Lithium Decreases Glial Fibrillary Acidic Protein in a Mouse Model of Alexander Disease.

Alexander disease is a fatal neurodegenerative disease caused by mutations in the astrocyte intermediate filament glial fibrillary acidic protein (GFAP). The disease is characterized by elevated levels of GFAP and the formation of protein aggregates, known as Rosenthal fibers, within astrocytes. Lithium has previously been shown to decrease protein aggregates by increasing the autophagy pathway for protein degradation. In addition, lithium has also been reported to decrease activation of the transcription factor STAT3, which is a regulator of GFAP transcription and astrogliogenesis. Here we tested whether lithium treatment would decrease levels of GFAP in a mouse model of Alexander disease. Mice with the Gfap-R236H point mutation were fed lithium food pellets for 4 to 8 weeks. Four weeks of treatment with LiCl at 0.5% in food pellets decreased GFAP protein and transcripts in several brain regions, although with mild side effects and some mortality. Extending the duration of treatment to 8 weeks resulted in higher mortality, and again with a decrease in GFAP in the surviving animals. Indicators of autophagy, such as LC3, were not increased, suggesting that lithium may decrease levels of GFAP through other pathways. Lithium reduced the levels of phosphorylated STAT3, suggesting this as one pathway mediating the effects on GFAP. In conclusion, lithium has the potential to decrease GFAP levels in Alexander disease, but with a narrow therapeutic window separating efficacy and toxicity

Structure based biophysical characterization of the PROPPIN Atg18 shows Atg18 oligomerization upon membrane binding

PROPPINs (β-propellers that bind polyphosphoinositides) are PtdIns3P and PtdIns(3,5)P2 binding autophagy related proteins. They contain two phosphatidylinositolphosphate (PIP) binding sites and a conserved FRRG motif is essential for PIP binding. Here we present the 2.0 Å resolution crystal structure of the PROPPIN Atg18 from Pichia angusta. We designed cysteine mutants for labelling with the fluorescence dyes to probe the distances of the mutants to the membrane. These measurements support a model for PROPPIN-membrane binding, where the PROPPIN sits in a perpendicular or slightly tilted orientation on the membrane. Stopped-flow measurements suggest that initial PROPPIN-membrane binding is driven by non-specific PIP interactions. The FRRG motif then retains the protein in the membrane by binding two PIP molecules as evident by a lower dissociation rate for Atg18 in comparison with its PIP binding deficient FTTG mutant. We demonstrate that the amine-specific cross-linker Bis(sulfosuccinimidyl)suberate (BS3), which is used for protein-protein cross-linking can also be applied for cross-linking proteins and phosphatidylethanolamine (PE). Cross-linking experiments with liposome bound Atg18 yielded several PE cross-linked peptides. We also observed intermolecular cross-linked peptides, which indicated Atg18 oligomerization. FRET-based stopped-flow measurements revealed that Atg18 rapidly oligomerizes upon membrane binding while it is mainly monomeric in solution

The music of lipids: how lipid composition orchestrates cellular behaviour

Background. Lipids are best known for their fundamental role in forming biological membranes and as intracellular signalling molecules. Interactions between proteins and lipids are central to nearly every cellular process yet these crucial relationships often go overlooked. Changes or switches in the lipid profile of a cell drastically affects cellular metabolism and signal transduction. In relationship to cancer, upregulation of lipid metabolism is often observed during the early stages of neoplasia and is a recognised hallmark of many types of cancer. Methods. We performed a comprehensive review of the literature using PubMed regarding lipid metabolism in cancer and the importance of protein-lipid interactions in the function of mitochondria. Results. An increase in the basal rate of de novo lipogenesis generates a substantial rise in the saturated fatty acid content of cellular membranes. The ensuing alteration in the acyl chain profile of phospholipids has severe consequences on the function of organelles and membrane-bound proteins, and result in a host of pathologies including the cardiac disorder Barth Syndrome. Conclusions. Although increased lipogenesis is specifically selected for during cellular transformation it remains unclear if it confers an advantage for survival or is a byproduct of more global changes in cellular metabolism. We discuss the current data regarding the potential of targeting the lipogenic switch as a cancer therapy. In addition, we describe the importance of mitochondrial phospholipid composition during a number mitochondria-driven events observed to have roles in cancer. We specifically highlight the function of cardiolipin in maintaining mitochondrial structure, regulating mitochondrial dynamics and bioenergetics as well as its contributions to mitophagy/autophagy and apoptosis

BAC-mediated transgenic expression of fluorescent autophagic protein Beclin 1 reveals a role for Beclin 1 in lymphocyte development.

Beclin 1/Atg6 is an essential component of the evolutionary conserved PtdIns(3)-kinase (Vps34) protein complex that regulates macroautophagy (autophagy) in eukaryotic cells and also interacts with antiapoptotic Bcl-2 family members, Bcl-2, and Bcl-x(L). To elucidate the physiological function of Beclin 1, we generated transgenic mice producing a green fluorescent Beclin 1 protein (Beclin 1-GFP) under Beclin 1 endogenous regulation. The beclin 1-GFP transgene is functional because it completely rescues early embryonic lethality in beclin 1-deficient mice. The transgenic mice appear normal, with undetected change in basal autophagy levels in different tissues, despite the additional expression of functional Beclin 1-GFP. Staining of Beclin 1-GFP shows mostly diffuse cytoplasmic distribution in various tissues. Detailed analysis of the transgene expression by flow cytometry reveals a Bcl-2-like biphasic expression pattern in developing T and B cells, as well as differential regulation of expression in mature versus immature thymocytes following in vitro stimulation. Moreover, thymocytes expressing high Beclin 1-GFP levels appear increasingly sensitive to glucocorticoid-induced apoptosis in vitro. Our results, therefore, support a role for Beclin 1 in lymphocyte development involving cross talk between autophagy and apoptosis