Research Projects

There are two main projects in the laboratory:

  • Cholesterol movement controlled by the Niemann-Pick C1 protein
  • Cholesterol movement from the plasma membrane to the endoplasmic reticulum

What is Niemann-Pick C Disease?

NPC is an autosomal recessive human genetic disease of lipid metabolism. Approximately one in 250,000 individuals are born with this disease. A typical affected individual exhibits:

  • Neonatal jaundice
  • Enlarged liver and/or spleen
  • Vertical gaze palsy
  • Progressive clumsiness, difficulty walking
  • Learning difficulties, slurred speech
  • Tremors, seizures, cataplexy
  • Hypersensitivity to touch

The first neurological signs usually appear in the preschool or early school years. With time, NPC children have difficulty eating and communicating. They are eventually confined to a wheelchair and typically die in their early teen years. Children who are affected at birth typically live 1 to 3 years, whereas adult-onset patients live for several decades.

Click HERE to view "One child's struggle with NPC".

Pathologically, NPC is a complex lipid storage disorder. Increases in sphingomyelin, cholesterol, lysobisphosphatidic acid (LBPA), neutral and acidic glycosphingolipids and phospholipids are seen in liver and spleen. However, the lipid storage pattern differs dramatically in the brain where glycolipids are the main storage material.

To learn more about Niemann-Pick C disease,
please go to the National Niemann-Pick Disease Foundation website
www.nnpdf.org
The NNPDF funds basic research on Niemann-Pick Disease.

This is an organization founded and run by the families of individuals with NPC. The NNPDF raises money to fund basic science on NPC disease and to fund support services for affected families.

• NPC fibroblasts store massive amounts of cholesterol.

The lipid storage in NPC fibroblasts can been visualized by staining cells with filipin, a fluorescent compound that binds cholesterol. In the photomicrographs shown below, the normal cells can barely be detected, whereas the fibroblasts taken from individuals with NPC show massive cholesterol accumulation. NPC fibroblasts can be studied in the lab to determine why cholesterol accumulates.

Normal Fibroblast
NPC Fibroblast
Normal Fibroblast
NPC Fibroblast

• What is the biochemical phenotype of NPC?

NPC is a disease of lipid transport. We study NPC in fibroblasts isolated from affected individuals.

Normal cells synthesize cholesterol in the endoplasmic reticulum (ER) and shuttle cholesterol between the ER and plasma membrane. They also internalize plasma lipoproteins, hydrolyze them in endosomes and lysosomes and then mobilize the released cholesterol to the plasma membrane and endoplasmic reticulum. NPC fibroblasts synthesize cholesterol normally in the ER and shuttle cholesterol between the plasma membrane and ER normally. But they are unable to transport LDL-derived cholesterol from lysosomes to the plasma membrane and ER. Thus, cholesterol and other lipids accumulate in lysosomes and storage bodies are formed.

These biochemical studies led to the hypothesis that NPC is a cholesterol transport molecule. How might such a molecule work? There are several possible mechanisms by which a protein might direct lipid movement. A cholesterol transport molecule could be a cytosolic carrier protein that binds cholesterol released from a donor membrane and releases it to an acceptor membrane. It could also be a membrane protein that directs the transport of cholesterol-rich vesicles, or a membrane protein that facilitates aqueous cholesterol diffusion by stimulating cholesterol desorption from donor membranes. The nature of the NPC cholesterol transport molecule became clearer when the gene for NPC was cloned.

• The NPC genes

The human and mouse NPC1 genes were identified using methods of positional cloning (Carstea et al., 1997; Loftus et al., 1997). NPC1 is a 47-kb gene found on human chromosome 18q11, which encodes a 1278 amino acid glycoprotein. It was named NPC1 because two separate genetic loci are responsible for NPC disease. NPC1 accounts for 95% of the clinical cases. In the general population, multiple distinct NPC1 gene mutations are found.

The second disease locus, NPC2, accounts for approximately 5% of known cases. NPC2 is clinically and biochemically indistinguishable from NPC1 and is, therefore, likely to be a constituent of the same transport pathway. NPC2 was cloned by a laboratory characterizing the lysosome proteome (Naureckiene et al., 2000). The NPC2 gene maps to human chromosome 14q24.3 and encodes a 170 amino acid protein.

Our focus is on NPC1. The human NPC1 gene encodes a 1278 amino acid membrane protein.

• The NPC1 protein

Here is the predicted topology of NPC1: The NPC1 protein has an N-terminal signal sequence (SS) followed by a leucine zipper domain. The protein is very hydrophobic, with at least 13 transmembrane domains (TM). Five of the transmembrane domains have high sequence similarity with HMG-CoA reductase and SREBP cleavage-activating protein, two endoplasmic reticulum proteins that are hypothesized to "sense" the sterol content of the membrane. One of the hydrophilic loops contains conserved cysteins reminiscent of a ring finger domain. NPC1 has a C-terminal dileucine motif (LL) that likely targets the protein to the late endosomes and lysosomes.

NPC1 has all of the hallmarks of a transmembrane protein and is, therefore, unlikely to be a cytosolic sterol carrier protein. Is it in the right location to mediate vesicular movement of LDL-cholesterol? Yes, since the protein has been localized by immunofluorescence microscopy to late endosomes and lysosomes.


• What does NPC1 do?

We don't know the precise function of NPC1. Is it a trafficking molecule responsible for the directed movement of cholesterol-rich vesicles? Is it a signaling molecule that activates cholesterol transporters?

Lysosomes function as the cell's recycling compartment. Lysosomes receive cellular and endocytosed proteins and lipids that need digesting. The metabolites that result are transported either by vesicles or directly across the membrane.

Vesicles deliver
lysosomal residents

Vesicles deliver
endocytosed nutrients

Damaged or unneeded
proteins are engulfed.

Vesicles bud
carrying metabolites
for reuse

Other metabolites are transported across
the membrane

Then the trash
gets compacted

Our hypothesis is that NPC1 and NPC2 are required for shuttling lipids between lysosomes, endocytic compartments, the trans Golgi network and plasma membrane. Cholesterol is not the only cargo shuttled by NPC1. The trafficking of fluid phase constituents from late endosomes is altered in NPC cells. Furthermore, NPC1 and NPC2 mutations affect the movement of glycosphingolipids.

Our model of NPC1 and NPC2 function is the following. LDL particles are digested in multivesicular late endosomes and lysosomes. Vesicles bud off of these compartments carrying fluid phase constituents and membrane lipids that have been retrieved from the internal vesicles of the multivesicular late endosomes. Functional NPC1 and NPC2 are required for retrieving lipids from the internal vesicles and shuttling transport vesicles to destinations such as the trans-Golgi network. Some mutations in NPC1 allow it to be targeted as normal to late endosomes but block its invagination into the late endosome interior. This may be due to NPC1 aggregation, binding to lipids or to other proteins. NPC1 or NPC2 dysfunction results in the accumulation of many membrane constituents (cholesterol, sphingomyelin, neutral and acidic glycosphingolipids) in multilamellar storage bodies.

To learn more about the research on NPC,
please go to the Ara Parseghian Medical Research Foundation website www.parseghian.org
APMRF funds basic research on NPC disease.

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Cholesterol movement controlled by the Niemann-Pick C1 protein

Our goal is to identify gene products involved in the directed intracellular movement of cholesterol. One clue that cholesterol transport must be protein mediated is the existence of the human genetic disease, Niemann-Pick C (NPC) , which is characterized by hepatosplenomegaly and neurodegeneration. NPC is caused by mutations in one of two genetic loci, NPC1 and NPC2. Our focus is on NPC1 because mutations in this locus are responsible for 95% of the clinical cases. The most striking consequence of NPC1 dysfunction is aberrant lipid movement, which results in lysosomal storage of both cholesterol and glycosphingolipids. The mechanism by which NPC1 facilitates lipid transport from late endosomes and lysosomes to other cellular membranes is unknown. Currently, there is no definitive therapy for NPC. Elucidation of cellular factors that suppress the NPC phenotype by stimulating cholesterol movement may reveal new therapeutic targets for NPC and other diseases of lipid metabolism.

We have isolated a somatic cell model of NPC disease with an unusual phenotype. Chinese hamster ovary (CHO) mutants 4-4-D (disease) and 4-4-S (suppressed) belong to the same complementation group as NPC fibroblasts and contain the identical base insertion in the NPC1 gene, which results in a frameshift and premature translational termination. Both cell lines have a stable phenotype. Mutant 4-4-D shows the classical NPC disease phenotype of lysosomal cholesterol storage; however, mutant 4-4-S shows no cholesterol storage by filipin fluorescence microscopy. The 4-4-S phenotype is likely due to expression of a gene that suppresses the mutant phenotype. Surprisingly, mutant 4-4-S still shows defective low density lipoprotein (LDL) stimulation of acyl-CoA/cholesterol acyltransferase (ACAT) in the endoplasmic reticulum (ER), which is characteristic of NPC.

Our hypothesis is that the 4-4-S suppressor is a protein that mobilizes cholesterol out of late endosomes and lysosomes, but fails to deliver the cholesterol to the ER. We will identify the gene product(s) that suppresses the 4-4 phenotype and determine the fate of the LDL-cholesterol. We will also investigate the intracellular trafficking of glycosphingolipids in mutant 4-4-S to determine if this other facet of the NPC phenotype has been corrected. Our work will elucidate a cellular factor capable of stimulating cholesterol movement to specific cellular destinations.

Specific Aim #1: To identify the gene product(s) that suppresses the phenotype of mutant 4-4. Complementation analysis between mutants 4-4-S and 4-4-D will reveal whether the 4-4-S phenotype is dominant (i.e. expression of a suppressing factor is increased) or recessive (i.e. an inhibiting factor is decreased). Comparison of 4-4-S and 4-4-D proteomes and gene expression patterns by two dimensional gel electrophoresis and microarray analysis, respectively, has revealed candidate suppressor proteins, some of which are involved in vesicle movement. Candidates from both approaches will be validated by expressing them in 4-4-S and 4-4-D cells.

Specific Aim #2: To determine the fate of LDL-cholesterol in mutant 4-4-S. Filipin fluorescence microscopy of 4-4-S cells shows no lysosomal cholesterol storage, yet cholesterol does not reach the ER. A preliminary experiment indicates that cholesterol does reach the plasma membrane. Cholesterol sequestration in transit vesicles or arrival at the plasma membrane will be thoroughly investigated using multiple approaches. The effect of candidate 4-4 suppressors on each cholesterol transport pathway will be evaluated.

Specific Aim #3: To examine intracellular trafficking of glycosphingolipids in 4-4-D and 4-4-S cells. Glycosphingolipids are the second major lipid class that accumulates in NPC lysosomes. Glycosphingolipid transport will be examined by fluorescence microscopy using BODIPY-LacCer to determine if cholesterol clearance is accompanied by restored trafficking of sphingolipids. Cellular levels and distribution of GM2 and GM3 gangliosides, which accumulate in NPC cells, will be determined. The effect of candidate 4-4 suppressors on glycosphingolipid trafficking will be evaluated.

We expect to elucidate the cellular factor responsible for mobilizing cholesterol from late endosomes and lysosomes. This will reveal potential therapeutic targets for NPC disease, and may also lead to innovative approaches to other diseases of lipid metabolism.

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Cholesterol movement from the plasma membrane to the endoplasmic reticulum

Elevated plasma cholesterol level is a major cause of coronary artery disease, which accounts for 550,000 deaths in the United States each year. Dietary fat is absorbed by intestinal enterocytes, and then transported from the enterocyte cell surface to the endoplasmic reticulum (ER) in order to be metabolized to cholesteryl esters and packaged into chylomicrons for delivery to the liver. In the liver, cholesterol movement to the ER is critical for its conversion to cholesteryl esters and subsequent incorporation into very low-density lipoproteins. It is also essential for metabolism of hepatic cholesterol to bile acids. The route and mechanism of cholesterol transport from the cell surface to the ER are unknown. Elucidation of cellular factors responsible for cholesterol movement to the ER may reveal new therapeutic targets for hypercholesterolemia.

We have isolated a unique somatic cell mutant with defective trafficking of plasma membrane cholesterol to the ER. Chinese hamster ovary (CHO) mutant 3-6 transports both newly synthesized and lipoprotein-derived cholesterol to the plasma membrane, but fails to mobilize cell surface cholesterol to the ER for metabolism or regulation of homeostatic responses. Despite increased levels of cholesterol in the 3-6 plasma membranes, 3-6 cells are resistant to amphotericin B, a polyene antibiotic that forms pores in cholesterol-rich membranes. Thus, the 3-6 phenotype is likely due to a change in the plasma membrane lipid composition.

Our hypothesis is that the gene responsible for the 3-6 phenotype encodes a protein that affects the lipid organization of the plasma membrane. Reduced 3-6 activity alters the plasma membrane lipid domain structure such that cholesterol is both hidden from amphotericin B and prevented from entering its endocytic pathway. In this study, we will identify the 3-6 gene and gene products that suppress the 3-6 phenotype. We will determine how cells adapt to reduced 3-6 activity to more fully understand the physiological roles of the 3-6 protein. We will also investigate changes in the plasma membrane lipid content and lipid trafficking that are induced by the 3-6 mutation. Our work will elucidate cellular factors responsible for maintaining the lipid organization of the plasma membrane and facilitating cholesterol movement to intracellular metabolic enzymes.

Specific Aim #1: To identify cDNAs that, when expressed in mutant 3-6 cells, correct the cholesterol transport defective phenotype. 3-6 cells expressing a retroviral receptor will be transfected with a mouse cDNA library in a retroviral vector. cDNAs that correct the phenotype will be identified. We will determine which of the correcting cDNAs encode the 3-6 protein.

Specific Aim #2: To identify proteins whose expression levels are altered by the 3-6 mutation. Comparison of CHO vs. 3-6 proteomes and gene expression patterns by two-dimensional gel electrophoresis and microarray analysis, respectively, has revealed candidate 3-6 proteins and pathways altered by the 3-6 mutation. Candidates from both approaches will be validated.

Specific Aim #3: To define the 3-6 induced changes in cellular lipid metabolism. We will determine how the 3-6 mutation alters the plasma membrane and/or ER lipid compositions. Sphingolipid transport will be examined in parental CHO and mutant 3-6 cells. We will investigate the mechanism by which candidate 3-6 proteins, and proteins that suppress the mutant phenotype, can modulate these membrane parameters.

Elucidation of cellular factor(s) responsible for cholesterol movement to the ER will reveal potential therapeutic targets. Future mouse genetic tests will determine if inhibition of these proteins could modulate intestinal cholesterol absorption and/or hepatic lipoprotein secretion.

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