Lipid oxidation and autophagy in yeast.

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Characterisation of endogenous Claudin-1 expression, motility and susceptibility to hepatitis C virus in CRISPR knock-in cells.

Background Information: Claudin‐1 (CLDN1) is a four‐span transmembrane protein localised at cell–cell tight junctions (TJs), playing an important role in epithelial impermeability and tissue homoeostasis under physiological conditions. Moreover, CLDN1 expression is up‐regulated in several cancers, and the level of CLDN1 expression has been proposed as a prognostic marker of patient survival.Results: Here, we generated and characterised a novel reporter cell line expressing endogenous fluorescent levels of CLDN‐1, allowing dynamic monitoring of CLDN‐1 expression levels. Specifically, a hepatocellular carcinoma Huh7.5.1 monoclonal cell line was bioengineered using CRISPR/Cas9 to endogenously express a fluorescent TagRFP‐T protein fused at the N‐terminus of the CLDN1 protein. These cells were proved useful to measure CLDN1 expression and distribution in live cells. However, the cells were resistant to hepatitis C virus (HCV) infection, of which CLDN1 is a viral receptor, while retaining permissiveness to VSV‐G‐decorated pseudoparticles. Nonetheless, the TagRFP‐CLDN1+/+ cell line showed expected CLDN1 protein localisation at TJs and the cell monolayer had similar impermeability and polarisation features as its wild‐type counterpart. Finally, using fluorescence recovery after photobleaching (FRAP) approaches, we measured that the majority of endogenous and overexpressed TagRFP‐CLDN1 diffuses rapidly within the TJ, whereas half of the overexpressed EGFP‐CLDN1 proteins were stalled at TJs.Conclusions: The Huh7.5.1 TagRFP‐CLDN1+/+ edited cell line showed physiological features comparable to that of non‐edited cells, but became resistant to HCV infection. Our data also highlight the important impact of the fluorescent protein chosen for endogenous tagging

Lysosomal dysfunction causes neurodegeneration in mucolipidosis II 'knock-in' mice

Kollmann K, Damme M, Markmann S, et al. Lysosomal dysfunction causes neurodegeneration in mucolipidosis II 'knock-in' mice. Brain. 2012;135(9):2661-2675.Mucolipidosis II is a neurometabolic lysosomal trafficking disorder of infancy caused by loss of mannose 6-phosphate targeting signals on lysosomal proteins, leading to lysosomal dysfunction and accumulation of non-degraded material. However, the identity of storage material and mechanisms of neurodegeneration in mucolipidosis II are unknown. We have generated 'knock-in' mice with a common mucolipidosis II patient mutation that show growth retardation, progressive brain atrophy, skeletal abnormalities, elevated lysosomal enzyme activities in serum, lysosomal storage in fibroblasts and brain and premature death, closely mimicking the mucolipidosis II disease in humans. The examination of affected mouse brains at different ages by immunohistochemistry, ultrastructural analysis, immunoblotting and mass spectrometric analyses of glycans and anionic lipids revealed that the expression and proteolytic processing of distinct lysosomal proteins such as alpha-l-fucosidase, beta-hexosaminidase, alpha-mannosidase or Niemann-Pick C2 protein are more significantly impacted by the loss of mannose 6-phosphate residues than enzymes reaching lysosomes independently of this targeting mechanism. As a consequence, fucosylated N-glycans, GM2 and GM3 gangliosides, cholesterol and bis(monoacylglycero)phosphate accumulate progressively in the brain of mucolipidosis II mice. Prominent astrogliosis and the accumulation of organelles and storage material in focally swollen axons were observed in the cerebellum and were accompanied by a loss of Purkinje cells. Moreover, an increased neuronal level of the microtubule-associated protein 1 light chain 3 and the formation of p62-positive neuronal aggregates indicate an impairment of constitutive autophagy in the mucolipidosis II brain. Our findings demonstrate the essential role of mannose 6-phosphate for selected lysosomal proteins to maintain the capability for degradation of sequestered components in lysosomes and autophagolysosomes and prevent neurodegeneration. These lysosomal proteins might be a potential target for a valid therapeutic approach for mucolipidosis II disease