A Short Synthetic Peptide Mimetic of Apolipoprotein A1 Mediates Cholesterol and Globotriaosylceramide Efflux from Fabry Fibroblasts.

Fabry disease is an X-linked sphingolipid storage disorder caused by a deficiency of the lysosomal enzyme α-galactosidase A (AGA, EC 3.2.1.22) resulting in the intracellular accumulation of globotriaosylceramide (Gb3). We found that Gb3 storage also correlates with accumulation of endosomal-lysosomal cholesterol in Fabry fibroblasts. This cholesterol accumulation may contribute to the phenotypic pathology of Fabry disease by slowing endosomal-lysosomal trafficking. We found that LDL receptor expression is not downregulated in Fabry fibroblasts resulting in accumulation of both cholesterol and Gb3. 5A-Palmitoyl oleoyl-phosphatidylcholine (5AP) is a phospholipid complex containing a short synthetic peptide that mimics apolipoprotein A1, the main protein component of high-density lipoprotein (HDL) that mediates the efflux of cholesterol from cells via the ATP-binding cassette transporter. We used 5AP and HDL to remove cholesterol from Fabry fibroblasts to examine the fate of accumulated cellular Gb3. Using immunostaining techniques, we found that 5AP is highly effective for depleting cholesterol and Gb3 in these cells. 5AP restores the ApoA-1-mediated cholesterol efflux leading to mobilization of cholesterol and reduction of Gb3 in Fabry fibroblasts.

Transduction of rat pancreatic islets with pseudotyped adeno-associated virus vectors.

BACKGROUND: Pancreatic islet transplantation is a promising treatment for type I diabetes mellitus, but current immunosuppressive strategies do not consistently provide long-term survival of transplanted islets. We are therefore investigating the use of adeno-associated viruses (AAVs) as gene therapy vectors to transduce rat islets with immunosuppressive genes prior to transplantation into diabetic mice. RESULTS: We compared the transduction efficiency of AAV2 vectors with an AAV2 capsid (AAV2/2) to AAV2 vectors pseudotyped with AAV5 (AAV2/5), AAV8 (AAV2/8) or bovine adeno-associated virus (BAAV) capsids, or an AAV2 capsid with an insertion of the low density lipoprotein receptor ligand from apolipoprotein E (AAV2apoE), on cultured islets, in the presence of helper adenovirus infection to speed expression of a GFP transgene. Confocal microscopy and flow cytometry were used. The AAV2/5 vector was superior to AAV2/2 and AAV2/8 in rat islets. Flow cytometry indicated AAV2/5-mediated gene expression in approximately 9% of rat islet cells and almost 12% of insulin-positive cells. The AAV2/8 vector had a higher dependence on the helper virus multiplicity of infection than the AAV 2/5 vector. In addition, the BAAV and AAV2apoE vectors were superior to AAV2/2 for transducing rat islets. Rat islets (300 per mouse) transduced with an AAV2/5 vector harboring the immunosuppressive transgene, tgf beta 1, retain the ability to correct hyperglycemia when transplanted into immune-deficient diabetic mice. CONCLUSION: AAV2/5 vectors may therefore be useful for pre-treating donor islets prior to transplantation.

Targeted mutation of the MLN64 START domain causes only modest alterations in cellular sterol metabolism.

The StAR-related lipid transfer (START) domain, first identified in the steroidogenic acute regulatory protein (StAR), is involved in the intracellular trafficking of lipids. Sixteen mammalian START domain-containing proteins have been identified to date. StAR, a protein targeted to mitochondria, stimulates the movement of cholesterol from the outer to the inner mitochondrial membranes, where it is metabolized into pregnenolone in steroidogenic cells. MLN64, the START domain protein most closely related to StAR, is localized to late endosomes along with other proteins involved in sterol trafficking, including NPC1 and NPC2, where it has been postulated to participate in sterol distribution to intracellular membranes. To investigate the role of MLN64 in sterol metabolism, we created mice with a targeted mutation in the Mln64 START domain, expecting to find a phenotype similar to that in humans and mice lacking NPC1 or NPC2 (progressive neurodegenerative symptoms, free cholesterol accumulation in lysosomes). Unexpectedly, mice homozygous for the Mln64 mutant allele were viable, neurologically intact, and fertile. No significant alterations in plasma lipid levels, liver lipid content and distribution, and expression of genes involved in sterol metabolism were observed, except for an increase in sterol ester storage in mutant mice fed a high fat diet. Embryonic fibroblast cells transfected with the cholesterol side-chain cleavage system and primary cultures of granulosa cells from Mln64 mutant mice showed defects in sterol trafficking as reflected in reduced conversion of endogenous cholesterol to steroid hormones. These observations suggest that the Mln64 START domain is largely dispensable for sterol metabolism in mice.

The ABCA1 transporter modulates late endocytic trafficking: insights from the correction of the genetic defect in Tangier disease.

We have previously established that the ABCA1 transporter, which plays a critical role in the lipidation of extracellular apolipoprotein acceptors, traffics between late endocytic vesicles and the cell surface (Neufeld, E. B., Remaley, A. T., Demosky, S. J., Jr., Stonik, J. A., Cooney, A. M., Comly, M., Dwyer, N. K., Zhang, M., Blanchette-Mackie, J., Santamarina-Fojo, S., and Brewer, H. B., Jr. (2001) J. Biol. Chem. 276, 27584-27590). The present study provides evidence that ABCA1 in late endocytic vesicles plays a role in cellular lipid efflux. Late endocytic trafficking was defective in Tangier disease fibroblasts that lack functional ABCA1. Consistent with a late endocytic protein trafficking defect, the hydrophobic amine U18666A retained NPC1 in abnormally tubulated, cholesterol-poor, Tangier disease late endosomes, rather than cholesterol-laden lysosomes, as in wild type fibroblasts. Consistent with a lipid trafficking defect, Tangier disease late endocytic vesicles accumulated both cholesterol and sphingomyelin and were immobilized in a perinuclear localization. The excess cholesterol in Tangier disease late endocytic vesicles retained massive amounts of NPC1, which traffics lysosomal cholesterol to other cellular sites. Exogenous apoA-I abrogated the cholesterol-induced retention of NPC1 in wild type but not in Tangier disease late endosomes. Adenovirally mediated ABCA1-GFP expression in Tangier disease fibroblasts corrected the late endocytic trafficking defects and restored apoA-I-mediated cholesterol efflux. ABCA1-GFP expression in wild type fibroblasts also reduced late endosome-associated NPC1, induced a marked uptake of fluorescent apoA-I into ABCA1-GFP-containing endosomes (that shuttled between late endosomes and the cell surface), and enhanced apoA-I-mediated cholesterol efflux. The combined results of this study suggest that ABCA1 converts pools of late endocytic lipids that retain NPC1 to pools that can associate with endocytosed apoA-I, and be released from the cell as nascent high density lipoprotein.

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 (DeltaSTART-MLN64), which contains N-terminal transmembrane domains but lacks the START domain, caused free cholesterol accumulation in lysosomes and inhibited late endocytic dynamics. The DeltaSTART-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 domain's role in cholesterol transport.