Faculty Bibliography

Connexins are integral membrane building blocks that form gap junctions, enabling direct cytoplasmic exchange of ions and low-molecular-mass metabolites between adjacent cells. In the heart, gap junctions mediate the propagation of cardiac action potentials and the maintenance of a regular beating rhythm. A number of connexin interacting proteins have been described and are known gap junction regulators either through direct effects (e.g., kinases) or the formation of larger multifunctional complexes (e.g., cytoskeleton scaffold proteins). Most connexin partners can be categorized as either proteins promoting coupling by stimulating forward trafficking and channel opening or inhibiting coupling by inducing channel closure, internalization, and degradation. While some interactions have only been implied through co-localization using immunohistochemistry, others have been confirmed by biophysical methods that allow detection of a direct interaction. Our understanding of these interactions is, by far, most well developed for connexin 43 (Cx43) and the scope of this review is to summarize our current knowledge of their functional and regulatory roles. The significance of these interactions is further exemplified by demonstrating their importance at the intercalated disc, a major hub for Cx43 regulation and Cx43 mediated effects.

OBJECTIVE:It has been reported that a disintegrin and metalloproteinase with thrombospondin motifs-12 (ADAMTS-12) is a susceptibility gene for rheumatoid arthritis (RA) development, and its level is significantly increased in RA patients. In addition, ADAMTS-12 was also reported to be required for normal inflammation. This study aims to determine the role of ADAMTS-12 and the underlying mechanisms in the pathogenesis of inflammatory arthritis. METHODS:The collagen-induced arthritis (CIA) model was established in ADAMTS-12-deficient mice and their control littermates to determine the role of ADAMTS-12 in vivo; microCT scanning was used to demonstrate the destruction of ankle joint; histological analysis illustrated synovitis, pannus formation, as well as bone and cartilage destruction; ELISA was performed to measure serum levels of inflammatory cytokines; Protein-protein interaction assays were performed to detect the interactions of ADAMTS-12 and its various deletion mutants with connective tissue growth factor (CTGF). RESULTS:Deficiency of ADAMTS-12 leads to accelerated inflammatory arthritis in the CIA mouse model. Loss of ADAMTS-12 causes enhanced osteoclastogenesis. In vitro and in vivo protein-protein interaction assays demonstrate CTGF, a previously unrecognized substrate of ADAMTS-12, binding and processing by ADAMTS-12. In addition, deletion of ADAMTS-12 enhances, while overexpression of ADMATS-12 reduces, CTGF-mediated inflammation. Furthermore, ADAMTS-12 regulation of inflammation is largely lost in CTGF deficient macrophages. Importantly, blocking CTGF attenuates elevated inflammatory arthritis seen in ADAMTS-12-deficient CIA mouse model. CONCLUSION/CONCLUSIONS:ADAMTS-12 proves to be a critical regulator of inflammatory arthritis through, at least in part, controlling CTGF turnover.

Worldwide there are over 46 million people living with dementia, and this number is expected to double every 20 years reaching about 131 million by 2050. The cost to the community and government health systems, as well as the stress on families and carers is incalculable. Over three decades of research into this disease have been undertaken by several research groups in Australia, including work by our original research group in Western Australia which was involved in the discovery and sequencing of the amyloid-β peptide (also known as Aβ or A4 peptide) extracted from cerebral amyloid plaques. This review discusses the journey from the discovery of the Aβ peptide in Alzheimer's disease (AD) brain to the establishment of pre-clinical AD using PET amyloid tracers, a method now serving as the gold standard for developing peripheral diagnostic approaches in the blood and the eye. The latter developments for early diagnosis have been largely achieved through the establishment of the Australian Imaging Biomarker and Lifestyle research group that has followed 1,100 Australians for 11 years. AIBL has also been instrumental in providing insight into the role of the major genetic risk factor apolipoprotein E ɛ4, as well as better understanding the role of lifestyle factors particularly diet, physical activity and sleep to cognitive decline and the accumulation of cerebral Aβ.

There is a great need for effective treatment options for post-traumatic stress disorder (PTSD). Neuropeptide Y (NPY) is associated with resilience to traumatic stress. MC4R antagonists, such as HS014, also reduce response to stress. Both regulate stress-responsive systems - the hypothalamic-pituitary-axis (HPA) and the noradrenergic nervous system and their associated behaviors. Therefore, we examined if their intranasal delivery to brain could attenuate development of PTSD-related symptoms in single prolonged stress (SPS) rodent PTSD model. Three regimens were used: (1) prophylactic treatment 30 min before SPS stressors, (2) early intervention right after SPS stressors, (3) therapeutic treatment when PTSD behaviors are manifested 1 wk or more after the traumatic stress. NPY delivered by regimen 1 or 2 prevented SPS-triggered elevation in anxiety, depressive-like behavior, and hyperarousal and reduced dysregulation of HPA axis. Hypothalamic CRH mRNA and GR in ventral hippocampus were significantly induced in vehicle- but not NPY-treated group. NPY also prevented hypersensitivity of LC/NE system to novel mild stressor and induction of CRH in amygdala. Some of these impairments were also reduced with HS014, alone or together with NPY. When given after symptoms were manifested (regiment 3), NPY attenuated anxiety and depressive behaviors. This demonstrates strong preclinical proof of concept for intranasal NPY, and perhaps MC4R antagonists, for non-invasive early pharmacological interventions for PTSD and comorbid disorders and possibly also as therapeutic strategy.

Alterations in ectopic lipid deposition and circulating lipids are major risk factors for developing cardiometabolic diseases. Angiopoietin-like protein 4 (ANGPTL4), a protein that inhibits lipoprotein lipase (LPL), controls fatty acid (FA) uptake in adipose and oxidative tissues and regulates circulating triacylglycerol-rich (TAG-rich) lipoproteins. Unfortunately, global depletion of ANGPTL4 results in severe metabolic abnormalities, inflammation, and fibrosis when mice are fed a high-fat diet (HFD), limiting our understanding of the contribution of ANGPTL4 in metabolic disorders. Here, we demonstrate that genetic ablation of ANGPTL4 in adipose tissue (AT) results in enhanced LPL activity, rapid clearance of circulating TAGs, increased AT lipolysis and FA oxidation, and decreased FA synthesis in AT. Most importantly, we found that absence of ANGPTL4 in AT prevents excessive ectopic lipid deposition in the liver and muscle, reducing novel PKC (nPKC) membrane translocation and enhancing insulin signaling. As a result, we observed a remarkable improvement in glucose tolerance in short-term HFD-fed AT-specific Angptl4-KO mice. Finally, lack of ANGPTL4 in AT enhances the clearance of proatherogenic lipoproteins, attenuates inflammation, and reduces atherosclerosis. Together, these findings uncovered an essential role of AT ANGPTL4 in regulating peripheral lipid deposition, influencing whole-body lipid and glucose metabolism and the progression of atherosclerosis.

The integrated stress response (ISR) is regulated by kinases that phosphorylate the α subunit of translation initiation factor 2 and phosphatases that dephosphorylate it. Genetic and biochemical observations indicate that the eIF2αP-directed holophosphatase - a therapeutic target in diseases of protein misfolding - is comprised of a regulatory, PPP1R15, and a catalytic, Protein Phosphatase 1 (PP1) subunit. In mammals, there are two isoforms of the regulatory subunit, PPP1R15A and PPP1R15B, with overlapping roles in the essential function of eIF2αP dephosphorylation. However, conflicting reports have appeared regarding the requirement for an additional co-factor, G-actin, in enabling substrate-specific dephosphorylation by PPP1R15-containing PP1 holoenzymes. An additional concern relates to the sensitivity of the holoenzyme to the [(o-chlorobenzylidene)amino]guanidines Sephin1 or Guanabenz, putative small molecule proteostasis modulators. It has been suggested that the source and method of purification of the PP1 catalytic subunit and the presence or absence of an N-terminal repeat-containing region in the PPP1R15A regulatory subunit might influence the requirement for G-actin and sensitivity of the holoenzyme to inhibitors. We find that eIF2αP-dephosphorylation by PP1 was moderately stimulated by repeat-containing PPP1R15A in an unphysiological low ionic strength buffer, whereas stimulation imparted by the co-presence of PPP1R15A and G-actin was observed under a broad range of conditions: low and physiological ionic strength; whether PPP1R15A regulatory subunit had or lacked the N-terminal repeat-containing region; and whether it was paired with native PP1 purified from rabbit muscle, or recombinant PP1 purified from bacteria. Furthermore, none of the PPP1R15A-containing holophosphatases tested was inhibited by Sephin1 or Guanabenz.

During embryogenesis, cells acquire distinct fates by transitioning through transcriptional states. To uncover these transcriptional trajectories during zebrafish embryogenesis, we sequenced 38,731 cells and developed URD, a simulated diffusion-based computational reconstruction method. URD identified the trajectories of 25 cell types through early somitogenesis, gene expression along them, and their spatial origin in the blastula. Analysis of Nodal signaling mutants revealed that their transcriptomes were canalized into a subset of wild-type transcriptional trajectories. Some wild-type developmental branchpoints contained cells expressing genes characteristic of multiple fates. These cells appeared to trans-specify from one fate to another. These findings reconstruct the transcriptional trajectories of a vertebrate embryo, highlight the concurrent canalization and plasticity of embryonic specification, and provide a framework to reconstruct complex developmental trees from single-cell transcriptomes.

Alterations in lipoprotein metabolism enhance the risk of cardiometabolic disorders including type-2 diabetes and atherosclerosis, the leading cause of death in Western societies. While the transcriptional regulation of lipid metabolism has been well characterized, recent studies have uncovered the importance of microRNAs (miRNAs), long-non-coding RNAs (lncRNAs) and RNA binding proteins (RBP) in regulating the expression of lipid-related genes at the posttranscriptional level. Work from several groups has identified a number of miRNAs, including miR-33, miR-122 and miR-148a, that play a prominent role in controlling cholesterol homeostasis and lipoprotein metabolism. Importantly, dysregulation of miRNA expression has been associated with dyslipidemia, suggesting that manipulating the expression of these miRNAs could be a useful therapeutic approach to ameliorate cardiovascular disease (CVD). The role of lncRNAs in regulating lipid metabolism has recently emerged and several groups have demonstrated their regulation of lipoprotein metabolism. However, given the high abundance of lncRNAs and the poor-genetic conservation between species, much work will be needed to elucidate the specific role of lncRNAs in controlling lipoprotein metabolism. In this review article, we summarize recent findings in the field and highlight the specific contribution of lncRNAs and RBPs in regulating lipid metabolism.

Stress responses promote obesity and insulin resistance, in part, by activating the stress-responsive mitogen-activated protein kinases (MAPKs), p38 MAPK, and c-Jun NH₂-terminal kinase (JNK). Stress also induces expression of MAPK phosphatase-1 (MKP-1), which inactivates both JNK and p38 MAPK. However, the equilibrium between JNK/p38 MAPK and MKP-1 signaling in the development of obesity and insulin resistance is unclear. Skeletal muscle is a major tissue involved in energy expenditure and glucose metabolism. In skeletal muscle, MKP-1 is upregulated in high-fat diet-fed mice and in skeletal muscle of obese humans. Mice lacking skeletal muscle expression of MKP-1 (MKP1-MKO) showed increased skeletal muscle p38 MAPK and JNK activities and were resistant to the development of diet-induced obesity. MKP1-MKO mice exhibited increased whole-body energy expenditure that was associated with elevated levels of myofiber-associated mitochondrial oxygen consumption. miR-21, a negative regulator of PTEN expression, was upregulated in skeletal muscle of MKP1-MKO mice, resulting in increased Akt activity consistent with enhanced insulin sensitivity. Our results demonstrate that skeletal muscle MKP-1 represents a critical signaling node through which inactivation of the p38 MAPK/JNK module promotes obesity and insulin resistance.

Osteoarthritis (OA) results from degenerative and abnormal function of joints, with localized biochemistry playing a critical role in its onset and progression. As high levels of all- trans retinoic acid (ATRA) in synovial fluid have been identified as a contributive factor to OA, the synthesis of de novo antagonists for retinoic acid receptors (RARs) has been exploited to interrupt the mechanism of ATRA action. BMS493, a pan-RAR inverse agonist, has been reported as an effective inhibitor of ATRA signaling pathway; however, it is unstable and rapidly degrades under physiological conditions. We employed an engineered cartilage oligomeric matrix protein coiled-coil (C_cc^S) protein for the encapsulation, protection, and delivery of BMS493. In this study, we determine the binding affinity of C_cc^S to BMS493 and the stimulator, ATRA, via competitive binding assay, in which ATRA exhibits approximately 5-fold superior association with C_cc^S than BMS493. Interrogation of the structure of C_cc^S indicates that ATRA causes about 10% loss in helicity, while BMS493 did not impact the structure. Furthermore, C_cc^S self-assembles into nanofibers when bound to BMS493 or ATRA as expected, displaying 11-15 nm in diameter. Treatment of human articular chondrocytes in vitro reveals that C_cc^S·BMS493 demonstrates a marked improvement in efficacy in reducing the mRNA levels of matrix metalloproteinase-13 (MMP-13), one of the main proteases responsible for the degradation of the extracellular cartilage matrix compared to BMS493 alone in the presence of ATRA, interleukin-1 beta (IL-1β), or IL-1 β together with ATRA. These results support the feasibility of utilizing coiled-coil proteins as drug delivery vehicles for compounds of relatively limited bioavailability for the potential treatment of OA.