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Protein transduction of Rab9 in Niemann-Pick C cells reduces cholesterol storage

Keishi Narita^*, Amit Choudhury^*, Kostantin Dobrenis, Deepak K. Sharma^*, Eileen L. Holicky^*, David L. Marks^*, Steven U. Walkley and Richard E. Pagano^*,,1

^* Department of Biochemistry and Molecular Biology Mayo Clinic and Foundation Rochester, Minnesota, USA;
Department of Neuroscience Albert Einstein College of Medicine Bronx, New York, USA; and
Molecular Neuroscience Program Mayo Clinic and Foundation Rochester, Minnesota, USA

¹ Correspondence: Mayo Clinic and Foundation, Stabile 8, 200 First St. S.W., Rochester, MN 55905, USA. E-mail: pagano.richard{at}mayo.edu

SPECIFIC AIMS

Niemann-Pick disease type C (NPC) is a genetic disorder in which patient cells exhibit lysosomal accumulation of cholesterol and sphingolipids caused by defects in either NPC1 or NPC2 proteins. We previously demonstrated that overexpression of endosomal Rab proteins (Rab7 or Rab9) in NPC1 mutant (^mut) human skin fibroblasts corrects lipid trafficking defects and reduces lipid storage. Here, we developed a protein transduction method for Rab9 to evaluate the therapeutic potential of this approach in cultured cells. We also used protein transduction to investigate the mechanism of the Rab9 correction of NPC cells.

PRINCIPAL FINDINGS

1. Preparation and purification of Rab9/transduction domain fusion protein
We constructed a plasmid to express a wild-type Rab9 fusion protein with the protein transduction domain, VP22, at its N terminus (designated VP22-Rab9). VP22-Rab9 was expressed in bacteria and purified by nickel chelate chromatography. As shown by immunoblot analysis, the purified protein fraction consisted of a 47 kDa polypeptide that represented the full-length fusion VP22-Rab9 protein and 22 kDa Rab9 peptide fragments. VP22-Rab9 was internalized by NPC^mut fibroblasts, as demonstrated by immunoblot analysis of cell lysates. A fraction of internalized VP22-Rab9 was shown to bind Rab GDP dissociation inhibitor, suggesting that this pool of VP22-Rab9 was prenylated and bound to GDP. Only the full-length polypeptide exhibited biological activity (i.e., reduced cholesterol content in NPC cells; see below). Treatment of cells with up to 5 µM of VP22-Rab9 showed no evidence of cytotoxicity.

2. Rab9 reduces filipin staining in NPC1^mut cells
Untreated NPC1^mut fibroblasts show intensely bright punctate filipin staining indicative of cholesterol accumulation, while normal human skin fibroblasts (HSFs) exhibit little filipin fluorescence. When NPC1^mut cells were incubated with 2.5 µM VP22-Rab9 for 48 h, fixed and stained with filipin, the intensity of the staining was reduced dramatically (Fig. 1 A). By contrast, when NPC1^mut cells were treated with the VP22 fragment alone (Fig. 1A ), under the same conditions, no significant reduction in filipin staining was observed. A dose-dependent decrease in the intensity of filipin staining was observed with increasing concentrations of VP22-Rab9, reaching a plateau at 2.5 µM (Fig. 1C ). Reduction in filipin staining by VP22-Rab9 treatment was time dependent, showing a maximum effect at 24-48 h (Fig. 1D ).

View larger version (42K): [in this window] [in a new window]   Figure 1. Correction of the NPC phenotype in NPC1mut fibroblasts treated with VP22-Rab9. Cells were incubated in 1% FBS ± VP22-Rab9 or VP22 alone (transduction protein control) at 2.5 µM for 48 h except as noted. A) Cells were treated ± VP22-Rab9 or VP22, and subsequently fixed and stained with filipin. Bar, 10 µm. B) Cells were treated ± VP22-Rab9 and biochemical quantification of free cholesterol was performed. Cellular lipids were extracted with chloroform/methanol (2/1), sterols were separated on TLC, stained, and quantified (4 replicates/sample). The data are presented as the mean ± SE. Values for VP22-Rab9-treated and untreated (UT) NPC cells were significantly different (< 0.05) in 2-tailed t tests. C) Dose dependency of VP22-Rab9 treatment. NPC1mut cells were incubated with different concentrations of VP22-Rab9 or VP22 only, fixed, stained with filipin, and the intensity quantified by image analysis. The values are expressed as % of those in untreated controls and are presented as the mean ± SE. D) Time course of VP22-Rab9 treatment. NPC1mut fibroblasts were cultured without (black bars) or with VP22-Rab9 continuously present (gray bars) for different time periods, and the intensity of filipin staining was quantified as in panel C. For open bars, cells were treated with VP22-Rab9 for 6 h, washed, and incubated for the rest of the time periods without VP22-Rab9 protein. E) Correction of intracellular lipid trafficking. NPC1mut cells ± VP22-Rab9 treatment were pulse labeled with BODIPY-LacCer. Bar, 10 µm. For quantification, 150 cells were counted per sample. White, % of cells showing punctate late endosomal/lysosomal stain; black, % of cells showing Golgi staining.

3. Rab9 reduces free cholesterol in NPC1^mut cells
Treatment of NPC1^mut fibroblasts with VP22-Rab9 resulted in a significant reduction in cellular free cholesterol levels, as shown by biochemical analysis of cellular lipids (Fig. 1B ). The levels of free cholesterol in untreated normal and NPC1^mut skin fibroblasts were 33 ± 4.8 and 48 ± 9.7 µg/mg of protein, respectively, whereas NPC1^mut cells treated with VP22-Rab9 showed significantly reduced free cholesterol levels (39±8.0 µg/mg protein) compared with untreated NPC1^mut fibroblasts (P<0.05) (Fig. 1B ).

4. VP22-Rab9 restores normal glycosphingolipid trafficking in NPC1 cells
We previously showed that BODIPY-LacCer is internalized by endocytosis and targeted predominantly to the Golgi apparatus in normal HSFs. In contrast, in NPC1^mut and many other sphingolipid storage disease (SLSD) fibroblasts, most of the fluorescent lipid accumulates in punctate endosomal structures as a result of elevated intracellular cholesterol. When NPC1^mut cells were treated with VP22-Rab9, many cells now showed a restoration of Golgi targeting of the LacCer analog (Fig. 1E ), similar to that seen in normal HSFs.

5. VP22-Rab9 treatment increases cholesterol esterification and sterol efflux
Treatment of NPC1^mut fibroblasts with VP22-Rab9 for 48 h resulted in higher Nile Red fluorescence than that observed in untreated NPC1^mut cells or NPC1^mut cells treated with VP22 only. Biochemical analysis showed a significant (100%) increase in cell-associated cholesterol ester levels in NPC1^mut fibroblasts treated with VP22-Rab9 (Fig. 2A ). After treatment with VP22-Rab9 for 48 h, there was a significant increase in both free cholesterol and cholesterol ester in the culture medium of NPC1^mut cells (Fig. 2B ), which could account for the bulk of free cholesterol lost from these cells. Immunoblot analysis showed that LDL-receptor levels were decreased in NPC1 cells after VP22-Rab9 treatment, suggesting that VP22-Rab9 treatment increased transport of some cholesterol to the endoplasmic reticulum (ER) (Fig. 2C ).

View larger version (22K): [in this window] [in a new window]   Figure 2. Fate of stored cholesterol after VP22-Rab9 treatment. Cultured HSFs or NPC1mut fibroblasts were either untreated (UT) or were incubated with 2.5 µM VP22-Rab9 or VP22 in (A, C) EMEM + 1% FBS for 48 h, or (B) McCoys 5A + 5% LPDS for 48 h, and used for the following. A) Quantitation of cholesterol ester (CE). Four or more samples were analyzed for each condition. Data are the mean ± SE. B) Cholesterol or CE content of cells vs. culture medium. The amount of each sterol in cells and medium was analyzed. Data are the mean of 4 independent assays. C) Reduction in LDLR expression. 50 µg of total protein was separated on SDS-PAGE gel and the LDLR detected and quantified by Western blot analysis. Data are presented as the mean ± SE. n = 3 independent experiments.

6. VP22-Rab9 treatment of other NPC cell types
The studies described above were performed on an NPC1^mut patient fibroblast sample with I1061T/P237S mutations in NPC1. When NPC fibroblast subtypes with more severe defects in cholesterol homeostasis (i.e., NPC1^null, NPC2^mut, and NPC2^null fibroblasts) were treated with VP22-Rab9 for 7 days in EMEM and 5% FBS, all treated NPC subtypes showed significant reduction in filipin staining relative to untreated controls. Cultured mouse cortical NPC1^null neurons incubated with 5 µM VP22-Rab9 in media with 1% FBS for 2 days showed significantly lower levels of filipin staining than untreated NPC1^null neurons.

CONCLUSIONS AND SIGNIFICANCE

We demonstrated that treatment with the VP22-Rab9 fusion protein could reduce accumulation of free cholesterol in NPC cells (Fig. 1) consistent with our previous studies using DNA transfection to overexpress Rab9. Protein transduction of Rab9 reduced cholesterol in nearly all the treated cells. Finally, VP22-Rab9 reduced cholesterol in a variety of NPC fibroblasts with different mutations as well as in cultured NPC1^null mouse neurons. Since VP22 fusion proteins have been successfully used in animal models in vivo, our studies raise the possibility that protein transduction of Rabs could be explored as a possible therapeutic approach for treatment of SLSDs.

Reduction in cellular free cholesterol in VP22-Rab9-treated NPC1^mut fibroblasts was accompanied by increased cholesterol esterification. Our data strongly suggest that VP22-Rab9 stimulates the transport of some free cholesterol to the ER, where most cholesterol esterification occurs (Fig. 3 ). The restoration of cholesterol delivery to the ER and consequent down-regulation of SREBP activation could be key steps in normalizing cholesterol homeostasis in NPC cells.

View larger version (23K): [in this window] [in a new window]   Figure 3. Effects of Rab9 protein transduction in NPC cells. Mutations in the NPC1 or NPC2 proteins lead to accumulation of unesterified cholesterol and other lipids in late endosomes, and to inhibition of transport of a glycosphingolipid analog (BODIPY-LacCer) from the PM via late endosomes to the Golgi apparatus. VP22-Rab9 treatment of NPC cells restored LacCer transport to the Golgi apparatus (Fig. 1E ), and lowered cellular cholesterol (Fig. 1A, B ). The reduction in stored cholesterol was accompanied by enhanced esterification of cholesterol (Fig. 2A ) and the appearance of sterols in the medium (Fig. 2B ). Based on the results with BODIPY-LacCer, we suggest that protein transduction of Rab9 stimulates vesicular transport of endogenous sphingolipids from late endosomes to the Golgi apparatus. Cholesterol may exit the late endosomes together with sphingolipids, or by an alternative mechanism bypassing the Golgi complex.

We noted that treatment of NPC cells with VP22-Rab9 stimulated the appearance of sterols in the culture medium (Fig. 2B ). The mechanism of sterol efflux is not yet identified. Further studies will be required to establish the role of specific transporters and carrier proteins in Rab9-stimulated sterol efflux. The mechanisms by which Rab9 transduction reduces cholesterol in the late endosomal compartment and increases delivery to the ER are not presently known. Two possible models (Fig. 3) for this process are 1) the direct stimulation of cholesterol vesicular transport such as was seen for sphingolipids; and 2) stimulation of sphingolipid vesicular transport out of the late endosome, which then "frees" cholesterol from its tight association with stored sphingolipids. Cholesterol could then be transported by either a vesicular or nonvesicular mechanism to the ER where most esterification occurs, possibly bypassing the Golgi complex. However, we have not ruled out the possibility that some cholesterol ester formation could take place in an endosomal compartment, or in the culture medium after cholesterol efflux.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2714fje;

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