Title: Development of a Rab9 Transgenic Mouse and Its Ability to Increase the Lifespan of a Murine Model of Niemann-Pick Type C Disease
Abstract: 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 ∼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 GM2 and GM3 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. 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 ∼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 GM2 and GM3 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. Niemann-Pick, type C (NP-C) is an autosomal recessive, neurodegenerative disease that is characterized by massive accumulation of cholesterol in peripheral tissues and glycosphingolipids in the brain. In humans, most cases of NP-C arise from mutations in NPC1 that encodes a large membrane protein with multiple transmembrane domains homologous to the sterol-sensing domains found in Patched, HMG-CoA reductase, and SCAP.1Patterson M Vanier M Suzuki K Morris J Carstea E Neufeld E Blanchette-Machie E Pentchev P Scriver CR Beaudet AL Sly WS Valle D Niemann-Pick Disease Type C: A Lipid Trafficking Disorder. McGraw Hill, New York2001: 3611-3634Google Scholar, 2Sturley SL Patterson MC Balch W Liscum L The pathophysiology and mechanisms of NP-C disease.Biochim Biophys Acta. 2004; 1685: 83-87Crossref PubMed Scopus (132) Google Scholar, 3Vanier MT Millat G Niemann-Pick disease type C.Clin Genet. 2003; 64: 269-281Crossref PubMed Scopus (507) Google Scholar Current therapeutic approaches for NP-C that are being explored using animal models include substrate reduction therapy to reduce the biosynthesis of stored glycosphingolipids,4Jeyakumar M Dwek RA Butters TD Platt FM Storage solutions: treating lysosomal disorders of the brain.Nat Rev Neurosci. 2005; 6: 713-725Crossref PubMed Scopus (154) Google Scholar, 5Patterson MC Vecchio D Prady H Abel L Wraith JE Miglustat for treatment of Niemann-Pick C disease: a randomised controlled study.Lancet Neurol. 2007; 6: 765-772Abstract Full Text Full Text PDF PubMed Scopus (490) Google Scholar, 6Zervas M Somers KL Thrall MA Walkley SU Critical role for glycosphingolipids in Niemann-Pick disease type C.Curr Biol. 2001; 11: 1283-1287Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar allopregnanolone therapy that seeks to correct the reduced neurosteroid levels seen in NP-C animals,7Griffin LD Gong W Verot L Mellon SH Niemann-Pick type C disease involves disrupted neurosteroidogenesis and responds to allopregnanolone.Nat Med. 2004; 10: 704-711Crossref PubMed Scopus (329) Google Scholar and treatment with a synthetic oxysterol that activates genes involved in cholesterol breakdown and removal.8Langmade SJ Gale SE Frolov A Mohri I Suzuki K Mellon SH Walkley SU Covey DF Schaffer JE Ory DS Pregnane X receptor (PXR) activation: a mechanism for neuroprotection in a mouse model of Niemann-Pick C disease.Proc Natl Acad Sci USA. 2006; 103: 13807-13812Crossref PubMed Scopus (139) Google Scholar We previously showed that overexpression of the small GTPase, Rab9, in cultured cells results in correction of lipid-trafficking defects associated with NP-C and significantly reduces lipid accumulation.9Choudhury A Dominguez M Puri V Sharma DK Narita K Wheatley CW Marks DL Pagano RE Rab proteins mediate Golgi transport of caveola-internalized glycosphingolipids and correct lipid trafficking in Niemann-Pick C cells.J Clin Invest. 2002; 109: 1541-1550Crossref PubMed Scopus (388) Google Scholar, 10Marks DL Pagano RE Endocytosis and sorting of glycosphingolipids in sphingolipid storage disease.Trends Cell Biol. 2002; 12: 605-613Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 11Narita K Choudhury A Dobrenis K Sharma DK Holicky EL Marks DL Walkley SU Pagano RE Protein transduction of Rab9 in Niemann-Pick C cells reduces cholesterol storage.FASEB J. 2005; 19: 1558-1560Crossref PubMed Scopus (73) Google Scholar Similar results have been obtained by overexpression of Rabs 4, 7, or 8 in cultured fibroblasts,12Choudhury A Marks D Pagano R Hirabayashi Y Igarashi Y Merrill JAH Endocytic Trafficking of Glycosphingolipids. Springer-Verlag, Tokyo2006: 295-307Google Scholar, 13Choudhury A Sharma DK Marks DL Pagano RE Elevated endosomal cholesterol levels in Niemann-Pick cells inhibit Rab4 and perturb membrane recycling.Mol Biol Cell. 2004; 15: 4500-4511Crossref PubMed Scopus (121) Google Scholar, 14Linder MD Uronen RL Holtta-Vuori M van der Sluijs P Peranen J Ikonen E Rab8-dependent recycling promotes endosomal cholesterol removal in normal and sphingolipidosis cells.Mol Biol Cell. 2007; 18: 47-56Crossref PubMed Scopus (82) Google Scholar or Rab9 in NP-C mouse neurons.11Narita K Choudhury A Dobrenis K Sharma DK Holicky EL Marks DL Walkley SU Pagano RE Protein transduction of Rab9 in Niemann-Pick C cells reduces cholesterol storage.FASEB J. 2005; 19: 1558-1560Crossref PubMed Scopus (73) Google Scholar Although the underlying mechanism for this correction is not completely understood, several studies have shown that elevated endosomal cholesterol interferes with the GDP dissociation inhibitor extraction of Rab proteins from endosomal membranes.13Choudhury A Sharma DK Marks DL Pagano RE Elevated endosomal cholesterol levels in Niemann-Pick cells inhibit Rab4 and perturb membrane recycling.Mol Biol Cell. 2004; 15: 4500-4511Crossref PubMed Scopus (121) Google Scholar, 15Ganley IG Pfeffer SR Cholesterol accumulation sequesters Rab9 and disrupts late endosome function in NPC1-deficient cells.J Biol Chem. 2006; 281: 17890-17899Crossref PubMed Scopus (101) Google Scholar, 16Lebrand C Corti M Goodson H Cosson P Cavalli V Mayran N Faure J Gruenberg J Late endosome motility depends on lipids via the small GTPase Rab7.EMBO J. 2002; 21: 1289-1300Crossref PubMed Scopus (281) Google Scholar Overexpression of Rab proteins may thus be sufficient to stimulate the intracellular transport that is otherwise blocked by the stored lipids. In the present study, we sought to test whether Rab overexpression might also have a beneficial effect in vivo. We developed strains of transgenic mice that ubiquitously overexpress Rab9 and then crossed these transgenic mouse strains into a mouse model of NP-C disease. We show that the overexpression of Rab9 increased the average lifespan by up to ∼22% and reduced some symptoms associated with this disease, suggesting that stimulation of intracellular transport might have therapeutic potential. All procedures involving mice were performed in accordance with the recommendations and approval of the Institutional Animal Care Use Committee of the Mayo Clinic and Foundation. Mice were weaned at postnatal day 21 and fed a standard chow diet. BALB/cNctr-Npc1m1N/+ breeding stock were purchased from The Jackson Laboratory (Bar Harbor, ME) and maintained as heterozygotes. Homozygous NP-C mutant mice are referred to throughout as NPCmut/mut mice. HA-tagged human Rab9 (I.M.A.G.E. Consortium) was inserted into the pCAGGS vector (generously provided by J. Miyazaki, Osaka University, Osaka, Japan), which contains the chicken actin promoter that is widely used for ubiquitous expression of proteins in transgenic mice.17Ikawa M Kominami K Yoshimura Y Tanaka K Nishimune Y Okabe M Green fluorescent protein as a marker in transgenic mice.Dev Growth Differ. 1995; 37: 455-459Crossref Scopus (106) Google Scholar DNA containing the promoter and the Rab9 transgene was then purified for microinjection into fertilized eggs. Transgenic mice were generated by the Mayo Clinic Transgenic Core by pronuclear microinjection of the foreign DNA fragments into one-cell-stage mouse embryos from FVB, C57BL/6J mice. Microinjected embryos were then transferred into surrogate mothers and progeny carrying the transgene were bred to establish the transgenic mouse lines. From this procedure we obtained ∼100 adult candidate mice. Tail biopsies were then assessed for proper integration of the transgene by polymerase chain reaction (PCR) and five transgenic strains (designated as strains 1, 100, 400, 500, and 800) were selected and further characterized (see text). Because mice homozygous for the NPC1 mutation are not fertile, we first crossed the Rab9 transgenic animals (Rab9TG+/TG−, NPCWT/WT) with NPC1 heterozygotes (NPCWT/mut). Offspring that were heterozygous for the transgene and for the NP-C mutation (Rab9TG+/TG−, NPCWT/mut) were identified by PCR of tail snips. These animals were then crossed with NPC1 heterozygotes to obtain offspring that were homozygous for the NP-C mutation and heterozygous for the Rab9 transgene (Rab9TG+/TG−, NPC1mut/mut), as well as NP-C disease control animals (Rab9TG−/TG−, NPC1mut/mut)). NPC1WT/WT animals ± Rab9TG were also used in some experiments. One hundred ng of total DNA, isolated from tail biopsies as described,18Miller SA Dykes DD Polesky HF A simple salting out procedure for extracting DNA from human nucleated cells.Nucleic Acids Res. 1988; 16: 1215Crossref PubMed Scopus (18345) Google Scholar were added to the reaction mixture [49 μl consisting of 10× PCR buffer (5 μl) (Denville Scientific Inc., South Plainfield, NJ), 10 mmol/L dNTPs (1 μl), 1 μl each of forward and reverse primers (25 μmol/L stock solutions; primers synthesized by IDT, Coralville, IA) for NPC-1 (forward: 5′-GGTGCTGGACAGCCAAGTA-3′; reverse: 5′-GATGGTCTGTTCTCCCATG-3′) or Rab9 (forward: 5′-GTACCCATACGATGTTCCGG-3′; reverse: 5′-GGCTTTCGGTGAAGATTGACTG-3′), TaqDNA polymerase (1 U) (Denville Scientific Inc.) and dH2O (41 μl)] and subjected to PCR. PCR conditions consisted of 95°C × 5 minutes, 94°C × 1 minute, 55°C × 2 minutes, and 72°C × 2 minutes, for 30 cycles and then 72°C × 10 minutes. PCR products were resolved on a 1% agarose gel by electrophoresis. Brain and liver samples from Rab9TG+/− founder mice and from NPCmut/mut mice expressing the Rab9 transgene were harvested and homogenized in 50 mmol/L Tris, pH 7.4, 25 mmol/L KCl, 0.25 mol/L sucrose with protease inhibitors. To visualize the HA-tagged Rab9 transgene, total brain and liver proteins (5 μg/lane) were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes, incubated with monoclonal anti-Rab9 Ab (diluted 1:1000, catalog no. R5404; Sigma Chemical Co., St. Louis, MO) followed by goat anti-mouse horseradish peroxidase (GE Health Care, Piscataway, NJ), and detected by chemiluminescence. To evaluate the levels of endogenous Rab9 protein, control animals that did not express the Rab9 transgene were processed as above, but using 20 μg of protein/lane. β-Actin levels on the same blots were detected using an anti-β-actin antibody (diluted 1:1000, catalog no. 4967; Cell Signaling Technologies Inc., Danvers, MA). For immunostaining and lipid analysis of GM2 and GM3 gangliosides, 70- or 80-day-old mice obtained from crosses of NP-C mice with Rab9 transgenic strain 500 or 800 were placed under deep anesthesia and perfused with saline. One cerebral hemisphere was immersion-fixed in 4% paraformaldehyde overnight at 4°C and subsequently immunostained for GM2 and GM3 gangliosides as described.19Zervas M Dobrenis K Walkley SU Neurons in Niemann-Pick disease type C accumulate gangliosides as well as unesterified cholesterol and undergo dendritic and axonal alterations.J Neuropath Exp Neurol. 2001; 60: 49-64PubMed Google Scholar In some experiments, one half of the brain (including cerebrum and cerebellum) was homogenized and used for lipid extraction and thin-layer chromatographic analysis essentially as described.6Zervas M Somers KL Thrall MA Walkley SU Critical role for glycosphingolipids in Niemann-Pick disease type C.Curr Biol. 2001; 11: 1283-1287Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, 20Ledeen RW Yu RK Gangliosides: structure, isolation, and analysis.Methods Enzymol. 1982; 83: 139-191Crossref PubMed Scopus (680) Google Scholar Immunostaining of the Rab9 transgene in the brain of NPCmut/mut animals was performed by the Mayo Tissue and Cell Molecular Analysis Core Facility. Mouse brains of 50-day-old animals were collected and fixed as described above. The samples were then embedded in paraffin and 4-μm sections were prepared. Sections were deparaffinized by three washes in xylene and rehydrated in a series of decreasing concentrations of alcohol. The slides were then placed in preheated (37°C) 1 mmol/L ethylenediaminetetraacetic acid, pH 8.0, for 30 minutes, cooled to room temperature in the same buffer for 5 minutes, and then rinsed for 5 minutes in running distilled water. Endogenous peroxide was blocked with 3% H2O2 for 5 minutes. Samples were then washed and incubated with an anti-HA Rat mAb (clone 3F10 used at 1:1000; Roche Applied Science, Indianapolis, IN) for 1 hour, washed, and further incubated with the secondary antibody (Rat-on-Mouse Polymer detection kit; Biocare Medical, Concord, CA) for 30 minutes. The sections were then washed, incubated in 3,3-diaminobenzidine for 5 minutes, counterstained with modified Schmidts' hematoxylin for 5 minutes, rinsed under tap water, and mounted with an aqueous mounting media. Animals were housed in standard cages and conditions. Rab9TG+/− NPCWT/mut (separate breeders for each strain) and Rab9TG−/− NPCWT/mut mice were crossed (see Supplemental Figure S1 at http://ajp.amjpathol.org). The date of birth of each litter was noted. Pups were genotyped by PCR and weaned at 3 weeks. Only Rab9TG+/− NPCmut/mut and Rab9TG−/− NPCmut/mut were kept. We then monitored the lifespan of Rab9TG+/− NPCmut/mut mice versus Rab9TG−/− NPCmut/mut mice obtained from crosses within the same strain, thus assuring that the backgrounds of the animals being compared were as close as possible. It was not possible to exclusively use littermates for these studies because of the small number of required genotypes occurring in each litter. Beginning at 5 weeks, NPCmut/mut mice were monitored daily for date of death. Mortality date was recorded when mice were found dead or judged to be in severe distress (eg, could not stand or exhibited paralysis of hind limbs). In the latter case, the moribund animals were euthanized according to Institutional Animal Care and Use Committee guidelines. We first generated transgenic mice (C57BL/6 background) that expressed HA-tagged human Rab9 ubiquitously and constitutively under the control of the chicken actin promoter (see Materials and Methods). We selected five founder mice that were positive for the Rab9 transgene (TG) by PCR. These animals were bred with normal C57BL/6 mice to create five separate strains of Rab9 transgenic mice. Each strain was perpetuated by breeding Rab9TG heterozygotes with normal mice. The five strains varied in their level of Rab9 overexpression (up to 30-fold higher than endogenous Rab9) and the Rab9 transgene was detected in all tissues tested (liver, brain, kidney, skin) for each strain; however, the relative distribution in different tissues varied among the different transgenic strains. For example, strain 1 exhibited high Rab9TG levels in liver, but an intermediate level in brain, whereas strain 500 had the highest levels of Rab9TG in both brain and liver (Figure 1A). This variation is seen more clearly in Figure 1B, in which the amount of Rab9TG in each transgenic strain was normalized to the level of endogenous Rab9 in the liver. No negative effects of the Rab9TG on breeding, longevity, or behavior of the mice were observed. In addition, Rab9TG mouse tissues were normal on examination for gross pathology. Each Rab9 transgenic strain was then crossed with animals from our NPC1 (BALB/c background) breeding colony. This required multiple crosses because mice homozygous for the NPC1 mutant allele are not fertile and must be maintained as heterozygotes. Thus the initial crosses were between a transgenic mouse (Rab9TG+/− NPCWT/WT) and an NPC heterozygote (Rab9TG−/− NPCWT/mut). Offspring were screened by PCR of tailsnips to identify NPC heterozygous animals expressing the Rab9 transgene (ie, Rab9TG+/− NPCWT/mut). Those animals were then backcrossed with the NPC heterozygous breeding stock, and their offspring were screened for animals homozygous for the NPC mutant allele, with (Rab9TG+/− NPCmut/mut) or without (Rab9TG−/− NPCmut/mut) the Rab9 transgene (see Supplemental Figure S1 at http://ajp.amjpathol.org). We first examined the tissue distribution of the Rab9 transgene in 88- to 113-day-old NPCmut/mut animals and found that although the levels of transgene overexpression in the brain were very similar in the NPCmut/mut animals and in the corresponding founder strains, the levels of expression in the liver in several strains were much lower than in the founders (Figure 1A). For example, in the founders from strain 500 the transgene expression was ∼30-fold higher than endogenous Rab9 in the liver, whereas in the Rab9TG+/− NPCmut/mut animals, the levels of the transgene in the liver were similar to that of endogenous Rab9 (Figure 1, A and B). One possible explanation for reduced expression of Rab9TG in livers of the experimental animals compared to the founder strains is that there is an effect of the NP-C disease state on expression of the transgene (eg, there is known to be considerable hepatocyte damage in the NP-C liver21Beltroy EP Liu B Dietschy JM Turley SD Lysosomal unesterified cholesterol content correlates with liver cell death in murine Niemann-Pick type C disease.J Lipid Res. 2007; 48: 869-881Crossref PubMed Scopus (57) Google Scholar). Thus, we investigated Rab9TG expression in livers from Rab9TG+/−NPCWT/WT mice of the 500 strain that were products of the same crosses as the Rab9TG+/− NPCmut/mut experimental animals (ie, had identical genetic backgrounds). We found that these animals had high Rab9 transgene expression in the livers (Figure 1C), much like the founder mice (Figure 1A). Thus, we conclude that the lower Rab9 transgene expression observed in the livers of NPCmut/mut mice of some strains is a result of an interaction of the NP-C disease state and Rab9 transgene expression. We also used immunohistochemistry to verify the expression of the HA-tagged Rab9 transgene in brain sections from 7-week-old Rab9TG+/− NPCmut/mut animals (strain 500) and readily detected the Rab9 transgene in the brain (Figure 1D). The HA-Rab9 transgene was expressed throughout the brain and was particularly enriched in the cerebellum (Figure 1D). The NPCmut/mut mice, with or without the Rab9 transgene, were next examined with respect to several parameters that are affected as the mutant mice age. i) Activation of microglia. Microglia are resident immune cells of the central nervous system that secrete potentially neurotoxic molecules such as tumor necrosis factor-α on activation. Inappropriate activation of microglia has been implicated in several neurodegenerative disorders including NP-C disease, and it has been suggested that lipid storage may cause microglial dysfunction.22Baudry M Yao Y Simmons D Liu J Bi X Postnatal development of inflammation in a murine model of Niemann-Pick type C disease: immunohistochemical observations of microglia and astroglia.Exp Neurol. 2003; 184: 887-903Crossref PubMed Scopus (146) Google Scholar, 23German DC Liang CL Song T Yazdani U Xie C Dietschy JM Neurodegeneration in the Niemann-Pick C mouse: glial involvement.Neuroscience. 2002; 109: 437-450Crossref PubMed Scopus (157) Google Scholar To examine the possibility that Rab9 expression has an effect on the activation of microglia, brain sections from the cerebellum were stained with a microglial-specific antibody, Iba1.24Schluesener HJ Seid K Kretzschmar J Meyermann R Allograft-inflammatory factor-1 in rat experimental autoimmune encephalomyelitis, neuritis, and uveitis: expression by activated macrophages and microglial cells.Glia. 1998; 24: 244-251Crossref PubMed Scopus (88) Google Scholar In agreement with previous reports, the NPCmut/mut sample showed a large accumulation of microglia with the typical amoeboid morphology characteristic of activated cells, whereas only ramified (resting) microglia were seen in NPCWT/WT animals.4Jeyakumar M Dwek RA Butters TD Platt FM Storage solutions: treating lysosomal disorders of the brain.Nat Rev Neurosci. 2005; 6: 713-725Crossref PubMed Scopus (154) Google Scholar, 22Baudry M Yao Y Simmons D Liu J Bi X Postnatal development of inflammation in a murine model of Niemann-Pick type C disease: immunohistochemical observations of microglia and astroglia.Exp Neurol. 2003; 184: 887-903Crossref PubMed Scopus (146) Google Scholar, 25Li H Repa JJ Valasek MA Beltroy EP Turley SD German DC Dietschy JM Molecular, anatomical, and biochemical events associated with neurodegeneration in mice with Niemann-Pick type C disease.J Neuropathol Exp Neurol. 2005; 64: 323-333PubMed Google Scholar However, there was no obvious difference in NPCmut/mut mice with or without expression of the Rab9 transgene in either 7- or 10-week-old animals (data not shown). ii) Weight loss. NP-C mice typically begin to lose weight 6 to 7 weeks after birth. To determine whether the Rab9 transgene had an effect on this parameter, we weighed mutant animals weekly throughout a 10- to 11-week period (Figure 2A). NP-C mice that were Rab9TG-negative began to lose weight at weeks 5 to 6, whereas Rab9TG-positive animals showed a trend toward increased weight retention. This was particularly evident for male offspring produced from NP-C animals crossed with transgenic strain 500 (Figure 2A). A similar trend was observed for other strains, but the effects were smaller (see Supplemental Figure S2 at http://ajp.amjpathol.org). At present we have no explanation for why the retardation in weight loss was more pronounced for males than females. iii) Lipid storage in the cerebral cortex. One hallmark of NP-C mice is the storage of gangliosides and cholesterol in the brain.1Patterson M Vanier M Suzuki K Morris J Carstea E Neufeld E Blanchette-Machie E Pentchev P Scriver CR Beaudet AL Sly WS Valle D Niemann-Pick Disease Type C: A Lipid Trafficking Disorder. McGraw Hill, New York2001: 3611-3634Google Scholar, 19Zervas M Dobrenis K Walkley SU Neurons in Niemann-Pick disease type C accumulate gangliosides as well as unesterified cholesterol and undergo dendritic and axonal alterations.J Neuropath Exp Neurol. 2001; 60: 49-64PubMed Google Scholar, 26Gondré-Lewis MC McGlynn R Walkley SU Cholesterol accumulation in NPC1-deficient neurons is ganglioside dependent.Curr Biol. 2003; 13: 1324-1329Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar To test the effect of Rab9 overexpression on this storage, we first performed ganglioside immunocytochemistry on sections of the cerebral cortex obtained from wild-type mice and NPCmut/mut mice ± Rab9TG expression.19Zervas M Dobrenis K Walkley SU Neurons in Niemann-Pick disease type C accumulate gangliosides as well as unesterified cholesterol and undergo dendritic and axonal alterations.J Neuropath Exp Neurol. 2001; 60: 49-64PubMed Google Scholar All samples were from 80-day-old male mice obtained from crosses with transgenic strain 500 because these gave the greatest increase in longevity (see below). Rab9TG overexpression decreased the levels of GM2 and GM3 gangliosides in the cerebral cortex as assessed qualitatively by immunostaining of three sets of animals of the indicated genotypes (Figure 2B). Gangliosides were also extracted from the whole brain of the mice and analyzed by thin-layer chromatography using resorcinol, a ganglioside-specific stain (Figure 2C).20Ledeen RW Yu RK Gangliosides: structure, isolation, and analysis.Methods Enzymol. 1982; 83: 139-191Crossref PubMed Scopus (680) Google Scholar In agreement with the results obtained by immunostaining, we found the highest levels of GM2 and GM3 storage in NPCmut/mut animals and a >30% reduction in this storage in NPCmut/mut mice expressing the Rab9 transgene. No storage of these gangliosides was seen in NPCWT/WT brain extracts (data not shown) as previously reported.6Zervas M Somers KL Thrall MA Walkley SU Critical role for glycosphingolipids in Niemann-Pick disease type C.Curr Biol. 2001; 11: 1283-1287Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar We also found that expression of the Rab9TG from strain 800 reduced ganglioside storage in the cerebral cortex and hippocampus in 70-day-old NPCmut/mut mice (see Supplemental Figure S3 at http://ajp.amjpathol.org). Thus, Rab9TG expression significantly reduced ganglioside storage in the brains of NP-C mice. In contrast, we could detect no effect of Rab9TG expression on filipin staining of unesterified cholesterol in brain sections obtained from NPCmut/mut animals (data not shown). Finally, we evaluated the effect of the Rab9 transgene on the longevity of the NP-C mutant mice by comparing the lifespan of animals homozygous for the NP-C mutant allele and expressing the Rab9 transgene to that in Rab9-negative animals obtained from the same strain. This assured that the backgrounds of the animals being compared were identical. This was important because the Rab9 transgenic founder strains were produced in a different background than the available NP-C mouse model (see above). Survival data for NP-C mice (±Rab9TG expression) were obtained for each transgenic strain and are displayed as Kaplan-Meier plots, which give the percentage of animals surviving at each day after birth. Survival curves and percent increase in lifespan for males, females, and both combined are shown in Figure 3, A and B, and Supplemental Figure S4 at http://ajp.amjpathol.org. All strains exhibited a significant increase in lifespan when the Rab9 transgene was expressed, with the largest increase (∼22%) seen for male mice (Rab9TG+/− NPCmut/mut) obtained from crosses with transgenic strain 500 (Figure 3A). Taken together, the results in Figure 2, Figure 3 demonstrate that expression of the Rab9 transgene reduced some of the symptoms seen in the NP-C mouse model and significantly extended the lifespan of these animals. The observation that lifespan was significantly extended in each of the five Rab9 transgenic strains suggests that life extension was attributable to Rab9 overexpression rather than interference with an unrelated gene during insertion of the transgene into the mouse DNA, because the Rab9 transgene is likely to be inserted at a different point in each mouse strain. Our previous in vitro studies using cultured NP-C fibroblasts and neurons showed that overexpression of Rab4, -7, or -9 corrected aberrant membrane trafficking and reduced lipid storage in cultured NP-C cells.9Choudhury A Dominguez M Puri V Sharma DK Narita K Wheatley CW Marks DL Pagano RE Rab proteins mediate Golgi transport of caveola-internalized glycosphingolipids and correct lipid trafficking in Niemann-Pick C cells.J Clin Invest. 2002; 109: 1541-1550Crossref PubMed Scopus (388) Google Scholar, 10Marks DL Pagano RE Endocytosis and sorting of glycosphingolipids in sphingolipid storage disease.Trends Cell Biol. 2002; 12: 605-613Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 13Choudhury A Sharma DK Marks DL Pagano RE Elevated endosomal cholesterol levels in Niemann-Pick cells inhibit Rab4 and perturb membrane recycling.Mol Biol Cell. 2004; 15: 4500-4511Crossref PubMed Scopus (121) Google Scholar Similar results have been demonstrated by other laboratories.14Linder MD Uronen RL Holtta-Vuori M van der Sluijs P Peranen J Ikonen E Rab8-dependent recycling promotes endosomal cholesterol removal in normal and sphingolipidosis cells.Mol Biol Cell. 2007; 18: 47-56Crossref PubMed Scopus (82) Google Scholar, 27Walter M Davies JP Ioannou YA Telomerase immortalization upregulates Rab9 expression and restores LDL cholesterol egress from Niemann-Pick C1 late endosomes.J Lipid Res. 2003; 44: 243-253Crossref PubMed Scopus (84) Google Scholar In the present study we extended this work to an animal model of NP-C disease and show that Rab9 overexpression can have a beneficial effect in vivo. Although modest, the effect of overexpression of Rab9 on life extension was similar to results obtained with substrate reduction therapy using Miglustat.6Zervas M Somers KL Thrall MA Walkley SU Critical role for glycosphingolipids in Niemann-Pick disease type C.Curr Biol. 2001; 11: 1283-1287Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar In addition, there are a number of considerations that might lead to further improvement of this approach. First, it is unknown whether the transgenic Rab9 is fully active in the mouse, since previous studies demonstrated that cholesterol can interfere with Rab9 function.12Choudhury A Marks D Pagano R Hirabayashi Y Igarashi Y Merrill JAH Endocytic Trafficking of Glycosphingolipids. Springer-Verlag, Tokyo2006: 295-307Google Scholar, 15Ganley IG Pfeffer SR Cholesterol accumulation sequesters Rab9 and disrupts late endosome function in NPC1-deficient cells.J Biol Chem. 2006; 281: 17890-17899Crossref PubMed Scopus (101) Google Scholar In addition, we found that the level of Rab9 overexpression was greatly reduced in the liver (Figure 1C), raising the possibility that some of the protein could be inactivated or degraded at a critical time during disease progression. These effects might be overcome using an inducible expression system so that the Rab9 transgene could be activated at a particular age and for a length of time chosen by the investigator. Second, the overexpression of other Rabs (eg, Rab4, -7, or -8) that control other pathways of intracellular transport and that have been shown to be effective in ameliorating the NP-C phenotype in vitro may be more effective than Rab9 in vivo.9Choudhury A Dominguez M Puri V Sharma DK Narita K Wheatley CW Marks DL Pagano RE Rab proteins mediate Golgi transport of caveola-internalized glycosphingolipids and correct lipid trafficking in Niemann-Pick C cells.J Clin Invest. 2002; 109: 1541-1550Crossref PubMed Scopus (388) Google Scholar, 13Choudhury A Sharma DK Marks DL Pagano RE Elevated endosomal cholesterol levels in Niemann-Pick cells inhibit Rab4 and perturb membrane recycling.Mol Biol Cell. 2004; 15: 4500-4511Crossref PubMed Scopus (121) Google Scholar, 14Linder MD Uronen RL Holtta-Vuori M van der Sluijs P Peranen J Ikonen E Rab8-dependent recycling promotes endosomal cholesterol removal in normal and sphingolipidosis cells.Mol Biol Cell. 2007; 18: 47-56Crossref PubMed Scopus (82) Google Scholar Third, targeting specific cell types such as the Purkinje cells for overexpression might give better results than ubiquitous overexpression of Rab9 as used in the current study. However, to fully develop the therapeutic potential of modulating membrane transport, future studies will need to concentrate on small molecule screens to identify compounds that can stimulate the intracellular transport machinery similar to Rab overexpression. Fourth, recent studies in treating NP-C in vivo have used multiple approaches with considerable success.8Langmade SJ Gale SE Frolov A Mohri I Suzuki K Mellon SH Walkley SU Covey DF Schaffer JE Ory DS Pregnane X receptor (PXR) activation: a mechanism for neuroprotection in a mouse model of Niemann-Pick C disease.Proc Natl Acad Sci USA. 2006; 103: 13807-13812Crossref PubMed Scopus (139) Google Scholar Thus, combined therapy using Rab overexpression and another approach such as substrate reduction may have a synergistic effect on outcome of this treatment. Finally, we note that the Rab9 transgenic mice developed in the current study may have other important applications beyond NP-C. These include studies of secretion in the intestine and/or liver, possible application to other lysosomal storage diseases, in vivo studies of the regulation of Rab9 expression and activity, and a source of various murine cell types with high levels of Rab9 expression. Download .zip (.6 MB) Help with zip files Supplementary Figures