Our current studies explore the phenotypic homogeneity of lysosomal storage diseases relative to their respective pathology in organelle function. Cholesterol and glycolipids are vital lipid molecules for the maintenance of cellular membranes, supplied to the cell by de novo synthesis and exogenous endocytic uptake of lipoproteins. The continuous recycling of these lipid molecules and maintenance of lipid equilibrium is crucial for cell viability. Receptors, enzymes and lipid transport molecules all play a role in maintenance of cellular membrane-lipid concentrations. In many inherited, human diseases there is an imbalance in trafficking of lipoprotein or plasma membrane-derived lipid molecules and they accumulate in cellular endocytic compartments resulting in a variety of lipidoses commonly called lysosomal storage diseases.? ? Study of Niemann Pick C disease brought us striking new knowledge about the dynamic and labyrinthine nature of the intracellular endocytic pathway through which internalized macromolecules move to their respective destinations. In spite of all its intricacies the endocytic pathway must be well organized to carry out its intrinsic transport and sorting functions. Macromolecules vital for cell function are internalized, enter an early tubular endosomal pathway that sorts ligands and receptors recycling to the plasma membrane from others destined to travel through the later arm of the endocytic pathway. It is the structure and dynamics of this late endosomal pathway that has been elucidated by studies on the NPC1 protein. When cells endocytose LDL, NPC1 protein is recruited into the late endosomal compartment. Trafficking studies in living cells using a functional chimera of NPC1-GFP, show that this late endocytic compartment consists of a dynamic, labyrinthine tubular system as well as vesicles and that the integrity of the dynamic nature of late endosomal tubules must be maintained for content exchange between late endosomes and lysosomes. Although appreciation of the existence of the late endosomal tubular reticulum came from visualization of NPC1-GFP in transfected, living normal and NPC mutant cells the dynamics and characteristics of this pathway can also be visualized in non-transfected normal and mutant cells with fluorescent dextrans. ? ? Immobility of late endosomal tubules is a pathology common to inherited glycolipid storage diseases. We also found that cholesterol accumulation causes cessation of dynamic late endosomal tubule trafficking and stasis of lysosomal content which led to the hypothesis that cells from other lysosomal lipid storage diseases such as Tay Sachs, Fabry and Gaucher might have similar defective late endosomal tubular mobility due to pathological accumulation of lipids in these inherited diseases. Using expression of NPC1-GFP or uptake of fluorescent dextran to mark the late endosomal tubular compartment, immobility of late endosomal tubules was found to be a pathology common to these inherited glycolipid storage diseases. As with Niemann Pick C cells, expression of NPC1 or nutritional depletion of cholesterol restored mobility of late endosomal tubules resulting in egress of accumulated glycolipids from lysosomes in the various mutant cells. Tays Sachs cells show increased synthesis of GM2 during LDL uptake and accumulation of GM2 in lysosomes and conversely decreased synthesis during the period of GM2 egress from lysosomes. Total GM2 in Tay sachs cells increased during LDL uptake and did not decrease during a 24 hour period after LDL uptake although GM2visualized immunocytochemically decreased in lysosomes. This indicates that GM2 can egress from lysosomes and is translocated to other sites in cells where late endosomal tubular mobility is reestablished.? ? Structure of Late Endosomal Tubules (LET). NPC1-GFP traffics between late endosomal vesicles and lysosomes via an extensive system of branching tubules with rapid rates of movement ranging between 1-5mm/s. Late endocytic tubules (LETS) move by a dynamic process involving tubulation and fission, followed by rapid anterograde and retrograde migration along microtubules. LET membranes are rich in unesterified cholesterol as shown by filipin cytochemistry. Electron microscopic analysis shows that LET's range dramatically in diameter (500-60nm) in size dependent on their content of luminal bilayered lamellae that appear to emanate from the internal luminal leaflet of the tubule bounding membrane. Further 3D EM analysis shows concentric lamellae accumulate in these tubules. A cholesterol-loaded endocytic compartment (CLC) containing mostly elongated membranous tubules with luminal lamellae (visualized with electron microscopy) has been isolated by density gradient centrifugation, from normal and mutant human diploid fibroblasts fed high levels of low density lipoprotein (LDL). We intend to characterize the cholesterol/phospholipid content of the CLC fraction. Understanding the structure, lipid and protein content of these anomolous LET tubules, containing luminal bilayers, can provide clues to the role of cholesterol and lipid transport proteins such as the NPC1 protein. Knowledge of lipids and proteins critical to preventing the pathological stasis of the late endosomal tubular/lysosomal interaction will have an impact on therapies designed to correct inherited metabolic diseases that disrupt cellular membrane lipid equilibrium.? Apolipoprotein D: role in cellular lipid trafficking. We showed that sterol accumulation in lysosomes is linked to a parallel enrichment of this organelle with apoD suggesting that apoD mediates a cellular function in the trafficking of LDL derived cholesterol through lysosomes. This issue was examined with mouse embryonic fibroblasts (MEFs) derived from normal and apoD (-/-) mice. ApoD-/-)cells accumulate cholesterol ester in addition to unesterified cholesterol in lysosomes. The mutant cells displayed a 3 fold higher level of cholesteryl-[3H] oleate synthesis during the period of LDL uptake as compared to normal cells suggesting that during this period of LDL uptake and unusual cholesteryl ester pool is being formed. In order to study the intracellular origin of the lysosomal cholesterol esters and their accumulation in the apoD deficient MEF, LDL uptake was studied in the presence of progesterone (progesterone blocks trafficking of cholesterol out of lysosomes) and in the presence of acyl cholesterol acyl transferase (ACAT) inhibition (ACAT inhibition, blocks synthesis of cholesterol ester in the endoplasmic reticulum ). Both progesterone and ACAT inhibition did not prevent the LDL induced accumulation of neutral lipid in lysosomes of apoD KO MEF's, showing that the origin of the lysosomal lipid was not synthesis by the endoplasmic reticulum. However, inhibition of ACAT did prevent the LDL induced synthesis of cholesterol-[3H] oleateby ApoD cells. Thus we conclude that the neutral lipid in lysosomes of ApoD (-/-) cells is unhydrolyzed LDL cholesterol ester.
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