Original article Sirt1-deficient mice exhibit an altered cartilage phenotype

tObjective: We previously demonstrated that Sirt1 regulates apoptosis in cartilage in vitro. Here weattempt to examine in vivo cartilage homeostasis, using Sirt1 total body knockout (KO) mice.Method: Articular cartilage was harvested from hind paws of 1-week and 3-week-old mice carrying wildtype (WT) or null Sirt1 gene. Knees of Sirt1 haploinsufficient mice also were examined, at 6 months. Jointcartilage was processed for histologic examination or biochemical analyses of chondrocyte cultures.Results: We found that articular cartilage tissue sections from Sirt1 KO mice up to 3 weeks of age exhibitedlow levels of type 2 collagen, aggrecan, and glycosaminoglycan content. In contrast, protein levels of MMP-13 were elevated in the Sirt1 KO mice, leading to a potential increase of cartilage breakdown, alreadyshown in the heterozygous mice. Additional results showed elevated chondrocyte apoptosis in Sirt1 KOmice, as compared to WT controls. In addition to these observations, PTP1b (protein tyrosine phosphataseb) was elevated in the Sirt1 KO mice, in line with previous reports.Conclusion: The findings from this animal model demonstrated that Sirt1 KO mice presented an alteredcartilage phenotype, with an elevated apoptotic process and a potential degradative cartilage process

Original article Sirt1-deficient mice exhibit an altered cartilage phenotype

tObjective: We previously demonstrated that Sirt1 regulates apoptosis in cartilage in vitro. Here weattempt to examine in vivo cartilage homeostasis, using Sirt1 total body knockout (KO) mice.Method: Articular cartilage was harvested from hind paws of 1-week and 3-week-old mice carrying wildtype (WT) or null Sirt1 gene. Knees of Sirt1 haploinsufficient mice also were examined, at 6 months. Jointcartilage was processed for histologic examination or biochemical analyses of chondrocyte cultures.Results: We found that articular cartilage tissue sections from Sirt1 KO mice up to 3 weeks of age exhibitedlow levels of type 2 collagen, aggrecan, and glycosaminoglycan content. In contrast, protein levels of MMP-13 were elevated in the Sirt1 KO mice, leading to a potential increase of cartilage breakdown, alreadyshown in the heterozygous mice. Additional results showed elevated chondrocyte apoptosis in Sirt1 KOmice, as compared to WT controls. In addition to these observations, PTP1b (protein tyrosine phosphataseb) was elevated in the Sirt1 KO mice, in line with previous reports.Conclusion: The findings from this animal model demonstrated that Sirt1 KO mice presented an alteredcartilage phenotype, with an elevated apoptotic process and a potential degradative cartilage process

Golgi Membranes Are Absorbed into and Reemerge from the ER during Mitosis

AbstractQuantitative imaging and photobleaching were used to measure ER/Golgi recycling of GFP-tagged Golgi proteins in interphase cells and to monitor the dissolution and reformation of the Golgi during mitosis. In interphase, recycling occurred every 1.5 hr, and blocking ER egress trapped cycling Golgi enzymes in the ER with loss of Golgi structure. In mitosis, when ER export stops, Golgi proteins redistributed into the ER as shown by quantitative imaging in vivo and immuno-EM. Comparison of the mobilities of Golgi proteins and lipids ruled out the persistence of a separate mitotic Golgi vesicle population and supported the idea that all Golgi components are absorbed into the ER. Moreover, reassembly of the Golgi complex after mitosis failed to occur when ER export was blocked. These results demonstrate that in mitosis the Golgi disperses and reforms through the intermediary of the ER, exploiting constitutive recycling pathways. They thus define a novel paradigm for Golgi genesis and inheritance

Loss-of-function mutations in TNFAIP3 leading to A20 haploinsufficiency cause an early-onset autoinflammatory disease

Systemic autoinflammatory diseases are driven by abnormal activation of innate immunity. Herein we describe a new disease caused by high-penetrance heterozygous germline mutations in TNFAIP3, which encodes the NF-B regulatory protein A20, in six unrelated families with early-onset systemic inflammation. The disorder resembles Behçet\u27s disease, which is typically considered a polygenic disorder with onset in early adulthood. A20 is a potent inhibitor of the NF-B signaling pathway. Mutant, truncated A20 proteins are likely to act through haploinsufficiency because they do not exert a dominant-negative effect in overexpression experiments. Patient-derived cells show increased degradation of IBα and nuclear translocation of the NF-B p65 subunit together with increased expression of NF-B-mediated proinflammatory cytokines. A20 restricts NF-B signals via its deubiquitinase activity. In cells expressing mutant A20 protein, there is defective removal of Lys63-linked ubiquitin from TRAF6, NEMO and RIP1 after stimulation with tumor necrosis factor (TNF). NF-B-dependent proinflammatory cytokines are potential therapeutic targets for the patients with this disease

Who Needs Microtubules? Myogenic Reorganization of MTOC, Golgi Complex and ER Exit Sites Persists Despite Lack of Normal Microtubule Tracks

A wave of structural reorganization involving centrosomes, microtubules, Golgi complex and ER exit sites takes place early during skeletal muscle differentiation and completely remodels the secretory pathway. The mechanism of these changes and their functional implications are still poorly understood, in large part because all changes occur seemingly simultaneously. In an effort to uncouple the reorganizations, we have used taxol, nocodazole, and the specific GSK3-β inhibitor DW12, to disrupt the dynamic microtubule network of differentiating cultures of the mouse skeletal muscle cell line C2. Despite strong effects on microtubules, cell shape and cell fusion, none of the treatments prevented early differentiation. Redistribution of centrosomal proteins, conditional on differentiation, was in fact increased by taxol and nocodazole and normal in DW12. Redistributions of Golgi complex and ER exit sites were incomplete but remained tightly linked under all circumstances, and conditional on centrosomal reorganization. We were therefore able to uncouple microtubule reorganization from the other events and to determine that centrosomal proteins lead the reorganization hierarchy. In addition, we have gained new insight into structural and functional aspects of the reorganization of microtubule nucleation during myogenesis

An activating NLRC4 inflammasome mutation causes autoinflammation with recurrent macrophage activation syndrome

Inflammasomes are innate immune sensors that respond to pathogen and damage-associated signals with caspase-1 activation, IL-1β and IL-18 secretion, and macrophage pyroptosis. The discovery that dominant gain-of-function mutations in NLRP3 cause the Cryopyrin Associated Periodic Syndromes (CAPS) and trigger spontaneous inflammasome activation and IL-1β oversecretion, led to successful treatment with IL-1 blocking agents1. Herein, we report a de novo missense mutation, c.1009A>T, p.Thr337Ser, in the nucleotide-binding domain of inflammasome component NLRC4 (IPAF/CARD12) that causes early-onset recurrent fever flares and Macrophage Activation Syndrome (MAS). Functional analyses demonstrated spontaneous inflammasome formation and production of the inflammasome-dependent cytokines IL-1β and IL-18, the latter exceeding levels in CAPS. The NLRC4 mutation caused constitutive caspase-1 cleavage in transduced cells and increased production of IL-18 by both patient and NLRC4 mutant macrophages. Thus, we describe a novel monoallelic inflammasome defect that expands the monogenic autoinflammatory disease spectrum to include MAS and suggests novel targets for therapy

Lipid Trafficking in Hepatocytes: Relevance to Biliary Lipid Secretion

The liver contains several cell types. Parenchymal cells, or hepatocytes, are in number and in volume (60% and 80% of all liver cells, respectively) the largest group. Hepatocytes are polarized cells in which three surface domains can be distinguished: (1) the basolateral surface (sinusoidal domain) that faces the liver sinusoid and is in contact with plasma, (2) the contiguous surface in among neighboring cells, and (3) the apical surface (bile canalicular domain) where bile secretion occurs. Bile contains a variety of solutes, including a substantial amount of cholesterol and (bile-specific) phospholipids. Hepatocytes are actively involved in uptake and secretion processes. Uptake takes place at the basolateral surface, secretion both at the apical and basolateral surface. Distinct differences in lipid composition exist between the various plasma and intracellular membranes. Moreover, in spite of the continuous flow of lipids among the membranes, resulting from vesicular trafficking among intracellular membranes (organelles), these differences are maintained. Thus, machinery must be operational in the hepatocyte to ensure the maintenance of the specific lipid composition and the appropriate selection of the lipids destined for bile secretion or to prevent nonbiliary lipids from entering such a pathway. This chapter focuses on the aspects of intracellular lipid trafficking involved in bile secretory processes: the lipid composition at different sites of the hepatocyte, the origin of biliary lipids, the transport processes through the hepatocytes, and the possible mechanisms of secretion