Faculty Bibliography

Progranulin (PGRN) is a growth factor with a unique beads-on-a-string structure that is involved in multiple pathophysiological processes, including anti-inflammation, tissue repair, wound healing, neurodegenerative diseases, and tumorigenesis. This review presents up-to-date information concerning recent studies on the role of PGRN in inflammatory arthritis and osteoarthritis, with a special focus on the involvement of the interactions and interplay between PGRN and tumor necrosis factor receptor (TNFR) family members in regulating such musculoskeletal diseases. In addition, this paper highlights the applications of atsttrin, an engineered protein comprising three TNFR-binding fragments of PGRN, as a promising intervention in treating arthritis.

Ras guanosine triphosphatases (GTPases) regulate signaling pathways only when associated with cellular membranes through their C-terminal prenylated regions. Ras proteins move between membrane compartments in part via diffusion-limited, fluid phase transfer through the cytosol, suggesting that chaperones sequester the polyisoprene lipid from the aqueous environment. In this study, we analyze the nature of the pool of endogenous Ras proteins found in the cytosol. The majority of the pool consists of farnesylated, but not palmitoylated, N-Ras that is associated with a high molecular weight (HMW) complex. Affinity purification and mass spectrographic identification revealed that among the proteins found in the HMW fraction is VPS35, a latent cytosolic component of the retromer coat. VPS35 bound to N-Ras in a farnesyl-dependent, but neither palmitoyl- nor guanosine triphosphate (GTP)-dependent, fashion. Silencing VPS35 increased N-Ras's association with cytoplasmic vesicles, diminished GTP loading of Ras, and inhibited mitogen-activated protein kinase signaling and growth of N-Ras-dependent melanoma cells.

The production of IL-21 by T follicular helper (Tfh) cells is vital in driving the germinal centre reaction and high affinity antibody formation. However, the degree of Tfh cell heterogeneity and function is not fully understood. We used a novel IL-21eGFP reporter mouse strain to analyze the diversity and role of Tfh cells. Through the analysis of GFP expression in lymphoid organs of IL-21eGFP mice, we identified a subpopulation of GFP(+), high IL-21 producing Tfh cells present only in Peyer's Patches. GFP(+)Tfh cells were found to be polyclonal and related to GFP(-)Tfh cells of Peyer's Patches in TCR repertoire composition and overall gene expression. Studies on the mechanisms of induction of GFP(+)Tfh cells demonstrated that they required the intestinal microbiota and a diverse repertoire of CD4(+) T cells and B cells. Importantly, ablation of GFP(+) cells resulted in a reduced frequency of Peyer's Patches IgG1 and germinal center B cells in addition to small but significant shifts in gut microbiome composition. Our work highlights the diversity among IL-21 producing CD4(+) Tfh cells, and the interrelationship between the intestinal bacteria and Tfh cell responses in the gut.

Purpose: Previous studies have shown the potential of diffusion tensor imaging (DTI) in capturing damage of the cartilage. DTI indices, such as mean diffusivity (MD) and fractional anisotropy (FA) are potential biomarkers for cartilage composition and structure. Our hypothesis is that the DTI biomarkers can detect early changes in articular cartilage after mechanical injury. We use a model to replicate the mechanism of injury in the cartilage during ACL rupture, and compare the DTI biomarkers against histology and biomechanics. The study is designed to make the results translational to clinical practice, as we use a clinical scanner, employ the same imaging sequence, and use a protocol which can be optimized for clinical studies. Materials and Methods: Nine cylindrical articular cartilage-on-bone plugs from a knee replacement surgery were incubated at 37C with culture medium. Over a 3-week long protocol, cartilage MRI scanning is combined with biomechanical and histological tests. We first perform a baseline MRI and stress-strain test on each sample. MRI was performed at 3 Twith an in-house butterfly coil specifically designed for the extracted ex-vivo samples. The MRI protocol included a radial spin echo DTI (RAISED) sequence that we normally use for human in-vivo applications. We acquired two b = 0 and 12-direction b = 300 s/mm2 images at a resolution of 0.18 x 0.18 x 1.2 mm3 and derived FA and MD parameters. After MRI and the biomechanical tests, we imparted a zero (n=3), intermediate (n = 3), or high (n = 3) overload to simulate the traumatic injury of cartilage. Two weeks after, the samples underwent a third MRI and biomechanical testing. Cartilage samples were then processed for histology with safranin-O to show the PG density and distribution. Safranin-O slides were graded via OARSI scoring, varying from 0 (heathy) to 6 (bone remodelling). Results: The DTI biomarkers are sensitive to the changes on articular cartilage after injury. In severely injured samples, the MD increased by 15 % in the 2 weeks, from an average of 1.36 mum2/ms at the baseline, whereas the FAdecreased by 43%froma baseline of 0.25.Mildly injured samples showed a MD trend towards higher values, increasing by 9 % frombaseline 1.28 mum2/ms, whereas the FA did not change. As expected, the biomechanics tests reflect a trend of decreased stiffness (Young's modulus) with increasing injury. The change in MD is correlated with the changes in the biomechanics with rho = 0.75 (p = 0.04), and correlated with the OARSI (rho = 0.65, p = 0.07). The OARSI score of the baseline samples was 0.83 +/- 0.44. Severe injury samples increased their OARSI score by 2.53 +/- 1.30, while the mild injury had only a moderate increase in OARSI (0.62 +/- 1.30). Zero injury samples neither changed their score, nor their biomechanical parameters. Conclusion: DTI biomarkers do capture the early signs of damage in articular cartilage. The use of clinically feasible MRI protocols provide new biomarkers for the early diagnosis and monitoring of early stages of post-traumatic OA

Following skeletal muscle injury, muscle stem cells (satellite cells) are activated, proliferate, and differentiate to form myofibers. We show that mRNA-decay protein AUF1 regulates satellite cell function through targeted degradation of specific mRNAs containing 3' AU-rich elements (AREs). auf1-/- mice undergo accelerated skeletal muscle wasting with age and impaired skeletal muscle repair following injury. Satellite cell mRNA analysis and regeneration studies demonstrate that auf1-/- satellite cell self-renewal is impaired due to increased stability and overexpression of ARE-mRNAs, including cell-autonomous overexpression of matrix metalloprotease MMP9. Secreted MMP9 degrades the skeletal muscle matrix, preventing satellite-cell-mediated regeneration and return to quiescence. Blocking MMP9 activity in auf1-/- mice restores skeletal muscle repair and maintenance of the satellite cell population. Control of ARE-mRNA decay by AUF1 represents a mechanism for adult stem cell regulation and is implicated in human skeletal muscle wasting diseases.

Lipid accumulation in macrophages has profound effects on macrophage gene expression and contributes to the development of atherosclerosis. Here, we report that angiopoietin-like protein 4 (ANGPTL4) is the most highly upregulated gene in foamy macrophages and it's absence in haematopoietic cells results in larger atherosclerotic plaques, characterized by bigger necrotic core areas and increased macrophage apoptosis. Furthermore, hyperlipidemic mice deficient in haematopoietic ANGPTL4 have higher blood leukocyte counts, which is associated with an increase in the common myeloid progenitor (CMP) population. ANGPTL4-deficient CMPs have higher lipid raft content, are more proliferative and less apoptotic compared with the wild-type (WT) CMPs. Finally, we observe that ANGPTL4 deficiency in macrophages promotes foam cell formation by enhancing CD36 expression and reducing ABCA1 localization in the cell surface. Altogether, these findings demonstrate that haematopoietic ANGPTL4 deficiency increases atherogenesis through regulating myeloid progenitor cell expansion and differentiation, foam cell formation and vascular inflammation.

Identification and characterization of molecular mechanisms that connect genetic risk factors to initiation and evolution of disease pathophysiology represent major goals and opportunities for improving therapeutic and diagnostic outcomes in Alzheimer's disease (AD). Integrative genomic analysis of the human AD brain transcriptome holds potential for revealing novel mechanisms of dysfunction that underlie the onset and/or progression of the disease. We performed an integrative genomic analysis of brain tissue-derived transcriptomes measured from two lines of mice expressing distinct mutant AD-related proteins. The first line expresses oligomerogenic mutant APP(E693Q) inside neurons, leading to the accumulation of amyloid beta (Abeta) oligomers and behavioral impairment, but never develops parenchymal fibrillar amyloid deposits. The second line expresses APP(KM670/671NL)/PSEN1(Deltaexon9) in neurons and accumulates fibrillar Abeta amyloid and amyloid plaques accompanied by neuritic dystrophy and behavioral impairment. We performed RNA sequencing analyses of the dentate gyrus and entorhinal cortex from each line and from wild-type mice. We then performed an integrative genomic analysis to identify dysregulated molecules and pathways, comparing transgenic mice with wild-type controls as well as to each other. We also compared these results with datasets derived from human AD brain. Differential gene and exon expression analysis revealed pervasive alterations in APP/Abeta metabolism, epigenetic control of neurogenesis, cytoskeletal organization and extracellular matrix (ECM) regulation. Comparative molecular analysis converged on FMR1 (Fragile X Mental Retardation 1), an important negative regulator of APP translation and oligomerogenesis in the post-synaptic space. Integration of these transcriptomic results with human postmortem AD gene networks, differential expression and differential splicing signatures identified significant similarities in pathway dysregulation, including ECM regulation and neurogenesis, as well as strong overlap with AD-associated co-expression network structures. The strong overlap in molecular systems features supports the relevance of these findings from the AD mouse models to human AD.

Use of low resolution single cell DNA FISH and population based high resolution chromosome conformation capture techniques have highlighted the importance of pairwise chromatin interactions in gene regulation. However, it is unlikely that associations involving regulatory elements act in isolation of other interacting partners that also influence their impact. Indeed, the influence of multi-loci interactions remains something of an enigma as beyond low-resolution DNA FISH we do not have the appropriate tools to analyze these. Here we present a method that uses standard 4C-seq data to identify multi-loci interactions from the same cell. We demonstrate the feasibility of our method using 4C-seq data sets that identify known pairwise and novel tri-loci interactions involving the Tcrb and Igk antigen receptor enhancers. We further show that the three Igk enhancers, MiEkappa, 3'Ekappa and Edkappa, interact simultaneously in this super-enhancer cluster, which add to our previous findings showing that loss of one element decreases interactions between all three elements as well as reducing their transcriptional output. These findings underscore the functional importance of simultaneous interactions and provide new insight into the relationship between enhancer elements. Our method opens the door for studying multi-loci interactions and their impact on gene regulation in other biological settings.

Atherosclerosis, caused in part by monocytes in plaques, continues to be a disease that afflicts the modern world. Whilst significant steps have been made in treating this chronic inflammatory disease, questions remain on how to prevent monocyte and macrophage accumulation in atherosclerotic plaques. Junctional Adhesion Molecule C (JAM-C) expressed by vascular endothelium directs monocyte transendothelial migration in a unidirectional manner leading to increased inflammation. Here we show that interfering with JAM-C allows reverse-transendothelial migration of monocyte-derived cells, opening the way back out of the inflamed environment. To study the role of JAM-C in plaque regression we used a mouse model of atherosclerosis, and tested the impact of vascular JAM-C expression levels on monocyte reverse transendothelial migration using human cells. Studies in-vitro under inflammatory conditions revealed that overexpression or gene silencing of JAM-C in human endothelium exposed to flow resulted in higher rates of monocyte reverse-transendothelial migration, similar to antibody blockade. We then transplanted atherosclerotic, plaque-containing aortic arches from hyperlipidemic ApoE-/- mice into wild-type normolipidemic recipient mice. JAM-C blockade in the recipients induced greater emigration of monocyte-derived cells and further diminished the size of atherosclerotic plaques. Our findings have shown that JAM-C forms a one-way vascular barrier for leukocyte transendothelial migration only when present at homeostatic copy numbers. We have also shown that blocking JAM-C can reduce the number of atherogenic monocytes/macrophages in plaques by emigration, providing a novel therapeutic strategy for chronic inflammatory pathologies.