Mammalian cells acquire cholesterol mainly from LDL. LDL enter the endosomes, allowing cholesteryl esters to be hydrolyzed by acid lipase. The hydrolyzed cholesterol (LDL-CHOL) enters the Niemann–Pick type C1 (NPC1)-containing endosomal compartment en route to various destinations. Whether the Golgi is involved in LDL-CHOL transport downstream of the NPC1 compartment has not been demonstrated. Using subcellular fractionation and immunoadsorption to enrich for specific membrane fractions, here we show that, when parental Chinese hamster ovary (CHO) cells are briefly exposed to 3 H-cholesteryl linoleate (CL) labeled-LDL, newly liberated 3 H-LDL-CHOL appears in membranes rich in trans-Golgi network (TGN) long before it becomes available for re-esterification at the endoplasmic reticulum (ER) or for efflux at the plasma membrane. In mutant cells lacking NPC1, the appearance of newly liberated 3 H-LDL-CHOL in the TGN-rich fractions is much reduced. We next report a reconstituted transport system that recapitulates the transport of LDL-CHOL to the TGN and to the ER. The transport system requires ATP and cytosolic factors and depends on functionality of NPC1. We demonstrate that knockdown by RNAi of 3 TGN-specific SNAREs (VAMP4, syntaxin 6, and syntaxin 16) reduces ≥50% of the LDL-CHOL transport in intact cells and in vitro. These results show that vesicular trafficking is involved in transporting a significant portion of LDL-CHOL from the NPC1-containing endosomal compartment to the TGN before its arrival at the ER.

Both genetic inactivation and pharmacological inhibition of the cholesteryl ester synthetic enzyme acyl-CoA:cholesterol acyltransferase 1 (ACAT1) have shown benefit in mouse models of Alzheimer's disease (AD). In this study, we aimed to test the potential therapeutic applications of adeno-associated virus (AAV)-mediated Acat1 gene knockdown in AD mice. We constructed recombinant AAVs expressing artificial microRNA (miRNA) sequences, which targeted Acat1 for knockdown. We demonstrated that our AAVs could infect cultured mouse neurons and glia and effectively knockdown ACAT activity in vitro. We next delivered the AAVs to mouse brains neurosurgically, and demonstrated that Acat1-targeting AAVs could express viral proteins and effectively diminish ACAT activity in vivo, without inducing appreciable inflammation. We delivered the AAVs to the brains of 10-month-old AD mice and analyzed the effects on the AD phenotype at 12 months of age. Acat1-targeting AAV delivered to the brains of AD mice decreased the levels of brain amyloid-β and full-length human amyloid precursor protein (hAPP), to levels similar to complete genetic ablation of Acat1. This study provides support for the potential therapeutic use of Acat1 knockdown gene therapy in AD.

Patients with Alzheimer’s disease (AD) display amyloidopathy and tauopathy. In mouse models of AD, pharmacological inhibition using small molecule enzyme inhibitors, or genetic inactivation of Acyl-CoA: cholesterol acyltransferase 1 (ACAT1) diminished amyloidopathy and restored cognitive deficits. In microglia, ACAT1 blockage increases autophagosome formation and stimulates amyloid β peptide1–42 degradation. Here we hypothesize that in neurons ACAT1 blockage augments autophagy and increases autophagy-mediated degradation of P301L-tau protein. We tested this possibility in murine neuroblastoma cells ectopically expressing human tau, and in primary neurons isolated from triple transgenic AD (3XTg-AD) mice that express mutant forms of APP, PS1, and human tau. The results show that ACAT1 blockage increases autophagosome formation and decreases P301L-tau protein content without affecting endogenous mouse tau protein content. In vivo, lacking Acat1 decreases P301L-tau protein content in the brains of young 3XTg-AD mice but not in those of old mice, where extensive hyperphosphorylations and aggregation of P301L-tau take place. These results suggest that, in addition to ameliorating amyloidopathy in both young and old AD mice, ACAT1 blockage may benefit AD by reducing tauopathy at early stage.

The FYVE domain binds with high specificity and avidity to phosphatidylinositol 3-phosphate. It is present in Ϸ30 proteins in humans, some of which have been implicated in functions ranging from early endosome fusion to signal transduction through the TGF-␤ receptor. To develop a further understanding of the biological roles of this protein family, we turned to the nematode Caenorhabditis elegans, which contains only 12 genes predicted to encode for phosphatidylinositol 3-phosphate binding, FYVE domain-containing proteins, all of which have homologs in the human genome. Each of these proteins was targeted individually by RNA interference. One protein, WDFY2, produced a strong inhibition of endocytosis when silenced. WDFY2 contains WD40 motifs and a FYVE domain, is highly conserved between species, and localizes to a set of small endosomes that reside within 100 nm from the plasma membrane. These endosomes are involved in transferrin uptake but lack the classical endosomal markers Rab5 and EEA1. Silencing of WDFY2 by siRNA in mammalian cells impaired transferrin endocytosis. These studies reveal the important, conserved role of WDFY2 in endocytosis, and the existence of a subset of early endosomes, closely associated with the plasma membrane, that may constitute the first stage of endocytic processing of internalized cargo.internalization ͉ phosphatidylinositol 3-kinase ͉ phosphoinositide ͉ total internal reflection fluorescence microscopy P hosphorylated phosphoinositides play critical roles in the process of endocytosis. Phosphatidylinositol 3-phosphate [PtdIns(3)P], a major product of PtdIns 3-kinase, is found almost exclusively on the surface of endosomes, where it can recruit proteins containing FYVE or PX domains (1, 2). The first protein found to be recruited onto endosomes in a PtdIns 3-kinasedependent manner was EEA1 (3-5). EEA1 also interacts with the GTPase Rab5 (6, 7), and calmodulin (8-10) and has been proposed to function as a tether to facilitate early endosome fusion (6,(11)(12)(13)(14).Many other proteins containing FYVE domains are recruited to early endosomes. Examples include the proteins Rabenosyn5 (15) and Rabip4 (16), which appear to coordinate the functions of the small GTPases Rab4 and Rab5, and Hrs, which is involved in ubiquitin-mediated lysosomal degradation (17, 18). In addition, Fab1p͞PIKfyve, which catalyzes the phosphorylation of PtdIns(3)P to PtdIns(3,5)P2 appears to have an important role in multivesicular body formation (19-21). FYVE domain-containing proteins also function in pathways not primarily related to endosomal trafficking. These include SARA (22-24), which mediates signal transduction through the TGF-␤ receptor. The human UniGene collection lists 30 different FYVE-domain-containing proteins, indicating that many more functions involving PtdIns(3)P and FYVE-domain interaction remain to be discovered. Of these, it is not known how many or which are involved in the control of trafficking in the endocytic pathway, or which are involved in other functions, such as specific ...

The earliest steps in nascent protein maturation greatly affect its overall efficiency. Constraints placed on maturing proteins at these early stages limit available conformations and help to direct the native maturation process. For type II membrane proteins, these cotranslational constraints include N-and C-terminal membrane tethering, chaperone binding, and disulfide bond formation. The cotranslational maturation process for the type II membrane glycoprotein influenza neuraminidase (NA) was investigated to provide a deeper understanding of these initial endoplasmic reticulum events. The type II orientation provides experimental advantages to monitor the first maturation steps. Calnexin was shown to cotranslationally interact with NA prior to calreticulin. These interactions were required for the efficient maturation of NA as it prematurely formed intramolecular disulfides and aggregated when calnexin and calreticulin interactions were abolished. Lectin chaperone binding slowed the NA maturation process, increasing its fidelity. Carbohydrates were required for NA maturation in a regiospecific manner. A subset of NA formed intermolecular disulfides and oligomerized cotranslationally. This fraction increased in the absence of calnexin and calreticulin binding. NA dimerization also occurred for an NA mutant lacking the critical large loop disulfide bond, indicating that dimerization did not require proper NA oxidation. The strict evaluation of proper maturation carried out by the quality control machinery was instilled at the tetramerization step. This study illustrates the type II membrane protein maturation process and shows how important cotranslational events contribute to the proper cellular maturation of glycoproteins.