Osteocytes and Paget's Disease of Bone.

PURPOSE OF REVIEW: To describe the contributions of osteocytes to the lesions in Paget's disease, which are characterized by locally overactive bone resorption and formation. RECENT FINDINGS: Osteocytes, the most abundant cells in bone, are altered in Paget's disease lesions, displaying increased size, decreased canalicular length, incomplete differentiation, and less sclerostin expression compared to controls in both patients and mouse models. Pagetic lesions show increased senescent osteocytes that express RANK ligand, which drives osteoclastic bone resorption. Abnormal osteoclasts in Paget's disease secrete abundant IGF1, which enhances osteocyte senescence, contributing to lesion formation. Recent data suggest that osteocytes contribute to lesion formation in Paget's disease by responding to high local IGF1 released from abnormal osteoclasts. Here we describe the characteristics of osteocytes in Paget's disease and their role in bone lesion formation based on recent results with mouse models and supported by patient data.

Establishment of a TNFRSF11B knock-out human induced pluripotent stem cell line (KSCBi002-B-2) via CRISPR/Cas9 system.

A TNFRSF11B (TNF Receptor Superfamily Member 11b) gene encodes a soluble decoy receptor, osteoprotegerin (OPG), which has a key role in repressing osteoclast differentiation. In this report, we generated a biallelic knock-out hiPSC line for the TNFRSF11B gene via CRISPR/Cas9. When TNFRSF11B Knock-out hiPSCs were differentiated into mesenchymal progenitor cells (MPCs), the expression level of OPG was significantly decreased compared to normal hiPSC-derived MPCs. This knock-out hiPSCs will provide a chance to study Paget disease of bone 5 (juvenile Paget disease).

Osteoclast-derived IGF1 induces RANKL production in osteocytes and contributes to pagetic lesion formation.

We previously reported that measles virus nucleocapsid protein (MVNP) expression in osteoclasts (OCLs) of patients with Paget disease (PD) or targeted to the OCL lineage in MVNP-transgenic mice (MVNP mice) increases IGF1 production in osteoclasts (OCL-IGF1) and leads to development of PD OCLs and pagetic bone lesions (PDLs). Conditional deletion of Igf1 in OCLs of MVNP mice fully blocked development of PDLs. In this study, we examined whether osteocytes (OCys), key regulators of normal bone remodeling, contribute to PD. OCys in PDLs of patients and of MVNP mice expressed less sclerostin, and had increased RANKL expression compared with OCys in bones from WT mice or normal patients. To test whether increased OCL-IGF1 is sufficient to induce PDLs and PD phenotypes, we generated TRAP-Igf1 (T-Igf1) transgenic mice to determine whether increased IGF1 expression in the absence of MVNP in OCLs is sufficient to induce PDLs and pagetic OCLs. We found that T-Igf1 mice at 16 months of age developed PD OCLs, PDLs, and OCys, with decreased sclerostin and increased RANKL, similar to MVNP mice. Thus, pagetic phenotypes could be induced by OCLs expressing increased IGF1. OCL-IGF1 in turn increased RANKL production in OCys to induce PD OCLs and PDLs.

Aged G Protein-Coupled Receptor Kinase 3 (Grk3)-Deficient Mice Exhibit Enhanced Osteoclastogenesis and Develop Bone Lesions Analogous to Human Paget's Disease of Bone.

Paget's Disease of Bone (PDB) is a metabolic bone disease that is characterized by dysregulated osteoclast function leading to focal abnormalities of bone remodeling. It can lead to pain, fracture, and bone deformity. G protein-coupled receptor kinase 3 (GRK3) is an important negative regulator of G protein-coupled receptor (GPCR) signaling. GRK3 is known to regulate GPCR function in osteoblasts and preosteoblasts, but its regulatory function in osteoclasts is not well defined. Here, we report that Grk3 expression increases during osteoclast differentiation in both human and mouse primary cells and established cell lines. We also show that aged mice deficient in Grk3 develop bone lesions similar to those seen in human PDB and other Paget's Disease mouse models. We show that a deficiency in Grk3 expression enhances osteoclastogenesis in vitro and proliferation of hematopoietic osteoclast precursors in vivo but does not affect the osteoclast-mediated bone resorption function or cellular senescence pathway. Notably, we also observe decreased Grk3 expression in peripheral blood mononuclear cells of patients with PDB compared with age- and gender-matched healthy controls. Our data suggest that GRK3 has relevance to the regulation of osteoclast differentiation and that it may have relevance to the pathogenesis of PDB and other metabolic bone diseases associated with osteoclast activation.

Optineurin regulates NRF2-mediated antioxidant response in a mouse model of Paget's disease of bone.

Degenerative diseases affecting the nervous and skeletal systems affect the health of millions of elderly people. Optineurin (OPTN) has been associated with numerous neurodegenerative diseases and Paget's disease of bone (PDB), a degenerative bone disease initiated by hyperactive osteoclastogenesis. In this study, we found age-related increase in OPTN and nuclear factor E2-related factor 2 (NRF2) in vivo. At the molecular level, OPTN could directly interact with both NRF2 and its negative regulator Kelch-like ECH-associated protein 1 (KEAP1) for up-regulating antioxidant response. At the cellular level, deletion of OPTN resulted in increased intracellular reactive oxygen species and increased osteoclastogenic potential. At the tissue level, deletion of OPTN resulted in substantially increased oxidative stress derived from leukocytes that further stimulate osteoclastogenesis. Last, curcumin attenuated hyperactive osteoclastogenesis induced by OPTN deficiency in aged mice. Collectively, our findings reveal an OPTN-NRF2 axis maintaining bone homeostasis and suggest that antioxidants have therapeutic potential for PDB.

Role of the Endocannabinoid/Endovanilloid System in the Modulation of Osteoclast Activity in Paget's Disease of Bone.

The role of the endocannabinoid/endovanilloid (EC/EV) system in bone metabolism has recently received attention. Current literature evidences the modulation of osteoclasts and osteoblasts through the activation or inhibition of cannabinoid receptors in various pathological conditions with secondary involvement of bone tissue. However, this role is still unclear in primary bone diseases. Paget's disease of the bone (PDB) could be considered a disease model for analyzing the role of the EC/EV system on osteoclasts (OCs), speculating the potential use of specific agents targeting this system for managing metabolic bone disorders. The aim of the study is to analyze OCs expression of EC/EV system in patients with PDB and to compare OCs activity between this population and healthy people. Finally, we investigate whether specific agents targeting EC/EV systems are able to modulate OCs activity in this metabolic bone disorder. We found a significant increase in cannabinoid receptor type 2 (CB2) protein expression in patients with PDB, compared to healthy controls. Moreover, we found a significant reduction in multi-nucleated tartrate-resistant acid phosphatase (TRAP)-positive OCs and resorption areas after treatment with JWH-133. CB2 could be a molecular target for reducing the activity of OCs in PDB, opening new therapeutic scenarios for the management of this condition.

Structural and Functional Analysis of Disease-Linked p97 ATPase Mutant Complexes.

IBMPFD/ALS is a genetic disorder caused by a single amino acid mutation on the p97 ATPase, promoting ATPase activity and cofactor dysregulation. The disease mechanism underlying p97 ATPase malfunction remains unclear. To understand how the mutation alters the ATPase regulation, we assembled a full-length p97R155H with its p47 cofactor and first visualized their structures using single-particle cryo-EM. More than one-third of the population was the dodecameric form. Nucleotide presence dissociates the dodecamer into two hexamers for its highly elevated function. The N-domains of the p97R155H mutant all show up configurations in ADP- or ATPγS-bound states. Our functional and structural analyses showed that the p47 binding is likely to impact the p97R155H ATPase activities via changing the conformations of arginine fingers. These functional and structural analyses underline the ATPase dysregulation with the miscommunication between the functional modules of the p97R155H.

Rhizomelia and Impaired Linear Growth in a Girl with Juvenile Paget Disease: The Natural History of the Condition.

In ultra-rare bone diseases, information on growth during childhood is sparse. Juvenile Paget disease (JPD) is an ultra-rare disease, characterized by loss of function of osteoprotegerin (OPG). OPG inhibits osteoclast activation via the receptor activator of nuclear factor-κB (RANK) pathway. In JPD, overactive osteoclasts result in inflammatory-like bone disease due to grossly elevated bone resorption. Knowledge on the natural history of JPD, including final height and growth, is limited. Most affected children receive long-term antiresorptive treatment, mostly with bisphosphonates, to contain bone resorption, which may affect growth. In this study, we report the follow-up of height, growth velocity, and skeletal maturation in a 16-year-old female patient with JPD. The patient was treated with cyclic doses of pamidronate starting at 2.5 years of age and with 2 doses of denosumab at the age of 8 years, when pamidronate was paused. In the following years, a sustainable decline in a height z-score and a stunted pubertal growth spurt; despite appropriate maturation of the epiphyseal plates of the left hand, the proximal right humerus and both femora were observed. Whether this reflects the growth pattern in JPD or might be associated to the antiresorptive treatments is unclear, since there is very limited information available on the effect of bisphosphonates and denosumab on growth and the growth plate in pediatric patients. Studies are needed to understand the natural history of an ultra-rare bone disease and to assess the effects of antiresorptive treatment on the growing skeleton.

Diffuse Paget's Disease of the Skull with Intense Uptake of Technetium-99m-Labeled Diphosphonate Tracer in Bone Scintigraphy.

Imaging in patients with Paget's disease of bone is very important clinically to show the presence of Pagetic abnormalities, assess disease progression, and identify adversely affected structures throughout disease course. Abnormalities and progression may be seen on radiographs, computed tomography, magnetic resonance imaging, and nuclear imaging. Herein, we report a case Paget's disease of bone showing diffuse characteristic pathology using technetium-99m-labelled diphosphonate tracer in bone scintigraphy (nuclear imaging). This case emphasizes the ability of nuclear imaging to rapidly visualize and assess progressive distribution of Pagetic involvement in a patient previously diagnosed with pituitary adenoma and mild Paget's disease of the skull.

Epigenetic analysis of Paget's disease of bone identifies differentially methylated loci that predict disease status.

Paget's disease of bone (PDB) is characterized by focal increases in disorganized bone remodeling. This study aims to characterize PDB-associated changes in DNA methylation profiles in patients' blood. Meta-analysis of data from the discovery and cross-validation set, each comprising 116 PDB cases and 130 controls, revealed significant differences in DNA methylation at 14 CpG sites, 4 CpG islands, and 6 gene-body regions. These loci, including two characterized as functional through expression quantitative trait-methylation analysis, were associated with functions related to osteoclast differentiation, mechanical loading, immune function, and viral infection. A multivariate classifier based on discovery samples was found to discriminate PDB cases and controls from the cross-validation with a sensitivity of 0.84, specificity of 0.81, and an area under curve of 92.8%. In conclusion, this study has shown for the first time that epigenetic factors contribute to the pathogenesis of PDB and may offer diagnostic markers for prediction of the disease.

Our skeleton stays healthy through an endless regeneration process, with specialized cells constantly absorbing and creating new bone tissue. Illnesses emerge when this breaking down and rebuilding cycle becomes imbalanced. For instance, in Paget's disease of bone (PDB for short) the skeleton becomes misshapen and fragile, with complications including pain, fractures, neurological problems, hearing loss and even cancer. For most patients however, symptoms are only present at an advanced stage, when irreversible damage to the skeleton has already occurred. Certain inherited genetic changes play a role in the development of PDB, but lifestyle and environmental factors are also thought to contribute. Indeed, accumulating evidence suggests that diet, pollution and infection may influence how genes involved in bone metabolism are activated. In this process, the environment may trigger chemical marks to be added onto DNA sequences, which ultimately switches specific genes on and off. To investigate whether the pattern of chemical marks in individuals with PDB may be characteristic, Diboun et al. scanned the genetic information of over 200 PDB patients, and compared it to healthy counterparts. Combining genomic analysis and machine learning revealed several chemical signatures that were remarkably different in the DNA of PDB individuals. These signatures affected sites close to genes involved in bone development, as well as response to mechanical loading and infection. This provides strong evidence that PDB could be, in part, triggered by the environment, as the placement of these marks is highly influenced by external factors. This research sheds light onto the underlying changes that trigger PDB. Future experiments should explore whether it may be possible to use these genetic changes to identify patients before the onset of irreversible and debilitating damage.

SVIP is a molecular determinant of lysosomal dynamic stability, neurodegeneration and lifespan.

Missense mutations in Valosin-Containing Protein (VCP) are linked to diverse degenerative diseases including IBMPFD, amyotrophic lateral sclerosis (ALS), muscular dystrophy and Parkinson's disease. Here, we characterize a VCP-binding co-factor (SVIP) that specifically recruits VCP to lysosomes. SVIP is essential for lysosomal dynamic stability and autophagosomal-lysosomal fusion. SVIP mutations cause muscle wasting and neuromuscular degeneration while muscle-specific SVIP over-expression increases lysosomal abundance and is sufficient to extend lifespan in a context, stress-dependent manner. We also establish multiple links between SVIP and VCP-dependent disease in our Drosophila model system. A biochemical screen identifies a disease-causing VCP mutation that prevents SVIP binding. Conversely, over-expression of an SVIP mutation that prevents VCP binding is deleterious. Finally, we identify a human SVIP mutation and confirm the pathogenicity of this mutation in our Drosophila model. We propose a model for VCP disease based on the differential, co-factor-dependent recruitment of VCP to intracellular organelles.

The role of autophagy in bone homeostasis.

Autophagy is an evolutionarily conserved intracellular process and is considered one of the main catabolism pathways. In the process of autophagy, cells are digested nonselectively or selectively to recover nutrients and energy, so it is regarded as an antiaging process. In addition to the essential role of autophagy in cellular homeostasis, autophagy is a stress response mechanism for cell survival. Here, we review recent literature describing the pathway of autophagy and its role in different bone cell types, including osteoblasts, osteoclasts, and osteocytes. Also discussed is the mechanism of autophagy in bone diseases associated with bone homeostasis, including osteoporosis and Paget's disease. Finally, we discuss the application of autophagy regulators in bone diseases. This review aims to introduce autophagy, summarize the understanding of its relevance in bone physiology, and discuss its role and therapeutic potential in the pathogenesis of bone diseases such as osteoporosis.

Increased S1P expression in osteoclasts enhances bone formation in an animal model of Paget's disease.

Paget's disease (PD) is characterized by increased numbers of abnormal osteoclasts (OCLs) that drive exuberant bone formation, but the mechanisms responsible for the increased bone formation remain unclear. We previously reported that OCLs from 70% of PD patients express measles virus nucleocapsid protein (MVNP), and that transgenic mice with targeted expression of MVNP in OCLs (MVNP mice) develop bone lesions and abnormal OCLs characteristic of PD. In this report, we examined if OCL-derived sphingosine-1-phosphate (S1P) contributed to the abnormal bone formation in PD, since OCL-derived S1P can act as a coupling factor to increase normal bone formation via binding S1P-receptor-3 (S1PR3) on osteoblasts (OBs). We report that OCLs from MVNP mice and PD patients expressed high levels of sphingosine kinase-1 (SphK-1) compared with wild-type (WT) mouse and normal donor OCLs. SphK-1 production by MVNP-OCLs was interleukin-6 (IL-6)-dependent since OCLs from MVNP/IL-6-/- mice expressed lower levels of SphK-1. Immunohistochemistry of bone biopsies from a normal donor, a PD patient, WT and MVNP mice confirmed increased expression levels of SphK-1 in OCLs and S1PR3 in OBs of the PD patient and MVNP mice compared with normal donor and WT mice. Further, MVNP-OCLs cocultured with OBs from MVNP or WT mice increased OB-S1PR3 expression and enhanced expression of OB differentiation markers in MVNP-OBs precursors compared with WT-OBs, which was mediated by IL-6 and insulin-like growth factor 1 secreted by MVNP-OCLs. Finally, the addition of an S1PR3 antagonist (VPC23019) to WT or MVNP-OBs treated with WT and MVNP-OCL-conditioned media (CM) blocked enhanced OB differentiation of MVNP-OBs treated with MVNP-OCL-CM. In contrast, the addition of the SIPR3 agonist, VPC24191, to the cultures enhanced osterix and Col-1A expression in MVNP-OBs treated with MVNP-OCL-CM compared with WT-OBs treated with WT-OCL-CM. These results suggest that IL-6 produced by PD-OCLs increases S1P in OCLs and S1PR3 on OBs, to increase bone formation in PD.

Increased Prevalence of Nephrolithiasis and Hyperoxaluria in Paget Disease of Bone.

CONTEXT: Nephrolithiasis (NL) and primary hyperparathyroidism (HPTH) are metabolic complications of Paget disease of bone (PDB), but recent data regarding their prevalence in PDB patients are lacking. OBJECTIVES: Study 1: To compare the prevalence of primary HPTH and NL in 708 patients with PDB and in 1803 controls. Study 2: To evaluate the prevalence of NL-metabolic risk factors in 97 patients with PDB and NL, 219 PDB patients without NL, 364 NL patients without PDB, and 219 controls, all of them without HPTH. DESIGN: Cross-sectional multicentric study. SETTING: Italian referral centers for metabolic bone disorders. PARTICIPANTS: Patients with PDB from the Associazione Italiana malati di osteodistrofia di Paget registry. Participants in the Olivetti Heart and the Siena Osteoporosis studies. MAIN OUTCOME MEASURES: HPTH; NL; NL-metabolic risk factors. RESULTS: Patients with PDB showed higher prevalence of primary HPTH and NL compared with controls (P < 0.01). The NL recurrence occurs more frequently in patients with polyostotic PDB. About one-half of patients with PDB but without NL showed 1 or more NL-related metabolic risk factors. The hyperoxaluria (HyperOx) prevalence was higher in patients with PDB and NL compared with patients with NL but without PDB and in patients with PDB without NL compared with controls (P = 0.01). Patients with PDB and HyperOx showed a longer lapse of time from the last aminobisphosphonate treatment. CONCLUSIONS: NL and HPTH are frequent metabolic complication of PDB. The NL occurrence should be evaluated in patients with PDB, particularly in those with polyostotic disease and/or after aminobisphosphonate treatment to apply an adequate prevention strategy.

Osteoclast-derived IGF1 is required for pagetic lesion formation in vivo.

We report that transgenic mice expressing measles virus nucleocapsid protein (MVNP) in osteoclasts (OCLs) (MVNP mice) are Paget's disease (PD) models and that OCLs from patients with PD and MVNP mice express high levels of OCL-derived IGF1 (OCL-IGF1). To determine OCL-IGF1's role in PD and normal bone remodeling, we generated WT and MVNP mice with targeted deletion of Igf1 in OCLs (Igf1-cKO) and MVNP/Igf1-cKO mice, and we assessed OCL-IGF1's effects on bone mass, bone formation rate, EphB2/EphB4 expression on OCLs and osteoblasts (OBs), and pagetic bone lesions (PDLs). A total of 40% of MVNP mice, but no MVNP/Igf1-cKO mice, had PDLs. Bone volume/tissue volume (BV/TV) was decreased by 60% in lumbar vertebrae and femurs of MVNP/Igf1-cKO versus MVNP mice with PDLs and by 45% versus all MVNP mice tested. Bone formation rates were decreased 50% in Igf1-cKO and MVNP/Igf1-cKO mice versus WT and MVNP mice. MVNP mice had increased EphB2 and EphB4 levels in OCLs/OBs versus WT and MVNP/Igf1-cKO, with none detectable in OCLs/OBs of Igf1-cKO mice. Mechanistically, IL-6 induced the increased OCL-IGF1 in MVNP mice. These results suggest that high OCL-IGF1 levels increase bone formation and PDLs in PD by enhancing EphB2/EphB4 expression in vivo and suggest OCL-IGF1 may contribute to normal bone remodeling.

Long term effects on biochemical bone markers of a single infusion of zoledronic acid in Paget disease of bone.

BACKGROUND: The aim of the present study is to evaluate long term biochemical response to a single dose of zoledronic acid in patients with Paget disease of bone, as well as evaluating the value of bone turnover markers in diagnosis and follow-up. METHODS: This is an observational, descriptive and prospective study. Included patients received a single-dose intravenous infusion of 5 mg zoledronic acid. Bone turnover markers were measured at baseline, and in every follow up visit. RESULTS: Thirty-nine patients with a mean follow-up of 56.49 months were included. At the time Paget disease was diagnosed, all of the patients (100%) had high serum procollagen type 1 amino-terminal propeptide values, but not all patients had high serum C-terminal telopeptide and alkaline phosphatase values (85% and 89% respectively). Biochemical response to therapy occurred in 38 out of 39 patients (97%). Two patients had partial response at 6 months but complete response thereafter. Only one patient relapsed (nadir procollagen type 1 amino-terminal propeptide 35.06 µg/l, value at relapse 75.2 µg/l) 4.5 years after treatment. Values of serum C-terminal telopeptide and alkaline phosphatase of this patient were normal despite P1NP relapse. CONCLUSIONS: We hence conclude that zoledronic acid is effective in inducing and maintaining biochemical remission and that procollagen type 1 amino-terminal propeptide is a better diagnostic and prognostic marker in PDB when compared to C-terminal telopeptide and alkaline phosphatase.

Uptake of 18F-Fluciclovine in Paget Disease.

Since its recent approval by the United States Food and Drug Administration, fluciclovine PET-CT has gained widespread use for imaging of recurrent prostate cancer patients. As an amino acid-based radiotracer transported by LAT-1 and ASCT-2 transporters, fluciclovine exploits the up-regulation of amino acid transporters in malignant cells. We present a rare case of fluciclovine uptake in Paget disease in a 58-year-old man with suspected recurrent prostate cancer and asymmetric increased left hemipelvic uptake on imaging.

Blockade of the angiotensin II type 1 receptor increases bone mineral density and left ventricular contractility in a mouse model of juvenile Paget disease.

Juvenile Paget disease (JPD1), an autosomal-recessive disorder, is characterized by extremely rapid bone turnover due to osteoprotegerin deficiency. Its extra-skeletal manifestations, such as hypertension and heart failure, suggest a pathogenesis with shared skeletal and cardiovascular system components. In spite of this, the effects of anti-hypertensive drugs on bone morphometry remain unknown. We administered an angiotensin II type 1 receptor blocker, olmesartan (5 mg/kg/day) to 8-week-old male mice lacking the osteoprotegerin gene, with and without 1 µg/kg/min of angiotensin II infusion for 14 days. Olmesartan treatment decreased systolic blood pressure, and echocardiography showed increased left ventricular systolic contractility. Three-dimensional micro-computed tomography scans demonstrated that olmesartan treatment increased trabecular bone volume (sham, +176%; angiotensin II infusion, +335%), mineral density (sham, +150%; angiotensin II infusion, +313%), and trabecular number (sham, +407%; angiotensin II infusion, +622%) in the tibia. Olmesartan increased cortical mineral density (sham, +19%; angiotensin II infusion, +24%), decreased the cortical bone section area (sham, -16%; angiotensin II infusion, -18%), decreased thickness (sham, -18%; angiotensin II infusion, -31%), and decreased the lacunar area (sham, -41%; angiotensin II infusion, -27%) in the tibia. Similar trend was observed in the femur. Moreover, olmesartan decreased angiotensin II-induced increases in tartrate-resistant acid phosphatase concentrations in plasma, but it affected neither type I procollagen N-terminal propeptides, nor the receptor activator of nuclear factor kappa-B ligand. Our data suggest that blockade of the angiotensin II type 1 receptor improves bone vulnerability, and helps to maintain the heart's structural integrity in osteoprotegerin-deficient mice.

VCP/p97 controls signals of the ERK1/2 pathway transmitted via the Shoc2 scaffolding complex: novel insights into IBMPFD pathology.

Valosin-containing protein (VCP), also named p97, is an essential hexameric AAA+ ATPase with diverse functions in the ubiquitin system. Here we demonstrate that VCP is critical in controlling signals transmitted via the essential Shoc2-ERK1/2 signaling axis. The ATPase activity of VCP modulates the stoichiometry of HUWE1 in the Shoc2 complex as well as HUWE1-mediated allosteric ubiquitination of the Shoc2 scaffold and the RAF-1 kinase. Abrogated ATPase activity leads to augmented ubiquitination of Shoc2/RAF-1 and altered phosphorylation of RAF-1. We found that in fibroblasts from patients with inclusion body myopathy with Paget's disease of bone and frontotemporal dementia (IBMPFD) that harbor germline mutations in VCP, the levels of Shoc2 ubiquitination and ERK1/2 phosphorylation are imbalanced. This study provides a mechanistic basis for the critical role of VCP in the regulation of the ERK1/2 pathway and reveals a previously unrecognized function of the ERK1/2 pathway in the pathogenesis of IBMPFD.

Molecular insights into an ancient form of Paget's disease of bone.

Paget's disease of bone (PDB) is a chronic skeletal disorder that can affect one or several bones in individuals older than 55 y of age. PDB-like changes have been reported in archaeological remains as old as Roman, although accurate diagnosis and natural history of the disease is lacking. Six skeletons from a collection of 130 excavated at Norton Priory in the North West of England, which dates to medieval times, show atypical and extensive pathological changes resembling contemporary PDB affecting as many as 75% of individual skeletons. Disease prevalence in the remaining collection is high, at least 16% of adults, with age at death estimations as low as 35 y. Despite these atypical features, paleoproteomic analysis identified sequestosome 1 (SQSTM1) or p62, a protein central to the pathological milieu of PDB, as one of the few noncollagenous human sequences preserved in skeletal samples. Targeted proteomic analysis detected >60% of the ancient p62 primary sequence, with Western blotting indicating p62 abnormalities, including in dentition. Direct sequencing of ancient DNA excluded contemporary PDB-associated SQSTM1 mutations. Our observations indicate that the ancient p62 protein is likely modified within its C-terminal ubiquitin-associated domain. Ancient miRNAs were remarkably preserved in an osteosarcoma from a skeleton with extensive disease, with miR-16 expression consistent with that reported in contemporary PDB-associated bone tumors. Our work displays the use of proteomics to inform diagnosis of ancient diseases such as atypical PDB, which has unusual features presumably potentiated by yet-unidentified environmental or genetic factors.