinase inhibitory function of TRxO237 may include inhibition of the ability of Tau to act as a signaling molecule to tether TTBK1 kinase to Tau at residue Ser422.

[0036] The amino acid sequence of Tau that has a Ser at position 422 is known in the art. The disclosure is pertinent to any isoform of Tau wherein a Ser is present at position 422 and may be phosphorylated. In an example, the Tau is a human Tau. In an example, the Tau is Tau isoform 2.

[0037] The disclosure also demonstrates that inflammatory arthritis can be treated by administering a combination of a GC, such as dexamethasone, and an agent that inhibits phosphorylation of Tau, such as TRxO237.

[0038] While aspects of the disclosure are demonstrated using dexamethasone, other GC’s that are associated with adverse effects are not necessarily excluded. Such GCs include but are not necessarily limited to prednisone, prednisilone, methylprednisilone, and betamethasone.

[0039] In examples, the individual to whom TRxO237 is administered has a condition that is associated with high-dose GC administration. Those skilled in the art will recognize the meaning of a “high dose” by taking into account the particular GC. Other factors that may be considered for determining a high dose GC administration include but are not necessarily limited to the duration of GC treatment, the frequency of GC administration, and the like. In non-limiting examples, a high dose of a GC comprises an amount that is more than 7.5 mg / day. In examples, a high dose of dexamethasone comprises an amount that is equal to or greater than 20mg / day. In examples, a high dose of a GC comprises an amount of the GC that is sufficient to induce bone loss. In a non-limiting example, lOmg / kg of body weight is a high dose of a GC.

[0040] In certain approaches, an effective amount of a TRxO237 is administered to an individual in need thereof. In examples, an effective amount of TRxO237 is administered to an individual who has or is at risk for developing GIO, or GC mediated osteoclastogenesis. In examples, the individual has or is at risk for developing inflammatory arthritis. In examples, the individual in need does not have Alzheimer's disease or frontotemporal dementia. In examples, an effective amount of a TRxO237, which may be administered concurrently or sequentially with a GC, is administered to an individual in need thereof. In examples, an effective amount is an amount that reduces, eliminates, or prevents one or more signs or symptoms of a disease and / or reduces the severity of the disease. An effective amount may also inhibit or prevent the onset of a disease or a disease relapse. A precise dosage can be selected by the individual physician in view of the patient to be treated. Dosage and administration can be adjusted to provide sufficient levels of TRxO237 and / or the particular GC to maintain the desired effect. Additional factors that may be taken into account include the severity and type of the disease state, age, weight, and gender of the patient, desired duration of treatment, method of administration, time and frequency of administration, drug combination(s), reaction sensitivities, and / or tolerance / response to therapy. In non-limiting examples, 0.10-10.00 mg / kg of TRxO237 is administered. In a nonlimiting example, 0.16 mg / kg, or at least 0.16 mg / kg, of TRxO237 is administered. In nonlimiting examples, 100-250 mg / day is administered.

[0041] The GC and the TRxO237 can be administered to an individual in need thereof using any suitable route, including but not necessarily limited to intravenous and oral routes.

[0042] The following Examples are intended to illustrate but not limit the disclosure.

[0043] Example 1

[0044] Tau is a low affinity receptor of glucocorticoids

[0045] To identify potential and previously unrecognized receptor(s) of glucocorticoids, with a focus on the low affinity receptor(s) that are distinct from the known high affinity receptor GR, we probed genome wide human protein arrays, containing over 20,000 individually printed proteins which represent more than 16,000 human genes, with low and high doses of biotin-labeled dexamethasone (FIG. 1A) (11-13). The results revealed that dexamethasone bound to GR and GPR97, two known receptors of dexamethasone (8, 14), with high affinity given that both low and high dose dexamethasone could bind to them (FIGS. IB and 1C). Considering that the interactions between dexamethasone and its low affinity receptor(s) occur only with high dose dexamethasone, we compared the binding profiles between low and high dose dexamethasone focusing on the proteins which selectively bound to high dose dexamethasone, which led to the identification of 8 proteins (FIGS. IB and 1C). High dose and low dose was 0.2 and 400pM dexamethasone, respectively. The disclosure includes identification of the binding partners of high dose dexamethasone under physiological conditions using a drug affinity responsive target stability (DARTS) (75) approach implementing Raw264.7 macrophages (FIG. ID). After incubation with high dose dexamethasone (lOpM), the cells were lysed and subjected to protease digestion, followed by the detection of the target bands via Coomassie blue staining. As shown in FIG. IE, two groups of protein bands were protected: one band was approximately 90kDa, which was close to the molecular weight of GR; whereas another group of protected bands was 50kDa or so. These results, together with the isolation of 8 high dose dexamethasone binding partners with the aforementioned proteome screening, led to the identification of Tau, a group of isoforms produced by alternative splicing with molecular weight of around 50kDa (16, 17, the disclosures of which are incoporated herein by reference), as the only candidate that may bind to high dose dexamethasone in both protein chips and live cells (FIG. IF).

[0046] The disclosure includes confirming and comparing the interactions of dexamethasone / Tau and dexamethasone / GR by treating Raw264.7 macrophages with serial concentrations of dexamethasone followed by a DARTS assay coupled with immunoblotting with antibodies specifically against GR and Tau, respectively. The DARTS assay demonstrated that 10 pM high dose dexamethasone was needed to obtain clear protection of Tau from protease mediated degradation, whereas protection of GR degradation could be seen from a dose as low as 10 nM dexamethasone (FIGS. 1G and 1H) The estimated dissociation constant (Ko) values of dexamethasone / Tau and dexamethasone / GR were 2.523 pM and 0.055 pM, respectively. Biophysical methods, including solid phase binding and one-step kinetic surface plasmon resonance (SPR) assay, further gave estimated Ko values of 206pM and 380pM for the interaction between dexamethasone and Tau, respectively (FIGS. II and 1 J). The SPR assay indicated that both endogenous GCs, cortisone and hydrocortisone, and synthetic GCs, including prednisolone and methylprednisone, displayed comparable binding affinity to dexamethasone with Tau (FIGS. 1J-1N) (18). Resveratrol, a chemical with a structure unrelated to that of GCs, was used as a negative control and did not show any binding to Tau, whereas methylene blue (MB), a chemical known to bind Tau (79), was chosen as a positive control and clearly bound to Tau in the SPR assay (FIGS. IO and IP). DARTS assay with live cells, solid phase binding and SPR assay with purified Tau protein gave different estimated binding affinity with dexamethasone, suggesting possible involvements of co-factors in promoting the binding of dexamethasone to Tau in the cells. The disclosure includes identification of the domain of Tau responsible for binding to dexamethasone. This was performed by generating serial deletion constructs of Tau fused to Flag tag (FIGS. 5A and 5B). DARTS assay with these constructs demonstrated that dexamethasone failed to protect the Tau deletion mutants lacking the proline-rich region from protease mediated degradation (FIG. 5C), indicating that the proline-rich region of Tau was required for its association with high dose dexamethasone (20). Subsequent molecular docking simulations revealed that dexamethasone and prednisolone had similar binding affinities and binding positions with Tau, exhibiting hydrophilic interactions with the amino acid residues 190-236 located within the proline-rich region of Tau (FIGS. 5D-5H). In addition, both GCs formed identical hydrogen bonding interactions with Aspl93, Thr212, Lys225, and Ser238 on Tau. Correspondingly, DARTS assay with Tau knockout Raw264.7 cells transfected with point mutations of these residues on Tau revealed that Thr212 on Tau was the most critical amino acid for interaction with high dose dexamethasone (FIGS. 51 and 5J). Taken together, these results demonstrate that Tau binds to high dose GCs as a previously unrecognized receptor with low affinity.

[0047] Example 2

[0048] Tau Deficiency abolishes dexamethasone induced bone resorption in inflammatory arthritis

[0049] Tau is known as a brain protein that promotes microtubule assembly. The disclosure shows that Tau was widely expressed in many tissues besides the brain, at relatively high levels in the heart, lung, kidney, bone and bone marrow, and at low levels in the liver and spleen (FIG. IQ). Long-term pharmacological use of GCs causes osteoporosis, the most common side effect of GCs therapy in rheumatoid arthritis (27). Therefore, we analyzed whether dexamethasone / Tau interaction was involved in dexamethasone-mediated osteoporosis with the collagen-induced arthritis (CIA) model, the widely-used, clinically- relevant inflammatory and autoimmune arthritis model (22) (FIG. 2A).

[0050] We generated inducible global GR knockout mice (GR- / -) by breeding GRflox / floxmice (23) with Rosa26a-CreERT2mice in which Cre-mediated recombination was induced by tamoxifen (24). Due to the unavailability of Tauflox / floxfor generating conditional Tau- / - mice, conventional global Tau- / - (25) mice were employed in the current study. GR deficiency rendered mice highly susceptible to CIA and impaired dexamethasone’s anti -inflammation action (FIGS. 2B-2E), which is in line with previous reports (26, 27). In contrast, Tau deficiency resulted in reduced inflammation in CIA mice and did not impair dexamethasone- mediated suppression of inflammation (FIGS. 2B-2E). Strikingly, dexamethasone induced significant bone loss in WT and GR- / - CIA mice, but not in Tau- / - CIA mice (FIG. 2F). Microcomputed tomography (pCT) analysis and TRAP assay revealed that dexamethasone led to significant femoral and tibia trabecular bone loss by promoting osteoclast activity in both WT and GR- / - CIA mice, and Tau deficiency abolished dexamethasone induced osteoclast activity and resultant bone loss (FIGS. 2G-2J). In contrast, osteoblast surface values were significantly decreased in both of WT and Tau- / - CIA mice treated with dexamethasone, but not in dexamethasone treated GR- / - CIA mice, indicating dexamethasone reduced osteoblast surface mainly through GR (FIGS. 2K and 2L). In agreement with the notion that the inflammatory process in RA is associated with bone loss independent of GCs (2S), CIA mice with various genetic backgrounds exhibited reduced bone mass. In addition, exaggerated inflammation in GR- / - CIA mice provoked more bone loss than that in WT and Tau- / - CIA mice (FIGS. 2G and 2H). Collectively, these data demonstrate that treatment of CIA mice with dexamethasone resembles a state of GIO with inflammatory arthritis. Additionally, dexamethasone exerts its anti-inflammatory action via GR and its adverse effect, i.e. bone resorption, via Tau.

[0051] Example 3

[0052] Tau is required for high dose dexamethasone induced bone resorption in glucocorticoid- induced osteoporosis model

[0053] The disclosure includes analysis of the influcence of Tau in high dose dexamethasone induced osteoporosis using a common GIO model with male mice treated with 10 mg / kg body weight dexamethasone for a total of 5 weeks. Dexamethasone significantly reduced bone mass in both WT and GR- / - mice, evidenced by significant lower femoral and tibia trabecular bone volume fraction, trabecular thickness and trabecular number, and this reduction was largely lost in dexamethasone treated Tau- / - mice (FIGS. 6A and 6B). In line with the observations in dexamethasone treated CIA mice, dexamethasone dramatically increased osteoclasts in femoral trabecular bone of WT and GR- / -, but not those of Tau- / - mice (FIGS. 6C and 6D). Consistent with these findings, Tau deficiency abolished dexamethasone-upregulated TRAP, RANKL and RANKL / OPG ratio observed in the femur of WT and GR- / - mice (FIGS. 6E- 6G). In contrast, dexamethasone significantly decreased osteoblast surface and bone formation rates in WT and Tau- / - mice, but not in GR- / - mice (FIGS. 6H-6J), implying that dexamethasone mediated inhibition of bone formation through GR. In addition, dexamethasone significantly increased serum levels of bone resorption marker CTX-1 and RANKL, while serum levels of OPG, the decoy receptor of RANKL, simultaneously decreased, thereby increasing the ratio of RANKL / OPG in WT and GR- / - mice (FIG. 6K). In contrast, in Tau- / - mice, serum levels of CTX-1 and RANKL were not increased; however, RANKL / OPG ratio was significantly decreased resultant of increased OPG levels after dexamethasone treatment (FIG. 6K). Meanwhile, dexamethasone decreased serum levels of bone formation marker PINP in both WT and Tau- / - mice, but not in GR- / - mice (FIG. 6K)

[0054] Since GIO predominantly affects the bone regions composed largely of cancellous bone, such as vertebrae and femur (29), we also evaluated the effects of Tau deficiency on bone quality in vertebrae. Similar to our observations in femur and tibia, Tau deficiency also ablated dexamethasone induced bone loss in vertebrae, whereas dexamethasone led to a significant reduction in trabecular bone volume fraction, trabecular thickness and number in WT and GR- / - vertebrae (FIGS. 6L and 6M).

[0055] To examine potential sex differences, we also repeated these assays with female mouse models and found that the findings with pCT, DEXA, Tartrate-resistant acid phosphatase (TRAP), osteoblast surface analysis and ELISA in male mice were recapitulated in female mice (FIGS. 7A-7H), indicating dexamethasone caused Tau-dependent bone resorption is sex independent.

[0056] Thus, and without intending to be bound by any particular theory, it is considered that GIO, modeled by high dose dexamethasone induced osteoporosis, is primarily resultant of Tau-mediated enhancement of osteoclast activity and subsequent bone resorption. GR dependent inhibition of bone formation also contributes to GIO, but it appears to be less important than Tau dependent bone resorption according to the described results.

[0057] Example 4

[0058] Tau is required for high dose dexamethasone mediated osteoclastogenesis

[0059] At low physiological concentrations, endogenous GCs are required for mesenchymal cells differentiation and function and relevant signaling is primarily mediated through GR (30, 31). However, at high pharmacological concentrations, long-term use of GCs leads to the development of GIO, the most common side effect conferred by GCs (32). In human subjects, GIO is characterized by a rapid increase in bone resorption followed by impaired bone formation (33). As such, the disclosure provides an analysis of whether Tau was also involved in high dose dexamethasone mediated perturbations in osteoblastogenesis and osteoclastogenesis in vitro. In the described experimental setting, both low and high dose dexamethasone did not affect osteoclast and osteoblast apoptosis obtained from the bone marrow of WT, Tau- / - (25) and GR- / - mice (FIGS. 8A and 8B). High dose dexamethasone significantly enhanced RANKL- and M-CSF-primed osteoclastogenesis of bone marrow mononuclear cells isolated from WT and GR- / - mice, but not those from Tau- / - mice (FIGS. 3A-3F). qRT-PCR analysis further disclosed that high dose dexamethasone significantly increased the expressions of osteoclast differentiation and bone resorption markers NFATcl, calcitonin receptor (CTR), Cathepsin K (CTSK) and TRAP in bone marrow cells isolated from WT and GR- / - mice, but not in those cells from Tau- / - mice (FIGS. 3G-3J), indicating that high dose dexamethasone- enhanced osteoclastogenesis depended on Tau. Consistently, knockout of Tau did, while knock out of GR did not, ablate dexamethasone-enhanced RANKL-primed osteoclastogenesis of mouse Raw264.7 macrophages and human THP-1 monocytes (FIGS. 3K-3Q). In contrast to high dose dexamethasone-enhanced osteoclastogenesis through Tau, we observed that high dose dexamethasone inhibited osteoblastogenesis largely through GR (FIGS. 8C-8H). Taken together, these data indicate that Tau is required for high dose dexamethasone-mediated osteoclastogenesis.

[0060] Example 5

[0061] TTBK1 phosphorylates Tau at residue Ser422 during high dose dexamethasone enhanced osteoclastogenesis

[0062] Phosphorylation is one of the most common post-translational modifications of Tau and may occur at many serine and threonine residues by a variety of kinases under physiological and pathological conditions (34, 35). To assess whether high dose dexamethasone was able to induce the phosphorylation of Tau, the total protein fraction of Raw264.7 cells treated with low or high dose dexamethasone were examined by immunoblotting with antibodies recognizing Tau phosphorylated at residues Ser202 / Thr205, Ser396 and Ser422, the well characterized phosphorylation sites in other diseases. High dose dexamethasone preferentially phosphorylated Tau at residue Ser422 (p-Tau Ser422) (FIG. 9 A). Re-introduction of Tau point mutations of these phosphorylated sites into Tau knockout Raw264.7 cells revealed that point mutation on Tau Ser422 failed to restore high dose dexamethasone mediated osteoclastogenesis, reinforcing the finding that p-Tau Ser422 is the critical phosphorylation site implicated in this process (FIGS. 9B-9D).

[0063] Tau is a microtubule-binding protein and associated with dynamic regulation of the cytoskeleton through direct binding to tubulin (36). Using confocal microscopy and fluorescent signal intensity plots of total Tau, p-Tau Ser422 and tubulin, we found that high dose dexamethasone activated p-Tau Ser422 and this phosphorylated Tau did not co-localize with tubulin, although total Tau co-localized with tubulin, particularly under the untreated conditions (FIGS. 9E and 9F). These findings indicate that p-Tau Ser422, which accounts for a very small percentage of total Tau, was not associated with microtubules in mediating high dose dexamethasone induced osteoclastogenesis.

[0064] To identify the kinase responsible for p-Tau Ser422, we performed biochemical copurification coupled with proteomics analysis with control and Tau KO Raw264.7 cells treated with high dose biotin-labeled dexamethasone, and identified 5 kinases that were recruited to Tau by high dose dexamethasone (FIGS. 9G and 9H). We next knocked down TTBK1 with its specific siRNA and inhibited 4 other kinases with their specific pharmacological inhibitors to determine the potential involvements of these kinases in high dose dexamethasone enhanced osteoclastogenesis. These assays led to the isolation of TTBK1, the kinase reported to phosphorylate Tau at residue Ser422 (37), as the only critical kinase responsible for phosphorylation of Tau Ser422 in mediation of high dose dexamethasone enhanced osteoclastogenesis (FIGS. 9I-9M). Imaging with confocal microscopy revealed that high dose dexamethasone induced the co-localization of TTBK1 with Tau (FIG. 9N). Additionally, co-immunoprecipitation assays revealed dexamethasone dependent interaction of TTBK1 with Tau. Furthermore, domain mapping experiment with serial Tau deletion mutants show that amino acids 396-441, which constitute Tau C-terminal region, were responsible for its binding to TTBK1 (FIGS. 90 and 9P). Taken together, these results indicate that high dose dexamethasone triggered the recruitment of TTBK1 kinase to Tau, leading to the phosphorylation of Tau at residue Ser422, thereby enhancing osteoclastogenesi s .

[0065] Example 6 Activated p-Tau Ser422 promotes the processing of NF-KB1 p!05 into p50 and enhances its nuclear translocation during high dose dexamethasone enhanced osteoclastogenesis

[0066] To isolate the transcription factor(s) downstream of p-Tau Ser422 that may mediate high dose dexamethasone induced osteoclastogenesis, we performed another biochemical copurification coupled with proteomics analysis using Tau knockout Raw264.7 cells transfected with or without Flag tagged Tau (FIG. 10A). Two transcription factors, NF-KB 1 plO5 and CTCF, were detected in the immunoprecipitated complexes of Tau in response to high dose dexamethasone treatment (FIG. 10B). NF-KB p50 is cleaved from its precursor plO5 through a proteasome mediated processing (38). We thus inhibited NF-KB p50 activity with andrographolide (39) or knocked down CTCF with its specific siRNA, and found that high dose dexamethasone mediated osteoclastogenesis was blocked once NF-KB p50 activity was inhibited, and was not affected by knockdown of CTCF (FIG. 10C-10G). In addition, coimmunoprecipitation demonstrated dexamethasone dependent interaction between Tau and pl05 / p50. In addition, deletion of amino acids 396-441 of the C-terminal region in Tau impaired the association between Tau and pl05 / p50 (FIGS. 10H-10I). Furthermore, high dose dexamethasone stimulated the processing of pl 05 into p50 (FIG. 10H, compare lanes 1 and 2), and deletion of Tau or knockdown of TTBK1 blocked this dexamethasone dependent processing of pl05 into p50 (FIGS. 10J-10M). In agreement with a previous report that NF- KB p50 preferentially formed heterodimers with p65 to translocate into nucleus and induce osteoclastogenesis in response to RANKL (40), stimulation of Raw264.7 cells with RANKL primed the nuclear translocation of p50 and dexamethasone further enhanced this process (FIGS. ION and 100). Collectively, high dose dexamethasone phosphorylates Tau to activate transcription factor p50, in turn, leading to enhanced osteoclastogenesis.

[0067] Example 7 Discovery of TRxO237 as a Tau inhibitor that inhibits dexamethasone induced p-Tau Ser422 and osteoclastogenesis

[0068] Since phosphorylation of Tau at Ser422 is demonstrated herein to be a significant molecular event required to mediate dexamethasone dependent osteoclastogenesis, we analyzed whether inhibiting p-Tau Ser422 could be used as a therapeutic approach for GIO. For this purpose, we screened an FDA-approved drug library including 958 drugs using an ELISA based high throughput method (FIG. 11 A), and identified 2 candicate drugs, raloxifene and TRxO237, that could inhibit dexamethasone induced p-Tau Ser422 (FIGS. 11B and 11C). Methylene blue (MB) and its derivatives are known Tau aggregation inhibitors that show positive preclinical results for Alzheimer’s disease (41, 42) and have advanced to clinical trials (43), although none have been approved for clinical use. TRxO237 is a derivative of MB in a stabilized and reduced form (42), failed to show clinical efficacy in Phase III clinical trials in Alzheimer’s disease patients (NCT01689246 and NCT01689233) (44). Here, we found that MB, TRxO237, and raloxifene significantly inhibited dexamethasone dependent activation of p-Tau Ser422 (FIGS. HD and HE). Secondary screening of these 3 drugs using TRAP staining revealed that only MB and TRxO237, but not raloxifene, blocked dexamethasone dependent osteoclastogenesis in a Tau dependent manner (FIGS. 11F-11J). Moreover, pit assay confirmed that both MB and Trx0237 ablated dexamethasone mediated bone resorption in a Tau dependent manner (FIGS. 11K-11M). Raloxifene is a selective estrogen receptor modulator that is an FDA approved drug for treating postmenopausal osteoporosis (45). But raloxifene failed to inhibit dexamethasone dependent osteoclastogenesis despite abolishing dexamethasone mediated activation of p-Tau Ser422, suggesting that additional post-translational modifications including phosphorylation at other residues beyond Ser422, ubiquitination, acetylation, methylation, oxidation and glycation, which were reported to be involved in Tau functions in other diseases (46), may also be involved in dexamethasone enhanced osteoclastogenesis. In summary, the data presented in this disclosure supports use of TRxO237 as a drug to antagonize dexamethasone induced Tau-dependent osteoclastogenesis.

[0069] Example 8

[0070] TRxO237 is therapeutic against glucocorticoid-induced osteoporosis through inhibition of p-Tau Ser422

[0071] The disclosure includes analysis of whether or not TRxO237 could be used as therapeutic against GIO in vivo. Indeed, TRxO237 treatment was associated with increased trabecular B V / TV and trabecular number in long bone and vertebra of dexamethasone treated WT and GR- / - mice, despite having no effects on trabecular bone thickness in long bone (FIGS. 12A-12E, 13H, and 131) Dexamethasone did not cause obvious bone loss in Tau- / - mice, and TRxO237 did not exert any effects on bone quality in terms of trabecular BV / TV, thickness and number (FIGS. 12B-12E). Histology analysis revealed that TRxO237 abolished high dose dexamethasone induced osteoclast activity, but did not affect osteoblast surface values in both long bones and vertebrae of WT and GR- / - mice (FIGS. 12F-12H and 13 J- 13L). Consistently, TRxO237 blocked the elevated serum levels of RANKL, the ratio of RANKL / OPG and CTX-1 in dexamethasone treated WT and GR- / - mice, but did not affect serum levels of the bone formation marker PINP (FIGS. 13A-13E). Additionally, TRxO237 prevented dexamethasone induced reduction of serum OPG level in WT and GR- / - mice, but not in Tau- / - mice (FIG. 13B). Consistent with the described observation that TRxO237 abolished dexamethasone mediated osteoclast activity through inhibition of p-Tau Ser422 in vitro, TRxO237 markedly reduced p-Tau Ser422 in bone tissues of WT and GR- / - GIO mice (FIGS. 13F and 13G). Taken together, the data of this disclosure demonstrate selection of TRxO237 from an FDA-approved drug library that protected against GIO in vivo. The disclosure includes analysis of the potential dose-dependent effects and preferred dosage of TRxO237 with WT GIO mouse model. All three dosage of TRxO237 employed were able to effectively protect against dexamethasone induced bone loss (FIG. 14A). pCT analysis of trabecular bone at femur and tibia demonstrated that even the all dosages of TRxO237 increased bone volume fraction, and trabecular number in GIO mice without affecting trabecular thickness (FIGS. 14B-14E). Nonetheless, even lowest dose of TRxO237 effectively decreased dexamethasone induced serum levels of bone resorption markers RANKL, RANKL / OPG ratio, and CTX1, simultaneously increased dexamethasone reduced OPG, despite having no effect on bone formation marker PINP (FIG. 14F). In addition, the lowest dose of TRxO237 blocked dexamethasone induced osteoclast activity (FIGS. 14G and 14H) Consistent with the described observation that TRx-237 abolished dexamethasone mediated osteoclast activity through inhibiting p-Tau Ser422 in vitro, all three doses of TRxO237 markedly reduced p-Tau Ser422 in bone tissue of GIO mice as compared to that of GIO mice without treatment. The highest dose of TRxO237 reduced dexamethasone activated p-Tau Ser422 to non-dexamethasone treated level (FIGS. 141 and 14J). Taken together, all the described dosages effectively ablated dexamethasone caused bone loss.

[0072] Example 9

[0073] Combinatorial treatment with dexamethasone and TRxO237 overcomes dexamethasone induced bone loss in treating inflammatory arthritis

[0074] Rheumatoid arthritis is a chronic inflammatory arthritis affecting 0.5-10% adults in developed countries (47). Despite adverse effects such as GIO, GCs continue to be an important component of rheumatoid arthritis therapy in clinic due to their broad-spectrum anti-inflammatory effects (48-50). In this disclsoure, we use CIA mice treated with dexamethasone as a clinically relevant inflammatory arthritis model to determine whether combinational treatment with anti-inflammatory dexamethasone and anti-GIO TRxO237 in CIA mice could overcome dexamethasone induced bone loss while retaining its antiinflammatory action. Combinational treatment elicited an anti-inflammatory effect un- distinguishable from that observed with dexamethasone monotherapy in CIA mice (FIGS. 4A-4C). In line with the described observation that genetic ablation of Tau prevents GIO, pharmacological administration of Tau inhibitor TRxO237 in dexamethasone treated CIA mice effectively protected against dexamethasone induced bone loss through inhibiting osteoclasts, without affecting osteoblasts (FIGS. 4D-4M). Immunohistochemistry staining revealed that dexamethasone-dependent p-Tau Ser422 was also abolished by TRxO237 in CIA mice (FIGS. 4N and 40), further supporting identification of p-Tau Ser422 as a therapeutic target in osteoporosis. Collectively, these data indicate that combinatorial treatment with dexamethasone and TRxO237 preserves the anti-inflammatory action while avoids the adverse effect of dexamethasone in treating inflammatory arthritis.

[0075] The disclosure also includes analysis of the association between p-Tau Ser422 and bone loss in bone tissues from healthy controls and patients with osteoporosis. Significantly higher p-Tau Ser422 levels were observed in bone tissues from osteoporosis patients than those from healthy individuals (FIGS. 4P and 4Q), demonstrating that a therapeutic drug, such as TRxO237, that targets / inhibits p-Tau Ser422, might prevent bone loss in general in addition to GIO in clinic.

[0076] It will be recognized from the foregoing Examples that GC ligand stimulation results in the nuclear translocation of its canonical high affinity receptor GR, where it 1) binds to glucocorticoid response elements or 2) tethers itself to other DNA-bound transcription factors and alters transcriptions of downstream genes (57), leading to anti-inflammation and immunosuppression (FIG. 15). The presence of and the need to identify previously unrecognized receptors have been actively discussed in the field. Starting from unbiased systematic proteome-wide target identification strategy coupled with DARTS screen, followed by confirmation with solid phase binding and SPR assays, the disclosure identifies Tau as a low affinity receptor of GCs. Serial biochemical co-purification coupled with proteomic analyses led to the isolation of TTBK1 kinase and transcriptional factor p50 as two key downstream mediators of GCs / Tau signaling to mediate GC dependent osteoclastogenesis and bone resorption. Although Tau is known as a microtubule-binding protein and its aggregation associated with various neurodegenerative diseases including Alzheimer disease and frontotemporal dementia (52, 53), it has many roles and can also function as a signaling molecule {34, 54). The disclosure demonstrates that, upon binding with high dose dexamethasone, non-microtubule-associated Tau acts as a signaling molecule to tether TTBK1 kinase, leading to the phosphorylation of Tau at residue Ser422, followed by the processing ofNF-xBl plO5 into p50 and its nuclear translocation, thereby, enhancing RANKL-primed osteoclastogenesis (FIG. 15). The demonstration that Tau is a low affinity receptor of GCs, and plays an important role in GC-induced bone resorption, is also pertinent to the long lasting debates / questions concerning the molecular events behind GCs osteoporosis adverse effect {32).

[0077] The disclosure also demonstrates that p-Tau Ser422 is a therapeutic target for treating GIO and shows that TRxO237 and its inhibition of Tau aggregation (55) can be repurposed to block dexamethasone induced bone loss. Combinatorial treatment with dexamethasone and TRxO237 enabled the beneficial anti-inflammatory action while averting the adverse bone resorption activity of dexamethasone in CIA, an inflammatory arthritis mouse model, resembling human rheumatoid arthritis. The disclosure also demonstrates that p-Tau Ser422 was significantly elevated in patients with osteoporosis, supporting the clinical inhibition of p-Tau Ser422 as a means to prevent bone loss in general in addition to GIO. Thus, the disclosure supports the use of TRxO237, a drug entering clinical phase III trial with known pharmacological and toxicological profiles, as a therapy for various GCs associated adverse effects, particularly GIO, p-Tau Ser422 associated Tauopathy and other pathological conditions, such as acute stress, in which GCs are markedly stimulated (56).

[0078] In summary, this disclosure identifies Tau as a GCs-binding receptor with low affinity. Additionally, the disclosure demonstrates this pathway plays a fundamental role in GIO, one of the several well-recognized substantial side effects of GCs. While this disclosure in part focuses on GIO, discovery of GCs / Tau pathway contributes to the understanding of diverse manifestations associated with high dose GCs, and the results of this disclosure may have a broader application to various GCs associated side effects and various Tau-related diseases and conditions. Thus, the disclosure demonstrates that TRxO237 is a drug that, through inhibiting GCs / Tau signaling, effectively overcomes dexamethasone induced bone loss. It is expected that TRxO237 may also be therapeutic against other side effects of GCs in various diseases and conditions, including Tauopathy. With the consideration that both GCs and Tau are involved in a many pathophysiological and disease processes, the identification of Tau as a low affinity receptor of GCs and manipulation of this newly described Tau / GCs pathway may also lead to therapeutics for various GCs- and Tau-associated pathologies and conditions.

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Claims

What is claimed is:

1. A method for inhibiting glucocorticoid caused bone loss in an individual, the method comprising administering to the individual an agent that inhibits phosphorylation of Tau.

2. The method of claim 1, wherein the phosphorylation of Tau is at Tau amino acid residue Ser 422.

3. The method of claim 2, wherein the phosphorylation of Tau is by TTBK1.

4. The method of any one of claims 1-3, wherein the agent is TRxO237.

5. The method of claim 4, wherein the individual has or is at risk for developing glucocorticoid-induced osteoporosis (GIO).

6. The method of claim 4, wherein the individual has or is at risk for developing dexamethasone mediated osteoclastogenesis.

7. The method of claim 4, wherein the individual is undergoing high dose glucocorticoid treatment.

8. The method of claim 5, wherein the glucocorticoid is dexamethasone.

9. The method of claim 7, wherein the glucocorticoid is dexamethasone.

10. A method for prophylaxis or treatment of inflammatory arthritis in an individual, the method comprising administering to the individual a glucocorticoid and an agent that inhibits phosphorylation of Tau.

11. The method of claim 10, wherein the phosphorylation of Tau is by TTBK1.

12. The method of claim 10, wherein the phosphorylation of Tau is at Tau amino acid residue Ser 422.

13. The method of any one of claims 10-12, wherein the glucocorticoid is dexamethasone.

14. The method of any one of claims 10-12, wherein the agent is TRxO237.

15. The method of any one of claims 10-12, wherein the glucocorticoid is dexamethasone and the agent is dexamethasone.

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