Runx Expression in Normal and Osteoarthritic Cartilage: Possible Functions of Runx Proteins in Chondrocytes: A Dissertation

The Runx family of transcription factors supports cell fate determination, cell cycle regulation, global protein synthesis control, and genetic as well as epigenetic regulation of target genes. Runx1, which is essential for hematopoiesis; Runx2, which is required for osteoblast differentiation; and Runx3, which is involved in neurologic and gut development; are expressed in the growth plate during chondrocyte maturation, and in the chondrocytes of permanent cartilage structures. While Runx2 is known to control genes that contribute to chondrocyte hypertrophy, the functions of Runx1 and Runx3 during chondrogenesis and in cartilage tissue have been less well studied. The goals of this project were to characterize expression of Runx proteins in articular cartilage and differentiating chondrocytes and to determine the contribution of Runx1 to osteoarthritis (OA). Here, the expression pattern of Runx1 and Runx2 was characterized in normal bovine articular cartilage. Runx2 is expressed at higher levels in deep zone chondrocytes, while Runx1 is primarily expressed in superficial zone chondrocytes, which is the single cell layer that lines the surface of articular cartilage. Based on this finding, the hypothesis was tested that Runx1 is involved in osteoarthritis, which is a disease characterized by degradation of articular cartilage and changes in chondrocytes. These studies showed that Runx1 is upregulated in articular cartilage explants in response to mechanical compression. Runx1 was also expressed in chondrocytes found at the periphery of OA lesions in the articular cartilage of mice that underwent an OA-inducing surgery. Runx1 was also upregulated in cartilage explants of human osteoarthritic knees, and IHC data showed that Runx1 is mainly expressed in chondrocyte “clones” characteristic of OA. To ascertain the potential function of the upregulation of Runx1 in these cartilage stress conditions and disease states, the hypothesis was tested that Runx1 is upregulated in very specific chondrocyte populations in response to the cartilage damage in osteoarthritis. These studies addressed the properties of these cells that related to functions in cell growth and differentiation. In both the surface layer of normal articular cartilage, and in OA cartilage, Runx1 expression by IF co-localized with markers of mesenchymal progenitor cells, as well as markers of proliferation Ki-67 and PCNA. This finding indicated that Runx1 is found in a population of cells that represent a proliferative population of mesenchymal progenitor cells in osteoarthritis. To further address Runx1 function and identify downstream targets of Runx proteins, a promoter analysis of genes that are known to be either downregulated or upregulated during chondrocyte maturation was done. These studies found that many of these genes have 1 or more Runx binding sites within 2kb of their transcription start site, indicating that they are potential downstream Runx target genes. Lastly, some preliminary experiments were done to characterize novel roles of Runx proteins in the chondrocyte. Runx proteins have been shown to epigenetically regulate their target genes by remaining bound to them throughout mitosis, “poising” them for transcription upon exit from mitosis. The hypothesis that Runx proteins also function by remaining bound to their target genes throughout mitosis in chondrocytes was tested. It was demonstrated by immunofluorescense imaging of Runx proteins on metaphase chromosomes of ATDC5 cells, that Runx2 remains bound to chromosomes during mitosis. Cell proliferation and hypertrophy are both linked to increases in protein synthesis. Runx factors, which regulate rates of global protein synthesis, are expressed in both proliferating and hypertrophic chondrocytes. Thus, it was hypothesized that Runx proteins regulate rates of global protein synthesis during chondrocyte maturation. These studies showed that the overexpression of Runx proteins in a chondrocyte cell line (ATDC5) did not affect protein synthesis rates or levels of protein synthesis machinery. Additionally, Runx proteins did not affect proliferation rates in this chondrocyte cell line

H3.1K27me1 maintains transcriptional silencing and genome stability by preventing GCN5-mediated histone acetylation

Epigenetic mechanisms play diverse roles in the regulation of genome stability in eukaryotes. In Arabidopsis thaliana, genome stability is maintained during DNA replication by the H3.1K27 methyltransferases ARABIDOPSIS TRITHORAX-RELATED PROTEIN 5 (ATXR5) and ATXR6, which catalyze the deposition of K27me1 on replication-dependent H3.1 variants. The loss of H3.1K27me1 in atxr5 atxr6 double mutants leads to heterochromatin defects, including transcriptional de-repression and genomic instability, but the molecular mechanisms involved remain largely unknown. In this study, we identified the transcriptional co-activator and conserved histone acetyltransferase GCN5 as a mediator of transcriptional de-repression and genomic instability in the absence of H3.1K27me1. GCN5 is part of a SAGA-like complex in plants that requires the GCN5-interacting protein ADA2b and the chromatin remodeler CHR6 to mediate the heterochromatic defects in atxr5 atxr6 mutants. Our results also indicate that Arabidopsis GCN5 acetylates multiple lysine residues on H3.1 variants, but H3.1K27 and H3.1K36 play essential functions in inducing genomic instability in the absence of H3.1K27me1. Finally, we show that H3.1K36 acetylation by GCN5 is negatively regulated by H3.1K27me1 in vitro. Overall, this work reveals a key molecular role for H3.1K27me1 in maintaining transcriptional silencing and genome stability in heterochromatin by restricting GCN5-mediated histone acetylation in plants

Measuring retention within the adolescent brain cognitive development (ABCD)SM study

The Adolescent Brain Cognitive Development (ABCD

Accelerating drug discovery for Alzheimer's disease: best practices for preclinical animal studies

Animal models have contributed significantly to our understanding of the underlying biological mechanisms of Alzheimer's disease (AD). As a result, over 300 interventions have been investigated and reported to mitigate pathological phenotypes or improve behavior in AD animal models or both. To date, however, very few of these findings have resulted in target validation in humans or successful translation to disease-modifying therapies. Challenges in translating preclinical studies to clinical trials include the inability of animal models to recapitulate the human disease, variations in breeding and colony maintenance, lack of standards in design, conduct and analysis of animal trials, and publication bias due to under-reporting of negative results in the scientific literature. The quality of animal model research on novel therapeutics can be improved by bringing the rigor of human clinical trials to animal studies. Research communities in several disease areas have developed recommendations for the conduct and reporting of preclinical studies in order to increase their validity, reproducibility, and predictive value. To address these issues in the AD community, the Alzheimer's Drug Discovery Foundation partnered with Charles River Discovery Services (Morrisville, NC, USA) and Cerebricon Ltd. (Kuopio, Finland) to convene an expert advisory panel of academic, industry, and government scientists to make recommendations on best practices for animal studies testing investigational AD therapies. The panel produced recommendations regarding the measurement, analysis, and reporting of relevant AD targets, th choice of animal model, quality control measures for breeding and colony maintenance, and preclinical animal study design. Major considerations to incorporate into preclinical study design include a priori hypotheses, pharmacokinetics-pharmacodynamics studies prior to proof-of-concept testing, biomarker measurements, sample size determination, and power analysis. The panel also recommended distinguishing between pilot 'exploratory' animal studies and more extensive 'therapeutic' studies to guide interpretation. Finally, the panel proposed infrastructure and resource development, such as the establishment of a public data repository in which both positive animal studies and negative ones could be reported. By promoting best practices, these recommendations can improve the methodological quality and predictive value of AD animal studies and make the translation to human clinical trials more efficient and reliable

Characteristics of undiagnosed diseases network applicants: implications for referring providers

Abstract Background The majority of undiagnosed diseases manifest with objective findings that warrant further investigation. The Undiagnosed Diseases Network (UDN) receives applications from patients whose symptoms and signs have been intractable to diagnosis; however, many UDN applicants are affected primarily by subjective symptoms such as pain and fatigue. We sought to characterize presenting symptoms, referral sources, and demographic factors of applicants to the UDN to identify factors that may determine application outcome and potentially differentiate between those with undiagnosed diseases (with more objective findings) and those who are less likely to have an undiagnosed disease (more subjective symptoms). Methods We used a systematic retrospective review of 151 consecutive Not Accepted and 50 randomly selected Accepted UDN applications. The primary outcome was whether an applicant was Accepted, or Not Accepted, and, if accepted, whether or not a diagnosis was made. Objective and subjective symptoms and information on prior specialty consultations were collected from provider referral letters. Demographic data and decision data on network acceptance were gathered from the UDN online portal. Results Fewer objective findings and more subjective symptoms were found in the Not Accepted applications. Not Accepted referrals also were from older individuals, reported a shorter period of illness, and were referred to the UDN by their primary care physicians. All of these differences reached statistical significance in comparison with Accepted applications. The frequency of subspecialty consults for diagnostic purposes prior to UDN application was similar in both groups. Conclusions The preponderance of subjective and lack of objective findings in the Not Accepted applications distinguish these from applicants that are accepted for evaluation and diagnostic efforts through the UDN. Not Accepted applicants are referred primarily by their primary care providers after multiple specialist consultations fail to yield answers. Distinguishing between patients with undiagnosed diseases with objective findings and those with primarily subjective findings can delineate patients who would benefit from further diagnostic processes from those who may have functional disorders and need alternative pathways for management of their symptoms. Trial registration clinicaltrials.gov NCT02450851 , posted May 21st 2015

Feasibility and safety of a 6-month exercise program to increase bone and muscle strength in children with juvenile idiopathic arthritis

Background: Arthritis in childhood can be associated with muscle weakness around affected joints, low bone mass and low bone strength. Exercise is recognized as an important part of management of children with juvenile idiopathic arthritis (JIA) but the exercise prescription to best promote bone and muscle health is unknown. We therefore aimed to: 1. assess feasibility and safety of a 6-month home- and group-based exercise program for children with JIA; 2. estimate the effect of program participation on bone mass and strength, muscle function and clinical outcomes and 3. determine if any positive changes in bone and muscle outcomes are maintained 6 months later. Methods: We recruited 24 children with JIA who were part of the Linking Exercise, Physical Activity and Pathophysiology in Childhood Arthritis (LEAP) study to participate in a 6-month home-based exercise program involving jumping and handgrip exercises, resistance training and one group exercise session per month. We assessed lumbar spine bone mass (dual energy X-ray absorptiometry), distal tibia and radius bone microarchitecture and strength (high-resolution peripheral quantitative computed tomography), muscle function (jumping mechanography, dynamometry) and clinical outcomes (joint assessment, function, health-related quality of life) at baseline, 6- and 12-months. Adherence was assessed using weekly activity logs. Results: Thirteen children completed the 6-month intervention. Participants reported 9 adverse events and post-exercise pain was rare (0.4%). Fatigue improved, but there were no other sustained improvements in muscle, bone or clinical outcomes. Adherence to the exercise program was low (47%) and decreased over time. Conclusion: Children with JIA safely participated in a home-based exercise program designed to enhance muscle and bone strength. Fatigue improved, which may in turn facilitate physical activity participation. Prescribed exercise posed adherence challenges and efforts are needed to address facilitators and barriers to participation in and adherence to exercise programs among children with JIA. Trial registration: Data of the children with JIA are from the LEAP study (Canadian Institutes of Health Research (CIHR; GRANT# 107535). http://www.leapjia.com/

Correspondence Between Perceived Pubertal Development and Hormone Levels in 9-10 Year-Olds From the Adolescent Brain Cognitive Development Study.

Aim: To examine individual variability between perceived physical features and hormones of pubertal maturation in 9-10-year-old children as a function of sociodemographic characteristics. Methods: Cross-sectional metrics of puberty were utilized from the baseline assessment of the Adolescent Brain Cognitive Development (ABCD) Study—a multi-site sample of 9–10 year-olds (n = 11,875)—and included perceived physical features via the pubertal development scale (PDS) and child salivary hormone levels (dehydroepiandrosterone and testosterone in all, and estradiol in females). Multi-level models examined the relationships among sociodemographic measures, physical features, and hormone levels. A group factor analysis (GFA) was implemented to extract latent variables of pubertal maturation that integrated both measures of perceived physical features and hormone levels. Results: PDS summary scores indicated more males (70%) than females (31%) were prepubertal. Perceived physical features and hormone levels were significantly associated with child\u27s weight status and income, such that more mature scores were observed among children that were overweight/obese or from households with low-income. Results from the GFA identified two latent factors that described individual differences in pubertal maturation among both females and males, with factor 1 driven by higher hormone levels, and factor 2 driven by perceived physical maturation. The correspondence between latent factor 1 scores (hormones) and latent factor 2 scores (perceived physical maturation) revealed synchronous and asynchronous relationships between hormones and concomitant physical features in this large young adolescent sample. Conclusions: Sociodemographic measures were associated with both objective hormone and self-report physical measures of pubertal maturation in a large, diverse sample of 9-10 year-olds. The latent variables of pubertal maturation described a complex interplay between perceived physical changes and hormone levels that hallmark sexual maturation, which future studies can examine in relation to trajectories of brain maturation, risk/resilience to substance use, and other mental health outcomes

A Tale of Two Addiction Theories: the effects of cocaine exposure on cue-induced motivation and action control

Cocaine addiction affects approximately 1.4 million Americans, costing the government billions of dollars and the addicted individual their life. Addiction is characterized by a continued desire to use a drug despite decreased enjoyment from taking it and a desire to abstain. Multiple theories attempt to explain how prolonged drug use induces the chronic brain changes that result in addiction. The incentive sensitization theory suggests that repeated exposure to drugs of abuse alters the neural circuitry that is involved in incentive motivation, the process that allows drugs and their associated stimuli to more strongly encourage drug-seeking behavior. Alternatively, the habit learning theory suggests that drugs of abuse pathologically subvert the reward-learning circuitry, leading to compulsive drug seeking triggered by drug-associated stimuli. Since both theories are based on the idea that drugs of abuse affect the dopaminergic system and alter normal reward processing, it has been proposed that drug exposure may also affect behavior for natural rewards. To understand the mechanisms by which cocaine use modifies reward-seeking behaviors, I have conducted experiments to investigate both addiction theories using cocaine and food rewards as the outcome. I have found that cocaine-paired cues promote cocaine-seeking and taking actions via a Pavlovian motivational mechanism. I have also explored the importance of the contingency of drug delivery and found that cocaine has a general effect on incentive motivation, with both self-administered and experimenter-delivered cocaine enhancing the ability of food-paired cues to motivate food-seeking behavior. This result was not obtained with animals that passively received cocaine infusions, suggesting a role of predictability in the effects of cocaine on incentive motivation. I have also found support for the habit learning theory, demonstrating that cocaine treatment can encourage habitual control of action selection for food rewards, even when feedback is given. However, experimenter-delivered cocaine does not prevent animals from learning to perform a behavior in a goal-directed fashion. Our results lend support to both theories, suggesting that drug induced changes to the dopaminergic systems may be affecting both incentive motivation and reward learning. These results have important implications for our comprehension of the neural processes involved in addiction