M E Comly

MLN64 Mediates Mobilization of Lysosomal Cholesterol to Steroidogenic Mitochondria

This study demonstrates that the steroidogenic acute regulatory protein-related lipid transfer (START) domain-containing protein, MLN64, participates in intracellular cholesterol trafficking. Analysis of the intracellular itinerary of MLN64 and MLN64 mutants tagged with green fluorescent protein showed that the N-terminal transmembrane domains mediate endocytosis of MLN64 from the plasma membrane to late endocytic compartments. MLN64 constitutively traffics via dynamic NPC1-containing late endosomal tubules in normal cells; this dynamic movement was inhibited in cholesterol-loaded cells, and MLN64 is trapped at the periphery of cholesterol-laden lysosomes. The MLN64 START domain stimulated free cholesterol transfer from donor to acceptor mitochondrial membranes and enhanced steroidogenesis by placental mitochondria. Expression of a truncated form of MLN64 (ΔSTART-MLN64), which contains N-terminal transmembrane domains but lacks the START domain, caused free cholesterol accumulation in lysosomes and inhibited late endocytic dynamics. The ΔSTART-MLN64 dominant negative protein was located at the surface of the cholesterol-laden lysosomes. This dominant negative mutant suppressed steroidogenesis in COS cells expressing the mitochondrial cholesterol side chain cleavage system. We conclude that MLN64 participates in mobilization and utilization of lysosomal cholesterol by virtue of the START domains role in cholesterol transport.

Cessation of rapid late endosomal tubulovesicular trafficking in Niemann–Pick type C1 disease

Niemann–Pick type C1 (NPC1) disease results from a defect in the NPC1 protein and is characterized by a pathological accumulation of cholesterol and glycolipids in endocytic organelles. We followed the biosynthesis and trafficking of NPC1 with the use of a functional green fluorescent protein-fused NPC1. Newly synthesized NPC1 is exported from the endoplasmic reticulum and requires transit through the Golgi before it is targeted to late endosomes. NPC1-containing late endosomes then move by a dynamic process involving tubulation and fission, followed by rapid retrograde and anterograde migration along microtubules. Cell fusion studies with normal and mutant NPC1 cells show that exchange of contents between late endosomes and lysosomes depends upon ongoing tubulovesicular late endocytic trafficking. In turn, rapid endosomal tubular movement requires an intact NPC1 sterol-sensing domain and is retarded by an elevated endosomal cholesterol content. We conclude that the neuropathology and cellular lysosomal lipid accumulation in NPC1 disease results, at least in part, from striking defects in late endosomal tubulovesicular trafficking.

Differential trafficking of the Niemann-Pick C1 and 2 proteins highlights distinct roles in late endocytic lipid trafficking

The cellular location of Niemann‐Pick C2 protein (NPC2) in cultured human fibroblasts and Chinese hamster ovary cells was examined immunocytochemically and in living cells by expression of a functional red fluorescent protein chimeric analogue. Results: NPC2 is present in the lysosomes of both cholesterol‐depleted and ‐replenished cells, unlike Niemann‐Pick C1 protein (NPC1) which is recruited to late endosomes only upon uptake of low‐density lipoprotein. With mobilization of cholesterol from lysosomes, immunocytochemical detection of NPC2 in lysosomes is greatly diminished, whereas NPC1 remains in the late endosomal compartment. We found a partial overlap in the trafficking and organellar sites of accumulation of NPC2 and NPC1. In living cells, NPC2 traffics with NPC1 in late endosomal tubules. However, in contrast to NPC1, which remains either in late endosomal vesicles and tubules or at the peripheries of cholesterol‐laden lysosomes, NPC2 moves into the central core of lysosomes. Glycolipid analysis reveals that, in contrast to null mutant NPC1 cells, which accumulate GM2 ganglioside only at the plasma membrane, with no endocytic storage, absence of NPC2 protein in null mutant NPC2 cells does not block internalization of GM2 into endocytic vesicles. This difference in the cellular distribution of GM2 in NPC1 and NPC2 null mutants is the first report of a variation in the phenotypic expression of these genotypically distinct lesions.

Nutrient-driven O-GlcNAc cycling impacts neurodevelopmental timing and metabolism

Nutrient-driven O-GlcNAcylation is strikingly abundant in the brain and has been linked to development and neurodegenerative disease. We selectively targeted the O-GlcNAcase (Oga) gene in the mouse brain to define the role of O-GlcNAc cycling in the central nervous system. Brain knockout animals exhibited dramatically increased brain O-GlcNAc levels and pleiotropic phenotypes, including early-onset obesity, growth defects, and metabolic dysregulation. Anatomical defects in the Oga knockout included delayed brain differentiation and neurogenesis as well as abnormal proliferation accompanying a developmental delay. The molecular basis for these defects included transcriptional changes accompanying differentiating embryonic stem cells. In Oga KO mouse ES cells, we observed pronounced changes in expression of pluripotency markers, including Sox2, Nanog, and Otx2. These findings link the O-GlcNAc modification to mammalian neurogenesis and highlight the role of this nutrient-sensing pathway in developmental plasticity and metabolic homeostasis.