Cholesterol Accumulation Sequesters Rab9 and Disrupts Late Endosome Function in NPC1-deficient Cells

Abstract

Niemann-Pick type C disease is an autosomal recessive disorder that leads to massive accumulation of cholesterol and glycosphingolipids in late endosomes and lysosomes. To understand how cholesterol accumulation influences late endosome function, we investigated the effect of elevated cholesterol on Rab9-dependent export of mannose 6-phosphate receptors from this compartment. Endogenous Rab9 levels were elevated 1.8-fold in Niemann-Pick type C cells relative to wild type cells, and its half-life increased 1.6-fold, suggesting that Rab9 accumulation is caused by impaired protein turnover. Reduced Rab9 degradation was accompanied by stabilization on endosome membranes, as shown by a reduction in the capacity of Rab9 for guanine nucleotide dissociation inhibitor-mediated extraction from Niemann-Pick type C membranes. Cholesterol appeared to stabilize Rab9 directly, as liposomes loaded with prenylated Rab9 showed decreased extractability with increasing cholesterol content. Rab9 is likely sequestered in an inactive form on Niemann-Pick type C membranes, as cation-dependent mannose 6-phosphate receptors were missorted to the lysosome for degradation, a process that was reversed by overexpression of GFPtagged Rab9. In addition to using primary fibroblasts isolated from Niemann-Pick type C patients, RNA interference was utilized to recapitulate the disease phenotype in cultured cells, greatly facilitating the analysis of cholesterol accumulation and late endosome function. We conclude that cholesterol contributes directly to the sequestration of Rab9 on Niemann-Pick type C cell membranes, which in turn, disrupts mannose 6-phosphate receptor trafficking. Niemann-Pick type C (NPC)2 is an autosomal recessive, neurodegenerative disorder. A hallmark of NPC is the massive accumulation of cholesterol and glycosphingolipids within late endosomes and lysosomes (reviewed in Refs. 1-3). In normal cells, endocytosed low density lipoproteins are delivered to endosomes, where they are hydrolyzed and free cholesterol is released. This cholesterol is transported rapidly out of endosomes to the plasma membrane and endoplasmic reticulum (4, 5). In NPC cells, the cholesterol does not exit the endocytic pathway and it accumulates within lysosomes.Approximately 95% of NPC patients harbor mutations in the NPC1 gene that encodes a large, late endosomal protein with 13 transmembrane domains (6 -8). Although NPC1 binds cholesterol weakly (9), the precise function of NPC1 is unknown; it may be involved in cholesterol export from late endosomes (10). The remainder of NPC patients carry mutations in the NPC2 gene that encodes a small, soluble protein present in the lumen of late endosomes and lysosomes (11). Unlike NPC1, NPC2 binds cholesterol with high affinity (12), but like NPC1, its precise role is unclear.Late endosomes act as sorting stations to deliver endocytosed molecules to lysosomes for degradation, while at the same time, retrieving other classes of proteins and lipids for transport back to non-degradative compartments. Mannose 6-phos...

Development of a Rab9 Transgenic Mouse and Its Ability to Increase the Lifespan of a Murine Model of Niemann-Pick Type C Disease

“…First, it is unknown whether the transgenic Rab9 is fully active in the mouse, since previous studies demonstrated that cholesterol can interfere with Rab9 function. 12,15 In addition, we found that the level of Rab9 overexpression was greatly reduced in the liver (Figure 1C), raising the possibility that some of the protein could be inactivated or degraded at a critical time during disease progression. These effects might be overcome using an inducible expression system so that the Rab9 transgene could be activated at a particular age and for a length of time chosen by the investigator.…”

“…11 Although the underlying mechanism for this correction is not completely understood, several studies have shown that elevated endosomal cholesterol interferes with the GDP dissociation inhibitor extraction of Rab proteins from endosomal membranes. 13,15,16 Overexpression of Rab proteins may thus be sufficient to stimulate the intracellular transport that is otherwise blocked by the stored lipids. In the present study, we sought to test whether Rab overexpression might also have a beneficial effect in vivo.…”

β-Amyloid Inhibits Protein Prenylation and Induces Cholesterol Sequestration by Impairing SREBP-2 Cleavage

“…4B). GDI capture of prenylated Rabs from cellular membranes may be physically hindered by cholesterol accumulation within membranes (Lebrand et al, 2002;Choudhury et al, 2004;Ganley and Pfeffer, 2006). Thus, to avoid confounding physical effects attributable to the accumulation of cholesterol in neurons treated with oA␤ 42 , we repeated the GDI Rab-capturing experiments in cytosolic and membrane fractions separately and confirmed that protein prenylation is reduced in oA␤ 42 -treated neurons (Fig.…”

Rab Proteins and the Compartmentalization of the Endosomal System

“…For example, Rab11-mediated endosomal recycling is central to cholesterol esterification and homeostasis, whereas excess cholesterol accumulation in endosomes abrogates Rab4-dependent recycling, sequesters Rab9, and causes immobilization of Rab7-positive late endosomes and redistribution of internalized cargo (Lebrand et al 2002;Choudhury et al 2004;Ganley and Pfeffer 2006;Chen et al 2008;Rocha et al 2009). Rab7 endosome motility on microtubules is selectively regulated by protein interactions that are sensitive to cholesterol levels (Chen et al 2008;Rocha et al 2009).…”