Christine L. Wheatley

Rab proteins mediate Golgi transport of caveola-internalized glycosphingolipids and correct lipid trafficking in Niemann-Pick C cells

We recently showed that human skin fibroblasts internalize fluorescent analogues of the glycosphingolipids lactosylceramide and globoside almost exclusively by a clathrin-independent mechanism involving caveolae. In contrast, a sphingomyelin analogue is internalized approximately equally via clathrin-dependent and caveolar routes. Here, we further characterized the caveolar pathway for glycosphingolipids, showing that Golgi targeting of sphingolipids internalized via caveolae required microtubules and phosphoinositol 3-kinases and was inhibited in cells expressing dominant-negative Rab7 and Rab9 constructs. In addition, overexpression of wild-type Rab7 or Rab9 (but not Rab11) in Niemann-Pick type C (NP-C) lipid storage disease fibroblasts resulted in correction of lipid trafficking defects, including restoration of Golgi targeting of fluorescent lactosylceramide and endogenous GM(1) ganglioside, and a dramatic reduction in intracellular cholesterol stores. Our results demonstrate a role for Rab7 and Rab9 in the Golgi targeting of glycosphingolipids and suggest a new therapeutic approach for restoring normal lipid trafficking in NP-C cells.

Clathrin-dependent and -independent internalization of plasma membrane sphingolipids initiates two Golgi targeting pathways.

Sphingolipids (SLs) are plasma membrane constituents in eukaryotic cells which play important roles in a wide variety of cellular functions. However, little is known about the mechanisms of their internalization from the plasma membrane or subsequent intracellular targeting. We have begun to study these issues in human skin fibroblasts using fluorescent SL analogues. Using selective endocytic inhibitors and dominant negative constructs of dynamin and epidermal growth factor receptor pathway substrate clone 15, we found that analogues of lactosylceramide and globoside were internalized almost exclusively by a clathrin-independent (“caveolar-like”) mechanism, whereas an analogue of sphingomyelin was taken up approximately equally by clathrin-dependent and -independent pathways. We also showed that the Golgi targeting of SL analogues internalized via the caveolar-like pathway was selectively perturbed by elevated intracellular cholesterol, demonstrating the existence of two discrete Golgi targeting pathways. Studies using SL-binding toxins internalized via clathrin-dependent or -independent mechanisms confirmed that endogenous SLs follow the same two pathways. These findings (a) provide a direct demonstration of differential SLs sorting into early endosomes in living cells, (b) provide a “vital marker” for endosomes derived from caveolar-like endocytosis, and (c) identify two independent pathways for lipid transport from the plasma membrane to the Golgi apparatus in human skin fibroblasts.

Cholesterol modulates membrane traffic along the endocytic pathway in sphingolipid-storage diseases.

Cholesterol modulates membrane traffic along the endocytic pathway in sphingolipid-storage diseases

Glycosphingolipids Internalized via Caveolar-related Endocytosis Rapidly Merge with the Clathrin Pathway in Early Endosomes and Form Microdomains for Recycling

We have previously demonstrated that glycosphingolipids are internalized from the plasma membrane of human skin fibroblasts by a clathrin-independent, caveolar-related mechanism and are subsequently transported to the Golgi apparatus by a process that is dependent on microtubules, phosphatidylinositol 3-kinase, Rab7, and Rab9. Here we characterized the early steps of intracellular transport of a fluorescent glycosphingolipid analog, BODIPY-lactosylceramide (LacCer), and compared this to fluorescent transferrin (Tfn), a well established marker for the clathrin pathway. Although these two markers were initially internalized into separate vesicles by distinct mechanisms, they became co-localized in early endosomes within 5 min. These results demonstrate that glycosphingolipid-containing vesicles derived from caveolar-related endocytosis fuse with the classical endosomal system. However, in contrast to Tfn, internalization and trafficking of LacCer was independent of Rab5a, a key regulator of transport to early endosomes. By taking advantage of the monomer/excimer properties of the fluorescent lipid analog, we were also able to visualize LacCer segregation into distinct microdomains of high (red emission) and low (green emission) concentrations in the early endosomes of living cells. Interestingly, the high concentration “red” microdomains co-localized with fluorescent Tfn upon exit from early endosomes and passed through Rab11-positive “recycling endosomes” prior to being transported back to the plasma membrane. These results together with our previous studies suggest that glycosphingolipids internalized by caveolar endocytosis are rapidly delivered to early endosomes where they are fractionated into two major pools, one that is transported via late endosomes to the Golgi apparatus and the other that is returned to the plasma membrane via the recycling compartment.

Broad screening test for sphingolipid-storage diseases.

BACKGROUND Lipid-storage diseases are collectively important because they cause substantial morbidity and mortality, and because they may present as dementia, major psychiatric illness, developmental delay, or cerebral palsy. At present, no single assay can be used as an initial general screen for lipid-storage diseases. METHODS We used a fluorescent analogue of lactosylceramide, called N-[5-(5,7-dimethylborondipyrromethenedifluoride)-1-pentanoyl]D- lactosylsphingosine (BODIPY-LacCer), the emission of which changes from green to red wavelengths with increasing concentrations in membranes, to examine the intracellular distribution of the lipid within living cells. FINDINGS During a brief pulse-chase experiment, the fluorescent lipid accumulated in the lysosomes of fibroblasts from patients with Fabrys disease, GM1 gangliosidosis, GM2 gangliosidosis (Tay-Sachs and Sandhoff forms), metachromatic leucodystrophy, mucolipidosis type IV, Niemann-Pick disease (types A, B, and C), and sphingolipid-activator-protein-precursor (prosaposin) deficiency. In control cells, the lipid was mainly confined to the Golgi complex. In a masked study, replicate samples of 25 of 26 unique cell lines representing ten different lipid-storage diseases, and 18 of 20 unique cell lines representing controls were correctly identified; the sensitivity was 96.2% (95% CI 80.4-99.9) and the specificity 90.0% (68.3-98.8). INTERPRETATION This method may be useful as an initial general screen for lipid-storage diseases, and, with modification, could be used for large-scale automated screening of drugs to abrogate lysosomal storage in various lipidoses. The unexpected accumulation of BODIPY-LacCer in several biochemically distinct diseases raises important questions about common mechanisms of cellular dysfunction in these disorders.

Inhibition of caveolar uptake, SV40 infection, and β1-integrin signaling by a nonnatural glycosphingolipid stereoisomer

Caveolar endocytosis is an important mechanism for the uptake of certain pathogens and toxins and also plays a role in the internalization of some plasma membrane (PM) lipids and proteins. However, the regulation of caveolar endocytosis is not well understood. We previously demonstrated that caveolar endocytosis and β1-integrin signaling are stimulated by exogenous glycosphingolipids (GSLs). In this study, we show that a synthetic GSL with nonnatural stereochemistry, β-d-lactosyl-N-octanoyl-l-threo-sphingosine, (1) selectively inhibits caveolar endocytosis and SV40 virus infection, (2) blocks the clustering of lipids and proteins into GSLs and cholesterol-enriched microdomains (rafts) at the PM, and (3) inhibits β1-integrin activation and downstream signaling. Finally, we show that small interfering RNA knockdown of β1 integrin in human skin fibroblasts blocks caveolar endocytosis and the stimulation of signaling by a GSL with natural stereochemistry. These experiments identify a new compound that can interfere with biological processes by inhibiting microdomain formation and also identify β1 integrin as a potential mediator of signaling by GSLs.

Caveolar endocytosis and microdomain association of a glycosphingolipid analog is dependent on its sphingosine stereochemistry

We have previously shown that glycosphingolipid analogs are internalized primarily via caveolae in various cell types. This selective internalization was not dependent on particular carbohydrate headgroups or sphingosine chain length. Here, we examine the role of sphingosine structure in the endocytosis of BODIPY™-tagged lactosylceramide (LacCer) analogs via caveolae. We found that whereas the LacCer analog with the natural (d-erythro) sphingosine stereochemistry is internalized mainly via caveolae, the non-natural (l-threo) LacCer analog is taken up via clathrin-, RhoA-, and Cdc42-dependent mechanisms and largely excluded from uptake via caveolae. Unlike the d-erythro-LacCer analog, the l-threo analog did not cluster in membrane microdomains when added at higher concentrations (5–20 μm). In vitro studies using small unilamellar vesicles and giant unilamellar vesicles demonstrated that l-threo-LacCer did not undergo a concentration-dependent excimer shift in fluorescence emission such as that seen with BODIPY™-sphingolipids with natural stereochemistry. Molecular modeling studies suggest that in d-erythro-LacCer, the disaccharide moiety extends above and in the same plane as the sphingosine hydrocarbon chain, while in l-threo-LacCer the carbohydrate group is nearly perpendicular to the hydrocarbon chain. Together, these results suggest that the altered stereochemistry of the sphingosine group in l-threo-LacCer results in a perturbed structure, which is unable to pack closely with natural membrane lipids, leading to a reduced inclusion in plasma membrane microdomains and decreased uptake by caveolar endocytosis. These findings demonstrate the importance of the sphingolipid stereochemistry in the formation of membrane microdomains.

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

Niemann-Pick, type C (NP-C) disease is an autosomal recessive neurovisceral storage disorder in which cholesterol and sphingolipids accumulate. There is no specific treatment for this disease, which is characterized by progressive neurological deterioration, sometimes accompanied by hepatosplenomegaly. We and others have shown that overexpression of certain Rab GTPases corrects defective membrane trafficking and reduces lipid storage in cultured NP-C fibroblasts. Here, we tested the possibility that Rab protein overexpression might also have beneficial effects in vivo using a murine model of NP-C. We first generated several lines of transgenic mice that ubiquitously overexpress Rab9 up to approximately 30-fold more than endogenous levels and found that the transgene expression had no obvious effects on fertility, behavior, or lifespan in normal mice. These transgenic strains were then crossed with NP-C mutant mice to produce NP-C homozygous recessive mice with and without the Rab9 transgene. Life expectancy of the NPC1 homozygous recessive animals was extended up to 22% depending on gender and the transgenic strain that was used. Histological studies and lipid analysis of brain sections indicated that the NP-C mice carrying the Rab9 transgene had dramatically reduced storage of GM(2) and GM(3) gangliosides relative to NP-C animals lacking the transgene. These results demonstrate that Rab9 overexpression has the potential to reduce stored lipids and prolong lifespan in vivo.

Co-regulation of caveolar and Cdc42-dependent fluid phase endocytosis by phosphocaveolin-1.

Several clathrin-independent endocytosis mechanisms have been identified that can be distinguished by specific requirements for certain proteins, such as caveolin-1 (Cav1) and the Rho GTPases, RhoA and Cdc42, as well as by specific cargo. Some endocytic pathways may be co-regulated such that disruption of one pathway leads to the up-regulation of another; however, the underlying mechanisms for this are unclear. Cav1 has been reported to function as a guanine nucleotide dissociation inhibitor (GDI), which inhibits Cdc42 activation. We tested the hypothesis that Cav1 can regulate Cdc42-dependent, fluid phase endocytosis. We demonstrate that Cav1 overexpression decreases fluid phase endocytosis, whereas silencing of Cav1 enhances this pathway. Enhancement of Cav1 phosphorylation using a phosphatase inhibitor reduces Cdc42-regulated pinocytosis while stimulating caveolar endocytosis. Fluid phase endocytosis was inhibited by expression of a putative phosphomimetic mutant, Cav1-Y14E, but not by the phospho-deficient mutant, Cav1-Y14F. Overexpression of Cav2, or a Cav1 mutant in which the GDI region was altered to the corresponding sequence in Cav2, did not suppress fluid phase endocytosis. These results suggest that the Cav1 expression level and phosphorylation state regulates fluid phase endocytosis via the interaction between the Cav1 GDI region and Cdc42. These data define a novel molecular mechanism for co-regulation of two distinct clathrin-independent endocytic pathways.

Enhancement of Heat Sensitivity and Modification of Repair of Potentially Lethal Heat Damage in Plateau-Phase Cultures of Mammalian Cells by Diethyldithiocarbamate

Diethyldithiocarbamate (DDC) is active both in vivo and in vitro in reducing the levels of enzymes such as superoxide dismutase (SOD) and glutathione peroxidase whose role in respiring cells is to remove toxic superoxide radicals and organic hydroperoxides. Although DDC, a copper-chelating agent, has been used to treat benign diseases, its potential as a heat sensitizer has not been fully explored. We have recently shown that the presence of 10(-3) M DDC for 2 hr causes a threefold reduction in the level of SOD in plateau-phase cultures of mammalian cells. At this concentration, the drug causes minimal toxicity but markedly affects both the shoulder and the slope of the heat survival curves. To explore another pathway of DDC sensitization, other than through reduced levels of SOD, we examined the repair of potentially lethal damage with and without DDC following exposure for 1 hr and 40 min at 43 degrees C. The repair, which progressed with a T 1/2 of about 10 hr, in either full medium or Hanks balanced salt solution (HBSS), in the absence of DDC, was completely blocked when DDC was added to the monolayers on completion of the heat exposure. DDC, in view of its ability to potentiate the effects of heat, is a potentially useful drug that could be used in an adjunctive setting with clinical hyperthermia.