Gladstone Institute of Cardiovascular Disease, Cardiovascular Research Institute, Department of Medicine, University of California, San Francisco San Francisco, California, USA syoung@gladstone.ucsf.edu
Niemann−Pick type C (NPC) disease is a fatal lipid-storage disorder caused by a 'lipid traffic jam' in late endosomes and lysosomes. A recent study indicates that overexpression of the vesicular transport proteins Rab7 or Rab9 increases the flow of traffic, allowing accumulated lipids to leave the endosomes and reach the Golgi apparatus and endoplasmic reticulum.
Niemann−Pick type C (NPC) disease is a recessive disorder characterized by the accumulation of lipids within late endosomes and lysosomes in many tissues, including the liver, spleen, and brain1,
2. The lipid accumulation in the brain causes a merciless neurodegenerative syndrome that is generally diagnosed in early preschool years and is fatal 5−10 years later. NPC disease is caused by mutations in NPC1 (95% of cases) or NPC2 (HE1) (5% of cases)3,
4, genes that encode proteins required for lipid trafficking through the late endosomal compartment. Defects in either protein cause a 'traffic jam'2 of lipids in the late endosome, converting that organelle into a veritable parking lot for LDL-derived cholesterol and sphingolipids. Accordingly, the challenge for NPC disease researchers is both clear-cut and daunting: Get the lipids moving! In June 17 issue of The Journal of Clinical Investigation, Choudhury, Dominquez and colleagues5 report exciting findings relevant to this challenge. Their data strongly suggest that the intracellular trafficking of cholesterol and sphingolipids in NPC disease fibroblasts can be dramatically improved by overexpressing Rab7 or Rab9, small GTPases involved in late endosome vesicular trafficking (Fig. 1).
Figure 1. Overexpression of Rab7 or Rab9 improves lipid homeostasis in NPC disease.
LDL particles, rich in cholesterol esters, are internalized by LDL receptors in clathrin-coated pits, and both sphingolipids and free cholesterol in plasma membrane rafts are internalized into endocytic compartments in a clathrin-independent fashion. In the setting of NPC1 or NPC2 mutations, late endosomal lipid trafficking is blocked, transforming those organelles into a 'parking lot' for free cholesterol and sphingolipids. Overexpression of Rab7 or Rab9 appears to ameliorate this lipid traffic jam by speeding up the delivery of sphingolipids to the Golgi and unesterified cholesterol to the endoplasmic reticulum.
The biochemical hallmark of NPC disease fibroblasts is the accumulation of unesterified cholesterol within endosomes, with a defect in moving that cholesterol pool to the endoplasmic reticulum (ER)1,
2. This trafficking defect can be readily documented by delayed esterification of internalized cholesterol and delayed suppression of de novo cholesterol synthesis and LDL receptor expression. However, NPC disease also involves an intracellular accumulation of sphingolipids. Sphingolipids in lipid rafts on the plasma membrane are continually being imported into cells via a clathrin-independent pathway, first appearing in the endosomes and then 15−45 minutes later appearing in the Golgi5. The endosome-to-Golgi transport of sphingolipids can be blocked by high levels of intracellular cholesterol6, such as those occurring in NPC disease.
The movement of sphingolipids from endosomes to Golgi can be tracked with a fluorescent glycosphingolipid, BODIPY-lactosylceramide (LacSer)5,
6,
7. In NPC1-deficient fibroblasts, BODIPY-LacCer trafficking is markedly abnormal, with the fluorescence remaining in endosomes and failing to reach the Golgi5,
7. Choudhury et al.5 found that transfection of wild-type fibroblasts with dominant-negative forms of Rab7 or Rab9 blocked the transport of BODIPY-LacCer from endosomes to the Golgi, mimicking the phenotype of NPC1-deficient cells. They then found that in NPC disease fibroblasts, overexpression of wild-type Rab7 or Rab9 restored Golgi targeting of BODIPY-LacCer. Finally, Rab7 or Rab9 overexpression caused a striking reduction in free cholesterol levels within cells (as judged by filipin staining) and a concomitant increase in Nile Red−staining neutral lipids (presumably cholesterol esters). This latter finding implies that the free cholesterol in the endosomal compartment made it to the ER for esterification. Overexpression of another Rab protein, Rab11, had no effect on BODIPY-LacCer transport or free cholesterol stores5.
NPC1 contains a sterol-sensing domain resembling those in other proteins that respond to sterol levels (HMG-Co-A reductase, SREBP cleavage-activating protein (SCAP) and Patched), and almost certainly plays an important role in lipid transport within the cell. Recent studies have demonstrated that NPC1 can transport fatty acids across membranes8, a process that could drag cholesterol and other lipids along for the ride and result in the biogenesis of the transport vesicles containing NPC1. NPC1-containing vesicles can be readily visualized by fluorescence videomicroscopy in wild-type cells9. Those vesicles move in a vectorial fashion between endosomes and other membrane compartments9, likely redistributing lipids in the process. This vesicular trafficking is virtually abolished when NPC1 is not functional9.
The nitty-gritty details of how Rab7 and Rab9 ameliorate the 'NPC traffic jam' have not been delineated, but the findings are nevertheless appealing, given that the Rab proteins have well-established roles in vesicular transport10. GDP-bound Rab proteins are normally transported to donor membranes by Rab GDP-dissociation inhibitor proteins (GDIs). Having reached their destination, the GDIs are released and the GDP on the Rab proteins is replaced by GTP through the action of Rab guanine nucleotide exchange factors, activating the Rab proteins. Activation enables Rabs to recruit effector proteins and influence many aspects of vesicular transport, including cargo sorting, vesicle formation, vesicle movement along cytoskeletal filaments and vesicle fusion with target membranes. In humans, a total of 60 Rabs have been identified, each playing some role in vesicular transport10. Rab7 and Rab9 have been implicated in the control of endosome-to-lysosome trafficking and endosome-to-Golgi transport, respectively. Rab11 is important in the recycling of early endosomes to the plasma membrane and also to the Golgi.
That Rab7 overexpression could accelerate the delivery of glycosphingolipids in endosomes to the Golgi seems consistent with recent findings of Bertram et al.11 They found that Rab7 overexpression in B lymphoma cells increased the rate at which antigens were processed and presented on MHC class II molecules at the cell surface, suggesting that Rab7 enhanced endosomal processing of antigens. However, the findings by Choudhury et al.5 in fibroblasts seem inconsistent with recent findings of Lebrand et al.12, who found that Rab7 overexpression in HeLa cells inhibited endosome motility and caused a perinuclear accumulation of endosomes. Perhaps Rab7 overexpression has different effects in HeLa cells and fibroblasts as a result of differences in the expression of Rab effectors. Alternatively, the movement of endosomes tracked by Lebrand et al.12 may simply not be relevant to the movement of lipids tracked by Choudhury et al.5.
The findings of Choudhury et al.5 are important because they further illuminate mechanisms of lipid trafficking in endosomes and because they suggest new strategies for developing NPC-disease therapies. Accordingly, their study is certain to spark a flurry of follow-up investigations, likely redirecting substantial effort within the NPC disease field. First and foremost, the findings by Choudhury et al.5 must be confirmed and extended. Their data on free cholesterol depletion and cholesterol ester accumulation were based on staining of lipids, not biochemical measurements. It will be important to demonstrate with biochemical assays that Rab overexpression reduces unesterified cholesterol levels in cells. Similarly, it will be essential to prove that the Nile Red−staining material represents cholesterol esters and that Rab overexpression truly increases cholesterol esterification rates. Also, it will be important to prove, unequivocally, that the apparent reduction in cell cholesterol levels is due to clearance of cholesterol from late endosomes rather than reductions in the entry of cholesterol into that compartment. Also, other questions remain: What levels of Rab overexpression are required to prevent the accumulation of lipids in endosomes/lysosomes? What are the levels of Rab expression in NPC1-deficient cells compared with wild-type cells? How does Rab overexpression in wild-type cells affect NPC1 expression and localization?
It will be intriguing to determine whether Rab overexpression works only in NPC1-deficient cells or whether it could work equally well for correcting lysosomal accumulation of sphingolipids and cholesterol in other sphingolipid storage diseases, such as those caused by deficiencies in enzymes that degrade lipids. Choudhury et al.5 used fluorescence microscopy to show that Rab overexpression in NPC disease fibroblasts improved the trafficking of BODIPY-LacCer. In the future, fluorescence videomicroscopy and electron microscopy need to be used to determine whether overexpression of Rab7 or Rab9 in NPC1-deficient cells truly normalizes vesicle movement and vesicle morphology—or whether subtle (or even not-so-subtle) differences in vesicular trafficking persist, compared with nontransfected wild-type cells.
The Rab overexpression studies are bound to focus more attention on how Rab GDIs affect lipid trafficking13. It will also be critical to define the biophysical relationship between the Rab proteins and NPC1. Does NPC1 interact directly with Rab proteins?
Finally, it will be necessary to overexpress Rab7 or Rab9 in Npc1-deficient mice, perhaps by using transgenes with inducible promoters, and then to determine whether the lipid-trafficking abnormality is ameliorated in the tissue where it matters the most—the central nervous system. Studies with genetically modified mice also should help to define if Rab overexpression causes unwanted side effects; for example, developmental abnormalities, tissue injury or an increased susceptibility to certain intracellular pathogens that pass through endosomes and lysosomes.
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